Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide.

Among the many somatic genome alterations present in cancer cells, changes in DNA methylation may represent reversible "epigenetic" lesions, rather than irreversible "genetic" alterations. Cancer cell DNA is typically characterized by increases in the methylation of CpG dinucleotides clustered into CpG islands, near the transcriptional regulatory regions of critical genes, and by an overall reduction in CpG dinucleotide methylation. The transcriptional "silencing" of gene expression associated with such CpG island DNA hypermethylation presents an attractive therapeutic target: restoration of "silenced" gene expression may be possible via therapeutic reversal of CpG island hypermethylation. 5-Aza-cytidine (5-aza-C) and 5-aza-deoxycytidine (5-aza-dC), nucleoside analogue inhibitors of DNA methyltransferases, have been widely used in attempts to reverse abnormal DNA hypermethylation in cancer cells and restore "silenced" gene expression. However, clinical utility of the nucleoside analogue DNA methyltransferase inhibitors has been limited somewhat by myelosuppression and other side effects. Many of these side effects are characteristic of nucleoside analogues that are not DNA methyltransferase inhibitors, offering the possibility that nonnucleoside analogue DNA methyltransferase inhibitors might not possess such side effects. Human prostate cancer (PCA) cells characteristically contain hypermethylated CpG island sequences encompassing the transcriptional regulatory region of GSTP1, the gene encoding the pi-class glutathione S-transferase (GSTP1), and fail to express GSTP1 as a consequence of transcriptional "silencing." Inactivation of GSTP1 by CpG island hypermethylation, the most common somatic genome alteration yet reported for human PCAs, occurs early during human prostatic carcinogenesis and results in a loss of GSTP1 "caretaker" function, leaving prostate cells with inadequate defenses against oxidant and electrophile carcinogens. We report here that the drug procainamide, a nonnucleoside inhibitor of DNA methyltransferases, reversed GSTP1 CpG island hypermethylation and restored GSTP1 expression in LNCaP human PCA cells propagated in vitro or in vivo as xenograft tumors in athymic nude mice.

Cyclooxygenase-2 is up-regulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma.

Cyclooxygenase-2 (COX-2) is the inducible isoform of the rate-limiting enzymes that convert arachidonic acid to proinflammatory prostaglandins as well as a primary target for nonsteroidal anti-inflammatory drugs. Accumulating evidence suggests that up-regulation of COX-2 is associated with carcinogenesis in multiple organ systems including the large bowel, lung, breast, and prostate. In this report, we examine the expression of COX-2 protein and mRNA in prostate tissue containing various lesions and in prostate cancer cell lines. In the cell lines, LNCaP, DU145, PC-3, and TSU, COX-2 protein expression was undetectable under basal conditions but could be induced transiently by phorbol ester treatment in PC-3 and TSU cells, but not in DU145 and LNCaP cells. Immunohistochemical analysis of 144 human prostate cancer cases suggested that, in contrast to several previous reports, there was no consistent overexpression of COX-2 in established prostate cancer or high-grade prostatic intraepithelial neoplasia, as compared with adjacent normal prostate tissue. Positive staining was seen only in scattered cells (<1%) in both tumor and normal tissue regions but was much more consistently observed in areas of proliferative inflammatory atrophy, lesions that have been implicated in prostatic carcinogenesis. Staining was also seen at times in macrophages. Western blotting and quantitative RT-PCR analyses confirmed these patterns of expression. These results suggest that if nonsteroidal anti-inflammatory drugs are indeed chemopreventive and/or chemotherapeutic for prostate cancer, their effects are likely to be mediated by modulating COX-2 activity in non-PCa cells (either inflammatory cells or atrophic epithelial cells) or by affecting a COX-2-independent pathway.

GSTP1 CpG island hypermethylation is responsible for the absence of GSTP1 expression in human prostate cancer cells.

GSTP1 CpG island hypermethylation is the most common somatic genome alteration described for human prostate cancer (PCA); lack of GSTP1 expression is characteristic of human PCA cells in vivo. We report here that loss of GSTP1 function may have been selected during the pathogenesis of human PCA. Using a variety of techniques to detect GSTP1 CpG island DNA hypermethylation in PCA DNA, we found only hypermethylated GSTP1 alleles in each PCA cell in all but two PCA cases studied. In these two cases, CpG island hypermethylation was present at only one of two GSTP1 alleles in PCA DNA. In one of the cases, DNA hypermethylation at one GSTP1 allele and deletion of the other GSTP1 allele were evident. In the other case, an unmethylated GSTP1 allele was detected, accompanied by abundant GSTP1 expression. GSTP1 CpG island DNA hypermethylation was responsible for lack of GSTP1 expression by LNCaP PCA cells: treatment of the cells with 5-azacytidine (5-aza-C), an inhibitor of DNA methyltransferases, reversed the GSTP1 promoter DNA hypermethylation, activated GSTP1 transcription, and restored GSTP1 expression. GSTP1 promoter activity, assessed via transfection of GSTP1 promoter-CAT reporter constructs in LNCaP cells, was inhibited by SssI-catalyzed CpG dinucleotide methylation. Remarkably, although selection for loss of GSTP1 function may be inferred for human PCA, GSTP1 did not act like a tumor suppressor gene, as LNCaP cells expressing GSTP1, either after 5-aza-C treatment or as a consequence of transfection with GSTP1 cDNA, grew well in vitro and in vivo. Perhaps, GSTP1 inactivation may render prostatic cells susceptible to additional genome alterations, caused by electrophilic or oxidant carcinogens, that provide a selective growth advantage.

