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Disruption of the regulatory communication from the stroma to the epithelium mediated by the FGF7/10-FGFR2 signaling axis in the prostate and expression of ectopic FGFR1 in prostatic epithelial cells often correlate with prostate cancer progression both in human and in experimental animals. Ectopic expression of constitutively active FGFR1 mutant (caFGFR1) at low levels in prostate epithelial cells induces low- to intermediate-grade prostatic intraepithelial neoplasia (PIN) within 6-8 months and high-grade PIN in 20-25 months. Depression of the FGFR2 signaling in the prostate also disturbs homeostasis in the prostate and induces prostate hyperplasia. To study whether PIN lesions induced by the caFGFR1 were expression-level dependent, and whether expression of the caFGFR1 and depression of the FGFR2 signaling in the prostate synergistically disturbed prostate homeostasis, we generated two new strains of ARR2PBi-caFGFR1 transgenic mice, which highly expressed caFGFR1 in prostatic epithelial cells. The mice were crossed with KDNR mice to generate ARR2PBi-caFGFR1/KDNR bigenic mice. The ARR2PBi-caFGFR1 mice developed high-grade PIN within 8 months, which was significantly faster than the mice expressing caFGFR1 at low levels. In addition, depression of the FGFR2 signaling clearly promoted perturbation of cellular homeostasis induced by the caFGFR1. The results demonstrated that the PIN development in caFGFR1 transgenic mice was caFGFR1 dosage-dependent, and indicated that the ectopic FGFR1 and the resident FGFR2 in epithelial cells had opposite impacts on intercompartmental homeostasis in the prostate. The bigenic mice provide a model with cooperative aberrations in the fibroblast growth factor signaling axis for evaluation of tumor-initiating events in prostate tumorigenesis.

The methylation status of 7 genes was examined in four cell lines, 36 samples of benign prostatic hyperplasia (BPH), 20 samples of prostatic intraepithelial neoplasia (PIN) and 109 samples of prostate cancer (PCa), using methylation-specific PCR (MSP): the pi-class glutathione S-transferase (GSTP1), retinoic acid receptor beta 2(RARbeta2), androgen receptor (AR), death-associated protein kinase (DAPK), tissue inhibitor of metalloproteinase-3 (TIMP-3), O(6)-methylguanine DNA methyltransferase (MGMT), and hypermethylated in cancer-1 (HIC-1). The frequencies of methylation in PCa were 88% for GSTP1, 78% for RARbeta2, 36% for DAPK, 15% for AR, 6% for TIMP-3, and 2% for MGMT, whereas the values were 11% for AR and DAPK, 6% for TIMP-3, 3% for GSTP1, and 0 for RARbeta2 and MGMT in BPH. Aberrant methylation of the GSTP1 and RARbeta2 genes was detected in 30% and 20% of PIN, respectively. Most samples of BPH and PCa were positive for HIC-1 methylation. Regarding accumulation of methylated cancer-related genes, there were significant correlations between PCa and BPH as well as PIN and BPH. In the present study, a high frequency of aberrant promoter methylation of the GSTP1 and RARbeta2 genes was noted in PCa. Our findings suggest that methylation of cancer-related genes may be involved in carcinogenesis of the prostate., (Copyright 2003 Wiley-Liss, Inc.)

The androgen-regulated enzyme fatty acid synthase (FAS), required for de novo lipogenesis, is overexpressed in several cancers including prostate carcinoma and has been associated with aggressive disease. FAS expression was assessed in 81 prostate carcinomas, both by immunohistochemistry in tissue microarrays and by Affymetrix Hu95Av2 oligonucleotide arrays. Both FAS mRNA and protein were significantly overexpressed in prostate carcinomas compared with the corresponding normal tissue. FAS mRNA and protein expression increased substantially from normal to prostatic intraepithelial neoplasia, to low grade, to high grade, and to androgen-independent bone metastases. A significant correlation between FAS mRNA and protein expression was found in two thirds of the cases. In 17% of the cases, FAS protein levels were high despite low mRNA levels, and these tumors exhibited a distinct molecular signature when compared with tumors that did not express FAS protein. Whereas the latter group of tumors expressed some proapoptotic genes, tumors with high FAS levels overexpressed, among other genes, its transcriptional regulator, steroid regulator binding protein, and apolipoprotein E. These data demonstrate (1) the consistent overexpression of FAS in prostate carcinoma compared with the adjacent normal tissue, (2) a strong association between FAS and prostate tumor initiation and progression, (3) the highest FAS expression occurring in androgen-independent bone metastases, (4) the transcriptional and posttranscriptional regulation of FAS in the majority and in a subset of prostate cancers, respectively, and (5) most importantly, the identification by FAS expression of prostate tumors with unique molecular signatures and potentially diverse biologic behavior.

To determine whether Akt activation was sufficient for the transformation of normal prostate epithelial cells, murine prostate restricted Akt kinase activity was generated in transgenic mice (MPAKT mice). Akt expression led to p70S6K activation, prostatic intraepithelial neoplasia (PIN), and bladder obstruction. mRNA expression profiles from MPAKT ventral prostate revealed similarities to human cancer and an angiogenic signature that included three angiogenin family members, one of which was found elevated in the plasma of men with prostate cancer. Thus, the MPAKT model may be useful in studying the role of Akt in prostate epithelial cell transformation and in the discovery of molecular markers relevant to human disease.

