Mutant Human Fibroblasts Research Articles

Toxoplasma gondii: Specific labeling of nucleic acids of intracellular parasites in Lesch-Nyhan cells

Lesch-Nyhan cells, which are incapable of incorporating hypoxanthine and guanine, supported the growth of Toxoplasma gondii. Massive infection of these cultured mutant human fibroblast cells resulted in a marked increase in the incorporation of radioactive hypoxanthine or guanine supplied in the medium. Autoradiography established that these radioactive purines supplied to infected Lesch-Nyhan cells were incorporated specifically into the intracellular parasites. No such specificity was seen in infected wild-type human fibroblast cells in which the nucleic acids of both the parasite and the host cell were labeled. Purine nucleotides within the intracellular parasite were not available for the synthesis of host nucleic acids. Toxoplasma gondii was less active than wild-type human fibroblast cells in the interconversion of purine nucleotides.

Regulation of purine nucleotide synthesis: Effects of inosine on normal and hypoxantine-guanine phosphoribosyltransferase-deficient fibroblasts

Incubation of normal and hypoxanthine-guanine phosphoribosyltransferase-deficient (mutant) human fibroblasts with inosine results in increased intracellular concentration of 5-phosphoribosyl 1-pyrophosphate ( PP-ribose- P). The magnitude of this increase is dependent on the concentration of the nucleoside and results from donation of the ribose moiety of inosine to the ribosyl phosphate moiety of PP-ribose- P through ribose phosphate intermediates. During incubation, rates of purine nucleotide synthesis de novo, estimated by incorporation of [ 14C] formate into formylglycinamide ribotide, are diminished in both normal and mutant cells: 5 mM inosine inhibits purine syntheiss by 60–80% in normal cells and 2–20% in hypoxanthine-guanine phosphoribosyltransferase-deficient cells. The rates of purine synthesis in both normal and mutant cells are increased, however, during incubation with methylene blue at concentrations (50–100 μM) which result in more modest increases in ribose 5-phosphate and PP-ribose- P concentrations than are observed with inosine. Saturation of the PP-ribose- P amidotransferase reaction by PP-ribose- P does not appear, therefore, to explain the failure of increased PP-ribose- P concentration to stimulate the rate of purine synthesis in either type of fibroblast during incubation with inosine. Although the dissociation between PP-ribose- P concentration and the rate of purine nucleotide synthesis in normal fibroblasts incubated with inosine may be explained at least in part by an accompanying increase in intracellular concentrations of purine nucleotide feedback inhibitors, purine nucleotide concentrations are unchanged in mutant cells during incubation with inosine; these cells, in addition, show minimal (<3% of normal) incorporation of labeled hypoxanthine or the hypoxanthine moiety of inosine into purine nucleotides. The effect of inosine on purine synthesis de novo in hypoxanthine-guanine phosphoribosyltransferasedeficient fibroblasts is not explained in full by consideration of the concentrations of purine nucleotides and of PP-ribose- P, the factors frequently invoked as antagonistic regulators controlling the rate of this process.

Use of mutant fibroblasts in the analysis of the regulation of cholesterol metabolism in human cells

Analysis of mutant human fibroblasts deficient in a cell surface receptor for low density lipoproteins (LDL) has led to the delineation of an important, hitherto unrecognized, regulatory process for cholesterol metabolism. On normal cells, binding of LDL to this receptor regulates cholesterol metabolism by two mechanisms: (a) suppression of cholesterol synthesis and (b) facilitation of the rate of proteolytic degradation of the lipoprotein. In cells from homozygotes with the autosomal dominant disorder Familial Hypercholesterolemia, a nearly total reduction in LDL receptors results in two secondary abnormalities: (a) overproduction of cholesterol due to an inability of LDL to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis, and (b) impairment in the rate of proteolytic degradation of LDL. Cells from heterozygotes possess about 50 per cent of the normal number of LDL recpetors; this leads to a concentration-dependent defect in regulation, so that attainment of rates of cholesterol synthesis and LDL degradation equal to that in normal cells requires a two to three-fold higher concentration of extracellular LDL in the heterozygote. The identification of this genetic regulatory defect in fibroblasts of heterozygotes with Familial Hypercholesterolemia makes available an in vitro system for studying the molecular mechanism by which a dominant mutation affects gene expression in mammalian cells.

Mutant human fibroblast clones able to grow on homocysteine instead of cyst(e)ine

Mutant clones able to grow on homocysteine or cystathionine in place of cyst(e)ine have been isolated from a diploid human fibroblast strain. These mutants are stable and retain normal fibroblast morphology and a near-diploid chromosome complement. Cystathionase activity does not appear to be increased, so that the biochemical nature of the mutation(s) is as yet unknown.