Coexpression of NRASQ61R and BRAFV600E in human melanoma cells activates senescence and increases susceptibility to cell-mediated cytotoxicity
Coexpression of NRASQ61R and BRAFV600E in human melanoma cells activates senescence and increases susceptibility to cell-mediated cytotoxicity
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DOI：
10.1158/0008-5472.can-05-4671
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发表时间：
2006-07-01
期刊：
影响因子：
11.2
通讯作者：
Sensi, Marialuisa
中科院分区：
文献类型：
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作者：
Petti, Carlotta;Molla, Alessandra;Sensi, Marialuisa
人类黑色素瘤中BRAF和NRAS癌基因的激活突变是相互排斥的。这一发现表明了上位关系，但甚至与合成致死性一致。为了评估后一种可能性，突变的NRAS（Q61 R）癌基因在组成型或多西环素调节的启动子下在携带活化的BRAF（V600 E）癌基因的转移性黑素瘤克隆（克隆21）中表达。在最初的10至12次体外传代后，组成型NRAS（Q61 R）转染子在细胞周期的G 0-G期显示进行性积累，并对衰老相关的β-半乳糖苷酶活性（SA-β-Gal）进行染色。在克隆21细胞（21 NRAS（610 N））中，通过Tet-Off系统诱导NRAS（Q61 R）的表达导致RAS/RAF/丝裂原活化蛋白激酶信号通路的过度激活，并且在体外第10代之后，导致衰老的促进。这通过增殖减少、扁平细胞形态、基质胶中生长减少、SA-β-Gal阳性染色以及AMP活化蛋白激酶和细胞周期抑制剂p21（waf 1/cip 1）的表达来证明。在具有沉默的NRAS（Q61 R）（21 NRAS（61 OFF））的21个细胞中或在用诱导型野生型ATRAS基因（21 NRAS（WTON））转染的细胞中均未检测到这些效应。此外，当与亲本21细胞或与21 NRAS（61 OFF）、21 NIRAS（61 ON）和组成型NRAS（Q61 R）转染子细胞相比时，显示对HLA I类抗原限制性和非特异性T细胞的细胞毒性的敏感性增加，以及几种MHC I类抗原加工机制组分的上调。这些结果表明NRAS和BRAF癌基因之间的合成致死性的关系，导致对“双突变”细胞的选择。
Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRAS(Q61R) oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAF(V600E) oncogene. After the first 10 to 12 in vitro passages, the constitutive NRAS(Q61R) transfectant displayed progressive accumulation in GO-G, phase of the cell cycle and stained for the senescence-associated beta-galactosidase activity (SA-beta-Gal). Inducible expression of NRAS(Q61R), by the Tet-Off system, in clone 21 cells (21NRAS(610N)) led to overactivation of the RAS/RAF/mitogenactivated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-beta-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21(waf1/cip1). These effects were detected neither in 21 cells with silenced NRAS(Q61R) (21NRAS(61OFF)) nor in cells transfected with an inducible wild-type ATRAS gene (21NRAS(WTON)). In addition, when compared with parental 21 cells, or with 21NRAS(61OFF), 21NIRAS(61ON) and constitutive NRAS(Q61R) transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen-restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against "double-mutant" cells.