Carcinomas of the transition zone (TZ) constitute approximately 20% of all prostate cancers. The TZ is the site of origin of grade 1 and grade 2 cancers, the most well-differentiated of the Gleason grade tumors, as well as for benign prostatic hyperplasia (BPH). In this regard, grade 1 carcinoma has architectural features that closely mimic gland-rich BPH nodules. Although a relationship between cancers arising in this zone and BPH has been suspected, such an association remains undefined. To gain insight into the origin, development, and progression of cancers arising in the TZ, we used a highly specific rabbit monoclonal antibody (P504S) directed against alpha-methylacyl-CoA racemase (AMACR) to study the expression of the enzyme in 25 cases of evolving and fully developed carcinomas of this zone. AMACR has been proposed as a new molecular marker for prostate cancer, because the enzyme is reportedly overexpressed in high-grade dysplasias, also termed prostatic intraepithelial neoplasia, a purported precursor of prostatic carcinoma, and in all grades of prostatic carcinoma of the peripheral zone. Using P504S, P63, or antikeratin 34beta E12 antibodies, we found it possible to define areas of transition from hyperplasia to carcinoma in 6 BPH nodules. In 3 other cancer-containing BPH nodules, staining for AMCAR was observed in benign hyperplastic glands that were juxtaposed to carcinoma. Enzyme expression was also evident in 5 additional cases in which BPH was found adjacent to cancer. In contrast; AMACR was not visualized in any other BPH nodules that we studied. Thus, using the enzyme as a marker, we document for the first time that some carcinomas of the TZ arise from an AMCAR-positive transition lesion within a subset of BPH nodules. Moreover, the finding of enhanced AMACR expression in benign glands within cancer-containing nodules as well as in BPH lesions adjacent to carcinoma suggests that in some cases, up-regulation of the enzyme may precede morphological evidence of neoplastic transformation. AMACR was lightly expressed in transition lesions and grade 1 carcinomas but more strongly expressed in higher-grade TZ cancers, suggesting that enzyme expression is enhanced with progression in this zone. Because AMACR is involved in the beta oxidation of branched fatty acids and their derivatives, enhanced expression of the enzyme in evolving carcinomas in BPH nodules, as well as its up-regulation in juxtaposed morphologically benign glands and grade 1 carcinomas, suggests that increased utilization of fatty acids may play an important role in carcinoma development and progression in the TZ., (Copyright 2003 Elsevier Inc.)

Background: Telomerase, a ribonucleoprotein complex that maintains telomeric DNA, has been detected in 67-93% of prostate carcinomas by telomeric repeat-amplification protocol assay (involving polymerase chain reaction). One study used in situ hybridization in nine patients; however, to date, no immunohistochemical results have been published., Methods: From two hospitals, the authors compiled data on 62 patients who underwent prostatectomy from January 1996 to May 2001. Representative tissue sections were immunostained with a polyclonal antibody to telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase. Staining was evaluated by two observers and was correlated with grade, stage, and biochemical failure. There were 28 sections from low-grade to intermediate-grade tumors (Gleason score, 3-6), 14 sections with a Gleason score of 3 + 4 = 7, 9 sections with a Gleason score of 4 + 3 = 7, and 11 sections from high-grade tumors (Gleason score, 8-10)., Results: From low-grade to high-grade tumors, the four groups described above disclosed nuclear reactivity in 64%, 100%, 100%, and 100% of sections, respectively. Mean percentages of 5%, 15%, 40%, and 51% of nuclei were reactive in the respective groups (P < 0.0001) with intratumoral heterogeneity. The percent of reactive tumor nuclei was not correlated with pathologic stage (P = 0.32) or margin status (P = 0.35). The basal cell layer in sections of high-grade prostatic intraepithelial neoplasia (HGPIN) and benign/atrophic acini was reactive; secretory cells were reactive in 13 of 34 HGPIN foci (38%) in 1-20% of nuclei and were never reactive in benign acini. Lymphocytes and skeletal muscle were reactive. Weak, nonspecific, cytoplasmic staining was noted in benign and tumor acini., Conclusions: Like cytokeratin 34betaE12, nuclear anti-TERT reactivity is a basal cell marker in nonneoplastic prostatic acini. Anti-TERT reactivity is acquired by secretory cells in tumorigenesis, but consistent reactivity is restricted to high-grade carcinoma (Gleason primary pattern >or=4). This histologic evidence suggests that higher grade tumors have maximally activated telomerase and may be most responsive to antitelomerase therapy., (Copyright 2002 American Cancer Society.DOI 10.1002/cncr.10988)

