Cytological studies on the infection of synchronized human tumor cells by Adenovirus type 1

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Content CYTOLOGICAL STUDIES ON THE INFECTION OF SYNCHRONIZED HUMAN TUMOR CELLS BY ADENOVIRUS TYPE 1 by Susan Frances House 1 11 A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (Anatomy) August 1966 UMI Number: EP54677 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publi: UMI EP54677 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 9 0 0 0 7 This thesisj written by (dl.O .o^.C....... under the direction of h.<r.V.~...Thesis Committee, and approved by all its members, has been pre sented to and accepted by the Dean of The Graduate School, in partial fulfillment of the requirements for the degree of Master of Science Dean Date. THESIS C O M M IT T E E _^__ ACKNOWLEDGMENT I v/ould like to express my appreciation to the members of my committee. Dr. P. H. Kasten, Dr. W. Yang, and Dr. R. Binggeli for their help in the preparation of this thesis I would also like to thank the members of the staff of the Pasadena Foundation for Medical Research for their help and assist ance . This work was supported by U. S. Public Health Service training grant number 5 T1 - GM 581-05. 11 TABLE OP CONTENTS Page INTRODUCTION .......................................... 1 MATERIALS............................................ 4 C e l l s ................. 4 V i r u s ........................................ 4 Solutions ...................................... 4 Fixatives and stains ................ 5 Autoradiography . ........................... 5 METHODS ............................................... 6 Synchronization of the cell population .... 6 The degree of synchronization..... ............ 7 Autoradiography . . . . . . ....... 8 Infection by adenovirus type 1.... .......... 9 Time-lapse phase-contrast cinematography . . . 10 RESULTS ..................................................12 Virus-induced alterations in nuclear morphology ........................... 12 Viral stages as seen with hematoxalin and eosin staining and with acridine orange staining ........... ..... 13 Cell infection curves ......................... 17 Characteristics of the cell infection curves for each phase of the cell cycle . . 19 DISCUSSION ............................................. 22 ill IV Page Viral-induced morphological changes . . . 22 The relation of the cell infection curve to viral development ......... 24 Resistivity and sensitivity ........... 29 BIBLIOGRAPHY 33 APPENDIX ........................................ 36 INTRODUCTION It is well known that cells constituting a dividing population go through a repeating pattern of biosynthetic events, starting from the formation of two daughter cells and progressing toward their division. The cell cycle has been subdivided into four phases (8 ): (1) M - or mitosis, where chromosomal segregation occurs; during this phase there is no DNA synthesis, and minimal RNA and protein formation (18); G1 - a post-mitotic period during which the cell resumes protein and RNA synthesis; (3) S - which is characterized by DNA synthesis (8 ); (4) g2 - a pre-mitotic phase devoid of DNA synthesis and active in RNA and protein synthesis. The S phase has been shown to consist of a DNA synthe sis with both a sequence and a time pattern. For instance, Kasten and Strasser (10), using tritiated thymidine and tritiated uridine, found that replication of a nucleolar- associated DNA was limited to an hour period in early S, while duplication of heterochromatin DNA took place late in the S phase. While the nucleolar-associated DNA was replicating, RNA synthesis throughout the nucleus and the appearance of labelled RNA in the cytoplasm stopped, indi cating perhaps, a key control function played by this DNA. 2 While a particular DNA is being replicated it cannot serve as a template for messenger RNA synthesis; so that there is a temporary, sequential turning off of various functions as the DNA controlling various processes is doubled. It would be interesting to see what effect the timing of synthetic events going on within the cell has on the susceptibility of the cell to virus infection. A virus might penetrate the cell and start influencing new virus replication more effectively when the 'machinery' for the synthesis of host RNA and DNA is already operating. In order to attack the problem of determining vulner able and resistant phases of the cell cycle to viral infection by a DNA virus, it was necessary to synchronize cultured cells. CMP human tumor cells were chosen because current work defining synthetic events during their cell cycle is being done in this laboratory. Also this cell line has been shown to be highly responsive to synchronization by the excess thymidine method of Bootsma (2) and is sus ceptible to infection by human adenoviruses. For the purpose of the investigation it was essential to introduce a rapid-acting DAN virus for short pulses during the cell cycle. In this study adenovirus type 1 was employed primarily because it required a short maximum adsorption time (13). Adenovirus type 1, which is non- oncogenic, would also make an interesting control for later 3 studies comparing oncogenic and non-oncogenic virus-cell interaction. The human adenoviruses comprise a group of about 30 types, some of which have been found to be onco genic in newborn hamsters (9), (14). In order to help answer the question as to whether or not some phases of the cell cycle are sensitive or resistant to viral infection, various cytological and cytochemical techniques were employed, as well as direct observation of living cells by phase-contrast time-lapse cinematography. MATERIALS Cells The stock CMP line used in these experiments was started by personnel of this laboratory in March, 1964, from a biopsy of an epithelioid adenocarcinoma. At the time the present work was initiated, the CMP cells had already undergone more than 50 passages in vitro. The cells were maintained in Eagle's basal medium plus 10 percent inactivated fetal bovine serum, penicillin G (10,000 units per liter), streptomycin (125,000 gamma per liter), and a 5 percent phenol red solution (2.5cc per liter). Virus Adenovirus type 1, Monte strain, was originally obtained from Dr. Robert McAllister of Children's Hospital in Los Angeles. The virus was propagated on HeLa and CMP cells by infecting cell monolayers with a high multiplicity of virus and allowing the cultures to grow for five days. At the end of five days any cells remaining on the glass were easily shaken loose and then the solution was frozen and thawed three times in order to release virus. The resultant suspension was stored at -80°C. Solutions Unlabeled thymidine was obtained from Cal Biochem. 