Marie‐France Robert

Nonselective inhibitors of human histone deacetylases (HDAC) are known to have antitumor activity in mice in vivo, and several of them are under clinical investigation. The first of these, Vorinostat (SAHA), has been approved for treatment of cutaneous T-cell lymphoma. Questions remain concerning which HDAC isotype(s) are the best to target for anticancer activity and whether increased efficacy and safety will result with an isotype-selective HDAC inhibitor. We have developed an isotype-selective HDAC inhibitor, MGCD0103, which potently targets human HDAC1 but also has inhibitory activity against HDAC2, HDAC3, and HDAC11 in vitro. In intact cells, MGCD0103 inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal. MGCD0103 induced hyperacetylation of histones, selectively induced apoptosis, and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner. MGCD0103 exhibited potent and selective antiproliferative activities against a broad spectrum of human cancer cell lines in vitro, and HDAC inhibitory activity was required for these effects. In vivo, MGCD0103 significantly inhibited growth of human tumor xenografts in nude mice in a dose-dependent manner and the antitumor activity correlated with induction of histone acetylation in tumors. Our findings suggest that the isotype-selective HDAC inhibition by MGCD0103 is sufficient for antitumor activity in vivo and that further clinical investigation is warranted.

OBJECTIVE: To directly ascertain the physiological roles in adipocytes of hormone-sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. RESEARCH METHODS AND PROCEDURES: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. RESULTS: Homozygous HSL-/- mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild-type and deficient littermates. HSL-deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL-/- mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL-/- adipocytes, lipolysis is not significantly increased by beta3-adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL-/- adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48-hour period at 4 degrees C was similar in HSL-/- mice and controls. Overnight fasting was well-tolerated clinically by HSL-/- mice, but after fasting, liver triglyceride content was significantly lower in HSL-/- mice compared with wild-type controls. CONCLUSIONS: In isolated fat cells, the lipolytic rate after beta-adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL-/- adipocytes suggests that HSL-independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.

Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B,de novo methyltransferases that are expressed in adult tissues. Here we have shown that depletion of DNMT3B, but not DNMT3A, induced apoptosis of human cancer cells but not normal cells. DNMT3B depletion reactivated methylation-silenced gene expression but did not induce global or juxtacentromeric satellite demethylation as did specific depletion of DNMT1. Furthermore, the effect of DNMT3B depletion was rescued by exogenous expression of either of the splice variants DNMT3B2 or DNMT3B3 but not DNMT1. These results indicate that DNMT3B has significant site selectivity that is distinct from DNMT1, regulates aberrant gene silencing, and is essential for cancer cell survival. Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B,de novo methyltransferases that are expressed in adult tissues. Here we have shown that depletion of DNMT3B, but not DNMT3A, induced apoptosis of human cancer cells but not normal cells. DNMT3B depletion reactivated methylation-silenced gene expression but did not induce global or juxtacentromeric satellite demethylation as did specific depletion of DNMT1. Furthermore, the effect of DNMT3B depletion was rescued by exogenous expression of either of the splice variants DNMT3B2 or DNMT3B3 but not DNMT1. These results indicate that DNMT3B has significant site selectivity that is distinct from DNMT1, regulates aberrant gene silencing, and is essential for cancer cell survival. DNA methyltransferase (mouse) mismatch enzyme-linked immunosorbent assay normal foreskin fibroblasts human mammary epithelial cells proliferating cell nuclear antigen