A Strategy For Isolating Growth-Suppressing Complementary DNAs From Human Esophageal Cancer Cell Line Treated With Retinoic Acid

Three cDNA libraries, designated Lib8501, Lib8712, and Lib8712RA, were constructed from human esophageal cancer cell lines EC8501,EC8712, and EC8712 treated continuously with retinoic acid (RA) at a concentration of 10^-5M for five days respectively. RA-treatment caused at first a drastic decrease in ³H-TdR uptake and expression level of c-myc of the cancer cell and then gave rise to their senescence after ten days action. Repeated subtractive hybridization (four rounds) of pooled Lib8501 and Lib8712 with cDNAs prepared from mRNA of the RA-treated cancer cell eliminated maximally the same genes expressed in both human esophageal cancer cells before and after RA-treatment. The unhybridized cDNAs represented, therefore, mainly the genes activated by RA-treatment. Using these enriched unhybridized cDNAs as probes 39 positive colonies were obtained from Lib8712RA. Taq I restriction analysis revealed that according to their incorporated sequences these clones could be grouped into four sets, representing at least 3 different inserts. Reintroducing these cDNA clones into the original human esophageal cancer cell line (EC8712) separately has found that two (RA526 and RA538) of these cDNA clones were capable of exerting growth-suppressing and senescence-inducing effects on the cancer cell.