title: Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane.
abstract: COS cells transiently expressing glutathione S-transferase (GST) pi, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (+/-)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 microM mClB for 30-35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 microM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST pi+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 +/- 0.3-fold greater than that of the control (80 +/- 4 nmol/min/mg protein). Upon a 5-fold purification of GST pi+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P less than 0.001).
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
authors.forename: R B
authors.initials: RB
authors.lastname: Puchalski
authors.forename: T H
authors.initials: TH
authors.lastname: Manoharan
authors.forename: A L
authors.initials: AL
authors.lastname: Lathrop
authors.forename: W E
authors.initials: WE
authors.lastname: Fahl
mesh_terms: D000818:Animals; D001580:Benzopyrenes / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line / Q000201:enzymology; D002470:Cell Survival; D003001:Cloning, Molecular; D003114:Colony-Forming Units Assay; D004351:Drug Resistance / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D005978:Glutathione / Q000032:analysis; D005982:Glutathione Transferase / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D011720:Pyrazoles / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D013539:Simian virus 40; D014170:Transformation, Genetic
chemical_list: D001580:Benzopyrenes; D011720:Pyrazoles; D011994:Recombinant Proteins; C006707:benzo(a)pyrene 4,5-epoxide; C059597:monochlorobimane; D005982:Glutathione Transferase; D005978:Glutathione