hemagglutinin trichostatin A RAS association domain family 1A gene methylation-specific polymerase chain reaction TdT-mediated dUTP nick-end labeling untranslated region DNA methylation is a highly plastic (1Yeivin A. Razin A. EXS. 1993; 64: 523-568PubMed Google Scholar) and critical component of mammalian development (2Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4348) Google Scholar, 3Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3159) Google Scholar). The maintenance DNA methyltransferase enzyme, Dnmt1,1 and thede novo methyltransferases, Dnmt3a and Dnmt3b, are indispensable for development because mice homozygous for the targeted disruption of any of these genes are not viable (2Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4348) Google Scholar, 3Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3159) Google Scholar). DNA methylation is also strongly implicated in tumorigenesis (4Esteller M. Corn P.G. Baylin S.B. Herman J.G. Cancer Res. 2001; 61: 3225-3229PubMed Google Scholar, 5Jones P.A. Laird P.W. Nat. Genet. 1999; 21: 163-167Crossref PubMed Scopus (2040) Google Scholar, 6Baylin S.B. Herman J.G. Trends Genet. 2000; 16: 168-174Abstract Full Text Full Text PDF PubMed Scopus (1388) Google Scholar).Dnmt1+/− mice develop fewer precancerous intestinal lesions than Dnmt1 wild type animals when bred with adenomatous polyposis coli (APC) multiple intestinal neoplasia (Min) animals predisposed to this neoplasia (7Laird P.W. Jackson-Grusby L. Fazeli A. Dickinson S.L. Jung W.E., Li, E. Weinberg R.A. Jaenisch R. Cell. 1995; 81: 197-205Abstract Full Text PDF PubMed Scopus (655) Google Scholar). Elevated levels of DNMT1 are also required to silence p16ink4a in bladder cancer cells (8Fournel M. Sapieha P. Beaulieu N. Besterman J.M. MacLeod A.R. J. Biol. Chem. 1999; 274: 24250-24256Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and to maintain the phenotype of fibroblasts transformed with the Fos oncogene (9Bakin A.V. Curran T. Science. 1999; 283: 387-390Crossref PubMed Scopus (214) Google Scholar). Overexpression of DNMT3A, DNMT3B, and various DNMT3B splice variants has also been reported in tumor cells (10Xie S. Wang Z. Okano M. Nogami M., Li, Y., He, W.W. Okumura K. Li E. Gene. 1999; 236: 87-95Crossref PubMed Scopus (331) Google Scholar, 11Robertson K.D. Uzvolgyi E. Liang G. Talmadge C. Sumegi J. Gonzales F.A. Jones P.A. Nucleic Acids Res. 1999; 27: 2291-2298Crossref PubMed Scopus (701) Google Scholar, 12Kanai Y. Ushijima S. Kondo Y. Nakanishi Y. Hirohashi S. Int. J. Cancer. 2001; 91: 205-212Crossref PubMed Scopus (184) Google Scholar); however, the extent to which they are involved in cancer remained to be investigated. In this report, we have investigated the role of DNMT3B in the aberrant methylation and inactivation of genes in human tumor cells as well as its role in the maintenance of the transformed phenotype. All cells were purchased from the American Type Culture Collection. Cells were transfected with the following phosphorothioate antisense oligonucleotides: DNMT1-AS, 5′-AAGCATGAGCACCGTTCTCC-3′; DNMT1-MM (mismatch), 5′-AACGATCAGGACCCTTGTCC-3′;DNMT3A-AS, 5′-CAGGAGATGATGTCCAACCC-3′; DNMT3A-MM, 5′-CACGACATCATCTCGAACGC-3′;DNMT3B-AS, 5′-CGTCGTGGCTCCAGTTACAA-3′; DNMT3B-MM, 5′-CCTCGTCGGTCGACTTAGAA-3′, as previously described (8Fournel M. Sapieha P. Beaulieu N. Besterman J.M. MacLeod A.R. J. Biol. Chem. 1999; 274: 24250-24256Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). For phenotypic rescue, HCT116 cells were seeded 48 h before transfection. Over the course of 72 h, cells were transfected with 75 nm DNMT3B-AS1 every 24 h (three treatments) and 5 μg of the DNMTpCGN constructs or empty vector every 48 h (two treatments). Surviving cells were stained with methylene blue 48 h after the last oligonucleotide treatment. Western blots were done following standard protocols. Preparation and use of polyclonal antibodies to DNMT1 were described previously (8Fournel M. Sapieha P. Beaulieu N. Besterman J.M. MacLeod A.R. J. Biol. Chem. 1999; 274: 24250-24256Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Antibodies raised in rabbits by the same method against purified glutathioneS-transferase fused