从人食管癌细胞系分离分化基因的新方法

本文报道一种从癌细胞中分离分化基因(抑癌基因)的全过程。用维甲酸(RA)处理食管癌细胞EC8712可使后者发生终末分化和衰老。从对数生长期的和RA处理5天后的EC8712各构建一cDNA文库,命名为Lib8712和Lib8712RA,后者为可在哺乳动物细胞内表达的质粒。用RA处理5天的EC8712制备cDNA探针,先同Lib8712的质粒DNA进行反复杂交,以除去探针中与之共有的DNA序列,将余下的、富集了的探针(代表RA激活的基因)同Lib8712RA进行集落杂交,以钓取其中的RA激活的序列。已获得若干个杂交阳性克隆,Northern分析表明,它们是经RA诱导后特异表达的基因。     

A STRATEGY FOR ISOLATING DIFFERENTIATIONINDUCING COMPLEMENTARY DNAs FROM HUMAN ESOPHAGEAL CANCER CELL LINE TREATED WITH RETINOIC ACID

Treatment of the human esophageal cancer cell line EC8712 with retinoic acid (RA) stopped the cell growth significantly and gave rise to terminal differentiation of the cells characterized by increased expression of involucrin gene. Two cDNA libraries were constructed from the parental and RA-treated cells respectively. Repeated subtractive hybridization of single-stranded plasmid DNA prepared from pooled colonies of cDNA library of the parental cells with cDNA probe generated from the RA-treated cells exhausted sequences common to both libraries of the cell. The unhybridized cDNA probe represented, therefore, the genes activated after RA-treatment. By using these enriched cDNAs as probe to screen the cDNA library constructed from the RA-treated cells thirty-nine positive colonies were obtained, of which two were specifically due to RA-induction. One of these two cDNA clones, designated as pRA538, has undergone further analysis and shown differentiation-inducing effect on parental cancer cells. A novel