Genetic changes and DNA damage responses in the prostate

The integrity of genomic DNA is challenged by genotoxic stress originating during normal cellular metabolism or by external insults. Cellular responses to DNA damage involve elegant checkpoint cascades enforcing cell cycle arrest, damage repair, apoptosis or cellular senescence. The loss or alterations of genes involved in the damage response pathways have been reported in many cancer susceptibility syndromes and in sporadic tumors. Furthermore, this surveillance pathway is activated during early tumourigenesis presumably due to uncontrolled replicative cycles and has been recognized as one of the main barriers against the development of cancer. This review discusses the relevance of prostatic epithelial cells in prostate tumourigenesis and highlights common molecular changes associated with prostate cancer. Furthermore, DNA damage responses of primary cultures of human prostatic epithelial cells and fresh human prostate tissues are discussed providing evidence for alterations in crucial DNA damage checkpoint molecules. New insights connecting prostate tumourigenesis to alterations and defects in the pathways maintaining genomic integrity will be discussed.     

Transgenic mouse with human mutant p53 expression in the prostate epithelium

BACKGROUND: Apoptosis is disrupted in prostate tumor cells, conferring a survival advantage. p53 is a nuclear protein believed to regulate cancer progression, in part by inducing apoptosis. To test this possibility in future studies, the objective of the present study was to generate a transgenic mouse model expressing mutant p53 in the prostate (PR). METHODS: Transgene incorporation was tested using Southern analysis. Expression of mutant p53 protein was examined using immunofluorescence microscopy. Apoptosis in the PR was evaluated using the Tunnel method. RESULTS: A construct, consisting of the rat probasin promoter and a mutant human p53 fragment, was prepared and used to generate transgenic mice. rPB-mutant p53 transgene incorporation, as well as nuclear accumulation of mutant human p53 protein, was demonstrated. Prostatic intraepithelial neoplasia (PIN) III and IV were found in PR of 52-week old transgenic mice, whereas no pathological changes were found in the other organs examined. PR ability to undergo apoptosis following castration was reduced in rPB-mutant p53 mice as compared to non transgenic littermates. CONCLUSIONS: Transgenic rPB-mutant p53 mice accumulate mutant p53 protein in PR, resulting in neoplastic lesions and reduced apoptotic potential in the PR. Breeding rPB-mutant p53 mice with mice expressing an oncogene in their PR will be useful in examining interactions of multiple genes that result in progression of slow growing prostate tumors expressing oncogenes alone to metastatic cancer.     

Detection of human papillomavirus DNA and p53 gene mutations in human prostate cancer

The relationship between integration with human papillomavirus (HPV) and p53 gene mutations in tissues of prostate cancer was examined. Tissue samples analyzed were obtained by total prostatectomy (29 stage B cancer cases) and from autopsy (22 endocrine therapy-resistant metastatic disease cases). HPV DNA was detected in 8 of 51 (16%, 5 in stage B and 3 in autopsy cases) by polymerase chain reaction (PCR) using consensus primers on L1 region. Genotypes of HPV were entirely type 16. Structural abnormalities of p53 gene were detected in 7 of the 22 autopsy cases (32%) by PCR-single-strand conformation polymorphism analysis and direct sequencing. No p53 gene mutation was found in stage B cancer cases. Analysis of mutation spectra revealed clear differences between Japanese and Westerners. There was a significant difference in the mutation frequency between stage B and autopsy cases (P < 0.01, Fisher's exact test). One case showed both integration of HPV and p53 gene mutation in different cancer foci. However, the other cases revealed an inverse correlation between the presence of HPV DNA and p53 gene mutations. These data show that p53 genetic alteration is correlated with the progression of prostate cancer, in contrast to the integration of HPV that may occur in a relatively early stage. In conclusion, this study may indicate that either p53 gene mutation or the presence of HPV's oncogenic protein E6 is involved in the development of prostate cancer. © 1996 Wiley-Liss, Inc.     

