Suppression of cell proliferation and deoxyribonucleic acid synthesis in cloned rat hepatoma H4-II-E cells overexpressing regucalcin
Suppression of cell proliferation and deoxyribonucleic acid synthesis in cloned rat hepatoma H4-II-E cells overexpressing regucalcin
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DOI：
10.1002/jcb.1274
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发表时间：
2002-01-01
影响因子：
4
通讯作者：
Yamaguchi, M
中科院分区：
文献类型：
--
作者：
Misawa, H;Inagaki, S;Yamaguchi, M
在稳定表达内源性调节素(RC)的克隆大鼠肝癌H4-II-E细胞中，研究了RC在细胞增殖调节中的作用。将RC/pCXN2载体导入H4-II-E细胞，筛选出稳定高表达RC的多个新霉素抗性克隆。与亲本野生型H4-II-E细胞相比，RC/pCXN_2转基因细胞的RC含量增加了19.7倍。Wi-ID型H4-II-E细胞、pCXN_2载体转染型细胞(模拟型)和RC/pCXN_2转染型细胞(转染型)分别在含胎牛血清(10%FBS)的条件下培养24、48、72h。随着培养时间的延长，野生型和模拟型细胞数均显著增加。与野生型和模拟型相比，转染体的细胞数明显减少。H4-II-E细胞培养12~48h后，细胞核DNA合成活性明显受到抑制，反应混合物中加入抗RC单抗(10~50 ng/ml)后，野生型和转染体细胞核DNA合成活性均显著升高，其中以转染组最为显著。抗RC单抗(50 ng/ml)增加DNA合成活性的作用可被加入1um的Regucalcin完全阻断。本研究表明，高表达RC的克隆性大鼠肝癌H4-II-E细胞增殖受到稳定抑制。(C)2001年Wiley-Liss，Inc.
The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4-II-E cells overexpressing RC stably. H4-II-E cells were transfected with RC/pCXN2 vector and the multiple neomycin-resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2-transfected cells used in this study was 19.7-fold as compared with that of the parental wild type H4-II-E cells. Wi Id type H4-II-E cells, pCXN2 vector-transfected cel Is (mock type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4-II-E cells was significantly suppressed in transfectants with culture for 12-48 h. The presence of anti-RC monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti-RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 muM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4-II-E overexpressing RC stably. (C) 2001 Wiley-Liss, Inc.