4 5 Tritiated thymidine with a specific activity of 13c/mM (Schwarz) was diluted to a concentration of l|Tc/ml in Eagle's medium. Fixatives and stains Coverslip preparations were fixed either in absolute methanol prior to staining with Jacobson's May-Grunwald Geimsa stain, or in Carnoy's 1:3 acetic alcohol before staining with Delafield's hematoxalin and counteretaining with eosin (H and E). Cell preparations were also specifi cally stained for nucleic acid patterns with 0 .01% acridine orange at pH 4.0 for five minutes, washed in buffer at pH 4.0, and studied by fluorescence microscopy using an ABO 200 mercury arc lamp in a Zeiss photomicro se ope . Autoradiography Kodak ARIO autoradiographic stripping film was used for all autoradiographic studies. METHODS Synchronization of the cell population Cells were synchronized following the method of Bootsma (2) using a 2.5mM double thymidine block. The procedure employed in this laboratory has been described by Kasten, Strasser, and Turner (10) but is given in detail here since it forms the main experimental tool for this study. Cells were harvested from T-flasks by treatment with a 0.25% trypsin solution and seeded onto coverslips in one and-a-half and two cc Yerganian tubes at an approxi mate dose of 25,000 cells/ml. Cells were allowed to grow for three days. The medium was then removed and replaced by a 2.5mM solution of thymidine in Eagle's medium. At the end of 24 hours the cells were washed with Gey's BSS and then allowed to grow for 15 hours in fresh normal medium. At the end of this time a second thymidine block was imposed for 24 hours. Following the release of this in hibition by replacement of the thymidine solution with normal Eagle's medium, the cells entered the S phase as a synchronized population. All experimental procedures were carried out in a 35'^C room and all solutions were pre warmed to 37^C to minimize temperature fluctuation and possible disturbance of synchrony. 6 7 The degree of synchronization The success of synchrony was evaluated through the use of two procedures. Autoradiography was used to record the percentage of cells which showed DNA synthesis indic ative of the S phase, and synchronization was evaluated by mitotic counts. A minimum of 1000 cells per slide was examined, either for mitotic figures or for labelling. After release of the second thymidine block (time O) synchronized coverslip samples were exposed to tritiated thymidine for 3 0 minutes every half hour up until the time of the mitotic peak at approximately 11 hours. Two more samples were taken at 1 and 4 hours after the mitotic burst. Random controls, seeded at the same time as the synchronized cells, were also exposed to labelled thymidine at 0 hour, 4 hours, 7 hours, approximately 11 hours, and at 15 hours post final reversal (post-wash). Following treatment with labelled thymidine, all samples were washed with Gey's BSS and the coverslip was broken in half. One half was fixed in Carnoy's fixative and saved for later autoradiography treatment. The other half was fixed in absolute methanol for 15 minutes and stained by Jacobson's May-Grunwald Geimsa stain to allow mitotic counts to be made. These counts were later correlated with the results shown by DNA labelling. Most of the random coverslips had mitotic counts of about 8 5 percent. The mitotic counts of the synchronized popula tions used in these experiments ranged from a 28 percent to a 60 percent mitotic peak (Table 2). Autoradiography After a minimum of 24 hours fixation in Carnoy's fluid, the coverslip samples were taken down to tap water through absolute, 90%, 7 0%, and 5 0% ethanol. They were then left in 5% trichloroacetic acid at 7°C for 15 minutes to remove acid—soluble precursors of nucleic acids, rinsed in distilled water, and transferred to 7 0% ethanol for 10 minutes. The coverslips were then air-dried, mounted cell side up with Permount on a microscope slide, and allowed to dry overnight in a 37°C oven before being covered with stripping film. Samples were left under the film in a refrigerator for 10 days and 2 weeks. After the film was developed in D19 and fixed, the cells were stained through the film with H and E. The slides were examined by bright field microscopy for the presence or absence of nuclear labelling. Cells showing uptake of labelled thymidine revealed well- defined silver grains over the nuclei. These nuclei were also examined for nucleolar labelling as an indication of nucleolar-associated DNA replication. Unlabelled samples preceding the mitotic peak were assumed to be in the G2 phase while unlabelled cells sampled after the mitotic peak were associated with the G1 phase. Infection by adenovirus type Previously grown viral stocks were diluted 1:10 in Eagle's medium; these were used as high multiplicity infecting mixtures. Three different stocks were grown as needed. Experiments 1-4, 5-10, and experiment 11 made use of the three stocks solutions respectively. Each experi ment was based on all the tube samples that were seeded at one time and therefore received identical synchrony treat ment. Each experiment had its own synchrony controls. Technical difficulties in handling a large number of samples within a short period of time usually limited to two the number of phases infected in a single experiment (Table 1). To initiate infection, the normal medium was removed from synchronized cells in Yerganian tubes and replaced by the viral suspension for 20 minutes. The viral medium was then removed and the cell monolayer washed two times with Gey's BSS. Fresh medium was added and was left unchanged during the subsequent viral development period of 72 hours. Cells were infected at the following times in their cell cycle: 4 hours post-wash (early S phase when nucleolar-associated DNA replicates); 5 hours post-wash (late S phase when heterochromatin doubles); 8 to 9 hours post-wash (G2 phase); at the peak of mitosis (M phase); 10 and 5 hours past the mitotic burst (G1 phase). A random population of cells was infected as a control. Cells were also infected while in the second thymidine block. For this purpose a solution of excess thymidine was used to dilute the virus stock and to wash the cells. The infected cells then remained in fresh blockage medium for the 7 2 hour viral growth period. In order to follow the viral-induced changes, individ ual coverslip samples were removed and fixed in Carnoy's fluid at approximately 8 , 12, 18, 24, 30, 35, 42, 48, 54 or 60, and 72 hours after the initial exposure to the virus. A minimum of 1000 stained cells per slide were examined microscopically for morphological changes caused by viral infection. Samples infected during the S and G2 phases were also fixed at the time of the mitotic burst of the uninfected but thymidine-treated synchrony controls. The coverslips were stained with H and E and examined for mitotic figures in an effort to see if the initial synchronization of the cell population would be disturbed after 1 to 8 hours in the presence of virus (Table 2). Time-lapse phase-contrast cinematography Cell suspensions were also seeded into Rose chambers, synchronized, and infected during each of the seyen time periods mentioned above. The progress of the infection was followed in living cells for the 7 2 hour experimental 11 time on five different units (12)• The same microscopic field was not followed for the full 72 hours because it did not always happen that the first cells followed were the ones that became infected. However, by 24 to 30 hours post-infection it was possible definitely to distinguish infected cells, and then they were followed either until the end of the 7 2 hour period or until apparent death. RESULTS Virus-induced alterations in nuclear morphology The adenovirus is a DNA virus that is synthesized in the nucleus (14)• Consequently the following discussion of morphological change is limited to nuclear changes. Examination of infected cells for abnormal nuclear changes indicative of viral infection revealed a progression of morphological changes. These viral lesions have been grouped into sequential stages on the basis of the order of first appearance on the stained slides of certain charac teristics representative of each stage. This proposed sequence of morphological development was substantiated by the use of time-lapse cinematography. Figure 6 diagrammatically illustrates the sequence of the morpho logical changes, and shows the defining characteristics of each stage. The rate of inclusion development and change