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Somatic mutations in the SRD5A2 gene associated with prostate cancer

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Somatic mutations in the SRD5A2 gene associated with prostate cancer

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Content SOMATIC MUTATIONS IN THE SRD5A2 GENE ASSOCIATED WITH PROSTATE CANCER by Patricia Sorina Sharpe A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements of the Degree MASTER OF SCIENCE (BIOCHEMISTRY AND MOLECULAR BIOLOGY) December 2002 Copyright 2002 Patricia Sorina Sharpe Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 1414855 UMI UMI Microform 1414855 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA 9 0 0 0 7 This thesis, written by under the direction of h£XT....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 j T)afe December 18, 2002 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dedication Mami & Tati, I am here only because of your constant love and support. You have sacrificed everything for me and I hope to make you proud each day. You define perseverance and success. I love you both very much Chris, you have been an inspiration and you have indeed paved the way for me. I followed in your footsteps and look where it took me. I love you more than words can say & congratulations to you, on your wedding with your beautiful wife Annik. Carmen, you have been so supportive even from 7,500 miles away. You were here to brighten my spirits through hardships and I was able to finish this work because of your help. I love you very much. Adam, you are everything to me. You have given me the truest friendship, happiness and love. I have accomplished so much because of your love and support and I will succeed because of your enduring love. You mean the world to me and I love you. My extended family and friends, you are my therapy. I have accomplished this through perseverance that I learned from you. My family and friends, with a Tittle’ help from upstairs .. .We did it! ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements I would like to thank Dr. Juergen Reichardt, my mentor, for supporting me and allowing me to learn so much in the course of my degree. I would also like to thank Dr. Zoltan Tokes and Dr. Nori Kasahara, my committee members, for guiding me and providing me with such valuable advice. Dr. Marcel Nimni and Dr. Zoltan Tokes thanks for believing in me enough so that I can be where I am today. I would like to thank Dr. David D’Argenio for supporting me through my whole USC career. In general, I would like to thank the whole Reichardt lab. Specifically, I would like to thank Dr. Lucio Ferraz for teaching me so much and being a constant source of support. Dr. Samira Kaissi, Dr. Patricia Favaro, and Ji Hee Kim, thanks for your support and knowledge. Your friendship is priceless, even from miles away. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents Contents Page b i. Dedication ii ii. Acknowledgements iii iii. List of Tables vi iv. List of Figures vii v. Abstract viii CHAPTER 1: PROSTATE CANCER 1.1 Cancer Epidemiology 1 1.2 Prostate Cancer 2 1.3 The Prostate Gland 3 1.4 Diagnosis Tools for Prostate Cancer 4 1.5 Risk Factors for Prostate Cancer 5 1.5.1 Age 5 1.5.2 Ethnicity 5 1.5.3 External Factors 6 1.5.3.1 Meat and Fat Intake 7 1.5.3.2 Lycopene 8 1.5.3.3 Vitamin C, Phosphorus, Vitamin D 9 1.5.3.4 Vitamin E and Vitamin A 10 1.5.4 Genetic Factors 12 1.5.4.1 Hereditary Prostate Cancer 12 1.5.4.2 Sporadic Prostate Cancer 14 CHAPTER 2: ANDROGENS 2.1 Role of Androgens in the Prostate 15 2.2 Androgen Metabolism 15 2.3 Androgen Activity in Prostate Cells 16 2.4 Testosterone, Ethnicity and Prostate Cancer 19 2.5 Prostate Cancer and Enzymes Involved in Androgen Metabolism 19 CHAPTER 3: STEROID 5a-REDUCTASE 3.1 Introduction 21 3.2 Testosterone to DHT Reaction 21 3.3 Genes Encoding for 5a-Reductase Isozymes 22 3.4 Two Functional Isozymes 23 3.5 Localization of the Steroid 5a-Reductase Type 2 24 3.6 SRD5A2 Gene 24 3.7 Disorders Associated with SRD5A2 26 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4: MATERIALS AND METHODS 4.1 Samples Analyzed 28 4.2 PCR Conditions 29 4.3 Template Purification 32 4.4 SNaPshot™ ddNTP Primer Extension 33 4.4.1 Optimization of SNaPshot™ Reaction 34 4.4.2 Post-Extension Treatment 35 4.4.3 Preparation of Samples for ABI Prism® 377 Sequencer 36 4.5 Automated Sequencing 37 4.5.1 Optimization of Sequencing Reaction 38 4.5.2 Removal of ddNTPs 39 4.5.3 Preparation of Samples for ABI Prism® Sequencers 39 4.5.3.1 Analysis using ABI Prism® 377 Sequencer 40 4.5.3.2 Analysis using ABI Prism® 3100 Sequencer 40 4.6 Single Strand Conformation Polymorphism (SSCP) 40 4.6.1 The Radioactive-PCR Reaction 41 4.6.2 Preparation of Samples for SSCR Run 42 4.6.3 SSCP Run 42 CHAPTER 5: RESULTS 5.1 Specific Aims 43 5.2 Optimization of PCR Conditions 43 5.3 Optimization of Dilution Factors for Individual DNA Samples 44 5.4 Genotyping using SSCP 45 5.5 Analysis of Samples with SSCP Shift 46 5.6 Analysis of Samples with no SSCP Shift 51 5.7 Review of All Results 52 CHAPTER 6: DISCUSSION/CONCLUSION 6.1 SRD5A2 and Prostate Cancer 55 6.2 Samples Analyzed 57 6.3 SSCP 58 6.4 Sequencing and SNaPshot as a Screening Technique 60 6.5 Substitutions Identified 61 6.5.1 Silent Substitutions 61 6.5.2 Missense Substitutions 62 6.5.2.1 Substitutions at Amino Acid 49 63 6.5.2.2 Substitution at Amino Acid 63 65 6.5.2.3 Novel Amino Acid Substitutions 66 6.6 Gleason Score Analysis 70 6.7 Future Directions 73 6.8 Conclusion 74 REFERENCES 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Tables Table Title Page Number 3.1 Tissue Distribution of 5a-Reductase Isozymes 23 3.2 Previously Published Mutations Found on Exon 1 of SRD5A2 25 3.3 Previously Published Mutations in Exon 1 of SRD5A2 25 4.1 PCR Primers used for Amplification of Exon 1 of SRD5 A2 29 4.2 Reagents for PCR Amplification 31 4.3 Temperature Profile for PCR Reactions 31 4.4 Fluorescent Dyes Assigned to Each ddNTP in the SNaPshot Reaction 34 4.5 Primers used for SNaPshot Method 34 4.6 Temperature Profile for SNaPshot Reaction 35 4.7 Fluorescent Dyes Assigned to each ddNTP used for Sequencing Reaction 38 4.8 Temperature Profile for Sequencing Reaction 39 5.1 Dilution Factors of DNA Samples 45 5.2 Substitutions Identified in Tumor DNA 53 5.3 Substitution Identified in Normal DNA 54 5.4 Substitutions in both Tumor and Normal DNA 54 6.1 Gleason Score of Patients with Nucleotide Substitutions 72 v i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Figure Title Page Number 1.1 Male Reproductive System 3 1.2 Phosphorus, Calcium, Vitamin D Axis 9 2.1 Androgen Metabolism from Cholesterol 16 2.2 Androgen Metabolism in Prostate Cells 17 2.3 Androgen Effect on Prostate Cells 18 3.1 Testosterone Conversion to DHT 22 3.2 Structure of the SRD5A2 Gene 24 3.3 Normal Male Development and Male Pseudohemaphroditism 27 4.1 Flow Chart of Experiments 29 4.2 Sequence of Exon 1 of SRD5A2 30 5.1 Optimized Conditions for Amplifiication of Tumor DNA 44 5.2 Negative and Positive Controls for SSCP 45 5.3 SSCP Shifts in Normal and Tumor DNA 46 5.4 Nucleotide Substitutions in Tumor Samples 47 5.5 Nucleotide Substitutions in Normal Samples 48 5.6 Nucleotide Substitutions in both Normal and Tumor Samples 49 5.7 SNaPshot Results for A49T and V63M Substitutions 50 5.8 Nucleotide Substitutions at Codon 49 51 5.9 Novel Nucleotide Substitutions Identified 52 v ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract Prostate cancer is the second leading cause of cancer-related deaths in men in the United States. The etiology of prostate cancer is largely unknown. However what is known is that both normal and tumor prostate cells are androgen dependent. Thus attractive genes to study are those that are involved in androgen metabolism. SRD5A2 is an attractive candidate gene in the study of prostate cancer because it encodes for 5a-reductase type 2 enzyme that converts testosterone to DHT, which are the two most active androgens in the prostate cell. The analysis was performed on 148 samples that corresponded to DNA extracted from normal and tumor prostate tissues from 73 prostate cancer patients. We found two already identified mutations, A49T in six samples and V63M was identified in four samples. Of these already identified mutations, most amino acid substitutions were in the tumor DNA, but there were also some found in normal DNA. In addition there were several novel somatic mutations identified in this study. These included one silent substitution, A19A that occurred in two different samples. Furthermore, one sample had the amino acid substitution A49V, and other missense substitutions include M il, A24T, G34E, A52T, S60F, and G66R. Two samples had double mutants, L35R and T45I, and a separate sample had A49T and A69A. From the total number of 19 prostate cancer patients that had a divergence from the consensus sequence of exon 1 of SRD5A2, 12 patients contained this nucleotide substitution in the tumor tissue, 5 patients only had the mutations detected in viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. normal tissue and 2 patients had the mutation identified in both the normal and tumor DNA. The analysis also includes a correlation between Gleason score of the tumor and the substitutions identified. The screening techniques were automated sequencing, which proved to be the most powerful technique, single strand conformation polymorphism (SSCP) and SNaPshot primer extension method. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1: PROSTATE CANCER 1.1 Cancer epidemiology Cancer is the second leading cause of death in the United States, surpassed only by heart disease. The occurrence of cancer drastically increases with age beginning with the middle ages. According to recent data published by the American Cancer Society (ACS), 77% of all cancers are diagnosed passed the age of 55 \ Besides age, sex is also an important predisposing component for cancer. Men have a 1 in 2 chance of developing cancer in their lifetime, while women have a risk of 1 in 31 . There are other risk factors for cancer such as environmental factors and many other have been identified. Statistical analysis has shown that an estimated 16 million cancer cases have been diagnosed since 1990 in the United States with an additional 1,284,900* new cancer cases this year alone1 . The mortality rate is equally frightening, with an estimated 555,000 deaths estimated for the year 20021 . Besides having a high impact on the population, cancer also has a very high financial burden on the United States. For the year 2001, treatment of cancer and other related costs were estimated to be as high as $156.7billion'. *Figure does not include basal and squamous cell skin carcinoma, as well as noninvasive cancer at any site, except for urinary bladder, not included. 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cancer is a major threat on the human population. The reason this is so is due to the nature of the disease being largely unknown. Thus it has been very difficult to pinpoint an exact cause of this complex disease. However, researchers have worked hard and have identified some possible risk factors, and additionally have identified parts of some pathways that play key roles in cancer predisposition, development and progression. A possible origin of cancer is the genetic alteration leading to malfunction or the loss of cellular cycle control. This thus involves genetic alterations that can be of two main types, hereditary or sporadic. These genetic alterations, whether they are hereditary or sporadic, have DNA alterations in the conserved sequence. These mutations can occur in tumor suppressor genes restraining their ability, and/or mutations activating oncogenes and/or mutations altering the conserved sequences and subsequently, altering the protein products. A cell becomes cancerous after multiple ‘hits’ into its DNA. Clearly, one can see the complexity of the disease. 1.2 Prostate cancer The leading diagnosed cancer is that of the prostate, with 189,000 expected new cases, and it is the second leading cause of cancer related deaths in the United States1 . Besides being a major malignancy in the United States, prostate cancer is one of the most common malignancies in the world. Prostate cancer has rapidly increased in other countries including East Asia7 1. 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.3 The prostate gland The prostate gland is about the size of a walnut and surrounds the neck of the male bladder and the urethra, as illustrated in Fig 1.1. P ro sta te G land V as d eferen s esticle Figure 1.1: Male Reproductive System (Reproducedfrom www.harhosp.org) The texture of the gland is partially muscular and partially glandular with ducts opening to the prostatic urethra. It is composed of two tissue components, one being the epithelium and the other the stroma. The prostate gland secretes a thin white milky fluid, which contains acid phosphatases, citric acid and proteolytic enzymes responsible for keeping the semen liquefied. The seminal vesicles are above the gland, which are the major contributors to the secretions. They secrete a yellowish discharge, slightly alkaline, high in fructose (this being the energy source for spermatozoa), and prostaglandins, which stimulates the activity of the spermatozoa. At the time of ejaculation, the sperm that is produced by the testis is carried through the vas deferens to the urethra, where it is mixed with the secretions from the seminal vesicles and the prostate before ejaculation occurs. 