J.P. O'Neill

In vitro induction, expression and selection of thioguanine-resistant mutants with human T-lymphocytes

Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G0 phase peripheral blood mononuclear cells from a single individual. After exposure to gamma-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TGr) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 X 10(4) cells/well. The development of this in vitro assay should allow direct study of susceptibility to gamma-irradiation in the human population in terms of both cytotoxicity and mutation induction.

Southern-blot analyses of human T-lymphocyte mutants induced in vitro by γ-irradiation

Abstract G0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of γ-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TGr) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR β and γ cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions ( 10 18 ) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5′ and 3′ regions of the hprt gene ( 4 18 and 4 18 , respectively). These results demonstrate that γ-irradiation primarily induces TGr mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.

Metabolic cooperation in the selection of thioguanine-resistant mutants does not occur with the human B-lymphoblastoid cell TK6

Selection for thioguanine resistant mutants in the human B‐lymphoblastoid cell TK6 yields the same results in both round and flat bottom 96‐well microtiter plates. These results suggest that metabolic cooperation is not an issue in these cells and show that round bottom wells can be used in place of flat bottom wells. Environ. Mol. Mutagen. 30:359–361, 1997.