varied greatly from one experiment to the next, so that it was difficult to give each stage a significant time period of predominance. It may be said that stage 1 was predominant during the first 18 hours of infection. However, except for the 8 hour sample, more than one stage was usually seen on the same slide. The rate of viral development 12 13 can change with variation in the infecting titer yet the sequence of changes related to viral development will not be altered (3). It was also observable in this present study that the morphological changes did not vary signif icantly between those seen in a random population and in cells infected during any of the phases of the cell cycle. Viral changes as seen with hematoxalin and eosin staining* and with acridine orange staining** The basic morphological change within the nucleus, brought about by viral infection, seems to be the re arrangement of cell chromatin and nucleoprotein coin ciding with the development of viral DNA and protein. These patterns of DNA rearrangement emphasized by selec tive acridine orange staining are very similar to the basophilic density patterns seen with H and E (Table 3). The normal CMP cell (figure 1-1 and figure 4-20) appears to be epithelioid. The nucleus usually has two to four distinct irregularily shaped nucleoli, and the nuclear * Hematoxalin is attracted to basophilic structures, while eosin tends to stain acidophilic substances. Consequently chromatin, nucleoli, nuclear membranes, and many viral components appear blue-black and the cytoplasm appears pink with H and E. ** Acridine orange is specific for RNA and DNA. With this stain DNA appears green to yellow-green, and RNA, con centrated in the nucleolus, appears orange. 14 membrane is smooth and either round or oval. The back ground nucleoplasm has varying densities but they are evenly spaced, so that the nucleus may be said to have a homogeneous granular texture. Stage 1 - The first change is the formation of abnormal, basophilic dark areas in the background nucleoplasm. This uneven background pattern will be termed "clumping". The chromatin may also become slightly fibrous, giving the nucleus a "patchy" appearance (figure l-2a). In some cases the "clumping" may be so pronounced that the nucleus appears "mottled" or "measly" (figure 1-3,3a,6 , and figure 3-16, and figure 4-19)• Large oval eosinophilic areas may fill the nucleus (figure 2-10, 10a). The nucleus may also appear normal except for one or two homogeneous eosinophilic areas (figure 1-2 and figure 3-14). The nucleus is not enlarged in this stage, but the nucleoli may be slightly rounder than usual. Stage 2 - This group is distinguished by the beginning of a separation of the nucleoplasm from the nuclear membrane leaving an empty margin, and by an enlargement of the nucleus (figure 3-4, figure 3-13, and figure 5-25,25), The background chromatin usually retains whatever type of pattern it displayed in stage 1 , whether it was "spotted" (see stage A), "patchy", or "mottled" (figure 1-5, and figure 5-25,26),. but the clumps may become more distinct 15 or fragmented, and interconnected by fibrous material. Stage 3- This stage shows the widest variety of morpholog ical abnormalities. It also accentuates changes begun in stage 2. The nucleus is quite swollen and irregularily shaped (figure 4-23,24). The condensing and rearranging nuclear material may group into a loose central mass (figure 1-5) or form smaller bodies of various sizes and shapes strewn throughout the nucleus (figure 1-3, figure 2-7, and figure 3-16), and it is sometimes difficult to distinguish between nucleoli, chromatin, and viral inclusions (figure 2-7). However, with acridine orange, the nucleolus appears as an abnormally round, large, intensely stained structure. In stages 2 and 3 the nucleolus is separated from the surrounding DNA-containing central mass by a distinct non-staining halo (figure 3-15, 16, 17). This clear area is not observed with H and E staining. Perhaps there may be protein present which would accept the stain. In stages 2, 3, and 4 acridine orange brings out a DNA substructure within the condensing DNA mass. In stage 2 each clumped area contains a cluster of small brilliant yellow-green dots (figure 3-15). In stage 3 the re arranged chromatin has coalesced into large homogeneous yellow-green bodies (figure 3-16,18), and in stage 4 these intense yellow-green entities are sometimes seen to be 16 embedded in a yellow-orange matrix (figure 3-18). This may indicate a breakdown and a dispersion of nucleolar material since the nucleus can no longer be seen by this stage. The nuclear background in stage 3 usually does not stain with either acridine orange or H and E. However, in advanced cases the background is glassy or hyaline, dis playing a purple tinge with H and E and a faint yellow- green glow with acridine orange (figure 2-7, and figure 3-17). Lesions with a glassy background are frequent at 54 and 7 2 hours post—infection• Stage 4 - This stage is exemplified by pycnotic nuclei : the central mass has condensed to form a solid, basophilic, or brilliant yellow-green body, the nucleus is small, and the background space is glassy (figure 1—5, figure 2—8, and figure 4-22). in most cases the nucleus is kidney bean shaped and the central mass is off-center,- touching the nuclear membrane at the point of indentation. Stage A - This stage is characterized by small dense basophilic or yellow-green spots in the nucleus. The morphological change may vary from that of a normal nucleus, except for scattered spots, to a prophase-like appearance (figure 2-11). No nucleoli can be distinguished in most cases, even with acridine orange (figure 3-13). Cells in this stage are thought to be associated 17 with recent mitosis. The only cells displaying this "spotted" morphology in the time-lapse films were the daughter cells produced by prolonged mitosis of a cell that showed signs of beginning viral development (figure 5-27,28,30). Stage B - This stage is characterized by a large, scalloped nucleus containing a combination of spots and fibers, both a brilliant yellow-green with acridine orange (figure 3-17). The large background area of the nucleus usually appears empty, and in smaller nuclei it is glassy (figure 2-12). Again, nucleoli are not visible with either H and E or with acridine orange. The first appearance of this stage often follows the appearance of stages A and 3. Stage B is often predominant at 7 2 hours, particularly in slides that previously or simultaneously showed many stage A lesions. Cell infection curves In order to show what will be called a "cell infection curve" for each phase of the cell cycle, graphs have been made of the percentage of cells showing morphologic signs of infection versus time after exposure to virus. For ease of comparison different parts of the curves have been assigned descriptive names. In order of appearance on the curve they are : initial slope, peak, dip or plateau, secondary rise (figure 7). Because of the wide range of 18 variation in infective dose from experiment to experiment it was not possible to run a statistical comparison be tween curves representing viral development in each of the phases of the cell cycle. It was thought that this experi mental variation was due to differences in the infecting titer between experiments. In order to assess the degree of susceptibility of each phase two type of comparison were made. First, and most important, was an observation of differences or similarities between phases infected during the same experiment. Within a single experiment the cell seeding and growth is identical for