Phospholipase A2 (PLA2) enzymes release arachidonic acid from cellular phospholipids in a variety of mammalian tissues, including prostate. Group IIa secretory PLA2 (sPLA2) can generate arachidonate from cellular phospholipids. We examined the group IIa sPLA2 expression in benign prostatic tissues, prostatic intraepithelial neoplasia (PIN), and adenocarcinoma to determine whether sPLA2 expression is altered in the carcinogenesis of human prostatic cancer. Thirty-three of 74 total cases (45%) of benign prostatic tissue showed positive immunohistochemical staining for group IIA sPLA2, whereas 63 of 69 total cases (91%) of high-grade PINs and 70 of 78 total cases (90%) of adenocarcinomas gave positive results. Four of 10 cases of low-grade PIN showed positive immunoreactivity for sPLA2. The number of cells staining for sPLA2 was significantly less in benign epithelium (4%) and low-grade PIN (4%) compared to high-grade PIN (40%) or adenocarcinoma (38%) (P < 0.001). There was no significant difference between high-grade PIN and adenocarcinoma in the number of cells staining positively for sPLA2. The intensity of sPLA2 immunoreactivity was also different among benign prostatic tissue, low-grade PIN, high-grade PIN, and prostatic adenocarcinoma specimens. The malignant cells demonstrated more intense immunohistochemical staining (moderate to strong staining in 81% and 69% cases for high-grade PIN and adenocarcinoma, respectively) than benign glands (moderate staining in 11% of cases). No strong staining was observed in benign glands or low-grade PIN. Our data are consistent with the contention that group IIA sPLA2 expression is elevated in neoplastic prostatic tissue and support the hypothesis that dysregulation of sPLA2 may play a role in prostatic carcinogenesis.

Environmental factors, especially the diet, play a prominent role in the epidemic of prostate cancer (PCA), in the United States. Many candidate dietary components have been proposed to influence human prostatic carcinogenesis, including fat, calories, fruits and vegetables, anti-oxidants, and various micronutrients, but the specific roles dietary agents play in promoting or preventing PCA remain controversial. We have collected evidence to suggest that GSTP1, the gene encoding the pi-class glutathione S-transferase (GST), may serve a "caretaker" function for prostatic cells. Although GSTP1 can be detected in normal prostatic epithelium, in almost all PCA cases, PCA cells fail to express GSTP1 polypeptides, and lack of GSTP1 expression most often appears to be the result of somatic "CpG island" DNA methylation changes. Loss of GSTP1 function also appears to be characteristic of prostatic epithelial neoplasia (PIN) lesions, thought to represent PCA precursors. We have recently learned that a new candidate early PCA precursor lesion, proliferative inflammatory atrophy (PIA), characterized by proliferating prostatic cells juxtaposed to inflammatory cells, contains epithelial cells that express high levels of GSTP1. These findings have formed the basis for a new model of prostatic carcinogenesis, in which prostatic cells in PIA lesions, subjected to a barrage of inflammatory oxidants, induce GSTP1 expression as a defense against oxidative genome damage. When cells with defective GSTP1 genes appear amongst the PIA cells, such cells become vulnerable to oxidants and electrophiles that inflict genome damage that tends to promote neoplastic transformation to PIN and PCA cells. Subsequently, PIN and PCA cells with defective GSTPI genes remain vulnerable to similar stresses tending to promote malignant progression. This new model for prostatic carcinogenesis has implications for the design of new prostate cancer prevention strategies. Rational prevention approaches might include: (i) restoration of GSTPI expression via treatment with inhibitors of CpG methylation, (ii) compensation for inadequate GSTPI activity via treatment with inducers of general GST activity, and (iii) abrogation of genome-damaging stresses via avoidance of exogenous carcinogens and/or reduction of endogenous carcinogenic (particularly oxidant) stresses.

Objective: To evaluate whether it is possible to detect telomerase activity in cells exfoliated from prostate biopsies immediately before fixation., Methods: A total of 115 transrectal biopsies of prostate tissue from 49 patients were touch-imprinted on an RNase-free microscope slide and then fixed. Touch imprints were immediately frozen and used to extract telomerase. Telomerase activity was determined by a telomeric repeat amplification protocol (TRAP) using a PCR-ELISA method. Inflammation and epithelial cells in each biopsy were quantitated by image cytometry., Results: A total of 90/115 extracts had a proteic content suitable for analysis. Telomerase activity was detected in 18/26 (70%) carcinomas, 2/9 (22%) low-grade prostatic intraepithelial neoplasia (PIN) lesions, and 1/3 (33%) high-grade PIN lesions. In 4 of 7 patients with telomerase-positive tumors, telomerase activity was also found in a distant site devoid of morphologically detectable cancer cells. Telomerase activity was detected in touch imprints from fragments with less than 1 mm(2) of epithelial tissue, and was not associated with the extent of inflammation., Conclusions: From the technical stand point, the touch-imprint method may provide a useful adjunct for telomerase detection in prostate biopsies. With this procedure the bioptic fragment is left intact for histological examination. Diagnostically, the presence of telomerase activity in sites distant from the original tumor might suggest the presence of tumor cells that are morphologically undetectable.

Cyclooxygenase-2 (COX-2) is the inducible isozyme of COX, a key enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids. COX-2 is highly expressed in a number of human cancers and cancer cell lines, including prostate cancer. We studied the immunohistochemical expression of COX-2 in the human prostate gland. The enzyme is strongly expressed in smooth muscle cells of both the normal and cancerous prostate. Its expression in noncancerous epithelial cells is limited to the basal cell layer. In prostatic inflammation, luminal epithelial cells surrounded by lymphocytes are induced to express the enzyme. COX-2 is expressed in the epithelial cells of high-grade prostatic intraepithelial neoplasia and cancer. We have demonstrated that treatment of human prostate-cancer cell lines with a selective COX-2 inhibitor induces apoptosis both in vitro and in vivo. The in vivo results also indicate that the COX-2 inhibitor decreases tumor microvessel density and angiogenesis. COX-2 inhibitors can prevent the hypoxic upregulation of a potent angiogenic factor, vascular endothelial growth factor. These results indicate that COX-2 inhibitors may, therefore, serve as effective chemopreventive and therapeutic agents in cancer of the prostate.

