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A Study Of The Mechanism Of Lead Chromate Induced Neoplastic Transformation Of C3H/10T1/2 Mouse Embryo Cells

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A Study Of The Mechanism Of Lead Chromate Induced Neoplastic Transformation Of C3H/10T1/2 Mouse Embryo Cells

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. H ie quality of this reproduction is dependent upon the quality of the copy submitted. B roken or indistinct print, colored or poor quality illustrations and photographs, print bleed through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright m aterial had to be removed, a note will indicate the deletion. Oversize m aterials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. H igher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 A STUDY OF THE MECHANISM OF LEAD CHROMATE INDUCED NEOPLASTIC TRANSFORMATION OF C3H/10T1/2 MOUSE EMBRYO CELLS by Michael Eric Dews A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Molecular Microbiology and Immunology) May 1995 Copyright 1995 Michael Eric Dews UMI Number: 9616950 UMI Microform 9616950 Copyright 1996, by UMI Company. AH rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA 90007 This dissertation, written by ............................Mi c . l ] a . ? . l . E r i c . D. e w5 . ..................... under the direction of h.is Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of re quirements for the degree of D O C TO R OF PH ILO SO PHY Dean of Graduate Studies Date .............. DISSERTATION COMMITTEE Chairperson DEDICATION This work is dedicated to my parents, Grace and Peter Dews, who inspired me to seek a career of learning and intellectual challenge, and who have always supported and encouraged my pursuits. ACKNOWLEDGMENTS I would like to thank my graduate advisor, Dr. Joseph Landolph, who facilitated my coming to USC and provided a great deal of guidance and support throughout my graduate studies. I deeply appreciate his significant effort in helping me develop my ability to think, to speak, and to write effectively and efficiently as a scientist. Dr. Landolph acted as a mentor in my development not only as a scientist but as a person, and provided a stimulating and congenial laboratory environment in which to work. I would like to thank the members of my dissertation committee, Dr. James Ou and Dr. Pradip Roy-Burman for their helpful discussions and suggestions throughout this work. I would also like to thank Dr. Stanley Tahara for his advice and support during my graduate studies at USC. I am grateful to my colleagues at USC for their friendship and scientific interaction, especially Drs. Lilliana Cerepnalkoski, Bushra Yasin, Steffan Ryter, and Josh Weng. I am grateful to Ed Little for his wit, wisdom, and skills in fermentation chemistry. I am particularly indebted to Laurent Ozbun, who collaborated with me to complete the m yc RNA half-life studies and the Northern Blotting survey analysis. I am grateful for the enthusiasm, dedication and excellent technical skill he brought to the laboratory, and for his friendship through trying times. I would also like to thank Dr. Kris Krishnan for his collaboration in performing m yc protein studies and his advice on Nuclear Run-on transcription analysis. I am most grateful to my wife, Christie, for her unwavering support, her patience and love throughout my graduate work. I appreciate her help editing this dissertation. I would like to thank my wife’s family for making me feel at home in Los Angeles. Finally, I am deeply thankful for the strong love and support of my parents and my family. TABLE OF CONTENTS Dedication ............................................................................................ ii Acknowledgments i i i List of Tables ........................................................................................ iv List of Figures ...................................................................................... v Chapter 1: Introduction ....................................................................... 1 Bibliography ....................................................................... 25 Chapter 2: General Survey Analysis of the Structure and Expression of Proto-Oncogenes and Tumor Suppressor Genes in the Transformed Cell Lines PbCr2 and PbCr3 Induced by Lead Chromate. Summary ......................................................................... 3 3 Introduction ..................................................................... 3 6 Materials and Methods .................................................. 45 Results ............................................................................. 5 3 Discussion ....................................................................... 1 01 Bibliography .................................................................... 106 Chapter 3: In Depth Analysis of c-myc Proto-Oncogene RNA and Protein Expression in Transformed Cell Lines PbCr2 and PbCr3. Summary ......................................................................... 11 7 i v Introduction ....................................................................... 119 Materials and Methods .................................................... 125 Results ............................................................................... 131 Discussion ................................ 153 Bibliography ....................................................................... 165 Chapter 4: Conclusions and Proposed Future Studies ............. 173 Bibliography ....................................................................... 179 v LIST OF TABLES 1 Chromium compounds................................................................. 4 2 Biological characterization of PbCr2 and PbCr3 cell lines........................................................................................ 44 3 Relative steady-state c-m yc RNA levels... .......................... 74 4 Relative steady-state RNA levels and transcript sizes of proto-oncogenes 7 9 5 Relative c-myc gene copy num ber.......................................... 83 6 Summary of restriction analysis of c-m y c ........................ 88 7 Relative steady-state RNA levels and transcript sizes of tumor suppressor g e n e s 9 8 8 Summary of survey of proto-oncogene and tumor suppressor gene expression and structure .......... 100 9 Nuclear run-on transcription analysis of c-m yc ........ 1 34 1 0 Estimated half-lives of c-m yc mRNAs in PbCr0 4 - and MCA-transformed cell lin e s 1 48 1 1 Steady-state levels of c-m yc protein ................................... 1 52 v i LIST OF FIGURES 1 Approaches for identifying and characterizing genes contributing to the transformed phenotype of PbCr0 4 -induced cell lines..................................................... 2 3 -2 4 2 Growth curves of 10T1/2 Cl 8, PbCr2 and PbCr3 cell lines in 850cm2 roller bottles .......................... 55 -5 6 3 Northern blot analysis of c-fos expression ...................... 59 -6 0 4 Northern blot analysis of c-abl expression ...................... 61 -6 2 5 Northern blot analysis of c-Ha-ras expression ............... 6 4 -6 5 6 Northern blot analysis of c-raflexpression ..................... 6 6 -6 7 7 Northern blot analysis of c-myc expression in cells at subconfluence................................................................. 69 -7 0 8 Northern blot analysis of c-myc expression in cells at mid-log phase................................................................. 72 -7 3 9 RNA slot blotting analysis of c-m yc RNA levels in cells at low and high density growth states ............... 7 6 -7 7 10 Partial restriction map of the mouse c-m yc gene ....... 80-81 1 1 Southern blot analysis of the c-m yc gene, BamH I & Hindi 1 1 digestio n .......................................................... 8 4 -8 5 1 2 Southern blot analysis of the c-m yc gene, Rsa\ & Xho\ digestion.................................................................. 8 6 -8 7 1 3 Northern blot analysis of p53 expression ............................. 9 1 -9 2 v i i 1 4 Southern blot analysis of p53 gene structure ................ 9 3 -9 4 1 5 Southern blot analysis of Rb gene structure ................... 96-97 1 6 Representative experiment of nuclear run-on transcription analysis of c-m y c .............................................. 1 32-1 33 1 7 Northern blot analysis of c-m yc RNA in PbCrO^and MCA transformed cell lines during actinomycin D chase........................................................................................... 1 37-1 39 1 8 Decay curves of c-myc mRNA in PbCr04 and MCA transformed cell lines................................................................. 1 40-1 42 1 9 Best-fit curves of the decay of separate fractions ofc-m ycm RNA........................................................................... 1 4 4 -1 4 6 2 0 Western Immunoblot analysis of c-m yc protein in transformed cell lines ............................................................... 1 49-1 50 v i i i Michael Eric Dews (Student) Joseph R, Landolph, Ph.D (Committee Chair) A Study of the Mechanisms of Lead Chromate Induced Neoplastic Transform ation of C3H/10T1/2 Mouse Embryo Cells The carcinogen lead chromate (PbC r04) induces neoplastic transformation of C3H/10T1/2 (10T1/2) mouse embryo cells. To understand the molecular mechanisms underlying this process, two PbCrC>4-transformed 10T1/2 cell lines, PbCr2 and PbCr3, were analyzed for alterations in the expression and/or structure of specific proto oncogenes and tumor suppressor genes which might contribute to their transformed phenotype. c -S is was undetectable in transformed or nontransformed 10T1/2 cell lines, c-Fos was expressed in 10T1/2 cells but its expression was greatly reduced in the PbCrCVtransformed cells. c-Abl, c-Ha-ras, and c- ra f expression were similar in PbCr0 4 -transformed and nontransformed 10T1/2 cell lines. PbCr2 and PbCr3 cell lines expressed 4.7-and 4-fold elevated steady-state levels of 2.3-kb c-m yc messenger RNA, without amplification or gross rearrangement of the c-m yc gene. Steady-state RNA levels and transcript sizes of tumor suppressor genes (Rb, p53, and APC) were similar in the lead chromate transformed cell lines. Transcription of c-m yc in PbCr2, PbCr3, and two 3- methylcholanthrene (MCA)-transformed cell lines which expressed elevated steady-state levels of c-m yc RNA was similar to that in 10T1/2 cells. A fraction (10-25%) of c-m yc RNA in PbCr04- and MCA-transformed cell lines had enhanced stability compared to the short-lived c-myc RNA of 10T1/2 cells. Elevated c-myc RNA levels were accompanied by higher steady-state levels of P 65Myc protein; 1.5-fold in PbCr2 cells and 1.3-fold in PbCr3 cells. MCA-transformed Cl 15 cells expressed 2.4-fold higher steady- state levels of P65Myc protein, but MCA Cl 16 cells expressed only 0.7-fold that of 10T1/2 cells. Therefore, enhanced stability of c-m yc transcripts causes elevated steady-state levels of c-myc RNA and protein, which likely contributes to the transformed phenotype. Furture work should address whether enhanced stabilization of myc transcripts is due to mutations in 375' mRNA destabilizing regions or alterations in trans-regulatory proteins which mediate c-myc RNA degradation. (Committee Chair) (Date) CHAPTER 1 INTRODUCTION Chromium is an essential trace element needed in minute quantities for insulin-related metabolic processes (1). Chromium deficiency from dietary intake can produce a condition similar to diabetes (1). When encountered at high concentrations it is also toxic, genotoxic, and carcinogenic in animal models and in humans (reviewed in 1-9). Indeed, the harmful effects associated with occupational exposure to chromium (Cr) compounds have been observed for more than 100 years (2). However, among the many useful chromium compounds, it is still unclear which pose the greatest toxic and carcinogenic risk and how these compounds exert their deleterious biological effects (reviewed in 1-9). It has long been observed that exposure to the extremely high concentrations of chromium compounds present in chromium- related industries is associated with a number of health risks. Excessive chromate exposure was first shown to cause large rounded perforations of the nasal septums of workers in the chromium industry (2). However, the most significant chromium- induced health risk is respiratory cancer (3). The first reported case of a chromate-exposed worker who developed cancer appeared in 1890. A 47-year-old worker exposed to chrome pigment was 1 diagnosed with adenocarcinoma of the left turbinate body (2). The first epidemiological study on cancer among chromium industry workers was carried out by Machle and Gregorius in 1948 (4). This study of workers in all U.S. plants producing chromates and bichromates found that 22% of all deaths were due to cancer of the respiratory system. The death rate for lung cancer was elevated 25-fold over that in workers not exposed to chromate, with a range of 18-50-fold for the different chromium processinf plants (4). A second study of these same plants by the Public Health Service in the 1950s found that a majority of the workers (57%) had perforated nasal septums, and there was a 29-fold increase in deaths from respiratory cancer (5). Since that time there have been dozens of epidemiological studies in several countries which have established a strong relationship between exposure to various chromium species and lung cancer in both chromate production and processing industries (reviewed in 2,6). Chromium, which can exist in oxidation states ranging from -2 to +6, occurs in nature mainly as iron dichromite or ferrous dichrom ite (FeO C^Oa), where it is in the trivalent state, designated C r3+ or Cr(lll), which is the most stable oxidation state (7). Cr also commonly occurs in the divalent or chromous state and in the hexavalent chromate state, Cr6+ or Cr(VI). Complexes containing the very unstable tetra- [Cr(IV)] and pentavalent Cr(V) states are known to exist as transitory intermediates in the reduction of Cr(Vl)(2). However, among these different valence states, only the 2 trivalent and hexavalent compounds are stable in the environment and therefore of importance in terms of human exposure (8). Both the trivalent and hexavalent chromium compounds presenting health risk in industrial settings occur in a wide range of solubilities, although the Cr(lll) compounds tend to be mostly of low solubility (T able 1)(9). In the occupational setting, exposure occurs by inhalation or skin contact with chromium-containing mist, dust, or fumes or via direct contact with aqueous chromium solutions (10). Potentially hazardous exposure to chromium compounds may occur in five main types of industry: (i) in the metal refining and chromate production industries; (ii) in the production of pigments for paints, dyes and tanning; (iii) in the electroplating industry; (iv) in the metallurgic industry producing ferrochromium alloys; and (v) in the manufacture of refractory materials, such as bricks, glass, ceramics, and certain ferrous metals (11). There is also exposure of welders of stainless steel to fumes containing chromium of several oxidation states (12). The vast majority of data on chromium carcinogenicity has come from the chromate production industry, which converts raw chromite ore (FeOC^Oa) into usable compounds (2). Exposures in this industry are to a wide variety of trivalent and hexavalent chromium compounds, most notably, chromite(lll) ore, sodium or potassium chromate(VI) and dichromate(VI), calcium chromate, and chromium(VI) trioxide (10). 3 Table 1 Chromium Compounds Compound Formula Valence Aqueous solubility Chromium Cr 0 Insoluble Chromium acetate Cr(COOCH3)3-H20 3 Soluble Chromium carbonate Cr203-C0 2 -H20 3 Slight Chromium phosphate CrP04 3 Insoluble Chromic Oxide C12O3 3 Insoluble Calcium chromate CaCr04 6 Slight Chromium trioxide 0 0 3 6 Soluble Lead chromate PbCr04 6 Insouble Potassium dichromate K2O2O 7 6 Soluble Potassium chromate K2C1O4 6 Soluble Sodium dichromate Na2Cr207 6 Soluble Sodium chromate Na2Cr04 6 Soluble Barium chromate BaCr0 4 6 Insoluble Strontium chromate SrCr04 6 Insoluble Zinc chromate ZnCr04 6 Insoluble 4 There is also epidemiological evidence that exposure of humans to compounds used in the chrome-pigment manufacturing industry is associated with an increased risk of bronchial carcinoma, although there are fewer studies documenting this risk (12). Workers involved in chromate pigment production are exposed primarily to hexavalent lead and zinc chromates, although other pigments containing strontium chromate, barium chromate, and hydrated chromic oxide are also produced, and worders are also exposed to these compounds (6). There is less information available from the chrome-plating, ferro-chromium production, and refractory material production industries, but the available data also supports an association between worker exposure and increased risk of lung cancer, although the risk appears to be smaller than in either the chromate production or pigment industries (9). Epidemiological data from industries that utilize chromium has clearly established a relationship between exposure to chromium salts and lung cancer (reviewed in 1-9). However, because workers in chromium-related industries are often exposed simultaneously to a number of compounds, such studies have not permitted the identification of specific Cr compounds as carcinogens (12). The data does support the hypothesis that Cr(VI) compounds are carcinogenic, and among these, chromates of low water solubility are the most carcinogenic, although a contribution by Cr(lll) cannot be ruled out by such studies (2,6,12,13). 5 The role of valence in this hypothesis is supported by knowledge of the cellular uptake and metabolism of chromium compounds. Hexavalent chromium readily enters cells, whereas cells are relatively impermeable to trivalent chromium (14). Cr(VI) as a tetrahedral chromate oxyanion enters cells through normal active transport carrier systems for sulfate and phosphate anions, which are somewhat non-specific (15). In contrast, the C r(lll) ion, being predominantly octahedral, crosses membranes only very slowly by simple diffusion (15). Once inside the cell, Cr(VI) is rapidly reduced through relatively unstable Cr(V) and Cr(IV) intermediates to Cr(lll), and stable C r(lll) complexes are formed (9,15). This suggests that the ultimate genotoxic agent resulting from exposure to Cr(VI) is either Cr(lll), the reactive intermediates Cr(V) or Cr(IV), active oxygen species generated during reduction of Cr(VI), or a combination of these factors (16). Further work is needed to define the ultimate carcinogens generated from Cr(VI). The solubility of Cr compounds is also believed play an important although less well defined role in determining carcinogenic potency. If inhaled Cr(VI) particles having low solubility are cleared less efficiently from the bronchial tree than those Cr(VI) compounds which are easily soluble in biological fluids, this might result in long-term exposure of bronchial epithelial cells to slowly dissolving particles and hence to higher doses of chromate ions (9,13,17,18). It is believed that prolonged delivery of an optimal concentration of chromate ions to target 6 cells is critical for the development of malignancy (19). This hypothesis is supported by the finding of deposits of chromium particles in the bronchial bifurcations of exchromate workers even 10 years after cessation of exposure (20). In order to test hypotheses concerning the roles of valence and solubility in chromium compound carcinogenicity and to gain insight into the potency of individual compounds, Cr(VI) and C r(lll) compounds of varying solubilities as well as chromium metal have been tested for carcinogenicity by a wide variety of routes in mice, rats and rabbits. The data from these experiments was recently reviewed by the IARC Working Group on the Evaluation of Carcinogenic Risks to Humans (12). They concluded that there is sufficient evidence for the carcinogenicity of calcium chromate, zinc chromates, strontium chromate and lead chromate in experimental animals. They found there was limited evidence for the carcinogenicity of chromium trioxide (chromic acid) and sodium dichromate. It was also determined that there was inadequate evidence for the carcinogenicity of metallic chromium, barium chromate and chrom ium (lll) compounds (12). These findings support the hypothesized inactivity of Cr(lll) compounds as carcinogens and the greater potency of the less soluble hexavalent chromates. Because of the strong evidence of carcinogenicity of certain chromium compounds from epidemiological studies and animal experiments, a significant effort has been made to understand the mechanisms of chromium-induced genotoxicity and carcinogenesis 7 using in vitro mutagenesis and mammalian cell transformation assays. An overall evaluation of this data supports the findings on the carcinogenicity of chromium in humans and experimental animals and also demonstrates the inactivity of Cr(0) and of C r(lll) compounds in contrast to the positive activity of Cr(VI) compounds (3,9,10,13,16, reviewed in 21-23,26,32). Chromium(VI) compounds are convincingly mutagenic in bacteria in a number of assay systems, whereas trivalent chromium salts are found to be non- mutagenic or very weakly mutagenic activity in bacterial systems (10). This difference is further supported by observations that the mutagenicity of Cr(VI) is decreased or abolished when hexavalent salts are reduced to the trivalent form with chemical or biological reducing agents (24). On the other hand, oxidation of the trivalent form to the hexavalent form greatly enhances its mutagenicity (25). In terms of solubility, the highly soluble hexavalent chromates are strongly mutagenic in bacteria, consistent with bioavailability of chromate ions resulting from the salt dissolution (21). Fewer tests of the poorly soluble chromates have been performed, and the results tend to depend on the degree of solubilization of the compound and thus the concentration of chromate ion. For instance, relatively insoluble lead chromate is not mutagenic in bacteria unless it is first dissolved in acid or alkali or unless the chelating agent nitrilotriacetic acid is added to the test medium to augment its solubility (12). 