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4 Diagnosis tools for prostate cancer Although the expected new cases for this year are the highest out of all cancers, the death rates have decreased quite a bit from previous years due to powerful new detection tools that allow early diagnosis. An early tool is physical examination called direct rectal examination (DRE), which proves to be effective, but not for early detection. In the United States, the rates increased steadily from 1981-1989, and then in the early 1990’s it increased steeply, due to the early diagnosis in men without symptoms via the prostate-specific antigen (PSA) blood test7 0. The prostate epithelial cells produce PSA, and if these cells are displaced away from the prostate they continue to synthesize PSA. Thus, if the prostate gland is damaged or enlarged, prostate epithelial cells are displaced and the levels of PSA will increase in the blood. The damage to the prostate gland can be a result of several events, including direct damage to the prostate by trauma, bacterial infection and cancer. Enlargement of the prostate is a natural course of events for this anatomical body up to a certain age. The male prostate gland, unlike other anatomical organs, continues to grow from early puberty when the size of the prostate gland almost doubles, and then 12 continues to grow through the middle ages of the man . This growth phase being continuous, and naturally varying from person to person can become problematic for some more than others. Benign prostatic hyperplasia (BPH) is the result of this continuous growth of the gland and it may cause symptoms usually after the age of 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 years old. This increases with age, and as high as 75% of men in the fifties and sixties have symptoms of BPH1 2 . Due to the continuous growth of the prostate, and many years, the possibility of DNA damage due to the constant proliferation of cells, plus some external factors discussed below and/or genetic predisposition to prostate cancer, the process of carcinogenesis can begin. Thus one can make the extended assumption that if a man lives long enough the prostate can cause serious problems and the enlargement of the gland along with other external factors may lead to the development of prostate cancer. Since not all men die of prostate fn cancer, it is most likely that men do not die from the disease, but rather with it. 1.5 Risk factors for prostate cancer 1.5.1 Age The remarkably sharp increase in incidence with age is a hallmark of this type of cancer. Prostate cancer is very rare before the age of 40 and drastically increases with age. Approximately 70% of prostate cancers cases are diagnosed in men older than 65 years, making age the highest risk factor for prostate cancer, in fact higher than any other type of cancer6 6. 1.5.2 Ethnicity Besides age, race is another risk factor for developing prostate cancer. Epidemiological studies have been done to see if there is any correlation between race and risk and/or severity of prostate cancer. African American men have a 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. larger volume of prostate cancer at autopsy than Caucasians, which may be due to a 80 higher rate of progression of the tumor manifested in this racial group . It has been suggested that this higher volume of cancer at the time of diagnosis might be due to lower socio-economic status of the African American population in the United States, resulting in decreased access to medical services. This hypothesis was dismissed by a study involving a relatively large multi-ethnic cohort of African-American male in the health profession5 9 . The results confirmed that in fact prostate cancer incidence in Caucasians was almost half of that of the African Americans and the rate of progression seemed to be different in these two racial populations5 9 . Organ-confined tumors were higher in African Americans than Caucasians, and the extraprostatic tumors were especially higher in African- Americans suggesting that the progression of the cancers may differ5 9 . Other races have been correlated with different risk factors for prostate cancer. The lowest incidence rates identified are in the American Indian/Alaskan Native followed by the Asian/Pacific Islander male population1 1.5.3 External factors Prostate cancer is incredibly complex due to its multifactorial nature. Besides age and ethnicity, other external factors that may influence the carcinogenesis pathway are lifestyle, physical activity, nutrition, environmental factors and many others. At a more specific molecular view, these external factors may be oxidative agents, 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. electrophilic and infectious agents that may enhance the genetically predisposed 28 patient, or may initiate the genetic damage that may occur sporadically . 1.5.3.1 Meat and fat intake In a relatively large population pool, the total intake of red meat was not associated with risk of total or advanced prostate cancer, however intake of certain meats on a daily basis such as processed meats (bacon and beef, pork or lamb) had increased risk of metastatic prostate cancer5 0 . Research has been done to study the effects of dietary protein on prostate cancer. A particularly interesting study transplanted a specific type of prostate adenocarcinomas into male rats1 7 . Although the tumors transplanted in the animals were very specific and represented only limited types of tumors in the human prostate, the findings are worth mentioning. It was identified that dietary protein restrictions reduce the growth rate of the transplantable 17 • tumors . Protein intake is important for all anatomical organs, including the prostate. Prostate cancer incidence is highest in westernized nations, in which their inhabitants consume the most meat out of all countries. Thus, one could make the extended assumption that in affluent countries, people eat more meat, and in less prosperous countries less meat is available, which is directly proportional with the incidence of prostate cancer. So perhaps, this trend might set a pattern for prostate cancer development from early stages in life of males, weather that is in childhood, 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. or perhaps during puberty when the prostate almost doubles in size. These dietary patterns during the male development may predispose or protect the patient from developing prostate cancer. These are questions that can only be solved by a large cohort study that involves young boys before puberty through adulthood. 1.5.3.2 Lycopene As discussed previously, some external factors influencing carcinogenesis are oxidative compounds such as singlet oxygen and scavenging free radicals. Thus, one good nutritional product would be one that quenches these compounds, such as 18 carotenoids, which are found in many fruits and vegetables . One of the naturally occurring carotenoids is lycopene found in tomatoes. Its efficiency to quench singlet oxygen and other free radicals is very high7 4 . Unlike other carotenoids, lycopene is a non-provitamin A. Rich diets in foods containing lycopene may reduce the risk of prostate cancer by protecting membrane lipids and DNA from 7 f \ being oxidized, and thus damaged . Lycopene has been identified to suppress the growth of human cancerous cells as well as inhibit spontaneous and induced tumors in animal models2 6. Findings indicate that the association between tomato sauce intake and men diagnosed at age 65 or older was statistically significant (P=0.001), and also there was a statistical significant difference in the risk of metastatic cancer formation between men consuming tomato sauce and ones that did not (P=0.01)3 °. However more work is warranted because some studies have shown no association between lycopene and prostate cancer risk4 3. 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.5.3.3 Vitamin C, Phosphorus, Vitamin D Although it is termed a vitamin, Vitamin D is actually a hormone. Vitamin D plays a key role in the regulation of Ca concentration in plasma. Calcitriol (1,25- dihydroxyvitamin D), which is the most active metabolite of Vitamin D, acts on the intestines, kidneys and bones to increase its concentration by stimulating the release of parathyroid hormone (PTH), which increases the activity of 1-a- hydroxylase responsible for converting 25-hydroxycholecalciferol (25-OHD) to the primary active form calcitriol (l,25-(OH)2D) as illustrated in Figure 1.2. GUT BONE Ca BLOOD Ca PTH High Serum Calcium haw Serum Caldum Ca Ca PTH Tpth Ca Figure 1.2: Phosphorus, Calcium, Vitamin D axis. When serum Ca is low, PTH increases and Ca bone resorption and kidney resorption increases and stimulates calcitriol production. Calcitriol increases Ca absorption from the gut. When serum Ca is high PTH decreases, Ca bone resorption and calcitriol production decreases. P is influenced by the kidneys, and PTH. (Reproduced from www.merck.com) A relative risk of prostate cancer is age, and since as people get older they are more sensitive to the sunrays, they avoid it, thus lowering the amount of vitamin D 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. gotten from the sunlight. Also race has a correlation with prostate cancer, with the African American population being the highest, which may also be correlated with vitamin D formation. Due to the dark pigmentation of the African Americans, vitamin D is not formed as a result of sun exposure5 7 , thus they have an endogenously lower vitamin D level. Furthermore, animal studies have shown that vitamin D is a potential growth inhibitor and may induce cellular differentiation in prostate cells5 7 . Calcium intake has also been studied in correlation with risk of prostate cancer. Following Figure 1.2, one would hypothesize, that the effect of calcium would be the inverse of vitamin D, since vitamin D levels are regulated by calcium. Based on this idea, dairy product intake, which is very high in calcium would be correlated with a higher prostate cancer risk, which is the case1 5 . Based on Figure 1.2, phosphorous should have a similar effect to that of calcium, since it regulates the levels of calcitriol. Indeed, increased fruit consumption that contains high fructose levels which in turn lowers phosphate serum levels, have a protective effect on prostate cancer.3 1 1.5.3.4 Vitamin E and Vitamin A Vitamin E was isolated in the mid 1930s and today there are eight naturally occurring tocopherols, its most common and most potent form being alpha- 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tocopherol. Tocopherols are redox agents, which enable them to act as antioxidants protecting cell membranes as well as DNA from damage from free radicals. In vitro4 0 and in vivo3 2 studies indicate that Vitamin E intake has a protective effect on prostate cancer. However contradictory views have been published1 6 , thus more research is necessary. Vitamin E intake increases cellular concentrations of vitamin A that could be due to the antioxidant effect of vitamin E. If this hypothesis is correct, then Vitamin A intake should be related with a decreased risk of prostate cancer, which indeed it • 58 ,29,63 I b i As far as other external factors that may influence the risk of prostate cancer, selenium, smoking, physical activity and others have also been studied with relation to the relative risk of prostate cancer. It is important to note that some epidemiological findings were statistically significant and others were not. Although all these correlations are interesting and may shine a new light on the pathway of prostate cancer development and progression, more work is necessary. The study of external factors and prostate cancer is continuous in hopes for finding an answer to the proposed question of whether there are any nutritional factors or other environmental factors that influence the predisposition of prostate cancer. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.5.4 Genetic factors Besides age and ethnicity, genetic factors are also risk factors for prostate cancer. Genetic factors are of two main types, hereditary and sporadic1 9 . 1.5.4.1 Hereditary prostate cancer It is estimated that approximately 5-10% of all prostate cancer cases are due to a positive family history.1 0 These cases differ from others in the average age at diagnosis, about 6 to 7 years younger than patients with sporadic cancer1 1 ,7 7 . Men with an affected father have the highest chance for the disease to be diagnosed at an earlier age, and African-Americans with the disease have a frequency of positive family history than Caucasians.2 0 Linkage analysis, although very useful, is very difficult to achieve due to the multifactorial nature of the disease. A major obstacle is the late onset of the disease and lack of informative family members. Finally, like in all pedigree studies the lack of informative families is one great obstacle. Nonetheless, genome wide linkage studies have identified 7 susceptibility loci. Studies vary in their conclusion on the basis of inheritance, and some studies conclude that the mode of inheritance may be autosomal recessive5 1 , or autosomal * 72 dominant . Furthermore, the consistent pattern of high risk in brothers of affected cases, indicate a possible X-linked model of inheritance to prostate cancer1 1 ,7 . The first susceptibility locus is Hereditary Prostate Cancer 1 (HPC1) gene, located on chromosome lq24-25. Linkage analysis indicates that approximately 6% of all 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. familial prostate cancer cases are linked to this region8 5 . Several criteria have been recognized for prostate cancer cases that may be linked to this region: age at diagnosis, number of individuals affected and male-to-male disease transmission. In some studies, however the linkage to this region was rather weak3 7 ’ 6, and other o studies have shown no linkage to this region. The predisposing gene for cancer of the prostate (PCAP) is located on chromosome lq42.2-43. Up to 40-50% of families were linked to this region8 . However, other studies found no significant evidence for linkage.6 Cancer of the prostate and brain (CAPB) gene is located on chromosome lq36 has been identified to be a susceptibility locus for a very rare prostate cancer-brain cancer combination2 7. Another susceptibility gene identified is hereditary prostate cancer X (HPCX) gene that is located on chromosome Xq27-28. It accounts for approximately 16% of hereditary prostate cancer cases.8 6 Men having HPCX as a susceptibility gene had no male-to-male transmission and a later age of diagnosis. The hereditary prostate cancer 20 (HPC20) gene is located on chromosome 20ql3, and 12% of all families studied were linked to familial prostate cancer.7 Findings from this study suggest that the model of inheritance may be autosomal. Lastly, there are two relatively new candidate genes discovered linked to prostate cancer. HPC2/ELAC2 gene located on chromosome 17p 117 5 is linked with 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. approximately 5% of prostate cancer families6 1. Other studies in the homogeneous population of Finland show that it might play a limited role in hereditary prostate cancer,6 4 ,7 8 and yet others found no linkage between prostate cancer and F1PC2/ELAC2 gene8 7 . Lastly, RNASEL gene, located on chromosome lq25 has been identified as a susceptibility gene for prostate cancer risk1 4 and has been 79 associated with an increased risk of hereditary prostate cancer . Although the findings are contradictory, the genes identified may be involved in the development and progression of prostate cancer. Recent genetic susceptibility studies indicate the possibility of multiple genes collaborating to yield the variable • • 7 Q phenotypic characteristics of prostate cancer. 