Oncogene (2001) 20, 7787 ± 7796 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Increased expression of unmethylated CDKN2D by 5-aza-2'-deoxycytidine in human lung cancer cells Wei-Guo Zhu1, Zunyan Dai2,3, Haiming Ding1, Kanur Srinivasan1, Julia Hall3, Wenrui Duan1, Miguel A Villalona-Calero1, Christoph Plass3 and Gregory A Otterson*,1 1Division of Hematology/Oncology, Department of Internal Medicine, The Ohio State University-Comprehensive Cancer Center, Columbus, Ohio, OH 43210, USA; 2Department of Pathology, The Ohio State University-Comprehensive Cancer Center, Columbus, Ohio, OH 43210, USA; 3Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University-Comprehensive Cancer Center, Columbus, Ohio, OH 43210, USA DNA hypermethylation of CpG islands in the promoter Introduction region of genes is associated with transcriptional silencing. Treatment with hypo-methylating agents can Methylation of cytosine residues in CpG sequences is a lead to expression of these silenced genes. However, DNA modi®cation that plays a role in normal whether inhibition of DNA methylation in¯uences the mammalian development (Costello and Plass, 2001; expression of unmethylated genes has not been exten- Li et al., 1992), imprinting (Li et al., 1993) and X sively studied. We analysed the methylation status of chromosome inactivation (Pfeifer et al., 1990). To date, CDKN2A and CDKN2D in human lung cancer cell lines four mammalian DNA methyltransferases (DNMT) and demonstrated that the CDKN2A CpG island is have been identi®ed (Bird and Wole, 1999). Disrup- methylated, whereas CDKN2D is unmethylated. Treat- tion of the balance in methylated DNA is a common ment of cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), alteration in cancer (Costello et al., 2000; Costello and an inhibitor of DNA methyltransferase 1, induced a dose Plass, 2001; Issa et al., 1993; Robertson et al., 1999). A and duration dependent increased expression of both proposed reason for this disruption seen in cancer is p16INK4a and p19INK4d, the products of CDKN2A and that genes that are methylated in the promoter regions CDKN2D, respectively. These data indicate that global (such as critical tumor suppressor genes) may be DNA demethylation not only in¯uences the expression of transcriptionally silenced in the tumors, thus providing methylated genes but also of unmethylated genes. a growth advantage for these cells (Jones and Laird, Histone acetylation is linked to methylation induced 1999). Usually, hypermethylation mediated gene silen- transcriptional silencing. Depsipeptide, an inhibitor of cing is limited to CpG islands in the promoter region histone deacetylase, acts synergistically with 5-Aza-CdR of genes (Baylin et al., 1998; Chan et al., 2000). In in inducing expression of p16INK4a and p19INK4d. However, contrast, gene exons are more frequently hypermethy- when cells were treated with higher concentrations of 5- lated at isolated CpGs (outside of CpG islands) and Aza-CdR and depsipeptide, p16INK4a expression was these methylated exons are not related to gene silencing decreased together with signi®cant suppression of cell (Herman and Baylin, 2000). There are some reports growth. Interestingly, p19INK4d expression was enhanced that the degree of methylation of CpG islands outside even more by the higher concentrations of 5-Aza-CdR of the promoter is positively correlated with an and depsipeptide. Our data suggest that p19INK4d plays a increase in gene expression (Chan et al., 2000; Glenn distinct role from other INK4 family members in et al., 1996; Gonzalgo et al., 1998). response to the cytotoxicity induced by inhibition of Recently, evidence has been accumulating that DNA methylation and histone deacetylation. Oncogene methylation inhibits gene transcription either directly (2001) 20, 7787 ± 7796. by interrupting the binding of transcription factors to promoters (Bird and Wole, 1999; Staiger et al., 1989), Keywords: DNA methylation; histone acetylation; or indirectly through methyl DNA binding proteins CDKN2D; p16INK4a; p19INK4d (MBPs) that bind preferentially to methylated DNA and recruit histone deacetylase enzymes (Nan et al., 1998; Ng and Bird, 1999). The binding sites of some transcription factors such as AP-2, E2F, and c-myc contain CpG dinucleotides (Tate and Bird, 1993). It *Correspondence: GA Otterson, Division of Hematology and has been postulated that methylation occurring at these Oncology, Department of Internal Medicine, The Ohio State binding sites may repress gene expression by blocking University, Room B415, Starling Loving Hall, 320 West 10th the access of transcription factors to the methylated Avenue, Columbus, OH 43210-1240, USA; DNA sequence (Kumari and Usdin, 2000; Staiger et E-mail: [email protected] Received 22 May 2001; revised 5 September 2001; accepted 13 al., 1989). Alternatively, or in addition to this September 2001 mechanism, MBPs have been shown to bind speci®cally 5-Aza-CdR increases expression of unmethylated CDKN2D W-G Zhu et al 7788 to methylated DNA sequences and may compete with tions of 