Carbamoylation of glutathione reductase and changes in cellular and chromosome morphology in a rat cell line resistant to nitrogen mustards but collaterally sensitive to nitrosoureas.

A Walker 256 rat carcinoma cell line (WR) with acquired resistance to nitrogen mustards has been found to lack cross-resistance to nitrosoureas. Although total cellular glutathione pools were similar in the parent (WS) and resistant cell lines (WS, 2.5 X 10(-6); WR, 2.0 X 10(-6) mol/mg protein), glutathione reductase activity was 3.98 in WR compared to 8.67 nmol reduced nicotinamide adenine dinucleotide phosphate oxidized per microgram protein per min in WS cells. Treatment of cells with a carbamoylating nitrosourea, N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea, produced a dose-dependent inhibition of glutathione reductase and depletion of thiols in both cell lines. The drug caused no direct DNA strand breakage, but a differential mitotic spindle-chromosome stain showed that spindle formation was inhibited in WR cells at N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea concentrations of greater than 50 microM. In WS cells, mitotic figures were still visible at 100 microM. Chromosomal damage was expressed in both cell lines at concentrations of 25 microM. The number and extent of these aberrations were greater in WR than WS. Observed karyotypic abnormalities included polyploidy, chromosome decondensation, and endoreduplication. In interphase cells, transmission electron microscopy showed that the most prevalent drug-induced lesions included (a) disappearance of plasma membrane filopodia, (b) appearance of membrane blebbing, and (c) development of irregular crescent-shaped nuclei. These morphological and cytogenetic changes correlate with cytotoxic responses of these cell lines to N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea and would be consistent with drug-induced inhibition of glutathione reductase.