8 The genotoxicity of both trivalent and hexavalent chromium compounds have been extensively studied in mammalian cells (reviewed in 22,26,32). These data strongly resemble the results of bacterial assays and indicate that the hexavalent salts are potent mutagens and clastogens and the trivalent compounds are usually inactive (12). The positive results seen with some C(lll) compounds tended to occur at concentrations 10- to 1,000-fold higher than required for Cr(VI) (21,27). Further, it is believed that positive results in tests of trivalent species are due to contamination with trace amounts of Cr(VI), nonspecific effects of high doses, and penetration of chromium(lll) by endocytosis after long exposure in vitro (12). However, Cr(lll) compounds did induce a variety of genetic effects, including DNA-protein crosslinks in acellular or subcellular targets. This is consistent with the hypothesis that Cr(VI) traverses the cell membrane into the cytoplasm, where it is reduced to Cr(lll), which exerts genetic effects. There is ample evidence for the genotoxic effects of Cr(VI) compounds in cultured cells, mostly from studies of the water soluble chromium compounds (27,28,29). Cr(VI) compounds of both medium and high solubility have been found to induce a broad range of genetic effects in cultured animal cells, including DNA damage, forward mutations, sister-chromatid exchanges, micronuclei and chromosomal aberrations (21). The limited studies on the less soluble Cr(VI) compounds, particularly lead chromate, suggests that they may be less mutagenic than the highly soluble chromates, in agreement 9 with results using particulate forms of these compounds in bacterial assays (21,29). However, like the soluble chromates, the less soluble Cr(VI) compounds are clastogenic, causing both chromosome aberrations and sister chromatid exchanges in cultured cells (21). The ability to induce morphological transformation of cells in culture provides some index of the carcinogenic potential of chemical agents, and at the same time provides a cellular system to investigate the molecular pathways and targets of particular chemicals (reviewed in 26, 30-32). A number of studies using a variety of cell systems and biological endpoints (eg. focus formation, anchorage independence), have demonstrated that soluble, moderately soluble, and insoluble Cr(VI) compounds, and some C r(lll) compounds, induce morphological transformation in rodent and human cells (reviewed in 21,22,26,32,33). Studies using several different mammalian cell systems have demonstrated the DNA damaging and transforming activity of soluble Cr(VI) compounds, in contrast to that of soluble C r(lll) compounds. Soluble K2Cr2 0 7 was able to transform mouse embryo cells more efficiently than trivalent CrCb (34). Both potassium dichromate and soluble hexavalent Cr0 3 induced a concentration- dependent incidence of morphological transformation in Syrian hamster embryo cells and were found to cause extensive chromosome damage (34). This DNA damage was effectively inhibited by the addition of a reducing agent to the medium (35). 1 0 Casto et al. found that soluble potassium chromate and moderately soluble calcium chromate significantly enhanced the adenovirus SA7 mediated viral transformation of Syrian hamster embryo cells, although not as well as the poorly soluble zinc chromate or lead chromate (36). Bianchi et al. showed that soluble l<2Cr20 7 and moderately soluble CaCrC>4, but not soluble trivalent CrCl3, induced a dose-dependent increase in anchorage-independent growth of BHK cells (37). K2Cr2 0 7 but not CrCb also increased the frequency of sister chromatid exchange (SCE) in hamster and mouse cells (37). Calcium chromate also induced morphological transformation of Balb/3T3 cells, Syrian hamster embryo cells, virus-infected rat embryo cells (38), and anchorage independence in BHK21 cells (39). Soluble sodium chromate induced morphological transformation of Syrian hamster cells (40). Previous work in our laboratory also showed that sodium chromate induced anchorage-independent (Al) transformation of normal human diploid fibroblasts (HFC) (27,41). This work also showed that Al was also induced by hexavalent Cr2 0 3 , CaCr0 4 , CaCr2 0 7 , K2Cr2C >7 and PbCrC>4. The soluble chromates, but not insoluble PbCr0 4 , were also weakly mutagenic at the hypoxanthine- guanine phosphoribosyl transferase (hgprt) locus, inducing mutation to 6-thioguanine resistance (6-TGr) (27). Interestingly, these studies also found that several trivalent chromium compounds, both soluble and insoluble, were able to induce cytotoxicity, Al transformation, and mutagenicity in HFC 1 1 over the same concentration range as the hexavalent compounds (27). Moreover, the solubility of the Cr(lll) compounds only slightly influenced their ability to induce Al in HFC, with an insoluble form of CrCl3 being 1.3- to 3-fold more active than soluble CrCl3(27). Both soluble and insoluble Cr(lll) compounds induced weak (2- to 5- fold) increases in mutation to 6-TGr in HFC. However, the weak mutation induced by the soluble Cr(lll) only occurred at concentrations that were 250-fold higher than those that induce Al or those of Cr(VI) compounds that induce cytotoxicity, mutation and Al (27). In addition, the mutagenicity of both soluble and insoluble Cr(lll) compounds occurred only at 1000-fold higher concentrations than that required for induction of Al (27). Thus, the mutations observed after treatment with Cr(lll) might not be representative of the DNA damage which induced transformation (27). Rather, it was hypothesized that chromium(lll), and perhaps insoluble, non- mutagenic PbCrC>4, may cause Al in HFC by inducing perturbations in membrane structure or membrane damage resulting in the generation of lipid metabolites or DNA damaging oxygen radicals (27, reviewed in 42). In evaluating the data from studies of cell transformation, it is clear that water-soluble chromates and some trivalent chromium compounds are far more active in vitro than when assayed for carcinogenicity in experimental animals. One explanation for the observed effects of soluble Cr(VI) is that high concentrations of C r6+ released by dissolved chromate that are achievable in cell 1 2 culture would be removed rapidly in vivo through urinary excretion (17). The effects of soluble Cr(lll) on cells in culture observed in some studies may result from non-specific effects of very high concentrations of Cr3+ and may not reflect the biological effects of these compounds in vivo (28). Cell culture studies have provided direct support for the model of uptake of Cr(VI) on the sulfate anion transport carrier and its reduction to C r(lll) mentioned previously. Cellular uptake of chromate in vitro is at least 10-fold greater than uptake of C r(lll) from equimolar solutions (43,44). These studies have also shown that intracellular reduction of chromate to C r(lll) also does occur (17,43). Several studies have used in vitro cytotoxicity and cell transformation assays to study the mechanisms of cell transformation by those compounds for which there is the strongest evidence for carcinogenicity in experimental animals. These compounds include the moderately soluble calcium chromate and the sparingly soluble Pb, Zn, and Sr chromates. Elias et al. found that all of these compounds induced morphological transformation of Syrian hamster embryo (SHE) cells in a dose dependent manner (45,46). In these studies, particles of Ca, Sr, and Zn chromates were completely solubilized in one day in cell culture conditions, giving rise to the chromate anion (CrC>42-> (45). Pb chromate was much less soluble, with only 20-36% of the Cr being released after 7 days in culture (46). The rates of solubilization of all chromates 1 3 was augmented in the presence of cells versus culture medium alone (46). The enhancements of transformation frequencies caused by treatm ents with the completely solubilized chromates were dependent on the concentration of total chromium per cell. This implies that the intracellular soluble total Cr concentration is responsible for the transforming activity, irrespective of the type of chromium salt (either Ca, Sr, or Zn chromate) (46). For cells treated with Pb chromate, the intracellular Cr concentration was much higher than that observed for the other Cr compounds, apparently due to intracellular phagocytosis of large amounts of chromium on unsolubilized PbCr0 4 (46). Interestingly, the transformation frequency induced by Pb chromate was, for the same concentrations of Cr/cell, 9-fold higher than that of the other compounds (46). This suggested a contributing role for Pb ions in the transform ation of cells by Pb chromate. This was further supported by the finding that a double treatment of Cr as CaCr0 4 and Pb as Pb(N0 3 )2 induced transformation of SHE cells at the same frequency as Pb chromate (46). C3H/10T1/2 Cl 8 (10T1/2) is an aneuploid, immortal, contact-inhibited, nontumorigenic mouse embryo cell line (47). 10T1/2 cells have a low frequency of spontaneous transformation and exhibit a high frequency of morphological transformation when treated with chemicals and radiations. These cells thus provide a very useful system for detecting carcinogens by the ability to induce m orphological transformation and for studying the molecular 1 4 mechanisms of chem ically induced neoplastic transformation (reviewed in 30,31). Taking advantage of this assay, our laboratory compared the cytotoxic, mutagenic, and cell transforming activities of particulate lead chromate with soluble potassium dichrom ate and moderately soluble calcium chromate and strontium chromate in 10T1/2 cells. Lead chromate induced a low but dose-dependent and reproducible frequency of type III morphological transform ation (29), the strongest type of morphological transformation in these cells (reviewed in 30). The transformed cells stably maintained a focus-forming phenotype, grew in soft agarose, and formed fibrosarcomas when injected subdermally into nude mice (29). Potassium dichromate, calcium chromate, and strontium chromate surprisingly did not induce morphological transform ation in 10T1/2 cells (29). Strontium chromate was slightly soluble in culture medium but eventually dissolved (29) as previously reported (46). Calcium chromate was assayed both in solubilized form and as a particulate (by suspending it in acetone), and neither form induced morphological transformation. Lead chromate was not very soluble, as determined by visual observation of many insoluble lead chromate particles, but lead chromate-treated cells had a large number of vacuoles and extruded cytoplasm over the lead chromate particles, suggesting that internalization of particles by phagocytosis did occur (29, reviewed in 22,26). It is significant that lead chrom ate-induced morphological transformation but did not induce mutation to either ouabain 1 5 resistance in either 10T1/2 or CHO cells or 6-thioguanine resistance in CHO cells (29). In contrast, calcium chromate, which did not induce morphological transformation in 10T1/2 cells, and did not induce mutation to ouabain resistance in 10T1/2 or CHO cells, did induce mutation to 6-thioguanine resistance in CHO cells (29, reviewed in 22,26). It appears from this work that unique physicochemical properties of lead chromate were responsible for its uptake, likely by phagocytosis, and its ability to induce cytotoxicity and cell transform ation (29, reviewed in 22,26). Cell transformation by lead chromate is not likely to be due solely to the result of chromate anion crossing the cell membrane, nor due to the types of mutations detected by the assays employed in this study, namely small deletion, fram eshift or point mutations (29, reviewed in 22,26). Therefore we asked the question, how does lead chromate induce neoplastic transform ation? One hypothesis to explain the transforming activity of lead chrom ate suggests that as cells attempt to internalize large particles of PbCr0 4 , the abortive phagocytosis causes the release of lysosomal hydrolases which may then damage chromatin structure and function (29). This idea is supported by the finding that human osteosarcoma cells (HOS) are transformed to anchorage independence and tumorigenicity by lead chromate. Sidhu et al. showed that cells treated with PbCr0 4 rapidly phagocytosed PbCr0 4 1 6 particles, which were later seen by electron microscopy within membrane bound vacuoles in the cytoplasm and the nucleus (48). There is evidence that hexavalent chromium compounds of all solubilities induce genotoxicity and transform cells in culture (reviewed in 22,26,32). Our present understanding of the mechanisms of these processes is based upon several observations. It is known that the chromate anion is readily taken up via the sulfate anion transport system (49) and, once inside, is reduced by one or more of the cellular reducing systems including ascorbate and glutathione reductase to chromium(V) and ultimately to chrom ium (lll) (50). Chromium(lll) binds to purified DNA in vitro, forming Cr-DNA adducts (51) and DNA-DNA cross-links (52,53). Chromium is also thought to interact directly with DNA in vivo, forming adducts (51), DNA interstrand cross-links (54) and DNA- protein cross-links (51,55). Chromium(V) has also been shown to bind to DNA (56). In addition, the process of reduction of chromium(VI) to chrom ium (lll) generates radical oxygen species, primarily hydroxyl radicals, which have the ability to cleave DNA, cause oxidative DNA base damage, influence DNA-protein cross- linking, or cause lipid peroxidation (57). However, It is not known what types of chromium-induced DNA damage correlate with in vitro cell transformation or carcinogenesis, nor is it clear which intracellular forms of chromium or byproducts of chromium reduction are the ultimate carcinogenic and/or mutagenic species (21,37, reviewed in 1 7 22,26,32). Furthermore, the intracellular reduction of chromate can occur by several different pathways, each of which may produce different endpoints (23). Because of its insoluble particulate nature and the possible contribution of lead ion toxicity to lead chromate toxicity, lead chromate poses yet further complexities upon the mechanistic considerations of soluble chromates. It is likely that the lead and chromium together account for the carcinogenicity of lead chromate (27,29, reviewed in 22,26,32). Although particulate lead chromate is inactive as a mutagen in cultured cells in most assays, several studies have shown it to be a potent clastogen. Lead chromate was found to damage chromosomes of both Chinese hamster ovary (CHO) and human foreskin fibroblasts in a dose dependent fashion (58). Analysis of the types of DNA damage induced in CHO cells revealed both single strand breaks (SSB) and DNA-protein crosslinks (DPC). The methods used in this study were not sensitive enough to detect low levels of DNA double-strand breaks (59). The authors noted that the generation of DPC were unlikely to result from a physical interaction between insoluble particles and DNA, suggesting that at least some of the genotoxicity of lead chromate could be attributed to the generation of a chemically reactive species. They further suggested that ionic chromate was most likely involved, although a role for reactive oxygen could not be excluded (59). 1 8 While it seems clear that cellular exposure to lead chromate leads to DNA damage, the mechanism or mechanisms that are responsible have not been defined. It appears likely that lead chromate particles may need to be internalized to induce genotoxicity (29, reviewed in 22,26,32). However, it is not known whether the physical interaction of the insoluble particle with the cell may cause such distortions as to disrupt chromatin structure or result in the release of DNA damaging enzymes. If internalization is necessary, it is not known whether the intracellular dissolution of lead chromate is responsible for genotoxicity. If intracellular dissolution occurs, it is not clear whether the Cr3+ ion is the DNA damaging agent or whether Cr5+, and/or reactive oxygen are involved. There is also evidence that both the Pb and Cr ions may contribute to the transforming potency of lead chromate (29,46, reviewed in 22,26,32). Pigments containing lead chromate are considered to be the most versatile of the inorganic pigments and are found in many formulations designed for a wide spectrum of uses (10). However, occupational exposure to lead chromate is associated with the development of human respiratory cancer (12). Lead chromate is also one of the most potent carcinogenic chromium compounds in experimental animals (60,61). Lead chromate is interesting for its ability to induce morphological transformation of cultured cells without causing detectable mutagenicity (29). It is therefore important to understand the molecular mechanisms of lead- 1 9 chromate induced morphological transformation as a step toward understanding lead chromate carcinogenesis. The current understanding of carcinogenesis is of a multistep process involving genetic alterations in a subset of genes whose expression either acts to drive cells toward replication (cellular proto-oncogenes) or to suppress cell division (tumor suppressor genes) (reviewed in 62-64). Carcinogens such as lead chromate are thought to act by inducing DNA damage at these specific loci, which leads to alterations in gene expression and eventually to altered growth characteristics (reviewed in 22,26,32). The mutational spectra of chemical and physical carcinogens is influenced by the nature of the compound. For instance, induction of skin carcinomas by UV light is indicated by the occurrence of mutations in the p53 gene involving pyrimidine dimers (65). Certain proto-oncogenes such as the ras GTPase can be activated by point mutations (66). Therefore, one might not expect that ras would be activated by "non-mutagenic" carcinogens such as lead chromate, unless oxidative DNA damage generated by chromium-induced oxygen radicals is involved (proposed in 27), and they cause deletions in 3' or 5' cis acting negative regulatory regions of proto-oncogenes activating them to oncogenes (reviewed in 22,26,32). At present, there is nothing known about the relationship between lead chromate or carcinogenic chromates in general and oncogenes (reviewed in 22,26). It is important to our understanding of the molecular mechanisms of carcinogenesis by these compounds 20 to determine whether they cause mutations which activate proto oncogenes or inactivate tumor suppressor genes to induce cell tra n s fo rm a tio n . Our laboratory has cloned two cell lines of morphologically transformed, anchorage independent and tumorigenic 10T1/2 cells induced by lead chromate. These cell lines, designated PbCr2 and PbCr3 have been biologically characterized in our laboratory by Patierno et al. (29,67). Both cell lines gave rise to foci when plated with nontransform ed 10T1/2 cells, grew to significantly higher saturation density than 10T1/2 cells, grew in soft agar, and gave rise to progressively growing fibrosarcomas when injected into nude mice (29). The purpose of this thesis project is to investigate the molecular mechanisms of lead chromate-induced neoplastic transformation of 10T1/2 cells. To this end, I tested the hypotheses that cell transform ation by lead chromate results from 1) amplification or 2) rearrangements of, or 3) activating deletions in single or multiple, specific proto-oncogenes, and/or 4) deletion and/or 5) point mutational inactivation of tumor suppressor genes, and 6) that these alterations are responsible for maintenance of the transformed phenotypes of the PbCr2 and PbCr3 ce lls. There is a rapidly expanding list of growth related genes which might be targets for the genotoxic and transforming effects of PbCrC>4 or its by-products. Thus, it might be difficult to detect 21 the particular targets of PbCrO/Hnduced cell transformation. In order to increase our chances of identifying relevant genetic alterations, we decided to employ several different approaches to pursue this problem. Our overall stratagem is depicted in figure 1. The first approach is to use Northern and Southern blot hybridization techniques to conduct a broad survey of tumor suppressor and proto-oncogenes for gross alterations in their structure or expression. If alterations are detected, they will be characterized in terms of their possible contribution to the transformed phenotype and implications for the mechanism of induction by PbCrC>4. I concentrated most of my effort on this approach. The second approach is to transfect genomic DNA from the PbCr0 4 -transformed 10T1/2 cells into an appropriate recipient cell line and identify activated oncogenes within DNA segments which induce transformation in the recipient cells or secondary transfectants. Our third approach is to identify affected genes by their differential expression (over-expressed proto-oncogenes, under-expressed tumor suppressor genes) between nontransformed and lead chromate transformed cells by constructing and screening subtracted cDNA libraries. 22 Figure 1 Approaches to identifying and characterizing genetic alterations contributing to the transformed phenotype of PbCr0 4 -induced morphologically transformed C3H/10T1/2 cell lines. 23 APPROACHES [1] [2] [3] Survey known jroto-oncogenes/ T. S. genes Construct cDNA libraries Northern Blot -transcript size -steady-state level Southern Blot -gene amplification -gross structural alterations Genomic DNA transfection -Assay for transf. -identify dominantly acting oncogenes (eg. ras ) Analyze mechanism of alterations Protein expression? Isolate altered cDNA clone biological activity Subtractive hybridization \ |PCR, Sequencel Clone & characterize overrepresented, underrepresented messages BIBLIOGRAPHY 1. Wallach, S. 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Res., 21: 1989. 32 CHAPTER 2 GENERAL SURVEY ANALYSIS OF THE STRUCTURE AND EXPRESSION OF PROTO-ONCOGENES AND TUMOR SUPPRESSOR GENES IN THE TRANSFORMED CELL LINES PbCr2 AND PbCr3 INDUCED BY LEAD CHROMATE SUMMARY Two morphologically transformed C3H/10T1/2 Cl 8 (10T1/2) cell lines derived from foci induced by the carcinogen lead chromate were evaluated to determine whether there were increased steady-state levels of RNA of specific cellular proto-oncogenes and/or decreased steady-state levels of RNA of specific tumor suppressor genes. We also studied whether specific proto-oncogenes were amplified or rearranged, or whether specific tumor suppressor genes were deleted in part or in whole in the transform ed cell lines. Expression of c-sis was not detected in the transformed cell lines or in nontransformed 10T1/2 cells, c-fos was expressed in 10T1/2 cells but was not detectable or greatly reduced in the PbC r04-tra n s fo rm e d cell lines. Expression of c-abl, c-H a-ras, and c-raf were 33 sim ilar in the PbCr0 4 -transform ed ceii lines and nontransform ed 10T1/2 cells. Interestingly, both PbCr0 4 -induced cell lines and two 3-m ethylcholanthrene (MCA) transform ed cell lines (MCA Cl 15 and MCA Cl 16) exhibited markedly elevated steady- state levels of the 2.3-kb c-myc messenger RNA. The PbCr2 cell line expressed 4.7-fold higher steady-state levels of c-myc RNA during log phase and 7-fold higher steady-state levels at subconfluence compared to 10T1/2 cells PbCr3 had 4-fold and 8-fold higher steady-state levels of c-myc RNA at low and high cell density respectively compared to 10T1/2 cells. The two MCA- transform ed cell lines expressed approxim ately 2-fold higher steady-state levels of c-myc mRNA during log phase and 8-fold higher steady-state levels at subconfluence. The size of the c-myc transcript was the same in nontransformed 10T1/2 cells and in the four transform ed cell lines, 2.3 kb. Southern blotting analysis showed no am plification or gross rearrangement of the c-myc gene in either PbCr(>4- induced cell line. An understanding of the mechanism responsible for enhanced expression of c-myc observed in this study will require exam ination of the regulation of myc RNA synthesis and degradation rates in the transform ed cells. 