Hypermethylation Can Selectively Silence Individual pltfnk4A AlÃ-elesin Neoplasia1

Inactivation of plf>'"l"IAand other tumor suppressor genes has been associated with promoter region hypermethylation in neoplasia. However, direct proof for aberrant DNA methylation as an independent event for loss of gene function has been difficult to obtain. We addressed this question in the colon carcinoma cell line HCT116, which contains one alÃ-eleof plSnk4A with a coding region fra mes hift mutation and one wildtype alÃ-ele.Neither alÃ-elecontains a mutation in the proximal promoter region. The promoter of the wild-type alÃ-ele,but not the mutant alÃ-ele,is hypermethylated, and only the mutant alÃ-eleis expressed. Transcription from the methylated/wild-type alÃ-elewas restored after cell treatment with the demethylating agent 5-aza-2'-deoxycytidine. Thus, in neoplastic cells, stable allele-specific loss of transcription may arise from aberrant meth ylation of a nonmutated promoter region, identifying hypermethylation as a direct mechanism for tumor suppressor gene inactivation. Introduction Inactivation of the pl6lnk4A cyclin-dependent kinase inhibitor is one of the most commonly observed abnormalities in human can cer, and inactivation of the cyclin D-pl6-Rb pathway could indeed be required for progression of most tumor types (1). Whereas genetic mechanisms, including point mutations (2-4) and, more commonly, homozygous deletions (2, 5), often inactivate pl6mk4A in tumors, loss of pl6'"k4A expression and several other tumor suppressor genes can also be associated with promoter region hypermethylation (6-13). This latter change accompanies a transcriptional repression that seems to be an alternative to coding region mutations for loss of gene function (6, 10, 14). However, molecular evidence that promoter region hypermethylation is di rectly associated with transcriptional loss has been difficult to establish. We now show that in the same cell, individual alÃ-elesof pl6mk4A are transcriptionally silenced by abnormal DNA methyl ation, demonstrating that DNA methylation can lead directly to loss of transcription, rather than simply being a mark for this process. Materials and Methods Southern Analysis and Cell Culture. Methylation analysis of the 5' CpG island of pl6 was performed exactly as described previously (7). Quantitation was carried out using ImageQuant software after exposure using a Phosphorlmager (Molecular Dynamics). Treatment with 5-aza-2'-deoxycytidine was at a concentration of 1 J^Mfor 5 days as described previously (10). MSP.3 Bisulfite treatment was as described previously (15). Unmethylated and methylated alÃ-elesof pl6mMA were specifically amplified from bisulfitemodified DNA with an anchored upstream primer Ul (5'-TTTTTAGAGGAl'1'1 GAGGGATAG) and methylation-specific antisense primers pl6-U2 Received 10/13/97; accepted 1/2/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by NIH Grants CA43318 and CA54396. S. K. M. is supported by The Academy of Finland, and J. G. H. is a V Foundation Scholar. 2 To whom reprint requests should be addressed, at The Oncology Center, The Johns Hopkins Medical Institutions, 424 North Bond Street, Baltimore, MD 21231. 3 The abbreviation used is: MSP, methylation-specific PCR. and pl6-M2 (15), respectively. A nested PCR was performed with biotinylated antisense primer BIO U/M (5'-AATCRACCTCCRACCRTAACTATT) and sense primer U2N (5'-GATTTGAGGGATAGGGTAGGAGG) to produce sin gle-stranded templates for DNA sequencing (16). Biotinylated PCR products were isolated using streptavidin-coated magnetic beads (Dynal AB, Oslo, Norway). Sequencing reactions were performed with Sequenase DNA se quencing kit (United States Biotechnology) after strand separation with alkali treatment. A nested, methylation nonspecific primer, ploseq (5'-TTTTGTTAGTATTAGGAGGAAGA), was used in sequencing. Genomic sequencing of normal colon was performed with two sets of primers that do not contain any CpG sites: Ul (15) and LI (5'-CTACCTAATTCCAATTCCCCTACA) for the first PCR; and U2N (15) and L2BIO (5'-TCCAATTCCCCTACAAACTTC) for the nested PCR. Primer ploseq was used as a sequencing primer (15). cDNA Analysis. cDNA synthesis was performed as described previously (10). Primers 5'-TGGAGCCTTCGGCTGACT and 5'-GGGACCTTCCGCGGCCAT from exon 1 and exon 2, respectively, were used to amplify a product of 428 bp. Primers for the nested PCR, exla (5'-TTCGGCTGACTGGCTGGC) and exlb (5'-CTGGATCGGCCTCCGAC), were both from exon 1. The lower-strand primer was labeled with biotin to facilitate solid-phase DNA sequencing of the amplified products. Primer ex 1a was used as a sequencing primer. Promoter Sequencing. Primers PIA (5'-TAGCTCCCTCCCCATTTTCCTAT) and P1B (5'-ACCCTCTACCCACCTGGATCG) were used to amplify a 1068-bp product of the piÃ3'"1"1*promoter region (-905 to +163 from ATG) followed by nested PCR with primers P2A (S'-TTTTCCTATCTGCCTACAGGC-3') and P2B (5'-GGCCTCCGACCGTAACTAT) that amplify a product of 1034 bp. The lower-strand primer in the nested PCR was biotin ylated. Sequencing was performed with primers P2A, SeqAl (5'-TGAACCCCGCGCTCCTG), SeqA (5'-TGGCAGTTAGGAGGTTGT), SeqB (5'TTCGCTAAGTGCTCGGAGT), and SeqC (5'-AGGAGGGGCTGGCTG-