Hypermethylation of the human glutathione S-transferase-pi gene (GSTP1) CpG island is present in a subset of proliferative inflammatory atrophy lesions but not in normal or hyperplastic epithelium of the prostate: a detailed study using laser-capture microdissection.

Somatic inactivation of the glutathione S-transferase-pi gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in approximately 70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-alpha (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA lesions. These data support the hypothesis that atrophic epithelium in a subset of PIA lesions may lead to high-grade PIN and/or adenocarcinoma. Because these atrophic lesions are so prevalent and extensive, even though only a small subset contains this somatic DNA alteration, the clinical impact may be substantial.

Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis.

Chromosomal instability appears to be key to the pathogenesis of malignant transformation in human cancers, yet the precise molecular mechanisms underlying chromosomal rearrangements remain largely unknown. Telomeres stabilize and protect the ends of chromosomes, but shorten because of cell division and/or oxidative damage. Critically short telomeres, in the setting of abrogated DNA damage checkpoints, have been shown to cause chromosomal instability in vitro and in animal models, leading to an increased cancer incidence as a result of chromosome fusions, subsequent breakage, and rearrangement. We present results from a quantitative, high-resolution, in situ method for telomere length assessment used to test the hypothesis that telomere shortening is an early contributor to human tumorigenesis. High-grade prostatic intraepithelial neoplasia (HGPIN) is a putative preinvasive precursor of prostatic adenocarcinoma, the most common noncutaneous malignancy in Western men. The telomere lengths of epithelial cells within HGPIN lesions were strikingly shorter than those of adjacent normal appearing epithelial cells in 93% (28 of 30) of lesions examined. This shortening is similar to what has been shown in fully invasive prostate adenocarcinomas. Interestingly, telomere shortening was restricted to the luminal epithelial cells of HGPIN and was not present in the underlying basal epithelial cells; this provides strong evidence that basal cells are most likely not the direct targets of neoplastic transformation. These findings reveal that telomere shortening is a defining somatic DNA alteration characterizing HGPIN. The implications of this are that the earliest phase of human prostate carcinogenesis may proceed as a consequence of chromosomal instability mediated by shortened, dysfunctional telomeres.

Smoking status and the human dopamine transporter variable number of tandem repeats (VNTR) polymorphism: failure to replicate and finding that never-smokers may be different.

Cigarette smoking, like many addictive behaviors, has been shown to have a genetic component. The dopamine transporter (DAT) gene (SLC6A3) encodes a protein that regulates synaptic levels of dopamine in the brain and is a candidate gene for addictive behaviors. We have collected smoking information from a national probability sample of 3383 adult volunteers contacted via a random-digit dialing telephone interview. A subset of individuals provided DNA from cheek swabs returned via the mail for subsequent genetic analysis of self-reported smoking behavior. DNA samples were genotyped at a variable number of tandem repeats (VNTR) polymorphism in the 3'-untranslated region of the DAT gene. If we classify smokers as non- (<100 cigarettes), former and current, we fail to replicate both Lerman et al. (Health Psychology 18:14-20, 1999) and Sabol et al. (Health Psychology 18:7-13, 1999) and support the absence of effects found by Jorm et al. (American Journal of Medical Genetics (Neuropsychiatric Genetics) 96:331-334, 2000). When we distinguish between never-smokers (no cigarettes ever) and non-smokers (1-99 in lifetime), we find a reliable trend essentially in the opposite direction from Lerman et al. (1999), with the 10-copy allele being more frequent in never-smokers. Biobehavioral research on cigarette smoking should distinguish between never- and non-smokers. We have also developed an improved set of polymerase chain reaction conditions to increase the frequency of successful amplification of DAT'sw VNTR, which is a long, G+C-rich repeat.

Alpha-methylacyl-CoA racemase: a new molecular marker for prostate cancer.

Identification of genes that are dysregulated in association with prostate carcinogenesis can provide disease markers and clues relevant to disease etiology. Of particular interest as candidate markers of disease are those genes that are frequently overexpressed. In this study, we describe a gene, alpha-methylacyl-CoA racemase (AMACR), whose expression is consistently up-regulated in prostate cancer. Analysis of mRNA levels of AMACR revealed an average up-regulation of approximately 9 fold in clinical prostate cancer specimens compared with normal. Western blot and immunohistochemical analysis confirms the up-regulation at the protein level and localizes the enzyme predominantly to the peroxisomal compartment of prostate cancer cells. A detailed immunohistochemical analysis of samples from 168 primary prostate cancer cases using both standard slides and tissue microarrays demonstrates that both prostate carcinomas and the presumed precursor lesion (high-grade prostatic intraepithelial neoplasia) consistently scored significantly higher than matched normal prostate epithelium; 88% of the carcinomas had a staining score higher than the highest score observed for any sample of normal prostate epithelium. Both untreated metastases (n = 32 patients) and hormone refractory prostate cancers (n = 14 patients) were generally strongly positive for AMACR. To extend the utility of this marker for prostate cancer diagnosis, we combined staining for cytoplasmic AMACR with staining for the nuclear protein, p63, a basal cell marker in the prostate that is absent in prostate cancer. In a simple assay that can be useful for the diagnosis of prostate cancer on both biopsy and surgical specimens, combined staining for p63 and AMACR resulted in a staining pattern that greatly facilitated the identification of malignant prostate cells. The enzyme encoded by the AMACR gene plays a critical role in peroxisomal beta oxidation of branched chain fatty acid molecules. These observations could have important epidemiological and preventive implications for prostate cancer, as the main sources of branched chain fatty acids are dairy products and beef, the consumption of which has been associated with an increased risk for prostate cancer in multiple studies. On the basis of its consistency and magnitude of cancer cell-specific expression, we propose AMACR as an important new marker of prostate cancer and that its use in combination with p63 staining will form the basis for an improved staining method for the identification of prostate carcinomas. Furthermore, the absence of AMACR staining in the vast majority of normal tissues coupled with its enzymatic activity makes AMACR the ideal candidate for development of molecular probes for the noninvasive identification of prostate cancer by imaging modalities.