Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells
Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells
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DOI：
10.1093/carcin/bgh014
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发表时间：
2004-03-01
期刊：
影响因子：
4.7
通讯作者：
Pascale, RM
中科院分区：
文献类型：
--
作者：
Simile, MM;De Miglio, MR;Pascale, RM
许多遗传相互作用参与了细胞周期的调控，但它们的作用和性质尚未完全阐明。了解这些相互作用在肝细胞癌中的行为，可以优化基于细胞周期抑制的预防和治疗策略。我们研究了在体外培养的人HepG 2和大鼠Morris 5123肝癌细胞中，通过脂质体递送的MYC和CYCLIN D1反义寡脱氧核苷酸（aODN（M），aODN（D1））抑制c-MYC和CYCLIN D1基因后的下游事件。0.5-20 μ M aODN（M）和aODN（D1）抑制两种细胞类型的体外生长。加扰寡聚体（SCR）和正义ODNs没有或相对较差的效果。10 μ mol的aODN（M）和aODN（D1）也能诱导HepG 2和5123细胞的凋亡指数显著增加，并抑制HepG 2细胞在软琼脂中的集落形成，而SCR则无此作用。用aODN（M）加aODN（D1）处理细胞对生长和凋亡没有累加效应。aODN（M）和aODN（D1）在mRNA和蛋白质水平上分别诱导c-MYC和CYCLIN D1基因表达降低>50%。aODNs对基因表达的抑制是高度特异性的，而SCR没有作用。aODN导致的c-MYC和细胞周期蛋白D1表达的减少与E2 F1 mRNA和蛋白质产生减少>50%有关，而细胞周期蛋白A和细胞周期蛋白E表达没有变化。这些结果表明c-MYC和CYCLIN D1均参与E2 F1基因的功能，并表明aODN（M）和aODN（D1）可能通过下调E2 F1基因来抑制肝癌细胞生长。E2 F1 aODN对E2 F1基因表达的抑制与HepG 2细胞的强烈生长抑制有关。因此，c-MYC和细胞周期蛋白D1与E2 F1基因的相互作用对于肝癌细胞中的细胞周期活性是必需的，并且它们的抑制可能具有治疗效果。
A number of genetic interactions are involved in the control of cell cycle, but their role and nature have not been completely clarified. The knowledge of the behavior of these interactions in hepatocellular carcinoma, could optimize preventive and therapeutic strategies based on cell cycle restraint. We studied downstream events following c-MYC and CYCLIN D1 gene inhibition, by lipoplex-delivered MYC and CYCLIN D1 antisense oligodeoxy nucleotides (aODN(M), aODN(D1)), in in vitro cultured human HepG2 and rat Morris 5123 hepatoma cells. 0.5-20 muM aODN(M) and aODN(D1) inhibited in vitro growth of both cell types. Scramble oligomer (SCR) and sense ODNs had no or relatively poor effect. Ten micromolar aODN(M) and aODN(D1), but not SCR, also induced a significant increase in the apoptotic index of HepG2 and 5123 cells, and inhibited colony formation in soft agar by HepG2 cells. Treatment of the cells with aODN(M) plus aODN(D1) had no additive effect on growth and apoptosis. aODN(M) and aODN(D1) induced >50% decrease in c-MYC and CYCLIN D1 gene expression, respectively, at both mRNA and protein level. The inhibition of gene expression by aODNs was highly specific, and SCR was without effect. The reduction in c-MYC and CYCLIN D1 expression by aODNs, was associated with a >50% decrease in E2F1 mRNA and protein production, without changes in CYCLIN A and CYCLIN E expression. These results suggest the involvement of both c-MYC and CYCLIN D1 on E2F1 gene function, and indicate that aODN(M) and aODN(D1) may inhibit hepatoma cell growth through down-regulation of the E2F1 gene. The inhibition of E2F1 gene expression by E2F1 aODN, was associated with strong growth restraint of HepG2 cells. Thus, interactions of c-MYC and CYCLIN D1 with E2F1 gene are essential for cell cycle activity in hepatoma cells, and their inhibition may have a therapeutic effect.