Measurement of Relative Copy Number of CDKN2A/ARF and CDKN2B in Bladder Cancer by Real-Time Quantitative PCR and Multiplex Ligation-Dependent Probe Amplification

Abstract

Many tumors have large homozygous deletions of the CDKN2A locus (encoding p14 ARF and p16) and of CDKN2B (p15). Our aim was to determine which gene is the major target in bladder cancer. We used quantitative real-time PCR (RTQ-PCR) to determine copy number of p15, of p14 ARF exon 1␤, and p16 exon 2 in 22 tumor cell lines and 83 bladder tumors, some of which had been assessed previously by duplex PCR. Titration experiments showed that homozygous deletion could be detected in the presence of up to 30% normal DNA. Results for cell lines were compatible with previous cytogenetic analyses. Ten cell lines and 32 tumors (38.5%) had homozygous deletion of at least one target. Thirteen tumors (15.7%) had deletion of all three targets. Two tumors had deletion of p14 ARF exon 1␤ alone and four of p16 exon 2 alone. RTQ-PCR detected more homozygous deletions than duplex PCR. Finally we used a multiplex ligation-dependent probe amplification kit to provide independent confirmation of results. We conclude that with appropriate controls RTQ-PCR is a sensitive and robust method to detect copy number changes in tumors even in the presence of contaminating normal cell DNA. More than 60% of transitional cell carcinomas (TCC) of the bladder of all stages and grades show loss of heterozygosity (LOH) of chromosome 9. 1-6 Although many tumors have LOH on both arms of the chromosome, a significant number (ϳ10% of tumors overall) have a region of interstitial LOH of 9p. 7 Many such deletions are small and this has allowed the identification of a critical region at 9p21 that contains CDKN2B and CDKN2A/ARF. These two loci encode three tumor suppressor genes, p15, p16, and p14 ARF , all of which are cyclin-dependent kinase inhibitors with key functions in cell cycle regulation. 8,9The CDKN2A locus encodes p16 and p14 ARF , both of which are capable of inducing cell cycle arrest. 10,11 p16 and p14 ARF have different first exons (1␣ and 1␤, respectively) approximately 13 kb apart, 12 giving rise to products in alternate reading frames with no homology at the protein level 13,14 (Figure 1). Exons 1␣, 2, and 3 encode p16, which induces G1 cell cycle arrest via the Rb pathway. Exons 1␤, 2, and 3 encode p14 ARF , which inhibits p53 degradation via binding to mdm2.The mechanism of CDKN2A/ARF inactivation in human cancers is somewhat tumor specific. Homozygous deletions at CDKN2A/9p21 are common in bladder tumors 6,15-17 and have been described in a variety of other sporadic tumors, including melanomas 18 and gliomas. 19 Pancreatic adenocarcinomas show inactivation of CDKN2A by either homozygous deletion or point mutation, 20 whereas esophageal tumors commonly show inactivation by point mutation. 21 A third mechanism of inactivation, transcriptional silencing by promoter hypermethylation, is commonly found in colorectal carcinoma. 22 Point mutations have only rarely been identified in bladder tumors 15,23,24 and promoter methylation, only infrequently. 25 Since the discovery of p14 ARF , 14 much interest has centered on the relative involvement of ...

Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway

“…There is evidence from deletion studies that this may be the key target at this locus in UCC (Aveyard and Knowles 2004;Chapman et al, 2005). The effect of modulation of the Rb pathway on NHUC lifespan was assessed either by expressing a mutant form of CDK4 (CDK4 R24C ) that is unable to bind p16 but retains cyclin-D-dependent kinase activity (Wolfel et al, 1995), or Id-1, a transcriptional repressor of p16 (Alani et al, 2001).…”

“…Terminal restriction fragment length was measured using the Telo TAGGG Telomere Length Assay kit (Roche, Hertfordshire, UK). CDKN2A status was analysed by MLPA using the P024 kit (MRC-Holland, Amsterdam, The Netherlands) (Aveyard and Knowles, 2004) and screened for exonic sequence variations (Chapman et al, 2005). TP53 was amplified in four fragments, comprising exons 2-4, exons 5-6, exons 7-9 and exons 10-11.…”

Chromosome 9p21 gene copy number and prognostic significance of p16 in ESFT

“…More recently, the technique has been applied to the analysis of copy number changes in tumours (Aveyard and Knowles, 2004).…”

Role of HPV in Urothelial Carcinogenesis: Current State of the Problem

“…Due to any of these events its expression at the level of protein p16 INK4a may become partly or fully lost. For example, homozygous INK4a deletions depriving cell of p16 INK4a synthesis were found in ~ 30-50 % of BC specimens (Aveyard, Knowles, 2004;Gallucci et al, 2007). Thus our data might prove unsuitability of p16 INK4a for role of the HPV-associated BC marker.…”

“…The point is, according to literature data, that factors determining INK4a expression in HPV-associated BC may differ in essence from those in HPV-positive CC. Thus in BC, in contrast to CC, INK4a undergoes frequent deletions, point mutations or promoter methylations (Ruas, Peters, 1998;Aveyard, Knowles, 2004;Gallucci et al, 2007). Due to any of these events its expression at the level of protein p16 INK4a may become partly or fully lost.…”