5-Aza-CdR and depsipeptide induce decreased transcription factors for their binding sites (Robertson p16INK4a expression but even higher p19INK4d expres- and Jones, 2000). MBPs have also been demonstrated sion. to recruit histone deacetylases to hypermethylated areas of the promoter, thereby increasing localized histone deacetylation (Bird and Wole, 1999; Nan et Results al., 1998), resulting in gene repression. However, some genes appear to be repressed by The CpG island in the promoter of CDKN2D is mechanisms distinct from methylation. For example, unmethylated in human lung cancer cells Loeb et al. (2001) analysed the promoter region of the Wilms' tumor suppressor 1 gene (WT1) in 20 samples Although evidence has shown that the promoter region of normal breast epithelium and 19 primary breast of CDKN2A is hypermethylated in many human carcinomas. They found that the promoter region was cancers and cancer derived cell lines (Herman et al., hypermethylated in six out of 19 tumors and in one out 1995; Otterson et al., 1995), there has been no report of 20 normal breast epithelial cells. However, WT1 studying the methylation status of CDKN2D.To mRNA or protein was expressed in the six tumors that investigate the CpG island in the promoter region of were hypermethylated, whereas WT1 protein was not CDKN2D, we used the WebGene computer program expressed in the 19 normal breast epithelial cells that (http://www.itba.mi.cnr.it/webgene/) to select CpG were unmethylated (Loeb et al., 2001). In addition, islands around the predicted promoter of CDKN2D. Evron et al. (2001) have reported that although most Brie¯y, as shown in Figure 1a, a non-repetitive region breast tumors showed a correlation between hyper- B (from 71100 to +1) was identi®ed as a CpG island. methylation of the cyclin D2 promoter and gene In this region the GC content is 67% and the observed/ silencing, some did not. By analysing methylation in expected presence of CpG is 0.84, consistent with this the promoter of cyclin D2 in 13 breast tumors by being a CpG island (Gardiner-Garden and Frommer, methylation speci®c PCR (MS-PCR), the investigators 1987). In order to further narrow the putative found that ®ve of 13 tumors were unmethylated in the promoter region of CDKN2D, three computer algo- promoter sequences. Among these samples, two rithms were used to predict the transcriptional start showed cyclin D2 mRNA and protein expression site. Using Promoter 2.0 (Knudsen, 1999), McPromoter whereas three did not. These observations suggest (Ohler et al., 2000) and Promoter Scan II (Prestridge, alternative transcriptional silencing pathways. 1995), the transcriptional start sites fall within a 350 bp Loss of p16INK4a and p15INK4b protein expression in region C that is between (7) 400 to (7)750 bp from cancers has been frequently related to DNA methyla- the translational start site (+1). Our estimate regarding tion (Herman et al., 1995, 1996b; Otterson et al., 1995). the promoter region of human CDKN2D is consistent The methylation status of the related INK4 family with that suggested by another group (Yue Xiong, members, p18INK4c and p19INK4d has been less well personal communication). studied. Among the four members of the INK4 family, The methylation status of the CpG island in the methylation induced inactivation of p16INK4a has been region B (71000 to +1) was investigated with extensively reported in almost all human cancer cells Southern blot hybridization using a methylation (Herman et al., 1995), whereas methylation mediated sensitive restriction enzyme NotI in 12 human lung inactivation of p15INK4b has been described principally cancer cell lines and normal human lung tissue. DNA in hematologic malignancies (Herman et al., 1996b). As from human normal lung tissue demonstrated bands of a member of the family of cyclin D kinase inhibitors, 24 and 5.8 kb following digestion with EcoRV or p19INK4d is reported to play a similar function to EcoRV/NotI, respectively (Figure 1b, lanes 1 and 2). p16INK4a (Guan et al., 1996; Okuda et al., 1995). This The 24 kb band after incubation with both enzymes functional similarity to p16INK4a implies that p19INK4d would be consistent with lack of NotI digestion, may functionally replace p16INK4a when there is suggesting methylation of the NotI site. DNA from deletion, mutation or methylation of CDKN2A. all of the human lung cancer cell lines tested was Here we planned to investigate the methylation completely digested with NotI (showing the 5.8 kb status of CDKN2D and determine if expression of band), indicating an unmethylated NotI site in all p19INK4d is in¯uenced by treatment of cells with a human lung cancer cell lines tested (Figure 1b). As a DNA demethylating agent (5-aza-2'-deoxycytidine (5- comparison, we also performed Southern hybridization Aza-CdR)) or histone deacetylase inhibitor in human using SacII as a methylation-sensitive restriction lung cancer cells. We also analysed methylation status enzyme to detect methylation status of CDKN2A in of CDKN2A and expression of p16INK4a in these cells.