Down-regulation of the human kallikrein gene 5 (KLK5) in prostate cancer tissues

BACKGROUND: Kallikreins are a subgroup of serine proteases with diverse physiological functions. Many kallikrein genes are differentially expressed in various malignancies and prostate specific antigen (PSA; encoded by the KLK3 gene) is the best tumor marker for prostate cancer. Human glandular kallikrein (hK2; encoded by the KLK2 gene) is an emerging tumor marker for prostate cancer. KLK5 is a newly discovered human kallikrein gene which shares a high degree of homology and is located adjacent to KLK2 and KLK3 genes on chromosome 19q13.4. Like KLK2 and KLK3, the KLK5 gene is regulated by steroid hormones in the BT-474 breast cancer cell line. We have previously shown that KLK5 is differentially expressed in ovarian and breast cancer. METHODS: We compared the expression of KLK5 in 29 pairs of histologically confirmed normal and prostate cancer tissues by quantitative RT-PCR using the LightCycler technology. RESULTS: KLK5 expression was significantly lower in cancer tissues compared to their normal counterparts. Lowest levels of expression were found in T3 stage tumors compared with T1 and T2. Also, a significant negative correlation was found between Gleason score and KLK5 expression. CONCLUSIONS: KLK5 should be further studied as a potential new prognostic marker in prostate cancer, whose expression is negatively correlated with cancer aggressiveness.     

Identification of single nucleotide polymorphisms in the human kallikrein 10 (KLK10) gene and their association with prostate, breast, testicular, and ovarian cancers

BACKGROUND: The KLK10 gene (also known as the normal epithelial cell-specific 1 gene) is a member of the expanded human kallikrein gene family. Recently, it has been reported that KLK10 is a tumor suppressor gene and that its expression is downregulated in various forms of cancer and cancer cell lines. KLK10 is also upregulated in ovarian cancer. We thus hypothesized that the KLK10 gene may be a target for mutations in various cancers. METHODS: We sequenced the five coding exons of the KLK10 gene using genomic DNA from various tumors, normal tissues, and blood, by PCR amplification and automated sequencing. RESULTS: In none of the tumor-derived DNAs, we identified somatic mutations that could inactivate this gene. However, we identified a prevalent germline single nucleotide variation at codon 50 (exon 3) of this gene [GCC (alanine) to TCC (serine)]. The GCC genotype was less prevalent in prostatic cancer patients in comparison to control subjects (P = 0.027) but no differences were seen with testicular, ovarian, and breast cancer. We also identified four genetic variations in exon 4, at codons106 [GGC (glycine) to GGA (glycine)], codon 112 [ACG (threonine) to ACC (threonine)], codon 141 [CTA (leucine) to CTG (leucine)], and at codon 149 [CCG (proline) to CTG (leucine)]. None of these variations was significantly different between normal subjects and cancer groups. CONCLUSIONS: We found no evidence for somatic mutations of the KLK10 gene in cancers of the prostate, breast, ovary, and testis. The single nucleotide variation at codon 50 appears to be associated with prostate cancer risk.     

Human kallikrein-related peptidase 12: antibody generation and immunohistochemical localization in prostatic tissues

