Previous studies have shown that DNA methyltransferase (Dnmt) 1 is required for maintenance of bulk DNA methylation and is essential for mouse development. However, somatic disruption of DNMT1 in the human cancer cell line HCT116 was not lethal and caused only minor decreases in methylation. Here, we report the identification of a truncated DNMT1 protein, which was generated by the disruption of DNMT1 in HCT116 cells. The truncated protein, which had parts of the regulatory N-terminal domain deleted but preserved the catalytic C-terminal domain, was present at different levels in all DNMT1 single-knockout and DNMT1͞DNMT3b doubleknockout cell lines tested and retained hemimethylase activity. DNMT1 RNAi resulted in decreased cell viability in WT and knockout cells and further loss of DNA methylation in DNMT1 knockout cells. Furthermore, we observed a delay in methylation after replication and an increase in hemimethylation of specific CpG sites in cells expressing the truncated protein. Remethylation studies after drug-induced hypomethylation suggest a putative role of DNMT1 in the de novo methylation of a subtelomeric repeat, D4Z4, which is lost in cells lacking full-length DNMT1. Our data suggest that DNMT1 might be essential for maintenance of DNA methylation, proliferation, and survival of cancer cells. DNA methylation ͉ epigeneticT he biological roles of the major mammalian DNA methyltransferase (Dnmt), DNMT1, have been enigmatic. Although gene-targeting studies in mice have clearly demonstrated an essential function of Dnmt1 in embryonic development, cell survival, and tumorigenesis (1), there have been controversial reports regarding the function of this enzyme in human cancer cells. In mice, Dnmt1 has been implicated in maintaining the majority of bulk DNA methylation, differentiation of ES cells, and imprinting (2, 3). Furthermore, deletion of Dnmt1 in mouse embryonic fibroblasts caused a decrease in genomic methylation, p53-dependent apoptosis, and deregulation of transcription (4). Heterozygosity for Dnmt1 in combination with administration of the DNMT inhibitor 5-aza-2Јdeoxycytidine (5-aza-CdR) greatly reduced the number of polyps in a mouse model for intestinal neoplasia (5).A series of RNAi experiments for DNMT1 have been described for various human cancer cell lines, which have shown apparently inconsistent results in regard to DNA methylation of tumor suppressor genes (6-10). The differences might be attributed to the techniques used but also to distinct sensitivities of individual cell lines (10). Furthermore, RNAi may not cause a complete depletion of the protein, so residual protein might still be available and capable of maintaining DNA methylation. Rhee et al. (11,12) generated a widely used series of HCT116 colon cancer cells with homozygous deletions for DNMT1 (DNMT1 Ϫ/Ϫ ) (11), DNMT3b (DNMT3b Ϫ/Ϫ ) (12), or both DNMT1 and DNMT3b (12) [double knockout (DKO)]. Surprisingly, somatic disruption of DNMT1 resulted in only a 20% decrease in overall genomic methylation with no discernible changes i...

Epigenetic drugs are in use in clinical trials of various human cancers and are potent at reactivating genes silenced by DNA methylation and chromatin modifications. We report here the analysis of a set of normal fibroblast and cancer cell lines after combination treatment with the DNA methyltransferase inhibitor 5-aza-2 ¶-deoxycytidine (5-aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA). Low doses of the drug combination caused cell cycle arrest, whereas high doses induced apoptosis in T24 bladder carcinoma cells. Both p16 (CDKN2A/INK4) and p21 (CIP1/SDI1/WAF1) expression were induced to similar levels in normal and cancer cells in a dose-dependent fashion after combination treatments. We detected a distinct increase of histone H3 acetylation at lysine 9/14 near the transcription start sites, in both LD419 normal fibroblasts and T24 bladder carcinoma cells, whereas the acetylation changes in the p21 locus were less apparent. Interestingly, the levels of trimethylation of histone H3 on lysine 9, which usually marks inactive chromatin regions and was associated with the p16 promoter in silenced T24 cells, did not change after drug treatments. Furthermore, we provide evidence that the remethylation of the p16 promoter CpG island in T24 cells after 5-aza-CdR treatment cannot be halted by subsequent continuous PBA treatment. The p16 gene is resilenced with kinetics similar to 5-aza-CdR onlytreated cells, which is also marked by a localized loss of histone acetylation at the transcription start site. Altogether, our data provide new insights into the mechanism of epigenetic drugs and have important implications for epigenetic therapy. [Cancer Res 2007;67(1):346-53]

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Despite key advances in cancer therapies, malignant tumors, such as melanoma, continue to be one of the leading causes of mortality. Recent debate on whether cancer can originate from a tumor-initiating subpopulation has permeated oncology and stem cell research. It has been well established that primary and immortalized tumor cells consist of heterogeneous cell populations. The profound effect of tumor heterogeneity on tumor growth and drug resistance remains elusive, but it is highly likely that subpopulations of cancer cells have different capabilities of self-renewal and drug resistance. Discrepancies between excellent in vitro potency and efficacy and poor patient response have been observed on multiple cancer therapeutics. Although this observation can be attributed to many factors, a better understanding of the contribution from subpopulations within a cancer will help bridge the gap between in vitro assay results and patient prognosis. To comprehend this impact, it is critical to isolate and characterize cancer subpopulations that possess higher growth and drug resistance properties so that novel therapeutics can be developed to eventually eradicate all cancer cells. In this article, we describe a method to enrich a subpopulation, CB4, from the melanoma cell line WM115. CB4 exhibited higher anchorage-independent growth, higher survival under serum starvation condition, and lower drug sensitivity to commonly used melanoma treatment compared with WM115. Details of functional properties and gene expression of CB4 compared with WM115 are reported. Our study demonstrates that it is feasible to isolate and enrich a subpopulation that exhibits higher growth capacity and treatment resistance from an immortalized tumor cell line.

Supplementary Table 1 from Inhibition of Histone Deacetylation Does Not Block Resilencing of p16 after 5-Aza-2′-Deoxycytidine Treatment