34 The steady-state RNA levels and transcript sizes of the tumor suppressor genes Rb, p53, and APC were the same in the two lead chromate transformed cell lines and the MCA transformed cell lines. Further, there were no gross structural alterations in the Rb or p53 genes in either the PbCrO Hnduced transformed cell lines or in the M CA-induced clones. These studies show that enhanced expression of c- m y c RNA correlate with the transformed state of 10T1/2 cells induced by PbCrC>4 and MCA. Elevated c-myc RNA levels may contribute to maintaining the transformed phenotype in these cell lines. 35 INTRODUCTION Chromium is an essential trace element and specific chromium compounds are needed at low levels for insulin metabolism (1). The daily intake of chromium from food of roughly 0.03-0.1 mg is adequate to fulfill this need (1). However, prolonged exposure to the very high concentrations of certain chromium compounds present in chromium-related industries (up to 2 mg/m3) is associated with a number of health risks, most notably lung cancer (2,3). In fact, numerous epidemiological studies have documented a high incidence of respiratory tract cancer in chromate workers in various countries (reviewed in 2, 4-6). Despite this evidence, such studies have not been able to identify individual species as carcinogens because workers are usually exposed sim ultaneously to several different chromium compounds. These studies have suggested that hexavalent compounds of medium or low solubility may be the more potent carcinogens (3,7). Experimental animal studies have confirmed that a number of chromium compounds are animal carcinogens and support the evidence implicating the less soluble chromium(VI) compounds as carcinogens (4). To further clarify the issues of oxidation state, solubility, and mechanisms of chromium carcinogenesis, the mutagenicity of chromium in bacteria and the mutagenicity and transformation of mammalian cells in culture has been investigated by a number of 36 laboratories, including our own. These studies showed that most hexavalent chromium salts are strong mutagens to bacterial and mammalian cells and induce morphological transformation of cultured cells, while the trivalent compounds are weak or inactive (reviewed in 7-9). The reason for this difference is that the oxyanion (CrCU2") of soluble chromium(VI) is readily taken up by cells through the sulfate anion transport system, while the soluble chrom ium (lll) is excluded from cells (6,10,11). For insoluble hexavalent Cr(VI) compounds, there is evidence that the compounds are phagocytosed, depositing a bolus of chromium in the cells (12) (reviewed in 8,9). Among the hexavalent compounds, animal studies have shown that those of medium or low water solubility are the most potent carcinogens (13). However, these data are not reflected so clearly by in vitro experiments which have shown that soluble Cr(VI) compounds are able to induce mutation and morphological transformation in a variety of cell types and using a number of different endpoints (reviewed in 8,9,13-16). Cells in vivo may be exposed to more Cr6+ from the slow dissolution of less soluble hexavalent chromate than from the highly soluble chromium compounds, which would likely be cleared from tissues rapidly. In contrast, it is likely that cultured cells would be subject to prolonged exposure to solubilized chromium(VI) (17). Upon uptake, chromium(VI) is eventually reduced intracellularly to chrom ium (lll) by cellular metabolizing systems, including the microsomal electron-transport cytochrome p-450 37 system (11). Intracellularly produced chrom ium (lll) binds to nucleic acids, proteins, and small molecules such as nucleotides (18). The chrom ium (lll) bound to DNA and/or protein induces damage to the DNA that eventually leads to mutation and hence cancer, and thus is considered to be an ultimate carcinogen (19). The intermediate chromium(V) has also been suggested to participate in chromium(VI) induced genotoxicity (7). In addition, recent evidence has shown that the reduction process may itself contribute to chromium carcinogenicity by the production of reactive oxygen species and resultant DNA damage (20-24). Lead chromate is a hexavalent compound of very low solubility (0.58 mg/1 at 25° C or 1.8 pM) which is extremely useful in the manufacture of orange and yellow pigments for paints (4). Similar to other hexavalent chromium salts of low solubility, lead chromate is a suspected human carcinogen (4). Indeed, an increased risk of respiratory cancer has been established for workers in the chrome- pigm ent producing industry, where workers are exposed primarily to lead chromate (25-28). Also, several studies have demonstrated that lead chromate is a potent carcinogen in rats when administered either intra-m uscularly or subcutaneously (29,30). Despite the carcinogenic potency implied by these studies, there is little knowledge of the molecular mechanisms of cell transformation caused by chromium(VI) compounds in general or by lead chromate in particular. 38 To gain a better understanding of how chromium compounds cause cancer, our laboratory examined and compared the in vitro cytotoxic, mutagenic, and cell transforming activities of several soluble and insoluble hexavalent chromium compounds, including lead chromate, in cultured C3H/10T1/2 mouse embryo cells (12). C3H/10T1/2 Cl 8 is a permanent aneuploid, mouse embryo cell line that has a low frequency of spontaneous transformation and can be transformed by many chemical carcinogens (31,32). In this study insoluble lead chromate, but not soluble calcium chromate or potassium dichromate, induced morphological transform ation of 10T1/2 Cl 8 mouse embryo cells (12). Several foci of m orphologically transformed cells arising from dishes of 10T1/2 cells treated with PbCrC> 4 were cloned and expanded. When biologically characterized, these clones grew to significantly higher saturation densities than 10T1/2 cells, grew in soft agar, and gave rise to progressively growing fibrosarcomas when injected into nude mice (12, reviewed in 8,9). Interestingly, lead chromate did not induce mutation to either 6-thioguanine resistance in 10T1/2 cells or ouabain resistance in CHO or 10T1/2 cells (12) or to 6-thioguanine resistance in diploid human fibroblasts (33) (reviewed in 8,9). Therefore, lead chromate induced morphologic, anchorage-independent and neoplastic transform ation of 10T1/2 cells by a mechanism not involving the base substitution, fram eshift, or small deletion mutations that would be measured in these mutations assays (reviewed in 8,9). We 39 are also considering mechanisms involving oxygen radical generated DNA damage and mutation which these assays do not measure well (33) (reviewed in 8,9). Other studies have shown that lead chrom ate induced morphological transformation of Syrian hamster embryo (SHE) cells (34) and human osteosarcoma cells (35) our laboratory has shown that lead chromate induced anchorage- independent growth in human diploid fibroblasts (21,33, reviewed in 8,9). These studies demonstrating the transforming ability of lead chromate, yet its lack of mutagenicity in assays for Ouar and 6TGr, suggest that due to its distinctive physicochemical properties it may exhibit unique mechanisms of genotoxicity. The insolubility of particulate lead chromate is such that a large intracellular accum ulation of intracellular chromium may result predominantly from phagocytosis of lead chromate particles by cells rather than by transport of the Cr6+ oxyanion (12). Electron micrographs of 10T1/2 cells treated with PbCr0 4 revealed cells extending plasma membrane over the particles, suggesting that cells were attempting to phagocytose the material (12). Cells were also seen with lead chrom ate particles within intracellular compartments. Others have shown that lead chromate is phagocytosed and accumulates within vacuoles in the cytoplasm of cultured human osteosarcoma cells (35). In addition, studies have shown that the DNA lesions produced by treating cells with lead chromate are not induced by media conditioned with lead chromate, implying a requirement for cell 40 contact with the particles (36). This does not, however, rule out the possible contribution of Cr6+ generated from lead chromate dissolution in close contact with the cell membrane. Although lead chromate is not mutagenic in some assays, several studies have shown it to be a potent clastogen. Lead chromate causes chromosome damage in both Chinese hamster ovary (CHO) and human foreskin fibroblasts in a dose dependent fashion (36). Analysis of the types of DNA damage induced in CHO cells revealed both single-strand breaks (SSB) and DNA-protein crosslinks (DPC) (36). While it appears clear that cellular exposure to lead chromate leads to DNA damage, the mechanisms that are responsible for DNA damage have not been defined. Lead chromate particles may likely need to be internalized to induce genotoxicity (12,33, reviewed in 8,9). It is not known whether the physical interaction of the insoluble particle with the cell may cause such distortions as to disrupt chromatin structure or result in the release of DNA damaging enzymes. It is also not known whether the intracellular dissolution of lead chromate is responsible for genotoxicity and whether the Cr3+ ion is the DNA damaging agent or whether Cr5+, and/or reactive oxygen species are involved. There is also evidence that following phagocytosis of a large bolus of PbCr0 4 in particulate form, both the Pb and Cr ions may contribute to the transforming potency of lead chromate (12,37, reviewed in 8,9). 41 Carcinogenesis is thought to occur by a multistep process involving accumulation of multiple genetic and epigenetic alterations (reviewed in 38,39). Deregulation and overexpression of proto-oncogenes that function to promote cellular proliferation (including genes which function to prevent programmed cell death), and inactivation of tumor suppressor genes that function to inhibit cellular proliferation (including genes which function to induce cell death) represent molecular alterations that are steps in this process, (reviewed in 38-40). Carcinogens such as lead chromate are believed to act by inducing DNA damage at these specific loci, which leads to alterations in gene expression and eventually to altered growth characteristics (reviewed in 8,9). The mutational spectra of chemical and physical carcinogens is influenced by the nature of the compound (41,42). For instance, induction of skin carcinomas by UV light is indicated by the occurrence of mutations in the p53 gene involving pyrimidine dimers (43). Certain proto oncogenes such as the GTPase ras can be activated by point mutations (44). Therefore, one might not expect that ras would be activated by non-mutagenic carcinogens such as lead chromate, unless oxidative DNA damage generated by chromium-induced oxygen radicals is involved. Our laboratory previously isolated and biologically characterized two clonal cell lines of morphologically transformed 10T1/2 cells derived from foci induced by lead chromate (12). These clones, designated PbCr2 and PbCr3, stably maintain the 42 focus-forming phenotype, grow to a significantly higher saturation density than 10T1/2 cells, grow in soft agar, and give rise to progressively growing fibrosarcomas when injected subcutaneously into nude mice (Table 2)(12). We are utilizing these cell lines to study the molecular mechanisms of lead chromate-induced transformation of 10T1/2 mouse embryo cells as a step toward understanding the molecular mechanisms of human neoplasia by carcinogenic lead chromate. In this study, we have initiated experiments to identify and characterize alterations in the structure and expression of growth regulatory proto-oncogenes and tumor suppressor genes in the PbCr2 and PbCr3 cell lines. In analyzing these PbCrCU-induced cell lines, we have also included for comparison the previously well- characterized 3-methylcholanthrene-induced cell lines MCA Cl 15 and MCA Cl 16 which serve as positive controls in some experiments (45-47). We also extend the molecular characterization of these two MCA-induced transformed clones in terms of the expression and structure of proto-oncogenes and tumor suppressor genes. 43 Table 2. Biological properties of PbCr0 4 - transformed C3H/10T1/2 cell lines.* Cell line Focus-forming efficiency (%) Focus morphology in reconstruction Efficiency of colony formation in soft agarose Saturation density (xl06) Tumorigenicity in nude mice C3H/10T1/2 Cl 8 0 None < 0.0005 1.06 0/4 PbCr2 102 Types II and III 37.2 4.75 1/4 PbCr3 28 Types II and III 19.3 3.73 3/3 * This data comes directly from reference #27, (Patierno, Banh and Landolph, 1988), from our laboratory. - p>- MATERIALS AND METHODS Cells and Cell Culture C3H/10T1/2 Cl 8 (10T1/2) cells are an aneuploid, immortal but otherwise nontransformed fibroblast cell line derived form the embryos of C3H mice (48). PbCr2 and PbCr3 are transformed cell lines which were derived in our laboratory by treating 10T1/2 cells with 50|j.M lead chromate for 24 hrs, then culturing cells for six weeks at which time foci of transformed cells were isolated by ring-cloning and expanded (12). These two cell lines have been biologically characterized (12). MCA Cl 15 and MCA Cl 16 are transformed cell lines derived from 10T1/2 cells treated with the chemical carcinogen 3-methylcholanthrene (45). These two cell lines have been characterized biologically and at the molecular level (46,47) and thus serve as positive controls in some of the experiments. Cells were cultured in Eagle's basal medium containing 10% fetal calf serum (Gemini Bio-Products, Calabasas, California, Upstate Biotechnology Incorporated, Lake Placid, New York) without antibiotics at 37°C in a 5% CO2 incubator in 75 cm2 tissue culture flasks, 100 mm dishes, or 850-cm2 roller bottles (Corning Glass Company, Corning, New York) as described (12,45,48, reviewed in 32). All cell lines were routinely screened and found to be negative for Mycoplasma by staining with Hoechst 33258 and fluorescence 45 microscopy (49). To obtain cells for DNA, RNA, and protein extractions, growth curves and nuclear run-on analysis, one million cells were seeded/850-cm 2 roller bottle in 150 ml of medium. To obtain cells in mid-log phase cells were harvested 3 days after seeding, otherwise medium was changed on day 4. Cell counts were obtained by trypsinizing and counting cells electronically using a Coulter counter, model ZF (Coulter Electronics, Hialeah, FL). Probes for Northern and Southern blot analysis To analyze c-myc structure and expression, we used a 2.5-kb Xba\-Hind\\\ fragment of the plasmid pSVc-m yc I obtained from the American Type Culture Collection (ATCC), Rockville, MD which encodes the second and third exons and 3' flanking sequences of the mouse myc gene (50) or a 1.7-kb Hind\\\ fragment of pMcmyc54, a nearly full length mouse myc cDNA clone supplied by Dr. Kenneth Marcu (State University of New York, Stony Brook, NY) (51). To analyze p53 structure and expression, we used a 1.0-kb Xho\-Ksp\ fragment of the p53 cDNA plasmid pll-4 generously provided by Dr. A. J. Levine (Princeton University, Princeton, New Jersey). (52). For studies of the retinoblastoma (Rb) gene we used a 3.7-kb EcoRI fragment of the human Rb cDNA clone PG3.8M (53), kindly provided by Dr. Y.-K. T. Fung (Childrens Hospital of Los Angeles, Los Angeles, CA). A 1.0-kilobase Psfl v-fos fragment was used to study c-fos expression (ATCC). For analysis of c-s/s we used a 1.2-kb Psfl fragment of pv-sis (ATCC). To detect c-abl expression, a purified 46 insert from the v-ab/ containing plasmid was obtained from Oncor (Gaithersburg, MD). c-Ha-ras was analyzed using a 6.6-kb BamHI fragment of the pUCEJ6.6 Ha-ras plasmid (50), provided by Dr. R. A. Weinberg (Massachusetts Institute of Technology, Cambridge, MA). To examine c-raf expression, a 2.9-kb EcoRI fragment of p627 was used (ATCC). For studies of the Adenomatous Polyposis Coli (APC) gene we used an EcoRI fragment the human APC cDNA clone 540 kindly provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD) (54). The actin probe was a 0.9-kb pst\ fragment of a vector containing the mouse skeletal a-actin cDNA clone J from Dr. N. Davidson (California Institute of Technology, Pasadena, CA). This plasmid is similar to the actin plasmids previously described (55). Preparation and Labeling of DNA Probes DNA probes for Northern and Southern blotting analysis consisted of gene fragments liberated from plasmid preparations by digestion with appropriate restriction enzymes according to the specifications of the manufacturer. Restriction enzymes (purchased from Boehringer Mannheim, Indianapolis, IN) were chosen to eliminate the inclusion of flanking plasmid sequences when liberating inserts for each probe purified. Digested plasmids were extracted with phenol/chloroform (1:1 v/v), and then inserts were separated from vector sequences by electrophoresis in low melting temperature agarose. Insert DNA was then cut out of the gel and 47 labeled within the low melt agarose. Probes were labeled to high specific activity (0.8-2 x I09 cpm/pg) with 32P-alpha-dCTP, (3000 Ci/mmole, ICN Biomedicals, Irvine, CA) using a random-primed DNA labeling kit (Boehringer Mannheim, Indianapolis IN). Labeling efficiency was determined by differential binding to DEAE cellulose filters. Incorporation efficiencies of 40-60% were routinely obtained. Unincorporated labeled nucleotides were removed using a sephadex G50 column hydrated with TE buffer (10mM Tris-HCI pH 8, 1mM EDTA pH 8). RNA Isolation and Blotting Total cellular RNA was isolated by the single-step acid- guanidinium-thiocyanate-phenol-chloroform method (56). All solutions except those containing Tris were treated with 0.2% diethylpyrocarbonate (depc), then autoclaved, and all glassware was baked for 8 hours at 190°C to remove RNase as recommended (57). Medium was removed from roller bottles by aspiration, and cells were lysed in situ by addition of 8 ml of 4M guanidinium thiocyanate. The lysate was then transferred to a polypropylene centrifuge tube, and 0.8 ml of 2M sodium acetate was added and the solution was mixed. 8 ml water-saturated phenol was then added and the solution was again mixed. Finally, 1.6 ml of chloroform/isoamyl alcohol (49:1 v/v) was added, and the solution was mixed vigorously. Samples were incubated for 15 min. on ice, then centrifuged at 3,000 x G for 20 min at 4°C. The supernatant 48 was then removed and mixed with an equal volume of isopropanol. Samples were stored at -20°C for several hours and then centrifuged as before. The pellet containing RNA was air dried briefly, dissolved in 1.5 ml of 4M guanidinium thiocyanate, and then transferred to microfuge tubes. An equal volume of isopropanol was added to each sample, the sample was then thoroughly mixed, and then samples were placed at -20°C for 1 hour. Samples were then pelleted by centrifuging them at 13,000 rpm for 15 min. The pellets were then washed twice with 70% ethanol and air dried. The RNA pellet was suspended in a small amount of depc-treated water, and the concentration was determined by spectrophotometry using the relationship 1.0 OD26o=40pg/ml. To check the integrity of the RNA, an aliquot of each sample (1-2pg) was electrophoresed in a 0.8% agarose gel in 0.5X Tris-borate buffer (pH 8.3) containing 0.5pg/m l ethidium bromide and the 18S and 28S ribosomal subunit RNAs visualized using a UV light box. For Northern blotting analysis, either Poly(A)-containing RNA which was purified by two passages through an oligodeoxythymidylic acid cellulose column as described by Aviv and Leder (58), or total cellular RNA, was electrophoresed in a 1% formaldehyde agarose gel. The gel was run in 0.2 M 3-(N- morpholino) propanesulfonic acid, pH 7.0-50 mM sodium acetate- ImM EDTA buffer. RNA was transferred to Nylon membranes (Hybond-N, Amersham Inc. Arlington Heights, IL) in 20X SSC (3M sodium chloride, 0.3 M sodium citrate pH 7.0) by the method of 49 Southern (59). RNA was then fixed to the membrane by UV cross- linking (Stratalinker, Stratagene Inc., La Jolla, CA). Ribosomal markers and a commercially available RNA size ladder (Bethesda Research Laboratories, Gaithersburg, MD) were used to determine the molecular sizes of gene transcripts in Northern gel analyses (57). To conduct RNA slot-blotting analysis, total RNA was transferred to nylon membranes using a slot blot apparatus (Schleicher & Schuell, Keene, NH) by the method of Sambrook, Fritsch and Maniatis (57). Briefly, RNA samples (10, or 20p.g) in IOjiI of water were mixed with 20pl of 100% formamide, 7p.l of formaldehyde (37%), and 2|il of 20X SSC. The RNA was denatured by heating it for 15 min. at 68°C and then cooled rapidly on ice. Samples were then mixed with 2 volumes of 20X SSC and loaded into slots, and the RNA was transferred by applying a gentle vacuum is across the membrane. The RNA was then fixed to the membrane by UV cross-linking (Stratalinker, Stratagene Inc., La Jolla, CA). Filters were prehybridized for 2 h at 65°C in 5X Denhardt's solution (0.1% ficoll, 0.1% poyvinylpyrollidine, 0.1% bovine serum albumin), 5X SSC, 100mM NaP0 4 , pH 6.5, 0.1% SDS and 100 pg/ml denatured salmon sperm DNA. Hybridizations were conducted overnight at 65°C in prehybridization buffer containing 1-2 x 106 cpm/ml of 32P-labeled probe. Filters were then washed twice for 15 min each in 2X SSC, 0.1% SDS at room temperature, followed by two washes for 20 min each in 0.1 X SSC, 0.1% SDS at 50 60°C. Blots were then exposed to XAR-2 film (Eastman Kodak, Rochester, NY) with intensifying screens at -70°C. Quantitation of Signals on RNA Blots To insure a linear response of the X-ray film to 32P in the presence of intensifying screens, all films were pre-flashed to obtain an increase in absorbance of 0.15 ( A 5 4 0 ) absorbance units above that of unexposed, developed film. Pre-flashing was performed using a standard electronic flash at a setting of 1msec. and an orange filter, as described by Laskey and Mills (60). The distance required to obtain sufficient exposure during pre-flashing was determined empirically for each box of film, because variations between lots of film were detected. Exposures in the presence of intensifying screens were carried out at -70°C. Autoradiograms were analyzed by quantitative densitometry using an LKB Ultroscan XL laser densitometer (Pharmacia LKB Biotechnology, Upsala, Sweden). The amount of signal for each band was measured as the area under the peak. Several exposures of each blot were scanned to insure that band intensities were within the linear range of the film. The data for oncogene expression was corrected for slight differences in RNA loading in Northern blots and counts hybridized in Nuclear Run-on analysis by comparison to the intensity of p-actin signals. 