Overexpression of Human Ku70/Ku80 in Rat Cells Resulting in Reduced DSB Repair Capacity with Appropriate Increase in Cell Radiosensitivity but with No Effect on Cell Recovery

Abstract

The effect of an overexpression of human Ku70/80 was studied using cells of the rat cell lines Rat-1 and R7080, the latter being transfected with the human cDNAs for Ku70 and Ku80. The overexpression was found to result in a 20% reduction of the DNA-PK activity. The kinetics of DSB repair, which was studied after exposure of the cells to 30 Gy of X rays, was biphasic and had identical half-times for Rat-1 and R7080 cells (tfast = 7 min and tslow = 135 min). However, there was a significant difference between the cell lines in the fractions of DSBs repaired with slow and fast kinetics. In R7080 cells, about twice as many DSBs were repaired with slow kinetics compared to Rat-1 cells (34% compared to 16%). A similar difference was found in the number of residual DSBs (3.6% compared to 2.0%). R7080 cells also showed a reduced capacity to repair chromosome damage as detected by the PCC technique. Concerning cell killing, R7080 cells were clearly more radiosensitive than Rat-1 cells (D0.1 = 6.4 compared to 10.5 Gy), and this increase in sensitivity correlated well with the increase in residual DSBs. The two cell lines, however, did not vary in cell recovery. For sublethal as well as potentially lethal damage, Rat-1 and R7080 cells showed identical recovery ratios. These data demonstrate that the overexpression of human Ku70/Ku80 led to a reduced capacity for DSB repair with an associated increase in cell sensitivity but with no effect on cell recovery.

Relationship between the radiosensitizing effect of wortmannin, DNA double‐strand break rejoining, and p21WAF1 induction in human normal and tumor‐derived cells

“…Although the approach of digitized image analysis of ethidium bromide stained gels has been recently used in several studies of others to determine the impact of DSBs on cellular radiosensitivity [33,36,37], we considered the possibility that a fraction of small size DNA released from the plugs may not be detected by ethidium bromide staining. Thus, in some experiments, after completion of electrophoresis, we transferred the DNA to support membranes by Southern protocol and visualized the DNA using the Cot‐1 DNA probe followed by autoradiography [38].…”

“…Induction and rejoining of DSBs following γ‐irradiation were analyzed by constant‐field gel electrophoresis (CFGE) according to the protocol of Kasten et al [33] with minor modifications. Briefly, to monitor initial DSB levels, fibroblasts were embedded in agarose plugs and exposed to γ rays on ice.…”

“…Electrophoresis was performed in special gel boxes containing wick compartments supplied with the AutoBase Electrophoresis System (Q‐Life Systems, Kingston, Ont., Canada). Electrophoresis was performed at a constant field strength of 0.6 V/cm for 36 h [33], after which each gel was stained with ethidium bromide and excited with 302‐nm light; the resultant images were acquired with a digitized image analyzer (BC Cancer Research Centre, Vancouver, BC, Canada) and used to quantify the amount of DNA that migrated into the gel. The relative level of DSBs was estimated from the amount of DNA migrating into the gel for irradiated compared to nonirradiated samples.…”

KUB3 amplification and overexpression in human gliomas

“…As for how overexpression of KUB3 effects these functions, there are no studies using human cells. However, a recent study on a rat cell line that constitutively overexpresses human Ku70 and Ku80 shows a 20% reduction of the DNA-PK activity (Kasten et al, 1999). The authors suggest that the reduction in activity results from a partial lack of homology of human and rat genes encoding Ku70/80 and a non-optimal complex formation due to differences in the protein structure.…”

Glioblastoma multiforme: the role of DSB repair between genotype and phenotype

“…This may also be the case for Ku70/ Ku80. Overexpression of Ku70/Ku80 that was found in rat cells resulted in reduced DSB repair capacity (Kasten et al, 1999).…”