Objective: To elucidate the incidence of inducible nitric oxide synthase (iNOS) expression in benign prostatic hyperplasia (BPH), low- and high-grade prostatic intraepithelial neoplasia (PIN) and prostatic carcinoma lesions, and to explore the role of iNOS in prostate tumorigenesis., Materials and Methods: Immunoreactivity for iNOS was examined in 20 samples each of BPH, high-grade PIN, low-grade PIN and prostatic carcinoma., Results: Positive iNOS immunostaining was detected in all samples from all patients; iNOS was detected in both basal epithelial cells and secretory cells of the glandular epithelium. High-grade PIN and prostatic carcinoma samples had more intense iNOS immunostaining than low-grade PIN and BPH samples. In all samples, smooth muscle cells showed weak or moderate iNOS immunoreactivity and endothelial cells showed moderate immunostaining., Conclusions: Nitric oxide generated by iNOS may be involved in prostate tumorigenesis and further studies with immunohistochemical and molecular biology are needed to determine the exact role of iNOS in the pathogenesis of prostatic carcinoma.

Telomerase is a ribonucleoprotein that stabilizes chromosomes by maintaining their telomeric ends. Although telomerase is normally expressed in reproductive tissues, it is virtually absent in most normal somatic tissues. During carcinogenesis, cells activate telomerase to protect chromosomal ends from the telomere erosion that occurs with replication. Prevention of telomere loss by activation of telomerase allows for the cellular immortalization that is a characteristic of cancer cells. Recent studies have shown that genetic instability arising from critical telomere shortening is a mechanism through which cancer cells attain multiple genetic aberrations that characterize a malignant clone. Thus, the timing of telomerase activation during carcinogenesis is likely to play an important role in modulating the genetic instability that determines the malignant phenotype. Earlier activation of telomerase should minimize genetic aberrations in neoplastic cells and lead to less aggressive tumors, or may prevent carcinogenesis. In this article, we discuss recent data on telomerase expression in prostate cancer, propose a model that relates the dynamics of telomerase activation to the evolution of different prostatic malignancies, and discuss the potential application of telomerase activation as a strategy for the prevention of prostate cancer.

Cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase-2 (NOS-2) each have an important role in angiogenesis. The expression of these genes was investigated in human prostate cancer by immunohistochemistry, the expression of COX-1 and COX-2 being confirmed by mRNA analysis. Prostate cancer specimens from 12 patients were compared to control prostates from 13 patients operated on for bladder carcinoma. The intensity of COX-2 and NOS-2 immunostaining was significantly stronger in prostate cancer cells than in the non-malignant glandular epithelium of the control prostates. COX-2 and NOS-2 were clearly also expressed in the lesions of prostatic intraepithelial neoplasia (PIN) in control prostates. COX-2 was detected in the muscle fibres of the hyperplastic stroma of some control prostates. No significant difference was detected in COX-1 expression between control and cancer prostates. These results indicate that the expression of COX-2 and NOS-2 is elevated in prostatic adenocarcinoma and in PIN.

Background: STAT3 phosphorylation is associated with the neoplastic state in many types of cancer, including prostate cancer. We investigated the role of IL-6 signaling and phosphorylation of STAT3 in 2 rat prostatic epithelial lines. NRP-152 and NRP-154 cells were derived from the same rat prostate, yet the NRP-152 cells are not tumorigenic while the NRP-154 cells are tumorigenic. These lines are believed to represent 2 of the stages in the development of prostate cancer, hyperplasia and neoplasia. Differences in signaling pathways should play a role in the 2 phenotypes, hyperplastic and neoplastic., Methods: We looked at the phosphorylation state of STAT3 by intracellular flow cytometry, using phospho-specific antibodies to STAT3. We used the same method to examine IL-6 production by the cell lines. We also measured apoptosis by binding of fluorescent annexin V to the cells., Results: Although both cells lines made IL-6 constitutively, phosphorylated-STAT3 was present in untreated NRP-154 cells, but not in NRP-152 cells. Treatment with dexamethasone inhibited the IL-6 production of NRP-152 cells, but enhanced that of NRP-154 cells. Treatment with the JAK2 inhibitor AG490 induced apoptosis in NRP-152, but not NRP-154 cells., Conclusions: We conclude from these experiments that STAT3 activity plays a role in the phenotype of NRP-154 cell, but not NRP-152 cells. The significance of alternative IL-6 signaling pathways in the different phenotypes of the 2 cell lines is discussed.

