Gene expression profiling during treatment of HT29 cells with selumetinib

Here, we investigate gene expression response of the BRAFV600E mutant cell line COLO205 to the MEK inhibitor selumetinib / AZD6244 / ARRY-142886. Although selumetinib causes long term G1 arrest, we observe cells stochastically entering the cell cycle without re-activation of ERK and initiation of a normal proliferative gene expression programme. Genes encoding DNA replication and repair factors are downregulated during G1 arrest, but many of these are transiently induced when cells escaping arrest enter S and G2. Nonetheless, mRNAs encoding key DNA replication factors including the MCM replicative helicase complex, PCNA and TIPIN remain at very low abundance. 1) HT29 cells either untreated or treated 24 hours with 1 µM selumetinib were glyoxal fixed and stained for CCNB1 then flow sorted into CCNB1 positive and CCNB1 negative fractions, RNA extracted and mRNA-seq performed.

本研究探究了BRAFV600E突变细胞系COLO205对MEK抑制剂司美替尼（selumetinib / AZD6244 / ARRY-142886）的基因表达应答。尽管司美替尼可诱导细胞产生长期G1期阻滞，但我们观察到细胞可在不重新激活ERK、不启动正常增殖性基因表达程序的情况下，随机进入细胞周期。编码DNA复制与修复因子的基因在G1期阻滞期间表达下调，但当逃逸阻滞的细胞进入S期与G2期时，其中多数基因会出现瞬时诱导表达。尽管如此，包括MCM复制解旋酶复合体（MCM replicative helicase complex）、PCNA及TIPIN在内的关键DNA复制因子的编码mRNA，其丰度仍维持在极低水平。1）将未接受处理或经1 µM司美替尼处理24小时的HT29细胞进行乙二醛固定，随后针对CCNB1进行染色，通过流式细胞分选分为CCNB1阳性与CCNB1阴性组分，提取RNA并开展mRNA测序（mRNA-seq）。