TGF-β mediated G1 arrest in a human melanoma cell line lacking p15INK4B: Evidence for cooperation between p21Cip1/WAF1 and p27Kip1

V. A. Flørenes, N. Bhattacharya, M. R. Bani, Y. Ben-David, R. S. Kerbel, J. M. Slingerland

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Abstract

We have studied TGF-β mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15(INK4B) expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-β caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21(Cip1/WAF1) and p27(Kip1). These findings were confirmed in cells released from quiescence in the presence of TGF-β, in which TGF-β inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21(Cip1/WAF1) and p27(Kip1) with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-β mediated arrest of asynchronously growing cells. Upregulation of p21(Cip1/WAF1) preceded that of p27(Kip1). Furthermore, p21(Cip1/WAF1) and p27(Kip1) were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21(Cip1/WAF1) and p27(Kip1) immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-β arrested cells. It has been proposed that in TGF-β arrested epithelial cells, up-regulation of p15(INK4B) and of p15(INK4B) binding to cdk4 serves to destabilize the association of p27(Kip1) With cyclin D1/cdk4, promoting p27(Kip1) binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-β. In TGF-β arrested WM35, which lack p15(INK4B), the increased p21(Cip1/WAF1) may serve a similar function to that of p15(INK4B): initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27(KiP1) on G1 cyclin/cdks.

Original languageEnglish (US)
Pages (from-to)2447-2457
Number of pages11
JournalOncogene
Volume13
Issue number11
StatePublished - Dec 1 1996
Externally publishedYes

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Keywords

  • G arrest
  • Melanoma
  • p15(INK4B)-negative
  • p21(Cip1/WAF1)
  • p27(Kip1)
  • TFG-β

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research
  • Genetics

Cite this

Flørenes, V. A., Bhattacharya, N., Bani, M. R., Ben-David, Y., Kerbel, R. S., & Slingerland, J. M. (1996). TGF-β mediated G1 arrest in a human melanoma cell line lacking p15INK4B: Evidence for cooperation between p21Cip1/WAF1 and p27Kip1. Oncogene, 13(11), 2447-2457.