p130 mediates TGF-beta-induced cell-cycle arrest in Rb mutant HT-3 cells.
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 E2F-4/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(1) phase. p21 protein level was elevated in TGF-beta-treated HT-3 cells, while other G(1) 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.
Choi
Hyun Ho
HH
Cancer Research Center, Seoul National University College of Medicine, Seoul, Republic of Korea.
Jong
Hyun Soon
HS
Hyun Song
Sang
S
You Kim
Tae
T
Kyeong Kim
Noe
N
Bang
Yung Jue
YJ
D001798:Blood Proteins / Q000378:metabolism*; D002453:Cell Cycle / Q000235:genetics; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016161:Genes, Retinoblastoma / Q000235:genetics*; D006131:Growth Inhibitors / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*
D001798:Blood Proteins; D006131:Growth Inhibitors; D011506:Proteins; C493837:RBL2 protein, human; D050717:Retinoblastoma-Like Protein p130; D016212:Transforming Growth Factor beta