Evidence for Transfer of Enzyme Product as the Basis of Metabolic Cooperation between Tissue Culture Fibroblasts of Lesch-Nyhan Disease and Normal Cells

Abstract

Tissue culture fibroblasts derived from patients with Lesch-Nyhan disease (congenital hyperuricosuria) have a reduced IMP:pyrophosphate phosphoribosyltransferase (EC 2.4.2.8) activity and therefore incorporate, as detected by radioautography, much smaller amounts of tritiated hypoxanthine or guanine into cell nuclei and cytoplasm than do normal cells. However, Lesch-Nyhan cells grown in close contact with normal fibroblasts incorporate these purines. This phenomenon, which requires cell to cell contact for correction of the mutant phenotype, has been called metabolic cooperation. After separation of Lesch-Nyhan cells from normal cells, there is a prompt reversion to the mutant phenotype although the transferase is stable under these conditions for many hours.These results are most compatible with the transfer from normal to mutant fibroblasts of the product of the normal enzyme, a nucleotide or a nucleotide derivative, rather than the transfer of the transferase or informational macromolecules leading to the synthesis of the enzyme. Metabolic cooperation may provide a mechanism for maintaining normal cell function in the heterozygote in vivo. Evidence has been presented previously that selection of normal cells, presumably during embryogenesis, also provides a means for achieving normal function in the heterozygote.

Effect of Leukocyte Transfusion in a Child with Type II Mucopolysaccharidosis

“…In view of the short survival of donor leukocytes in a recipient, a further requirement for treatment with leukocyte transfusion may be the susceptibility of the abnormal cells to correction by the appropriate "factors" even in absence of contact with the normal cells. Such conditions are met in the case of various mucopolysaccharidoses (6) but not in the case of the Lesch-Nyhan syndrome, where metabolic cooperation seems to require intimate contact between normal and abnormal cells (14).…”

Gap Junctions

“…Impaired HGPRTase results in an elevated concentration of phosphoribosyl pyrophosphate, a marked increase in the rate of purine biosynthesis, and an overproduction of urate. Mutant fibroblasts from patients with Lesch-Nyhan syndrome can be metabolically rescued in cell culture by gap junction formation with normal cells (Cox et al 1970), a process termed metabolic cooperation (Subak-Sharpe et al 1969). Furthermore, metabolic cooperation likely accounts for the lack of symptoms in heterozygous female Lesch-Nyhan carriers.…”

Comparison of contact-mediated communication in normal and transformed human cells in culture.

“…We have previously shown that the decrease in recovery of 6TGr clones is contact-dependent (19). Others have established, by autoradiography, that the labeled molecules transferred from HPRT+ to HPRT-cells are the nucleotide products of the enzymatic reaction (32), and that this transfer is contact-dependent (15-17, 27, 32) and is not affected (3,25). As expected if our assay were measuring gap junction-mediated communication, the recovery of 6TGr clones (Table 3) is not influenced by the presence, as donors, of the L cell, known to be noncommunicating by all criteria [lacking gap junctions (25,26) as well as the ability to transfer ions (25,26) and nucleotides (17,25,27) over, the dosage effect, or severe reduction in communication, observed when both donor and recipient are transformed (T:T) is expected because, in this case, both cell types manifest junctional defects.…”