p130 mediates TGF-β-induced cell-cycle arrest in Rb mutant HT-3 cells

Abstract

Objective. The retinoblastoma proteins include Rb and the functionally and structurally related proteins p107 and p130. It has been reported that HT-3 cells are sensitive to TGF-beta growth inhibition, despite the Rb mutation. The purpose of this study was to elucidate the growth-inhibitory mechanism of TGF-beta in Rb mutant HT-3 cells. Methods. Growth inhibition by TGF-beta in cervical carcinoma cell lines was evaluated by counting cell numbers. Cell-cycle distribution was determined by staining DNA with propidium iodide (PI) and measured using a flow cytometer. The level of each protein expression was determined by Western blot analysis. To evaluate the assembly of cdk2/p21, cdk2/cyclin E, and E2174/p130 complexes by TGF-beta, immunoprecipitation was performed. Results. TGF-beta inhibited the proliferation of HT-3 cells expressing mutant Rb protein and efficiently induced cell-cycle arrest at G, phase. p21 protein level was elevated in TGF-beta-treated HT-3 cells, while other G, regulatory protein levels were unaltered. TGF-beta markedly enhanced the binding of p21 with cdk2 but decreased that of cdk2 with cyclin E and inhibited the phosphorylation of p130 but did not change Rb and p107 protein status. We also found that E2F-1 protein level was lower in TGF-beta-treated cells and suggest that this might be the result of enhanced binding between E2F-4 and p130. Conclusions. Our results demonstrate that p130, instead of Rb, can mediate growth inhibition by TGF-beta in Rb mutant HT-3 cells. (C) 2002 Elsevier Science (USA).