ELEVATION OF PI-CLASS GLUTATHIONE S-TRANSFERASE ACTIVITY IN HUMAN-BREAST CANCER-CELLS BY TRANSFECTION OF THE GST-PI GENE AND ITS EFFECT ON SENSITIVITY TO TOXINS

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Abstract
Increased expression of the glutathione S-transferase (GDT; E.C. 2.5.1.18) .pi. class isozyme is associated with both malignant transformation and drug resistance, as well as with decreased estrogen receptor content in breast cancer. In order to further characterize the role of this enzyme in drug resistance, we cloned the cDNA encoding the human isozyme GST.pi. and developed two eukaryotic expression vectors using this cDNA and either the human metallothionein IIa or cytomegalavirus immediate-early promoters. These GST.pi. expression vectors were cotransfected with pSV2neo into drug-sensitive MCF-7 human breast cancer cells, which have low amounts of GST activity and which do not express GST.pi.. The transfected cells were selected for G418 resistance and individual clones were screened for GST activity. Three clones that demonstrated increased GST activity were selected for further study. Immunoprecipitation studies demonstrated that the increase in GST activity in these clones was due to expression of GST.pi.. Although the total GST activity of the positive clones was increased as much as 15-fold over that in wild-type MCF-7 cells, there was no change in glutathione peroxidase activity, as measured using cumenehydroperoxide as a substrate. Immunoblot studies revealed that the increased GST enzyme produced in the transfected cells was identical in size to endogenous GST.pi.. Southern -lot analysis demonstrated the incorporation of The GST.pi. expression vector into the genome of the positive clones and Northern blot analysis showed that the transfected genes made a hybrid GST.pi. RNA that was slightly larger than the endogenous GST.pi. RNA. Primer extension studies demonstrated that this increase in length corresponded to the added length of the 5''leader sequence of the expression vector. The effect of increased GST.pi. activity on the sensitivity of the transfected clones to several cytotoxic agents was assessed by colony-forming assay. The transfected clones were slightly more resistant (1.3-4.1-fold) to benzo(a)pyrene and its toxic metabolite benzo(a)pyrene-(anti)-7,8-dihydrodiol-9,10-expoxide , as well as to ethacrynic acid (3.1- to 4.4-fold). Although increased GST.pi. expression is found in MCF-7 cells selected for doxorubicin resistance, the transfected clones were not consistently more resistant to doxorubicin than control cells. In addition, the transfected cells were not resistant to either melphalan or (cis)-platinum, even though conjugation with glutathione is known to play a role in the detoxification of both of these drugs. Thus, increased GST.pi. expression, in isolated from other intracellular changes, provides a modest levels of protection from the cytotoxic effects of some lipophilic carcinogens but does not markedly contribute to resistace against doxorubicin, melphalan, or (cis)-platinum.