1.5.4.2 Sporadic prostate cancer Sporadic prostate cancer accounts for most of the prostate cancer cases. In the study of sporadic prostate cancer, researchers have tried to concentrate on key elements that play an important role in the development and other elements that contribute to the cellular control of prostatic cells. Besides the genes described in section 1.5.4.1 potentially associated with hereditary prostate cancer, some other genes studied are oncogenes and tumor suppressor genes, but also genes that encode for molecules that play a key role in androgen metabolism. Androgens have been of interest because the prostate is an androgen-regulated organ3 1. 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2: ANDROGENS 2.1 Role of androgens in the prostate Cancer is a very complex disease, and thus there are different ideas for carcinogenesis one of which is hormones and their effect on regulatory processes of cell. One idea is that if levels of androgens, which affect cell division and proliferation, are changed than the chances of random genetic errors increase3 5 . Alternatively androgens also protect cancerous cells, in particular prostate cancer cells from apoptosis by blocking the cellular caspase activation42. The prostate gland is an androgen-regulated organ. It requires brief stimulation by the androgens to develop during the embryologic stages, and then after the prostate gland reaches its adult size, it needs continuous stimulation for the growth and the 1 T 9 T maintenance of the gland ’ . As a result, androgens not only regulate growth, but also cellular differentiation. Androgens are also necessary for survival of both the normal and the cancer cells. Prostate cancer cell growth depends on the activity of the androgens because they regulate the progression of the tumor, by controlling cellular proliferation and apoptosis2 4. 2.2 Androgen metabolism Testosterone is an important and abundant androgen in the human male body. Approximately 95% (6-7mg/day) of circulating testosterone levels is synthesized in 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the testes4, and the rest is made in the adrenal glands and the peripheral tissue as seen in Fig 2.1. Cholesterol D HEAS cholesterol dcsmolasc sulfuryl transferase 17^-hydroxysteroitl dehydrogenase P re g n e n o lo n e 17a-hy<lrtnyiaK^ p reg n e n o lo n e " 17’20' dcsm° lasl: > - D H E A > ............................................ 5 -A n d ro ste n e d lo I 3S3-hvdroxysteroid 3p-hydroxysteroid 3(J“ hydroxystcroul dchydrogenase-i^’ Msomerase dehydrogenase -A^Visomeme dehydrogenase -65 ,4 -isomcrase * * 17B-hydroxysteroid 17,20- dehydrogenase Progesterone 17° Hylir<lliyl!' »- 17“ -H ydroxy- — dcsm“la-e ► A ndrostenedione 4 ......... progesterone Testosterone D HEA = D ehydroepiandrosterone D HEAS = D ehydroepiandrosterone sulfate E strone l?P-hydroxysteroid dehydrogenase E stradiol Figure 2.1: Androgen Metabolism from Cholesterol. (Reproduced from Epidemiol Rev 23: 42-58, 2001) 2.3 Androgen activity in prostate cells The majority of circulating testosterone in the blood stream is bound to albumin or sex-hormone-binding globulin (SHBG) and a small percentage travels freely. Testosterone freely diffuses into the prostate cell, and is rapidly and irreversibly converted to dihydro testosterone (DHT) by the 5(X-reductase enzyme. DHT is the metabolically more active androgen in the prostate cell6 6 . Like testosterone, some DHT is synthesized in the testes and circulates in the body bound to serum proteins, and a small amount circulates freely. The majority of the DHT produced is 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in synthesized from the conversion of testosterone in the peripheral tissues as illustrated in Fig 2.2. sulfurvl tra n sfe rs p-A ndro stan cd io l sulfate glucurony) tra n sfe ra se^ . 3p-A ndrostanediol glu curonide 3p-A ndrostanediol 3P-hydroxystcroid^ dehydrogenase Testosterone 5a**rcductase type 2 DHT 3a-hydroxysteroUI dehydrogenase 17p-hydroxysteroid dehydrogenase 3a-A ndrostanediol su lfu rvi transfers* glucurony! transferase 3a-A n d ro stan ed io I sulfate 17p-hyctroxysteroid dehydrogenase A ndrostenedlone 5a-reductase type 2 A n d ro stan ed io n e 3a-hydroxysteroid dehydrogenase 17{i-liydroxysteroid dehydrogenase A ndrosterone glucuronyl transferase sulfury! transferase 3a-A n d ro stan cd io l g lu cu ro n id e (3 a -d io l G) i 17{J-hydroxysteroid dehydrogenase r A n d ro stero n e glucuronide A n d ro stero u e sulfate Figure 2.2: Androgen Metabolism in Prostate Cells. DHT, dihydrotestosterone. (Reproduced from Epidemiol Rev 23: 42-58, 2001) Once testosterone undergoes the irreversible conversion by the membrane bound enzyme 5(X-reductase to DHT in the prostate cell, DHT may be either inactivated through the pathways illustrated in Fig 2.2 or bind the androgen receptor (AR). The AR can also bind testosterone, but DHT has a five fold higher affinity for the receptor than does testosterone2. Upon binding of DHT to the AR subsequent reactions lead to activation of target genes involved in synthesis of prostate specific antigen (PSA), cell growth and survival as illustrated in Fig 2.3. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 I Testosterone Sa-reductase *► OKI Androgen-respcxave cbI I Liganc binding Dimerization and pftospbor/iat-on < ’ HSP Co-activator recruitment DNA { AR AFtA/ landing Artdrogen-resconse element Target gene activation t t " " " \ ■ fP SA ' : : tGrowth / ^ f Survival * Biological responses Figure 2.3: Androgen effect on Prostate Cells. Testosterone circulates in the blood bound to sex-hormone-binding globulin (SHBG) and freely diffuses in prostate cell. 5a-reductase converts testosterone to DHT, which binds the androgen receptor (AR) and subsequent heat shock protein (HSP) dissociation occurs. Dimmerization o f the hormone-receptor complex occurs and phosphorylation follows. The dimmer penetrates the nuclear membrane and binds to the androgen-response elements. Upon co-activator (such as ARA70) or co-repressor (not shown) binding, the general transcription apparatus (GTA) is recruited and subsequent gene transcription activation occurs o f genes involved in prostate specific antigen (PSA) production as well as genes involved in cellular growth and survival. (Reproduced from Nature Rev Cancer1: 34, 2001) 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Testosterone and DHT are the most active androgens in the prostate cell, and their role has been elucidated. In vivo studies have shown that 75% DHT is retained in the prostate cell’s nuclei, while testosterone is a minor component in the nuclei2 , thus illustrating that the majority of the DHT converted from testosterone binds the AR and affects cellular processes. 2.4 Testosterone, ethnicity and prostate cancer As described in section 2.1, androgens are important in both the function and maintenance of normal and cancer prostate cells. Researchers have tried to find a correlation between androgens and other risk factors for prostate cancer, such as race. The race having the highest risk factor for prostate cancer is African American. Young healthy black men had a 15% higher mean testosterone levels and 13% higher free testosterone when compared with white counterparts6 5. It is actually the increased free testosterone levels that are associated with a higher prostate cancer risk.2 5 Further evidence indicates that DHT:testosterone ratio is highest in African American men, intermediate in Caucasians and lowest in Asian 84 Americans , which is directly correlated with prostate cancer risk. 2.5 Prostate cancer and enzymes involved in androgen metabolism Since androgens play a key role in the prostate cell, the genes encoding enzymes in androgen metabolism, Fig 2.1 and Fig 2.2 have been attractive candidates for the study of sporadic prostate cancer. 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. One such example is the HSD3B gene, which encodes for 3(1-Hydroxy steroid Dehydrogenase. There are two functional isozymes in humans: type I is primarily expressed in placenta and skin, and it is 5 times less active than type II which is expressed in adrenals and gonads45. It is an enzyme with a dual function. The dehydrogenase-isomerase reaction converts androstenediol to testosterone using NAD+ as a cofactor, and the other reaction being the ketosteroid reductase activity reducing DHT to 5a-androstenediol with NADH as a cofactor. Nonsense, frameshift and missense mutations have been found to be associated with severe salt wasting, adrenal hyperplasia and a type of male pseudohemaphroditism49. Besides the genes encoding for the two different types of 3P-hydroxysteroid dehydrogenase, studies are also looking at the genes encoding for 17P- hydroxysteroid dehydrogenase enzymes and many other genes that encode for enzymes specified in Fig 2.1 and Fig 2.2. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3: STEROID 5a-REDUCTASE 3.1 Introduction The steroid 5a-reductase enzyme (3-oxo-5a-steroid A4 -reductase) plays a critical role in androgen metabolism. Its role is the 5a-reduction of 4-ene-3-keto-steroids to their respective 5a-dihydro-3-keto-steroids. The A4-steroid reduction is of androstenedione, progesterone and testosterone, and yields 5a-androstane-3,17- dione, 5a-pregnane-3,20-dione and 5a-dihydrotestosterone (DHT) respectively4. 3.2 Testosterone to DHT reaction As indicated in Section 2.3, testosterone and DHT are the most active androgens in the prostate cell, thus testosterone to DHT conversion is of main interest in the study involving prostate cancer. Testosterone is converted to DHT by the 5a- reductase enzyme aided by the reduced form of the nicotinamide adenine dinucleotide phosphate (NADP+) as seen in Fig 3.1. Since this enzyme has never been purified, hypotheses can be made about the binding site of the cofactor. A possible such mechanism of binding of the cofactor to the enzyme is by the insertion of the 4S-hydrogen of the nicotinamide ring into the 5a configuration of O 'y the steroid substrate . 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. OH OH O NADPH + H ' NADP+ 5i /-Reductase O H Testosterone (T) 5«-Dihydrotestosterone (DHT) Figure 3.1: Testosterone Conversion to DHT. Testosterone is converted to DHT by the irreversible reaction o f 5 a-reductase using NADPH as it’ s cofactor. (Reproduced from TEM 9: 317, 19983 () 3.3 Genes encoding for 5a-reductase isozymes Two functional isozymes have been identified in the steroid 5a-reductase family. These functional isozymes have never been purified. Two different, non-syntenic genes, SRD5A1 and SRD5A2 encode the two isozymes. SRD5A1 encodes for the type I isozyme located on chromosome 5pl5 and SRD5A2 encoding for the type II isozyme is located on chromosome 2p23— >p225 2 . A pseudogene, SRD5AP1 has been identified having the highest homology to type I isozyme and has been mapped to the long arm of chromosome X41. Two additional sequences mapped to chromosome 6 and 8 have been recently identified to have high homology to the SRD5A1 gene, but are not characterized as pseudogenes or functional isozymes2 1. The amino acid components of the genes of the functional isozymes are mostly hydrophobic, and have 49% sequence identity. These two genes have a higher sequence identity with the rat homolog than they do with each other. Human 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SRD5A1 gene has 60% sequence identity with the rat homolog and SRD5A2 gene /"O has 77% sequence identity with the respective rat homolog . Both genes have 5 exons, and the coding region of SRD5A1 gene is slightly larger. SRD5A1 gene encodes for 259 amino acids, while SRD5A2 encodes for 254 amino acids44. 3.4 Two Functional Isozymes The pH optimum of the two enzymes differs with the type 1 active over an alkaline range, pH 6.0-8.5, and type 2 enzyme is active over a specific pH range, around pH 56 9 . The Km for the type 1 isozyme is 25-fold higher than that of type 23 4. The enzyme expression also differs, as illustrated in Table 3.1. 5a-reductase type 2 enzyme is also transiently expressed in newborn skin and scalp and in benign prostatic hyperplasia and adenocarcinoma of the prostate7 6. Thus, in the study of prostate cancer, SRD5 A2 is the gene of interest encoding for 5a-reductase type 2. Table 3.1: Tissue Distribution o f 5 a-reductase isozymes. The two functional isozymes are type 1 and type 2 isozymes.