BACKGROUND: Human tissue kallikrein-related peptidases (genes, KLKs; proteins, KLKs) are a subgroup of serine proteases present in a variety of tissues and biological fluids. A number of human tissue KLKs are established or candidate serologic biomarkers for prostate cancer. Human kallikrein-related peptidase 12 (KLK12, KLK12), recently identified in our laboratory, is a novel member of the KLK gene family. Here, we report generation of antibodies against the full-length recombinant KLK12 (classical form) and the immunohistological localization of this KLK in normal and malignant prostate tissues. METHODS: The mature form of KLK12 cDNA was amplified using PCR and cloned into a plasmid vector for protein production in E. coli. Following identification by mass spectroscopy, recombinant KLK12 was purified and used as immunogen in rabbits. Anti- KLK12 antibody was used for immunostaining of paraffin-embedded sections of human prostate tissue. Immunoexpression of KLK12 in benign and malignant prostate tissue was evaluated using a prostate cancer tissue array. RESULTS: Anti-KLK12 antibody showed a predominantly apical and membranous staining of the luminal cells of the normal prostate in contrast with the predominantly diffuse cytoplasmic staining observed in both prostatic intra-epithelial neoplasia and adenocarcinomas. This was occasionally associated with an intense granular supranuclear staining. More than 95% of the prostate cancers on the tissue microarray were KLK12 positive. CONCLUSION: Higher levels of KLK12 in malignant prostatic glands, and the shift in subcellular localization of KLK12 in prostate cancer observed in this study point to the potential role of this kallikrein during prostate carcinogenesis.     

Detection of human papillomavirus (HPV) DNA in human prostatic tissues by polymerase chain reaction (PCR)

Human papillomavirus (HPV) infections are strongly linked to the pathogenesis of uterine cervical neoplasms, and have been implicated in other cancers of the female genital tract. In contrast, the association of HPV with the cancers of the male urogenital tract is less evident, except in anal and penile cancers. However, recent studies reporting the prevalence of HPV infections in human prostate cancers (60–100% HPV 16 positive vs. no infection of HPV) have raised controversies regarding the prevalence of HPV in benign and neoplastic human prostate. We investigated the prevalence of HPV infections in prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinomas in 23 surgically resected prostates. Polymerase chain reaction (PCR) was used to amplify HPV 6b/11, 16 and 18 specific DNA sequences, using type specific HPV primers selected from the transforming gene E6-E7. The areas of PIN and cancer in 6 p.m H&E stained tissue sections were identified, and respective areas of PIN and cancer were isolated from the adjacent serial sections and used for DNA amplification and HPV detection (Fig. 1). Our results demonstrated the presence of HPV 16 in three carcinomas (13%), using type specific primers in PCR amplified samples. We were not able to demonstrate the presence of other HPV types (HPV 6b/11 or HPV 18) in any of the samples using specific primers. Two of these prostates showed relatively strong positive signals by dot blot analysis, when hybridized with a 32P-labeled HPV 16 type specific oligonucleotide probe. One more sample showed weak positivity, when hybridized with a 32P-labeled HPV 16 type specific oligonucleotide probe. Subsequently, we have confirmed these results by Southern hybridization of the samples transferred to nylon membrane after agarose gel electrophoresis and detected by HPV 16 type specific oligonucleotide probe, using chemiluminescent assay. We, therefore, conclude that HPV infections of the prostate in general are not as common as has been previously claimed by other investigators. © 1993 Wiley-Liss, Inc.     

Prostate specific antigen and human glandular kallikrein 2 in early detection of prostate cancer

PURPOSE: Several tumor markers have recently been applied for prostate cancer screening. We analyze the effectiveness of prostate specific antigen (PSA), age specific PSA, PSA velocity, volume adjusted PSA densities, change in PSA level following antibacterial therapy, free-to-total PSA ratio, alpha1-antichymotrypsin bound PSA, alpha2-macroglobulin bound PSA, alpha1-protease inhibitor bound PSA and human glandular kallikrein 2 in detecting prostate cancer. MATERIALS AND METHODS: We conducted a review of the literature between September 2000 and February 2001. A total of 7,250 abstracts and articles published during the previous 12 years were retrieved from MEDLINE using the key words PSA and human glandular kallikrein 2. Of these reports 135 are included in this review. RESULTS: We analyzed and systematized data from studies regarding the effectiveness of PSA and human glandular kallikrein 2 and their derivatives in the detection of prostate cancer. CONCLUSIONS: Improvement in the specificity and sensitivity of PSA is imperative. Free-to-total PSA ratio, transition zone PSA density and change in PSA level increase the specificity of PSA to some extent. Protocols investigating the effectiveness of different combinations of these 3 measurements seem necessary for improving the effectiveness of prostate cancer screening among men within the diagnostic "gray zone." PSA velocity, age adjusted PSA levels and PSA density might be used in limited cases. alpha1-Antichymotrypsin, alpha2-macroglobulin and alpha1-protease inhibitor bound PSA, and human glandular kallikrein 2 are promising experimental methods.     

