Point mutations can inactivate in vitro and in vivo activities of p16INK4a/CDKN2A in human glioma

Abstract

Deletions of chromosomal region 9p21 are among the most common genetic alterations observed during the clonal evolution of high grade malignant gliomas. Structural and functional evidence has suggested that homozygous deletion involving CDKN2A (the genetic locus encoding the cyclin-dependent kinase inhibitor p16 INK4a ) is a mechanism of inactivation of this gene and that it can be a growth suppressor in human gliomas. However, the presence of other potential suppressor genes in the 9p21 region and the relatively large sizes of the deletions has made it di cult to be certain that the CDKN2A gene is their actual target. Here, we tested this hypothesis by determining the growth suppressive e ects, cell cycle inhibitions, and the activities of seven naturally occurring glioma-derived CDKN2A alleles carrying point mutations and found that two of them were functionally compromised. To resolve discrepancies among the di erent existing functional assays, we developed an assay for p16 INK4a function that allowed us to demonstrate that the expression of wild-type CDKN2A, but not alleles with inactivating mutations, prevents pRB phosphorylation in vivo in human glioma cells. These data suggest that CDKN2A is a critical target for mutational inactivation in human malignant gliomas.

Functional evaluation of tumour-specific variants of p16INK4a/CDKN2A: correlation with protein structure information

“…Similarly, our functional data imply that H98Y is not a mutant and the same conclusion was drawn recently by Arap et al in a survey of glioma-associated sequence variants (Arap et al, 1997). This contrasts with H98P which we and others have shown to be functionally defective (Koh et al, 1995;Parry and Peters, 1996;Zhang and Peng, 1996).…”

“…To try to address this possibility, we explored alternative assays based on ectopic expression of p16 INK4a sequences in eukaryotic cells. Several procedures have been described that use two types of readout: the ability of p16 INK4a to cause a G1 cell cycle arrest in transfected cells (Koh et al, 1995;Lukas et al, 1995;Shapiro et al, 1995;Parry and Peters, 1996;Arap et al, 1997) and the interaction of p16 INK4a with either endogenous or exogenous CDKs (Reymond and Brent, 1995;Shapiro et al, 1995;Yang et al, 1995;Castellano et al, 1997b). One example of the latter approach would be the yeast two hybrid system, which has been used to some e ect both in the initial identi®cation of p16 INK4a (Serrano et al, 1993) and in the functional evaluation of mutants (Reymond and Brent, 1995;Yang et al, 1995).…”

“…Only a minority of the known missense variants have been functionally analysed and although the severe loss-offunction mutants are easily detected, a substantial proportion of the variants tested thus far behave ambiguously in di erent assay systems or have given inconsistent results when analysed by di erent laboratories (Koh et al, 1995;Lukas et al, 1995;Ranade et al, 1995;Reymond and Brent, 1995;Shapiro et al, 1995;Wick et al, 1995;Yang et al, 1995;Enders et al, 1996;Lilischkis et al, 1996;Parry and Peters, 1996;Tevelev et al, 1996;Yarbrough et al, 1996;Zhang and Peng, 1996;Arap et al, 1997;Harland et al, 1997;Sun et al, 1997). There are a number of possible reasons.…”

The INK4 family of cell cycle inhibitors in cancer

“…However, some germline mutations a ecting p16 INK4a in melanoma-prone persons (Hussussian et al, 1994;Gruis et al, 1995;Washimi et al, 1995;Fitzgerald et al, 1996), exclusively target exon 2 of the INK4a gene, apparently without a ecting p19 ARF function . These mutations prevent p16 INK4a from binding CDK4 (Ranade et al, 1995;Koh et al, 1995;Yang et al, 1995;Arap et al, 1997). Conversely, mutations in CDK4 that prevent INK4 binding have also been found in human melanomas (WoÈ lfel et al, 1995;Ruas and Peters, 1998).…”

Translation of p15.5INK4B, an N-terminally extended and fully active form of p15INK4B, is initiated from an upstream GUG codon

“…CDKN2B is frequently homozygously or heterozygously deleted together with CDKN2A in a variety of tumour cell lines and primary tumours (Hirama and Koe er, 1995). Overexpression of p15 is able to block cell cycle progression in G1 (Arap et al, 1997;McConnell et al, 1998;Stone et al, 1995). We have recently found that overexpression of p15 from an adenovirus vector, blocks cell proliferation, induces replicative senescence, and downregulates telomerase activity similarly to p16 (Fuxe et al, 2000 unpublished results).…”