( Reproduced from Annu Rev Biochem 63: 25-61, 1994) Tissue 5 a-reductase type I 5 a-reductase type II prostate, epididymis, seminal vesicle, genital skin - ++ testis, ovary, adrenal, brain1 , kidney - - liver + + nongenital skin ++ - * method of detection: mRNA and protein; cerebellum, hypothalamus, medulla oblongata, pituitary, pons. 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.5 Localization of the steroid 5a-reductase type 2 There are two types of tissue components in the prostate as reviewed in Section 1.3. In tissues extracted from normal and benign prostatic hyperplasia tissues, the DHT concentration is highest in the nuclei of the stroma5. This indicates that although steroid 5a-reductase type II is expressed in epithelial cells in both the nuclei and the stroma, it is highest in the nuclei of the stroma. 5a-reductase type II enzyme is a membrane bound enzyme as reviewed in Section 2.3. 3.6 SRD5A2 gene SRD5A2 gene is composed of 5 exons, which encodes for a 254 amino acids protein as shown in Fig 3.2. It contains 4 introns that are of the following approximate sizes: 46.8kb, 2.13kb, 1.91kb, and 3.04kb6 2 . 151 1 0 2 164 1695 2sl TA Repeat Figure 3.2: Structure o f the SRD5A2 gene. The grey boxes symbolize the coding region o f the exons while the white boxes symbolize untranslated regions. The corresponding exon sizes in bp are noted. The coding regions for exon 1 and exon 5 are listed below the red boxes. The introns are the dark bands connecting the exons. The TA Repeat in the 3 ’UTR corresponds to a useful polymorphic marker. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Besides the TA repeat indicated in Figure 3.2, other polymorphisms and mutations have been identified in SRD5A2. Mutant enzyme studies have indicated enzyme kinetic changes due to the mutations as seen in Table 3.2 and Table 3.3. Table 3.2: Previously Published Mutations Found in exon 1 o f SRD5A2. (Reproduced from Annu Rev Biochem 63: 25-61, 19946 9 ) Substitution V v m ax (nmol/min/mg) TK m(|JM ) NADPH K m(MM) pH Optimum Protein Half Life(h) WT 2.0-5.0 0.5-1.0 10.0-20.0 5.0 > 20 G34R 0.5 10.0-12.0 8.0-15.0 5.2-6.0 > 20 L55Q no enzyme activity < 6 P59R no enzyme activity < 6 Y91D no enzyme activity < 6 Q56R no enzyme activity 15-20 Substitution TK m(PM ) NADPH K m(MM) V v m ax (nmol/min/mg) pH Optimum Protein (%) WT 0.9 (0.7-1.0) 8(6-14) 1.9 (1.7-2.2) 6.0 (6.0) 100(91-114) C5R (1.2% of 428) 0.9 8 1.8 6.0 103 P30L (0.2% of 428) 2.1 21 0.5 5.5 102 P48R (0.5% of 428) 2.2 12 1.2 6.0 127 A49T (2.0% of 1308) 2.7 7 9.9 6.0 98 A51T(0.2%of428) 0.7 12 1.1 6.0 92 Table 3.3: Previously Published Mutations in exon 1 o f SRD5A2. ( Reproduced from Pharmacogenetics 10: 407-413, 2000) a i Q56R is another substitution found in exon which inactivates the enzyme . V89L has been studied with the hypothesis that the Leu allele, most prevalent allele in Asians, has a protective role in prostate cancer46. The Val allele was associated with a 2-fold increase in both progression and risk of prostate cancer development when compared to men with the Leu allele5 4 . However, recent data suggests that there is no correlation between the V89L polymorphism and prostate cancer5 6 . 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Since the 5a-reductase type 2 has never been purified, a way to study important sites of the gene is by mutagenesis. Researchers have reconstructed mutations into expressible cDNAs and transfected these into cells with the use of a vector plasmid. Thus, putative substrate and cofactor binding sites have been identified. From Tables 3.2 and 3.3, and other publications2, a putative substrate-binding region may include exon 1 and 5, while the putative cofactor binding site lies in the middle of the protein. The carboxyl-terminal holds a high conservation between type I and type II and also has a 75% conservation between the rat 5a-reductase and human type I3 , which may therefore represent a key part of the enzyme. 3.7 Disorders associated with SRD5A2 Besides association with prostate cancer and BPH, SRD5A2 has been implicated in hirutism, acne, male pattern baldness, and a form of male pseudohemaphroditism6 0. At the time of conception, the sex chromosomes dictate the development of either ovaries or testes. If the testes develop during embryogenesis two key androgens, testosterone and DHT are involved in sexual maturation. The virilization of the Wolffian ducts and the subsequent formation of the epididymis, vas deferens, the ejaculatory ducts and seminal vesicles are under the control of testosterone, while DHT is involved in the development of the urogenital sinus and the external genitalia, specifically the urethra and the prostate8 3 . Upon 5a-reductase deficiency, the internal organs develop normally, while those that are dependent on DHT do not, i.e. external genitalia. This is the phenotype of male pseudohemaphroditism, 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. an inherited rare autosomal recessive disorder due to the deficiency of 5a-reductase type 2 enzyme3 4. The effects of this deficiency are illustrated in Fig 3.4. XY—^Indifferent gonads—G'estes XY—^Indifferent gonads—>Testes I I DHT i I i Male i i i DHT I i I External f Genetalia J . I t Wolffian Duct Little J . Effect | 1 I | Wolffian Duct Normal Development Male Pseudohemaphroditism Figure 3.3: Normal Male Development and Male Pseudohemaphroditism. The abnormal male development due to 5a-reductase type 2 deficiency has the clinical aspects o f male pseudohemaphroditism (Reproduced from Bailliere’ s Clin Endocrinol Metabolism 8: 405-431, 1994) The genotype of these patients is 46, XY males with an external female phenotype with bilateral testes, and virilized Wolffian structures leading to a vagina. Although most patients are assigned a female gender, this is only temporary, until puberty, when they undergo male pubertal development. At puberty, their voices deepen, increased muscle mass is observed, the phallus enlarges, the testes descent from the inguinal canal into the bifid scrotum, and spermatogenesis occurs6 0 , thus being normal pubertal development due to normal testosterone levels. However, the prostate remains small, there is very little hair growth, no temporal recession of the hairline and no acne3 9 . 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4: MATERIALS AND METHODS 4.1 Samples analyzed The 148 samples analyzed were extracted from prostate cancer patients. The DNA was extracted from patient matched normal (N) and tumor (T) prostate tissues. The DNA samples analyzed were extracted from 73 different prostate cancer patients. The information provided with each sample was the Gleason score of the tumor. The samples were previously formalin fixed, and then paraffin embedded. There was no xylene deparaffmization performed, and the slides were compared to stained slides to avoid working with flammable material (xylene and the subsequent ethanol cleanup). The samples were scraped off of the slide, and compared to an identical stained slide, which allowed identification of the normal and tumor tissue. Subsequently the tissue was digested with Proteinase K, and our laboratory subsequently received the collection of tissues. The experiments performed on these samples are illustrated in Fig 4.1. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DNA I * I^ R Hot-PCR Agarose Gel T Load SSCP I Gel Extraction PCR Product Purification / \ Sequencing SNaPshot Figure 4.1: Flow Chart o f Experiments. 4.2 PCR Conditions The primers used to amplify exon 1 of SRD5A2 were custom Ordered Invitrogen (Carlsbad, CA) primers, as described previously4 7 and are shown in Table 4.1. Table 4.1: PCR Primers used for Amplification o f Exon 1 o f SRD5A2. The forward and reverse primers are described. Forward PCR Primer GCA GCG GCC ACC GGC G Reverse PCR Primer GTG GAA GTA ATG TAC GCA GAA The forward and reverse primers yielded a product of 309bp representing exon 1. These primers annealed at the locations as shown in Figure 4.2, in which the sequence of the template, and the respective amino acid sequences are shown. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ttg cgg gg g ccg cg ctctcttctgg gaggG C A G C G G C C A C C G G C G a g g aac ac g g cg cg 1 10 Met Gin Val Gin Cys Gin Gin Ser Pro Val Leu ATG CAG GTT CAG TGC CAG CAG AGC CCA GTG CTG Ala Gly Ser Ala Thr Leu Val Ala Leu Gly Ala GCA GGC AGC GCC ACT TTG GTC GCC CTT GGG GCA Leu Ala Leu Tyr Val Ala Lys Pro Ser Gly Tyr CTG GCC TTG TAC GTC GCG AAG CCC TCC GGC TAC Gly Lys His Thr Glu Ser Leu Lys Pro Ala Ala GGG AAG CAC ACG GAG AGC CTG AAG CCG GCG GCT Thr Arg Leu Pro Ala Arg Ala Ala Trp Phe Leu ACC CGC CTG CCA GCC CGC GCC GCC TGG TTC CTG Gin Glu Leu Pro Ser Phe Ala Val Pro Ala Gly CAG GAG CTG CCT TCC TTC GCG GTG CCC GCG GGG lie Leu Ala Arg Gin Pro Leu Ser Leu Phe Gly ATC CTC GCC CGG CAG CCC CTC TCC CTC TTC GGG Pro Pro Gly Thr Val Leu Leu Gly Leu Phe Cys CCA CCT GGG ACG GTA CTT CTG GGC CTC TTC TGC Leu* His Tyr Phe His Ar CTA CAT TAC TTC CAC AG GTAGCGTT Figure 4.2: Sequence o f exon 1 o f SRD5A2. The intronic sequence is shown in italic letters and smaller font. The primer annealing sites are illustrated by bold and underline letters. (Reproducedfrom Endo 131: 1571-1573, 1992) AmpliTaq Gold® with Gene Amp® (Perkin-Elmer Corp, Foster City, CA) was used in the PCR reaction and the manufacturer also provided the 10X PCR Buffer II and MgCl2 solution. The reagents in the PCR reaction are shown in Table 4.2. 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.2: Reagents for PCR Amplification. The volumes and concentrations are indicated. The stock concentration o f the PCR Buffer and the DMSO signifies the concentration provided by the manufacturer._________________________ Reagent Volume/Concentration Primers (Reverse/Forward) 0.4 pi / 150pmol/pl dNTP (Takara, Otsu, Japan) 0.6 pi / 2mM 10X PCR Buffer II 7.2 pi / Stock MgCl2 2.2 pi / 25mM DMSO(Sigma, St. Louis, MO) 2.5 pi / Stock AmpliTaq Gold 0.4 pi / 5U/pl DNA Variable sm m rnm Final Volume (Fill with Sterile H2O) 50pl The samples were amplified using a Programmable Thermal Controller 100 (PTC- 100) TM (MJ Research Inc, Watertown, MA) machine in 200pl tubes with individual caps. No mineral oil was necessary, since the machine is equipped with a heated lid. The temperature profile for the PCR reaction is shown in Table 4.3. Table 4.3: Temperature Profile for PCR Reactions. Exon 1 o f SRD5A2 was amplified using the PTC-100 PCR machine with an initial denaturing step, the 35 cycles represent the denaturation, annealing and extension steps, and a final extension step at 72°C is necessary. PCR Profile Temp (°C) Time (min) Cycles 95 9 1 95 1 — V 60 1 72 1 72 10 1 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3 Template purification After the 309bp template was amplified it was loaded on a 2% Ultra Pure Agarose gel (GibcoBRL, Grand Island, NY) in a gel electrophoresis device (Owl Separation Systems, NH). The running buffer for the template purification of samples analyzed with the ABI Prism 377 was TBE and that of the samples analyzed using the ABI Prism 3100 was TAE. The 10X TBE buffer was prepared by mixing 54.0g Tris Base (ICN Biomedicals, Aurora, OH), 27.5g Boric Acid (ICN Biomedicals, Aurora, OH), 3.75g Disodium EDTA Electrophoresis Grade (Fisher Scientific, Fairlawn, NJ) in a final volume of 500ml. This solution was stirred for >4 hours to ensure complete dissolution. The 25X TAE Buffer (CLP Inc., San Diego, CA) was made by the dilution of the concentrated TAE powder with water. All further steps had no change in protocol due to the change of TAE to TBE as the main buffer. The PCR reaction, 50pl, was loaded into the wells of the 2% agarose combined with 4pl of loading buffer, prepared by 0.25% Bromophenol Blue (Fisher Scientific, Fairlawn, NJ) and 40% sucrose (Fisher Scientific, Fairlawn, NJ). The PCR reaction was next to 1.05pg/pl of lOObp ladder (Invitrogen, Carlsbad, CA). The bands were exposed to 254 nm UV light (Dual Light™ Transilluminator, Ultra-Lum Inc., Claremont, CA) and excised out of the gel using industrial steel blades. DNA purification was achieved using the QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA). The protocol provided by the company was followed 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. closely with minor alterations as is reviewed: • Gel pieces were placed in 2.0ml microcentrifuge tube and weighed • 3 volumes of QG buffer added to 1 volume of agarose gel containing the DNA template (the volume of the agarose gel ranged from 0.1g-0.23g) • Incubated at 55°C ± 5°C for 10 minutes and vortexed • Added 1 volume of isopropanol to sample and vortexed • Placed QIAquick spin column in the 2ml collection tube. To bind DNA, 700pl of the sample was added to the column and spun for 1 min at 13,500 rpm (all subsequent steps were performed at this rpm) • Discarded the flow-through and placed back into the collection tube. Added 500pl QG buffer to column and centrifuged for 1 min • To wash, added 750pl of PE buffer and centrifuged for 1 min • For complete washing of column, re-centrifuged for 1 minute • Placed QIAquick column in a 1.5-ml microcentrifuge tube. To elute DNA added 3 5 pi EB buffer to the center of the column (without touching it) and let the column stand for 1.5 minutes and then centrifuged for 1 min • To concentrate the template DNA, centrifuged in DNA SpeedVac DNA 110 (Savant Instrument, Holbrook, NY) at medium heat for 28 minutes, and then resuspended in 24 pi sterile H20. • To quantify the template DNA, 4pL of purified DNA was loaded into a 2% agarose gel, with 2pl of loading buffer next to 0.53pg/pl of lOObp ladder. 