51 DNA Isolation and Southern Blotting High molecular weight cellular DNA was extracted from 2 x 10 6 cells/cell line grown in 850-cm2 roller bottles. Cells were removed with 0.1% trypsin, rinsed 3X with PBS, and then suspended in 14 ml of ice-cold lysis buffer ( 0.32 M sucrose, 10 mM Tris-HCI pH 7.5, 5 mM MgCl2, 1% Triton X-100). After standing for 10 minutes on ice in lysis buffer, nuclei were pelleted by centrifugation at 4°C, 1000 X G for 10 minutes, resuspended in 10 m! lysis buffer, and then repelleted. The nuclei were then suspended in 4 ml of nuclei dropping buffer (75 mM NaCI, 24 mM EDTA, pH 8.0) at room temperature. To this was added 1 ml of 1 mg/ml proteinase K in nuclei dropping buffer. After mixing, 0.26 ml of 10% SDS was added, and the samples were incubated at 37°C overnight with gentle shaking. Samples were then extracted thoroughly with an equal volume of Tris-buffered phenol, followed by extraction with one volume of chloroform:isoamyl alcohol (24:1). DNA was then precipitated by adding 0.1 volume of 3 M sodium acetate and 1.5 volumes of isopropanol. DNA was spooled out and dissolved in 1-3 ml of TE buffer (10mM Tris-HCI pH 8, 1mM EDTA pH 8). After the DNA was completely dissolved, 0.5 volume of 7.5 M ammonium acetate was added, and the solution was mixed. Then, 2 volumes of cold absolute ethanol was added and the solution was mixed to precipitate the DNA. The DNA was again spooled out, rinsed in 95% ethanol, dried briefly, and dissolved in 1 ml T E '2 buffer (10 mM Tris-HCI, pH 8, 0.1 mM EDTA, pH 8). 52 DNA samples (20 |ig) were digested for 12-15 hrs with 5-10 U /jig of the appropriate restriction enzyme (Boehringer Mannheim, Indianapolis, IN) and then electrophoresed in 0.8% agarose gels in 0.5X Tris-borate buffer (pH 8.3). DNA containing gels were then soaked briefly in 0.2N HCI to depurinate the DNA and then rinsed with water. Gels were then denatured in 1.5M NaCI, 0.5N NaOH followed by neutralization in 1M Tris (pH 7.4), 1.5M NaCI. DNA fragments were then transferred to nylon membranes by capillary action using 20X SSC (59). /-//nc/lll-digested lambda DNA was used to provide molecular weight standards. After transfer, the lane containing the lambda DNA was removed and stained with methylene blue to visualize bands. Hybridization and washes were carried out as described above for RNA blotting. RESULTS Expression of Proto-oncogene RNAs in P b C r04- transform ed and M CA-transform ed cell lines com pared to n on transform ed 10T1/2 cells. To identify genetic alterations which might be responsible for the induction or maintenance of the transformed phenotype of P b C r0 4 -transformed cells, we compared the steady-state levels and transcript sizes of the proto-oncogenes c-s/s, c-fos, c-abl, c-Ha- ras, c-raf, and c-myc in the PbCr2 and PbCr3 cell lines with those 53 of 10T1/2 cells. We also employed Southern blotting analysis to examine the structure of the c-myc gene for alterations such as gene amplification, or rearrangement, or change of restriction sites. Previous studies from our laboratory have shown that the expression of proto-oncogenes such as c-myc and c-fos are dependent upon the growth state in 10T1/2 cells (46,47). Therefore, to analyze oncogene expression in PbCrCVtransformed cell lines, we first constructed growth curves to determine the cell densities at which 10T1/2 cells and the PbCr0 4 -transformed cell lines were in logarithmic growth and at confluence (Fig. 2). Both nontransformed and PbCr0 4 transformed cell lines were in log- phase of growth from day 1 to day 4. Nontransformed 10T1/2 cells become confluent by day 5. We therefore extracted Poly(A)- selected RNA from growing cells cultured in 850 cm2 roller bottles on day 3 after seeding, to insure that they were in exponential growth phase. Purified RNAs were then size-fractionated on 1% agarose formaldehyde gels, transferred to nylon membranes, and hybridized to the various 32P-labeled oncogene DNA probes. To account for slight variations in loading of the RNA, membranes were then stripped and rehybridized with a mouse skeletal a-actin cDNA probe. We found that the a-actin probe recognized both the 1.6-kb a-actin message and the 2.1-kb p-actin mRNA. This is not surprising considering the high degree of homology between the two genes. The 2.1-kb p-actin mRNA was found to be expressed at 54 Figure 2. Growth curves of 10T1/2 Cl 8 cells and PbCr0 4 - transformed cell lines in roller bottles under the conditions of this study. Cells were seeded at 1 x 106/8 5 0 -cm 2 roller bottle and medium was changed on day 4. Points, average of cell counts ± standard error from two roller bottles for 10T1/2 cells, one roller bottle for transformed cells in one experiment. To determine the cell number, the cells were trypsinized and counted electronically in a Coulter Counter. 55 Number o f cells / 850cm2 Growth curve of 10T1/2 Cl 8 cells and lead chromate transformed cell lines 7 6 10T1/2CI8 PbCr2 PbCr3 5 0 1 Day after seeding 56 higher levels in our cells than a-actin making its quantitation easier, p-actin mRNA was also expressed at similar levels in both the transformed and nontransformed cell lines and was therefore used to normalize levels of oncogene and tumor suppressor gene RNAs for slight variations in the loading of RNA on Northern gels. The oncogene RNA levels were determined from densitometric scans of autoradiographs within the linear range of the x-ray film. These values were then normalized to actin and expressed relative to the levels of expression in 10T1/2 cells. Northern blotting analysis failed to detect expression of the c-sis proto-oncogene RNA in nontransformed, PbCrCVtransform ed or MCA-transformed 10T1/2 cells. A previous study of 10T1/2 cells and transformed clones from our laboratory also did not detect expression of this gene in eight radiation and chemically transformed cell lines, including MCA Cl 15 and MCA Cl 16 (data not shown) (46). The c-fos gene encodes a protein product which associates with the c-jun protein product in a nucleoprotein complex that activates or represses transcription from promoters containing AP- 1 binding sites (61). c-fos is normally expressed at very low basal levels in growing cells but is transiently induced to high levels by a variety of factors that promote growth and/or differentiation (62, reviewed in 63). Constitutive overexpression of c-fos can lead to cell transformation (64). A 2.2-kb c-fos transcript was expressed in 10T1/2 cells but was not detectable or reduced to less than 10% 57 of its expression 10T1/2 cells in the PbCr2 and PbCr3 cell lines (Fig. 3). This finding is consistent with the low relative expression of c-fos mRNA our laboratory has observed previously in other transformed 10T1/2 cell lines, including MCA Cl 15 and MCA Cl 16 (46), so our data on 10T1/2, MCA Cl 15 and MCA Cl 16 is consistent with our previous work. The c-abl gene encodes a phosphotyrosine kinase protein which becomes oncogenic in mice when the gene is transduced by the Moloney murine leukemia virus and contributes to chronic myelocytic leukemia in humans when the gene is translocated and fused to the bcr gene (65). Both nontransformed and the four transformed 10T1/2 cells lines expressed two c-abl transcripts, one of 4.6-kb and a second, weaker band of 6.0-kb. Densitometric quantitation of the 4.6-kb transcript revealed no significant differences in abundance between either the lead chromate transformed clones or the MCA induced cells and 10T1/2 cells when the data was normalized to p-actin expression to account for any RNA loading or transfer variations (Fig. 4). The c-Ha-ras oncoprotein belongs to a family membrane associated GTP binding proteins which relay mitogenic and developmental signals initiated by cell surface receptors into the cytoplasm and nucleus (reviewed in 66). Activating mutations of ras genes are found in a nearly one third of all human cancers (66). Transfection of mutated ras into 10T1/2 cells causes weak morphological transformation (Type I foci) (67), B. Yasin and 58 Figure 3. Steady-state levels of c-fos mRNA in nontransformed 10T1/2 cells, P bC r04-transformed cells (PbCr2 and PbCr3) and MCA-transformed cells (MCA Cl 15 and MCA Cl 16). Cells were grown to late-log phase in roller bottles and RNA was extracted and poly(A) selected as described in materials and methods. Two (ig of poly(A)- containing RNAs was electrophoresed in a 1.0% agarose formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized with a 32P-labeled v-fos probe. To control for RNA loading the blot was stripped and rehybridized with an a-actin cDNA probe. Relative densitom etric readings were determined using a laser scanning densitometer, the value for 10T1/2 cells was arbitrarily set to 1.0. kb, kilobase. 59 c- fos actin c-fos 1 .0 <0.1 <0.1 <0.1 <0.1 actin_____________1.0 0.5 0.4 0.1 0.7 c-fos! actin 1.0 <0.1 <0.1 <0.1 <0.1 60 Figure 4. Steady-state levels of c-abl mRNA in nontransformed 10T1/2 cells, PbCr0 4 -transformed cells (PbCr2 and PbCr3) and MCA-transformed cells (MCA Cl 15 and MCA Cl 16). Cells were grown to mid-log phase (day 3) in roller bottles and RNA was extracted and poly (A) selected as described in materials and methods. Five jig of poly(A)-containing RNAs was electrophoresed in a 1.0% agarose formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized with a 32P-labeled v-ab/probe. To control for RNA loading, the blot was stripped and rehybridized with an a-actin cDNA probe, kb, kilobase. 61 c-abl actin '. ; • ? - ■ , tLtS'^'f^ J’-Lz *< X /.>* *± i't,«;:,-.'..- ?t?Rc« c -a b l 1.0 3.1 2.0 1.8 2.6 actin_______________1.0 2.5 1.8 1.8 2.9 c-abll actin 1 .0 1 .2 1.1 1 .0 0 .9 62 J. R. Landolph, personal observations. Northern blotting analysis of c-H a-ras expression revealed a 1.4-kb message, which was present at approximately the same level in all cell lines (Fig. 5). C-ra/1 gene encodes a cytoplasmic serine/threonine kinase which is binds to the ras oncoprotein (68). Raf transduces signals from ras by phosphorylating downstream targets such as the MAP kinases (69). We also detected expression of a 3.1-kb c-ra/-specific transcript in nontransformed 10T1/2 cells in the PbCrCU-transformed 10T1/2 cell lines and in the two MCA induced clones. All of the cell lines had the same-sized transcripts. The steady-state level of c-ra f mRNA was not increased in the four transformed cell lines, and was actually decreased 40-50% in three cell lines (Fig. 6). Steady-state Levels of c-m y c RNA in Lead Chrom ate- transform ed Cell lines Compared to 10T1/2 Cl 8 Cells. Previous work in our laboratory has revealed that twelve chem ically and radiation transformed 10T1/2 cell lines contain elevated steady-state levels of mRNA transcribed from the c-m yc proto-oncogene relative to nontransformed 10T1/2 cells (46,47). In addition, transformed 10T1/2 cell lines induced by nickel oxide, nickel subsulfide, and sodium arsenite also have elevated steady- state levels of c-m yc RNA (70,71). It was therefore of particular interest to determ ine whether induction of transformation by lead chromate might result in higher steady-state levels of c-m yc mRNA or alterations in the structure of c-m yc transcripts or gene. RNA was therefore extracted from cells in late exponential growth 63 Figure 5. Steady-state levels of c-Ha-ras mRNA in nontransformed 10T1/2 cells, PbCr0 4 -transform ed cells (PbCr2 and PbCr3) and MCA-transformed cells (MCA Cl 15 and MCA Cl 16). Cells were grown to mid-log phase (day 3) in roller bottles and RNA was extracted and poly (A) selected as described in materials and methods. Five |ig of poly(A)-containing RNAs was electrophoresed in a 1.0% agarose formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized with a 32p-labeled BamH\ fragment of the pUCEJ6.6 Ha-ras plasmid. To control for RNA loading, the blot was stripped and rehybridized with an a-actin cDNA probe. Relative densitom etric readings were determined using a laser scanning densitometer, the value for 10T1/2 cells was arbitrarily set to 1.0. kb, kilobase. 64 c-Ha -ras act in c-Ha -ras 1.0 0.9 0.8 1.0 1.7 act in 1.0 0.7 0.7 0.9 1.3 c -H -ra s /a c tin 1.0 1.3 1.1 1.1 1.3 Figure 6. Steady-state levels of c-raf 1 mRNA in nontransformed 10T1/2 cells, PbCr0 4 -transformed cells (PbCr2 and PbCr3) and MCA-transformed cells {MCA Cl 15 and MCA Cl 16). Cells were grown to mid-log phase (day 3) in roller bottles and RNA was extracted and poly (A) selected as described in materials and methods. Five mg of poly(A)-containing RNAs was electrophoresed in a 1.0% agarose formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized with a 32p-labeled 2.9-kb EcoRI fragment of p627 plasmid. To control for RNA loading, the blot was stripped and rehybridized with an a-actin cDNA probe. Relative densitometric readings were determined using a laser scanning densitometer, the value for 10T1/2 cells was arbitrarily set to 1.0. kb, kilobase. 66 o £ D o 1 O i rt* -t Q > 5’ 0 ) -ji ■ — 0 ) o F + 5' . © b b © o o b o n 0 0 o o o b 0 0 In o o o b 0 0 © o o ’ ■ > 1 b b I O 05 ■vj actin 10T1/2 PbCr2 PbCr3 C I 15 C I 16 0 1 &> phase, and analyzed so that we might detect small alterations in the abundance of c-myc message in the transformed cell lines. Northern blotting analysis revealed a c-m yc transcript of 2.3 kilobases in both nontransformed 10T1/2 cells and in the PbCr2 and PbCr3 cell lines, indicating no gross alterations in the size of the c-myc message in the PbCr0 4 -transformed cells. However, quantitation of the autoradiographic signal intensities indicated that the PbCr2 and PbCr3 cell lines expressed 7-8-fold higher steady-state levels of c-myc mRNA compared to 10T1/2 Cl 8 cells (Fig. 7). In this experiment, we saw a significant difference in the amount of p-actin RNA between nontransformed and transformed cell lines (Fig. 7). It is not known whether this is due to differential regulation of actin in transformed cells at high cell density or simply to differences in RNA loading on this particular gel. Therefore, the 7-8 fold higher steady state c-myc RNA levels represent non-normalized expression. Normalizing the expression results in 12-56-fold increased steady-state levels of c-myc RNA in the transformed cell lines. The two 3-methylcholanthrene (MCA)-transformed 10T1/2 cell lines, MCA C1 15 and MCA C1 16, included as a positive control, also showed approximately 8-fold higher steady-state levels of c-myc mRNA without normalization. The enhanced levels of c-myc RNA in MCA C1 15 and MCA C1 16 are somewhat greater than previously observed, but may reflect differences in the growth state of cells when RNAs were extracted (46). 68 Figure 7. Steady-state levels of c-myc mRNA at late-log phase in nontransformed 10T1/2 cells, PbCr0 4 -transformed cells (PbCr2 and PbCr3) and MCA-transformed cells (MCA Cl 15 and MCA Cl 16). Cells were cultured in roller bottles and RNA was extracted on day 6 and poly (A) selected as described in materials and methods. Two pg of poly(A)- containing RNAs was electrophoresed in a 1.0% agarose formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized with a 32p-labeled 4.8 kb fragment of the pSVc-myc plasmid encoding exons II and III of the mouse c-myc gene. To control for RNA loading, the blot was stripped and rehybridized with an a-a ctin cDNA probe. Relative densitometric readings were determined using a laser scanning densitometer, the value for 10T1/2 ceils was arbitrarily set to 1.0. kb, kilobase. 69 c-myc 2.3 K b act in 2.1 K b c-myc / Q > K S' & & o* . <o < T O 1 .0 7.1 8.1 7 .8 8.2 actin 1.0 0.5 0.4 0.1 0.7 c-myc/actin 1.0 14 22 56 12 70 Nontransformed 10T1/2 cells are contact inhibited and cease to divide when their density reaches that of a confluent monolayer (48). The PbCr2 and PbCr3 cell lines, in contrast, grow to 4.8- and 3.7-fold higher saturation densities, respectively, and continue to divide after reaching confluence (12). Therefore, at subconfluence, it is likely that a larger proportion of the transformed cells are dividing than the nontransformed 10T1/2 cells. We wanted to asses whether the amount of cell cycling occurring in PbCr2 and PbCr3 cells versus 10T1/2 cells at late log phase was responsible for the higher steady-state levels of c-m yc mRNA in the transformed cells. Therefore, we extracted poly(A)+ RNA from 10T1/2 cells and P b C r0 4 -transformed cell lines at mid log phase and the level of c- myc RNA was analyzed by Northern blotting. The RNA blot shown in fig. 8 demonstrates that a 6.1-fold and 4.2-fold enhanced expression of c-myc in the PbCr2 and PbCr3 cell lines was also apparent during exponential growth. Quantitative densitom etric analysis averaged from three separate experiments indicated that PbCr2 and PbCr3 cell lines exhibit 4.7 and 4.0-fold higher steady- state levels of c-m yc mRNA, respectively, compared with logarithm ically growing 10T1/2 cells (table 3). In this set of experiments, we also found that the MCA Cl 15 and MCA Cl 16 cell lines expressed 2.0 and 1.8-fold higher levels of myc RNA than the exponentially growing 10T1/2 cells (Table 3). This overexpression is less pronounced than previously observed for these two clones (46). This may be because in previous experiments, 10T1/2 cells 71 Figure 8. Steady-state levels of c-m yc mRNA at mid-log phase in nontransform ed 10T1/2 cells, PbCrCU-transformed cells (PbCr2 and PbCr3) and MCA-transformed cells (MCA Cl 15 and MCA Cl 16). Cells were cultured in roller bottles and RNA was extracted on day 3 and poly (A) selected as described in materials and methods. Five pg of poly(A)- containing RNAs was electrophoresed in a 1.0% agarose formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized with a 32P-labeled murine c-m yc cDNA probe. To control for RNA loading, the blot was stripped and rehybridized with an a-actin cDNA probe. Relative densitom etric readings were determined using a laser scanning densitometer, the value for 10T1/2 cells was arbitrarily set to 1.0. kb, kilobase. 72 c -myc * * ■ * < ‘ ,r * w r * ■ j ' actin c-myc 1.0 4.4 3.0 1.8 2.7 actin 1.0 0.7 0.7 0.9 1.3 c-m yd actin 1.0 6.1 4.2 2.0 2.0 Table 3. Steady-state levels of c-myc m RNA# Cell line_________Exp.t 1 _____ Expt. 2 Expt. 3 Avg ± SE 10T1/2* 1.0 1.0 1.0 1.0 ± 0 PbCr2 6.6 4.8 2.8 4.7 ± 1.9 PbCr3 6.2 3.8 2.0 4.0 ± 2.1 MCA Cl 15 2.4 2.6 1.1 2.0 ± 0.8 MCA Cl 16 2.2 2.2 0.9 1.8 ± 0.8 * 10T1/2 mRNA level arbitrarily set to 1.0. # Steady-state c-myc RNA levels normalized to actin. 74 were analyzed at a higher density, which would have resulted in partial down regulation of c-myc expression in the control cells, leading to a higher ratio for the value of c-myc in transformed cells versus c-myc RNA in nontransformed cells. This indicates that overexpression of c-myc RNA in the two P b C r0 4 -transformed cell lines relative to 10T1/2 cells is likely not simply a consequence of the growth state of the cells. However, the differences in c-myc RNA abundance between the PbCrC>4- transformed cell lines and the nontransformed 10T1/2 cells was less pronounced during mid-log phase than at late log phase, suggesting a difference in the way c-myc is regulated in 10T1/2 cells versus the PbCr2 and PbCr3 cells lines as the cells approach confluence. To explore this further, total RNA was extracted from early log phase cultures (< 30% confluent density) and post confluent cultures of 10T1/2, PbCr2 and PbCr3 cells. Relative c- m yc expression was then examined as a function of growth state in each cell line by RNA slot-blotting analysis (Fig. 9a). Averages of densitom etric quantitation of two slots for each cell line showed that 10T1/2 cells down-regulate c-myc RNA levels by approximately 75% at confluence (Fig. 9b). In contrast, c-myc expression is repressed by only about 25% in the PbCr2 cell line and by roughly 50% in PbCr3 cell line. This suggest that the PbCr04 - transformed cell lines have a defect in their ability to down- regulate c-m yc expression in response to the influence of cell contacts. However, even in low density cultures there is an 75 Figure 9. Effect of cell density on steady-state levels of c-m yc RNA. Cells were cultured in 100-mm2 dishes and total RNA extracted from each cell line at 30% confluent density and at post-confluent density. A, Ten and 20 |ig of RNA was analyzed for c-myc expression by slot- blotting analysis. B, Relative repression of c-m yc expression at high cell density as determined from densitometric quantitation of RNA slot-blots. Expression at high cell density averaged from two experiments + standard error. 