Pretreatment plasma levels of testosterone and sex hormone binding globulin binding capacity in relation to clinical staging and survival in prostatic cancer patients

Pretreatment plasma concentrations of total testosterone (T), sex hormone binding globulin binding capacity (SHBG). T/SHBG ratio, and free testosterone (fT) were measured in 123 patients with prostatic cancer categorized into groups according to the UICC classification. The patients were randomized to orchiectomy or estrogen therapy and the mean follow-up time was 48 months. The mean plasma levels of T were higher in patients without metastases and with intracapsular cancer, but the differences were not statistically significant. The calculated ratio of T/SHBG was noticed to be significantly higher (p less than 0.05) in the M0 category. The prognostic significance of pretreatment T and, more impressively, T/SHBG ratio and fT was confirmed. Low pretreatment values indicated poorer prognosis. This study supports the view that there are differences in the pretreatment T and fT levels in prostatic cancer patients in relation to the stage of tumor and that these hormone assays could be used as prognostic factors.     

Increased spermine oxidase expression in human prostate cancer and prostatic intraepithelial neoplasia tissues

BACKGROUND: Inflammation has been strongly implicated in prostate carcinogenesis, but the precise molecular mechanisms linking inflammation and carcinogenic DNA damage are not known. Induction of the polyamine catabolic enzyme, spermine oxidase (SMO) has been linked to increased reactive oxygen species (ROS) and DNA damage in human gastric and lung epithelial cells and suggest direct mechanistic links between inflammation, SMO activity, ROS production, and epithelial carcinogenesis that are likely relevant in prostate cancer. METHODS: Tissue microarrays consisting of matched normal and diseased specimens from patients diagnosed with prostate cancer, prostatic intraepithelial neoplasia (PIN), or proliferative inflammatory atrophy (PIA), as well as unaffected individuals, were stained for SMO expression and analyzed using image analysis techniques and TMAJ software tools. RESULTS: Average SMO staining was significantly higher in prostate cancer and PIN tissues compared to patient-matched benign tissues. Benign tissues from prostate cancer, PIN, and PIA patients also exhibited significantly higher mean SMO expression versus tissues from prostate disease-free patients. CONCLUSIONS: Tissues from patients diagnosed with prostate cancer and PIN exhibit, on average, locally increased SMO expression in regions of prostatic disease and higher overall SMO expression in prostatic epithelial cells compared to healthy individuals. Further studies are warranted to directly examine the role of SMO-produced ROS in prostate carcinogenesis.     

Differential expression of the estrogen receptor beta (ERbeta) in human prostate tissue,premalignant changes,and in primary,metastatic, and recurrent prostatic adenocarcinoma

