Within the human prostate epithelium four cell populations can be discriminated based on their expression of keratins (K). Basal cells express high levels ofThe phenotypic identification and cell biological characterization of epithelial stem cells has gained increasing interest in the study of human malignancies. Within rapidly proliferating tissues such as bone marrow, intestinal tract, and squamous epithelia, cell turn-over is governed by stem cells.

Understanding the molecular etiology of prostate cancer (CaP) progression is paramount for broadening current diagnostic and therapeutic modalities. Current interest in the role of wnt pathway signaling in prostate tumorigenesis was generated with the ®nding of b-catenin mutation and corresponding nuclear localization in primary lesions. The recent ®nding of b-catenin-induced enhancement of androgen receptor (AR) function potentially ties b-catenin to key regulatory steps of prostate cell growth, di erentiation, and transformation. By immunohistological analysis of metastatic tumors, we detected nuclear bcatenin in 20% of lethal CaP cases, suggesting a more common role for b-catenin in advanced disease than would be predicted by its mutation rate. Interestingly, bcatenin nuclear localization was found to occur concomitantly with androgen-induced regrowth of normal rat prostate. These in vivo observations likely implicate b-catenin involvement in both normal and neoplastic prostate physiology, thus prompting our interest in further characterizing modes of b-catenin signaling in prostate cells. Extending our previous ®ndings, we demonstrate that transient b-catenin over-expression stimulates T cell factor (TCF) signaling in most CaP cell lines. Further, this activity is not subject to crossregulation by phosphoinositide-3-kinase (PI3-K)/Akt signaling, a stimulatory pathway often upregulated in CaP upon PTEN inactivation. Consistent with a previous report, we observed that transient b-catenin overexpression enhances AR-mediated transcription o two natural target gene promoters. However, we were unable to recapitulate b-catenin-induced stimulation of ectopically expressed AR in AR-negative cells, suggesting that other AR-associated factors are required for this activity. Although LNCaP cells are capable of this mode of AR co-stimulation, stable expression of mutant b-catenin did not alter their proliferative response to androgen. In total, our characterization of b-catenin signaling in CaP reveals the complex nature of its activity in prostate tissue, indicating that b-catenin potentially contributes to multiple stimulatory inputs required for disease progression.

GSTP1CpG 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,

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Fatty liver is an important cause of morbidity in humans and is linked to impaired liver regeneration after liver injury, but the mechanisms for impaired liver regeneration remain unknown. In the normal liver, the interleukin (IL)-6/STAT-3 pathway is thought to play a central role in regeneration because this pathway is disrupted in IL-6-deficient mice that exhibit impaired liver regeneration after 70% partial hepatectomy (PH). To determine whether inhibition of STAT-3 is involved in fatty liver-related mitoinhibition, regenerative induction of STAT-3 was compared in normal mice and leptin-deficient ob/ob mice that have fatty livers and markedly impaired liver regeneration after PH. In both groups, two waves of STAT-3 activation were observed, the first in endothelia and the second in hepatocytes. Before PH, a significantly higher percentage of ob/ob endothelial and hepatocyte nuclei expressed phosphorylated (activated) STAT-3. After PH, phospho-STAT-3 accumulated in liver nuclei of lean mice and this response was markedly exaggerated in ob/ob mice. Moreover, a striking inverse correlation was noted between hepatocyte nuclear accumulation of phospho-STAT-3 and DNA synthesis (as assessed by bromodeoxyuridine labeling), as well as cyclin D1 mRNA induction and protein expression. In contrast, STAT-3 activation was positively correlated with p21 protein expression in both groups of mice. Because these results link exaggerated STAT-3 activation with impaired hepatocyte proliferation, STAT-3 inhibition cannot be a growth-arrest mechanism in ob/ob fatty livers. Rather, hyperinduction of this factor may promote mitoinhibition by up-regulating mechanisms that impede cell cycle progression.

A method was developed to assess human telomere lengths at the individual cell level in tissue sections from standard formalin-fixed paraffin-embedded tissues. We coupled this method with immunofluorescence to allow the simultaneous identification of specific cell types. Validation of this in situ quantification method showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. The assay requires very few cells ( approximately 10 to 15). Thus, small tissue samples, including clinical biopsies, can be easily accommodated. In addition, the cells under study need not be actively cycling and there is no requirement for tissue disaggregation or cell culture. This method provides a more accurate assessment of telomere lengths than Southern blotting because confounding contributions from undesired cell types within tissue samples are avoided. Using this technique, we were able to perform the first comparison of relative telomere lengths in matched tumor versus normal epithelial cells within archival human prostate tissues.

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.

Hepatic adenomas are strongly linked to excess hormonal exposure, but little else is known about their pathogenesis. The Wnt signaling pathway, which is activated in both hepatocellular carcinomas and hepatoblastomas, has not been studied in hepatic adenomas. Fifteen hepatic adenomas were studied by immunohistochemistry for estrogen, progesterone, and androgen receptors (ER, PR, AR, respectively) and correlated with the results of immunostaining for ␤-catenin. Direct sequencing was performed to look for mutations in key genes involved in the Wnt signaling pathway: Exon 3 of ␤-catenin encompassing the glycogen synthase kinase 3␤ (GSK-3␤) phosphorylation region and the mutational cluster region of the adenomatosis polyposis coli protein (APC). Analysis for loss of heterozygosity (LOH) at chromosome 5q was also performed. Immunostaining for both ER and PR was present in 11/15 (73%) adenomas, and staining with one hormone receptor was positively associated with staining for the other receptor. AR positivity was present in 3/15 cases. Nuclear accumulation of ␤-catenin was present in 7/15 (46%) of adenomas, indicating activation of the Wnt signaling pathway. However, no ␤-catenin mutations, no APC mutations in the mutational cluster region, and no 5q LOH were detected. Two APC polymorphisms of unknown significance were seen. No clear association between ␤-catenin nuclear accumulation and hormone receptor positivity was discerned. Activation of the Wnt signaling pathway appears to be important in a subset of hepatic adenomas but does not result from common ␤-catenin or APC mutations and does not appear to be directly linked to hormonal receptor status.