Phenotypic and molecular changes in normal human cells following knockdown of DNA-PKcs by RNA interference

Abstract

A procedure based on the newly discovered RNA interference (RNAi) phenomenon was used to study the genetic control of cellular radiation responses in normal human cells. The aim was to study molecular and phenotypic effects in cultured normal human cells following knockdown of a single gene, Prkdc (DNA dependent protein kinase catalytic subunit), with small interfering double stranded RNAs (siRNAs). Prkdc is a key component of the non-homologous end joining process involved in rejoining DNA double strand breaks. This would enable the comparison of a single change on human cells with otherwise identical genetic backgrounds. The mRNA and protein products were successfully knocked down by transfection of cells with either of two siRNA sequences applied in nanomolar concentrations. The knockdown resulted in radiosensitization for cell killing, chromosomal aberration induction, and reduced the rejoining of interphase chromosome breaks after irradiation. It also resulted in a later reduction of ATM (mutated in Ataxia-Telangiectasia). Prkdc mRNA was reduced within a few hours after anti-Prkdc siRNA transfection, and DNA-PKcs protein began to decrease about 1.5 to 2 days later. Only after the DNA-PKcs had decreased did the ATM mRNA level drop, followed by a drop in the ATM protein starting at about 3.5 days. The mRNA and protein returned to pre-transfection levels at later sampling times since the transfections are transient. Control siRNA transfections using siRNAs with a slightly altered sequence did not produce any such molecular or phenotypic changes. No evidence was seen of the induction of an interferon response. Reduced ATM protein was also observed in a Prkdc mutant hamster cell line, and the ATM deficiency was restored in a line stably transfected with a human Prkdc-containing vector. Another hamster line with reduced (but kinase-dead) DNA-PKcs did not display reduced ATM. This is the first report of a phenotypic change, following knockdown of any DNA repair protein by RNA interference, and the first study showing, directly, that the levels of DNA-PKcs can influence the levels of ATM which in turn control a vast network of DNA damage checkpoint responses, apoptosis, and homology directed repair of DNA double strand breaks.