all tubes^ and the same viral suspension is used, so that differences in viral develop ment directly reflect the influence of the phases of the cell cycle. Secondly, conclusions formed from comparing curves from the same experiment were extended to all phases of all experiments by comparing the results of other experiments in which there was a common phase. For example, in both experiments 5 and 7 (figure 8 - A, B) the G2 curve shows a higher percentage of infection than G1; while in experi ment 8 (figure 9-A) S4 is slightly more susceptible than G2. Therefore it must be assumed that S4 is also more sensitive than Gl. It was also noted that curves representing the same 19 phase were similar so that a representative curve was drawn for each phase (figure 1 1 ). Characteristics of the cell infection curves for each phase of the cell cycle Sg - This is the only curye that does not show a dip or a plateau followed by a second rapid rise in the percentage of infected cells. The curye is characterized by a steep initial slope that within 30 hours reaches a plateau level of 7 0 percent or 80 percent infected cells. The population shows less than a 2 0 percent increase in infected cells during the remainder of the experiment (figure 9-B). Gl - Cells infected during this phase seem to be more resistant to viral development than cells in any other phase. In the two experiments 5 and 7 (figure 8 -A,.B,) the development of viral lesions is significantly lower for Gl than for G2. In experiment 11 (figure 10-A) it is indicated that the infecting titer of virus may be much higher than in all preceding experiments, since the initial viral exposure yielded signs of infection in almost 1 0 0 percent of the cells within 35 hours after infection. This experi ment was valuable because it allowed a comparison of the varying time intervals during which a maximum number of cells showed morphological indication of viral infection during three phases of the cell cycle. It is assumed that the initial rise in the number of infected cells is 20 primarily due to the manifestation of the original in fection, if this is the case then G1 shows a marked lag period compared to M and S^. S4 , G2, and M - The S^, G2, and M curves all show simi lar peaks and dips (figure 9-A, and figure 11). After comparing the initial slope and the peak of each curve with other curves in several experiments (figure 8 -A,B, figure 9-A, and figure 10-A), it may be concluded that S^, G2, and M are also "sensitive" phases. Random - An infected random population gives rise to a two-step growth curve, with a rather long time interval between the first and second periods of increase in the number of infected cells (figure 10-B). However, it must be remembered that a random population consists of cells in all phases of the cell cycle. The sum of their indi vidual responses to viral infection results in the random cell infection curve. The number of cells in each phase is in proportion to the amount of time the cells are in each phase within the 28 hour growth cycle. If each curve produced by synchro nized cells infected during the various phases of the cell cycle is weighted according to the length of each phase in the 28 hour period, and then the points on the curves are added and averaged, an infection curve is obtained which is very similar to the cell infection curve produced by 21 the random population (figure 10-B). Block - Infected cells left under the influence of 2.5mM excess thymidine show viral lesions (figure 10-B), while mitotic counts taken on infected samples consistently show less than 0 .1% mitotic figures. The appearance of viral changes in the presence of excess thymidine is compatible with Green's observation that little viable adenovirus is produced in the presence of 10”"^M thymidine (6 ) . Green followed viable virus, while this study only followed viral induced morphological change. The sudden rise of infected cells at approximately 50 hours may indicate a sudden synthesis of protein or RJSfA components in the larger percent of the initially infected cells. None of the viral stages observed prog ressed beyond an early stage 2, even by 7 2 hours. DISCUSSION Because of the broad range of observations and results presented in this study, the discussion will be broken into three parts. The first part will deal with viral-induced morphological changes; the second will discuss possible relation of the cell infection curve to viral development ; and the third will discuss the sensitivity or resistivity of the phases of the cell cycle. Viral-induced morphological changes The morphological changes produced by viral develop ment occur in a continuous progression. In order to simplify these changes into a few major stages all varia tions seen by different researchers cannot be presented, and there may appear to be some discrepency between the observations of different workers. The use of different cell types and different viral strains may also lead to variation in dominant stages. The stages of viral develop ment outlined in this study are similar to sequential changes seen by other workers (3),(1),(7). However, certain features of the abnormal changes mentioned in this study should be discussed, Boyer (3) and Hamada (7), using different types of 22 23 cells than the CMP cells of this study describe the presence of small eosinophilic areas surrounded by a "baso philic rim" as representing early viral changes. This type of inclusion was not seen in CMP cells. However the middle and late stages described by Hamada correspond respectively to stages 2 and 3, and to stage 4 described in this study. The morphological changes observed in this work with acridine orange correspond quite closely to the descriptions of acridine orange stained nuclear changes seen by Armstrong (1). Some morphological changes presented in this study have not been described by other workers. These are seen in stages A and B. The hypothesis that stage A arises from the division of an infected parent cell may seem surprising. It is possible that this type of inclusion may only be produced by the division of a cell in stage 1. This is compatible with the observations of Green. Green states that in a population which was 1 0 0 percent infected he still obtained a 20-30 percent increase in the number of cells, but only within the first 12 hours after infection (5). Cells with very early viral development, which appeared normal when stained in this study, can divide, as is seen by cells infected during S and G2. These infected populations still show a high mitotic burst (Table 2) within 10 hours post- 24 infection. Cells with more advanced inclusions which may be seen at 1 2 hours post-infection on the stained slides are not able to divide (5). It is conceivable that after the division of a cell in stage 1 the condensed chromosomes and rearranged nuclear material do not spread out again but remain as small dense bodies, and that the nucleolus does not reappear. This explanation would account for the characteristic lesion of stage A. This stage is first observed following or in conjunction with the appearance of stages 2 and 3, which in turn usually appear after 13 hours post-infection, the time which corresponds to the beginning of viral maturation (15). The relation of the cell infection curve to viral develop ment The cell infection curves do not directly represent changes in initially infected cells when the cell popula tion is not static; with a non-static population two vari ables must be realized and the degree of their influence taken into consideration. The first variable is the contin ued division