INTRACELLULAR GLUTATHIONE HETEROGENEITY IN L1210 MURINE LEUKEMIA SUBLINES MADE RESISTANT TO DNA-INTERACTING ANTINEOPLASTIC AGENTS

Intracellular glutathione (GSH) content was measured by flow cytometry using monochlorobimane (mBCl) and by the enzymatic assay in a set of 6 sublines of murine L1210 leukemia cells made resistant to DNA-intera cting agents having distinct mechanisms of action: L-phenylalanine mus tard (L-PAM), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin ( DDP), N-deformyl-N-(4-N,N-bis(2-chloroethylamino) benzoyl) distamycin A (FCE 24517), doxorubicin (DX) and 3'-deamino-3'(2-methoxy-4-morpholi nyl)-doxorubicin (FCE 23762). A significant correlation was demonstrat ed between the mean intracellular mBCl fluorescence values measured by flow cytometry and levels of GSH measured by the classical enzymatic assay, despite the possible influence of glutathione-S-transferases an d of other thiols on the mBCl fluorescence. Although less specific, th e flow cytometric method is more informative than the enzymatic assay, allowing detection of fluorescence distributions, which we proved to be characteristic of each subline. In order to assess a procedure enab ling a quantitative analysis to be made of intercellular GSH heterogen eity, we propose the use of appropriate thresholds and parameters of t he mBCl flow cytometric distribution. By use of this analysis procedur e, distinct types of alterations, with respect to the heterogeneity di stribution of the parental L1210 cell line, have been evidenced in res istant cells. A uniform increase in mBCl fluorescence was observed amo ng cells of the sublines resistant to L-PAM and FCE-24517. The mean mB Cl fluorescence increase in sublines resistant to DX and DDP was due t o a higher number of cells with fairly high mBCl fluorescence, but sti ll within the range spanned by the parental cell line. A less heteroge neous mBCl fluorescence distribution was found in the L1210 subline re sistant to FCE 23762, which was, however, similar to a cloned sensitiv e line. Though GSH was linked to the principal cause of drug resistanc e only in the L-PAM-resistant cell line, alterations in heterogeneity, as detected by mBCl fluorescence distributions, were found in 5 out o f 6 resistant lines.