Evidence for a Role for P16 in Replicative Senescence and Immortality in Human Keratinocytes

Loughran, Oonagh; Malliri, Angeliki; Owens, David M.; Ozanne, Brad; Frame, Margaret C.; Parkinson, E.K.

doi:10.1042/bst024550sa

Cited by 14 publications

(20 citation statements)

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“…The very low turnover of p16 mRNA and the observations of high levels of p16 in late passage number ®broblasts and keratinocytes are both in line with the idea that once p16 expression is induced it will lead to an irreversible exit from the cell cycle as part of the senescence mechanism Loughran et al, 1996;Alcorta et al, 1996). This hypothesis is supported by data suggesting the existence of a gene involved in senescence mapping to the 9p21 locus (Loughran et al, 1994) and the loss of senescence in p16 7/7 mouse embryo ®broblasts .…”

Section: Introductionsupporting

confidence: 73%

“…Di erent signalling pathways have been suggested to control the levels of INK4a proteins in the cells. p15 expression is induced after the cells have been treated with TGF-b (Hannon and Beach 1995) and increased levels of p16 and p18 are observed in cells of late passage numbers (Hara et al, 1996;Alcorta et al, 1996;Loughran et al, 1996) and in skeletal muscle cells undergoing di erentiation (Franklin and Xiong, 1996), respectively. This might suggest that in spite of similar biochemical activity, it is possible that the e ect of CDK inhibition in the cell by the INK proteins can vary depending on the cell phenotype.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule

et al. 1998

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We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat of the p16 CDKN2/INK4a (p16) tumour suppressor protein (residues 84 ± 103 of the human p16 protein) can bind to cdk4 and cdk6 and inhibit cdk4-cyclin D1 kinase activity in vitro as well as block cell cycle progression through G1. Substitution of two valine residues corresponding to amino acids 95 and 96 (V95A and V96A) of the p16 peptide reduces the binding to cdk4 and cdk6 and increases its IC 0.5 for kinase inhibition approximately threefold when linked to the Antennapedia homeodomain carrier sequence. The same mutations increase the IC 0.5 approximately ®vefold in the p16 protein. Substitution of aspartic acid 92 by alanine instead increases the binding of the peptide to cdk4 and cdk6 and the kinase inhibitory activity. The p16 peptide blocks S-phase entry in nonsynchronized human HaCaT cells by approximately 90% at a 24 mM concentration. The V95A and V96A double substitution minimizes the cell cycle inhibitory capacity of the peptide whereas the D92A substitution increases its capacity to block cell cycle progression. A deletion series of the p16 derived peptide shows that a 10 residue peptide still retains cdk4-cyclin D1 kinase and cell cycle inhibitory activity. The p16 peptide inhibited S-phase entry in ®ve cell lines tested, varying between 47 ± 75%, but had only a limited (11%) inhibitory e ect in the pRb negative Saos-2 cells at a concentration of 24 mM. Like the full length p16 protein, the p16 peptide does not inhibit cyclin E dependent cdk2 kinase activity in vitro. These data suggest that acute inhibition of CDKcyclin D activity by a peptide derived from the INK4 family will stop cells in late G1 in a pRb dependent fashion.

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Section: Introductionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule

et al. 1998

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“…The p16 ink4a protein has been linked to senescence (Alcorta et al, 1996;Loughran et al, 1996;Serrano et al, 1997;Foster et al, 1998), and as high levels of p16 ink4a were re-expressed in SCC15 after 5-Aza-CdR treatment, we investigated whether re-expression of p16 ink4a in this cancer cell line would prompt the cells to undergo senescence.…”

Section: Resultsmentioning

confidence: 99%

“…Deletions and mutations of the p16 ink4a gene have been described in several oral cancers and cell lines (Zhang et al, 1994;Lydiatt et al, 1995;Reed et al, 1996), and recently, loss of p16 ink4a protein expression in oral carcinomas has also been linked to promoter hypermethylation (Loughran et al, 1996). One speci®c CpG island in the p16 ink4a gene was found methylated in 20% of primary head and neck cancers (Gonzalez et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2′-deoxycytidine treatment induces a senescence-like state

1998

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We have previously reported that a set of oral squamous cell carcinoma lines express speci®cally elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 ampli®cation and expresses functional p16 ink4a , the other cell lines express undetectable levels of p16 ink4a , despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hypermethylated p16 ink4A promoter and a third cell line, SCC9, has a mutation in the p16 ink4a promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16 ink4a protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16 ink4a . In this line, cdk6 activity was decreased after 5-aza-2'deoxycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein pRB was detected. Expression of p16 ink4a persisted, even after the drug was removed and the cells expressed senescence-associated b-galactosidase activity. Ectopic expression of p16 ink4a with a recombinant retrovirus in this cell line also induced a similar senescence-like phenotype. Hence, it was possible to restore a functional pRB pathway in an oral squamous cell carcinoma line by inducing re-expression of endogenous p16 ink4a in response to treatment with a demethylating agent.

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“…All four proteins bind directly to CDK4 and CDK6 in vitro but, despite their biochemical and structural similarities, only p16 INK4a has the credentials of a tumour suppressor. A likely explanation is that p16 INK4a accumulates as cells reach the end of their replicative lifespan (senescence) so that inactivation of p16 INK4a favours the emergence of immortal cell clones (Alcorta et al, 1996;Hara et al, 1996;Loughran et al, 1996;Rezniko et al, 1996;Yeager et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 1999

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Evidence for a Role for P16 in Replicative Senescence and Immortality in Human Keratinocytes

Cited by 14 publications

References 0 publications

Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule

Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule

Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2′-deoxycytidine treatment induces a senescence-like state

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

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