76 c-myc RNA Expression 10T1/2 PbCr2 PbCr3 Low Density High Density m m jug RNA 10 20 10 20 10 20 Ftelative c-myc RNA level Cell line Low Density High Density % Repression 10T1/2 1 0 .2 5 ± 0 .0 8 75 PbCr2 1 0 .7 4 + 0 .0 6 26 PbCr3 1 0 .5 2 + 0 .0 2 48 Value of c-myc expression at low denstiy arbitrarily set to 1. aberrant regulation of c-myc RNA expression in PbCrC>4- transformed cell lines, which accounts for the elevated steady- state RNA levels observed in log phase cultures and contributes to most of the overexpression detected in cultures at high cell d e n sitie s. Our findings on c-myc expression in log phase cultures and the expression of the other proto-oncogenes examined is summarized in Table 4. Structure of the c-m yc gene in PbCrC>4-transform ed and M C A -transform ed cell lines. We next investigated whether there were gross alterations in the structure or copy number of the m yc gene which might be contributing to the higher steady-state levels of c-m yc RNA in the P b C r04 -transformed cell lines and that could easily be visualized by Southern gel analysis. Genomic DNA was extracted from each cell line and then digested with a variety of restriction endonucleases which cut within or outside the c-m yc coding region. In particular, enzymes were used which are known to cut within the immediate 3' and 5’ flanking regions of the c-myc gene (Fig. 10). These regions were targeted because they are known to harbor regulatory signals for c-myc RNA synthesis and degradation (reviewed in 72). The enzyme EcoRl, which cuts only in the flanking regions at a significant distance 5 and 3’ of the m yc exons, was used to identify rearrangements or translocations occurring well outside the coding region. The restricted DNA was size fractionated on 0.8% agarose 78 Table 4. Relative steady-state levels and transcript sizes of proto-oncogene RNAs in transformed cells lines. Transf. /N ontransf. a Cell line c-myc c-fos c-Ha-ras c -raf-1 c-sis c-abl 10T1/2 1.0 1.0 1.0 1.0 — 1.0 PbCr2 4.7 ±1.1 <0.1 1.3 0.8 ±0.2 — 1.2 PbCr3 4.0 ±1.2 <0.1 1.1 0.6 ±0.0 — 1.1 MCA Cl 15 2.0 ±0.5 <0.1 1.1 0.6 ±0.2 — 1.0 MCA Cl 16 1.8 ±0.4 <0.1 1.3 0.5 ±0.2 — 0.9 Transript size (kilobases) 2.3 2.2 1.4 3.1 — 6.0 4.6 Altered size in Transformed cell lines No No No No — No a Northern blots were hybridized with indicated probes and specific bands quantified by densitomtry. Values are expressed as a ratio of autoradiographic signals between 10T1/2 cells (Nontransf.) and transformed clones (Transf.), each having been normalized to actin. No detectable expression. Values for c-myc are averaged from three experiments, values for c-rafl averaged form two experiments (± standard error), values for other oncogenes from one experiment each. C O Figure 10. Partial restriction map of the mouse c-myc gene. Shaded areas indicate untranslated regions. Compiled from previously published restriction maps and sequence data. 80 c-myc R BH SX B S RR R RR !i_______ U ___ [ U — Exon 1 Exon 2 B= Bam HI E= Eco Rl H= Hind III S= Sac I R= Rsa I X=Xho I 00 X H E Exon 3 \ 1 1 Kb gels, transferred to nylon filters, and hybridized with the 32P- labeled murine c-myc cDNA probe. We first determined whether amplification of the c-myc gene might be in part responsible for increased steady-state levels of c- myc RNA in transformed cells. Several Southern blots were scanned using a soft laser densitometer, and the c-myc band intensities were compared to that of 10T1/2 cells. No amplification of the myc gene was detected in the PbCr2 and PbCr3 cell lines, nor in the two MCA-transformed cell lines (Table 5). In each of the Southern blots we observed similar hybridization patterns and fragment sites and intensities for the PbCr04 -transformed cell lines, and for the MCA-transformed cell lines, relative to nontransformed 10T1/2 cells (Figs. 11 and 12, summarized in Table 6). Therefore, translocation, gross rearrangement, or large deletions were not observed and do not account for the observed increase in c-myc RNA levels in the PbCr2 and PbCr3 cell lines. The results obtained for the two MCA-transformed clones agrees with previously published restriction analysis and extends these findings (46,47). The PbCrCV and MCA-induced cell lines have acquired a number of transformation-related phenotypes including growth to elevated saturation density, stable focus formation in reconstruction assays, anchorage independence and tumorigenicity (12,45,73). There is substantial evidence from our laboratory and other laboratories that the individual transformation-associated phenotypes acquired by 10T1/2 cells segregate independently 82 Table 5 Relative copy number c-myc gene in genomic DNA of nontransformed and chemically transformed 10T1/2 cells. Cell line Expt. 1 Expt. 2 Expt. 3 Avg. ±SE 10T1/2* 1.0 1.0 1.0 1.0+0 PbCr2 1 .3 1 .0 0 .8 1.0 ±0.1 PbCr3 0 .8 0 .7 0 .5 0.7 ±0.1 MCA Cl 15 1.0 1.6 0.9 1.2 ±0.2 MCA Cl 16 0.7 1.4 0.7 0.9 ±0.2 * 10T1/2 relative copy number arbitrarily set to 1. Relative DNA content calculated from quantitative densitometry of Southern blot autoradiograms. Figure 11. Southern blotting analysis of the c-myc gene in nontransformed 10T1/2, PbCr04 -transformed and MCA- transformed ceil lines. Approximately 20pg of genomic DNA from each cell line was digested with Bam HI or Hind III and size fractionated on a 0.8% agarose gel, transferred to a nylon membrane and hybridized with a 32P-labeled mouse c-myc cDNA probe (pMc-myc54). H/ndlll-digested lambda DNA was used as the molecular weight marker, kb, kilobases. 84 V I S ’ >-&.:• * * ■ ^;v.^^:ir ' - ' ^ : ' ^ - ' <.v 'y ' . $ & ' ^ r V : / . ' : IO fO 00 O l ..« I I I O to ■ a © w 10T1/2 PbCr2 fij’’ ,a j? e jF ? ? PbCr3 MCA CM 5 MCA CM 6 10T1/2 PbCr2 PbCr3 MCA CM 5 MCA CM 6 0) 0 ) 3 o = 5 2 (Q A W A a 3: 3 ‘ o l i s (fi A 0 ) A a o 3 ' S I I 0 > (O C > to w X O " Figure 12. Southern blotting analysis of the c-m yc gene in nontransformed 10T1/2, PbCr0 4 -transformed and MCA- transformed cell lines. Approximately 20pg of genomic DNA from each cell line was digested with X ho I or Rsa\ and size fractionated on a 0.8% agarose gel, transferred to a nylon membrane and hybridized with a 32P -labeled mouse c-myc cDNA probe (pMc-myc54). Hindi 11-digested lambda DNA was used as the molecular weight marker. kb, kilobases. 86 c-myc R s a A Digested DNA i n I D T“ T “ o o < < Xhct Digested DNA lo <o mm ' & & & * 7-r * ■ * vmm & * $ S r- i , . - * - / ' * J & p / i i f u v -df ?§L, 8 S 1 S ^ v u fU 'm s kb 2 3 9 .4 6.6 4 .3 2.0 87 Table 6 Summary of c-myc Southern blot restriction analysis Cell line EcoRI BamH\ Hind\\\ Xho\ Rsa\ Sacl 10T1/2 23 kb 7.0 kb 1.1 kb 4.5 kb 4.0 kb 1.9 kb 0.6 kb 0.2 kb 11.5 kb 1.5 kb PbCr2 23 kb 7.0 kb 1.1 kb 4.5 kb 4.0 kb 1.9 kb 0.6 kb 0.2 kb 11.5 kb 1.5 kb PbCr3 23 kb 7.0 kb 1.1 kb 4.5 kb 4.0 kb 1.9 kb 0.6 kb 0.2 kb 11.5 kb 1.5 kb MCA Cl 15 23 kb 7.0 kb 1.1 kb 4.5 kb 4.0 kb 1.9 kb 0.6 kb 0.2 kb 11.5 kb 1.5 kb MCA Cl 16 23 kb 7.0 kb 1.1 kb 4.5 kb 4.0 kb 1.9 kb 0.6 kb 0.2 kb 11.5 kb 1.5 kb (12,74, reviewed in 8,9,32). This strongly suggests that the transformed cell lines in this study harbor multiple genetic alterations in growth regulatory genes. Expression and structure of tumor suppressor genes in PbCr0 4 - and MCA-transformed 10T1/2 cell lines. One class of genes which has rapidly gained prominence and contributes significanly to human cancer is that of tumor suppressor genes (75). We therefore examined the steady-state levels of RNA expression and transcript sizes of the tumor suppressor genes p53, RB and APC in the PbCr2 and PbCr3 cell lines to determine whether there were alterations in their expression, specifically decreases which would suggest a deletion or inactivating mutational event. For p53 and Rb, we also employed Southern blotting analysis to examine the structure of the genes for alterations such as deletion, or rearrangement, or change of restriction sites. The p53 tumor suppressor gene encodes a nuclear protein which mediates cell cycle arrest and apoptosis in response to DNA damage in part by transcriptional activation of a cell cycle inhibitor (reviewed in 76,77). Loss of wild type p53 function contributes to immortalization, cell transformation and tumorigenesis (reviewed in 78). Northern blotting analysis of mRNA isolated from nontransformed 10T1/2 cells demonstrated a 1.85-kb mRNA species that hybridized to a mouse p53 cDNA probe, consistent with the 89 reported size for mouse p53 RNA (79) (Fig. 13). Analysis of p53 expression in the two MCA and the PbCrCU transformed cell lines also revealed the presence of a single 1.85-kb transcript (Fig. 13). Quantitative densitometry of several Northern blots showed no reduction in the steady-level of the p53 mRNA in any of the four transformed cell lines. In fact, the PbCr2 and PbCr3 cell lines expressed 1.3- and 1.6-fold higher levels of p53 message than did the 10T1/2 cells (Table 7). The significance of this overabundance of p53 mRNA is not known. Because of the importance of p53 as a target for mutation in a wide spectrum tumors and transformed cell lines (80,81), we also examined the genomic structure of p53 for restriction polymorphisms which might not be detected by Northern blotting. High molecular weight DNA from nontransformed 10T1/2 cells and from PbCr04 - and MCA-induced cell lines was digested with EcoR\, Hind\\\, BamH\, Sac\, Xho\, and Rsa\. The restricted DNA was fractionated on 0.8% agarose sizing gels, transferred to nylon filters, and hybridized with the 32P-labeled murine p53 cDNA probe. No rearrangements or deletions of p53 were found in digested DNA from any of the transformed cell lines. The intensity of hybridization to the p53 DNA was roughly equivalent in all cell lines, indicating no loss of p53 alleles in any of the cell lines examined (Fig. 14). We also examined the well studied retinoblastoma tumor suppressor gene (Rb) in the transformed cell lines. The Rb gene 90 Figure 13. Steady-state levels of p53 mRNA in nontransformed 10T1/2 cells, PbCrC>4-transformed cells (PbCr2 and PbCr3) and MCA-transformed cells (MCA Cl 15 and MCA Cl 16). Cells were grown to mid-log phase (day 3) in roller bottles and RNA was extracted and poly (A) selected as described in materials and methods. Five jig of poly(A)-containing RNAs was electrophoresed in a 1.0% agarose formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized with a 32P-labeled murine p53 cDNA probe. To control for RNA loading, the blot was stripped and rehybridized with an a-actin cDNA probe. Relative densitometric readings were determined using a laser scanning densitometer, the value for 10T1/2 cells was arbitrarily set to 1.0. kb, kilobase. 91 P 5 3 re act in p53 1.0 0.7 1.1 1.0 0.9 actin 1.0 0.5 0.8 0.8 0.9 p 53 /a c tin 1 .0 1 .4 1 .4 1.2 1 .0 92 Figure 14. Restriction endonuclease-Southern blotting analysis of DNAs from nontransformed 10T1/2, PbCrCU-transformed and MCA-transformed cell lines. Approximately 20pg of genomic DNA from each cell line was digested with Hind\\\ and size-fractionated on a 0.8% agarose gel, transferred to a nylon membrane and hybridized with a 32P-labeled murine p53 cDNA probe, kb, kilobases. 93 p53 h- o O n C L 2 in id ° z i £ 0 0 i? 4 * ^-yv(< r f Kb ■ 2 3 ■9.4 •6.6 •4.4 i— 2.3 1 — 2 .0 94 encodes a nuclear phosphoprotein of 105 kDa which acts to inhibits cell cycle progression at the G1/S boundary by inhibiting the activity of members of the E2F family of transcription factors (reviewed in 77,82). Rb is mutated or deleted in human tumors and transformed rodent cell lines (81, reviewed in 83). In C3H/10T1/2 cells in our laboratory, both the nontransformed and transformed cell lines expressed a 4.7-kb message, but at barely detectable levels, allowing only a qualitative analysis of Rb expression (Table 7). We are now endeavoring to obtain a quantitative measure of Rb transcript levels in the nontransformed and transformed cell lines under study. Southern blotting analysis of the Rb gene in DNA samples digested with Hind\\\ showed no gross alterations, here we did not find evidence for mutation of restriction sites or large deletion within the Rb gene in the transformed cell lines using these crude methods (Fig. 15). Finally, we examined the expression of the adenomatous polyposis coli gene (APC) in the PbCr2, PbCr3, MCA Cl 15, and MCA Cl 16 cell lines. The APC protein associates with the cytoskeleton and may mediate intercellular communication through cell surface molecules such as the cadherins (84,85). Mutations in APC are involved in the development of colon cancer in humans (86) and mice (87). We found that a 9.4-kb APC transcript was expressed at barely detectable levels in both the nontransformed and transformed 10T1/2 cells (Table 7). There was no detectable alteration in the size of the APC gene message in any of the 95 Figure 15. Restriction endonuclease-Southern blotting analysis of DNAs from nontransformed 10T1/2, PbCr0 4 -transform ed and MCA-transformed cell lines. Approximately 20pg of genomic DNA from each cell line was digested with Hind\\\ and size-fractionated on a 0.8% agarose gel, transferred to a nylon membrane and hybridized with a 32p-labeled human retinoblastoma (Rb) cDNA probe (PG3.8M). kb, kilobases 96 ID 2.0 £ » o) < o ro c o 10T1/2 * PbCr2 PbCr3 lh 0 " * Cl 15 E W * t J l , Q 16 JJ O ' T ab le 7. Relative Steady-state levels and Transcript sizes of tumor suppressor gene RNAs in transformed cells lines. Transf. / Nontransf. a Cell line p53 Rb APC 10T1/2 1.0 + b + PbCr2 1.3 ±0.1 + + PbCr3 1.6 ±0.2 + + MCA Cl 15 1.0 ±0.2 + + MCA Cl 16 0.9 ±0.1 + + Transript size (kilobases) 1.85 4.7 9.5 Altered size in Transformed cell lines No No No a Northern blots were hybridized with indicated probes and specific bands quantified by densitometry. Values are expressed as a ratio of autoradiographic signals between 10T1/2 cells (Nontransf.) and transformed clones (Transf.) each having been normalized to actin. b Northern blotting established the presence of mRNA for this gene but the expression was too low to quantitate accurately. p53 expression is averaged from two expperiments ± standard error. 98 transformed cell lines, but signal intensities on autoradiographs were not sufficient to determine relative expression levels accurately. We are optimizing the sensitivity of our RNA analysis to obtain a quantitative measure of APC transcript levels in the 10T1/2 cells and the transformed cell lines under study. A summary of the results of our survey analysis of the expression and structure of proto-oncogenes and tumor suppressor genes is presented in Table 8. Of the genes we have examined so far, we found elevated steady-state levels of the 2.3-kb c-m yc proto-oncogene messenger RNA in two transformed 10T1/2 cell lines induced by lead chromate and two transformed 10T1/2 cell lines induced by MCA. Higher steady-state levels of c-myc RNA expression occurs without amplification or gross rearrangement of the c-myc gene. We also observed that c-fos mRNA is expressed at reduced levels or is absent in all of the transformed cell lines relative to 10T1/2 Cl 8 cells. We detected no alterations in the expression of the proto-oncogenes c-abl, c-Ha-ras or c-raf, nor in the expression of the tumor suppressor genes Rb, p53 APC, nor in the structure of Rb or p53. 99 Table 8 Summary of the survey of proto-oncogene and tumor suppressor gene expression and structure in PbCrCk- and MCA- transformed 10T1/2 cell lines vs.lO Tl/2 Cl 8. Amplification Deletion/ Rearrangement Altered Transcript size Enhanced Steady-state Level Loss of Expression c-sis ND* ND* c-fos — — + c-abl — ■ — c-H -ras . . . — c-raf — — c-myc ----- ----- — _ |_ ----- B APC p53 Rb ------ ------ ------ ------ ------ ------ + / — ------ ------ ------ ------ ------ *ND No transcript detected in non-transformed or transformed cell lines. + Transformed cells display this characteristic. “ No change compared to nontransformed 10T1/2 cells. DISCUSSION There is evidence for the carcinogenic potency of hexavalent chromium compounds of low water solubility (1). However, the molecular mechanisms by which these compounds exert their effects remains obscure (8,9). Our laboratory previously showed that insoluble lead chromate induced a low, but concentration- dependent induction of type III morphological transformation in C3H/10T1/2 cells without inducing mutation to ouabain resistance in 10T1/2 cells or CHO cells or mutation to 6-thioguanine resistance (in CHO cells) (12). These observations are consistent with a mechanism of cell transformation resulting from the potent clastogenic properties of lead chromate (36,88). However, the critical genetic or epigenetic targets involved in lead chromate- induced cell transformation are unknown, nor are the types of lesions produced in such targets. Here, we examined two transformed cell lines (PbCr2 and PbCr3) cloned from PbCr0 4 -induced type III foci for alterations in growth regulatory genes which might be mechanistically related to the induction and/or maintenance of their transformed phenotype. We tested the hypotheses that morphological and neoplastic transformation induced by lead chromate results from rearrangements of, or amplification of, or small deletions in negative regulatory elements of, single or multiple specific proto oncogenes and/or deletion or inactivation of tumor suppressor 101 genes, or by a combination of these mechanisms. To test these hypotheses, and ultimately to elucidate the molecular mechanisms of lead chromate-induced cell transformation we utilized the approach of screening known growth-related oncogenes and tumor suppressors by Northern and Southern blotting for alterations in structure or expression level. This chapter describes the results we have obtained so far by this approach. Northern blotting analysis showed no detectable expression of the proto-oncogene c-sis in the transformed cell lines or nontransformed 10T1/2 cells. The transcript sizes and steady- state levels of c-fos, c-abl, c-Ha-ras, and c-raf transcripts was unaltered in the PbCr0 4 -transformed cell lines, c-fos transcript sizes were the same in transformed cell lines, but the steady-state levels of c-fos RNA were decreased in transformed cells. Expression of the tumor suppressor genes Rb, p53 and APC, were the same in the non transformed and in the transformed cell lines. Southern blotting analysis showed no gross changes in the genomic structures of the tumor suppressor genes 53 and Rb in the transformed cells compared to 10T1/2 cells. This general survey has now widened, because the MCA and PbCr04 induced clones express multiple transformation-associated phenotypes, which therefore suggests alterations in at least 2 and perhaps as many as 5 separate genes are responsible for neoplastic transformation. Northern blotting analysis did, however, reveal a difference in the steady-state levels of the c-myc proto-oncogene RNA in both 1 02 PbCr0 4 -induced clones. When analyzed at late log phase, the PbCr2 and PbCr3 cells were found to express 7-8-fold higher steady-state levels of c-myc mRNA than 10T1/2 cells. Analysis of cells in early exponential growth showed 4.7-fold and 4.0-fold higher steady- state levels of c-myc mRNA in the PbCr2 and PbCr3 cell lines respectively relative to 10T1/2 cells. This finding is particularly interesting, because elevated levels of myc RNA have been found in 10T1/2 cells transformed by chemicals (3-methylcholanthrene, bleomycin, 7,12 dimethyl benz(a)anthracene), X rays, and neutrons. This finding also suggests that in 10T1/2, cells tight regulation of c-m yc RNA expression is important for maintaining normal growth characteristics such as contact inhibition (46,47). In nontransformed 10T1/2 cells, our laboratory has shown that c-m yc is closely associated with the growth state of the cells (46,47). At high cell density, the steady-state level of c-myc mRNA is decreased by 2 to 10-fold and cells cease dividing (47,89). In contrast, the MCA-transformed Cl 15 and Cl 16 cells do not down regulate c-myc expression to the same extent at high cell density suggesting that cell density dependent repression of c-myc is deregulated in these cell lines. RNA slot-blotting analysis showed that at high cell density PbCr2 and PbCr3 cells do not down- regulate c-myc mRNA to the same extent as 10T1/2 cells, and the cells continue to divide. This pattern of c-myc deregulation has also been shown in other transformed 10T1/2 cell clones examined in our laboratory (46). 1 03 To investigate whether structural alterations in the myc gene underlie the pattern of deregulated myc expression in the PbCrCV transformed 10T1/2 cells, genomic DNA was isolated from 10T1/2, PbCr2, PbCr3, MCA Cl 15, and MCA Cl 16 cells and digested with restriction enzymes, and the separated fragments were probed with the mouse c-myc cDNA. Densitometric quantitation of Southern blots showed no amplification of the c-myc gene in the PbCr2, PbCr3, MCA Cl 15 or MCA Cl 16 cell lines. Further, digestion of the c-myc gene in the transformed cell lines with a number of enzymes which are known to cut both outside the three myc exons (EcoRI)as well as within the 5' (BamHI, Hind\\\, Xho\ and Sacl) and 3' (Sacl and Xho I) regulatory regions revealed no structural alterations. Hence, we did not find evidence for translocations, rearrangements, or large deletions in the myc genes of the PbCr2, PbCr3, MCA Cl 15, and MCA Cl 16 cell lines. The absence of amplification or gross rearrangement of the c- myc gene raises the question of what are the mechanisms responsible for the presence of higher steady-state levels of c-m yc mRNA in the PbCr2 and PbCr3 cell lines. Our laboratory has also observed higher (2-8 fold) steady-state levels of c-myc RNA expression in other transformed 10T1/2 cell lines without finding amplification or rearrangement of the c-myc gene (46,47). Others have found overexpression of c-myc RNA in transformed human cell lines in the absence of gross gene alterations (90). Further, human colon carcinomas and colon carcinoma cell lines frequently exhibit 104 overexpression of c-myc RNA and protein without amplification or rearrangement of the gene (91). We suggest that if there are structural alterations in the myc genes of the PbCr04-induced cell lines which contribute to c-m yc overexpression, they consist of small deletions or mutations which can not be easily detected by Southern blotting analysis. Such deletions or mutations might affect c-myc RNA synthesis by removing sites of binding for negative regulators of transcription or enhancing the binding of positive trans-regulatory proteins (92). Small deletions might affect the sequences involved in regulating attenuation of transcription of myc known to be an important control mechanisms under some conditions (93). Small structural alterations which disrupt control elements in the 3' or 5' untranslated regions of m yc might contribute to stabilization of the normally short-lived message and thus to enhanced steady-state RNA levels. 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Further, because no explanation yet exists for the elevated levels of c-m yc RNA in the 3-m ethylcholanthrene transformed clones MCA Cl 15 and MCA Cl 16, these cell lines were included in these studies. Nuclear run-on analysis showed that the transcription rate of the c-myc gene was not enhanced in three of four transformed cell lines, while the PbCr3 cell line showed a small (1.5-fold ±0.2) increase in transcription compared to 10T1/2 Cl 8. Actinomycin D chase experim ents revealed that a fraction (approxim ately 10-25% depending on the cell line) of c-myc RNA in both the lead chromate-induced cell lines and the MCA-induced 117 cell lines had an enhanced stability compared with the very short lived c-myc RNA expressed in 10T1/2 cells. Elevated c-myc RNA levels were accompanied by 1.5-fold higher steady-state levels of P65Myc protein in the PbCr2 cells and 1.3-fold higher levels of P65Myc in the PbCr3 cell line. The MCA Cl 15 cell line expressed 2.4-fold higher steady-state levels of the p65 c-myc protein, whereas in the MCA Cl 16 cells, the level was only 0.7- fold that of 10T1/2 Cl 8 cells. Taken together with the findings of the previous chapter, the results suggest that the enhanced stability of the c-myc message transcribed from one altered c-myc gene results in elevated levels of RNA and often protein, which likely contributes to m aintaining the transformed phenotype of these cells. Efforts are in progress to determine whether the enhanced stabilization of the altered c-myc transcript is due to mutations in the 3’ or 5' mRNA destabilizing regions or to alterations in trans-regulatory proteins which target c- myc RNA for degradation. 