Frequent p16-independent inactivation of p14ARF in human melanoma.
BACKGROUND
The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene.


METHODS
We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided.


RESULTS
We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases.


CONCLUSIONS
Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16.
Freedberg
Daniel E
DE
Department of Dermatology, New York University School of Medicine, 550 First Ave, New York, NY 10016, USA.
Rigas
Sushila H
SH
Russak
Julie
J
Gai
Weiming
W
Kaplow
Margarita
M
Osman
Iman
I
Turner
Faye
F
Randerson-Moor
Juliette A
JA
Houghton
Alan
A
Busam
Klaus
K
Timothy Bishop
D
D
Bastian
Boris C
BC
Newton-Bishop
Julia A
JA
Polsky
David
D
D000818:Animals; D045744:Cell Line, Tumor; D002899:Chromosomes, Human, Pair 9; D019941:Cyclin-Dependent Kinase Inhibitor p16; D019175:DNA Methylation; D017353:Gene Deletion; D020868:Gene Silencing; D019942:Genes, p16; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008545:Melanoma / Q000235:genetics* / Q000473:pathology*; D008546:Melanoma, Experimental / Q000235:genetics / Q000473:pathology; D051379:Mice; D018895:Microsatellite Repeats; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics*
C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009363:Neoplasm Proteins; D025803:Tumor Suppressor Protein p14ARF