Human colon carcinoma cell-line HCT116 transfected by antisense cDNA as a tool to study the Ku86 involvement in cell proliferation

In this work, we used colon carcinoma cell-line HCT116 to study the involve ment of the 86-kDa subunit (Ku86) of DNA-protein kinase (DNA-PK) in human t umoural cell proliferation. We transfected these cells with a 639-bp cDNA e ncoding a Ku86 portion inserted into pcDNA3.1 vector in the antisense orien tation. After selection by neomycin, we obtained more than 300 resistant co lonies. In the Y'A5 colony that we chose as total population, we showed by PCR and RT-PCR that pcDNA3/Ku86 antisense was integrated in genomic DNA and that transcript was present. After cloning, we selected two clones, A20 an d A23, which contained significatively reduced level of Ku86 protein. These mio clones displayed a reduced DNA-PK activity from 44% to 71% and a slowe r growth than control cells. These results suggest that the HCT116 cell-lin e is a useful tool to investigate the role of Ku86 in the regulation of hum an tumoural cell growth. (C) 2000 Elsevier Science Inc. All rights reserved .