4.4 SNaPshot™ ddNTP primer extension The ABI PRISM® SNaPshot™ ddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA) provides the majority of the reagents necessary for detecting single nucleotide substitutions. The method requires the purified PCR template, a 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. designed primer, either forward or reverse, and the reaction mixture provided by the manufacturer. The primer designed binds to the region around the mutation, leaving the mutation at the 3’ end for the extension of one nucleotide by the AmpliTaq® Polymerase in the presence of fluorecently labeled ddNTPs. The kit provided by Applied Biosystems contains the SNaPshot Ready Reaction mix, which contains the polymerase, the fluorescently labeled ddNTPs and the reaction buffer. The ddNTPs in the Ready Reaction mix are individually tagged with different fluorescent dyes, as described in the Table 4.4. Table 4.4: Fluorescent Dyes Assigned to each ddNTP in the SNaPshot Reaction. (Reproduced from ABI Prism® SNaPshot™ ddNTP Primer Extension Kit Protocol) ddNTP Color of Peaks A Green C Black G Blue T(U) Red 4.4.1 Optimization of SNaPshot™ reaction The primers for A49T and V63 M screening are illustrated in Table 4.5. Table 4.5: Primers used for SNaPshot™ method. Used to screen for mutations at nucleotide 145 (corresponding to A49T mutation) and 187 (corresponding to V63M mutation). SNaPshot A49T-F GAA GCC GGC GGC TAC CCG CCT GCC A SNaPshot V63M-F G CAG GAG CTG CCT TCC TTC GCG SNaPshot V63M-R TG CCC GCG GGG ATC CTC GCC 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The amount of the reagents used for the SNaPshot reaction are as follows: PCR template (varied amount according to the concentration of the amplified template), 0.15pmol/pl of each primer (145F, 187F, 187R), 5pl Ready Reaction mix in lOpl final volume. The PTC-100 machine (described in section 4.2) was used. For the conservation of reagents and time, a multiplex SNaPshot reaction was performed. The manufacturer designed the kit for a single reaction, thus optimization was necessary. The multiplex reaction detected the G145A substitution (corresponding to A49T) and G187A (V63M). The optimized conditions are listed in Table 4.6. Table 4.6: Temperature Profile for SNaPshot Reaction. There were 25 cycles o f the initial denaturing step at 96°C, followed by the annealing temperature o f 55°C and the extension step at 60°C. Temperature (°C) Time (sec) Cycles 96 10 ► 25 55 5 60 30 4.4.2 Post-extension treatment The primers used to screen for the two known mutations are 25 bp (A49T-F), 22bp (V63M-F) and 20bp (V63M-R). Thus, the resulting fragments are quite small and the unincorporated ddNTP will co-migrate with them. Thus, the unincorporated ddNTPs are removed by treatment of the solution with a 1.0 Unit of the calf intestinal phosphatase, CIP (New England BioLabs® Inc, Beverly, MA). The CIP 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. alters the structure of the unincorporated ddNTP by removing the 5’ phosphate group and this results in alteration of the migration pattern. Firstly, the CIP master solution is prepared by mixing 4pl of CIP (lOU/pl), 4pl Buffer 3 (lOOmM NaCl, 50mM Tris-HCl, lOmM MgCF, ImM dithithreitol pH 7.9 @ 25°C) and 32pl of H2O, in a final volume of 40pl. To attain 1.0 Units of CIP, lpl of this master solution was added to the SNaPshot reaction. The reaction was incubated at 37°C for 1 hour, after which the enzyme was inactivated at 72°C for 15 minutes. The reaction was stored at 4°C. 4.4.3 Preparation of samples for ABI Prism® 377 sequencer Before loading the reaction in the sequencer, a loading buffer containing 5 parts deionized formamide, 1 part 25mM EDTA and 50mg/ml blue dextran (Applied Biosystems, Foster City, CA) is added. 3pl of this loading buffer is added to 3pi of the SNaPshot reaction and is heat denatured at 97±5°C for 5 minutes. The solution is then immediately placed on ice and is slightly centrifuged to collect all the sample. 2 pl of the denatured solution is loaded into the prepared denaturing gel. The denaturing gel was prepared as follows: 60ml of 4.63% Long Ranger, 42pl TEMED (Fisher Scientific, Fairlawn, NJ) and 10% Ammonium Persuflate (Fisher- Scientific, Fairlawn, NJ). The Ammonium Persuflate was no more than 14 days old to ensure optimal polymerization of the denaturing gel. The 4.63% Long Ranger was prepared by adding 108g Urea (Fisher Scientific, Fairlawn, NJ), 30ml 10X TBE (prepared as described in section 4.3), 30ml of 50% Long Range 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (BioWhittaker Molecular Application, Rockland, ME) in a final volume of 324 ml which was accomplished by the addition of sterile water. The plates were thoroughly cleaned before each use and were acid washed every 10-15 uses with 3M Hydrochloric acid. The gel was poured on these plates and was left to polymerize with square tooth combs to form the wells for approximately 2 hours. The plates were subsequently washed again, dried and placed in the machine, with IX TBE as the running buffer. The ABI Prism 377 Sequencer machine was ran under the Gene Scan program. Before the samples were loaded into the machine, the machine underwent a pre-run step to ensure that all the necessary parameters were correct for collection of data. When the temperature of the plates and electronic parameters were optimal, the machine was paused, the samples were loaded in the designated wells and collection of data began. 4.5 Automated sequencing The necessary reagents for a sequencing reaction are the purified template of interest, primers and the reaction mixture containing all other reagents. Applied Biosystems furnishes their own BigDye™ Terminator Cycle Sequencing Ready Reaction Kit version 2.0 and 3.0. The BigDye™ ready-reaction mixture contains AmpliTaq® DNA Polymerase, MgCl, buffers and the fluorecently labeled ddNTPs. The ddNTPs are fluorecently labeled with dye terminators and they give off colors as described in Table 4.7. 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.7: Fluorescent Dyes assigned to each ddNTP used in the Sequencing Reaction. (Reproduced from Applied Biosystems Protocol manual for ABI PRISM® BigDye ™ Terminator Cycle Sequencing Ready Reaction Kits for the Original and Version 2.0. Copyright 2000) ddNTP Color of Raw Data on ABI Prism 377 Electrophorograms A Green C Red G Blue T Black 4.5.1 Optimization of sequencing reaction The sequencing reaction contained 3.2pmol/pl of either the forward or reverse primers described in Table 4.1. Other reagents included 2pi of 5X buffer (400mM Tris-HCl pH 9.0, lOmM MgCf), 4pl BigDye™ (v 2.0 or 3.0), and the template DNA amount was variable, ranging from 35-90 ng/pl in a 20pl final volume. The temperature profile, as well as the PCR machine used required some optimization. Several different conditions were tried, and two different thermal cyclers were tested: the Robocycler Gradient 40 (Stratagene, La Jolla, CA) and the PTC-200 (MJ Research Inc, Watertown, MA) machine. The PTC-200 was chosen for all reactions due to the heated lid feature that avoids evaporation, without the aid of mineral oil addition. The temperature profile of the sequencing reaction is described in Table 4.8. 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.8: Temperature Profile for Sequencing Reaction. The initial denaturation step was performed for 5 min at 95°C, followed by 35 cycles o f 95°C denaturation, 50°C annealing and 60°C extension. Temperature (°C) Time (min) Cycles 95 5 1 95 0.5 I 35 50 0.25 60 4 4.5.2 Removal of ddNTPs Much like the SNaPshot reaction, the removal of ddNTPs is a necessary step in the sequencing reaction. To remove any unincorporated ddNTPs the sequencing reaction was loaded in the Auto Seq™ G-50 (Amersham Pharmacia Biotech Inc, Piscataway, NJ). The preparation of the columns follows the protocol suggested by the manufacture with some alterations to yield best results: • Vortexed to ensure homogeneous mixture and break the bottom off • Place in a 2ml collection tube and vortex at 4,500 rpm for 3 minutes. • Added the reaction to the center of the column (without touching it) • Placed columns in a labeled 1.5ml centrifuged for 1 min at 4,500 rpm (keeping the column orientation to avoid it from crashing over itself) • The flow-through was the cleaned reaction, which is then concentrated by using the SpeedVac (described in section 4.3) at medium heat for 8 minutes 4.5.3 Preparation of Samples for ABI Prism® Sequencers The analysis was completed on the ABI Prism® 377 and the ABI Prism® 3100 Sequencing Machines. 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.5.3.1 Analysis using ABI Prism® 377 sequencer After the samples were purified as described in section 4.5.2, the dried concentrated reaction is resuspended in 4pl of loading buffer (described in section 4.4.3). The sequencer underwent a pre-run, to ensure that the parameters were ideal for gathering data using the Sequencing Analysis Software™ v3.4.1 (Applied Biosystems, Foster City, CA). During this time the DNA samples were heat denatured at 97±5°C for 10 minutes and then 2 pl of the denatured reaction was loaded into the square wells of the gel (described in section 4.4.3). The data was analyzed using Sequence Navigator® (Applied Biosystems, Foster City, CA) and Factura™ software v2.2.2 (Applied Biosystems, Foster City, CA). 4.5.3.2 Analysis using ABI Prism® 3100 sequencer For the ABI Prism® 3100 Sequencer, the reactions were purified as described in section 4.5.2. The concentrated sample was then resuspended in 10pl formamide and was placed in a 96-well plate. The samples were heat denatured at 97±5°C for 1 0 minutes, after which the plate was loaded in the machine and the sequencer automatically loaded the reactions. The sequencing machine gathered the data and it was analyzed by SeqScape™ Software (Applied Biosystems, Foster City, CA). 4 .6 S in g le S tr a n d C o n fo r m a tio n P o ly m o r p h is m (S S C P ) SSCP is a method that analyzes the secondary structure of the DNA strand. Due to a nucleotide substitution, the secondary conformation of the DNA would be altered 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. which would cause a shift in the migration pattern visible on an acrylamide gel. The alteration of the migration pattern is detected by a radioactive-PCR reaction, due to a radioactively-labeled primer in the PCR reaction. 4.6.1 The radioactive-PCR reaction The radioactive-PCR reaction is gotten by addition of a radioactively labeled primer in the PCR reaction. The forward primer described in Table 4.1 was labeled with 3 2 P (Perkin Elmer, Foster City, CA). The primer was labeled as follows: 4pl of the forward primer (150pmol/pl) with sterile FECXvariable, adjusted to a final volume of 40pl) was incubated at 95°C for lOmin to heat denature the primer. This mixture was then immediately placed on ice, and 8 pi of Forward Reaction Buffer, 0.5pl of T4 Kinase (lOU/pl) ( GibcoBRL, Grand Island, NY), and 0.5pl (double the amount for each half life passed) of [y32-P]ATP (150pCi/pl) was added. The solution was incubated at 37°C for 2 hours, after which an inactivation step at 57°C for 5 minutes was performed. The primer was subsequently stored at -20°C in a plexi-glass container. The radioactive-PCR reaction was identical to the one described in Table 4.2 with 0.308pl of 15pmol/pl 32P-forward primer, with an adjusted H2O amount to yield the a final volum e o f 50p l. The reaction m ixture w as placed in a P T C -100 for amplification of the fragment of interest. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.6.2 Preparation of samples for SSCP run After the template was amplified using the PTC-100 machine, the solution was prepared to be loaded into the SSCP gel, by adding 8pl of the radioactive-PCR solution to 4pl of the loading buffer (95% formamide and 0.05% bromophenol blue). The samples were heat denatured at 97±5°C for 10 minutes, after which they were placed on ice and were ready to load into the prepared acrylamide gel. 4.6.3 SSCP run Firstly, the stock solution of the 10% glycerol (59.2:1 acrylamide:bisacrylamide) solution was prepared. This was accomplished by adding 0.5g of bis-acrylamide (Bio-Rad, Hercules, CA), 29.6g Acrylamide (Kodak Laboratory and Research, New Haven, CT), 30mL of 100% Glycerol (ICN Biomedicals, Aurora, OH), and 22.5ml 10XTBE (as described in Section 4.3). To polymerize the acrylamide gel, 60ml of the stock acrylamide solution was mixed with 500pl of fresh 10% APS and 50pl TEMED. The gel was poured on clean plates, and left to polymerize with square tooth combs for approximately 2 hours. After polymerization, the plates were cleaned and mounted on the horizontal apparatus. Subsequently 8 (0 .1 of the denatured radioactive-PCR solution as described in section 4.6.2, was loaded. The samples were ran at 2000 Volts, 300 mAmps and 12 Watts for 22 hours. 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 5: RESULTS 5.1 Specific aims As discussed previously, there is ample evidence to show that SRD5A2 is an attractive candidate for the study of prostate cancer because of its role in androgen metabolism. 5a-Reductase type 2 has never been purified, therefore there is much to learn about this enzyme. SRD5A2 has been intensely studied, and several mutations and polymorphisms have been identified. 5.2 Optimization of PCR conditions The 148 samples that were analyzed were DNA samples extracted from prostate cancer patients. The DNA was extracted from patient matched normal and tumor prostate tissue. The negative controls used for all experiments were genomic DNA extracted from galactosemia patients with a wild-type SRD5A2 gene sequence. The positive controls for already identified mutations were DNA extracted from BPH patients. A significant amount of time was dedicated for the optimization of the PCR conditions for the amplification of the normal and tumor DNA samples with no non-specific bands. Different parameters were changed, including the type of polymerase used, the temperature as well as the time variants were manipulated. Finally the optimal conditions were found and yielded amplification of both the 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. controls (not shown) as well as the samples of interest with no non-specific bands, as illustrated in Fig 5.1. Band of interest 51T 100 bp ladder Figure 5.1: Optimized conditions for amplification o f tumor DNA. Samples amplified are 5 IT and 53T. Blank o f the PCR reaction is not shown. 