A novel class of unstable 6-thioguanine-resistant cells from dog and human kidneys

Abstract

Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion. In medium containing a relatively high concentration (10 micrograms/ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10(-4) to 10(-5). A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in thioguanine medium, consistent with a reduction in hypoxanthine phosphoribosyltransferase activity. When these thioguanine-resistant primary clones were subsequently grown in the absence of thioguanine and assayed for the thioguanine-resistant phenotype and hypoxanthine phosphoribosyltransferase activity, it was found that most were now thioguanine-sensitive and yielded cell-free extracts with substantial amounts of hypoxanthine phosphoribosyltransferase activity. In contrast, thioguanine-resistant human clones grown continuously in the presence of thioguanine yielded cell-free extracts with little or no detectable hypoxanthine phosphoribosyltransferase activity. Southern blot analysis demonstrated no structural alterations in the hypoxanthine phosphoribosyltransferase gene in thioguanine-resistant primary human kidney clones. These results suggest that a novel mechanism(s) for thioguanine resistance and the control of hypoxanthine phosphoribosyltransferase expression may occur in dog and human kidney cells.

Somatic Mutations Are Frequent and Increase with Age in Human Kidney Epithelial Cells

“…This apparent exponential increase, and comparable age-dependent increases in TG-resistant mutant frequencies in human and mouse T-lymphocytes (20,(22)(23)(24)(25), are compatible with 'error catastrophe' and other somatic mutation-based theories of aging (26). One as yet unexplained finding is that we have seen comparable age-dependent increases in TG-resistant mutant frequency in human and dog kidney, though not in mouse kidney or mouse skeletal muscle fibroblasts (18,27). This difference is unlikely to reflect strong, species-specific selection against HPRT-deficient cells: the frequency of TG-resistant T-lymphocytes increases in aged mice in tissue where the selection against HPRT-deficient variants appears to be more stringent than in slowly dividing tissues such as kidney (25,28,29).…”

Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

“…The expression of WT and mutant forms of the human PDGFR # subunit in TRMP cells (Turker et al, 1988) has been previously described (Kazlauskas and Cooper, 1989;. For most experiments TRMP cells expressing -1 0 PDGFRs/cell were used, whereas the PLC'yl studies were performed on cells expressing -104…”

The stress response resolution assay. I. Quantitative assessment of environmental agent/condition effects on cellular stress resolution outcomes in epithelium

“…Quantitative assays for mutations in endogenous reporter genes have become useful tools for investigating the mutagenic potency and specificity of chemicals in several cell types [Albertini et al, 1985[Albertini et al, , 1996O'Neill et al, 1987]. However, limited success has been achieved in developing quantitative assays for mutagenic responses in epithelial cells, the cell type most prone to neoplastic transformation in humans and rodent models [Turker et al, 1988;Gould, 1991, 1992;Driscoll et al, 1995Driscoll et al, , 1996Martin et al, 1996;Suzuki and Hei, 1996;Colgin et al, 2002;Hoenerhoff et al, 2009], and these assays are not designed to inform for outcomes of cellular stress responses [Toussaint et al, 2000a;Simmons et al, 2009].…”