The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity to MUC1 by immunization with peptides have generally failed partly because of tolerance. In this study, we have developed chemoenzymatic synthesis of extended MUC1 TR glycopeptides with cancer-associated O-glycosylation using a panel of recombinant human glycosyltransferases. MUC1 glycopeptides with different densities of Tn and STn glycoforms conjugated to KLH were used as immunogens to evaluate an optimal vaccine design. Glycopeptides with complete O-glycan occupancy (five sites per repeat) elicited the strongest antibody response reacting with MUC1 expressed in breast cancer cell lines in both Balb/c and MUC1.Tg mice. The elicited humoral immune response showed remarkable specificity for cancer cells suggesting that the glycopeptide design holds promise as a cancer vaccine. The elicited immune responses were directed to combined glycopeptide epitopes, and both peptide sequence and carbohydrate structures were important for the antigen. A MAb (5E5) with similar specificity as the elicited immune response was generated and shown to have the same remarkable cancer specificity. This antibody may hold promise in diagnostic and immunopreventive measures.

Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. It is important to know the genomic targets of DNA-binding proteins and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation. Chromatin immunoprecipitation (ChIP) is an important technique in the study of protein-gene interactions. Using ChIP, DNA-protein interactions are studied within the context of the cell. The basic steps in this technique are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a very versatile tool, the procedure requires the optimization of reaction conditions. Several modifications to the original ChIP technique have been published to improve the success and to enhance the utility of this procedure. This review addresses the critical parameters and the variants of ChiP as well as the different analytical tools that can be combined with ChIP to enable better understanding of DNA-protein interactions in vivo.

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Defects in apoptotic pathway contribute to uncontrolled proliferation of cancer cells and confer resistance to chemotherapy. Growth arrest and DNA damage inducible, alpha (GADD45a) is up-regulated on docetaxel treatment and may contribute to docetaxel-mediated cytotoxicity. We examined the mechanism of regulation of GADD45a in prostate cancer cells and the effect of its up-regulation on sensitivity to docetaxel chemotherapy. Expression of GADD45a in PC3 cells was higher than that in Du145 and LNCaP cells (17-and 12-fold, respectively; P < 0.05). Although the proximal promoter region was unmethylated in all three cell lines, methylation of a 4 CpG region upstream of the proximal promoter correlated inversely with gene expression levels. Methylation was reversed by treatment of Du145 and LNCaP cells with DNA methyltransferase inhibitors, leading to reactivation of GADD45a expression in these cells. The 5 ¶ 4 CpG region was also frequently methylated in prostate cancer tissues. Methylation of this region correlated inversely with gene expression in prostate cancer and benign prostate tissues. The methyl binding protein MeCP2 was associated with the methylated 4 CpGs in Du145 cells, and knockdown of MeCP2 in these cells (Du145 MeCP2 À ) led to a significantly increased expression of GADD45a (3-fold; P = 0.035) without affecting the methylation status of the gene. Enhanced sensitivity to docetaxel was observed by up-regulation of GADD45a in Du145 cells by recombinant expression of GADD45a or pretreatment with 5-azacytidine. Our results show that GADD45a is epigenetically repressed and is a potential target for treatment of prostate cancer. [Cancer Res 2009;69(4):1527-35]

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Background:Prostate-specific antigen (PSA) screening for prostate cancer results in a large number of unnecessary prostate biopsies. There is a need for specific molecular markers that can be used in combination with PSA to improve the specificity of PSA screening. We examined GADD45a methylation in blood DNA as a molecular marker for prostate cancer diagnosis.Methods:The study included 82 men, with PSA levels >4 ng ml−1 and/or abnormal digital rectal exam, who underwent prostate biopsy. We compared GADD45a methylation in DNA from serum and buffy coat in 44 patients (22 prostate cancer and 22 benign). GADD45a methylation in serum DNA was examined in 82 patients (34 cancer and 48 benign).Results:There was no significant difference in buffy coat GADD45a methylation between cancer and benign patients. Serum GADD45a methylation was significantly higher in cancer than in benign patients. Classification and regression tree predictive model for prostate cancer including risk groups defined by PSA, free circulating DNA (fcDNA) level and GADD45a methylation yielded specificity of 87.5%, sensitivity of 94.1% and receiver operator characteristic curve area of 0.937.Conclusions:Serum GADD45a methylation in combination with PSA and fcDNA level was useful in distinguishing benign from prostate cancer patients.

BackgroundTranscriptional silencing associated with aberrant promoter methylation has been established as an alternate pathway for the development of cancer by inactivating tumor suppressor genes. TMS1 (Target of Methylation induced Silencing), also known as ASC (Apoptosis Speck like protein containing a CARD) is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to promote apoptosis directly and by activation of downstream caspases. This study describes the methylation induced silencing of TMS1/ASC gene in prostate cancer cell lines. We also examined the prevalence of TMS1/ASC gene methylation in prostate cancer tissue samples in an effort to correlate race and clinico-pathological features with TMS1/ASC gene methylation.ResultsLoss of TMS1/ASC gene expression associated with complete methylation of the promoter region was observed in LNCaP cells. Gene expression was restored by a demethylating agent, 5-aza-2'deoxycytidine, but not by a histone deacetylase inhibitor, Trichostatin A. Chromatin Immunoprecipitation (ChIP) assay showed enrichment of MBD3 (methyl binding domain protein 3) to a higher degree than commonly associated MBDs and MeCP2. We evaluated the methylation pattern in 66 prostate cancer and 34 benign prostatic hyperplasia tissue samples. TMS1/ASC gene methylation was more prevalent in prostate cancer cases than controls in White patients (OR 7.6, p 0.002) while no difference between the cases and controls was seen in Black patients (OR 1.1, p 0.91).ConclusionOur study demonstrates that methylation-mediated silencing of TMS1/ASC is a frequent event in prostate cancer, thus identifying a new potential diagnostic and prognostic marker for the treatment of the disease. Racial differences in TMS1/ASC methylation patterns implicate the probable role of molecular markers in determining in susceptibility to prostate cancer in different ethnic groups.

Background: Free circulating DNA (fcDNA) has been shown to be elevated in serum of prostate cancer patients compared with benign controls. However, studies evaluating the role of fcDNA as a biomarker in a "representative" patient group who have undergone prostate cancer screening are lacking. Our study examined the use of serum fcDNA levels as a biomarker of prostate cancer in such a setting.Methods: The study included 252 men, with prostate-specific antigen (PSA) levels >4 ng/mL and/or abnormal digital rectal exam. fcDNA levels in serum before prostate biopsy were quantitated by real-time PCR amplification of the glutathione S-transferase, pi, gene.Results: Patients with PSA ≤ 10 ng/mL with fcDNA > 180 ng/mL were at increased risk for prostate cancer compared with those with fcDNA ≤180 ng/mL (odds ratio, 4.27; 95% confidence interval, 2.05-8.88; P < 0.001; area under the curve, 0.742). The multivariate model including age, race, PSA, fcDNA, and interaction between fcDNA and PSA yielded a high negative predictive value of 93.1% and increased specificity of 33.1% compared with negative predictive value of 73.3% and specificity of 6.7% in the model excluding fcDNA.Conclusions: Our results indicate that fcDNA may improve the specificity of prostate cancer screening. Impact: Our study shows that adding fcDNA to prostate cancer screening can reduce the number of unnecessary prostate biopsies. Cancer Epidemiol Biomarkers Prev; 19(8); 1984-91. ©2010 AACR.