17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells.
17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells.
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17beta-Estradiol 在 G(1) 阻滞的人乳腺癌细胞的有丝分裂刺激过程中诱导细胞周期蛋白 D1 基因转录、p36D1-p34cdk4 复合物激活和 p105Rb 磷酸化。
作者：
L. Altucci;R. Addeo;L. Cicatiello;S. Dauvois;M. Parker;M. Truss;M. Beato;Sica;F. Bresciani;A. Weisz
MCF-7人乳腺癌细胞表达功能性雌激素受体并在雌激素刺激下生长。G(1)同步的MCF-7细胞，在不含雌激素的培养基中暴露于HMG-CoA还原酶抑制剂辛伐他汀而处于静止状态，即使在培养基中持续存在辛伐他汀抑制多肽生长因子触发的信号转导途径的情况下，在17 - β -雌二醇的刺激下（E(2)）也容易恢复细胞周期进程。在这些条件下，细胞周期蛋白D(1)基因转录在刺激后的前1-9小时内被短暂诱导，表现为细胞周期蛋白D(1) mRNA和蛋白(p36（D(1)）在细胞内的积累，以及稳定转染的D(1)启动子-荧光素酶杂交基因的表达增强。雌激素诱导的p36（D(1)）容易与p32（cdk2）和p34（cdk4）结合，但不与p31（cdk5）结合，然而p31（cdk5）在这些细胞中大量表达。只有p36(D(1))-p34（cdk4）复合物被E(2)激活，通过抗D(1)抗体免疫沉淀在细胞提取物中检测到，随后评估了视网膜母细胞瘤（Rb）基因产物的磷酸转移酶活性，并分析了体内p105（Rb）磷酸化。在人类D(1)基因转录起始位点上游的第一个944 bp处发现了一个雌激素应答调节区。该DNA区域的序列分析显示，在这种情况下，对雌激素反应的顺式作用元件可能与典型的EREs不同。
MCF-7 human breast cancer cells express functional estrogen receptor and grow in response to estrogen stimulation. G(1)-synchronized MCF-7 cells, made quiescent by exposure to the HMG-CoA reductase inhibitor Simvastatin in estrogen-free medium, readily resume cell cycle progression upon stimulation with 17beta-estradiol (E(2)), even under conditions where polypeptide growth factor-triggered signal transduction pathways are inhibited by the continuous presence of Simvastatin in the culture medium. Under these conditions, cyclin D(1) gene transcription is transiently induced within the first 1-9 h of stimulation, as shown by the accumulation of cyclin D(1) mRNA and protein (p36(D(1))) in the cell and by enhanced expression of stably transfected D(1) promoter-luciferase hybrid genes. Estrogen-induced p36(D(1)) associates readily with p32(cdk2) and p34(cdk4), but not with p31(cdk5), which is however abundantly expressed in these cells. Only p36(D(1))-p34(cdk4) complexes are activated by E(2), as detected in cell extracts by immunoprecipitation with anti-D(1) antibodies followed by assessment of phosphotransferase activity toward the retinoblastoma (Rb) gene product and by analysis of p105(Rb) phosphorylation in vivo. An estrogen-responsive regulatory region has been mapped within the first 944 bp upstream of the transcriptional startsite of the human D(1) gene. Sequence analysis of this DNA region reveals that the cis-acting elements responsive to estrogen are likely to be different in this case from the canonical EREs.