of uninfected cells. Mitotic counts from in fected slides show an average of 1% or 2% of the cells in mitosis. The mitotic count rises to 4% or 5% at 36 or 42 hours post-infection. This "peak" coincides in time with the appearance of a dip or plateau in most of the cell in fection curves. It is also interesting to note that this 25 "peak" comes about 28 hours after the first mitotic burst at 1 0 or 12 hours post—wash, and that the time between divisions, as followed in time-lapse cinematography, for CMP cells is 28 hours (11)♦ After 50 hours post-infection the number of mitotic figures are never above 0.5%. These observations indicate that the increase in uninfected cells through division will not alter the basic interpretation of the cell infection curves* The second variable influencing the analysis of the degree of initial viral infection or subsequent viral development is the contribution of secondary infection to the percentage of cells showing a viral lesion. Polasa (15), has shown that adenovirus type 2 begin to mature as early as 13 hours post-infection, but maximum maturation is not reached until approximately 28 hours post-infection. Hamada*s work with adenovirus type 1 also supports this finding (7). Assuming approximately an 8-12 hour lag be tween secondary infection and an indicative morphological change, secondary infection may begin to add to the number of infected cells at approximately 23 hours post-infection. However, in view of the fact that adenovirus is released gradually (4), it is unlikely that secondary infection significantly adds to the percentage of cells showing infection before 40 hours. It is even possible that the small early increase in the number of infected cells 26 due to secondary infection may balance out the decrease in the percentage of infected cells caused by cell division. This, however, is a speculation, since the per centages are not known. Keeping in mind the two variables - (a) the continued division of uninfected cells, and (b) the contribution of secondary infection to the percentage of cells showing viral lesions - viral development may be related to the various parts of the cell infection curve. In considering the experiments presented here it must be remembered that the formation of viable virus has not been followed; viral development is inferred from the morphological changes induced by viral infection. The initial rise of the cell infection curve reflects the increase of viral induced nuclear changes. The slope of this increase is related to differences in the time that the virus takes to produce an observable change in different cells. The different time intervals may result from the state of the cells (the phase the cell is in) and the variation of physiological state of cells within a single phase. In general, the height of the first peak or plateau must be close to the percentage of initially infected cells. The post-infective time needed to reach this peak is related to two factorss first, the interval necessary 27 for maximum manifestation of the initial viral infection; and second, the infecting dose (4), which varies in differ ent experiments. Within the same experiment, where the infective titer is the same, differences in the heights of the first peak reflect important differences in cell sus ceptibility to viral produced changes. In the same experi ment identical populations of cells in two different phases are exposed to the same viral suspension for the same length of time, yet a population in one phase of the cell cycle does not show as many infected cells as in the other phase - indicating a difference in cell susceptibility or cell resistivity. If the height of the first peak is related to the percentage of initially infected cells, then in experiments 5 and 7 (figure 8-A>B) it may be seen that G2 is more sensitive than is Gl to initial viral infection. A dip or plateau probably represents a time of occurrence of viral release and secondary infection plus the lag between secondary infection and observable change. Depending on the time of viral maturation, the amount and rate of viral release, and the number of dividing cells, this part of the infection curve appears as a dip (figure 8 -A,B, and figure 9-A), or a plateau (figure 10-B), or as a "rising plateau". For example, Sg shows an early steep increase in the number of infected cells and reaches the beginning of a "rising plateau" at approximately 30 hours 28 post-infection (figure 9-B). This may be explained by a rapid and synchronous development of viral changes follow ing viral infection, accompanied and followed by an ex tremely slow or low release of viable virus, permitting little secondary infection. The plateau was also buffered because of non—infected cell mitosis and therefore non infected cell increase. In experiment 11 (figure 10-A) there is little secondary infection because almost 1 0 0% of the cells became infected initially. The second rise in the percentage of cells showing inclusions is probably the result of a second cycle of infection by released virus. During the second rise the cells are no longer in synchrony. However, the slope of this rise is affected by the quantity and the time of virus release by cells that were initially infected in a synchronized state. In summary, when one applies the interpretation of this discussion to infection curves for each phase, one obtains the following conclusions: 1. The Gl phase is the most resistive to viral infection. 2. The S5 phase is the most vulnerable to infection, as indicated by the most rapid viral development and highest percentages of cells initially becoming infected. 3. The S^, G2, and M phases also show a relatively 29 high degree of sensitivity to viral infection, and are listed in probable order of decreasing sensitivity. Resistivity and sensitivity The original intent of this work was to answer the question: are certain phases of the cell cycle more sensi tive to viral infection than others? This study has shown that the Gl phase is more resistant to viral infection than any of the other phases. However, "Resistivity" may be involved in any one of the three steps of viral formation; viral attachment, viral penetration, aid viral synthesis. Not knowing the initial infective titer or the titer of the suspension after 20 minutes absoprtion time, it is difficult to evaluate the degree of attachment or penetration. But it may be sur mised, considering experiment 11 (figure 10-A) for example, that the lag in morphological change shown by Gl is the result of slow viral development, since all three phases reach 85% infection by 30 or 40 hours. This period is sufficiently short so that it may be assumed that the number of infected cells is a reflection of the actual proportion of cells that were initially infected. There fore it would seem that the Gl phase was not more resist ant than the Sg or M phases to viral attachment, but that the virus induced cell changes were delayed. 