Intracellular glutathione heterogeneity in L1210 murine leukemia sublines made resistant to dna‐interacting anti‐neoplastic agents

Abstract

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-interacting agents having distinct mechanisms of action: L-phenylalanine mustard (L-PAM), 1,3-bis(2-chloroethyl)-I-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-morpholinyl)-doxorubicin (FCE 23762). A significant correlation was demonstrated 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 and of other thiols on the mBCl fluorescence. Although less specific, the 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 enabling a quantitative analysis to be made of intercellular GSH heterogeneity, we propose the use of appropriate thresholds and parameters of the mBCl flow cytometric distribution. By use of this analysis procedure, distinct types of alterations, with respect to the heterogeneity distribution of the parental L1210 cell line, have been evidenced in resistant cells. A uniform increase in mBCl fluorescence was observed among cells of the sublines resistant to L-PAM and FCE-24517. The mean mBCl fluorescence increase in sublines resistant to DX and DDP was due to a higher number of cells with fairly high mBCl fluorescence, but still within the range spanned by the parental cell line. A less heterogeneous mBCl fluorescence distribution was found in the L1210 subline resistant to FCE 23762, which was, however, similar to a cloned sensitive line. Though GSH was linked to the principal cause of drug resistance only in the L-PAM-resistant cell line, alterations in heterogeneity, as detected by mBCl fluorescence distributions, were found in 5 out of 6 resistant lines.

Glutathione content as a potential mediator of the vulnerability of cultured fetal cortical neurons to ethanol‐induced apoptosis

“…Oddly, there is a lack of literature addressing the concept that subpopulations of specific cells that normally possess low GSH content are predisposed to cytotoxicant damage. In nonneural cells, heterogeneity of GSH distribution in thymocytes can connect to sensitivity to apoptosis (Voehringer et al, 1998), and chemotherapeutic drug resistance can likewise be related to intracellular glutathione heterogeneity in murine leukemia (Tagliabue et al, 1993) and sarcoma cell lines (Richardson and Siemann, 1997). One of the most definitive studies of a heterogeneous content of GSH mediating toxicant responses utilized alveolar Clara cells in which GSH varies fourfold within the controls (West et al, 2000).…”

Sensitization of human colon adenocarcinoma cells (LoVo) to reactive oxygen species by a lysosomotropic compound

“…Since leukemia cells have a lower glutathione content (22) than SW 620 colon adenocarcinoma cells (23), i.e. their intrinsic defense system to oxidative stress is considerably weaker, differences in glutathione content may partly explain the difference in sensitivity to MDL 72527 between leukemia and adenocarcinoma cell lines.…”

Mechanism of Resistance to Oxidative Stress in Doxorubicin Resistant Cells.

“…One milliliter of PBS was added and cellular GSH analysis was performed by flow cytometry. 16) Enzymatic assay: Total GSH (reducedϩ oxidized) was determined enzymatically according to the method of Tietze. 17) Cells were collected by centrifugation, washed twice with ice-cold PBS, and sonicated in 10 mM HCl.…”