Sarkar Devanand, Su Zao-Zhong, Lebedeva Irina V, Sauane Moira, Gopalkrishnan Rahul V, Valerie Kristoffer, Dent Paul, Fisher Paul B
Department of Pathology, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):10054-9. doi: 10.1073/pnas.152327199. Epub 2002 Jul 11.
Subtraction hybridization identified melanoma differentiation-associated gene-7 (mda-7) as a gene induced during terminal differentiation in human melanoma cells. On the basis of structure, chromosomal localization and cytokine-like properties, mda-7 is classified as IL-24. Administration of mda-7/IL-24 by means of a replication-incompetent adenovirus (Ad.mda-7) induces apoptosis selectively in diverse human cancer cells without inducing harmful effects in normal fibroblast or epithelial cells. The present studies investigated the mechanism underlying this differential apoptotic effect. Infection of melanoma cells, but not normal immortal melanocytes, with Ad.mda-7 induced a time- and dose-dependent increase in expression, mRNA and protein, of a family of growth arrest and DNA damage (GADD)-inducible genes, which correlated with induction of apoptosis. Among the members of the GADD family of genes, GADD153, GADD45 alpha, and GADD34 displayed marked, and GADD45 gamma showed minimal induction. Treatment of melanoma cells with SB203580, a selective inhibitor of the p38 mitogen-activated protein kinase (MAPK) pathway, effectively inhibited Ad.mda-7-induced apoptosis. Additional support for an involvement of the p38 MAPK pathway in Ad.mda-7-mediated apoptosis was documented by using an adenovirus expressing a dominant negative mutant of p38 MAPK. Infection with Ad.mda-7 increased the phosphorylation of p38 MAPK and heat shock protein 27 in melanoma cells but not in normal immortal melanocytes. In addition, SB203580 effectively inhibited Ad.mda-7-mediated induction of the GADD family of genes in a time- and dose-dependent manner, and it effectively blocked Ad.mda-7-mediated down-regulation of the antiapoptotic protein BCL-2. Inhibition of GADD genes by an antisense approach either alone or in combination also effectively blocked Ad.mda-7-induced apoptosis in melanoma cells. These results support the hypothesis that Ad.mda-7 mediates induction of the GADD family of genes by means of the p38 MAPK pathway, thereby resulting in the selective induction of apoptosis in human melanoma cells.
消减杂交技术鉴定出黑色素瘤分化相关基因-7(mda-7)是人类黑色素瘤细胞终末分化过程中诱导产生的一个基因。基于其结构、染色体定位及细胞因子样特性,mda-7被归类为IL-24。通过无复制能力的腺病毒(Ad.mda-7)给予mda-7/IL-24可在多种人类癌细胞中选择性诱导凋亡,而对正常成纤维细胞或上皮细胞无有害影响。本研究探讨了这种差异性凋亡效应背后的机制。用Ad.mda-7感染黑色素瘤细胞而非正常永生化黑色素细胞,可诱导生长停滞和DNA损伤(GADD)诱导基因家族的表达、mRNA及蛋白质呈时间和剂量依赖性增加,这与凋亡诱导相关。在GADD基因家族成员中,GADD153、GADD45α和GADD34表现出明显诱导,而GADD45γ诱导程度最小。用p38丝裂原活化蛋白激酶(MAPK)途径的选择性抑制剂SB203580处理黑色素瘤细胞,可有效抑制Ad.mda-7诱导的凋亡。使用表达p38 MAPK显性负性突变体的腺病毒进一步证明了p38 MAPK途径参与Ad.mda-7介导的凋亡。用Ad.mda-7感染可增加黑色素瘤细胞中p38 MAPK和热休克蛋白27的磷酸化,但对正常永生化黑色素细胞无此作用。此外,SB203580可有效抑制Ad.mda-7介导的GADD基因家族的诱导,且呈时间和剂量依赖性,还可有效阻断Ad.mda-7介导的抗凋亡蛋白BCL-2的下调。单独或联合使用反义方法抑制GADD基因也可有效阻断Ad.mda-7诱导的黑色素瘤细胞凋亡。这些结果支持以下假说:Ad.mda-7通过p38 MAPK途径介导GADD基因家族的诱导,从而导致人类黑色素瘤细胞中凋亡的选择性诱导。