Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway.
Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway.
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发表时间：
2002-12
期刊：
影响因子：
11.2
通讯作者：
J. Bao;X. Le;Rui-yu Wang;Jiuhong Yuan;Lin Wang;E. Atkinson;R. Lapushin;M. Andreeff;B. Fang;Yin-hua Yu;R. Bast
中科院分区：
文献类型：
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作者：
J. Bao;X. Le;Rui-yu Wang;Jiuhong Yuan;Lin Wang;E. Atkinson;R. Lapushin;M. Andreeff;B. Fang;Yin-hua Yu;R. Bast
ARHI是一种印迹的肿瘤抑制基因，编码一种M（r）26,000 GTP结合蛋白，与ras和rap有60%的同源性，但功能截然不同。ARHI表达在大多数乳腺癌和卵巢癌中下调。使用双重腺病毒系统，我们已经在卵巢癌和乳腺癌细胞中重新表达ARHI，这些细胞已经失去了ARHI表达。ARHI的重新表达抑制生长，降低侵袭性，并诱导凋亡。在用ARHI腺病毒感染后5天，通过末端脱氧核苷酸转移酶介导的切口末端标记测定和通过用流式细胞术分析的膜联蛋白V染色判断，30-45%的MDA-MB-231乳腺癌细胞和5-11%的SKOv 3卵巢癌细胞凋亡。虽然聚（ADP-核糖）聚合酶可以在凋亡细胞的细胞核中检测到，没有激活的效应半胱天冬酶（半胱天冬酶3，6，7，或12）或启动半胱天冬酶（半胱天冬酶8或9）可以检测到细胞裂解液中使用蛋白质印迹。当在含有2304个已知基因的定制cDNA阵列上分析基因表达时，相对于用LacZ腺病毒感染的对照细胞，用ARHI腺病毒感染上调了15个基因。在智人钙蛋白酶样蛋白酶中观察到最大程度的mRNA上调。在Western印迹分析中，在用ARHI腺病毒感染后3-5天，钙蛋白酶蛋白增加2-3倍。LacZ腺病毒感染后，钙蛋白酶蛋白没有增加。钙蛋白酶裂解后，ARHI再表达，钙蛋白酶的抑制剂，但不是半胱天冬酶的抑制剂，部分防止ARHI诱导的细胞凋亡。因此，ARHI在乳腺癌和卵巢癌细胞中的再表达似乎通过半胱天冬酶非依赖性、钙蛋白酶依赖性机制诱导细胞凋亡。
ARHI, an imprinted putative tumor suppressor gene, encodes a M(r) 26,000 GTP-binding protein that is 60% homologous to ras and rap but has a dramatically different function. ARHI expression is down-regulated in a majority of breast and ovarian cancers. Using a dual adenovirus system, we have reexpressed ARHI in ovarian cancer and breast cancer cells that have lost ARHI expression. Reexpression of ARHI inhibited growth, decreased invasiveness, and induced apoptosis. At 5 days after infection with ARHI adenovirus, 30-45% of MDA-MB-231 breast cancer cells and 5-11% of SKOv3 ovarian cancer cells were apoptotic as judged by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay and by Annexin V staining with flow cytometric analysis. Although poly(ADP-ribose) polymerase could be detected immunohistochemically in the nuclei of apoptotic cells, no activation of the effector caspases (caspase 3, 6, 7, or 12) or the initiator caspases (caspase 8 or 9) could be detected in cell lysates using Western blotting. When gene expression was analyzed on a custom cDNA array that contained 2304 known genes, infection with ARHI adenovirus up-regulated 15 genes relative to control cells infected with LacZ adenovirus. The greatest degree of mRNA up-regulation was observed in a Homo sapiens calpain-like protease. On Western blot analysis, calpain protein was increased 2-3-fold at 3-5 days after infection with ARHI adenovirus. No increase in calpain protein was observed after LacZ adenovirus infection. Calpain cleavage could be detected after ARHI reexpression, and inhibitors of calpain, but not inhibitors of caspase, partially prevented ARHI-induced apoptosis. Consequently, reexpression of ARHI in breast and ovarian cancer cells appears to induce apoptosis through a caspase-independent, calpain-dependent mechanism.