Combined DNA repair defects in testicular metastasis from prostate cancer sensitize to immune checkpoint blockade

Testicular metastasis in prostate cancer is uncommon and may be a culprit of widespread disease which portends a worse prognosis. Little is known about the molecular biology of metastases to the testicles and whether there is any role for targeted therapeutics. Here we report a case of prostate cancer recurring with testicular and lung metastases. Targeted sequencing of the patient''s left testicular tumor after orchiectomy disclosed inactivating mutations in the CDK12 and ARID1A genes, and MSH2 and MSH6 loss resulting in high microsatellite instability and high tumor mutational burden. The patient experienced a complete radiographic and prostate-specific antigen response at 12 weeks of PD-1 immune checkpoint blockade with pembrolizumab and continues uneventfully on treatment. Molecular characterization of this rare phenotypic subtype of prostate cancer in larger studies may help deliver precision therapies with the potential to improve outcomes.     

Two-dimensional electrophoresis of proteins from different lobes of the rat prostate and seminal vesicle

Two-dimensional ISO-DALT electrophoresis of cytosols and secretions from various lobes of the rat prostate gland and seminal vesicle reveals major differences in intracellular and secretory protein patterns. This study confirms a previous study utilizing one-dimensional electrophoresis. The dorsal lobe and coagulating gland appear to secrete predominantly relatively basic proteins (molecular mass, MM, 70,000–200,000 daltons, pI > 7) in contrast to the ventral lobe, which secretes proteins of a more acidic character (pI 4–5). The majority of proteins in the latter case appear to represent subunits of the major secretory protein of the rat ventral prostate, prostatein. At least one secretory protein is relatively specific for the lateral lobe (MM 16,000 daltons, pI 4.5). The vesicular secretion also contains relatively greater quantities of basic proteins than acidic but with varying molecular mass (12,000–100,000 daltons). This study extends the search for specific protein markers for different lobes of the rat accessory sex glands and provides additional biochemical data which can be exploited in the future to isolate selected secretory proteins on a large scale for antibody production.     

Comparison of the zones of the human prostate with the seminal vesicle: morphology, immunohistochemistry, and cell kinetics

BACKGROUND: The prostate contains three glandular zones (central, peripheral, transition) with widely differing susceptibilities to cancer and benign prostatic hyperplasia (BPH). Most of the prostate is derived from urogenital sinus, but the central zone may be derived from Wolffian duct, in common with the seminal vesicles (SV). The peripheral zone is the most frequent site of cancer and the transition zone is the almost exclusive site of BPH. METHOD: We compared the histology and immunohistochemistry of the SV with those of the prostate zones in order to identify differences associated with susceptibility to disease or different embryological origins. Sections from the prostates of nine organ donors (aged 15-36) were stained for tissue-specific markers, antigens previously shown to stain differentially between the zones and markers of cell proliferation and cell death. RESULTS: Neuroendocrine cells were absent from the SV and significantly fewer neuroendocrine cells were seen in the central zone compared to the peripheral zone. Most of the SV epithelium stained for lactoferrin, compared to approximately one-third of central zone and only 2% of peripheral zone epithelial cells. The proliferative index of the central zone was approximately 50% lower and the incidence of apoptotic cells approximately half that of the peripheral and transition zones. CONCLUSIONS: The central zone has features in common with both the SV and the other zones of the prostate. The higher incidence of proliferative diseases in the transition and peripheral zones may be associated with the higher rate of cell turnover observed in these zones.     

Sperm DNA damage and seminal antioxidant activity in subfertile men

Supraphysiological ROS levels can lead to apoptosis, lipid peroxidation, and DNA and protein damage. This pilot study aimed to investigate the sperm oxidative damage in subfertile men, to describe the relationship between the antioxidant system and ROS. Sixty-four semen samples were categorised according to the evaluated routine parameters (WHO, WHO laboratory manual for the examination and processing of human semen, 2010). Results were cross-referenced with the DNA damage [Comet (n = 53) and TUNEL (n = 49) assays], antioxidant enzyme activity [SOD (n = 51), CAT (n = 48) and GST (n = 48)], and content of total thiols (n = 36), lipid hydroperoxides (n = 35) and MDA (n = 31). Compared to pathospermic samples, normozoospermic presented 40%–45% fewer spermatozoa with fragmented DNA, 19% fewer hydroperoxides, and slightly higher total thiols and MDA levels. Asthenozoospermic/asthenoteratozoospermic samples had the lowest GST activity. SOD and CAT showed a similar trend. Our results evidenced significant positive correlations between DNA damage and immotile spermatozoa; SOD and CAT, GST and total thiols; CAT and GST; total thiols and sperm concentration; and MDA levels and head/midpiece abnormalities and hydroperoxides. This work contributes to the existing body of knowledge by showing that the oxidative status correlates with the classic sperm analysis parameters. Oxidative stress and DNA damage evaluation might be a valuable diagnostic and prognostic tool in cases of idiopathic male subfertility.     

