G(1) arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637

被引:61
作者
Schnier, JB
Nishi, K
Goodrich, DW
Bradbury, EM
机构
[1] UNIV TEXAS,MD ANDERSON CANC CTR,DEPT TUMOR BIOL,HOUSTON,TX 77030
[2] LOS ALAMOS NATL LAB,DIV LIFE SCI,LOS ALAMOS,NM 87545
关键词
cell cycle; p21(Waf1)/(Cip1); p27(Kip1);
D O I
10.1073/pnas.93.12.5941
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The protein kinase inhibitor staurosporine has been shown to induce G(1) phase arrest in normal cells but not in most transformed cells, Staurosporine did not induce G(1) phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB(-)). However, when infected with a pRB-expressing retrovirus [Goodrich, D, W., Chen, Y,, Scully, P, & Lee, W.-H. (1992) Cancer Res. 52, 1968-1973], these cells, now pRB(+), were arrested by staurosporine in G(1) phase, This arrest was accompanied by the accumulation of hypophosphorylated pRB, In both the pRB(+) and pRB(-) cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment, In contrast, cyclin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB(+) cells, Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E, The CDK inhibitor proteins p21((Waf1/Clp1)) and p27((Kip1)) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p21 from staurosporine-treated pRB(+) cells revealed a 2.5- to 3-fold higher ratio of p21 bound to CDK2 compared with staurosporine-treated pRB(-) cells, In pRB(+) cells, p21 was preferentially associated with Thr160 phosphorylated active CDK2, In pRB(-) cells, however, p21 was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant, This is the first evidence suggesting that G(1) arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB-dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p21 and p27.
引用
收藏
页码:5941 / 5946
页数:6
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