Arachidonic acid (AA) metabolites are implicated in the oncogenesis of several tumors, including prostate cancer. 15-Lipoxygenase-2 (15-LOX-2) is a novel AA-metabolizing enzyme with a limited tissue distribution, which includes prostate, lung, skin, and cornea. Previous studies have shown that 15-LOX-2 is present in benign prostate secretory cells and reduced in prostate adenocarcinoma and that production of the 15-LOX-2 metabolite 15S-hydroxyeicosatetraenoic acid is reduced in malignant compared with benign prostate. The objective of this study was to determine the frequency with which 15-LOX-2 immunostaining is reduced in prostate carcinoma and to correlate reduced expression with tumor differentiation (grade) and other pathologic parameters in radical prostatectomy specimens. Paraffin immunoperoxidase with a polyclonal antibody specific for 15-LOX-2 was performed on tumors and benign portions from 70 cases, and the percentage of tumor immunostaining for 15-LOX-2 was assessed. Whereas uniform 15-LOX-2 immunostaining was observed in secretory cells of benign glands, it was markedly reduced or absent in most adenocarcinomas: 23 of 70 tumors showed completely absent 15-LOX-2 immunostaining, and 45 of 70 cases showed negative immunostaining in more than 50% of the tumor. The extent of reduced 15-LOX-2 immunostaining correlated with tumor differentiation, with retained expression particularly in Gleason score 5 tumors versus a significant reduction of 15-LOX-2 in higher-grade tumors (mean +/- SD tumor 15-LOX-2 positive: Gleason score 5 = 67%+/-30%, Gleason score 6 = 16%+/-30%, Gleason score 7 = 23%+/-28%, Gleason score > or =8 = 41%+/-46%). In 16 cases with multifocal tumors or different foci of the same tumor with different grades, the higher-grade foci had significantly reduced 15-LOX-2 expression compared with the lower-grade foci. In peripheral zone tumors without complete loss of 15-LOX-2 expression, there was a significant inverse relationship between 15-LOX-2 immunostaining and tumor volume. There was not a significant correlation between 15-LOX-2 immunostaining and serum PSA or pathologic stage. In a subset of 27 cases, 15-LOX-2 expression in high-grade prostatic intraepithelial neoplasia (HGPIN) glands was significantly reduced compared with benign glands. These data show that in contrast to the uniform expression of 15-LOX-2 in differentiated secretory cells of benign prostate, reduced 15-LOX-2 is a common alteration in prostate carcinoma, and this correlates with tumor cell differentiation. That reduced expression is seen in HGPIN suggests that this may be an early alteration in carcinoma development.

Background: Oxidative stress results in damage to cellular structures and has been linked to many diseases, including cancer. The authors sought to determine whether the expression of three major antioxidant enzymes, copper-zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), and catalase, was altered in human prostate carcinoma and its likely precursor, high grade prostatic intraepithelial neoplasia (PIN). The level of reactive oxygen species damage was evaluated by measuring the expression of the DNA adduct 8-hydroxydeoxyguanosine., Methods: The authors evaluated the tissue expression of the antioxidant enzymes in prostate carcinoma by immunohistochemistry, immunogold electron microscopy, and enzymatic assay. The polymerase chain reaction was used to amplify and screen tissue specimens for the genes of SOD1, SOD2, and extracellular SOD (SOD3). Matched paraffin embedded tissue sections were evaluated by RNA in situ hybridization for expression of SOD1 and immunohistochemically for the DNA adduct 8-hydroxydeoxyguanosine., Results: All prostatic tissues, including cancer, displayed immunoreactivity for the three antioxidant enzymes in epithelial cells, with no staining of the stroma, inflammatory cells, or endothelial cells. The number of immunoreactive cells was greater in benign epithelium than in PIN and cancer for each enzyme. The mean percentage and intensity of immunoreactive cells was greatest for SOD2, intermediate for SOD1, and lower for catalase. Staining in cancer was heterogeneous. Immunogold ultrasound studies revealed strong mitochondrial labeling for SOD2, which was greater in benign epithelium than in cancer; SOD1 labeling was invariably weaker, with nuclear labeling in benign epithelium and cytoplasmic labeling in cancer cells. There was no difference in enzyme activity for the three antioxidant enzymes between benign epithelium and cancer. No mutations were found in the 5 exons of SOD1, 5 exons of SOD2, and 3 exons of SOD3, except for 3 of 20 cases with polymorphisms for exon 3 of SOD1. Intense nuclear immunoreactivity for 8-hydroxydeoxyguanosine was present in fewer than 3% of epithelial cells, with no apparent differences among benign epithelium, PIN, and cancer., Conclusions: SOD1, SOD2, and catalase had lower expression in PIN and prostate carcinoma than in benign epithelium. The number of immunoreactive cells in PIN was similar to cancer, indicating that these are closely related. Enzyme activities were variable, with no difference between benign epithelial cells and cancer, although this lack of change in enzyme activity could have been due to the presence of contaminating benign cells within the cancer specimens. The results of reactive oxygen species damage were found only in the epithelium and not in the stroma. Expression of the DNA adduct 8-hydroxydeoxyguanosine was present in fewer than 3% of cells, with no apparent differences among benign epithelium, PIN, and cancer. These findings suggest that oxidative stress is an early event in carcinogenesis., (Copyright 2000 American Cancer Society.)

