Low frequency of p16/CDKN2A methylation in sporadic melanoma: Comparative approaches for methylation analysis of primary tumors

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methy...

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Veröffentlicht in:Cancer research (Chicago, Ill.) Ill.), 1997-12, Vol.57 (23), p.5336-5347
Hauptverfasser: GONZALGO, M. L, BENDER, C. M, YOU, E. H, GLENDENING, J. M, FLORES, J. F, WALKER, G. J, HAYWARD, N. K, JONES, P. A, FOUNTAIN, J. W
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container_issue 23
container_start_page 5336
container_title Cancer research (Chicago, Ill.)
container_volume 57
creator GONZALGO, M. L
BENDER, C. M
YOU, E. H
GLENDENING, J. M
FLORES, J. F
WALKER, G. J
HAYWARD, N. K
JONES, P. A
FOUNTAIN, J. W
description Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.
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All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. 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source MEDLINE; American Association for Cancer Research; EZB-FREE-00999 freely available EZB journals
subjects Biological and medical sciences
Chromosome Deletion
Chromosome Mapping
Chromosomes, Human, Pair 9
Dermatology
Dinucleoside Phosphates
DNA Methylation
DNA Primers
DNA, Neoplasm - chemistry
Genes, p16
Humans
Medical sciences
Melanoma - genetics
Melanoma - metabolism
Melanoma - pathology
Mutation
Polymerase Chain Reaction
Promoter Regions, Genetic
Restriction Mapping
Skin Neoplasms - genetics
Skin Neoplasms - metabolism
Skin Neoplasms - pathology
Tumor Cells, Cultured
Tumors of the skin and soft tissue. Premalignant lesions
title Low frequency of p16/CDKN2A methylation in sporadic melanoma: Comparative approaches for methylation analysis of primary tumors
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