30 Interpretation of cell vulnerability relates to the interaction of the cell's biosynthetic processes with the virus directed synthetic events leading to the formation of viable virus or virus induced pathological changes with in the cells. The main synthetic processes of each phase of the cell cycle have already been mentioned. The probable sequence of synthetic steps necessary for viral formation is as follows : 4 hours— cell protein necessary for later viral DNA synthesis is made (15); 7 hours- viral DNA synthesis is initiated (6 ); 9 hours— viral RNA synthesis starts (16); 9 or 10 hours- viral structural protein synthesis begins (l5); 13 hours- mature virus first appears and reaches a maximum at 28 hours (15). How does this viral sequence superimpose itself on the biosynthetic events of the cell's? The Sg phase seems to be most sensitive to virus because a rise of Infected cells reaches a high peak within 35 hours post-infection. In several experiments thymidine labelled controls indicated that the nucleolar- associated DNA synthesis took place 3 to 5 hours post—wash. Therefore, infection of a cell at 6 hours post-wash may coincide with the re-establishment of RNA synthesis (lO). The first step toward viral formation is the synthesis of cell protein. The formation of this substance may be hastened by the concurrent m-RNA and consequent protein 31 synthesis already going on in the cell at 6 hours post- wash. Immediate establishment of viral development may lead to the apparent sensitivity of the S5 phase. In view of the fact that essentially no protein or RNA synthesis occurs during mitosis, one might expect the M phase to be more resistant than the Gl to viral infec tion. However, this study indicates that the M phase is not more resistant than the Gl phase. Interpretation of this finding requires further experimental work. The results presented here only followed sequential pathological nuclear changes after cell infection, and these changes do not always coincide with actual viral formation. It is likely, however, that viable virus was formed, as indicated by the secondary infection rise seen in all experiments. A comparison of viable virus produced by cells infected during different phases of the cell cycle would also be valuable in judging resistivity or sensitivity. A knowledge of the infecting titer of virus is also necessary for full interpretation of the results of this study. Even within the same experiment, where the titer used to infect each phase is the same, one still does not know the ratio of virus to cells. The results in this paper are based on a maximum of three different experiments for each phase. It would be 32 desirable to repeat the experiments with some of the vari- ables eliminated, or at least known. This would also add to the number of experiments confirming the findings of this study. BIBLIOGRAPHY LITERATURE CITED 1. Armstrong, J.A., and Hopper, P.K. 1959 Pluorenscent and phase contrast microscopy of human cell cultures infected with adenovirus. Exptl. Cell Res. 15: 584 2. Bootsma, D., Budke, L., and Vos, O. 1964 Studies on synchronous division of tissue culture cells initi ated by excess thymidine. Exptl. Cell Res, 33: 301 3. Boyer, G.S., Leuchtenberger, C., and Ginsberg, H.S, 1957. Cytological and cytochemical studies of hela cells infected with adenoviruses. J. Exptl. Med. 105: 195 4. Ginsberg, H.S. 1958 Characteristics of adenovirus III, Reproductive cycle of types 1 to 4. J. Exptl. Med. 107: 133 5. Green, M., and Daesch, G.E. 1961 Biochemical studies on adenovirus multiplication II. Kinetics of nucleic acid and protein synthesis in suspension cultures. Virology 13:169 6 . Green, M. 1962 Biochemical studies on adenovirus multiplication III. Requirement for DNA synthesis. Virology 18: 601 7. Hamada, C. 1963 Studies on adenovirus (c) Immuno- fluorescent and cytochemical studies on the growth of adenovirus type 1 in hela cells. Ann. Rep. of the Inst, for Virus Res. Kyoto Univ. 6 : 12 8 . Howard, A., and Pelc, S.R, 1953 Synthesis of desoxy ribonucleic acid in normal and irradiated cells and its relation to chromosome breakage. Heredity Suppl. 6 : 261 9. Huebner, R.J., Rowe, W.P., and Lane, W.T. 1962 Oncogenic effects in hamsters of human adenovirus types 12 and 18. Proc. Natl. Acad. Sci. 48: 2051 10. Kasten, F.H., Strasser, F.F., and Turner, M. 1965 Nucleolar and cytoplasmic ribonucleic acid in hibition by excess thymidine. Nature 207: 161 34 35 11. Kasten, F.H, 1966 Personal communication. 12. Lefeber, C.G. 1963 Modular design for time-lapse cinemicrography. Cinemicrography in Cell Biology, Pt. I, 3-26. Ed. by G»G# Rose. Academic Press, Inc., New York. 13. McAllister,R#M. 1965 Personal communication. 14. Pereira, H.G., Huebner, R.J., Ginsberg, H.S., and Van Der Veen. 1963 A short description of the adenovirus group. Virology 20:613 15. Polasa, H., and Green, M. 1964 Biochemical studies on adenovirus multiplication VIII, Analysis of protein synthesis. Virology 25:68 16. Rose, J.A., Reich, P.R., and Weissman, S.M. 1965 RNA production in adenovirus-infected KB cells Virology 27:571 17. Taylor, J.H, 1960 Nucleic acid synthesis in relation to the cell division cycle. Ann. N.Y. Acad. Sci. 90:409 APPENDIX 37 FIGURE 1 1. A field of random, untreated CMP cells. H and E stain. X 600 2. All the cells show a large eosinophilic mass typical of one form of Stage 1 (arrows). The very dark bodies seen in the nuclei are nucleoli. H and E stain. Experiment 1; Random; 36 hours post-infection X 7 20 2a. Shows two nuclei with the "patchy" Stage 1 lesion. Arrows point to nucleoli. H and E stain. Experiment 7; Gl; 72 hours post-infection X 800 3. Cell 'a' shows a mottled Stage 1. Cells 'b' and 'd' show Stage 3 lesions. Cell 'c ' appears to be unin fected. H and E stain. Experiment 2; M; 36 hours post-infection X 800 3a. These two cells also demonstrate the mottled or measly Stage 1 morphology. H and E stain. Experiment 2; M; 36 hours post-infection X 800 4. The cells in the center of the field show Stage 2 morphology: the nucleoplasm has started to withdraw from the nuclear membrane leaving a clear margin around the inside edge of the nucleus. Two or three nucleoli may be seen in each nucleus. H and E stain. Experiment 1; Random; 36 hours post-infection X 600 5. Arrows point to uninfected cells. Cells 'b ' show Stage 2 inclusions with a mottled clumping retained from the previous Stage 1. One can see : oval eosinophilic clumps (c) grouped toward the center of the nucleus, a peripheral margin (m), and nucleoli (n). Cells 'a' represent Stage 3 ; the dense nuclear material has scattered and the nucleus has enlarged. The black triangle points to a cell in Stage 4. H and E stain. Experiment 6; S6; 38 hours post-infection X 7 00 6. Cell 'a' is an example of a typical Stage 3. Cells 'c' show a mottled Stage 1 morphology. Cell 'b' has fragmented nucleoli. Cell 'd ' appears to be uninfected. H and E stain. Experiment 1; Random; 50 hours post infection X 800 F I G U R E I © \ & © . 4 « y # • i cm?:. 39 FIGURE 2 7. Cells 'a' show the glassy Stage 3 : the nucleus is dis torted and the nuclear material has withdrawn into a dense central mass leaving behind only a few scattered clumps. The nuclear background is a glassy purple. The arrow points to an earlier Stage 3. Cell ’b' is in Stage 2, and shows margination, H and E stain. Experiment 1; Random; 5 0 hours post-infection X 7 20 8 . All of the cells in this field except the two center cells show advanced Stage 4 lesions. The nuclei are small, and have an off-center dense central mass (arrow). H and E stain. Experiment 8 ; S4 ; 73 hours post-infection X 800 9. Cells in this field demonstrate progressive nuclear changes. Cell ‘d ' is uninfected. Cell 'a' shows a beginning chromatin clumping, cell 'b ' shows late Stage 1 morphological changes, and cell 'c ' is in Stage 2, showing margination (m) and a round nucleolus (n). Cell 'e ' shows another type of Stage 1 lesion (arrow), H and E stain. Experiment 1; Random; 35 hours post-infection X 1000 10. Shows the slightly atypical Stage 1 morphology formed by cells infected in a blocked state. The arrow points to large oval eosinophilic bodies. No nucleoli are observable. H and E stain. Experiment 7; Block; 43 hours post-infection X 800 10a. The blocked cell has formed a mottled Stage 1, how ever, the small eosinophilic masses are vacuolated. Two nucleoli may be seen (arrows). H and E stain. Experiment 7; Block; 43 hours post-infection X 800 11. Shows Stage A lesions. The upper four cells show a combination of spots and clumps, nucleoli may be seen (n). The cells in the lower half of the field show a prophase-like morphology; no nucleoli are present in these cells. H and E stain. Experiment 5; Gl; 40 hours post-infection X 800 12. Most of the cells in this field represent Stage B. The background is glassy with scattered spots and fibers. H and E stain. Experiment 6 ; S^; 39 hours post-infection X 700 FIGURE © © m i "%,-' e a k" • - V . “ . : © 4 t M v - : i ü y . . ^ . s ? ■ ^ ‘ ® 41 FIGURE 3 The arrows in this figure point to nucleoli. All cells in these pictures were stained with acridine orange. 