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

Ruas, Margarida; Brookes, Sharon; McDonald, Neil Q.; Peters, Gordon

doi:10.1038/sj.onc.1202918

Cited by 70 publications

(57 citation statements)

References 69 publications

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“…The mixed proteins were immunoprecipitated with an antiserum against p16 and analysed by SDS-PAGE. As revealed in Figure 3A, the P48T variant was indistinguishable from wild-type p16 in its ability to interact with CDK4 or CDK6, while the previously documented mutant A20P (Ruas et al, 1999) showed no detectable binding to CDK4 or CDK6. Neither the wild-type nor the variant p16 proteins interacted with CDK2 (data not shown).…”

Section: Functional Analysis Of the P48t Mutant Proteinmentioning

confidence: 81%

“…We found that the P48T variant of p16 is functionally impaired in its ability to inhibit the cell growth and cell cycle progression, thus suggesting a causal role for this mutation. However, the in vitro CDK binding assay has shown that the P48T variant retains some activity, placing it in the category of mutants that are partially impaired (Ruas et al, 1999). Interestingly, the P48 residue has been shown to be structurally buried, contributing to the hydrophobic core of the protein, and previous analysis of a different alteration at this residue, P48L, revealed a p16 variant defective in both CDK binding in vitro and cell proliferation inhibition (Ruas et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

“…Two copies of the HA tag were incorporated at the amino terminus of the wild-type p16 construct in pBABEpuro. Retroviral infection of TIG-ER fibroblasts and analysis of cell proliferation effects were performed according to the previously described procedure (Ruas et al, 1999). For cell cycle inhibition assay, the CDKN2A cDNA was cloned into the BamHI site of pCMVneoBam expression vector (Baker et al, 1990), and transfected in human osteosarcoma cell line U2OS, negative for p16 expression, together with a plasmid encoding the CD20 cell surface marker (Koh et al, 1995).…”

Section: Functional Assaysmentioning

confidence: 99%

“…In vitro binding to CDK4/6 represents a relatively simple but non-quantitative test of p16 function and previous investigations have revealed a number of cancer-associated p16 variants that retain CDK-binding potential in vitro yet appear functionally impaired in vivo (Ruas et al, 1999). To determine whether the P48T variant belonged to this category, we used a recombinant retrovirus to express this variant in human diploid fibroblasts engineered to express the receptor for ecotropic viruses (TIG-ER cells) (Ruas et al, 1999).…”

Section: Functional Analysis Of the P48t Mutant Proteinmentioning

confidence: 99%

“…To determine whether the P48T variant belonged to this category, we used a recombinant retrovirus to express this variant in human diploid fibroblasts engineered to express the receptor for ecotropic viruses (TIG-ER cells) (Ruas et al, 1999). By monitoring the proliferation of TIG-ER cells expressing the P48T variant ( Figure 3B), we found that this mutant protein had a diminished ability to inhibit cell growth, whereas wild-type p16 caused growth arrest and the empty vector had no effect on cell proliferation.…”

Section: Functional Analysis Of the P48t Mutant Proteinmentioning

confidence: 99%

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CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

et al. 2001

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Summary Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a noncarrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.

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Section: Functional Analysis Of the P48t Mutant Proteinmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Functional Assaysmentioning

confidence: 99%

Section: Functional Analysis Of the P48t Mutant Proteinmentioning

confidence: 99%

Section: Functional Analysis Of the P48t Mutant Proteinmentioning

confidence: 99%

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CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

et al. 2001

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Sporadic multiple primary melanoma cases: CDKN2A germline mutations with a founder effect

Auroy

Avril

Chompret

et al. 2001

Genes Chromosomes & Cancer

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Multiple primary cancers are one of the hallmarks of inherited predisposition. Outside the familial context, multiple primary tumors could be related either to germline de novo mutations or to low-penetrance mutations, in predisposing genes. We selected 100 patients who displayed multiple primary melanoma (MPM) without any known melanoma cases recorded within their families and looked for germline mutations in the two melanoma-predisposing genes identified to date, CDKN2A and CDK4 exon 2. Nine patients (9%) had germline mutations in CDKN2A, whereas none carried germline mutations in exon 2 of CDK4. Seven cases displayed a recurrent missense mutation, G101W, already described in more than 20 melanoma-prone families; one case carried a missense mutation never reported to date (P114S), and the last case was a carrier of a 6 bp insertion at nucleotide 57 resulting in a duplication of codons 18 and 19. To ascertain whether the G101W was a mutational hot spot for de novo mutations or a common founder mutation, we genotyped eight microsatellite markers flanking the CDKN2A gene. After allowing for recombination over time, haplotype sharing provided evidence for an original G101W mutation common to 6 out of 7 sporadic MPM cases. Therefore, it can be concluded that de novo germline CDKN2A mutations associated with MPM are rare.

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Frequent loss of the CDKN2C (p18^INK4c) gene product in pituitary adenomas

Kirsch

Mörz

Pinzer

et al. 2008

Genes Chromosomes & Cancer

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Genomic alterations of cyclin-dependent kinase inhibitors have been demonstrated in a variety of tumor types including brain tumors. Among them, the cyclin-dependent kinase inhibitor 2A (CDKN2A or p16(INK4a)) gene has been shown to be frequently deleted or inactivated in astrocytic tumors. The CDKN2C (p18(INK4c)) gene is functionally related to CDKN2A. Moreover, mice with targeted disruption of CDKN2C alone or combined CDKN2C and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27(Kip1)), or CDKN2C and TP53 gene disruption develop pituitary adenomas (PA) at high frequencies. The purpose of our study was to investigate genetic alterations of the CDKN2C gene by analysis of loss of heterozygosity (LOH), screening for mutations, analysis of promoter methylation, and protein expression in 38 PAs. In addition, genomic alterations and protein expression of the cell cycle genes CDKN2A and its alternatively spliced form, p14(ARF), as well as the retinoblastoma RB1 gene were investigated. LOH at the CDKN2C gene locus was detected in 25% of pituitary adenomas, whereas the RB1 and CDKN2A loci were altered in only 10%. No mutations were detected within the coding regions of the CDKN2C gene. However, 39.5% of adenomas displayed CDKN2C promoter methylation. The absence of CDKN2C protein was correlated with LOH of the CDKN2C locus on chromosome 1 and with methylation of the CDKN2C promoter. This is the first report to describe that the tumor suppressor gene CDKN2C is frequently targeted by genomic alterations in pituitary adenoma. The most common genetic alteration was promoter methylation suggesting that inactivation of CDKN2C by this mechanism may play an important role in pituitary adenoma development. Additional Supporting Information may be found in the online version of this article.

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

Cited by 70 publications

References 69 publications

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

Sporadic multiple primary melanoma cases: CDKN2A germline mutations with a founder effect

Frequent loss of the CDKN2C (p18^INK4c) gene product in pituitary adenomas

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

Cited by 70 publications

References 69 publications

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

Sporadic multiple primary melanoma cases: CDKN2A germline mutations with a founder effect

Frequent loss of the CDKN2C (p18INK4c) gene product in pituitary adenomas

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Frequent loss of the CDKN2C (p18^INK4c) gene product in pituitary adenomas