to residues 10–118 of DNMT3A and 4–101 of DNMT3B were used at a dilution of 1:2000. Horseradish peroxidase-linked antibodies (Sigma) and ECL (Amersham Biosciences) were used for chemiluminescent detection. Northern blot analysis using 10–15 μg of total RNA/sample was performed according to standard protocols. Detection of histone H4 employed an end-labeled antisense oligonucleotide probe as previously described (13Robertson K.D. Keyomarsi K. Gonzales F.A. Velicescu M. Jones P.A. Nucleic Acids Res. 2000; 28: 2108-2113Crossref PubMed Scopus (171) Google Scholar). Detection ofDNMT1, DNMT3A, or DNMT3B mRNA was done as previously described (11Robertson K.D. Uzvolgyi E. Liang G. Talmadge C. Sumegi J. Gonzales F.A. Jones P.A. Nucleic Acids Res. 1999; 27: 2291-2298Crossref PubMed Scopus (701) Google Scholar). Northern blots were scanned and quantified using an alpha imager (Alpha Innovotech). Cells were fixed with paraformaldehyde and processed for TUNEL (TdT-mediated dUTP nick-end labeling) analysis using a fluorescein apoptosis detection system (Promega) or cell-death detection ELISA Plus kit (Roche Molecular Biochemicals) performed according to the manufacturers' protocols. Cytoplasmic histone DNA assay was performed using the cell-death detection ELISA Plus kit (Roche) according to the manufacturer's protocol. Standard protocols were used. Briefly, 5 μg of DNA were digested with either HpaII orMspI (Invitrogen) using the manufacturer's conditions. Samples were loaded on a 1% agarose gel and transferred to a Zeta-Probe nylon membrane (Bio-Rad). Blots were hybridized to a classic satellite 2 oligonucleotide as previously described (14Szyf M. Bozovic V. Tanigawa G. J. Biol. Chem. 1991; 266: 10027-10030Abstract Full Text PDF PubMed Google Scholar, 15Rhee I. Jair K.W. Yen R.W. Lengauer C. Herman J.G. Kinzler K.W. Vogelstein B. Baylin S.B. Schuebel K.E. Nature. 2000; 404: 1003-1007Crossref PubMed Scopus (366) Google Scholar). RT-PCR for RASSF1A was performed as described (16Dammann R., Li, C. Yoon J.H. Chin P.L. Bates S. Pfeifer G.P. Nat. Genet. 2000; 25: 315-319Crossref PubMed Scopus (996) Google Scholar). As a sample loading control, 100 ng of the same reverse-transcribed cDNA was used to amplify α-actin using the forward primer 5′-ACGAAACTACCTTCAACTCCATC-3′ and the reverse primer 5′-TGGTCTCAAGTCAGTGTACAGGT-3′. Specificity of RASSF1Aamplification was verified by Southern blot using as a probe the32P-labeled internal oligonucleotide probe 5′-GCAACCTCTTCATGAGCTTG-3′. Genomic DNA from lipofectin control and antisense-treated cells were bisulfite treated (Intergen Company) and used to amplify the RASSF1A promoter region as described in Ref. 16Dammann R., Li, C. Yoon J.H. Chin P.L. Bates S. Pfeifer G.P. Nat. Genet. 2000; 25: 315-319Crossref PubMed Scopus (996) Google Scholar. PCR products were cloned into TA cloning vectors (Invitrogen) and sequenced. Atlas human cancer cDNA expression arrays (CLONTECH Laboratories, Inc.) were performed according to the manufacturer's protocols. DNA samples were digested with HpaII and AciI or withMspI (normalization control) and incubated with 1×Taq buffer, 1.0 mm MgCl2, 0.25 unit of AmpliTaq (PerkinElmer), and [3H]dCTP at 56 °C for 1 h as described in Ref. 17Pogribny I., Yi, P. James S.J. Biochem. Biophys. Res. Comm. 1999; 262: 624-628Crossref PubMed Scopus (177) Google Scholar. Samples were precipitated in trichloroacetic acid, applied to nitrocellulose filters, and processed for scintillation counting. To test the roles of the DNMT isoforms in cancer cells, we identified potent isotype-specific antisense inhibitors. We screened numerous antisense phosphorothioate oligodeoxynucleotides (AS p-ODN, 20 bases in length) complementary to 5′- and 3′-untranslated regions as well as coding sequences of the respective mRNA for the ability to specifically reduce the mRNA of the target isotype after 24 h of treatment. The location of the most potent of these inhibitors within their target mRNA is shown in Fig.1 A. Potency and dose dependence of the inhibition was assessed by treating human cancer cells growing in culture with increasing concentrations (0–75 nm) of inhibitors DNMT1-AS1,DNMT3A-AS1, DNMT3B-AS1 (antisense