13. Cell 'a' displays a Stage 2 lesion. Cells surrounding the letter 'b' appear to be in Stage A. In most of these cells no nucleoli are visible, and the appearance of the DNA pattern is patchy and spotted. Experiment 11; M; 44 hours post-infection X 600 14. The large right-hand cell shows Stage 1 morphology, with one large inclusion body (arrow). Two brilliant round nucleoli are observed (o). Experiment 11; G2; 24 hours post-infection X 800 15. Cell 'a ' is uninfected. Cell 'b ' shows a late Stage 2 morphology. Note that the nucleoli are surrounded by a non-staining 'halo' or clear area. The clumped nuclear material shows an internal substructure : each large coalesced mass contains clusters of small, very bright yellow-green bodies, perhaps representing condensations of actual viral material. Cell 'c' shows the beginning of a viral infection. The background chromatin is staining more intensely than normal and the nucleolus has become large and round. Experiment 11; S5 ; 47 hours post-infection X 1200 16. Cells on either side of letter 'a' and cell 'b ' show examples of Stage 3. The chromatin has withdrawn from the edges of the nucleus leaving a wide margin. Nucleoli have a non-staining halo around them. Cell 'c' represents a late mottled Stage 1. Experiment 11; S5 ; 47 hours post-infection X 720 17.The lower cell is another example of Stage 3 where the chromatin material has separated from the nuclear membrane, leaving behind a glassy background. The upper cell represents Stage B ; all that can be seen are scattered spots and fibers, and a small nucleolus. Experiment 11; S^; 47 hours post-infection X 2000 18. The condensed central mass of the lower cell, which is in Stage 3. displays a substructure of oval, homo geneous, intensely staining bodies (arrow). The two top cells are just beginning to show chromatin clumping. Nucleoli (o) may be seen in all the cells. Experiment 11; S^; 47 hours post-infection X 1200 FIGURE A - c 43 FIGURE 4 All cells in this figure were taken with phase- contrast optics. The pictures were abstracted from time-lapse films of experiments 3 and 4. 19. This nucleus displays mottled Stage 1 morphology. Dense round nucleoli are indicated by the letter 'N '. G2 phase; 44 hours post-infection X 3000 20. Random, uninfected CMP cells in their 100^^ passage. X 600 21. Same nucleus as seen in picture 19. The condensing nuclear material has separated from the nuclear membrane leaving an empty margin (m); this nucleus is now in Stage 2. Nucleoli (N) are seen. G2 phase; 48 hours post-infection X 3000 22.This nucleus appears to be in Stage 4. The nuclear material has condensed into two dark masses ; the rest of the nucleus is empty and shows no density changes in the movie. Random; 65 hours post-infection X 2000 23. The upper right nucleus is the same nucleus seen in pictures 19 and 21. However, the nuclear membrane has become very puckered, and the chromatin has condensed further. It is not possible to definitely distinguish nucleoli; Stage 3. The lower left cell (arrow) shows clumping throughout the nucleus ; late Stage 1. G2 phase; 56 hours post-infection X 3000 24. This nucleus is the same one pointed out by the arrow in picture 23. Here the arrows point to the edge of the nuclear membrane which has become puckered. The nuclear material has also withdrawn to the center of the nucleus. A possible nucleolus is indicated by the letter 'N'; Stage 3 (late). G2 phase ; 72 hours post infection X 3000 FIGURE 4 45 FIGURE 5 All cells in this figure were taken with phase- contrast optics; the pictures were absracted from the time-lapse movie of experiments 3 and 4. 25. The nucleus indicated by the black arrow shows an early Stage 2 lesion; the clumped nucleoplasm is beginning to separate from the nuclear membrane. Two nucleoli may be seen (n). S4 phase; 51 hours post infection X 1200 26. The arrow points to the same nucleus seen in picture 25. The chromatin material has condensed further, and a wide peripheral margin may be seen (m). One of the nucleoli is still visible (n). 8 4 phase ; 55 hours post-infection X 1400 27. The center cell (black arrow) will soon enter into a prolonged mitosis. It shows signs of early viral in fection: irregular nuclear membrane, slight background clumping, and spots; Stage 1. The two cells on either side of the center cell also appear to be infected. The upper right cell (hollow arrow) is in Stage 2. One may observe margination, an irregular nuclear membrane, and large, round, vacuolated nucleoli. M phase ; 40 hours post-infection X 1200 28. The nuclei indicated by the black arrows are the daughter huclei from the center cell of picture 27. These nuclei show no nucleoli, but are prophase-like. They show typical Stage A morphology. The upper right cell (hollow arrow) is the same cell so indicated in picture 27. However it has formed a more advanced viral lesion. M phase ; 54 hours post-infection X 1200 29. This nucleus is in Stage 2. One may see a faint margin (m). The condensed nuclear material is indicated by the letter 'M'. Arrows point to two enlarged nucleoli (n). 3 0. The two cells in this field are the daughter cells from a parent cell that showed signs of early viral infection, and then went into an abnormally long mitosis. Arrows point to small dense spots typical of Stage A . The four or five large dense bodies seen in each nucleus may be nucleoli. G2 phase ; 72 hours post-infection X 1300 I FIGURE 5 s f , m , V f I k i T FIGURE 6 47 Stage B 1 or # V IR A L IN D U C ED N U C LEA R CHANGES N orm al + V irus Stage A Stage I Stage 2 Stage 3 i Stage 4 I e arly dum ping of background chrom atin retention of clumping enlargem ent of nucleus m argination enlargem ent of nucleus condensation and re a r rangem ent of chrom atin empty or glassy background pycnotic nucleus dense o ff-c en ter mass glassy background TABLE 1 48 E X P E R IM E N T NU M BER PHASES IN F E C T E D 1 ------------ -- Random 2 ----------------- M 3 --------------- a ll phases - used for tim e-la p se cinem atography 4 - — — - sam e as experim ent number 3 5 ----------- Gl, G2, and Block 6 --------------- Block and Sg 7 ----------------- Gl and G2 8 ----------------- 84 and G2 9 --------------- 84 and 86 1 0 ----------------- 84 1 1 --------------- M, Gl, and G2 F I G U R E 7 REPRESENTATIVE CELL INFECTION CURVE peak plateau secondary rise initial slope dip a. hours p o s t-in fe c tio n 49 FIGURE 8 A. Shows the Gl and G2 cell infection curves from Experiment 7. B. Shows the Gl and G2 cell infection curves from Experiment 5. 