Expression of p16INK4a/p16α and p19ARF/p16β is frequently altered in non-small cell lung cancer and correlates with p53 overexpression

The CDKN2 locus expresses two different mRNA transcripts, designated α and β. The protein product of the α transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The β transcript is translated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p16INK4a has shown that the protein is downregulated in a significant number of tumours, but less is known on the expression of the p19ARF. We have examined the expression of p16INK4a and p19ARF in resectable non-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiplex RT–PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1α methylation was analysed in a PCR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tumours expressed p16INK4a and p19ARF at nil to low levels, respectively. p19ARF was localized primarily to the nuclei of tumour cells, but was also seen to varying degrees in nuclei of lymphocytes, chondrocytes, fibroblasts, and epithelial cells. No tumour with normal p16INK4a had decreased p19ARF expression. Among 16 tumours with nil to low p16INK4a expression, 11 tumours exhibited full methylation of at least one site within exon 1α and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1α was observed in five tumours which also lacked p19ARF. In normal lung, p16INK4a and p19ARF were not expressed at detectable levels, the multiplex RT–PCR results were balanced, and sites within exon 1α were strongly methylated. In tumours, imbalanced multiplex RT–PCR data (p16INK4a<p19ARF) predicted methylation of exon 1α (P=0.0006) as well as downregulation of p16INK4a. p19ARF downregulation was inversely correlated with p53 overexpression (P=0.025), whilst negative immunostaining for p16INK4a was inversely correlated with pRb downregulation (P=0.003) and directly correlated with p53 overexpression as assessed by immunostaining (P=0.015). Our results show that: (1) p16INK4a and p19ARF expression are altered in almost half of resectable NSCLC; (2) methylation within exon 1α is a frequent, but not the only mechanism of p16INK4a downregulation; and that (3) the inverse association of p19ARF and p53 alteration is consistent with a linked pathway.