Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Chemical Agents Selective for Mutant or Normal Cultured Fibroblasts in Mixed and Heterozygote Cultures

Abstract

Cultured fibroblasts established from skin biopsies from patients with the Lesch-Nyhan syndrome are deficient in hypoxanthine-guanine phosphoribosyl-transferase (EC 2.4.2.8) activity. This deficiency makes possible the use of chemicals that select either for or against deficient variants in cultured fibroblasts. Two-way selection has been achieved by the use of 6-thioguanine, which selects for the deficient mutant, and azaserine, which selects to some extent for the normal allele in mixed cultures, as well as in cultures from heterozygotes. Theoretical considerations predict that the phenomenon of metabolic cooperation would tend to reinforce the former and to weaken the latter type of selection, and this is in accordance with the experimental findings.

Bystander effect of purine nucleoside analogues in HSV-1tk suicide gene therapy is superior to that of pyrimidine nucleoside analogues

“…Metabolic cooperation was initially demonstrated by Subak-Sharpe et al, 28,29 who showed that hypoxanthine could only be incorporated in hypoxanthine/guanine phosphoribosyl transferase-deficient (HGPRT − ) cells when cocultured with HGPRT-positive cells, a process later shown to be mediated by gap junctional communication. Later, Fujimoto et al 30 demonstrated that HGPRT − cells became sensitive to the purine analogue 6-thioguanine in cocultures with HGPRT + cells. The same effect was observed with 8-azaguanine and adenine analogs with HGPRT − and adenine phosphoribosyl transferase-deficient (APRT − ) cells, respectively.…”

The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro.

“…These HGPT-cells, normally resistant to 6-TG, became sensitive to 6-TG when cocultured with HGPT+ fibroblasts. They suggested that metabolic coupling resulted in the transfer of toxic metabolites to the HGPT-cells, a process they termed "the kiss of death" (27). Electron microscopy and microinjection experiments have shown that intercellular gap junctions are present in cells demonstrating metabolic coupling (28,29 Previous studies have found no injury to normal cells surrounding tumors treated with GCV after HSV-tk gene transfer (31).…”

“…The concept of metabolic coupling was proposed in 1969 (26) to describe intercellular transfer of molecules that alter the metabolism of recipient cells. Fujimoto et al (27) used 6-thioguanine (6-TG) to select fibroblasts lacking hypoxanthine/guanine phosphoribosyltransferase (HGPT). These HGPT-cells, normally resistant to 6-TG, became sensitive to 6-TG when cocultured with HGPT+ fibroblasts.…”

The use of genetic marking to assess the interaction of sensitive and multidrug-resistant cells in mixed culture

“…(Loewenstein & Kanno, 1964). The reports of Fujimoto et al (1971) and Corsaro and Migeon (1977) provide ample evidence that the communication which exists between coupled cells in monolayer culture is sufficiently extensive to allow transfer of 6-thioguanine sensitivity or oubain resistance.…”

“…In some cases where the interaction is between drug-sensitive and -resistant subpopulations, the mechanism underlying this phenomenon is clear, as in the cases of transfer of thioguanine sensitivity by cell-to-cell transfer of thioguanine nucleotides (Fujimoto et al, 1971) or of ouabain resistance by transfer of cytoplasmic Na+ and K+ (Corsaro & Migeon, 1977). In other cases the mechanism of interaction remains obsure or poorly defined (Miller et al, 1981;Tofilon et al, 1984Tofilon et al, , 1987.…”