5.3 Optimization of dilution factors for individual DNA samples A total of 1481 DNA samples were analyzed, labeled 51-123 (corresponding to 1-73 from the original batch). 74 tumor samples represent DNA extracted from prostate cancer tissues and 74 normal samples represent DNA extracted from matching patient normal prostate tissues. Several fold dilutions were necessary to be made in order for the DNA to amplify, as seen in the Table 5.1. 1 Note: Samples were extracted from 72 different patients, and 4 samples were extracted from 1 patient. 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TableS. 1: Dilution Factors o f DNA Samples. The dilution factors for each individual tumor and normal sample was optimized. The dilution factors are listed, and the stock DNA solution was the original undiluted DNA received from the previous laboratory. DNA source No amplification 1:50 dilution 1:10 dilution 1:4 dilution 2pl of stock DNA Tumor tissue 14 5 4 46 5 Normal tissue 10 0 5 57 2 Total (148) 24 5 9 103 7 5.4 Genotyping using SSCP After optimizing of the PCR conditions and achieving the proper dilution factors for each sample, the DNA samples were genotyped using SSCP. The only positive controls available for previously identified mutations were for A49T. The heterozygous mutant AT (corresponding to nucleotides GA) and the homozygous mutant TT (corresponding to nucleotides AA position 145) were used as positive controls extracted from BPH patients. As one can see from Fig 5.2, both negative and positive controls for A49T are detectable. pBH* ' AA AT TT Fig 5.2: Negative and Positive Controls for SSCP. The previously identified mutation, A49T was used as a positive control. AA is the wild-type, the two separate distinct bands identify the heterozygous mutant AT, and the homozygous mutant TT has a slower migration pattern than the AA. 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. All tumor and normal DNA samples were subjected to SSCP analysis. Although the conditions for amplification were optimized, some samples still did not amplify. In total, 14 normal and 12 tumor samples had a detectable SSCP shift. All of these samples were later sequenced to find what is responsible for the shift. An example of an interesting shift is illustrated in Fig 5.3. Figure 5.3: SSCP Shifts in Normal and Tumor DNA. AA is the wild-type sequence, and has the corresponding shift, and TT is the homozygous mutant at codon 49. As one can clearly see sample number 5IN and 57T have a different migration pattern than the wild-type. 5.5 Analysis of samples with SSCP shift All 26 normal and tumor DNA samples that had abnormal migrating DNA bands such as those illustrated in Fig 5.3 were subsequently sequenced. 8 tumor samples and 4 normal samples that were found to have an SSCP shift had a corresponding nucleotide substitution detectable by sequencing. The SSCP shift and the sequencing of tumor samples are illustrated in Fig 5.4. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sample SSCP Shifts Sequencing- Forward Reaction Sequencing- Reverse Reaction 55T ■ 55T AA N/A M il T G N A T C G->A Mil 56T AA 56T G C N C T C— > T A19A A G N G C aaAa a G-*A A19A 58T AA 58T C A N C C TCwdS G G N T G G— »A A49T C— *T A49T 72T 72T AA T T N C T 'VWNa. C-^T S60F GN A AG W V A , G— > A S60F 87T ■ *. 87T AA C G N T C A N C G S & V b G— > A V63M C— > T V63M 106T AA 106 C G N G A G— »A G34E T C N C G C— »T G34E Figure 5.4: Nucleotide Substitutions in Tumor Samples. The sample, the SSCP shift and the forward and reverse sequencing reactions are illustrated. The nucleotide and amino acid substitution is provided under each sequence. The forward sequencing reaction in 55T is not available. N denotes the substitution. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Some mutations were also found in normal samples, and these mutations were confirmed by both SSCP and sequencing. Sample SSCP Shift Forward Sequencing Reaction Reverse Sequencing Reaction G C NT GG N T G A N G : T A N C C 78N 78N AA A —>G L35R C— >T T45I T— >C L35R G— >A T45I C G N T G C A N C G 99N AA 99N G— >A V 63M C -*T V 63M Figure 5.5: Nucleotide Substitutions in Normal Samples. The corresponding sample number, the SSCP shift visible and the forward and reverse sequencing reaction is illustrated. The nucleotide substitution is provided under each sequence. Sample 78N is an exception, since it has a double mutant that is separated by the dotted line; each forward and reverse sequencing reaction is represented correspondingly. N denotes the particular nucleotide substitution. Some other mutations were found in both the normal and the tumor DNA. These nucleotide substitutions were confirmed by both SSCP and sequencing as illustrated in Fig 5.6. 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sample SSCP Shift Forward Sequencing Reaction Reverse Sequencing Reaction 71T 71T AA WT A49T GGN TG /W # C— > T A49T 71N 71N AA C ANCC G— > A A49T G GN TG C— » T A49T 86T 86T AA C G NTG tN % f) G— *A V63M C AN C G M f l i s C— > T V63M 86N N/A CGNTG i c & k i Q i G— > A V63M CANCG Ike C— > T V63M Figure 5.6: Nucleotide Substitutions in both Normal and Tumor DNA. Patient 71 and 86 contained the above mutations in both the DNA extracted from normal and tumor prostate tissue. The forward sequencing reaction fo r sample 7IT was not available. N corresponds to the particular nucleotide substitution. The substitution seen in Fig 5.4 of sample 55T was only detected in the reverse strand, because the mutation is so close to the forward primer. The mutation in sample 71T is only slightly visible in the reverse strand, and looks like the wild- type sequence. These very low peaks are expected when analyzing tu'mor DNA. 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The tumor DNA mutations are not like constitutional DNA where the heterogeneous nucleotide substitution is visible with 50% mutant and 50% normal. Since the tumor DNA is very heterogeneous, the mutant peaks can be as low as 10%, or even lower in the case of sample 7IT. Indeed the nucleotide substitution is hardly seen in sample 7IT, however this particular case requires further discussion in section 6.5.2.1. Besides SSCP and sequencing, SNaPshot was also used as a genotyping tool for the two already identified mutations in exon 1 of SRD5A2, A49T and V63M. The SNaPshot primers designed and used for the reaction are described in Table 4.5. The results for some of the SNaPshot reactions for the A49T and V63M amino acid substitutions are illustrated in Fig 5.7. Sample/Amino Acid Substitution SNaPshot Results & Primers Used 58T/A49T k G( 1)— ► A(2)=Forward 86TW63M , A « / H A G(3)— >A(4)=Forward C( 1)— >T(2)=Reverse Figure 5.7: SNaPshot results for A49T and V63M substitutions. For the A49T substitution, the forward SNaPshot primer was used in the reaction and yielded the G to A nucleotide substitution. For V63M substitution, the forward primer resulted in the G to A substitution and for the reverse, the complimentary strand is seen, corresponding to a C to T substitution. The respective peak numbers are indicated. 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.6 Analysis of samples with no SSCP shift All samples were subsequently analyzed using sequencing. In this study the previously identified mutation A49T was detected in several samples, as seen in Fig 5.4 and Fig 5.8. All the samples with exception of sample 95N and 90N, which had a double mutant and a homozygous TT mutant at codon 49 and an A to V amino acid substitution, contained the heterozygous AT mutant. Samples Example of Forward Sequencing Reaction Example of Reverse Sequencing Reaction C ANCC G GN TG i 52T, 61T, 81T t u b G— *A C— > T A49T A49T CAACC ▼ GGTTG 95N N S h P t. G— > A C— > T A49T-homozygous A49T -homozygous GCNCG CGNGC 95N iA /V v x ^ N sfsa C— »T G— > A A69A A69A AGNCC G G NCT 90N /WV\ C-*T G— > A A49V A49V Figure 5.8: Nucleotide Substitutions at Codon 49. The A49T recurrent mutation was found in sample 52T, 61T and 8IT. The homozygous mutant at codon 49 was identified in sample 95N along with another novel mutation A69A. The corresponding forward and reverse sequencing reaction is illustrated. N represents the nucleotide substitution. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Besides the nucleotide substitutions already mentioned, there have been other novel nucleotide substitutions identified in both normal and tumor DNA. These samples with the corresponding substitutions are illustrated in Fig 5.9. Samples Forward Sequencing Reaction Reverse Sequencing Reaction 103T G C N C T C— > T A19A A G N G C G— > A A19A 108T C C N C C G— > A A52T G G N G G VvAJW C— > T A52T 107T C G N G G aA aaA G— > A G66R C N C G C a. C— > T G66R 114N G G T C A C— »T A24T ACC T T V G— > A A24T Figure 5.9: Novel Nucleotide Substitutions Identified. The sample number and the corresponding sequencing reactions are noted. N represents base substitutions. 5.7 Review of all results In Fig 5.4 thru 5.9, all the nucleotide substitutions are noted. Some substitutions observed are found strictly in the tumor tissue as seen in Table 5.2. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.2: Substitutions Identified in Tumor DNA. Nucleotide and amino acid substitutions are indicated. The normal DNA o f all samples was sequenced and had the wild-type sequence with the exception o f sample 55N that was N/A. Sam ple N u cleotid e Substitution A m ino A cid Substitution 56T C 57T A la 19 Ala 56N normal Normal 103T C 57T A la 19 Ala 103N normal Normal 106T G 101A G ly34G lu 106N normal Normal 52T G145A Ala49Thr 52N Normal Normal 58T G145A Ala49Thr 58N Normal Normal 61T G145A Ala49Thr 61N Normal Normal 81T G145A Ala49Thr 81N Normal Normal 108T G 154A A la52T hr 108N Normal Normal 72T C 179T Ser60P he 72N Normal Normal 107T G 196A G ly66A rg 107N Normal Normal 87T G187A Val63Met 87N Normal Normal 55T G 3A M e tl He 55N N/A N/A N/A= samples that did not amplify. Bold letters= novel nucleotide substitutions identified in this study 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A recap of all the samples and the corresponding substitutions found only in the DNA extracted from normal prostate tissue is provided in Table 5.3. Table 5.3: Substitutions Identified in Normal DNA. DNA extracted from patient 114 and 99 had a somatic mutation identified only in the normal sample and not the tumor DNA. Sam ple N ucleotide Substitutions A m ino A cid Substitutions 114N G 70A A la24T hr 114T Normal Normal 99N G187A Val 63Met 99T Normal Normal 78N A 104G L ys35A rg C 134T T hr45Ile 78T N/A N/A 95N G 145A A la49T hr C 207T A la69A la 95T N/A N/A 90N C 146T A la49V aI 90T N/A N/A N/A= samples that did not amplify. Bold letters= novel nucleotide substitutions identified in this study Table 5.4 illustrates the two patients that contain the recurrent mutations, A49T and V63M in DNA extracted from both the normal and the tumor DNA. Table 5.4: Substitutions in Both Tumor and Normal DNA. Both A49T and V63M mutations have been previously identified. Sam ple N ucleotide Substitutions A m ino A cid Substitutions 71T G145A Ala49Thr 71N G145A Ala49Thr 86T G187A Val63Met 86N G187A Val63Met 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 6: DISCUSSION/ CONCLUSION 6.1. SRD5A2 and prostate cancer The prostate gland is an androgen-regulated organ. The DNA samples analyzed were extracted from prostate cancer patients. The main goal of the study was to investigate if any mutations occur in exon 1 of SRD5A2 in DNA extracted from prostate cancer patients, and compare it to the patient matched DNA extracted from normal prostate tissue. SRD5A2 is an attractive candidate gene for prostate cancer predisposition and/or progression because it encodes for 5a-reductase type 2 enzyme. 5a-reductase type 2 enzyme plays a key role in androgen metabolism, specifically it converts testosterone to DHT, which are the two most active androgens in prostate cells. This study is important for two major reasons. Firstly it is critical at the clinical level. 5a-reductase type 2 is an active enzyme in prostate cells, and like most enzymes it is highly regulated. If the enzyme is not properly regulated this will have serious consequences on the cell, due to the effects of the androgens on the prostate cell, such as on growth, survival and the synthesis of proteins. If a mutation is identified which reduces the 5a-reductase activity, not enough testosterone will be converted to DHT. This might have a drastic effect on the cell, more specifically it may retard growth, decrease the survival of the cell, which may ultimately affect the function of the prostate as a whole. This may also have a 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. protective effect on developing prostate cancer, and this hypothesis is strengthened by the fact that there are no prostate cancer cases in patients with complete 5a- reductase deficiency. On the other hand if a mutation in the SRD5A2 gene increases the enzyme’s activity, more testosterone will be converted to DHT, which will affect all the downstream components. If there are increased amounts of DHT in the cell, then more DHT will bind to the androgen receptor and all downstream events will occur as described in Figure 2.3. This will result in increased transcription of genes involved in cellular growth, increased survival of the cell, which is part of the definition of a cancerous cell. Thus, SRD5A2 is a very attractive gene for the study of prostate cancer predisposition, perhaps development and progression. Conclusively, upon the identification of a recurrent mutation in prostate cancer patients this would allow early screening of the disease and thus prevention of the disease in the male population. Besides the clinical aspect of these findings, further experiments with the mutations identified in this study may discover important structural characteristics of the enzyme since it has never been purified. Through biochemical analysis of the mutant enzymes the active sites of the enzyme can be identified. This is how researchers have found putative substrate and cofactor binding sites. Combining the results from Table 3.2 and Table 3.3, one can see that the majority of exon 1 is involved in substrate binding, since mutations altered the Km of testosterone. Some other observations have been that like the rest of the protein coding region, exon 1 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. encodes for mostly hydrophobic amino acids. In fact, 67.7% of amino acids are hydrophobic. Therefore conversion of this particular pattern may disrupt the delicate structure of the enzyme and alter its function or completely inactivate the enzyme. Enzyme kinetic studies have shown that mutations that completely inactivate the enzyme are at amino acid position 55, 56, 59 and 91, which indicate that these sites play a critical role in enzyme activity. Therefore mutagenesis analysis of the mutations identified in this study can show new regions that may be involved in cofactor and substrate binding, and perhaps also reveal a little more of the secondary structure of the protein. 