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) m, Ya, or Yb, (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 benzoblpyrene (*)-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 p M mClB for 3035 s during transit before being analyzed for fluorescence intensity and sorted. The apparent K , for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 pM. Stained GST Ya+ or Yb,+ cells catalyzed the conjugation 2 or 5 times more effectively than GST n+ 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 f 4 nmoYmin/mg protein). Upon a 5-fold purification of GST n+ 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 <0.001).Key terms: Resistance to carcinogens, reversion of transient expression, COS cells Glutathione S-transferases (GST,3 EC 2.5.1.18) are a family of isozymes that can catalyze the covalent addition of the tripeptide glutathione, present at 0.5-10 mM in mammalian cells (35), to a structurally diverse array of physiological and xenobiotic electrophiles (21,25,32,37,41,52,53). GSTs are ubiquitous in the animal and plant kingdoms (53), and Mannervik (32) has classified the mammalian cytosolic GSTs into three classes (Pi, Alpha, and Mu) based on structural, immunological, and enzymatic properties. Mammalian cytosolic GSTs constitute 1-10% of total cytosolic protein (21) and are composed of two subunits (23-28 kilodaltons each) (32) that dimerize by noncovalent interactions (51). As demonstrated with in vitro kinetic analyses, GSTs can conjugate glutathione to anticancer alkylating agents (2,3,9,49,59) [1606][1607][1608][1609][1610][1611][1612] 1991) and Ublacker et al. (Cancer Res 51: 1783-1788, 1991, based upon in vitro kinetic analyses, support our findings done in whole cells which show that the rat )I and a class GSTs catalyze the conjugation of GSH to monochlorobimane more efficiently than 71 class GSTs.

Treatment with a JNK inhibitor increases, whereas treatment with a p38 inhibitor decreases, H2O2-induced calf pulmonary arterial endothelial cell death

“…The increased GSH levels in the control cells following MAPK inhibitor treatment may be a direct response to ROS generated by these inhibitors. GSH levels are high in typical cells (≤10 mM) and GSH transferase is ubiquitously present (48). Thus, measuring CMF fluorescence, which is produced upon reacting with thiol groups via a glutathione S-transferase-mediated reaction, is useful to evaluate GSH levels (49).…”

“…Thus, measuring CMF fluorescence, which is produced upon reacting with thiol groups via a glutathione S-transferase-mediated reaction, is useful to evaluate GSH levels (49). However, CMF dye has limitations in accurately determining whole GSH content and GSH:glutathione disulfide (GSSG, the oxidized form of GSH) ratios in cells, as this dye may also detect other thiol groups (48). The determination of exact GSH levels and GSH:GSSG ratios in H 2 O 2 -treated CPAECs with or without each MAPK inhibitor are further required in order to understand the precise role of GSH in the regulation of CPAEC redox status.…”

Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane

“…Monochlorobimane is a lipophilic probe that passively diffuses across the cell membrane. Once in the cytoplasm it reacts specifically with GSH, leading to the formation of B-SG adducts (Puchalski et al 1991). The reaction of mBCl with GSH is very specific because it is catalysed by glutathione S-transferase (GST; Puchalski et al 1991).…”

“…Once in the cytoplasm it reacts specifically with GSH, leading to the formation of B-SG adducts (Puchalski et al 1991). The reaction of mBCl with GSH is very specific because it is catalysed by glutathione S-transferase (GST; Puchalski et al 1991). For this reason mBCl is the most specific of the currently available GSH probes (Haugland 2005).…”

Assessment of fluorochromes for cellular structure and function studies by flow cytometry

“…This UV excited dye is less reactive and more specific, but monochlorobimane/flow cytometry techniques for GSH quantitation must be applied cautiously, particularly with human tumor cells [185]. Glutathione-S-transferase (GSTs) catalyzes intracellular conjugation of GSH to the dye, and this reaction is isoenzyme dependent [29,144,185]. The rat/s and ~ class GSTs catalyse the conjugation more efficiently than ~ class GSTs.…”