p38alfa and ATF2 act differentially depending on DUSP1 expression in NCSCL in response to cisplatin

DUSP1 is involved in different cellular pathways including cancer cell proliferation, angiogenesis, invasion and resistance to chemotherapy. To gain insight into the cellular signaling pathways involving DUSP1 actions and the response to Cisplatin (CDDP) in non small cell lung cancer (NSCLC), we have used a double strategy that combines microarray and SiRNA technology. This strategy provided a differential expression profile of genes involved in CDDP response in NSCLC cell line regulated by DUSP1 using H460 and H460cri and a time course to CDDP. KEYWORDS: Expression profiling by array in cells with genetic modification in response to CCDP treatment The human non small lung cancer cell line H460 used in this study was from ATCC (American Type Culture Collection). The cell line was maintained in RPMI (Gibco, Invitrogen) supplemented with 10% bovine serum and transfected by Lipofectamine Plus Reagent from Life Technologies as directed by the manufacturer. The DUSP1 pSuperRetro-derived vectors were constructed as described (Chattoppadhyay et al., 2006). Clones expressing siRNA for DUSP1 were selected for their ability to grow in the presence of puromycin and kept for selection. Stable transfection was confirmed by Western blotting. We analyzed the differences in expression of 47000 genes in H460 cells expressing DUSP1SiRNA (H460cri) compared with the parental cell line H460pSuperRetro vector (H460v) after 0, 1, 3 and 6 hours of CDDP exposure, in order to know the different pathways that were activated by CDDP treatment dependent on expression of DUSP1. RNA was extracted and quality control was checked before hybridization on Affymetrix microarrays.

双特异性磷酸酶1（DUSP1）参与多种细胞通路，包括癌细胞增殖、血管生成、侵袭及化疗耐药。为深入解析非小细胞肺癌（non small cell lung cancer, NSCLC）中涉及DUSP1作用的细胞信号通路，以及细胞对顺铂（Cisplatin, CDDP）的应答机制，本研究采用了结合微阵列（microarray）与小干扰RNA（Small interfering RNA, SiRNA）技术的双重研究策略。该策略借助H460与H460cri细胞系，并设置顺铂处理时间梯度，分析了由DUSP1调控的、参与非小细胞肺癌细胞顺铂应答的基因差异表达谱。
关键词：经基因修饰的细胞响应CCDP处理的阵列表达谱分析
本研究使用的人非小细胞肺癌细胞系H460购自ATCC（American Type Culture Collection，美国典型培养物保藏中心）。该细胞系于添加10%牛血清的RPMI培养基（Gibco、Invitrogen）中培养，并按照制造商说明书，使用Life Technologies公司的Lipofectamine Plus Reagent进行转染。DUSP1的pSuperRetro载体构建方法参照已发表文献（Chattoppadhyay等，2006）。针对DUSP1的小干扰RNA表达克隆通过嘌呤霉素筛选获得并保留用于后续实验。稳定转染效果通过蛋白质印迹法（Western blotting）验证。为明确依赖DUSP1表达、由CDDP处理激活的各类细胞通路，我们分析了分别经0、1、3、6小时CDDP处理后，表达DUSP1小干扰RNA的H460细胞（H460cri）与转染H460pSuperRetro空载体的亲本细胞系（H460v）中47000个基因的表达差异。在Affymetrix微阵列（Affymetrix microarrays）进行杂交前，我们提取了总RNA并完成质量检测。