Isolation of variants of Chinese hamster ovary cells with abnormally low levels of GSH: decreased ability to cleave endocytosed disulfide bonds.

Mutants of Chinese hamster ovary cells were selected for resistance to a 3 hour exposure to 4 microgram/ml N-methyl-N'-nitro-N-nitrosoguanidine and tested for glutathione (GSH) levels. Six of eight clones that survived the initial treatment had reduced GSH levels ranging from 26 to 61% of wild-type values. These eight cell lines were tested for their susceptibility to a drug conjugate in which methotrexate (MTX) is disulfide-linked to poly(D-lysine) (MTX-SS-PDL) to test their capacity to cleave the endocytosed disulfide bond and release free MTX from this otherwise undegradable drug conjugate. We had shown that wild-type cells were killed by approximately 1 x 10(-7) M MTX given as free drug, as MTX-poly(L-lysine) or as MTX-SS-PDL, but were not affected by MTX-poly(D-lysine). All six lines with abnormally low levels of GSH were resistant to MTX-SS-PDL. The variants with the lowest levels of GSH (MNR-5 and MNR-10) were tested further and showed near-normal sensitivity to MTX and MTX poly(L-lysine). As expected, both lines were hypersensitive to melphalan. They were, however, normally sensitive to diphtheria toxin and ricin, indicating that some cleavage of the interchain disulfides in these protein toxins occurs even when cellular GSH is abnormally low. The lesser GSH requirement for toxin activation may be due to their extraordinary potency.