oligonucleotides), or mismatch (MM) (Fig. 1 B). All inhibitors had IC50 values for mRNA inhibition of less than 50 nm. To determine the specificity of the inhibition, Northern blots with total RNA isolated from treated cells were hybridized with probes for the given target DNMT isoform as well as with probes for non-target DNMT mRNA and the non-target mRNA glyceraldehyde-3-phosphate dehydrogenase. Non-target mRNA were not affected by any of the inhibitors tested, and, as expected, mismatch control oligonucleotides had no effect on any of the mRNA examined (Fig. 1 B, left panel). Several human tumor cell lines of various tissue origins were similarly tested with essentially identical results (data not shown). The DNA methyltransferases (DNMT1,DNMT3A, and DNMT3B) are S-phase-specific genes. Therefore, to control for possible indirect effects onDNMT3B mRNA levels, Northern blots with mRNA from DNMT3B-depleted A549 cells were also hybridized with probes for another S-phase-specific gene, histone H4. DNMT3B depletion had no effect on histone H4 expression or on DNMT1 mRNA levels (Fig.1 B, middle panel). The ability to depleteDNMT3B from two normal cells, human mammary epithelial cells (HMEC) and normal foreskin fibroblasts (MRHF), was also shown (Fig.1 B, right panel). To demonstrate reduction in target protein levels, we raised antibodies specific for DNMT1 (8Fournel M. Sapieha P. Beaulieu N. Besterman J.M. MacLeod A.R. J. Biol. Chem. 1999; 274: 24250-24256Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), DNMT3A, and DNMT3B. All antibodies were shown to specifically recognize target human proteins (expressed in baculovirus or mammalian systems) with high affinity; however, endogenous levels of DNMT3A protein were below the level of detection in all cell lines tested (data not shown). As expected from the mRNA inhibition, Western blot analysis with anti-DNMT1 and anti-DNMT3B antibodies on nuclear extracts prepared from cancer cells treated with increasing doses of DNMT1, DNMT3A, or DNMT3B inhibitors for 48 h showed selective dose-dependent inhibition of only the target DNMT isoform (Fig. 1 C). DNMT1 has been shown to be in a complex with proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication. This interaction can be disrupted by the cell cycle regulator p21WAF1/CIP1 (18Chuang L.S. Ian H.I. Koh T.W., Ng, H.H., Xu, G. Li B.F. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (775) Google Scholar), suggesting that DNMT1 may play an active role in replication in addition to its role in regulation of gene expression. In fact, Dnmt1 mutant fibroblasts proliferate at only one-third the rate of control cells (19Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. E. Jaenisch R. Nat. Genet. 2001; Scopus Google Scholar). To determine the effect of DNMT3A or DNMT3B depletion on the of human cancer cells, we treated cancer cells or cancer cells with increasing doses (0–75 nm) of the inhibitors or DNMT3B-AS1 A potent dose-dependent effect DNMT3B depletion was in cancer cell lines In DNMT3A depletion had no significant effect on the of either cell type (Fig. 2 The effect was of because cells mutant Y. S. M. 1999; PubMed Scopus Google Scholar) and the wild type A549 cell Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) were to DNMT3B In the effect of DNMT3B depletion was also in human bladder cancer cells that protein T. Y. V. M. K. T. Int. J. Cancer. PubMed Scopus Google Scholar) (data not as well as the wild type HCT116 human cancer cell Y. B. J. 1999; PubMed Scopus Google Scholar) (Fig. 2 panel). within the DNMT3A and family because splice variants for have been identified in and human (10Xie S. Wang Z. Okano M. Nogami M., Li, Y., He, W.W. Okumura K. Li E. Gene. 1999; 236: 87-95Crossref PubMed Scopus (331) Google Scholar, 11Robertson K.D. Uzvolgyi E. Liang G. Talmadge C. Sumegi J. Gonzales F.A. Jones P.A. Nucleic Acids Res. 1999; 27: 2291-2298Crossref PubMed Scopus (701) Google Scholar, M. S. Li E. Nat. Genet. PubMed Scopus Google Scholar). These splice variants have the to proteins with for and and a protein with methyltransferase and in the domain (11Robertson K.D. Uzvolgyi E. Liang G. Talmadge C. Sumegi J. Gonzales