50 FIGURE 8 lüü 90 80 5 S 60 50 I 40 I 30 20 10 30 hours post-infection 100 90 80 70 3 60 u 1 50 40 Eiqperiment 5 30 o / 20 L G2 42 hours post-infection 51 FIGURE 9 A. Compares the G2 and S4 cell infection from Experiment 8 . B. Compares two Sg phase cell infection curves from two different experiments. The last point for each curve represents the last sample taken because of cells coming of the coverslip. F I G U R E 9 52 lüü 90 80 70 CO o > u 60 TJ B 50 u a ; 40 30 20 30 48 54 hours post-infection 3 u TS î "2 § ï 100 90 80 70 60 50 40 30 20 10 0 o / Sg Phase Experiment 11 I I I o— — — — — Ejqpe riment 6 1 I 1 I ______ 4 8 12 18 24 30 36 42 48 54 60 hours post-infection 72 53 FIGURE 10 A. Shows the cell infection curves produced by the three phases that were infected in experiment 11. The last point for each curve is the last sample taken. After this point all cells had come loose from the glass. B. Block and Random refer to cell infection curves from experiments 6 and 1 respectively. The average of the composite curves was made by weighting each representative curve from figure 11 according to the duration of the phase in the cell cycle, and then adding the five composite curves (S4 , 8 5 , G2, M, and Gl) together. The curve of each phase was multi plied by a fraction: 8 4 - 4/28; 8 ^ - 4/28; G2 - 2/28; M - 2/28; and Gl - 16/28. The numerator is the approximate time spent by the cell in the phase, and the denominator is the total length of one cell cycle. The three curves shown in this figure were plotted on the same graph because of their similarity to each other. FIGURE 10 54 100 90 80 70 (0 a > u 60 "V I 0) s 50 40 a 0) u f i a E}q>eriment 11 30 20 Gl 10 8 12 18 62 24 30 48 36 54 42 100 90 80 u 60 •o S o 50 0) "2 z 40 S ^ 30 0) o. 20 10 0 hours post-infection / y • ----------------- Block - Experiment 6 Average of composite curves • — • — Random - Experiment 1 8 12 18 24 30 36 42 48 hours post-infection 54 60 72 55 FIGUKE 11 The curves for similar phases from all experi ments were added and averaged to give one cell infection curve representing each phase. (Exceptions were experiment 11 where the infecting titer was very high^ and experiment 9 where the infecting titer was very low.1 56 C O 00 m vp ui C O C O CO o CO I—I 00 o • W _ <M 00 o o o o o o o o o o o G a o ( Ü 1 4 - ) o cu cc i - t Z 3 Jg Oi oo CO lO C O CM sjjao pa;oajui ;uaojad 57 FIGURE 12 comparison OF THE 84^ Sq , and G2 CEIoL INFECTION CURVES TO THE Gl CURVE The Gl curves from experiments 5 and 7 were averaged to give a curve representing Gl. The G2 curves were similarly averaged. The difference in percentages of infected cells between Gl and G2 was plotted, using the Gl curve as a baseline. To compare 84 with Gl the difference between G2 and 84 in experiment 8 was calculated. The percentage difference of 84 subtracted from G2 was then plotted against the representative G2 curve from experiments 5 and 7- 85 and 84 from experiment 9 were compared in the same manner and the percentage differecne of 8^ subtracted from 84 was plotted using the 84 phase as a common curve. 58 c v i F - 4 M o; Ü cg o CD O CD m O m c . 2 s I CD OO O OO CM OO CM 00 C Q a C Q k , I O O O O o o o o o o o 0 5 OO b— CD ID Tf OO CM + + + + + + + + + ! ? o OO I lO xuojj aouaaajjip ;uaojad 59 TABLE 2 ESTIMATE OP SYNCHRONY The post-wash times representing each experi ment were chosen to give a brief but general estimation of the degree of synchrony at any time. All possible post-wash times are not in cluded. Some experiments show no labelled slides, and some labelled slides appear to be missing, be cause of an error the autoradiographic film was exposed to light before development and these samples were no longer usable. Symbols : C . . . means random control * . . . indicates the time of infection with virus CO d r-4 a , m C M X! > - 4 X 3 O C T \ r — I J - i X c c J C O00 M < D > Ü C L ) M ■X X C M g o 0 ) J - i M C OX g J - l < 0 ) H A - i m C C j X C O 3 O x> M X X X- o X 3 U X 3 60 m c n •3C < ± m x > 00 0 ) d in o • C Î C O •H C O % — i O < U 4 - 1 X » ' r 4 C C J S r4 < N C u in c s j 1 —I in cn < N n C M t - H CO C T i C M X) C M 00 C M • r - l c n C O C M C O C M t — 4 •H O C O 4 - 1 o • r - l 4 - 1 Q •H B O CO C M C M X C O X C C S in C O - K 1 2 e g O I c n 3 r - 4 •w t — 4 C v J 1 C O 4 - 1 o C O c u o X C O in u « C O 0 c n M o 3 X O X C M c n T — I C M n 1 — 4 4 - 1 c c S X e g C O u C O < y \ 0 CO M o X 3 X O X X r# ic o c n t — 4 1 ^ c n 00 CO u o o M - l iH C M X X in C O 0 0 C O c n 3 3 a\ X •H • 1 - 4 > > f X c n + c n C U ( U C O to c c S e g X X X. X 1 — 4 o o C M X X O CQ C O C O • 1 - 4 •H C O 1 — 1 C O 1 — 1 o <u o c u 4 J X 4 - 1 X •H c c J ' , 4 e g s 1 — 4 s 1 — 4 in X . • c u X • 3 C 00 r > . CO 00 • C M <± C M T — i o\ cn C M CM cn • o\ O C O •H C O 1 — I o o > - w X •H c c J S r-l n C M C M m I—I I — I o c n i-j 4 c C M 00 O in • i c m c n O O C M X) C O •H C O O 4 - 1 •H B P l , 00 a. <M in 1 — 1 cn CM <M o in I t o in C O T --1 I t CM •x C O C O o •X X C O •X o X S •X B cn in i - H m X C O X 3 C O 1 2 e g 1 in 1 2 X in 1 C O X o C O o X X C O X M 3 3 O O X X S 1 —4 X • X e g 3 « t « C O C O u X 3 3 o o X X X O 1 - 4 X X o o X X - X C O C O X 3 3 X X 1^ •X •X > > + + - K • K T — 4 ( U O 3 C O C O e g 3 X X X X X c n CQ CQ Q ) 3 3 3 O o o 3 o 3 C O C O • 1 - 4 •X C O1 — 1 C O I—1 o 0 ) O 3 X X X X •X e g •X e g B 1 —1 S 1 - 4 O o\ r — 4 , • X X X J X w M o M t - r- 4 C M c n c n ■ a Mf • c n < |- r — I C M C M c n c n • c r , in C M in c n o in r — I r — I & r — I 0 > X c c J 61 TABLE 3 APPEARANCE OF NUCLEAR CHANGES SEEN WITH HEMATOXALIN AND EOSIN AND WITH ACRIDINE ORANGE Summary of symbols used: A.O. - - - aridine orange H.E. - - - hematoxalin and eosin * — — - green color, indicative of DNA ** - - — bright green *** - - - brilliant yellow-green + - - — indicates the presence of the nucle olus (+) - - - nucleolus sometimes present - - - — nucleolus not seen CO I <ü u 3 • -P O U a -H P4 m • 3 -P W rH 3 • O (U M < ü m I —1 Q ) U U 3 CU • S O • 5 ■H U O I—I O u H Tî 3 f O M -P <ü 03 <ü U * PaO g < p ( L ) U d -P f d C D M -1 U •H in o I —1 o -p > 1 U C D Cn fd X M r o rH e n CN - s CN - fd CN e n o C D 1 —i 1 —i C N ro O ^ * s • s CN ro V X > o LO - « . *• 1 —1 C N ro ro CN r - fd - *. CN 1 —1 CN 1 —l ro m e n n - CN « » » . » . fd - » - CD '— i I —1 CN CN ro (N u o CD r -l 00 I —1 1 —1 rH + + I + I I ! + ! + 62 rM U o -H fd tH I —I S i* eu C D O 3 0 3 r -l X fd ^ 3 ' - r 4 c u • ü - a * 4 ( ^ •je - J e ■ J e ^ ■ j e • J e 4e -je 03 03 fd g 4e 4e 4e 4e 4e 4e 03 P C D C D I —1 C U p .X n U U u u u cu fd fd fd fd fd rH 1 —1 iH 1 —1 rH O rQ rQ C D C D rb rb ib -P > 1 I 1 t-1 .M rH I I l ■P C D C D C U u cu C D C D C D C D C U P 3 P fd p n 0 n d g rH rH p M ri rH iH 1 — 1 1 — 1 C D rb rQ cu b cu ib A A rQ 4e 4e ■ J e ■ J e p u p < D 0 3 p p P 0 3 P g -p p Q )•H 3 n ( U w p m I — ! U H •• > 1 0 C D 1 — 1 0 3 1 — 1 P < D p P Ü n -H I — 1 3 u I— 1 U -H Q ) P c O -P u C D P p C U 1 — 1 P p nj -H ib f d p C n C D p o nj P P U nj p e u c 3 f d X Q ) P m P P -P p P g u -H -P -P p I — 1 P P -P P O p P p P p •H C n U H 0 3 f d p U O C D P O C U C U -P O r H 1 — 1 p o eu I — 1 p P 1 — 1 P P m p O o P u -H f d U H p p C n u ü C n r H C U C n rb g -p O C n tn p 1 X U H • r H 1 — 1 p . M t t C U Q C D u p U H U o 1 — 1 P U q > 1 C D 0 C n C D 3 -H p p P U H P 1 U P p b 1 - -1 3 g O e u p Cn^ u O r P Q ) Q ) P P rP o u -P -H -H C D-H g •H P -H o m P > 1 p -p -P 0 3 3 C n -P p P >1 4 J • » > 1 p P » . Q ) n f d O O p C r— 1 C D P m O P m Q ) iP Q ) -P P A f : C U g C D -H f d C D U C n P m P m m m C U e n -P P u tn I — 1 X p P P U P p Q ) O p P P f d C D 3 C D p Q ) I — 1 > 1 P I — 1 p p p U 1 — 1 m m H Pa (P P d O e u Q Ü eu e u a w Ü I — 1 CN ro < C Q

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Core Title Cytological studies on the infection of synchronized human tumor cells by Adenovirus type 1

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