The stereoselectivities of three biochemically distinct human glutathione transferases, the acidic isoenzyme (pi) purified from placenta and the basic (alpha-epsilon) and the near-neutral (mu) isoenzymes purified from liver, were determined with (+/-)-benzo(a)pyrene-4,5-oxide, pyrene-4,5-oxide, and (+/-)-styrene-7,8-oxide as substrates. Transferase mu was highly selective (greater than 95%) for reaction of glutathione with R-configured oxirane carbon atoms of (+/-)-benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide, whereas transferase pi was highly stereoselective (greater than 95%) for S-configured epoxide carbon atoms of (+/-)-benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide. The basic transferases (alpha-epsilon) showed relatively low stereoselectivity with these polycyclic arene oxide substrates; glutathione reaction at R-configured oxirane carbons was preferred, but only by about 2-fold. With (+/-)-benzo(a)pyrene-4,5-oxide as substrate, transferases mu and alpha-epsilon were enantioselective for (4R,5S)-benzo(a)pyrene-4,5-oxide (about 6-fold), whereas transferase pi showed little enantioselectivity. With (+/-)-styrene-7,8-oxide as substrate, transferases mu and pi were selective for (7S)-styrene-7,8-oxide, but this enantioselectivity was not great (1.3- to 1.8-fold); enantioselectivity could not be accurately determined with alpha-epsilon due to the low enzymatic turnover. Transferase pi selectively catalyzed the reaction of glutathione with the benzylic oxirane carbon (C-7) of (+/-)-styrene-7,8-oxide whereas alpha-epsilon preferentially catalyzed reaction with the terminal epoxide carbon (C-8) atom.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkenes↗

PubMedDate not supplied

Characterization of multidrug resistance in SEWA mouse tumor cells: increased glutathione transferase activity and reversal of resistance with verapamil.

The glutathione transferase (EC 2.5.1.18) activity of multidrug-resistant and drug-sensitive SEWA mouse tumor cells was determined. Increase in activity was found in all multidrug-resistant sublines. Electrophoretic separation of cytosolic proteins and immunodetection, using antisera against Class Alpha, Class Mu and Class Pi glutathione transferase, revealed a two-fold increase of the Class Pi transferase in the resistant cells. The role of glutathione transferase of Class Pi in the multidrug resistance and its distinction from the over-produced 21,000 dalton protein p21 is discussed. Reversal of the resistance, using the calcium antagonist verapamil, and different sensitivity to verapamil between different sublines are also reported.

Animals↗

PubMedDate not supplied

Characterization of four doxorubicin adapted human breast cancer cell lines with respect to chemotherapeutic drug sensitivity, drug resistance associated membrane proteins and glutathione transferases.

Four human breast cancer cell lines with or without estrogen and progesterone receptors were adapted to growth in the continuous presence of doxorubicin (Dox) at 10 (Zr-75-1), 15 (HTB-122), or 50 (MDA-MB-231 and Hs578T) ng/ml. The sublines of Zr-75-1, MDA-MB-231 and Hs578T showed 5-10-fold Dox resistance and also cross-resistance to vincristine (VCR) and etoposide (VP16). The sublines of Zr-75-1, MDA-MB-231 and Hs578T showed 5-10-fold Dox resistance and also cross-resistance to vincristine (VCR) and etoposide (VP16). The sublines maintained or slightly increased their cis-platinum (CDDP) sensitivity. The sublines of HTB-122 showed resistance only to VP16 combined with a paradoxical increased sensitivity to VCR. The phenotypic alteration in the sublines with respect to Dox sensitivity was maintained for at least two months in the absence of Dox. The glutathione depletor buthionine sulfoximine (BSO) and the calcium channel blocker verapamil (Ver) increased the Dox sensitivity slightly only in the MDA-MB-231 and Hs578T sublines, respectively. Ver also tended to protect some of the sublines from CDDP. The sublines of Zr-75-1 and Hs578T showed increased expression of the 170-kDa permeability glycoprotein (P-gp), whereas expression of a 85-kDa membrane protein determined by the MRK20 antibody was increased in the sublines of Zr-75-1, and HTB-122. Class pi glutathione transferase (GST) levels varied greatly between the cell lines but increased during Dox selection only in the subline of Zr-75-1. Class mu GST was detectable in the MDA-MB-231, Hs578T and HTB-122 cell lines, whereas class alpha GST was detectable in these sublines but undetectable in their parental cell lines. The Zr-75-1 subline showed a 5-fold increase in the class alpha concentration. Except for a correlation between increased P-gp expression and resistance to Dox, VCR and VP16, no obvious correlations between receptor status, increased P-gp expression, membrane proteins, GST levels and acquired drug resistance were found. Thus, except for a possible role for P-gp in multidrug-resistance, these findings indicate a pronounced mechanistic heterogeneity responsible for cytotoxic drug sensitivity also in cells with a common histologic origin and exposed to the same drug.

ATP Binding Cassette Transporter, Subfamily B, Mem↗