F.A. Jones P.A. Nucleic Acids Res. 1999; 27: 2291-2298Crossref PubMed Scopus (701) Google Scholar). DNMT3B2 only which the and to proteins the and (11Robertson K.D. Uzvolgyi E. Liang G. Talmadge C. Sumegi J. Gonzales F.A. Jones P.A. Nucleic Acids Res. 1999; 27: 2291-2298Crossref PubMed Scopus (701) Google Scholar), which a The of these splice variants from to human suggests that they The antisense inhibitors described target sequences of DNMT3A and to all the splice the inhibitors tested to a depletion of all DNMT3A and DNMT3B mRNA and proteins (Fig. and C). To test the effects of DNMT3B depletion be to of a DNMT3B splice we evaluated the ability of expressed DNMT3B splice variants to reverse or the phenotype by depletion of all DNMT3B is to that the expressed DNMT isoforms not the target of the antisense which target sequences in the 3′-untranslated regions and not for and are not Several cell lines were tested for the ability to levels of exogenous and DNMT1 after HCT116 cells were used because they were the only cell type to exogenous DNMT3B protein 48 h after in a significant of the of the and DNMT1 proteins was 48 h after of HCT116 cells by Western blot with and anti-DNMT3B antibodies B, left panel). HCT116 cells were treated for 72 h with a of either DNMT3B-AS1 nm) and the empty vector nm) and or the cells were left for an 24 h at which cell were by methylene blue The effect of DNMT3B depletion was rescued by exogenous expression of either DNMT3B2 or DNMT3B3 but not by DNMT1 or vector sequences (Fig. 2 B, right panel). These results demonstrate the selectivity of the inhibition for the DNMT3B and that DNMT3B2 and DNMT3B3 roles in cancer cells distinct from of DNMT1. In an to the gene expression induced by DNMT3B depletion h we performed cDNA human cancer DNMT3B depletion the expression of a of genes on the of The genes that showed the are shown in left The in gene expression are with the inhibition of of and is of of an Western blot analysis the of p21WAF1/CIP1 and of at the protein level (data not shown). because these genes are not to be by these may either be to the of methylation-silenced genes or may be by the levels of DNMT3B In addition to the analysis of gene expression by cDNA we also a analysis of specific genes previously shown to be by methylation at high in cancers. methylation and silencing of protein is associated with cell cancer 2001; PubMed Scopus Google Scholar). methylation-specific PCR analysis that is not the region of the in either A549 or cells (data not shown). The RAS RASSF1A is a tumor suppressor gene that has been shown to be by methylation in a high of and (16Dammann R., Li, C. Yoon J.H. Chin P.L. Bates S. Pfeifer G.P. Nat. Genet. 2000; 25: 315-319Crossref PubMed Scopus (996) Google Scholar, E. S. L. K. B. D. Kondo M. A. S. Y. S. S. E. M. J. Cancer 2001; PubMed Scopus Google Scholar, R. G. Pfeifer G.P. Cancer Res. 2001; 61: Google Scholar). RASSF1A has been shown to be by methylation in A549 and cells (16Dammann R., Li, C. Yoon J.H. Chin P.L. Bates S. Pfeifer G.P. Nat. Genet. 2000; 25: 315-319Crossref PubMed Scopus (996) Google Scholar). We examined DNMT3B depletion to of RT-PCR analysis expression was induced in a dose-dependent by DNMT3B depletion in (data not and A549 cells (Fig. 2 the of DNMT3B for active of this gene in these cancer cells. doses of DNMT3B-AS1 were required to RASSF1A in A549 cells when with cells. This is with the observation that doses of are also required for its in A549 cells to cells (16Dammann R., Li, C. Yoon J.H. Chin P.L. Bates S. Pfeifer G.P. Nat. Genet. 2000; 25: 315-319Crossref PubMed Scopus (996) Google Scholar). DNMT3A and DNMT3B have been shown to be that in by the histone K.E. Baylin S.B. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, N. M. T. J. 2001; PubMed Scopus Google Scholar). Therefore, gene may and To the involvement of in the of RASSF1A induced by we treated cells with increasing doses of the trichostatin A or with a of of cells with did not RASSF1A as expected, DNMT3B depletion did (Fig. 2 right panel). The of and DNMT3B depletion in levels of RASSF1A expression (Fig. 2 right suggesting that the of RASSF1A silencing in these cells is inhibition of with DNMT3B that the RASSF1A promoter was highly in A549 cells and demethylation of within the RASSF1A promoter in DNMT3B-depleted A549 cells The demethylation was to fewer than that by either demethylation of or by specific inhibition of the maintenance methyltransferase DNMT1 by antisense demethylation of most within is (8Fournel M. Sapieha P. Beaulieu N. Besterman J.M. MacLeod A.R. J. Biol. Chem. 1999; 274: 24250-24256Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). This suggests that DNMT3B from DNMT1 in of its site selectivity and regulates within DNMT1 is required to maintain methylation of all because DNMT3B depletion in significant possible that the most affected cells are in to expression of RASSF1A is associated with of and apoptosis of cancer cells (16Dammann R., Li, C. Yoon J.H. Chin P.L. Bates S. Pfeifer G.P. Nat. Genet. 2000; 25: 315-319Crossref PubMed Scopus (996) Google Scholar, Bell A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), we investigated the that cell by apoptosis was a factor to the on DNMT3B-depleted A549 cells cell cycle with an of cells in a and, most a in a of cells. or cells showed no (Fig. panel). A549 cells treated for 24 h with DNMT3B inhibitors showed suggesting that DNMT3B depletion is required to the TUNEL right and (data not that DNMT3B depletion induced cell of cancer cells. The of apoptosis was on the dose and the of the DNMT3B inhibitors tested (data not shown). To determine normal cells to DNMT3B depletion to that of the cancer cells, we treated and cells with DNMT3B inhibitors. DNMT3B depletion did not in cell cycle or induce apoptosis in these normal cells (Fig. B, left panel). was by the histone apoptosis assay in DNMT3B-depleted A549 cells control) but not in similarly treated normal cells (Fig. B, right panel). The apoptosis is with a of genes in cancer cells. of the is the of of a of genes or is the effect of of many genes in be of for as be of the tissue specificity of genes. demethylation of fibroblasts can induce apoptosis (19Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. E. Jaenisch R. Nat. Genet. 2001; Scopus Google Scholar). the apoptosis described is we the effect of DNMT3B depletion on global methylation levels and juxtacentromeric satellite methylation was evaluated by [3H]dCTP on DNA from A549 cells treated with DNMT3B inhibitors for to DNMT3B depletion did not significant global demethylation at any of these B, left panel). classic satellite DNA is but DNA from mice or with in DNMT3B and no methylation in this region Bestor T.H. D. N. M. M., E. Nature. 1999; PubMed Scopus Google Scholar, C. P. S. A. 1999; PubMed Scopus Google Scholar). cells showed demethylation of the classic satellite 2 region (Fig. B, right to that by the global In demethylation was not in DNMT3B-depleted cells (Fig. B, right panel). These results demonstrate that the apoptosis is not a of and that DNMT3B is required to but not to methylation of classic in cancer cells. inhibition of DNMT3B in cancer cells DNMT3B depletion with doses of DNMT3B-AS1 in is to that genes the of cancer cells are to DNMT3B depletion and levels of DNMT3B the of of this I. K.E. Jair K.W. Yen R.W. Schuebel K.E. Lengauer C. Kinzler K.W. Baylin S.B. Vogelstein B. Nature. PubMed Scopus Google Scholar) reported the on two HCT116 cancer that they following two of also that DNMT3B, in with DNMT1, is required for with the of DNMT3B did not induce demethylation of juxtacentromeric satellite In however, the did not or This most results from the in employed to DNMT3B disruption by two of to This against cells that apoptosis or had any other for to of tumor suppressor genes by The method described in this specifically DNMT3B in an treated of cells and is not by because the only 2 of the than 20 of gene, the antisense inhibitors were depleted of DNMT3B possible that other or DNMT3B mRNA DNMT3B In results that DNA methyltransferase isoforms significant methylation site specificity and demonstrate that the novo methyltransferase DNMT3B is required for the active of genes and for the of cancer cells. These results also that DNMT3B inhibitors may have as cancer We Besterman for critical of the and for and for