title: Expression of p16INK4a/p16alpha and p19ARF/p16beta is frequently altered in non-small cell lung cancer and correlates with p53 overexpression.
abstract: The CDKN2 locus expresses two different mRNA transcripts, designated alpha and beta. The protein product of the alpha transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The beta 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 1alpha 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 1alpha and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1alpha 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 1alpha were strongly methylated. In tumours, imbalanced multiplex RT-PCR data (p16INK4a<p19ARF) predicted methylation of exon 1alpha (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 down-regulation (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 1alpha 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.
authors.affiliation: Department of Clinical Research, University of Bern, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Vonlanthen
authors.forename: J
authors.initials: J
authors.lastname: Heighway
authors.forename: M P
authors.initials: MP
authors.lastname: Tschan
authors.forename: M M
authors.initials: MM
authors.lastname: Borner
authors.forename: H J
authors.initials: HJ
authors.lastname: Altermatt
authors.forename: A
authors.initials: A
authors.lastname: Kappeler
authors.forename: A
authors.initials: A
authors.lastname: Tobler
authors.forename: M F
authors.initials: MF
authors.lastname: Fey
authors.forename: N
authors.initials: N
authors.lastname: Thatcher
authors.forename: W G
authors.initials: WG
authors.lastname: Yarbrough
authors.forename: D C
authors.initials: DC
authors.lastname: Betticher
mesh_terms: D000368:Aged; D000818:Animals; D019556:COS Cells; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D019938:Cyclin D1 / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005260:Female; D016193:G1 Phase / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D005807:Genes, Overlapping; D016147:Genes, Tumor Suppressor; D019942:Genes, p16; D016158:Genes, p53; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D016160:Retinoblastoma Protein / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D011506:Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016160:Retinoblastoma Protein; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1