Comparisons Of Induced Mutations At TK And HPRT Loci, Using Two Human Lymphoblastoid Cell Lines, TK6 And TK6-E6, Respectively

Human cells have species superiority over bacteria and animal cells in gene mutation assay, for the results make more sense and can be easily extrapolated to humans. The most often used loci in mammalian mutation assay are tk and hprt. There are several tk+/-genotype human cell lines were built for tk gene mutation assay and the WI-L2 lymphoblastoid family is the primary one. The major members of WI-L2 are TK6 (wild type p53), TK6-E6 (abrogated p53), and WTK1 (mutant p53); they are the same in all but the p53 status. WI-L2 family members are mainly used in ionizing radiation-induced mutation researches aimed at p53 functions at present, but seldom in the genotoxicity assay fields.In this study, four positive chemical mutagens different in structures and mutation mechanism from each other, including methyl methanesulfonate(MMS), ethyl methanesulfonate(EMS),o-phenylenediamine(OPD), glyoxal(GLY), were selected for mutation test at tk and hprt loci in human lymphoblastoid with different p53 status, TK6 and TK6-E6, respectively. And the test procedure is the same as MLA using nticrowell method. Induced mutations at tk or hprt locus in two different human lymphoblastoid cell lines by different mutagens were compared systemically to accumulate data for the further use of WI-L2 family members in mutation assay. The results are showed as below:TK6-E6 is more durable to toxicity of test agents than TK6. RS(Relative survival) is appropriate to indicating the cytotoxic, but RSG (Relative Suspension Growth) is not a bad choice with convenience and efficiency in designing dose groups, because it needs much shorter time than RS in mensuration. SMF (spontaneous mutation frequency) in TK6 at tk and hprt locus is 5.48-10.72X 10"6 and 5.33-9.55 X 10'6, respectively. SMF in TK6-E6 at tk and hprt locus is 15.09-19.75 X 10'6 and 11.76-19.78 X 10"6, respectively. All test agents induced dose-dependent mutation responses at two loci in two cell lines. The most sensitive mutation test is the test at tk locus in TK6-E6. Compared between cell lines, the mutation sensitivity is higher in TK6-E6 than that in TK6 at tk locus, but the same at hprt locus. Compared between loci, the mutation sensitivity is higher at tk locus than at hprt locus. The RSC (Rate of Slow Growth colonies) in TK6 is 0.38-0.50 and much lower than in TK6-E6 which is 0.84-0.90, RSC decreased with concentration increasing in TK6 whereas still at high level without tendential alteration in TK6-E6. RSC in both cell lines lack the ability of distinguishing agents with different mutation mechanisms.Considering aspects of cytotoxic durability, mutation sensitivity, and tumor genetics that p53 function lost at first, TK6-E6 is superior to TK6 when used in mutation assay with higher sensitivity, dosage treatment allowance and the more correlations between results and tumor generation.