EXPRESSION OF P16INK4A P16-ALPHA AND P19ARF/P16-BETA IS FREQUENTLY ALTERED IN NONSMALL CELL LUNG-CANCER AND CORRELATES WITH P53 OVEREXPRESSION/

The CDKN2 locus expresses two different mRNA transcripts, designated a lpha and beta. The protein product of the or transcript is the cell cy cle inhibitor and tumour suppressor p16INK4a. The beta transcript is t ranslated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p 16INK4a has shown that the protein is downregulated in a significant n umber of tumours, but less is known on the expression of the p19ARF, W e have examined the expression of p16INK4a and p19ARF in resectable no n-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiple x RT-PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1 alpha methylation was analysed in a P CR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tu mours expressed p16INK4a and p19ARF at nil to low levels, respectively , p19ARF was localized primarily to the nuclei of tumour cells, but wa s 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 p16INK4 a expression, 11 tumours exhibited full methylation of at least one si te within exon 1 alpha and these tumours showed normal p19ARF expressi on. In contrast, no methylation of exon 1 alpha was observed in five t umours which also lacked p19ARF, In normal lung, p16INK4a and p19ARF w ere not expressed at detectable levels, the multiplex RT-PCR results w ere balanced, and sites within exon 1 alpha were strongly methylated. In tumours, imbalanced multiplex RT-PCR data (p16INK4a < p19ARF) predi cted methylation of exon 1 alpha (P = 0.0006) as well as downregulatio n of p16INK4a, p19ARF downregulation was inversely correlated with p53 overexpression (P = 0.025), whilst negative immunostaining for p16INK 4a was inversely correlated with pRb downregulation (P = 0.003) and di rectly correlated with p53 overexpression as assessed by immunostainin g (P = 0.015), Our results show that: (1) p16INK4a and p19ARF expressi on are altered in almost half of resectable NSCLC; (2) methylation wit hin exon 1 alpha is a frequent, but not the only mechanism of p16INK4a downregulation; and that (3) the inverse association of p19ARF and p5 3 alteration is consistent with a linked pathway.