Transcriptional silencing of tumor suppressor genes by DNA methylation occurs in cancer cell lines and in human tumors. This has led to the pursuit of DNA methyltransferase inhibition as a drug target. 5-Aza-2Ј-deoxycytidine [5-aza-CdR (decitabine)], a potent inhibitor of DNA methyltransferase, is a drug currently in clinical trials for the treatment of solid tumors and leukemia. The efficacy of 5-aza-CdR may be related to the induction of methylation-silenced tumor suppressor genes, genomic hypomethylation, and/or enzyme-DNA adduct formation. Here, we test the hypothesis that 5-aza-CdR treatment is perceived as DNA damage, as assessed by the activation of the tumor suppressor p53. We show that 1) colon tumor cell lines expressing wild-type p53 are more sensitive to 5-aza-CdR mediated growth arrest and cytotoxicity; 2) the response to 5-aza-CdR treatment includes the induction and activation of wild-type but not mutant p53 protein; and 3) the induction of the downstream p53 target gene p21 is partially p53-dependent. The induction of p53 protein after 5-aza-CdR treatment did not correlate with an increase in p53 transcripts, indicating that hypomethylation at the p53 promoter does not account for the p53 response. It is relevant that 5-aza-CdR has shown the greatest promise in clinical trials for the treatment of chronic myelogenous leukemia, a malignancy in which functional p53 is often retained. Our data raise the hypothesis that p53 activation may contribute to the clinical efficacy and/or toxicity of 5-azaCdR.

Mutations in the human adenomatous polyposis coli (APC) gene are thought to initiate colorectal tumorigenesis. The tumor suppressor function of APC is attributed primarily to its ability to regulate the WNT pathway by targeting the destruction of ␤-catenin. We report here a novel role for APC in regulating degradation of the transcriptional co-repressor C-terminalbinding protein-1 (CtBP1) through a proteasome-dependent process. Further, CtBP1 suppresses the expression of intestinal retinol dehydrogenases, which are required for retinoic acid production and intestinal differentiation. In support of a role for CtBP1 in initiation of colorectal cancer, adenomas taken from individuals with familial adenomatous polyposis contain high levels of CtBP1 protein in comparison with matched, uninvolved tissue. The relationship between APC and CtBP1 is conserved between humans and zebrafish and provides a mechanistic model explaining APC control of intestinal retinoic acid biosynthesis.Germline mutations in the adenomatous polyposis coli (APC) 2,3 tumor suppressor invariably result in familial adenomatous polyposis coli (FAP), a syndrome characterized by early onset colorectal cancer (1). The mechanism by which APC mutations cause colon tumorigenesis is attributed primarily to its role in negatively regulating canonical WNT signaling (2, 3). In this role, APC functions by targeting the transcriptional coactivator ␤-catenin for intracellular degradation through a proteasome-dependent pathway, thereby limiting its ability to associate with T cell factor/lymphoid enhancer factor nuclear transcription factors. Current evidence indicates that following APC mutation, ␤-catenin accumulates and translocates into the nucleus, where it partners with T cell factor/lymphoid enhancer factors to drive a program of cellular proliferation. As evidence for a genetic relationship between APC and WNT signaling, some studies cite the existence of rare, ␤-cateninactivating mutations in colon adenocarcinomas (4, 5). Importantly, however, these mutations do not appear to fully recapitulate the clinical phenotypes associated with APC mutation (6). This discrepancy raises the possibility of additional, ␤-cateninindependent functions for APC.A number of reports suggest that the functions of APC are not limited to its well established role in regulating canonical WNT signaling. For example, APC is reported to bind to microtubules, to regulate asymmetric cell division in Drosophila male germline stem cells, and to promote proper T-cell differentiation in mice (7-11). Further, we recently demonstrated that sporadic human colorectal carcinomas lack retinol dehydrogenases and that introduction of APC into human colon carcinoma cells lines induced the expression of the retinol dehydrogenase DHRS9 in a ␤-catenin-independent manner (12). In addition, apc mcr zebrafish lack expression of intestinal enzymes, such as rdh1l, that are required for retinoic acid production. Injection of apc mcr zebrafish embryos with mRNA encoding rdh1l or treatment with exogenous...

Chronic lymphocytic leukemia is a heterogenous B lymphocyte disorder with a variable natural history. Like other Malignancies, key regulatory genes like the tumor suppressor, p16, and the mismatch repair gene, hMLH1, are frequently silenced by DNA methylation in patients with chronic lymphocytic leukemia (CLL). Although focal hypermethylation is found, global genomic DNA is hypomethylated compared to genomic DNA from healthy volunteers. Inhibition of DNA methylation may help CLL cells regain normal cellular function. Few studies have been done evaluating DNA methylation of CLL cells likely in part due to the difficulty in establishing a CLL cell line without altering DNA methylation. In addition, the primary cells do not divide in vitro, making the assessment of effect of a DNA methylation inhibitor very difficult. Of the ten samples tested from patients with CLL, we have not found p16 or hMLH1 expression by western blotting. Since 2-Chlorodeoxyadenosine (Cladribine) is already used clinically for treatment of CLL and may deplete available methyl donors, we asked if Cladribine is effective because of its inhibitory effect on DNA methylation in leukemia cells. To further evaluate the role of DNA methylation in CLL, we assessed the effect of 5-aza-2′-deoxycitidine (Decitabine), a DNA methyltransferase inhibitor compared to Cladribine in HT-29 cells. HT-29 cells do not express p16 and MAGE because of DNA methylation of the promoters. Assessment of global DNA methylation is done using reversed-phase high-performance liquid chromatography. Our results reveal that daily doses of 300nM Cladribine modestly inhibited global DNA methylation by 20+/−14% at 72 hours (n=3), while daily doses of 500nM Decitabine inhibited global DNA methylation by 75+/−9% at 72 hours (n=3). This results in re-expression of MAGE-1, a gene silenced by DNA methylation in all somatic tissues and p16, only in the Decitabine treated cells. We also evaluated the Cdx-2 promoter, an intestinal restricted gene silenced by DNA methylation in HT-29 cells. Decreased methylation of two regions of the Cdx-2 promoter was seen in the cells treated with Decitabine compared to the control cells. Cladribine treated cells had no effect on the Cdx-2, and may actually increase DNA methylation (result of 20 sequences). These results could be explained by the marked growth inhibitory effect of Cladribine at 300nM. We chose to evaluate Cladribine at 300nM because this plasma concentration is obtainable in patients treated with 0.1mg/kg of Cladribine, a dose frequently used in treatment of hairy cell leukemia. Cells with decreased DNA methylation after treatment with Cladribine may have died at the 72 hour time point.