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

Abstract

The CDKN2 locus expresses two di erent mRNA transcripts, designated a and b. The protein product of the a transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The b 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 signi®cant 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 1a 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, ®broblasts, 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 1a and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1a was observed in ®ve 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 1a were strongly methylated. In tumours, imbalanced multiplex RT ± PCR data (p16INK4a5p19ARF) predicted methylation of exon 1a (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 1a 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.

utImmunohistochemical Detection of the Alternate INK4a-Encoded Tumor Suppressor Protein p14ARF in Archival Human Cancers and Cell Lines Using Commercial Antibodies: Correlation with p16INK4a Expression

“…Although we optimized the assay for only one of the five antibodies tested, monoclonal 14PO2 from NeoMarkers, which has been used before in immunofluorescence assays (19), it is quite possible that comparable results may be obtained with other anti-p14 ARF antibodies. In agreement with earlier studies, the presence of immunoreactive p14 ARF was indicated by granular or diffuse nuclear staining, with or without nucleolar accentuation (17,18); cytoplasmic staining was observed even in cells with homozygous deletions of the INK4a gene and may thus be nonspecific. In some normal cells, cell lines and tumors, the staining pattern was predominantly nucleolar, consistent with the recently described nucleolar sequestration of MDM2 by p14 ARF (7,19,20).…”

“…All four studies agree that p14 ARF loss is less common than down-regulation of p16 INK4a . Five of our lung cancers were negative for p16 INK4a but positive for p14 ARF , and this phenotype was observed in two earlier studies (17,18 (13). Interestingly, nine of 30 pancreatic carcinomas were positive for p14 ARF , suggesting that inactivation of this protein in this tumor type may not be as critical as abrogation of p16 INK4a .…”

“…IHC, immunohistochemistry; HD, homozygous deletion; ϩ, positive nuclear staining; Ϫ, no nuclear staining. studies on this tumor type (17,18,22). All four studies agree that p14 ARF loss is less common than down-regulation of p16 INK4a .…”

“…IHC allows evaluation of protein expression in specific cells and is applicable to most archival tissues. We are aware of only two previous immunohistochemical studies on p14 ARF expression in human cancers (17,18). Both of these used rabbit polyclonal antibodies, which are not commercially available, and only one of them was performed on paraffin sections (17).…”

Aberrant p16 promoter methylation in smokers and former smokers with nonsmall cell lung cancer

“…4 The tumour suppressor gene p16 (p16 INK4a /CDKN2A) is a cell cycle regulator that is frequently inactivated in many different types of malignancies, including lung cancer. [5][6][7] It has been demonstrated that p16 gene may be transcriptionally inactivated by aberrant promoter methylation during tumorigenesis. 8 -10 The gene product, p16 protein, is a molecular component of the retinoblastoma protein (pRB) regulatory pathway, functioning as a specific inhibitor of cyclin-dependant kinases 4 and 6.…”