Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytotoxicity of Adriamycin on human colon adenocarcinoma cell lines was investigated. Concentrations of Adriamycin producing 50% inhibition were very similar in HT29, Sw480, Sw620, and Sw1116 cells, whereas Caco-2 cells were relatively insensitive. As compared to the Sw1116 cell line, Caco-2 cells were also insensitive to mitoxantrone. Sensitivity to cisplatin, 5-fluorouracil, or ethacrynic acid was comparable in both cell lines. To find the mechanism for this mitoxantrone and Adriamycin resistance, several potential Adriamycin-detoxifying systems were characterized and quantified in both Sw1116 and Caco-2 cells. No dramatic differences in glutathione content and expression of both selenium dependent- and independent glutathione peroxidase, UDP-glucuronyltransferase, and cytochrome P-450 were found. However, highly significant differences in glutathione S-transferase activity were present, the expression of both class pi and class alpha glutathione S-transferases being much higher in the Caco-2 cell line. In addition, a slightly higher content of P-170 glycoprotein was present in the Caco-2 cells. These findings suggest that glutathione S-transferases, and to a lesser extent the P-170 glycoprotein, may be involved in mitoxantrone and Adriamycin resistance of Caco-2 colon carcinoma cells.
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PMID:Biochemical characterization of resistance to mitoxantrone and adriamycin in Caco-2 human colon adenocarcinoma cells: a possible role for glutathione S-transferases. 134 15

Resistance to chemotherapy is a significant problem in the treatment of colorectal carcinomas. To obtain insight into the mechanism of drug resistance, the expression of P-170 glycoprotein and biotransformation enzymes that are potentially able to contribute to drug resistance were investigated in paired samples of normal mucosa and tumors from 24 patients with colorectal cancer. In the tumors, glutathione S-transferase (GST) enzyme activity and content of GST-pi and P-170 glycoprotein were increased significantly compared with normal mucosa (P less than 0.03, P less than 0.003, and P less than 0.02, respectively). In contrast, GST-alpha and -mu, present in minor amounts compared with GST-pi, were downregulated in the tumor. Cytochrome P-450(4,5,6) and UDP-glucuronyltransferase (towards 4-nitrophenol and bilirubin) levels were significantly lower in the tumors (P less than 0.0001 and P less than 0.0002, respectively). Because decreased expression of cytochrome P-450 and increased levels of GST-pi and the P-170 glycoprotein have been implicated in (multi)drug resistance, these findings strongly suggest that in colorectal tumors the inherent resistance is multifactorial. Research to overcome this resistance should therefore be directed toward a combined treatment that eliminates all of these different mechanisms.
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PMID:Expression of drug-metabolizing enzymes and P-170 glycoprotein in colorectal carcinoma and normal mucosa. 135 41

The development of tumor drug resistance is the major obstacle to successful systemic chemotherapy. Therefore, devising methods for reversing drug resistance is a high priority and could lead to significant improvements in cancer treatment. The mechanisms of tumor drug resistance are manifold and are not well understood. The phenomenon of multidrug resistance (MDR) represents the development of resistance to most drugs, regardless of their chemical structure. Several types of MDR are known, for example, the overexpression of a cell membrane glycoprotein (P-170), increased activity of glutathione S-transferase, elevated levels of glutathione (GSH), and alterations in topoisomerase action. A partial reversal of tumor drug resistance has been achieved by the use of competitive inhibitors for the function of glycoprotein P-170, or by the inhibition of GSH synthesis; however, this strategy has not been substantially successful for improving the response of human tumors to clinical therapy. We have recently used electroporation, in conjunction with the cytotoxic drug, cisplatin (cDDP), in an attempt to circumvent drug resistance in cDDP-resistant mouse tumor cells (RIF/Ptr1). Electroporation is the application of a high-voltage electric shock which is known to create transient pores in plasma membranes of cultured cells. Electroporation plus cDDP treatment increased intracellular cDDP concentration and reversed cellular resistance to cDDP-induced cell killing.
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PMID:New approaches to the study of tumor drug resistance. 136 47