p63 protein expression is rare in prostate adenocarcinoma: implications for cancer diagnosis and carcinogenesis.

OBJECTIVES: To examine the expression of the p63 protein in normal, preneoplastic, and neoplastic human prostatic tissue. The p63 gene, a member of the p53 gene family, is expressed in the basal epithelial cells of multiple organs. Irregularities in p63 expression have been associated with epithelial carcinogenesis. METHODS: We performed immunohistochemistry with an anti-p63 antibody on specimens from radical prostatectomies, prostate needle biopsies, and metastatic prostate adenocarcinoma. We analyzed p63 expression in regions of normal prostate, benign prostatic hyperplasia, proliferative inflammatory atrophy (PIA), high-grade intraepithelial neoplasia, and adenocarcinoma. RESULTS: Basal epithelial cells in normal, benign prostatic hyperplasia, and high-grade intraepithelial neoplasia tissue stained intensely for the p63 polypeptide, but the vast majority of adenocarcinoma specimens from 233 patients-66 (94%) of 70 radical prostatectomies, 132 (89%) of 148 prostate needle biopsies, and 14 (93%) of 15 metastases-did not. In tumors in which the adenocarcinoma cells were positive, the staining intensity was weak and occurred in less than 1% of the cells. Tumors that stained positive for p63 were more likely to be high grade than those that did not (P <0.0001). Basal cells in PIA expressed p63, but these cells were sparsely distributed relative to the basal cells in the normal glands. Luminal cells in PIA were, in general, negative for p63. CONCLUSIONS: In contrast to normal and preneoplastic prostatic tissue, the vast majority of prostate adenocarcinomas do not express p63. Therefore, p63 immunohistochemistry represents a potential novel adjuvant method for facilitating the pathologic diagnosis of prostate cancer in prostate needle biopsies. The selective expression of p63 in normal basal cells, coupled with the finding that p63 null mice fail to develop prostates, provides strong evidence that the basal cells represent prostatic epithelial stem cells. In addition, these findings suggest that p63 may protect prostatic epithelial cells against neoplastic transformation and support the hypothesis that intermediately differentiated cells in the luminal epithelium of PIA are the targets of neoplastic transformation in the prostate.

GSTA1 expression in normal, preneoplastic, and neoplastic human prostate tissue.

BACKGROUND: Glutathione S-transferases (GSTs), inducible enzymes that catalyze the detoxification of reactive electrophiles and oxidants, protect against neoplastic transformation. Prostatic adenocarcinoma and high-grade prostatic intraepithelial neoplasia (HGPIN) fail to express GSTP1, a major class of GST. This failure of expression is associated with methlyation of the GSTP1 promoter, a somatic alteration proposed to be a critical step in prostatic carcinogenesis. However, simple atrophy and post-atrophic hyperplasia-proliferative lesions associated with chronic inflammation, which we have termed "proliferative inflammatory atrophy" (PIA)-express elevated levels of GSTP1. We postulated that this increase in GSTP1 expression in PIA occurs in response to increased oxidative stress. We examined the expression of another major class of GST, GSTA1, in the human prostate. METHODS: We performed immunohistochemistry against GSTA1 on formalin-fixed radical prostatectomies (n = 45). A stereological grid point counting method was used to estimate the percent of cells staining positive for GSTA1 in normal prostate, PIA, HGPIN, and adenocarcinoma. RESULTS: In contrast to GSTP1, normal peripheral zone epithelium was virtually devoid of GSTA1. Strikingly, though, epithelial cells in PIA demonstrated strong staining for GSTA1 (median of percent of cells staining positive = 44) as compared to those in normal peripheral zone (median = 3.0, P <.00001), HGPIN (median = 2.9, P <.00001), and adenocarcinoma (median = 3.8, P <.00001). Variations in GSTA1 were also detected between normal anatomic zones: the central zone showed an increase in the percentage of cells staining positive (median = 20.9) as compared to the transition (median = 0.47, P <.0002) and the peripheral (P <.0001) zones. CONCLUSIONS: Expression of GSTA1 is increased in PIA, supporting the concept that cells within these lesions are subject to localized increases in oxidative stress. Low levels of GSTA1 and GSTP1 in HGPIN and adenocarcinoma suggest a broad lack of detoxification activity in these cells, which may be associated with carcinogenesis in the prostate.