118 INTRODUCTION In the previous chapter, we began investigating the mechanism s responsible for the transformed phenotype in two PbC r0 4 -transformed cell lines by examining whether there were alterations in the expression or structure of proto-oncogenes and tumor suppressor genes. We found that both PbCr2 and PbCr3 cell lines exhibited markedly higher steady-state levels of the same sized 2.3 kb c-myc RNA compared to nontransformed 10T1/2 cells. c-myc RNA expression was approximately 4-fold higher in early exponential growth phase and 8-fold higher in late exponential growth in the transformed cells. Southern blotting analysis indicated that neither amplification or gross structural alteration of the myc gene could account for the aberrantly high transcript levels in the PbCr2 and PbCr3 cell lines. In this chapter, I describe our efforts to understand the mechanism(s) responsible for the elevated steady-state levels of c-myc RNA and to determine whether the overabundance of c-myc RNA gives rise to an overabundance of c-myc protein in the transformed cells. The c-myc oncogene is the cellular homologue of the oncogene v-m yc found in several avian and feline retroviruses (1,2). Regulated c-myc expression is critical for controlled cell proliferation, whereas deregulated, constitutive over-expression of c-myc is a frequent hallmark of many tumor-derived cells (3,4). The c-myc gene is expressed at constitutively elevated levels in a 119 variety of tumors, suggesting that it plays an important role in multistage carcinogenesis (5,6). Introduction of c-myc into primary cells in culture under most circumstances does not cause morphological or neoplastic transformation (7). Only in conjunction with other oncogenes, such as src or ras (which by themselves are also unable to fully transform primary cells), is full neoplastic transformation observed (7,8). In non-malignant cells, there is compelling evidence for the involvement of c-myc in the regulation of proliferation, mitogenesis, differentiation, and programmed cell death (9-11). The c-myc protein is a transcription factor (12) and contains basic region-helix-loop- helix and leucine zipper structural motifs (13). Site-specific DNA binding activity, however, is only found in a heterodimeric complex with another cellular protein, designated Max (12,14). The expression of c-myc is generally correlated with cellular proliferation. The gene is expressed at a low constitutive level in growing cells and is down-regulated in both quiescent and differentiating cells (5,15). Mitogenic stimulation of quiescent cells elicits a rapid rise in c-myc mRNA and protein, a peak within 3-4 h, and then a decay to the lower steady-state level found in exponentially growing cells (15,16). c-myc is thus often grouped into the category of immediate early genes, and it has been suggested that it participates in the cascade of events that follows mitogenic stimulation of quiescent cells (17). 120 However, unlike a number serum induced genes which accompany cell cycle entry, c-myc appears to have an important role in actually controlling the cell's decision to divide (18). Ectopic induction of Myc is sufficient to drive quiescent growth factor-deprived fibroblasts into the cell cycle (19). This suggests that Myc may mediate the mitogenic response through transactivation of genes necessary for Gi transition. It has been shown that Myc, with its partner Max, can specifically bind to and activate expression from promoters containing an E-box motif with the central sequence CACGTG (20,21) At present, the critical target genes of Myc transcriptional activation required for entry into S phase have not been identified. Two genes reported to be induced by Myc are the ornithine decarboxylase gene and the a - prothymosin gene (19,22). However, it is unclear whether such genes function as down-stream regulators of the mitogenic cascade. More recently, it was shown that activation of Myc can activate expression of cyclins E and A in quiescent cells (23). Expression of cyclin A is closely linked to and a prerequisite for entry into the S phase of the cell cycle (24), thus induction of this gene could reflect the mitogenic potential of Myc. The three exons of the c-myc gene are transcribed from two promoters P1 and P2, which give rise to transcripts of 2.4 and 2.2 kb, respectively, and account for approximately 10 and 90% of myc transcripts in normal cells, respectively (10). The major open reading frame begins at an AUG codon at the 5' end of exon 2 and 121 codes for a protein of approximately 65-kDa in mouse cells. A second translation product, p68, initiates from a CUG codon at the 3' end of exon 1 (25). Expression of the c-myc gene is tightly regulated at the transcriptional, post-transcriptional, and post- translational levels (10). Both the c-myc mRNA and protein have very short half-lives, on the order of 10 minutes for the RNA and approximately 25 minutes for the c-m yc protein (10). In addition to regulation of transcriptional initiation, control of transcript elongation through a site at the 3' end of exon 1 regulates the synthesis of full length message in response to differentiation inducing agents (26,27). The c-myc proto-oncogene may be activated to its oncogenic form by genetic alterations disrupting normal regulatory mechanisms controlling c-myc expression (reviewed in 10). In some tumors, c-myc activation can be attributed to gross structural alterations of the c-m yc locus. These include retroviral transduction, promoter and enhancer element insertion, gene am plification, chromosomal translocation, and mutations or deletions of regulatory sequences (5,10,28,29,). However, many tumors and cell lines with deregulated c-myc do not demonstrate such structural alterations, indicating the existence of alternative mechanisms of c-myc activation (30-33). In either case, oncogenic activation is manifested by one or more of increased RNA levels, constitutive synthesis, or alterations in the ratios between the c- m yc products (10). Mutation of the myc protein product itself has 122 not been considered to be a major factor contributing m yc-in duced cell proliferation. However, there are reports of a high frequency of mutations in the second or third exons of the c-m yc gene in Burkitt's lymphoma cell lines (34,35). In addition, it was recently shown that cells expressing transfected myc genes containing mutations at sites of protein phosphorylation display increased growth potential compared with cells expressing transfected wild type m yc (36). The altered transforming ability of the mutated Myc did not correlate with a specific change in transactivating ability, leaving the functional significance of such changes unclear (36). We (3,4) and others (15) have shown that the c-myc gene is tightly regulated by extracellular signals, such as serum growth factors and cell contact in mouse fibroblast cell lines, including C3H/10T1/2 cells (3,4). At confluence, nontransformed 10T1/2 cells respond to signals presumably generated by cell-cell contacts which cause them to cease dividing. This is accompanied by an approximately 10-fold reduction in c-m yc RNA levels compared with logarithm ically growing cells (3). In contrast, one of the hallmarks of m orphologically transformed 10T1/2 cells is a loss of contact inhibition of cell division, which results in m ultilayered growth in culture and a higher saturation density than nontransformed 10T1/2 cells (37). Our studies of the expression of proto-oncogenes in fifteen morphologically transform ed 10T1/2 cell lines induced by chemical carcinogens and radiations, have shown that expression of the 2.3 kb c-myc mRNA is elevated in 13 1 23 of these cell lines by approximately 2-8 fold (3,4,38,39). Coleman et al., in examining 42 m orphologically transformed 10T1/2 cell lines either spontaneously arising or induced by MCA, N-methyl-N'- nitro-N-nitrosoguanidine (MNNG), or benzo[a]pyrene diol epoxide (BPDE), found overexpression of a normal c-myc transcript (>1.5- fold over 10T1/2) in 79% of the cell lines (40). Together with these findings, our recent demonstration that c-m yc is also expressed at higher steady-state in two lead chromate transformed 10T1/2 cell lines compared to 10T1/2 Cl 8 cells, suggest an important role for c-m yc in the control of normal growth and its dysregulation in the genesis of a transformed phenotype in 10T1/2 cells. However, little is known about the mechanism of c-m yc dysregulation in the transformed 10T1/2 cell lines having elevated c-m yc RNA, nor is it known whether m yc protein is overabundant in such cell lines. This chapter describes efforts to investigate these two questions in transformed 10T1/2 cell lines induced by lead chromate and by MCA. 1 24 MATERIALS AND METHODS Preparation of target DNA for nuclear run-on transcription assays To analyze c-myc transcription, the plasmid pSVc-myc I, which encodes the second and third exons and 3' flanking sequences of the mouse myc gene, (7) was used. To asses GAPDH transcription, the plasmid pHcGAP encoding the human glyceraldehyde-3-phosphate dehydrogenase cDNA clone from ATCC {Rockville MD) was used. We used the mouse skeletal a-actin cDNA clone J to analyze actin transcriptional activity. As a control for non-specific hybridization, we used the pBluescript vector from Statagene (La Jolla, CA). Plasmid DNAs were isolated from large scale bacterial cultures by cesium chloride gradient centrifugation (41). Plasmids were then linearized by digestion with appropriate restriction enzymes, and then the enzymes were removed by phenol-chloroform extraction. To denature linearized plasmids, DNA samples in TE (10mM Tris-HCI pH 8, 1mM EDTA pH 8) were mixed with 1/10 vol of 1N NaOH and incubated at room temperature for 30 min. Samples were then mixed with 10 vol of 6x SSC and chilled on ice. Aliquots containing 10 pg DNA were then slot blotted onto nylon membranes using a vacuum manifold. DNA was then fixed to the membrane by UV cross-linking using a Stratalinker on automatic setting, Stratagene (La Jolla, CA). Preliminary experiments using serial 125 dilutions of linearized plasmids showed that loading 10pg of DNA per slot was adequate to provide an excess of target DNA for hybridization. Nuclear run-on transcription assays To harvest nuclei for transcription assays, cells were seeded into 850-cm2 roller bottles at 1x106 cells/bottle and harvested at mid-log phase (3 days after initiation of cultures) as for Northern blot analysis. To isolate nuclei, the medium was removed, the cells were washed twice with ice cold phosphate buffered saline (PBS), and then the cells (2 x 107 cells/ceil line) were scraped from bottles into 40 ml of PBS with a rubber policeman. Cells were sedimented by centrifugation (500x g, 5 min, 4°C). For cell lysis, 4 ml NP-40 lysis buffer (10mM Tris pH7.4, 10mM NaCI, 3mM M gCl2, 0.5% NP-40) were added to the cell pellet, and the mixture vortexed gently for 10 secs and incubated on ice for 5 min. Nuclei from the broken cells were pellet by centrifugaton (500x g, 5 min, 4°C), resuspended in 4 ml lysis buffer, and centrifuged again. The final pellet was resuspended in 200 ^l of glycerol storage buffer (50 mM Tris-HCI, pH 8.3, 40% [by volume] glycerol, 5 mM MgCl2, 0.1 mM EDTA) and quickly frozen in liquid N2. For nuclear run-on reactions, 200 jil of frozen nuclei were quickly thawed and transferred to a 12 ml round bottom culture 126 tube. To this was added 200 pi of 2x reaction mixture (10 mM Tris-HCI, pH 8.0, 5 mM MgCI2, 300mM KCI, 1 mM each of ATP, CTP GTP and 5 mM dithiothreitol) and 10 |il (100 |iCi) of [<x-32P]UTP (New England Nuclear; aprox. 760 Ci/mmol). The mixture was incubated at 30°C for 30 min in a Dubnoff metabolic shaking incubator (CGA/Precission Scientific, Chicago, IL) at 100 revolutions per min. For control reactions 2 |ig/ml of a-am anitin (Sigma) was added to each reaction mixture before incubation. After labeling nuclei, the nuclear suspension was transferred to a microcentrifuge tube, and the nuclei were pelleted by a brief spin. The pellet was then dissolved in 1 ml RNAzol (Biogentex, Houston, TX) by pipetting it up and down in a 1000 |il pipet tip. Next, 100(xl of chloroform was added, and the mixture was shaken vigorously, then incubated on ice for 15 min. The mixture was then centrifuged at full speed for 15 min in microcentrifuge, after which the supernatant was transferred to a clean microcentrifuge tube, and an equal volume of ice cold isopropanol added. The resultant mixture was maintained at -20°C for 20 min, and then it was centrifuged for 15 min at 4°C in a microcentrifuge to pellet the lableled RNA. The pellet was rinsed once with cold 70% ethanol, dried briefly and resuspended in 200 |il RNase free TE. To separate labeled RNA from unincorporated [a-32P]UTP, samples were loaded onto a Sephadex G-50 column. The amount of labeled RNA in each sample was then determined by liquid scintilation counting of aliquots of the column eluents. Equal counts per minute of each 127 sample were diluted in hybridization buffer (10 mM TES, pH 7.4, 10mM EDTA, 0.2% SDS, 0.6 M NaCI and 5x Denhardt's Solution) for hybridization (usually 5 x 106 cpm in 5 ml). Prior to hybridization, membrane strips containing slot blotted DNA were placed in 7 ml plastic scintilation vials and prehybridized with hybridization solution without labeled RNA for 2 h at 65°C in a shaking water bath. This was then replaced with hybridization solution containing labeled RNA and incubated for 40 h at 65°C with shaking. After hybridization, membranes were removed and washed once in 2x SSC, 0.1% SDS for 20 min at room temperature and twice in 0.2x SSC, 0.1% SDS for 15 min at 50°C and exposed to preflashed Kodak XAR film at-70°C. The relative amount of hybridization was determined by densitometry of each band as for Northern blot analysis (see chapter 2). To account for possible variations in the number of counts per minute hybridized to each membrane, the m yc signal intensities on each membrane were normalized to that of actin on the same membrane, and the value for myc in 10T1/2 cells was arbitrarily defined as 1.0. Determ ination of half-iife of the c-myc RNA Cells were cultured in 850-cm2 roller bottles exactly as for RNA steady-state analysis (see chapter 2). At mid-exponential growth phase, (day 3 after seeding) actinomycin D (Sigma Chemical Co., St. Louis, MO) dissolved in BME plus 10% fetal bovine serum was added to the cells at a final concentration of 5 pg/ml. Total 128 cellular RNA was extracted by the GITC method (as described in chapter 2) either before or at various times after addition of actinomycin D (15, 30, 60, 90, 120, 150 min). Aliquots of 30 pg of total RNA from each time point were analyzed by Northern blotting as performed for steady-state RNA analysis. Blots were first hybridized with the 1.7-kb Hind\\\ fragment of pMc-myc54 and then stripped and rehybridized with the a-actin clone cDNA probe to control for RNA loading. Decay curves for c-myc RNA were determined by densitometric scanning of the bands visualized on the autoradiograms, normalized against values for actin RNA, and then plotted using the Cricket Graph program (Cricket Software Co., Malvern, PA). Half-life estimates of c-myc RNA were calculated from the slopes of best-fit curves of myc RNA decay using time points at 15 min and later to obviate the initial lag phase before transcription was completely halted. Fractions of myc RNA with differential stabilities were estimated by extrapolation from best fit curves of c-myc RNA decay to the ordinate. Western Blot Analysis of Myc Protein To analyze the sizes and steady-state levels of c-myc proteins in transformed versus nontransformed 10T1/2 cells, the cells were seeded into roller bottles at 1X106 cells/ roller bottle, and nuclear proteins were extracted from mid-log phase cells (day 3 post- seeding). To isolate proteins, cells were harvested by rinsing them in 129 each roller bottle with ice-cold isotonic phosphate-buffered saline (PBS), scraping cells from bottles into 20 ml of PBS with a rubber policeman, and centrifuging cells at 500 g for 5 min at 4°. Cells were then lysed in 2 ml NP-40 lysis buffer (10mM Tris pH7.4, 10mM NaCI, 3mM MgCl2, 0.5% NP-40.) per roller bottle for 5 min on ice, and then nuclei were pelleted by centrifugation at 500 g for 5 min at 4°. Nuclei were then either frozen at -70°C for later processing or lysed immediately in Laemmli lysis buffer (o.1 M Tris pH 6.8, 25% glycerol, 2.0% SDS, 10% 2-mercaptoethanol and 0.01% bromphenol blue) at a concentration of 8x105 nuclei per 60pl. Samples were then boiled 5 min to clarify the supernatant and spun by microcentrifuge for 15 min. Supernatants were transferred to a new tube, and then stored at -70° C for future use. Samples containing protein from 8x105 cells were subjected to SDS-PAGE (8.5% acrylamide) and electrotransferred to nitrocellulose membrane. Immunodetection was performed using an affinity-purified polyclonal antibody directed against full-length murine c-Myc peptide, which was kindly provide by Dr. Stephen Hann (Vanderbilt University, Nashville, TN). Proteins were visualized on Hyperfilm using the Enhanced Chemiluminescence (ECL) immunodetection system (Ammersham) according to the manufacturer's protocols. Quantitation of signal intensities was performed as for Northern blot autoradiographs. 130 RESULTS Transcriptional activity of c-myc in transform ed clones. We first tested the hypothesis that the higher steady-state levels of c-myc RNAs in PbCrCXj-transformed and MCA-transformed cell lines might result from increased transcriptional activity of the gene. To do this, we assayed the relative amounts of nascent c-m yc transcripts in these cell lines by nuclear run-on analysis of nuclei isolated from logarithmically growing cells. Since it has been shown that c-myc transcription can be regulated by a block to elongation at the 3' end of the first exon (27,29), we used a probe containing only exons 2 and 3 in order to only compare productive read-through transcripts. in four separate experiments, equivalent amounts of total radioactive mRNA were hybridized to denatured DNA bound to Nylon filters. Transcription of the (3-actin gene was monitored using a cDNA probe and was used as an internal standard. We detected no hybridization to the Bluescript plasmid lacking an insert demonstrating an absence of non-specific hybridization. A representative experiment out of the four we conducted is shown in figure 16. Densitometer scanning of the autoradiographs allowed us to estimate the relative transcriptional activities of the c-m yc gene in the four transformed cell lines compared with 10T1/2 cells, and the results of four experiments are averaged in table 9. 131 Figure 16. Nuclear run-on transcription assay of the c-myc gene. Nuclear transcripts were generated as described in Materials and methods. Five ml of hybridization solution containing aproximately 1 x 106 cpm/ml of 32P -la be le d nascent transcripts were hybridized to membranes bearing denatured, linearized plasmids. In the right lane nuclei of 10T1/2 cells were incubated with 2(xg/ml a- am anitin. 132 Nuclear Run-on V ________________________________ 10T1/2 PbCr2 PbCr3 c-myc ^ V * GAPDH it ia a ^ g r actin B luescript v Analysis c-myc ___________________ J MCA a 1 5 MCA a 16 a-aman. Table 9 Nuclear run-on experiments showing the transcription of the c-myc gene normalized to transcription of the actin gene. Cell line Expt 1 Expt 2 Expt 3 Expt 4 Avg. ± SE 10T1/2 1.00 1.00 1.00 1.00 1 .0 0 ± 0.00 PbCr2 1.56 0.45 0.91 0.79 0 .9 3 ± 0.23 PbCr3 1.57 1.64 1.89 0.81 1 .48 ± 0.23 MCA Cl 15 1.07 0.88 1.45 0.57 0 .9 9 ± 0.18 MCA Cl 16 0.45 0.44 1.59 1.34 0 .7 6 ± 0.30 When the amount of c-myc transcripts from exons 2 and 3 of the myc gene were normalized to the amount transcribed from the actin gene, there was found to be no elevation in c-myc transcription rate in the MCA-induced CI-15, MCA CI-16, or the P bC r0 4 -induced PbCr2 cell line compared with 10T1/2 cells (Fig. 16, Table 9). The PbCr3 cell line showed a small (1.6-1.9-fold) increase in m yc transcription in 3 of 4 experiments, but the averaged value of four experiments was 1.5 ± 0.2, indicating a large error in the measurement. While it is possible that this difference in c-myc transcription might be contributory to enhanced steady- state levels of c-m yc RNA in transformed cell lines, it likely would not be sufficient in itself to account for the elevated steady-state levels of myc RNA in PbCr3 cells. This observation and the lack of increased transcription of c-myc in the three other transformed cell lines examined strongly suggest that transcriptional mechanisms likely do not contribute to the increased myc RNA levels in all four transformed cell lines. Half-lives of c-myc RNAs in transformed cell lines. It is well documented that c-myc gene expression can be regulated at the level of RNA stability (reviewed in 10). To determine whether overabundance of c-myc RNA in the MCA- and P bC r0 4 -transformed cell lines was due to alterations in the stability of the c-m yc transcript, we compared the half-lives of m yc RNAs in the transformed cell lines with those of 135 nontransformed 10T1/2 cells. Cultures of exponentially growing cells were incubated for various times in the presence of actinomycin D (Act. D) to block transcription, and then total RNA was extracted and analyzed for the amount of c-myc RNA remaining by Northern blotting using the c-myc cDNA probe. As a control, these blots were stripped and rehybridized with the probe to detect the p-actin transcript, which is a very stable transcript (42). Analysis of actin expression showed that all lanes had equal amounts of hybridizable RNA and also ruled out the possibility that the rapid turnover of c-myc RNA in 10T1/2 cells could be due to nonspecific destabilization induced by actinomycin D. Representative Northern blots of c-m yc RNA levels after addition of act. D are shown in figures 17a and 17b. Kinetic curves of RNA degradation were generated from quantitative densitometry of several Northern blots for each cell line (Fig. 18a and 18b) . The value at each time point represents the average c-myc RNA peak areas normalized to actin RNA peak areas from 3-5 separate experiments and expressed as a percentage of the c-myc RNA at T=0 min. Figure 18a shows that after a short lag phase (10-15 min), c- m yc RNA in nontransformed 10T1/2 cells was rapidly degraded and was undetectable (less than 1% of the amount at t=0) by 90 min after addition of actinomycin D. To estimate the half-lives of c- m yc RNAs in the cell lines under study we used the Cricket Graph program (Cricket Software Co., Malvern, PA) to construct best-fit 136 Figure 17. Northern blotting analysis of c-myc mRNA during an actinomycin D chase. Cells were grown to mid-log phase in 850-cm2 roller bottles, medium was changed 24 hr before treatment and then cells were incubated in the presence of actinomycin D (5pg/ml). At various intervals medium containing the act. D was removed and cells were lysed immediately by the addition of 4M guanidinium thiocyanate. RNA was processed and 30pg was electrophoresed in 1% agarose formalehyde gel and transfered to a nylon membrane. Blots were hybridized with a 32P-labeled c-myc cDNA probe and subsequently with an actin cDNA probe. A) 10T1/2 and lead chromate transformed-transformed cell lines. B) 10T1/2 and MCA-transformed cell lines. 137 A 10T1/2 PbCr2 PbCr3 O' 15' 30' 60' 90' 1 20' 1 5 O' c-myc i ♦ r -8 n ^ < r Act in O' 15' 30' 60' 90' 120' 1 5 O' msssmts^ m x j j * „ , z v / t , 2 i- Act in O' 15' 30' 60' 90' 1 20' 1 5 0 1 c-myc i 5 W M W . Act in 138 c-myc 10T1/2 Act in c-myc MCA a 15 A ctin c-myc MCA a 16 Act in Figure 18. Half-lives of c-m yc transcripts (ti/2) from nontransform ed 10T1/2 cells, A) PbCrCM-transformed 10T1/2 cells (PbCr2, PbCr3) and B) MCA-transformed 10T1/2 cells (MCA Cl 15, MCA Cl 16) plotted from densitometric readings of Northern blots. The average of at least three separate experiments is plotted for each point with the standard error of the mean. The a indicates that c-m yc RNA was not detectable at this tim e point. 