Background/aims: Patients with high grade prostatic intraepithelial neoplasia of the transition zone appear to be at increased risk of developing prostatic carcinoma, although not to the same degree as patients with high grade prostatic intraepithelial neoplasia of the peripheral/central zone. Previous investigations have shown loss of expression of pi-class glutathione S-transferase (GST-pi; an enzyme that protects against electrophilic carcinogens) in prostatic carcinoma and in high grade prostatic intraepithelial neoplasia. The aim of this study was to compare the expression of GST-pi in high grade prostatic intraepithelial neoplasia of the transition zone with that in high grade prostatic intraepithelial neoplasia of the peripheral/central zone (that is, non-transition zone)., Methods: Immunostaining with the anti-GST-pi antibody was performed on 20 high grade prostatic intraepithelial neoplasia samples of the transition zone, either isolated or associated with prostatic carcinoma (groups 1 and 2, respectively; 10 cases each) and on 20 high grade prostatic intraepithelial neoplasia samples of the non-transition zone, either isolated or associated with prostatic carcinoma (groups 3 and 4, respectively; 10 cases each). This study also included six samples of high grade prostatic intraepithelial neoplasia simultaneously present in the transition and non-transition zones and not associated with prostatic carcinoma (group 5). The presence of immunostaining, staining intensity, and the distribution of immunostaining were evaluated in the high grade prostatic intraepithelial neoplasia lesions and in the normal tissue and cancer areas., Results: The GST-pi antibody stained the cytoplasm of the cells lining the ducts and acini of normal prostate tissue. Staining was stronger and more diffuse in the basal cell layer than in the luminal (or secretory) cell layer. Immunohistochemical staining with anti-GST-pi antibodies failed to detect the enzyme in all prostatic carcinoma foci but one. Two patterns were detected in high grade prostatic intraepithelial neoplasia. One was represented by GST-pi staining similar to that of the normal tissue (pattern A). The other deviated from it and was characterised by absence of GST-pi expression in the secretory cells and abundant expression in scattered basal cells (pattern B). Pattern A staining was seen more frequently in the transition than in the non-transition zone. Pattern B staining was seen mainly in high grade prostatic intraepithelial neoplasia of non-transition zone associated with cancer., Conclusions: The differential expression of GST-pi in the transition and non-transition zones indicates the existence of two populations with the morphological appearance of high grade prostatic intraepithelial neoplasia that might have different associations with carcinoma.

Background: The DNA topoisomerase II-alpha (topo II-alpha)-targeting drug etoposide was recently shown to be an active agent in the combined chemotherapy of hormone-insensitive prostatic carcinoma. Aside from being the molecular target of etoposide, topo II-alpha is also a cell proliferation marker. Much experimental data indicate that cells sensitive to topo II-targeting chemotherapeutic drugs are rapidly proliferating and show elevated topo II expression. There is little information concerning topo II expression in lesions of the prostate., Methods: Paraffin blocks from cases of invasive prostatic carcinoma, prostatic intraepithelial neoplasia, and prostatic nodular hyperplasia were retrieved from the surgical pathology files at the University of Utah Health Sciences Center. Using a new immunohistochemical stain, specific for the alpha isoform of DNA topo II, enzyme expression was evaluated in 54 prostatic adenocarcinomas, 22 lesions of high-grade prostatic intraepithelial neoplasia (PIN), and 10 cases of benign prostatic nodular hyperplasia. Results were semiquantitated by determining for each case a topo II-alpha index, which represented the percent of positively staining cells., Results: The average topo II-alpha index for well-differentiated prostatic adenocarcinomas (Gleason scores 2-4) was 1.5 +/- 0.9; for moderately differentiated tumors (Gleason scores 5-7), 3.1 +/- 2.4; and for poorly differentiated tumors (Gleason scores 8-10), 6.7 +/- 5.5. The average topo II-alpha index for all invasive prostatic adenocarcinomas was 4.0 (range, 0-19.0). Benign prostatic nodular hyperplasia had the lowest average topo II-alpha index, of 0.54 (range, 0.2-1.0). The average topo II-alpha index of 2.3 (range, 0-8.6) for high-grade prostatic intraepithelial neoplasia was intermediate between the invasive tumors and benign prostate., Conclusions: Topo II-alpha expression in carcinoma of the prostate correlates with Gleason score. The carcinomas with the highest expression of enzyme are more poorly differentiated and have the highest Gleason scores. Prostatic nodular hyperplasia shows little expression of topo II-alpha. Prostatic intraepithelial neoplasia has an average topo II-alpha index intermediate between nodular hyperplasia and carcinoma., (Copyright 2000 Wiley-Liss, Inc.)