In situ hybridization-theory and practice.

In situ hybridization is a technique to determine and localize target nucleic acids in morphologically preserved tissue sections. Recent advances in methods have greatly increased the sensitivity of the technique, and it is currently possible to detect extremely few copies of any given target sequence with nonisotopic methods. In this teaching review, we integrate theoretical background, technical considerations, and guidelines for usage for this important component of molecular diagnosis.

Immunohistochemical detection of chlamydial antigens in association with cystitis.

To investigate the etiologic role of Chlamydia trachomatis in cystitis, the authors used the immunoperoxidase technique with a monoclonal antibody against Chlamydia and examined paraffin sections from 36 cases of histologically proven cystitis. The average patients' age was 60 (range, 2-85) years. Biopsies were taken for follow-up of treated bladder carcinoma (19), hematuria (8), and other nonneoplastic conditions (9). Chlamydial antigens were detected by immunohistochemistry in 12 (33%) of these 36 cases. Staining for Chlamydia occurred in the upper layers of the transitional epithelium and involved long stretches of epithelium. Underlying inflammation was usually chronic but did not have specific distinguishing features. Eight of the Chlamydia-positive biopsies were taken for follow-up of treated carcinoma, two were for hematuria, one for neurogenic bladder, and one for evaluation of sterile pyuria. Eleven (92%) of these 12 positive cases had a history of recent urologic instrumentation, in contrast to only 11 (46%) of 24 negative cases (P less than 0.02). There was no significant difference in the age or sex distribution between the two groups. The authors conclude that Chlamydia trachomatis can ascend the urethra and infect the bladder urothelium. Urologic instrumentation enhances the ability of Chlamydia to reach the bladder. Chlamydia trachomatis may play an etiologic role in cystitis.

Rapid immunoperoxidase demonstration of Rickettsia rickettsii in fixed cutaneous specimens from patients with Rocky Mountain spotted fever.

Immunofluorescence (IF) of skin biopsies for detection of Rickettsia rickettsii (RR) has proven useful as a rapid test for confirmation of Rocky Mountain spotted fever (RMSF). However, IF lacks sensitivity, requires special equipment and training, and is difficult to interpret. The authors have developed an indirect avidin-biotin immunoperoxidase (IP) technique to detect RR in fixed and frozen tissue sections. The technique was evaluated on fixed cutaneous specimens from patients dying of RMSF and compared to specimens from control patients dying of an acute febrile illness with skin manifestations and vasculitis. IP correctly identified RR in 9 of 12 cases with probable identification in 2 additional cases. Of 11 controls, 10 were negative and one was uninterpretable. RR was easily visualized within cytoplasm and nuclei of endothelial cells in association with perivascular lymphocytic infiltrates and less frequently with vasculitis or non-inflamed vasculature. IP is rapid, amplifies small quantities of antigen, gives excellent histologic detail as compared with IF, and is easily adapted for use in hospitals with immunoperoxidase capabilities.

Comparison of immunoperoxidase and DNA in situ hybridization techniques in the diagnosis of cytomegalovirus colitis.

Cytomegalovirus (CMV) infection of the colon occurs in immunocompromised patients and in patients with ulcerative colitis. In the former, it can cause serious bleeding or colonic perforation and in the latter, it may precipitate a fulminant phase of illness. The authors compared the immunoperoxidase technique with a monoclonal antibody (Mab) against CMV early antigen and DNA in situ hybridization using a biotinylated probe in the identification of virus-infected cells in colon samples from patients with known CMV colitis and with fulminant ulcerative colitis without histologic evidence of infection. The Mab stained the nuclei of infected cells without recognizable viral inclusions particularly in cases showing few characteristic viral inclusions. In well-developed CMV infections, with many inclusions, immunoperoxidase staining was less prominent. The biotinylated DNA probe recognized cytopathic cells with prominent inclusions but was only rarely positive in smaller cells. In specimens with few inclusions, there was no staining with in situ hybridization. No positivity was observed by either technique in cases of fulminant ulcerative colitis with no morphologic evidence of CMV infection. The authors conclude that immunoperoxidase using monoclonal anti-CMV early antigen adds useful information in the evaluation of early or focal cases of CMV colitis. DNA in situ hybridization is usually positive in cytopathic cells and is less useful for diagnosis. CMV could not be demonstrated in cases of fulminant ulcerative colitis when viral inclusions were not observed in routine preparations.