Characterization of melphalan-glutathione adducts whose formation is catalyzed by glutathione transferases

Summary We have quantitated the levels of mRNAs in bone marrow samples from patients with multiple myeloma of the mdrl gene (responsible for the Multidrug Resistance phenotype) and for two of the glutathione S-transferase gene. GST-2 and GST-3 (which can also inactivate a wide variety of cytotoxic drugs) and examined the relationship between the levels of expression of these genes and response to subsequent chemotherapy. From a total of 47 patients. 37 were treated with chemotherapy with 34 evaluable for response. Twenty-nine of the patients treated had not received any treatment prior to the marrow sampling while eight had previously received chemotherapy. Patients who failed to respond to initial chemotherapy had significantly higher levels of mdrl than patients who responded (P = 0.01). In the total myeloma patient data set. mRNA levels for mdrl and GST-2 were significantly correlated (Spearman rank correlation coefficient (r) = 0.54. P = 0.0004) as were expression levels of GST-2 with GST-3 (r = 0.43. P = 0.017). GST-3 and mdrl levels were more weekly associated (r = 0.16. P = 0.4). These data would suggest a significant relationship between failure of chemotherapy in multiple myeloma patients and increases in expression of the mdrl gene together with other genes whose products will generate additional mechanisms of resistance to chemotherapeutic agents.

Section: Patient Populationmentioning

confidence: 68%

Levels of expression of the mdr1 gene and glutathione S-transferase genes 2 and 3 and response to chemotherapy in multiple myeloma

Linsenmeyer

Jefferson²,

Wolf³

et al. 1992

“…Three different glutathione S-conjugates of these two nitrogen mustards can be produced: the monoglutathionyl S-conjugate, the monohydroxy glutathionyl S-conjugate and the bisglutathionyl S-conjugate (Dulik et al, 1986(Dulik et al, , 1990Ciaccio et al, 1990). This is exemplified for chlorambucil in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

“…The a-isoform of the glutathione transferases (GST), the most efficient enzyme catalysing the glutathione conjugation to these drugs, has been isolated and purified from cell lines selected for resistance to melphalan or chlorambucil, as well as from human and mouse liver cytosol and from human kidney cytosol (Lewis et al, 1988;Ciaccio et al, 1990;Meyer et al, 1993). In addition, the glutathione S-conjugates of both drugs have been characterized by fast atom bombardment mass spectrometry (Dulik et al, 1986(Dulik et al, , 1990.…”

mentioning

confidence: 99%

Multidrug resistance protein-mediated transport of chlorambucil and melphalan conjugated to glutathione

Barnouin

Leier²,

Jedlitschky³

et al. 1998

Summary The human multidrug resistance protein (MRP1) confers resistance of cells to a number of different cytostatic drugs and functions as an export pump for glutathione S-conjugates, glucuronides and other amphiphilic anions. The present study details for the first time MRP1 -mediated ATP-dependent transport of various glutathione S-conjugates of the bifunctional alkylating agents chlorambucil and melphalan. In membrane vesicles prepared from cells expressing recombinant MRP1, the conjugates were transported at rates in the following order: monoglutathionyl chlorambucil > bisglutathionyl chlorambucil > monohydroxy monoglutathionyl chlorambucil and monoglutathionyl melphalan > monohydroxy monoglutathionyl melphalan. In addition, we show that membranes from chlorambucil-resistant GST-a-overexpressing CHO cells as well as from their parental cells express the hamster homologue of MRP1. With both CHO cell membrane preparations, we observed ATP-dependent transport of monoglutathionyl chlorambucil and of leukotriene C4, a glutathione S-conjugate and high-affinity substrate of MRP1. The transport rates measured in the resistant cells were only two-to three-fold higher than those measured in the control cells. These results together with cytotoxicity assays comparing MRP1-overexpressing cell pairs with the CHO cell pair indicate that, although MRP1-mediated transport is active, it may not be the rate-limiting step in chlorambucil resistance in these cell lines.

“…Although the direct involvement of these enzymes in the detoxification of anticancer drugs has not been clearly established, the GSTmediated conjugation of melphalan has been reported (Dulik et al, 1986). This implies that other nitrogen mustards of similar structure will also be substrates for these enzymes.…”

Section: Discussionmentioning

confidence: 99%

Glutathione S-transferase isoenzymes in human tumours and tumour derived cell lines

Lewis

Forrester²,

Hayes³

et al. 1989

Edinburgh, Edinburgh Royal Infirmary, Edinburgh EH3 9 YW; and 3Department of Clinical Oncology, Newcastle General Hospital, Newcastle-upon-Tyne, UK.Summary An increasing body of evidence indicates that glutathione S-transferases play a role in the intrinsic and acquired resistance of tumours to anticancer drugs. In view of the wide use of tumour cell lines to understand the factors which confer either sensitivity or resistance to chemotherapeutic agents we have determined glutathione S-transferase (GST) activity and isozyme composition in nine human cell lines. These data have been compared with the values obtained in solid tumours. In most cases overall GST activity was higher in the tumours than in the cell lines. This was most pronounced for the breast tumour samples relative to MCF7 cell line. The pi class GST subunit was present at similar concentration in the cell lines and the tumours, and in most cases was the most abundant subunit present. The alpha and mu class GST were expressed in most of the cell lines but at much lower concentration than the pi class subunit. Also considerable variability particularly in the expression of the mu subunits was observed. This was also the case for the expression of these subunits in the solid tumour samples. The levels of these GSTs (when expressed) in the solid tumours was invariably higher than that observed in the cell lines. There are therefore several similarities but also some significant differences in GST expression in solid tumours and cell lines. Whether the differences are because expression is lost during the generation of the cell lines or whether it reflects the individuality of human tumours remains to be clearly established.The glutathione S-transferases (GST) are a multigene family of dimeric proteins which play a central role in the detoxification of electrophilic xenobiotics (Chasseaud, 1979). In man, cytosolic GSTs have been divided into three major classes termed alpha (basic), mu (neutral) and pi (acidic) (Mannervik, 1985;Stockman et al., 1987). Proteins within these groups have marked differences in their substrate specificity. There is a growing body of evidence which indicates that GST play an important role in both carcinogenesis and drug resistance. For example, certain compounds which inhibit chemical carcinogenesis are often inducers of the GST in the target tissue (Benson et al., 1978;Benson & Barretto, 1985). These proteins are also overexpressed in preneoplastic lesions (Kitahara et al., 1984;Pickett et al., 1984; Buchmann et al., 1985) and, in addition, have been demonstrated to be increased in both normal and tumour cells exposed to cytotoxic drugs (Adams et al., 1985;Wang & Tew, 1985;Carmichael et al., 1986;Batist et al., 1986;Evans et al., 1987;Robson et al., 1987;Wolf et al., 1987a;.It is likely that glutathione S-transferase levels and isoenzyme composition will play a role in both the intrinsic and acquired resistance to cancer chemotherapy (McGowan & Fox, 1986;Wolf et al., 1987b;Buller et al., 1987;Lewis et al., 1988a). Surpris...