Objective: To detect telomerase activity inprostate needle-biopsy samples and its role in diagnosis and prognosis of prostate cancer., Methods: 20 tissue samples of prostate cancer tissues and 16 samples of adjacent tissues were obtained by needle-biopsy guided by transrectal B ultrasonography. 16 samples of benign prostatic hyperplasia tissues were obtained by prostatectomy and were confirmed pathologically. Telomerase activity was revealed by modified PCR-based telomeric repeat amplication protocol (TRAP)-silver staining assay., Results: Telomerase activity was revealed in 18 cases of prostate cancer. In tissues adjacent to prostate cancer, telomerase activity was found in 7 of 11 cases of prostatic intraepithelial neoplasia (PIN), and in 2 of 5 cases of BPH. None of tissue samples from patients with BPH showed telomerase activity., Conclusions: Telomerase activity in prostate needle-biopsy samples might be an useful marker for detecting prostate cancer and biological malignancy.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a known activator of latent MMP-2 (pro-MMP-2), and increased MMP-2 expression has been associated with tumor aggressiveness in prostate cancer. However, expression of MT1-MMP in human prostate tissue has not been described. We investigated the expression and immunolocalization of MT1-MMP and MMP-2 in the epithelial components of benign prostate epithelium, high-grade prostatic intraepithelial neoplasia (HGPIN), and prostate cancer. Tissue sections from the peripheral zone of 50 prostates (radical prostatectomy specimens) were chosen based on their containing benign glands, HGPIN, and prostate cancer glands. All 50 sections were immunostained for MT1-MMP and MMP-2 and were evaluated for staining pattern, uniformity, and intensity. Western blotting and gelatin zymography were done to confirm expression of MT1-MMP and activity of MMP-2, respectively. Comparisons were made between benign epithelium, HGPIN, and cancer. In benign glands, basal cells (BCs) uniformly stained intensely for MT1-MMP, whereas secretory cells (SCs) were rarely positive (P < 0.0001). Conversely in HGPIN, SCs showed consistent cytoplasmic staining (P < 0.0001). In cancer cells, staining was heterogeneous and varied from no staining to very intense staining in select glands. MMP-2 in normal tissue stained both BCs and the apical region of SCs, whereas in HGPIN, staining was observed in the SC in a predominantly cytoplasmic pattern. Similar to MT1-MMP, staining in cancer tissue for MMP-2 was heterogeneous; however, there was a significant association between the pattern of MMP-2 and MT1-MMP staining within the epithelial components of the cancer glands in individual specimens (P < 0.001). Finally, MMP-2 and MT1-MMP were confirmed to be expressed in the prostate tissues by gelatin zymography and Western blotting. In conclusion, we found that consistent changes in localization and intracellular distribution of MMP-2 and MT1-MMP were associated with the transition from benign prostate epithelium to HGPIN, suggesting that regulation of these enzymes is altered during the earliest stages of prostate cancer.

Background: In prostatic neoplasia, the time from tumour initiation and progression to invasive carcinoma often begins in men in the fourth and fifth decades of life and extends across decades. Until recently, the events initiating the process and the developments concomitant with the evolution towards invasive disease were largely unknown., Methods: Quantitative and analytical methods are applied to provide insights into certain individual molecular events and their effects on the complex multiple feedback system of cellular metabolism and regulation in prostate neoplasia., Results: Prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) are associated with or possibly preceded by changes in the chromatin of secretory cell nuclei. The changes are detectable with a Bayesian belief network and quantifiable by computer image analysis in prostatic tissue that still appears histologically normal. In addition, normal looking prostate epithelium shows some molecular changes similar to those present in the associated preneoplastic and neoplastic lesions. Such changes are also occasionally present in normal prostate glands without PIN and cancer., Conclusions: The subtle morphological and molecular changes of normal looking epithelium might be seen as the onset of the development of prostatic neoplasia.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)-based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non-radioactive in situ hybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin-embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situ hybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate., (Copyright 1999 John Wiley & Sons, Ltd.)

Purpose: To investigate the relationship of telomerase activity, telomere length, and DNA ploidy in high grade prostatic intraepithelial neoplasia (PIN)., Materials and Methods: Tissue samples were carefully microdissected to obtain adenocarcinoma or PIN-containing tissue free of cancer. Telomerase activity was measured using the PCR-based telomeric repeat amplification protocol (TRAP). Telomere length was estimated from Southern blots of telomere restriction fragments (TRFs). DNA ploidy of PIN and carcinoma was determined by image analysis of adjacent Feulgen stained tissue sections., Results: Telomerase activity was found in 4 of 25 samples (16%) of high grade PIN. All telomerase positive PIN foci had a diploid DNA content. Although 5 of 25 samples (25%) of high grade PIN foci analyzed were DNA aneuploid, none of these demonstrated telomerase activity. Telomerase positive foci of prostate carcinoma (69% of all cancer foci analyzed) displayed heterogeneity in TRF length, with a mean TRF length two kilobase pairs shorter than that of telomerase negative specimens., Conclusions: Telomerase activity is present in a low percentage of high-grade PIN foci, which are diploid by DNA content measurements.

Prostate intraepithelial neoplasia (PIN) is a purported prostate cancer precursor lesion and a candidate biomarker for efficacy assessment in prostate cancer chemoprevention trials. Loss of expression of the pi-class glutathione S-transferase enzyme GSTP1, which is associated with the hypermethylation of deoxycytidine residues in the 5'-regulatory CG island region of the GSTP1 gene, is a near-universal finding in human prostate cancer. GSTP1 expression was assessed by immunohistochemistry in 60 high-grade PIN samples adjacent to and distant from prostate adenocarcinoma. Whereas abundant enzyme polypeptide expression was evident in all normal prostatic tissues, all samples of high-grade PIN and adenocarcinoma were completely devoid of GSTP1. DNA from 10 high-grade PIN lesions was analyzed for GSTP1 CG island methylation changes using a PCR technique targeting a polymorphic (ATAAA)n repeat sequence in the promoter region of the GSTP1 gene. Somatic GSTP1 CG island methylation changes were detected in DNA from 7 of the 10 PIN lesions. Allele discrimination was possible for 5 of the 10 DNA samples: 2 of the 5 samples exhibited DNA methylation changes at both alleles; whereas 3 samples displayed no DNA methylation changes at either allele. GSTP1 CG island methylation changes were present in each of the five homozygous samples. Hypermethylation of the 5'-regulatory region of the GSTP1 gene may serve as an important molecular genetic biomarker for both prostate cancer and PIN. The finding of frequent GSTP1 methylation changes in PIN and prostate cancer supports a role for PIN lesions as a prostate cancer precursor and may provide insight to the molecular pathogenesis of prostate cancer.