140 % c-myc mRNA Remaining Decay of c-myc mRNA in 10T1/2 Cl 8 and lead chromate-induced cell lines 1000 1 10T1/2 Cl 8 PbCr2 PbCr3 100 10 - 15 30 45 60 75 90 105 120 135 150 165 0 Tim e (minutes) 141 % c-myc mRNA Remaining Decay of c-myc mRNA in 10T1/2 Cl 8 and MCA-induced cell lines 1000 q 10T1/2 Cl 8 MCA Cl 15 MCA Cl 16 00 0 15 30 45 60 75 90 105 120 135 150 165 Tim e (minutes) 142 curves of RNA levels after the initial lag phase following act. D treatment (Fig. 19a and 19b). From the slope of the best-fit curve, the c-myc RNA in 10T1/2 Cl 8 cells was estimated to decay with a half-life of 9.5 min. This value is in agreement with the findings of Startwout and Kinniburgh for 10T1/2 cells (43). When the RNA decay curves for the PbCr2 and PbCr3 cell lines were compared with those of 10T1/2 cells, there was a marked difference in the rate of c-myc RNA decay after the initial lag phase (Fig 18a). This difference indicated an increased stability of the myc message in the PbCrO/Hnduced cell lines. In the PbCr2 cell line, the c-m yc specific transcripts appeared to undergo a biphasic decay. The initial phase, comprising most of the myc RNA, had an estimated half-life of 15.5 min. In addition, there appeared to be a second component comprising approximately 8% of the myc RNA with a half-life of 64 min as estimated by extrapolation from best fit curves of myc RNA decay to the ordinate (Fig. 19a, middle). The c- myc RNA in the PbCr3 cell line decayed as a single component with a half-life of 17 min (Fig. 19a, bottom). RNA decay curves generated from Northern blot analysis of the MCA-CI 15 and MCA-CI 16 cell lines also revealed a stabilization of c-m yc RNA, similar to that observed for the PbCr0 4 -induced clones (Fig 18b). MCA-CI 15, like PbCr3, displayed a biphasic decay of myc RNA, with the initial component having a half-life of 19 min, while a second fraction comprising approximately 11% of the total m yc RNA had a half-life of 83 mtn 143 Figure 19 Best-fit curves of the decay of separate fractions of c- myc mRNA after actinomycin D treatment. Data was averaged from time points from 15 min and therafter following act. D treatment and before c-myc mRNA became undetectable. Curves were plotted using Cricket Graph software. 144 % c-myc RNA Remaining % c-myc RNA Remaining 15.5 m i n Pb C r 2 % c-myc RNA Remaining § o (Jj o k * M o (- * O l o 0 0 o % om yc R N A R em aining % c * m y c R N A Rem aining % c *m y c R N A Remaining IO O O 1 0 T 1 /2 Cl 8 1 0 0 = 9.5 min 1/2 l O 60 150 O 30 90 120 180 IO O O MCA Cl 15 lO O 19 min 1/2 lO = 83 min 1/2 150 0 30 60 90 120 180 IO O O MCA Cl 16 lO O = 22.5 min 1/2 l O 0 150 60 90 180 30 120 146 (Fig 19b, middle). The myc RNA in the MCA-CI 16 cells appeared to consist of a single component with an apparent half-life of 22.5 min (Fig. 19b, bottom). The estimated half-lives and relative amounts of each fraction of c-m yc RNA in the 10T1/2 cells and the transformed cell lines is summarized in table 10. Myc protein expression in PbCr0 4 - and MCA- transform ed cell lines. W estern blotting analysis with a mouse M yc-sp e cific antiserum was next performed to determine whether elevated steady-state levels of c-myc RNA in the PbCrCU-transformed cell lines also resulted in elevated levels of c-myc protein or expression of altered myc protein products. The MCA Cl 15 and MCA Cl 16 cell lines were also evaluated for m yc protein expression, because previous studies and this report have shown elevated m yc RNA levels in these cell lines. For Western blotting analysis, 1 X 10 6 cells were seeded into 850 cm2 roller bottles, and nuclear protein was extracted at mid-log phase, on day 3, exactly as was done for RNA extraction and Northern blotting analysis. Nuclear proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with an antiserum to full m yc peptide. M yc specific bands were visualized on light sensitive film by enhanced chemiluminescence, and thus were able to be quantitated by laser scanning densitometry. Fig. 20 shows a prominent c-Myc specific band of approximately 65-kDa in all cell 147 Table 10 Estimated Half-lives of c-myc RNAs in nontransformed 10T1/2 cells and in PbCr2, Pbcr3, MCA Cl 15 and MCA Cl 16. Component 1 Component 2 Cell line % of Total half-life (min)* % of Total half-life (min)* 10T1/2 100 9.5 - - PbCr2 92 15.5 8 64 PbCr3 100 17 - - MCA Cl 15 89 19 11 83 MCA Cl 16 100 22.5 - - Note : Actual half-lives in the table should have 15 min added to them to be read directly from the actual data. Figure 20. Western immunoblot analysis of p65-c-Myc in nontransformed 10T1/2, PbCr0 4 -transformed and MCA- transformed cell lines. Nuclear protein extracts from equal numbers of logarithmically growing cells were analyzed by 8.5% SDS-PAGE. Immunodetection was performed using an affinity purified polyclonal antibody directed against full-lenth murine c-Myc peptide. Proteins were visualized by enhanced chemiluminescence. The migration of protein molecular weight standards (BioRad) is indicated at right. kDa, kiloDaltons. 149 c-Myc tn to cm _ _ - . 0 0 5 3 3 T - S S C M 1 _ o A a. 00 l_ O A Q . kDa - 139.9 ; / — 33.3 “ 28.6 150 lines. This band correlates with the major murine c-Myc species, which is initiated from the first AUG codon at the 5' end of exon II (25). A second, less abundant Myc protein, which migrated at a molecular weight of approximately 68 kDa on several gels, likely corresponds to a second Myc protein known to be initiated from a CUG codon within exon I (25). We detected no alterations in the electrophoretic mobility of the two c-Myc protein species in either the PbCr0 4 -transformed cell lines or the MCA-transformed cell lines compared with c-myc proteins expressed by nontransformed 10T1/2 cells by this one-dimensional analysis. However, densitometric measurements of immunoblots averaged from three separate experiments show that the steady-state levels of c-m yc protein are elevated in both PbCr2 and PbCr3 cell lines, and especially in the MCA Cl 15 cell line (Table 11). The PbCrCV induced clones express moderately higher amounts of c-Myc (1.3- fold and 1.5-fold), while in MCA Cl 15 the overabundance of c-Myc is more obvious at a 2.4-fold increase. The MCA Cl 16 does not appear to overexpress c-myc protein during log phase growth when compared to 10T1/2 cells, despite expressing higher steady-state levels of c-myc RNA during exponential growth. In addition to our finding higher steady-state levels of c-m yc RNA, the over-abundance of c-myc protein we found in two PbCrC>4- transformed cell lines and one MCA-transformed cell line suggests that c-m yc may be mechanistically involved in mediating the transformed phenotype of these cell lines. The lack of 1 51 Table 11 Relative Steady-state levels of c-Myc protein. Cell line Expt. 1 Expt. 2 Expt. 3 Avg. iS E 1 0 T 1 /2 1.0 1.0 1.0 1.0 ± 0 PbCr2 1.5 2 .7 0 .4 1.5 ± 0 .7 PbCr3 1.6 0 .5 1.8 1.3 ± 0 .4 MCA Cl 15 3 .2 2 .8 1.3 2.4 ± 0 .6 MCA Cl 16 0 .6 1.2 0 .4 0.7 ± 0 .2 overexpression of c-m yc protein in the MCA Cl 16 cells may reflect a m echanism of post-transcriptional regulation which counteracts the overexpression of m yc RNA and which is ineffective in the other cell lines. Alternatively, deregulation of c-m yc in the form of an inability to down-regulate c-m yc protein levels in response to environmental signals might not be detected as an increase in the quantity of c-Myc in the cell during log phase growth. DISCUSSION In the previous chapter, we examined the expression and structure of proto-oncogenes and tumor suppressor genes in two P bC r0 4 -transformed 10T1/2 cell lines, PbCr2 and PbCr3, to gain insight into molecular mechanisms of lead chromate induced cell transform ation. We found that both clones expressed significantly (2-6 fold) elevated steady-state levels of c-m yc mRNA and slightly (1.2-2.4 fold) elevated levels of the m yc protein product. Over expression of m yc was not accompanied by detectable alterations in the size of the 2.3-kb c-m yc transcript or of the p65 m yc protein. Furthermore, DNA restriction digest Southern blotting analysis revealed no amplification, rearrangements, or other gross structural alterations in the m yc gene in either transformed clone. We also found that among two 3-methlylcholanthrene (MCA) 153 transformed cell lines, MCA Cl 15 and MCA Cl 16, which express elevated levels of c-myc RNA, one (MCA Cl 15) also contained 2-4 fold higher steady-state levels of c-m yc protein. However, it was not known what mechanisms were responsible for the elevated steady-state levels of c-myc RNA in either the PbCr0 4 - transform ed or MCA-transformed cell lines. In this chapter we investigated the contributions of transcriptional and posttranscriptional mechanisms to deregulated c-m yc expression in these two sets of transformed 10T1/2 cell clones. We found no increase in rate of transcription of exons 2 and 3 of the myc gene in isolated nuclei from three of the four transformed cell lines compared to that of 10T1/2. The PbCr3 cells showed a slight (1.5-fold) increase in myc transcription. When we blocked transcription with actinomycin D and examined the rate of RNA decay, we found a slower turnover of c-myc RNA in both the MCA and PbCr0 4 transformed cell lines. Therefore, we believe that alterations, likely loss of posttranscriptional mechanisms which control degradation of this RNA, contribute to the elevated steady-state levels of c-m yc mRNA in the transform ed cell lines studied. Numerous lines of evidence have identified the c-myc gene as a central regulator in the control of proliferation, differentiation, and apoptosis in mammalian cells (11,44). In order to fulfill this important role, the expression of m yc must be exquisitely tuned to respond rapidly to a variety of different signals. It is not 154 surprising, then, that myc expression is governed by a complex set of controls which act both at the transcriptional and posttranscriptional levels. This includes those of transcript initiation, elongation, messenger RNA stability, regulation of translation, protein stability, phosphorylation status, and negative feed-back mechanisms (44). There is evidence that the multiplicity of signals such as those generated by growth factors, cytokines, extracellular matrix, and cell-cell contact which affect c-myc expression, do so by specifically affecting one or more of these mechanisms (10). The regulatory mechanism utilized may depend upon how rapid, how large or how long-lasting a change in myc expression is required. Interestingly, evidence from experiments in vitro and in vivo suggest that for the myc gene, posttranscriptional mechanisms play a particularly prominent role in myc reguation a wide variety of cell types and contexts. For example, studies using BALB/c 3T3 mouse fibroblasts have demonstrated the importance of posttranscriptional mechanisms in regulating c-myc RNA levels in response to growth conditions in culture. It has been shown that in the presence of serum growth factors subconfluent cultures expressed about 10-fold higher levels c-myc mRNA than density arrested cells. Similarly, our laboratory has found that C3H/10T1/2 cells down regulate the level of c-myc RNA when the cells are grown to confluence (3). However, the level of c-myc gene transcription in the 3T3 cells was approximately the same in 155 both low and high density cultures (15). This suggests that the down regulation of c-myc induced by cell contact in mouse fibroblasts is controlled at the posttranscriptional level, possibly through regulation of mRNA stability. Other studies have shown the importance of posttranscriptional mechanisms in the control of c-myc expression by a variety of factors, including serum. It is well known that when serum starved cells are stimulated with fresh serum, the steady-state levels of c-myc RNA rise more than 20-fold. However, it was shown that serum stimulation caused only a 5-fold increase in the transcription activity of the c-myc gene (45). Thus, the up-regulation of myc RNA levels in respnse to serum stim ulation must involve posttranscriptional controls such as stabilization of the c-m yc transcript. In WEHI 231 mouse B- lymphoma cells, stabilization of the mRNA is partly responsible for a rapid induction of c-myc expression after incubation with antisera to cell surface immunoglobulin (46). Posttranscriptional mechanisms are believed to account for much of the regulation of c-m yc message levels in murine erythroleukemia cells induced to differentiate with HMBA. Interestingly, it is thought that intra nuclear events such as RNA maturation and/or transport may play an important role in this process rather than alterations in RNA stability (45). Interferon treatment was shown to markedly increase the stability of c-myc mRNA in 3T3 fibroblasts (47). In quiescent organs in mice, the steady-state level of c-myc mRNA 156 varies dramatically from one tissue to the other, it is high in lymphoid organs, low in the liver, although c-m yc transcription is similar in these organs (48). Finally, reduction of c-myc RNA levels by dexamethasone in human lymphoma cells and by sodium butyrate in a human colon carcinoma cell line were both suggested to be the result of specific destabilization of the c-m yc transcript (49,50). Given the importance of posttranscriptional control of myc, it is not surprising that alterations in the mechanisms regulating messenger RNA degradation are associated with elevated steady- state levels of myc RNA and with unrestrained growth in human and rodent cells (51-57). In our studies, aberrant posttranscriptional controls manifested as a longer half-life of c-myc RNA appear to be largely responsible for elevated c-myc RNA levels in PbCr0 4 and MCA-transformed cell lines. At mid-log phase, PbCr2 cells expressed 4.7-fold higher steady-state levels of c-m yc RNA than nontransformed 10T1/2 cells, although both cell lines had equivalent rates of c-m yc transcription. Actinomycin D chase experiments revealed that enhanced stabilization of c-m yc transcripts contribute to the overabundance of this RNA. Analysis of the myc RNA decay curve suggests that the pool of stabilized transcripts contains two populations; one comprises most of the RNA and has a half-life 1.5- fold greater than that of 10T1/2 m yc RNA, and the other fraction of about 10% has a half-life nearly 7-fold longer than the control. If 1 57 we assume that the contribution of increased stability to elevated steady-state levels is directly proportional to the increases in half-life and to the ratios of the two components, we would expect the steady-state level of m yc RNA in the PbCr2 cells to be slightly more than 2-fold greater than that of 10T1/2 cells in logarithmic growth. This suggests that other posttranscriptional mechanisms, perhaps involving mRNA processing or transport, may be involved in the altered levels of myc RNA in this clone. The myc RNA in the PbCr3 cell line exhibited a single decay com ponent with a half-life 1.8-fold longer than that found in 10T1/2 cells. This clone also showed a small (1.5-fold) increase in c-m yc transcription when averaged over four separate experiments. These two factors might be expected to cause approxim ately a 2.7-fold elevation in m yc RNA steady-state expression. Here, again, additional regulatory elements besides RNA transcription and stabilization may contribute to the 4.0-fold higher steady-state level of c-m yc RNA. Our studies of c-myc regulation in the two MCA-transformed cell lines suggested that elevated m yc RNA levels were not the result of increased transcription of the gene. However, the increased half-lives of the two components of decay in MCA Cl 15 (89% of myc RNA had a 19 min ti/2, 11% of myc RNA had an 83 min ti/ 2, so combined approximately 2.7-fold longer t i/ 2) and the single species of c-m yc transcript in MCA Cl 16 (2.4-fold longer t i/ 2) 158 appear to be sufficient to account for the roughly 2-fold higher steady state levels of c-myc RNA in these cell lines. What is the mechanism responsible for the altered stabilities of c-myc mRNAs in the PbCr0 4 - and MCA-transformed cell lines? Alteration of c-myc RNA metabolism has been shown to occur in certain tumors and transformed cell lines, usually as the result of changes in the structure of the transcript (10). For instance, in both mouse plasmacytomas and human Burkitt's lymphomas, translocations frequently occur between the largely noncoding first exon and coding second exon of c-myc, leading to mRNAs that initiate at cryptic promoters in the first intron and hence lack the normal 5' untranslated region (UTR). The resulting tumor specific mRNAs have half-lives on the order of 2-4 h instead of 15-20 min (51-53). Loss of a 61 nucleotide AT-rich sequence in the 3' UTR of the c-myc gene by chromosomal translocation in human T-cell leukemia increases mRNA stability by 5-fold (56). The presence of intron 1 in mature c-myc mRNAs of two mouse plasmacytoma cell lines causes stabilization of the message despite the presence of normal 5' and 3' untranslated regions (54). Northern and Southern blotting analysis revealed no gross alterations in the RNA or DNA of the c-myc gene in the four transformed 10T1/2 cell lines exhibiting defective RNA turnover. This suggests that either mutations exist in sequences important for myc RNA instability that are undetectable by the crude methods employed, or that there are alterations in trans-acting factors 159 which specifically mediate c-myc RNA degradation rates. Such factors could act either positively to enhance turnover as occurs when mouse 3T3 fibroblasts are grown to confluence (15), or negatively to control myc RNA levels through stabilization as is seen mouse liver cells after partial hepatectomy (58). Our next step will be to determine whether there are undetected structural alterations causing the stabilization of c- m yc transcripts, and if so, to determine how large a sequence alteration would be required to increase the stability of the message by 2-3-fold. Interestingly, Bonnieu et al. found that a deletion of 130 nucleotides in the 3’ UTR of a transfected pSvc- myc construct caused the transcribed RNAs to have 2-3-fold longer half-lives (59). However, it was not determined whether this was the smallest alteration that would produce such an effect. Studies using hybrid transcripts and deletion mutants have revealed the presence of at least two instability determinants. One element lies in the 3' untranslated region and presumably consists of (A+U) rich sequences, and the other lies in the carboxy-terminal portion of the coding region and colocalizes with sequences encoding protein-dimerization motifs (60). The exact sequences which are responsible for the instability of c-myc mRNA have yet to be defined. Moreover, evidence from tumors cells in which only the first exon and intron are altered shown that addition or deletion 5' sequences can interfere with the degradation signal (60). The message stabilization resulting from loss of 61 nucleotides within 160 the 3' UTR mentioned above indicates an alteration which might not be detected by Northern or Southern blotting analysis. There is also some evidence that m yc RNA degradation signals might be associated RNA loop structures (60). It is therefore conceivable that small alterations within stretches im portant for base pairing might disrupt RNA secondary structures and thereby interfere with message instability signals. Several lines of evidence support the possibility that alterations in trans-regulators might be causing aberrant stabilization of c-m yc transcripts in the transformed cell lines examined in this study. First, we see no structural alterations in the m yc transcripts by Northern blotting analysis or in the gene by Southern blotting analysis, although these are relatively crude methods. Secondly, there is both indirect and direct evidence for the existence of proteins which bind to and regulate c-myc mRNA stability. The alterations in m yc RNA stability during normal physiological processes such as cell cycle entry, growth arrest, and differentiation all occur without changes in the sequence of the transcript. Further, several putative regulatory factors have been purified which bind specifically to m yc RNA in vitro and reduce the half-life of m yc mRNA by 3- to 6-fold (61-63). Finally, there is evidence from studies of other transformed cells that elevated steady-state levels of c-m yc resulting from mRNA stabilization can occur in the absence of structural alteration of the transcript. Baer et al. found greater than a 2-fold enhancement 161 in c-myc mRNA half-lives in cells from two of eight patients with acute myeloid leukemia without an alteration in transcript size (57). The normally short-lived c-myb transcript was also found to be stabilized but not c-fos mRNA (57). This is interesting because one of the putative destabilizing proteins mentioned above also destabilizes c-myb RNA but not whole cell polyadenylated RNA (61). Furthermore, studies with human B lymphoma cell lines have demonstrated that c-myc mRNA half-lives in cells immortalized with Epstein-Barr virus are more than double that of EBV-negative cell lines during late exponential phase of growth (55). This difference occurs in the absence of apparent structural alterations in the c-myc transcript (64). Because we have not yet identified the cause of the altered stability of c-myc transcripts in the transformed 10T1/2 cell lines, it is difficult to speculate why we observed biphasic decay curves in two of the four cell lines and single component curves in the others. The 10T1/2 Cl 8 cell line has 81 chromosomes. The transformed clones we studied are presumably slightly less than tetraploid. Therefore, they might be expected to each harbor four copies of the c-myc gene. If one copy was mutated and gave rise to longer lived transcripts, a biphasic decay curve might be expected, wherein 75% of the mRNA would decay with kinetics similar to 10T1/2 cells, and 25% of the mRNA would display altered kinetics. In fact, it was observed that in the PbCr2 and MCA Cl 15 cell lines both components displayed extra stability, and the longer lived 162 component only comprised about 10% of the myc mRNA pool. This suggests that additional factors may be involved in determining the overall kinetics of decay in these cell lines. The chromosome harboring the transforming c-myc gene might be present in only one half of the cells, or it might be transcribed more slowly than the wild-type c-myc gene. It may be that a single copy of myc with a 6-fold longer half-life is transcribed at 1/2 of the rate of the remaining copies, and the remaining copies express transcripts that are stabilized by only 2-fold. Interpretations from the mRNA decay curves about differences in the metabolism of c-m yc transcripts between the nontransformed 10T1/2 and transformed of cell lines is limited by the fact that we don't know whether the cells actually have four copies of the c-myc gene or whether all copies are actively transcribed. Studies now in progress are aimed at determining the mechanism of mRNA stabilization in the PbCrCU-transformed and MCA-transformed cell lines. To asses the significance of elevated c-myc mRNA levels in PbCrC>4- and MCA-induced cell lines in terms of contributing to their transformed phenotype, we quantitated the relative steady-state levels and molecular weights of c-myc proteins in each of the cell lines. Results averaged from several western immunoblotting experiments demonstrated a modest (10%-140%) elevation of c-myc protein in the PbCr2 and PbCr3 cell lines. The molecular weight of the c-m yc protein was 65,000 in the nontransformed and four 163 transformed cell lines. The c-m yc protein has a short half-life, but in most cases the steady-state levels of c-myc protein correlate roughly with the steady-state levels of mRNA (65). Therefore, it is somewhat surprising not to find a greater elevation in m yc protein levels in the transformed cells studied here. However, there is significant evidence that c-myc protein levels can be regulated at the post- transcriptional level. For example, overexpression of a transfected human c-myc gene caused phenotypic changes of malignant transformation in NIH3T3 cells (66). Yet, despite the expression of high level of m yc RNA, the cells contained a relatively low level of m yc protein, suggesting that expression was controlled at a post- transcriptional level (66). Also, while the overabundance of m yc protein in PbCrCVtransformed cell lines is not very dramatic, recent evidence suggests that during exponential cell growth, cells are very sensitive to c-myc expression levels (67). It was reported that even small changes in expression, whether positive or negative, caused alterations in growth rate (67). In addition, very high levels of c-m yc protein are known to be toxic to cells. Hence, if the c-m yc protein level in the immortalized nontransformed 10T1/2 cells is relatively high, significant overexpression above this level might be not be tolerated by transformed derivative cell lines. Too high c-m yc protein levels may be cytotoxic by over-stimulating the signal transduction pathways. 