6.2 Samples analyzed 148 samples were screened for mutations in exon 1 of SRD5A2 via single strand conformation polymorphism (SSCP), sequencing and SNaPshot. These samples were extracted from the tumor and normal tissue of 73 prostate cancer patients. There is only one sample extracted from each individual, except for one individual that had 2 normal and 2 tumor tissue DNA examined. Because of the nature of the tumor DNA samples the amplification step required optimization of the parameters. Unlike genomic (constitutional) DNA, tumor tissue is much harder to work with since the tumor cell is a rather chaotic system. Firstly, the tumor cell may lose cell cycle control, the proofreading mechanism of the polymerases may not be functionable, and therefore it may contain many 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mistakes. Secondly, there could be extra components, such as proteins, RNA, DNA, that were not compartmentalized properly, and this would get in the way of the PCR. Thus, the amplification steps for this type of DNA, are quite different from ones that are used for genomic DNA. Therefore a vast amount of time was invested in optimizing the concentration of PCR reagents, and the conditions for amplification. The dilution factor of the individual DNA samples was also necessary to be optimized. This is partially due to the extraction step of the DNA from the tissue, since it was Proteinase K digested, and the DNA was not cleaned up using any phenol or any DNA purification kit, and was not quantified. Thus, 124 samples out of 148 were amplified and analyzed as seen in Table 5.1. Of these, 21 normal and tumor samples combined had nucleotide substitutions, which corresponds to 19 different patients. Thus, two samples had the substitutions in both normal and tumor tissue. The method by which these substitutions were detected is by single strand conformation polymorphism (SSCP), which analyzes the secondary structure of the template, SNaPshot which yields the extension of a primer by one nucleotide at the site of the mutation. Finally, the most powerful technique to detect the substitutions was sequencing. 6.3 SSCP At the beginning of the project, samples were subjected to SSCP. This is a quick method to detect amino acid substitutions due to the effects of the substitution on 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the secondary structure of the coding sequence of interest. After detection of shifts by the migration of the bands, the samples were re-amplified, and then analyzed by sequencing. In a total of 26 samples that had a SSCP shift, 11 samples had a nucleotide substitution that was detected by sequencing, while the rest of the samples had the consensus sequence. Furthermore, of the 10 samples that contained a nucleotide substitution detected by sequencing, 6 samples had the wild- type SSCP shift. Thus although SSCP is a fast screening tool, if the conditions for the particular mutations are not optimal, the mutation may not be detected. There is previous data that indicates this, for example when screening for mutation on exon 1 in SRD5A2 the detection of Y91D was not identified when using SSCP8 1. There are several possible reasons that some of the mutations detected using sequencing were not detectable by SSCP. Firstly, the gel matrices are important parameters for the detection of a mutation, and they are not ideal, this may lead to altered migration. Therefore optimization of gel matrices has to be established for each specific mutation in order to be detected. If this is the case, than this method is only useful for screening for already known mutations. The reason behind this is because if there is a positive control, this sample can be ran on different gel matrices to see which is the ideal gel matrix for the detection of the shift. This method should therefore be reserved for screening of already known mutations, because if ran on a gel matrix that is known to work for several mutations, many more can be missed because the gel preparation was not ideal for a particular shift. SSCP analyzes the secondary structure of the DNA, which is firstly not only hard 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to predict, but also very easy to lose or change under different conditions. These conditions are the temperature of the room, the amount of time taken to load the samples into the acrylamide gel, and other such factors. Some of the parameters were kept relatively constant, and these were the temperature and time of denaturation, the denaturing solution, and amount of time the samples were kept on ice to ensure the single strand conformation is kept. However, even though these elements were tightly supervised, the secondary structure of the DNA is very delicate and minute elements can make a big difference. To try to avoid some of these problems, an SSCP gel was done at a relatively constant temperature of 4°C, and 2 samples with substitutions were detected this way, however 7 other samples with substitution were missed. Besides these factors, the size of the fragment might also cause differential results. It has been estimated that the detection rate of SSCP fragments larger than 200bp falls to 80%5 5 , however not all published data agrees that the effective size limit is in the 200 bp range22. Other elements affecting the ability of SSCP to recognize a mutant allele, is the position of the mutation, whether it is towards the middle of the template amplified or towards the extremes. 6.4 Sequencing and SNaPshot as a screening technique After demonstrating which samples had a particular SSCP shift, the samples underwent sequencing analysis. This, obviously is a very powerful technique, however it also requires optimization to ensure clean sequences. After detection of the nucleotide substitutions by sequencing some of the samples underwent another 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. technique to be confirmed. SNaPshot allowed genotyping of A49T and V63M, using specific primers for each mutation. SNaPshot proved to be a powerful technique for genotyping already identified mutations. 6.5 Substitutions identified Two previously identified mutations have been found in this study. These mutations are A49T and V63M, and the recurrence rate of these two mutations was high in the 124 samples analyzed. There were several novel substitutions identified in this study as described in Table 5.2, Table 5.3, and Table 5.4. 6.5.1 Silent substitutions Silent mutations change a particular nucleotide at a location, but do not alter the coding amino acid. These types of mutations are just as important to study as other types. Although there is no alteration of the amino acid, which can directly influence the structure and function of the protein, the translation rate can be altered. The cause of the change is due to codon usage preference of the cell, or perhaps even unavailability of a certain tRNA that recognizes the codon, however in most cases this is solved by the versatility of the tRNA molecule. The versatility of the molecule in this context is achieved by the wobble effect, where the tRNA recognizes a codon with different bases at its 3rd position of the anticodon, at its 5’ end. Ser and Leu are exceptions because additional changes to other bases within the codon still encode for the same amino acid. Met and Trp are the only two 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. amino acids that have only one codon for the respective amino acid. Ala is an amino acid that is encoded by 4 different codons, with a varying 3rd nucleotide. The tumor DNA extracted from patient 56 and 103, have a silent substitution at amino acid position 19 in the tumor DNA. The two mutations are both encoded for by the nucleotide substitution of Cytosine (C) to Thymine (T) at nucleotide position 57. The substitution was confirmed in sample 56T using SSCP, however there was no SSCP shifts detected for sample 103T. The DNA extracted from normal prostate tissue from both patients was wild-type. The translation rate for this particular somatic mutation may be altered because of the codon usage. The translation of the mutant protein may be retarded because the codon for the wild- type has a higher frequency than that of the mutant . 6.5.2 Missense Substitutions Missense mutations are one of two different classes of mutations, which have no affect on the reading frame. The other class, frameshift mutations alter the reading frame by inserting or deleting nucleotide(s). Missense mutations alter a particular nucleotide and the result is an amino acid substitution. There are two different types of missense mutations, transversion and transition mutations. Transversions change a pyrimidine (C or T) to a purine (A or G) or vice versa. Transition mutations exchange a purine for a purine, or a pyrimidine for a pyrimidine. 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6.5.2.1 Substitutions at amino acid 49 Of the 21 normal and tumor samples that contained nucleotide substitutions 8 samples had them at codon 49. The previously identified mutation, A49T was found in 7 DNA samples extracted from 6 patients. Thus one patient contained this mutation in both the normal and tumor DNA. A49T involves the nucleotide conversion of a G to A at position 145. A49T is a previously identified and well- studied mutation. In vitro enzyme kinetic studies indicate that the Vm a x of the • 48 mutant is much higher than that of the normal, 9.9 vs. 1.9 nmol/mm/mg . This indicates that the rate of conversion of testosterone to DHT is much higher in the mutant enzyme. This mutation has also been correlated with race. African American men who carry this mutation have a 7.2-fold increased risk to develop clinically significant prostate cancer, while Latinos have a 3.6-fold increased risk47. In this study 6 samples had the heterozygous GA mutation and 1 sample had the homozygous AA mutant. The respective normal samples of the 6 tumors containing the heterozygous mutations was wild-type. A special case that warrants more discussion is that of patient 71. Samples 71T and 71N represent the DNA extracted from tumor and normal prostate cells, respectively. The mutation in this patient was identified in the normal sample and sequencing of the tumor DNA proved to show only traces of the mutant that could have been interpreted as background. These results were quite perplexing, and the explanation for the normal DNA containing a mutation and not the tumor, is due to the loss of the 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mutant allele. Loss of heterozygosity (LOH) is an event that occurs in tumor cells and symbolizes the loss of one allele. A parallel experiment done by a colleague on this patient showed that in fact the tumor undergoes LOH. The marker used for the LOH study is the TA repeat, which is located in the 3’UTR and it is very polymorphic, ranging from TAo to TA9 to TAis, with minor variability in the exact number of repeats9. Thus, it is interesting that the allele lost is the mutant. Perhaps this mutation occurs along with another in the tumor suppressor gene that may cause LOH? Patient 95 had the homozygous AA mutation at nucleotide position 145 in the DNA extracted from normal prostate tissue. This sample, however, had another observed mutation. This was a silent mutation that altered nucleotides G to T at position 207, causing a silent mutation at codon 69. This sample was confirmed by SSCP. The tumor DNA was not amplified, even after several dilutions. It would be of great interest to amplify the tumor sample in order to check for LOH. However, here one can hypothesize the following scenario: since this mutation is homozygous, both alleles are altered. Therefore, from the data on DNA extracted from patient 71, perhaps for the homozygous mutant, both alleles are lost, and this is the reason that the tumor DNA does not amplify. Sample 90N was another sample that was extracted from normal prostate tissue that had a particular nucleotide substitution, however the tumor DNA was not able to be 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. amplified. The nucleotide substitution was C to T at position 146 and a corresponding Ala to Val substitution at codon 49. The SSCP of this sample had a wild-type band migration. Although the amino acids involved have the same hydrophobic properties, the Val contains two additional methyl groups (-C H 3 ), which might affect the structure of the protein, and thus alter the enzymatic properties. Considering that 8 samples contained a substitution at codon 49, perhaps this could indicate a particular hot spot for mutations in exon 1 of SRD5 A2 gene. 6.5.2.2 Substitutions at amino acid 63 The V63M mutation has been previously identified and it corresponds to the nucleotide substitution of G to A at position 187. Both Val and Met are hydrophobic, but there is one major structural difference, which may result in the alteration of the structure as well as the function of the enzyme. Met contains a thioester (-S -C H 3 ) in its side chain, which might hold an important role in converting the stability of the protein, because of the presence of sulfur. The samples, which contained the mutation, were sample 86T and 87T, and sample 86N also had this mutation, while 87N was wild-type. The third patient having this amino acid substitution was patient 99, and it was found only in the normal DNA, and not the tumor. The mutation in sample 86T and 87T were confirmed by SNaPshot and SSCP, which also confirmed the mutation in sample 99N. 