30. Effect of Deficiency of DNA-dependent Protein Kinase Catalytic Subunit on Mutational Properties in Cultured Mouse Mammary Tumor cells

Yuko Hoki-Fujimori, Ryoko Araki, Ryutaro Fukumura, Akira Fujimori, Ikuko Furuno-Fukushi, Koki Sato Masumi Abe and Kouichi Tatsumi

Keywords: adenine phosphoribosyltransferase (APRT) hypoxanthin phosphoribosyltransferase (HPRT), non homologous end joining (NHEJ), 8-aza-adenine (8AA)

DNA-dependent protein kinase (DNA-PK) composed of catalytic subunit (as), Ku7O and Ku86 is indispensable for repair of DNA double-strand breaks and V(D)J recombinatton. As it binds to DNA double-strand breaks and requires such structural alterations for its enzymatic activity, DNA-PK has been presumed as being one of the key molecules for sensing DNA damages. However, neither true in vivo substrates for the kinase activity nor the precise mechanisms for controlling the signal transduction triggered by DNA double-strand breaks have been clarified yet. Recently we have found that SX9, a radiosensitive subline of mouse mammary tumor FM3A cells, bears a T-C transition at 9572 of the Dna-phcs gene that causes a Leu-Pro substitution at codon 3191 and results in a complete loss of the DNA-PKcs functions. The effect of total deprivation of DNA-PKcs on mutational properties was examined by comparing the mutabilities at the hemizygous X-linked Hprt focus and the autosomal Aprt focus between the null mutant SX9 cells and their parental SR-1 cells that are heterozygously inactivated at the Aprt gene (Aprt +/-). SX9 cells were approximately 4-fold more sensitive than SR-1 cells to X-ray cytotoxicity with the lost shoulder and the steeper slope in the survival curve. Spontaneous mutant fractions in SX9 cells were 4-fold (8 x 10-4) and 9f old (1.7 x 10 -4) higher than in SR1 cells f or 8 azaadenine (8AA) resistance and 6 thio-guanine (6TG) resnstance, respectively. While a statistically signifi cant increase was induced by X-rays for 8AA resistant mutant fraction in both SX9 and SR-1 cells, 6TG resistant mutant fractions were clearly increased only in SR-1 cells. When increased mutant fractions were replotted against surviving fractions instead of exposure dose of X-rays, the hypomutability of SX9 cells for the two markers became conspicuous at least in the dose range allowing survival above 1O%. The impairment of non-homologous end-joining of DNA double-strand breaks appeared to result in the hypomutability to X-rays by'render lllg cells with potentially mutagenic DNA insults more vulnerable to cell killing.