164 BIBLIOGRAPHY 1. Vennstrom, B., Sheiness, D., Zabielski, J., and Bishop, J. M. Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29. J. Virol.., 42: 773-779, 1982. 2. Neil, J. C., et at. Transduction and rearrangement of the m yc gene by feline leukaemia virus in naturally occurring T-cell leukaem ias. Nature (London), 308: 814-820, 1984. 3. Shuin, T., Billings, P. C., Lillehaug, J. R., Patierno, S. R., Roy- Burman, P., and Landolph, J. R. Enhanced expression of c-myc and decreased expression c-fos protooncogenes in chemically and radiation-transformed C3H/10T1/2 CI8 mouse embryo cell lines. Cancer Res., 46: 1986. 4. Billings, P. C., Shuin, T., Lillehaug, K., Miura, T., Roy-Burman, P., and Landolph, J. R. Enhanced expression and state of the c- myc oncogene in chemically and x-ray-transformed C3H/10T1/2 Cl 8 mouse embryo fibroblasts. Cancer Res., 47: 3643-3649, 1987. 5. Cole, M. D. The myc oncogene: its role in transformation and differentiation. Ann. Rev. Genet., 20: 361-384, 1986. 6. Moore, J. P., Hancock, D. C., Littlewood, T. D., and Evan, G. I. A sensitive and quantitative enzyme-linked immunosorbance assay for the c-myc and N-myc oncoproteins. Oncogene Res., 2: 65-80, 1987. 7. Land, H., Parada, L. F., and Weinberg, R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature (London), 304: 596-601, 1983. 165 8. Ruley, E. H. Adenovirus early region 1A enables viral and cellular genes to transform primary cells in culture. N ature (Lond.), 304: 602-606, 1983. 9. Luscher, B. and Eisennman, R. N. New light on Myc and Myb. Part I Myc. Genes & Dev., 4: 2025-2035, 1990. 10. Spencer, C. A. and Groudine, M. Control of c-myc regulation in normal and neoplastic cells. Adv. Cancer Res., 56: 1-48, 1991. 11. Evan, G. I., et al. Induction of apoptosis in fibroblasts by c- Myc protein. Cell, 69: 119-128, 1992. 12. Blackwood, E. M. and Eisenman, R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science (Wash. DC), 251: 1211-1217, 1991. 13. Murre, C., McCraw, P. S., and Baltimore, D. A new DNA-binding and dimerization motif in immunoglobulin enhancer binding, daughter-less, MyoD and Myc proteins. Cell, 56: 777-783, 1989. 14. Blackwell, T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N., and Weintraub, H. Sequence-specific DNA binding by the c-Myc protein. Science (Wash. DC), 250: 1149-1151, 1990. 15. Dean, M., Levine, R. A., Ran, W., Kindy, M. S., Sonnenshein, G. E., and Campisi, J. Regulation of c-myc transcription and mRNA abbundance by serum growth factors and cell contact. J. Biol. Chem., 261: 9161-9166, 1986. 16. Waters, C. M., Littlewood, T. D., Hancock, D. C., Moore, J. P., and Evan, G.l. c-Myc protein expression in untransfomed fibroblasts. Oncogene, 6: 797-805, 1991. 17. Almendral, J. M., Sommer, D., MacDonald-Bravo, H., Burckhardt, J., Perera, J., and Bravo, R. Complexity of the early genetic response to growth factors in mouse embryo fibroblasts. Mol. Cell. Biol., 8: 2140-2148, 1988. 166 18. Baserga, R. The cell cycle: myths and realities. Cancer Res., 50: 6769-6771, 1990. 19. Eilers, M., Schirm, S., and Bishop, J.M. The Myc protein activates transcription of the alpha-prothymosin gene. EM BO J., 10: 133-141, 1991. 20. Amati, B., Dalton, S., Brooks, M.W., Littlewood, T.D., Evan, G.I., and Land, H. Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max. N ature (Lond.), 359: 423-426, 1992. 21. Kretzner, L., Blackwood, E.M., and Eisenman, R.N. Myc and Max proteins posses distinct transcriptional activities. N a tu re (Lond.), 359: 426-429, 1992. 22. Bello-Fernandez, C., Packham, G., and Cleveland, J. L. The ornithine decarboxylase gene is a transcriptional target of c- Myc. Proc. Natl. Acad. Sci. USA, 90: 7804-7808, 1993. 23. Jansen-Durr, P., et al. Differential modulation of cyclin gene expression by MYC. Proc. Natl. Acad. Sci. USA, 90: 3685-3689, 1993. 24. Pagano, M., Pepperkok, R., Verde, F., Ansorge, W., and Draetta, G. Cyclin A is required at two points in the human cell cycle. EMBO J., 11: 961-971, 1992. 25. Spotts, G. D. and Hann, S. R. Enhanced translation and increased turnover of c-myc proteins occur during differentiation of murine erythroleukemia cells. Mol. Cell. Biol., 10: 3952-3964, 1990. 26. Bentley, D. L. and Groudine, M. A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells. Nature (Lond.), 321: 702-706, 1986. 167 27. Eick, D. and Bornkamm, G. W. Transcriptional arrest within the first exon is a fast control mechanism in c-myc gene expression. Nucleic Acids Res., 14: 8331-8346, 1986. 28. Kelly, K. and Siebenlist, U. The regulation and expression of c- myc in normal and malignant cells. Ann. Rev. Immunol., 4: 317-338, 1986. 29. Cesarman, E., Dalla-Favera, R., Bentley, D., and Groudine, M. Mutations in the first exon are associated with altered transcription of c-myc in Burkitt lymphoma. Science (Wash. DC), 238: 1272-1275, 1987. 30. Erisman, M. D., Rothberg, P. G., Diehl, R. E., Morse, C. C., Spandorfer, J.M., and Astrin, S.M. Deregulation of c-myc gene expression in human colon carcinoma is not accompanied by amplification or rearrangement of the gene. Mol. Cell. Biol., 5: 1969-1976, 1985. 31. Yoshimoto, K., Hirohashi, S., and Sekiya, T. Increased expression of the c-myc gene without gene amplification in human lung cancer and colon cancer cell lines. Jpn. J. Cancer Res., 77: 540-545, 1986. 32. Terrier, P., et al. Structure and expression of c-myc and c-fos proto-oncogenes in thyroid carcinomas. Br. J. Cancer, 57: 43- 47, 1988. 33. Stacey, S. N., Nielson, I., Skouv, J., Hansen, C., and Autrup, H. Deregulation in trans of c-myc expression in immortalized human urothelial cells and in T24 bladder carcinoma cells. Mol. Carcinogenesis, 3: 216-225, 1990. 34. Rabbitts, T. H., Forster, A., Hamlyn, P., and Baer, R. Effect of somatic mutation within the translocated c-m yc genes in Burkitt’s lymphoma. Nature (Lond.), 309: 592-597, 1984. 35. Yano, T., Sander, C. A., Clark, H. M., Dolezal, M. V., Jaffe, E. S., and Raffeld, M. Clustered mutations in the second exon of the 168 MYC gene in sporadic Burkitt's lymphoma. Oncogene, 8: 2741- 2748, 1993. 36. Henriksson, M., Bakardjiev, A., Klein, G., and Luscher, B. Phosphorylation sites mapping in the N-terminal domain of c- m yc modulate its trasforming potential. Oncogene, 8: 3199- 3209, 1993. 37. Reznikoff, C. A., Bertram, J. S., Brankow, D. W., and Heidelberger, C. Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. C a n ce r Res., 33: 3239-3249, 1973. 38. Lillehaug, J. R., Evans, D. P., and Landolph, J. R. Increased expression and increased half-life of c-myc RNA in 10T1/2 cell lines transformed by sodium arsenite. {in preparation). 39. Sakuramoto, T., Evans, D. P., Miura, T., Boone, S., and Landolph, J.R. Enhanced expression of the c-Ha-ras and c-myc proto oncogenes in 10T1/2 mouse embryo cell lines transformed by crystalline nickel monosulfide and nickel oxide, (in preparation). 40. Coleman, W. B., Throneburg, D. B., Grisham, J. W., and Smith, G. J. Overexpression of c-K-ras, c-N-ras and transforming growth factor |3 co-segregate with tumorigenicity in morphologically transformed C3H 10T1/2 cell lines. Carcinogenesis, 15: 1005-1012, 1994. 41. Maniatis, T., Fritsch, E. F., and Sambrook, J. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Publications, 1990. 42. Krowczynska, A., Yenofsky, R., and Brawerman, G. Regulation of messenger RNA stability in mouse erythroleukemia cells. J. Mol. Biol., 181: 231-239, 1985. 169 43. Startwout, S. G. and Kinniburgh, A. J. c-myc RNA degradation in growing and differentiating cells: possible alternative pathw ays. Mol. Cell. Bio., 9: 288-295, 1989. 44. Marcu, K. B., Bossone, S. A., and Patel, A. J. myc function and regulation. Annu. Rev. Biochem., 61: 809-860, 1992. 45. Nepveu, A., Levine, R. A., Campisi, J., Greenberg, M. E., Ziff, E. B., and Marcu, K. B. Alternative modes of c-myc regulation in frowth factor-stim ulated and differentiating cells. O n co g e n e , 1: 243-250, 1987. 46. Levine, R. A., McCormack, J. E., Buckler, A., and Sonenshein, G. A. Transcriptional and posttranscriptionai control of c-m yc gene expression in WEHI 231 cells. Mol. Cell. Biol., 6: 4112- 4116, 1986. 47. Levine, R. A., Seshardri, T., Hann, S. R., and Campisi, J. Posttranscriptionai changes in grow th-factor-inducible gene regulation caused by antiproliferative interferons. C ell Regulation, 1: 215-226, 1990. 48. Morello, D., Asselin, C., Lavenu, A., Marcu, K. B., and Babinet, C. Tissue-specific post-transcriptional regulation of c-m yc expression in normal and H-2K/human c-myc transgenic mice. Oncogene, 4: 955-961, 1989. 49. Maroder, M., et al. Post-transcriptional control of c-m yc proto-oncogene expression by glucocorticoid hormones in human T lymphoblastic leukemic cells. Nuc. Acids Res., 18: 1153-1857, 1990. 50. Souleimani, A. and Asselin, C. Regulation of c-myc expression by sodium butyrate in the colon carcinoma cell line Caco-2. FEBS Lett., 326: 45-50, 1993. 51. Eick, D., et al. Aberrant c-myc RNA species of Burkitt's lymphoma cells have longer half-lives. EMBO J., 4: 3717- 3726, 1985. 170 52. Piechaczyk, M., Yang, J. Q., Blanchard, J. M., Phillippe, J., and Marcu, K. B. Posttranscriptionai mechanisms are responsible for accumulation of truncated c-myc RNAs in murine plasma cell tumors. 42: 589-597, 1985. 53. Rabbitts, P. H., Forster, A., Stinson, M. A., and Rabbitts, T. H. Truncation of exon 1 from the c-myc gene results in a prolonged c-myc mRNA stability. EMBO J., 4: 3727-3733, 1985. 54. Bauer, S. R., et al. Altered myc gene transcription and intron- induced stabilization of myc RNAs in two mouse plasmacytomas. 4: 615-623, 1988. 55. Lacy, J., Summers, W. P., and Summers, W. C. Post transcriptionai mechanisms of deregulation of MYC following conversion of a human B cell line by Epstein-Barr virus. EMBO J., 8: 1973-1980, 1989. 56. Aghib, D. F., Bishop, J. M., Ottolinghi, S., Guerrasio, A., Serra, A., and Saglio, G. A 3' truncation of MYC caused by chromosomal translocation in a human T-cell leukemia increases mRNA stability. 5: 707-711, 1990. 57. Baer, M. R., Augustinos, P., and Kinniburgh, A. J. Defective c- myc and c-myb RNA turnover in acute myeloid leukemia cells. Blood, 79: 1319-1326, 1992. 58. Morello, D., Fitzgerald, M. J., Babinet, C., and Fausto, N. c-myc, c-fos, and c-jun regulation in the regenerating livers of normal and H-2K/C-myc transgenic mice. Mol. Cell. Biol., 10: 3185-3193, 1990. 59. Bonnieu, A., Roux, P., Marty, L., Jaentreur, P., and Piechaczyk, M. AUUUA motifs are dispensible for rapid degradation of the mouse c-myc RNA. Oncogene, 5: 1585-1588, 1990. 171 60. Laird-Offringa, I. A. What determines the instability of c-m yc proto-oncogene mRNA. BioEssays, 14: 119-124, 1992. 61. Brewer, G. and Ross, J. Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid com ponent. Mol. Cell. Biol., 9: 1969-2006, 1989. 62. Brewer, G. An A+U-rich element RNA-binding binding factor regulates c-myc mRNA stability in vitro. Mol. Cell. Biol., 11: 2460-2466, 1991. 63. Bernstein, P. L., Herrick, D. J., Prokipcak, R. D., and Ross, J. Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant. G enes Dev., 6: 642-654, 1992. 64. Lacy, J., Summers, W. P., Watson, M., Glaser, P. M., and Summers, W. C. Amplification and deregulation of MYC following Epstein-Barr virus infection of a human B-cell line. Proc. Natl. Acad. Sci. USA, 84: 5838-5842, 1987. 65. Hann, S. R. and Eisenmann, R. N. Proteins encoded by the c-myc oncogene: differential expression in neoplastc cells. Mol. Cell. Biol., 4: 2486-2497, 1984. 66. Ray, R., Thomas, S., and Miller, D. M. Mouse fibroblasts transformed with the human c-myc gene express a high level of mRNA but a low level of c-myc protein and are non- tumorigenic in nude mice. Oncogene, 4: 593-600, 1989. 67. Shichiri, M., Hanson, K. D., and Sedivy, J. M. Effects of c-myc expression on proliferation, quiescence, and the G0 to G1 transition in nontransformed cells. Cell Growth and Diff., 4: 93-104, 1993. 172 CHAPTER 4 CONCLUSIONS AND PROPOSED FUTURE STUDIES There is a lack of knowledge about the mechanisms by which chromium compounds cause human cancer. Our laboratory has used model cell culture systems to study the cellular and molecular mechanisms of oncogenesis by carcinogenic metal salts. A previous publication from our laboratory (1), detailed studies of chromate compounds using the C3H/10T1/2 Cl 8 (10T1/2) cell transformation assay. We showed that insoluble lead chromate induced morphological, anchorage-independent, and neoplastic transformation of 10T1/2 cells but did not induce mutation to ouabain resistance in 10T1/2 or CHO cells or 6-thioguanine resistance (in CHO cells) (1). We also cloned and biologically characterized two cell lines derived from type III foci of lead chromate treated 10T1/2 cell cultures. The isolation and biological characterization of two lead chromate transformed cell lines (PbCr2 and PbCr3) presented me with an excellent opportunity to study induction of morphological transformation by this compound at the molecular genetic level. We hypothesized that transformation of 10T1/2 cells by lead chromate resulted from deletion or inactivation of tumor suppressor genes and/or amplification and/or activation of proto 173 oncogenes. We also hypothesized that such alterations contributing to morphological transformation by lead chromate could be identified by comparing the PbCr2 and PbCr3 ceil lines with 10T1/2 Cl 8 cells at the molecular level. The large number of growth-related genes, and therefore putative targets for interaction with PbCrC>4 or its by-products, made the scope of this project rather broad. Therefore, we employed Northern and Southern blot hybridization techniques to conduct a rapid, broad survey of tumor suppressor genes and proto oncogenes for gross alterations in structure or expression to increase our chances of finding molecular lesions causative of cell transformation. Our choice of genes to examine first was guided by studies previously conducted with other transformed 10T1/2 cell lines which established interesting patterns of gene expression to which we could also compare the PbCr2 and PbCr3 cell lines. This approach proved fruitful in that we identified substantial differences in c-myc expression without having surveyed many different genes. The eventual goal in following all of these above approaches was to characterize the genetic lesions identified, understand their contribution to the transformed phenotype, and if possible gain insight into mechanism by which they arose as it relates to lead chromate exposure. Upon identification of altered steady- state levels of c-myc in the PbCrCVtransformed cell lines, the remainder of my dissertation focused on characterizing the 174 apparent lesion responsible this dysregulation of myc expression. It was found that aberrant posttranscriptionai regulation including mRNA stabilization was responsible for elevated c-m yc transcript levels in the transformed cells. It was also shown that overabundance c-myc mRNA resulted in elevated myc protein levels but these levels were not exactly in proportion to that of the mRNA. These findings of higher steady-state levels of c-myc RNA reveal an interesting and important phenomenon which we believe contributes partly to induction and maintenance of the transformed phenotype. The specific lesion of the level of the c-myc RNA and c- myc DNA which accounts for the observed differences in myc RNA metabolism awaits more detailed analysis of the myc gene stru ctu re . In future work our second approach will be to transfect genomic DNA from the PbCrCVtransformed 10T1/2 cells into an appropriate recipient cell line and identify activated oncogenes within DNA segments which induce transformation in the recipient cells or secondary transfectants. Because of the early success of the Northern and Southern blotting first approach, we have not yet employed this second strategy. In addition, this approach is highly specific for small genes like c-Ha-ras and c-ki-ras, and not sensitive for large genes. The third approach will be to identify the differential expression of genes between nontransformed and lead chromate transformed cells by constructing and screening subtracted cDNA libraries. Again, the success of the first approach 175 postponed the need for this strategy. However, Laurent Ozbun, who collaborated with me on some studies described in this dissertation, is employing a similar strategy involving differential mRNA display to identify additional genes besides c-myc which might contribute to the transformed phenotype of the PbCrCV induced clones. Experiments utilizing the polymerase chain reaction, DNA sequence analysis, and single strand conformation polymorphism analysis should be conducted to determine whether alterations of c-m yc expression reflect changes in the nucleotide sequence of the gene. Efforts should also concentrate on whether there are differences in the intracellular environments of transformed cells that confer altered regulation of exogenous myc genes, c-myc mRNAs from nontransformed and transformed cell lines might also be compared for the binding of putative destabilizing factors. It will also be important to asses the biological significance of alterations in myc genes if discovered. RNA hybrid experiments could analyze the effects of identified mutations on RNA stabilities. In addition, if c-m yc cDNAs are identified in the transformed cells they should be tested for the ability to induce foci in 10T1/2 or NIH3T3 cells when co-transfected with an activated ras gene, c-myc expression is dramatically reduced in nontransformed 10T1/2 cells at high cell density, whereas c-m yc expression remains high in the transformed cell lines. It would also be of interest to know whether the down-regulation of myc in 1 76 the 10T1/2 cells occurs by transcriptional control and whether this is blocked in the transformed clones or whether transcriptional repression is abrogated by a further increase in RNA stabilization in the transformed clones. Finally, studies of 10T1/2 cells transformed by a variety of mechanisms indicates that those cells which have progressed to the tumorigenic phenotype (such as PbCr2 and PbCr3) have undergone multiple (probably 3 or more) alterations in growth regulatory genes or epigenetic processes (2). In fact, experiments with transfected oncogenes have shown that over-expression of c- m yc is not sufficient to induce morphological transformation in C3H/10T1/2 cells, but that mutated c-Ha-ras and mutated c-m yc genes together cause strong morphological transformation. (3). Therefore, we hypothesize that there are alterations in proto-oncogenes and/or tumor suppressor genes in addition to c- m yc which also contribute to the transformed phenotype of the PbCr2 and PbCr3 cell lines. Furthermore, it is likely that such additional alterations might affect pathways which would complement those activated by overexpression of c-myc. One such pathway is thought to include Ras because an Ha-ras gene is able to cooperate with c-m yc to transform primary fibroblasts (4). In fact the 3-methylcholanthrene transformed 10T1/2 Cl 15 and Cl 16 cell lines have a mutation in codon 12 of the c-Ki-ras gene (5). Our studies found no overexpression of the c-Ha-ras gene in the lead chromate transformed cell lines. However, it is not yet known 1 77 whether ras genes including Ha -ras, K-ras or N-ras might harbor activating point mutations in the PbCrC>4-transformed cells. Future studies should address this possibility and should seek to identify alterations in additional proto-oncogenes or tumor suppressor genes which might cooperate with c-myc in maintaining the oncogenic phenotype of the PbCr2 and PbCr3 cell lines. 178 BIBLIOGRAPHY 1. Patierno, S. R., Banh, D. and Landolph, J. R. Transformation of C3H/10T1/2 mouse embryo cells to focus formation and anchorage independence by insoluble lead chromate but not soluble calcium chromate: relationship to mutagenesis and internalization of lead chromate particles. Cancer Res., 48: 5280-5288, 1988. 2. Smith, G. J., Bell, W., and Grisham, J. W. Clonal analysis of the expression of multiple transformation phenotypes and tumorigenicity by morphologically transformed 10T1/2 cells. Cancer Res., 53: 500-508, 1993. 3. Taparowsky, E. J., Heaney, M. L., and Parsons, J. T. Oncogene- mediated multistep transformation of C3H10T1/2 cells. Cancer Res., 47: 4125-4129, 1987. 4. Land, H., Parada, L. F., and Weinberg, R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature (London), 304: 596-601, 1983. 5. Chen, A. C. and Herschman, H. R. Tumorigenic methylcholanthrene transformants of C3H10T1/2 cells have a common nucleotide alteration in the c-ki-ras gene. Proc. Natl. Acad. Sci. USA, 86: 1608-1611, 1989. 179

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Creator Dews, Michael Eric (author)

Core Title A Study Of The Mechanism Of Lead Chromate Induced Neoplastic Transformation Of C3H/10T1/2 Mouse Embryo Cells

Degree Doctor of Philosophy

Degree Program Molecular Microbiology and Immunology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Landolph, Joseph R. (committee chair), Ou, Jing-Hsiung James (committee member), Roy-Burman, Pradip (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c20-602143

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Rights Dews, Michael Eric

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA