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Abstract
Genomic locus at chromosome 9p21 that contains the CDKN2A and CDKN2B tumor suppressor genes is inactivated through mutations, deletions and promoter methylation in multiple human cancers. Additionally, the locus encodes an anti-sense RNA (ANRIL). Both hemizygous and homozygous deletions at the locus targeting multiple genes are fairly common in different cancers. We in this study investigated breakpoints in five melanoma cell lines, derived from metastasized tumors, with previously identified homozygous deletions using array comparative genomic hybridization (aCGH). For breakpoint mapping, we used primer approximation multiplex PCR (PAMP) and inverse PCR techniques. Our results showed that three cell lines carried complex rearrangements. In two other cell lines, with focal deletions of 141 kb and 181 kb, we identified fusion gene products, involving MTAP and ANRIL. We also confirmed the complex rearrangements and focal deletions in DNA from tumor tissues corresponding to three cell lines. The rapid amplification of 3′cDNA ends (3′RACE) carried out on transcripts resulted in identification of three isoforms of MTAP-ANRIL fusion gene. Screening of cDNA from 64 melanoma cell lines resulted in detection of fusion transcripts in 13 (20%) cell lines that involved exons 4-7 of the MTAP and exon 2 or 5 of the ANRIL genes. We also detected fusion transcripts involving MTAP and ANRIL in two of the seven primary melanoma tumors with focal deletion at the locus. The results from the study, besides identifying complex rearrangements involving CDKN2A locus, show frequent occurrence of fusion transcripts involving MTAP and ANRIL genes.
Highlights
The cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) locus on chromosome 9p21, a target of frequent inactivation in various human cancers, encodes three tumor suppressors [1,2,3,4,5,6]
The pair wise copy-number data from 44 metastatic melanoma cell lines and corresponding peripheral blood mononuclear cells from a previous study was used to select cell lines with homozygous deletions (HD) [22]. 12 of the 44 cell lines carried HD at the 9p21 locus that ranged between 139 kb to 5.6 Mb (Supplementary Figure S1)
Three cell lines (MaMel-30, MaMel-95, MaMel-19) with deletions less than 500 kb and two cell lines (MaMel103a and MaMel-08a) with more than 2 Mb deletions were selected for mapping of breakpoints using primer approximation multiplex PCR (PAMP) or inverse PCR techniques (Supplementary Figure S2)
Summary
The cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) locus on chromosome 9p21, a target of frequent inactivation in various human cancers, encodes three tumor suppressors [1,2,3,4,5,6]. The locus transcribes a long intergenic noncoding RNA, termed as antisense non-coding RNA in the INK4 locus (ANRIL), involved in the repression of CDKN2A and 2B [7, 8]. Both p16 and p15 disrupt cell cycle progression through inhibition of retinoblastoma phosphorylation via disruption of CDK4/Cyclin D1 complex. Elevated expression of any of the three proteins encoded from the CDKN2A/2B locus results in cell cycle arrest leading to cellular senescence [10]. Germline mutations specific to p16 and to some extent to www.impactjournals.com/oncotarget
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