44. Functional Complementation of the Radiation Sensitive Mutant M10 Cell Line by Human XRCC4 cDNA Expression

Masahiko Mori, Hiromi Itsukaichi and Koki Sato^*
(^*Kinki Univ.)

Keywords: DNA -double strand break repair, XRCC genes

Nine complementation groups have been reported for Chinese hamster and mouse cell mutants hypersensitive to the lethal effect of ionizing radiation. We isolated several ionizing radiation sensitive mutants from mouse cells. The mouse lymphoma L5178Y cell mutant M10 is defective in DNA double-strand break repairs and is hypersensitive to ionizing radiation. Stackhouse et al (Mutation Research 323, 47-52, 1994) reported the gene that corrects the radiation hypersensitivity of M10 cells is located on chromosome 5 and it is tentatively assigned to the 5q14 to 5pter region. The XRCC4 gene is located on chromosome 5 and is required for the repairs of DNA double-strand break in mammalian cells. Without XRCC4, cells are hypersensitive to ionizing radiation and defective for V(D)J recombination.

To analyze complementation of the radiosensitivity characteristics of M10 cell, the pME18S vector carrying the GFP, GFP-XRCC4 ORF or XRCC4 ORF-GFP was introduced into M10 cells by DNA transfection, and stable transfectants were selected by use of the blasticidin S resistant marker cotransfected pMAM2BSD. The clones, XRCC4 sequences and expressing GFP fusion protein, were corrected for radiation sensitivity similar to that of parental cell, L5178Y (Fig. 17). pME18S GFP showed radiation sensitivity of M10 cells.

Our results demonstrate that human XRCC4 cDNA complemented the ionizing radiation hypersensitivity of M10 cells.

Fig.17. Cell survival after ionizing radiation of transformants. X-ray survival of wild type L5178Y cell, mutant M10 cells, mutant M10 cells transformed with GFP human XRCC4 fused cDNA, or with human XRCC4 GFP fused cDNA, or GFP cDNA.
Error bars represent standard deviation derived from triplicate experiments.