精子DNA损伤、核蛋白组型转换与顶体酶活性及精液参数的相关性分析

目的:探讨精子DNA损伤、精子核蛋白组型转换与顶体酶活性及精液参数的相关性。方法:收集535例精液标本,采用精子染色质扩散(sperm chromatin dispersion,SCD)检测精子DNA损伤,并与精子核蛋白组型转换、顶体酶活性和WHO手册(第4版)精液参数进行相关性分析。结果:精子DNA损伤与精子核蛋白组型转换、顶体酶活性、精子浓度及前向运动精子这些指标之间比较均具有显著性差异(P<0.01);精子DNA损伤与年龄、核蛋白组型转换、精子浓度和D级精子比例之间呈显著正相关(P<0.01或P<0.05),而与顶体酶活性呈显著负相关(P<0.001),多元逐步回归分析显示年龄、精子浓度、D级精子比例、核蛋白组型转换及顶体酶活性是5个独立的相关变量。精子核蛋白组型转换、顶体酶活性、精子密度和前向运动精子这4个指标的异常率在精子DNA异常组(DFI≥30%)中均显著的高于正常组(DFI<30%)。结论:精子DNA损伤与精子核蛋白组型转换、顶体酶活性及WHO(第4版)精液各参数之间存在密切的联系,可能是评价精子质量的另一项重要的指标。     

Apoptosis and DNA damage in human spermatozoa

DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis, resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.     

Tumor suppressor gene P53 mutations in human prostate cancer

The genetic background underlying the growth and development of human prostatic cancer is not yet clear. Here we searched for possible mutations in the entire coding region of tumor suppressor gene p53 in primary human prostatic carcinomas, using polymerase chain reaction and single-strand conformational polymorphism analysis of RNA. We found p53 gene mutations in 4 of 21 cases (19%). DNA sequencing of the polymerase chain reaction products revealed missense point mutations that resulted in amino acid changes in exon 5 or 3 in three cases and single base deletions in exon 7 in two cases. One case contained both a missense point mutation and a single base deletion. Three of these four cases were pathologically diagnosed as poorly differentiated adenocarcinomas, and three of the four cases were clinically localized to stage C or D. None of seven noncancerous prostate tissues nor three well-differentiated adenocarcinoma tissues showed any mutations. The present results suggest that p53 gene mutation is involved in the late progression steps of human prostate carcinogenesis. © 1995 Wiley-Liss, Inc.     

Cell cycle checkpoint efficiency and cellular response to paclitaxel in prostate cancer cells

BACKGROUND: Defects in the cell cycle machinery of prostate cancer cells might impair the efficiency of cell cycle checkpoints and affect the cell response to chemotherapeutic drugs. We examined the relationship between the status of microtubule damage-activated checkpoints and the response of hormone-refractory prostate cancer cells to paclitaxel. METHODS: The two cell lines DU145 and PC3 harboring defects at proteins involved in the regulation of checkpoints activated by microtubule damage were examined for cell sensitivity, apoptotic response, and efficiency of checkpoints in response to paclitaxel. RESULTS: In spite of a comparable sensitivity to the antiproliferative effects of paclitaxel, DU145 and PC3 cells exhibited different cell cycle control at checkpoints activated by microtubule damage. A transient mitotic arrest was induced by the taxane in both cell lines. However, PC3 cells underwent a rapid mitotic slippage and displayed a defective postmitotic checkpoint as evidenced by the appearance of polyploid cells. In this cell line, paclitaxel-induced cell death was a slow and delayed event, occurring also after S-phase re-entry. The mitotic checkpoint appeared to be more stringent in DU145 cells compared to PC3 cells. Moreover, despite the expression of mutated proteins involved in the prevention of DNA endoreduplication (p53, pRb, and p16(INK4A)), these cells did not progress into the cell cycle but efficiently underwent apoptosis by 24 hr. Such a response of DU145 cells was associated with phosphorylation of the p21(WAF1) protein. CONCLUSIONS: These observations evidence that activation of checkpoints following microtubule damage in prostate cancer may be regulated through complex mechanisms possibly involving p21(WAF1). Our findings support that the status of cell cycle checkpoints might affect the modality of cell death. However, the relevance of the mode of cell death for the sensitivity to taxanes remains to be determined.