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Therefore, the mutation in patient 86 exists in both tumor and normal DNA, which is the first time that this mutation was documented in normal tissue, and this is also the case for sample 99N. A possible reason, as described in section 6.5.2.1, may be due to the loss of the mutant allele, and in the sequence, only the consensus sequence is represented, which may be the wild-type allele. 6.5.2.3 Novel amino acid substitutions Seven novel amino acid substitutions were identified, 4 of which were confirmed by SSCP. The first somatic mutation is a G to A nucleotide substitution at position 3, which converts Met to lie at amino acid position 1. The mutation was found in the tumor DNA and the normal sample of this patient was not amplified. If the starting codon is altered, then the protein that is translated will be truncated. The next Met in SRD5A2 coding region is at codon 157, and thus this may alter or completely inactivate the enzyme. This mutation was seen in the sequencing reaction using the reverse primer and not in the forward sequencing reaction because the position of the mutation is very close to the forward primer used. Another novel substitution detected with with SSCP and sequencing is sample 72T, of which the corresponding normal sample is wild-type. The alteration was Ser to Phe at codon 60, a nucleotide change of C to T at position 179. The mutation converts Ser, which is hydrophilic to Phe, a very hydrophobic amino acid. This may result in the alteration of the localization and function of the enzyme. 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Since the somatic mutations identified in sample 55T and 72T were rather hard to see, better sequencing results would be gotten from the design of new primers that are located farther away from the starting codon and also farther away into the intron, to ensure amplification of the exon. To try to get better results for 72T in the reverse reaction, an alternate reverse primer was designed. Although the conditions that were tried worked for the genomic DNA, it did not work for the tumor DNA(data not shown). Thus, further optimization was necessary, and this would be the case for an alternate forward primer as well. Thus in order to re confirm using sequencing, newly designed primers are necessary to confirm these two mutations. A mutation detected in sample 106T was identified by automated sequencing and was confirmed by SSCP. The DNA extracted from normal prostate tissue was sequenced and the sequence was wild-type. The missense mutation is Gly to Glu at position 34, and a respective nucleotide substitution of G to A at position 101. Other mutations have been identified at this codon. The previously identified mutation, converted the wild-type Gly to an Arg. The results of this mutation lead to a decreased Vm a x as described in Table 3.2. The mutation also resulted in an increased the Km for the substrate, thus indicating that it could be a putative testosterone binding site. This region has also been considered to be involved in finasteride binding, which is a competitive inhibitor of the substrate. The missense mutation identified in this study alters Glycine, the amino acid with the smallest 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. side chain, to Glutamic Acid. A possible hypothesis of the effect of the mutation on the enzyme activity is that if altering the small Gly, and having a Tyr at amino acid 33 (which is very hydrophilic and active), Lys35 (basic amino acid) and His36 (which has the imidazole ring, which is most often found in the reactive centers of the protein because of its versatility) it might hinder the activities of the reactive center and thus may inhibit substrate binding. Sample 108T had a somatic mutation at codon 52 which substituted the wild-type Ala to Thr due to a nucleotide alteration of G to A at position 154. The normal sample extracted from this patient had the wild-type sequence. Much like the A49T mutation, the A52T mutation converts a hydrophobic to a hydrophilic amino acid. Because Thr contains a hydroxyl group, it makes the respective amino acid more reactive. This conversion in the properties of the amino acids is important because it is very likely that the function may be altered, perhaps the mutation converts this amino acid from being within the membrane to the aqueous filled environment, thus altering its function. Another novel somatic mutation identified had a nucleotide substitution of G to A at position 196, which resulted in the amino acid conversion of Gly to Arg at codon 66. This substitution was confirmed by SSCP. The mutation was found in sample 107T and the matching normal sample was analyzed and had the wild-type sequence. Gly, a hydrophobic amino acid is the smallest amino acid with an -H as 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. its side chain. Arg on the other hand, is a basic amino acid. This is an important substitution, and again might have some effect on the structure of the enzyme. Since Gly is such a small amino acid, and it is hydrophobic, it may be structurally positioned in the middle of other amino acids. A hypothetical structural prediction may be that at amino acid 64 which is a Pro, the protein may bend, and the Gly at 66 and Ala at 65 allow a sharp bend to occur since they are relatively small molecules. However, if it is converted to Arg, which is a rather large amino acid, it may certainly change its conformation, and thus the function as well. Another novel somatic mutation was identified in sample 114N, specifically the Ala to Thr amino acid substitution at position 24, which corresponds to G to A nucleotide substitution at position 70. It was only identified in DNA extracted from normal prostate tissue, while the tumor DNA had the wild-type sequence. As reviewed in Table 5.3, those samples had the substitutions only in the normal DNA. This may be due to loss of the mutant allele, which would result in the sequence of the tumor sample representing only one allele, the wild-type allele. Sample 78N contained 2 separate novel nucleotide substitutions. The tumor DNA was not amplified and this leaves only the normal DNA carrying the alterations, which was sequenced and was confirmed by SSCP. One of the 2 substitutions was Lys 35 to Arg, a respective nucleotide substitution of A to G at position 104. As discussed for the mutation at codon 34, this is a putative substrate binding site. If 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the Lys at amino acid position 35, which is a basic amino acid, is substituted with an Arg, which is also a basic amino acid, of similar size, it might not have such a large impact on the structure or function of the enzyme. The 2n d substitution was at amino acid 45 from the wild-type Thr to lie, representing a C to T alteration at nucleotide position 134. Thr is hydrophilic and lie is a hydrophobic amino acid. A hypothesis is that this double mutant may cooperate to yield a certain effect on the enzyme. The majority of this work is based on sequencing data. Although other methods were performed, there were some limitations and sequencing proved to be the most effective. SNaPshot is a powerful technique for already known mutations since one must design primers around the nucleotide substitution. SSCP is also a good technique however it proved to be effective for already identified mutations. 6.6 Gleason score analysis In this study, there are several novel nucleotide substitutions. The pathologists provided the Gleason score for each tumor sample from each specific patient. This is an important tool to assess the degree of tumor progression. The Gleason score is the sum of the Gleason grade, which is a way to measure the integrity and progression of the tumor. The lowest score is 1, where the tumor is very well differentiated, and the highest is 5 which signifies poorly differentiated cells. A Gleason score of 6 is a Gleason grade of 3+3, which is the most common grade, a 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Gleason score of 7 is a Gleason grade of 3+4 and so on, with a maximum of a Gleason score of 10. Table 6.1 shows the Gleason score for each patient with a Table 6.1: Gleason Score o f Patients with Nucleotide Substitutions. The Tissue where the mutations was found and the amino acid substitution o f the sample is provided. The patient Gleason Score is the sum o f the Gleason grade o f the tumor. Patient Tissue type Somatic Mutation Gleason Score 55 Tumor Metl lie 6 56 Tumor Ala 19 Ala 6 103 Tumor Alal9Ala 6 114 Normal Ala24Thr 6 106 Tumor Gly34Glu 6 78 Normal Lys35Arg/ Thr45Ile 6 52 Tumor Ala49Thr 7 58 Tumor Ala49Thr 6 61 Tumor Ala49Thr 6 71 Normal/Tumor Ala49Thr 9 81 Tumor Ala49Thr 9 95 Normal Ala49Thr/Ala69Ala 7 90 Normal Ala49Val 9 108 Tumor Ala52Thr 6 72 Tumor Ser60Phe 6 107 Tumor Gly66Arg 6 86 Normal/Tumor Val63Met 6 87 Tumor Val63Met 6 99 Normal Val63Met 6 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. As far as a correlation between the grade of the tumor and the individual somatic mutation, none was found. Most of the tumor samples with a substitution had a Gleason score of 6, which represented fairly well differentiated cells. The Gleason score of 6 also represented the average of all samples analyzed. However, 3 samples, all containing a substitution at codon 49, two A49T mutants and the A49V had a Gleason score of 9. A possible explanation may be that due to alterations at this amino acid the rate of tumor progression may be increased, and thus the cells may subsequently become less differentiated and thus increasing the Gleason score. However the inverse may also be possible; the mutations at codon 49 may be a result of the increased Gleason score. Thus, if there is another cause that increases the Gleason score, the resultant less differentiated tumor cells may have an effect at amino acid 49, which may aid the carcinogenesis process. Thus, codon 49 of SRD5A2 may be a potential hot spot for mutations. 6.7 Future directions This study identified several new somatic mutations that have not yet been published. For 5 of the samples analyzed, the alterations were in the DNA extracted from normal prostate tissue. The tumor samples extracted from the corresponding patients had either very weak heterogeneous mutations, none at all or the sample simply did not amplify. Thus, the possible cause is LOH of the mutant allele. LOH analysis, therefore should be done on all samples. 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In order to study the effect of these mutations on the function of the enzyme, kinetic studies are necessary. Thus, firstly it is necessary to reconstruct the mutation of interest with the help of a mutagenesis kit. This mutation would be reconstructed into expressible cDNAs and ligated into a vector that would be transfected into appropriate cells with little or no endogenous SRD5A2 expression. Thus the mutant enzyme would be expressed, and kinetic studies can be performed. The kinetic studies would yield the Vm a x of the enzyme and the Km of the substrate. If these values are altered, one can hypothesize how this mutation could lead to possible development or progression of prostate cancer due to improper function of this enzyme, which would lead to different levels of androgens in the cell, leading to tumor formation. Besides this result, studies such as these would also indicate if the particular amino acid is important in substrate or cofactor binding, which is important in characterizing the enzyme, since it has never been purified. 6.8 Conclusion 124 samples were screened for mutations in the SRD5A2 gene in somatic tissue. The analysis of DNA representing tumor and normal prostate tissue found two previously identified mutations, along with several novel nucleotide substitutions. All somatic mutations were analyzed using automated sequencing and most were confirmed with SSCP and some with SNaPshot. 12 out of 19 somatic mutations were identified in DNA extracted from prostate tumor tissue and 5 samples contained substitutions strictly in the normal DNA. The remaining 2 mutations, 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. both the normal and the tumor DNA had the previously identified mutations. Further studies in LOH are warranted to prove the reason that the mutations are only found in normal and not tumor prostate cells, and also the screening of the genomic DNA from the respective patients is necessary. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES 1. American Cancer Society. Cancer Facts & Figures, 2002. Retrieved on 9/4/02. http://www.cancer.org/downloads/STT/CancerFacts&Figures2002TM.pdf. 2. Anderson, K.M., Liao, S. 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Creator Sharpe, Patricia Sorina (author)

Core Title Somatic mutations in the SRD5A2 gene associated with prostate cancer

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

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

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