TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1.
We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta 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 p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.
Flørenes
V A
VA
Department of Medicine, University of Toronto, Sunnybrook Health Science Centre, Ontario, Canada.
Bhattacharya
N
N
Bani
M R
MR
Ben-David
Y
Y
Kerbel
R S
RS
Slingerland
J M
JM
D002352:Carrier Proteins / Q000235:genetics; D018797:Cell Cycle Proteins; D002899:Chromosomes, Human, Pair 9 / Q000235:genetics*; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016193:G1 Phase / Q000187:drug effects* / Q000502:physiology; D015321:Gene Rearrangement; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation
C493956:CDKN1A protein, human; C493966:CDKN2B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D016160:Retinoblastoma Protein; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27