BACKGROUND: Estrogen signaling mediated by the estrogen receptor beta (ERbeta) has potential implications in normal and abnormal prostate growth. Few studies have addressed this issue in human prostate tissue leaving conflicting results on the immunolocalization of the ERbeta in benign and neoplastic lesions. METHODS: Using a new monoclonal antibody, the current study reports on the differential expression of the ERbeta in tissue sections from 132 patients with prostate cancer. RESULTS: The prostatic epithelium expressed the ERbeta extensively in secretory luminal cell types and at lower levels in basal cells. Atrophic changes of the peripheral zone (PZ) were more immunoreactive than hyperplastic lesions of the transition zone (TZ). When compared with glandular tissue of the PZ, high-grade prostatic intraepithelial neoplasia (HGPIN) revealed decreased levels of the ERbeta in 30 of 47 cases and was unreactive in six lesions. In informative cases with suitable internal controls, all primary tumors (n = 60), lymph node (n = 7), and bone metastases (n = 5) expressed the ERbeta at variable degree. No correlation was found between the ERbeta status, the primary Gleason grade (P = 0.254), and the pathological stage (P = 0.157). Recurrent adenocarcinoma revealed markedly decreased levels in 15 of 40 cases and was ERbeta negative in five recurrent lesions. CONCLUSIONS: The secretory epithelium is a major target of ERbeta-mediated estrogen signaling in the human prostate. Its downregulation in HGPIN is consistent with chemopreventive effects that the ERbeta may exert on the prostatic epithelium. The continuous expression of the receptor protein at significant levels in untreated primary and metastatic adenocarcinoma indicates that these tumors can use estrogens through an ERbeta-mediated pathway. The partial loss of the ERbeta in recurrent tumors after androgen-deprivation may reflect the androgen-dependence of ERbeta gene expression in human prostate cancer.     

Tissue polypeptide antigen (TPA) as a prognostic aid in human prostatic carcinoma

Serum levels of tissue polypeptide antigen (TPA) and prostatic acid phosphatase (PAP) in serum, the presence or absence of skeletal metastases, tumor grade, patient age, and erythrocyte sedimentation rate (ESR) were determined in 50 patients with prostatic adenocarcinoma before onset of any therapy. Crude survival rates were estimated for a 5-year period after the time of diagnosis. The prognostic value was estimated by means of the log rank test and multivariate life table analysis. The TPA, PAP, tumor stage, and ESR all appeared to be useful as prognostic markers. Tumor grade and patient age were not significantly related to crude survival. The TPA proved to be the most reliable prognostic marker in single test estimates as well as in a multivariate life table analysis (p less than 0.01).     

Differential effects of growth factor antagonists on neoplastic and normal prostatic cells

Growth factors such as epidermal growth factor and fibroblast growth factor have been suggested to be involved as paracrine or autocrine mediators of androgen action in normal and malignant prostatic cells. Suramin and protamine are potent in vitro growth factor antagonists. To evaluate the effect of growth factor antagonists on prostatic growth, suramin and protamine were administered to castrated rats simultaneously receiving exogenous testosterone replacement in an attempt to block androgen-dependent restoration of the normal rat ventral prostate. Using this prostatic restoration model, there was no statistical difference in the prostate wet weight or total DNA content attained between rats given testosterone alone and those given testosterone in combination with either suramin or protamine. In intact rats, suramin administration caused an 80% reduction in serum testosterone; concomitantly, these rats had a 40% reduction in mean prostate weight. This decrease in size could be blocked with androgen supplementation. To examine the effects of growth factor antagonists on neoplastic prostatic cell growth, rats bearing the androgen-independent AT-2 subline of the Dunning R-3327 tumor were treated with either suramin or protamine. The same dosing regimen found to be ineffective in blocking the restoration of the involuted prostate of castrated rats resulted in a significant reduction in the growth rate of AT-2 tumors. These results suggest that the growth factor antagonists suramin and protamine given in vivo have a differential ability to slow the growth of malignant vs. normal prostatic cells.     

Allelic loss of the retinoblastoma gene in primary human prostatic adenocarcinomas

Inactivation of the retinoblastoma (Rb) gene has been implicated in the genesis and progression of a number of tumor types, including prostatic adenocarcinomas. We have analyzed a series of 46 surgically-resected human prostatic adenocarcinomas for allelic loss of the Rb gene with PCR amplification of a highly polymorphic region of the gene. 41 of 46 tumors (89%) were informative and 11 of these (27%) had lost one Rb allele. The relative frequency of this occurrence suggests that inactivation of the retinoblastoma gene may be an important event in prostate carcinogenesis.