(CANCER RESEARCH 49,1422-1428, March 15,1989] Expression of Anionic Glutathione-5-transferase and P-Glycoprotein Genes in Human Tissues and Tumors Jeffrey A. Moscow,1 Craig R. Fairchild, Mary Jane Madden, David T. Ransom, Harry S. Wieand, Erin E. O'Brien, David G. Poplack, Jeffrey Cossman, Charles E. Myers, and Kenneth H. Cowan Medicine Branch [J. A. M., C. R. F., M. J. M. E. E. O., C. E. M., K. H. C.], PediatrìeOncology Branch [D. G. P.], and Laboratory of Pathology fJ. C.J, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892; and théDepartment of Medical Oncology [D. T. R., H. S. W.], Mayo Clinic, Rochester, Minnesota 55905 ABSTRACT The development of multidrug resistance in MCF-7 human breast cancer cells and the acquisition of broad resistance to xenobiotics in rat hyperplastic nodules are both associated with increased P-glycoprotein (mar) gene expression as well as changes in activities of intracellular (Intoxication enzymes; among these changes is a significant increase in the activity of the anionic isozyme of glutathione-S-transferase (GST). We have isolated a cDNA encoding the human anionic glutathione-5- transferase, (ìS'1'ir-l, from a cDNA library constructed from multidrug- resistant MCF-7 cells. The deduced amino acid sequence of GSTr-1 shows that while the human anionic GST displays 85% nucleotide and amino acid sequence homology to the rat anionic isozyme, it is markedly less related to human basic GST isozymes. We have examined the expression of GST* and P-glycoprotein in 170 specimens of human tissues and tumors. P-Glycoprotein RNA expression was positive in eight of 23 lymphomas and two of 12 colon tumors; however, many other normal and malignant tissues, including lung, bladder, and breast tumors, had low or undetectable levels of P-glycoprotein RNA expression. In contrast, GST»was readily detected in a wide variety of normal and malignant tissues. The level of GSTr mRNA expression in normal tissues was heterogeneous, with lowest levels found in liver and the highest levels found in lung, esophagus, and placenta. GSTr was also variably expressed in human tumors, with the lowest relative levels occurring in lymphoma and breast cancer and the highest levels found in lung cancer and head and neck tumors. In addition, comparison of paired specimens from the same patient indicated that GST n-expression was increased in many tumors relative to matched normal tissue. INTRODUCTION The phenomenon of MDR,2 the ability of malignant cells in vitro to develop cross-resistance to a broad range of structurally dissimilar toxins, has been associated with decreased intracel lular drug accumulation and with overexpression of a high- molecular weight (A/r 130,000-180,000) membrane-associated glycoprotein (P-glycoprotein) (1-5). Recent studies have shown that MDR can be conferred upon drug-sensitive cells by transfer of genes encoding P-glycoprotein (6-8). However, despite re ports of increased P-glycoprotein expression in some human tumors in the past few years (9-12), the role of P-glycoprotein in clinical drug resistance is not yet clear. We have recently reported the development of MDR in a human breast cancer cell line selected for resistance to Adria- mycin (Adr* MCF-7) (13). MDR in this cell line is associated with a number of phenotypic and biochemical changes that are remarkably similar to those reported to occur in vivo in rat liver HNs (14), a well-characterized model of chemical carcinogen- esis in which the transformed hepatocytes demonstrate broad- spectrum cross-resistance to hepatic toxins (15). Both Adr* Received 7/7/88; revised 11/18/88; accepted 11/29/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom correspondence should be addressed at Building 10, Room 12C112, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. 1The abbreviations used are: MDR, multidrug resistance; HNs, hyperplastic nodules; GST, glutathione-S-transferase. MCF-7 cells and rat HNs demonstrate decreased toxin accu mulation accompanied by overexpression of the P-glycoprotein gene as a manifestation of the MDR phenotype (16, 17). In addition, however, broad spectrum resistance in both models is associated with similar changes in the regulation of intracellular detoxication reactions, including marked increases in glutathi- one-S-transferase activity (13, 18-20). The glutathione-S-transferases are a family of enzymes which are involved in the metabolism of a broad range of xenobiotics (21). These enzymes exist as homodimers or heterodimers. The cytosolic forms of these enzymes are the products of three separate gene families which are distinguished on the basis of distinctive isoelectric focussing properties; basic, neutral, and anionic. GST isozymes have in common their ability to detoxify electrophilic and Hydrophobie substances with reduced gluta- thione. In addition, GST isozymes have been shown to bind covalenti) and noncovalently to toxins and to detoxify organic peroxides to less reactive alcohols (21). Isozymes composed of subunits from these different groups have different substrate specificities and are immunologically distinct. Recent studies have shown that the activity of the anionic GST isozyme is increased in both rat HNs and Adr* MCF-7 cells. The rat anionic GST, GST-P, an isozyme usually present only in low levels in liver, is markedly elevated in xenobiotic- resistant rat HNs (18, 19). In addition, our laboratory has reported that the human form of the anionic GST, GST*-, is increased in multidrug-resistant Adr* MCF-7 cells when com pared with the parental cell line (WT MCF-7) (13, 20). In order to further explore the association between GSTir and drug-resistance, and to determine whether GSTTr could be a useful marker for clinical drug resistance, we have cloned the cDNA encoding this enzyme and used this cDNA as a probe to examine GSTjr expression in human tissues and tumors. We also recently reported isolation of a human P-glycoprotein sequence cDNA from Adr* MCF-7 cells (22) and have exam ined the same specimens for P-glycoprotein expression in order to compare the expression of these two genes in a variety of human tumors. MATERIALS AND METHODS Nucleotide Sequence Analysis. GST«is a 725-base pair cDNA cloned from a cDNA library constructed from RNA isolated from multidrug- resistant Adr" MCF-7 cells and which was screened by hybridization with a radiolabeled rat anionic GST probe, pGP5 (23), which was kindly provided by M. Muramatsu. GSTi-l fragments were subcloned into an M 13 vector (Bethesda Research Laboratories) and sequenced using the dideoxy method with 3sS-labeled adenosine triphosphates. Standard M-13 primers were used to sequence from the ends of the fragments (Bethesda Research Laboratories) according to the manufac turer's instructions. Additional oligonucleotide primers were con structed to sequence areas from within the fragments (Midland Certified Reagent Company, Midland, TX). Hybridization Studies. Tumors and normal tissues for RNA analysis from surgical pathological specimens were quick-frozen in liquid nitro 1422 on March 10, 2016. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from