Mitogen-activated protein (MAP) kinases are key elements of the signalling systems needed to transduce different extracellular messages into cellular responses. At least three parallel MAP kinase pathways have been identified: one, stimulated by serum and growth factors to activate extracellular signal-regulated protein kinases (ERKs) by dual tyrosine and threonine phosphorylation, triggers cell proliferation or differentiation; the other two, induced by a variety of cellular stresses to activate c-jun N-terminal kinases (JNKs) and reactivating kinase (p38/RK), result in growth arrest and induction of apoptosis. Mitogen-activated protein kinase phosphatases (MKPs) inactivate MAP kinases through dephosphorylation and, thus, can modulate the MAP kinase pathways. Expression of JNK-1, ERK-1, p38/RK and MKP-1 proteins was investigated by immunohistochemistry and expression of MKP-1 mRNA by in situ hybridisation in 50 cases of high-grade prostatic intraepithelial neoplasia (PIN), thought to represent the precursor of prostate cancer. The frequency of apoptotic cells was also determined in these cases. Overexpression of the three MAP kinases and MKP-1 mRNA was found in all cases of high-grade PIN compared with normal prostate. Immunoreactivity for MKP-1 protein was found to be as intense as in normal glands in 30% and weaker in 56% of the PIN cases. Fourteen per cent of PIN cases did not stain with MKP-1 antibody. The proportion of apoptosis was significantly higher (P < 0.008) in PIN lesions that did not express MKP-1 protein than in those that did. These results are consistent with our previous demonstration of preferential inhibition of the apoptosis-related kinases by MKP-1 and further support the contention that MKP-1, even in PIN, may shift the balance existing between cell proliferation and death. When expressed, it may inhibiting those pathways that lead to apoptosis.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. Telomerase activation has been seen in many immortal cell lines and cancers. Telomerase activity was analyzed in prostate carcinoma; in coexistent prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), atrophy and normal tissue; and in benign prostate glands. Telomerase activity was detected in 80 of 87 (92%) prostate cancers. Forty-one matched samples (from a total of 32 cases) were available for comparative analysis. The presence of telomerase activity in adjacent PIN, BPH, and normal tissue was correlated with telomerase activity in the malignant epithelium. In these adjacent tissues, telomerase activity was found in 11 of 15 (73%) PINs, 13 of 26 (50%) BPHs, and 1 of 6 (16%) atrophy and 4 of 11 (36%) normal tissues. In contrast to the BPH tissue from cancer-bearing glands, all 16 BPH specimens from patients only diagnosed with BPH were telomerase activity negative. In cancer samples, there was no correlation between telomerase activity and Gleason grade or preoperation prostate-specific antigen level. Our data indicate that telomerase activity is present in most prostate cancers. The high rate of telomerase activity in the benign-appearing areas of these glands may be attributed either to the presence of occult cancer cells or to early molecular alterations of cancer that were histologically inapparent.

Basal cells lining prostatic acini have unique morphologic and immunophenotypic characteristics. The role of these uncommitted cells in the genesis of cancer in the prostate is intriguing. Here, we discuss immunophenotypic and molecular features of basal cells of prostatic acini, and compare them with those of cytologically transformed cells of prostatic intraepithelial neoplasia (PIN) in both human tissues and animal models. Following a summary of the current concepts of molecular events in prostatic cancer, we will discuss the role of the ras-dependent pathway in early prostate carcinogenesis with a special emphasis on mitogen-activated protein kinase phosphatase (MKP-1). We will also outline the importance of techniques such as differential display-polymerase chain reaction (ddPCR) followed by in situ hybridization in the characterization of genes which may have a critical role in early prostate carcinogenesis. Finally, we will underscore the role of animal models in understanding the early events leading to neoplastic transformation of prostate cells.

Preneoplastic lesions, early neoplastic lesions and carcinomas in situ have been demonstrated to be of great value for many purposes in many organ systems. Their recognition can be useful in epidemiological studies and can facilitate the selection of patients for therapeutic interventions. They can be used as "surrogate endpoint biomarkers" in studies aimed at the chemoprevention of cancers. In the lung, colon and various other organs, such markers are well recognized to be associated with the development of cancer in man. In the livers and colons of experimental animals, there has been detailed characterization of "enzyme-altered foci" (EAF) as "putative preneoplastic markers." The words "surrogate" and "putative" are important; the biological potential of these lesions needs to be elucidated in much greater detail. The quantification of early lesions that are associated with and sometimes precursors of neoplasia is of particular value because they are much more numerous, in most organ systems, than the carcinomas that develop in the same organs. The most abundant of these lesions show minimal or no morphological alterations. For example, EAF and aberrant crypts are more numerous than polyps in the colons of patients and experimental animals with cancer or precancerous conditions that affect the colon. Currently, there are few well documented putative or surrogate markers that are highly associated with the development of prostatic carcinoma in man. The best documented among these is prostatic intraepithelial neoplasia. We have recently reported the identification of EAF in the human prostate. While they share many phenotypic alterations with prostatic intraepithelial neoplasia, much remains to be accomplished if their biological fate is to be understood.