Mechanisms for resistance were studied in three classic type, human small cell lung cancer cell lines, GLC14, GLC16, and GLC19, that were established from one patient during clinical follow-up. Clinically the tumor changed from sensitive (GLC14) to completely resistant to (chemo)therapy (GLC19) during this period. The stain with JSB-1 antibody, detecting the Mr 170,000 multidrug resistance associated glycoprotein, was most pronounced in GLC16 and absent in GLC19. Intracellular Adriamycin (Adr) concentrations were decreased in GLC16 and GLC19 versus GLC14. Glutathione levels were 12.9, 15.5, and 16.6 micrograms/mg protein; total sulfhydryl groups were 36.5, 45.7, and 48.8 micrograms/mg protein; and glutathione S-transferase activity was 13, 29, and 43 nmol I-chloro-2,4-dinitrobenzene/min/mg protein for GLC14, GLC16, and GLC19, respectively. Incubation with DL-buthionine-S,R-sulfoximine increased Adr and cisplatin induced cytotoxicity, whereas X-ray induced cytotoxicity remained the same. Catalase activity increased from 0.88 to 1.73 to 3.83 mumol H2O2/min/mg protein in, respectively, GLC14, GLC16, and GLC19. Compared to GLC14 and GLC16, Adr induced a higher amount of DNA strand breaks in GLC19. In none of the three cell lines could Adr induced DNA strand breaks be repaired. X-ray induced a comparable amount of DNA strand breaks in all three cell lines but all cell lines were capable of repairing the X-ray induced DNA strand breaks within 90 min. It is concluded that a number of different mechanisms are operative and that some but not all of the observed changes in mechanisms for drug resistance in these lines correlate with the clinical data.
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PMID:Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow-up. 254 37

Double-stranded cDNA sequences for rat alpha 1-acid glycoprotein and rat glutathione S-transferase mRNAs were inserted into the Pst I site of bacteriophage M13mp7 and used to develop a new method for preparing specific cDNA hybridization probes directly from cloned template DNA. A palindrome sequence surrounding the Pst I site in the vector DNA permitted single-stranded DNA isolated from the recombinant phage to fold back, thus forming a stable hybrid bounded on the ends by a large loop of M13mp7 single-stranded DNA and a small loop of inserted foreign DNA. A primer corresponding to an internal sequence of the foreign DNA was hybridized, then Escherichia coli DNA polymerase I was used to synthesize a 32P-labeled complementary DNA copy of the cloned inserted DNA. The single-stranded cDNA reaction product was easily isolated by subsequent sedimentation through alkaline sucrose gradients. Gel electrophoresis of the labeled cDNA product, after denaturation with glyoxal, indicated a single discrete band with an electrophoretic mobility corresponding to the length of the inserted DNA sequence. About 95% of the cDNA product formed S1 nuclease-resistant hybrids in hybridization reactions with excess RNA in solution. DNA sequences complementary to the M13mp7 vector DNA were not detected in the cDNA product. Thus, these M13mp7-derived probes are the functional equivalent of cDNA copies to mRNAs and can be employed for quantitative measurements of mRNA concentration. This simple, rapid method probably can be used for most cloned DNA sequences to yield single-stranded radioactively labeled DNA, without contaminating vector DNA sequences, for virtually any hybridization requirement.
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PMID:Simple rapid method for the synthesis of radioactively labeled cDNA hybridization probes utilizing bacteriophage M13mp7. 627 92

Identification of cell surface viral binding proteins is important for understanding viral attachment and internalization. We have fused the pre-S domain of the duck hepatitis B virus (DHBV) large envelope protein to glutathione S-transferase and demonstrated a 170-kDa binding protein (p170) in [35S]methionine-labeled duck hepatocyte lysates. This glycoprotein was found abundantly in all extrahepatic tissues infectible with DHBV and in some noninfectible tissues, though it is not secreted into the blood. The interaction of pre-S fusion protein with p170 was competitively inhibited by wild-type DHBV in a dose-dependent manner. In addition, infection of hepatocytes with DHBV blocked the binding of pre-S fusion protein to p170, which suggests a biological role for p170 during natural infection. The p170 binding site was mapped to a conserved sequence of 16 amino acid residues (positions 87 to 102) by using 24 pre-S deletion mutants; this binding domain coincides with a major virus-neutralizing antibody epitope. Furthermore, site-directed mutagenesis revealed that an arginine residue at position 97 is critical for p170 binding. p170 was purified by a combination of ion-exchange and affinity chromatographies, and four peptide sequences were obtained. Two peptides showed significant similarities to human and animal carboxypeptides H, M, and N. Taken together, these results raise the possibility that the p170 binding protein is important during the replication cycle of DHBV.
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PMID:Interaction between duck hepatitis B virus and a 170-kilodalton cellular protein is mediated through a neutralizing epitope of the pre-S region and occurs during viral infection. 747 30

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N-terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.
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PMID:Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2. 751 75

A series of overlapping fragments spanning the entire coding sequence of the gH gene of murine cytomegalovirus (MCMV) were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST) using the pGEX expression system. A region of antibody-binding was mapped to the NH2-terminus of glycoprotein H (gH) between amino acid residues 26 and 90 on the basis of the reactivity of GST-gH fusion proteins with polyclonal antibodies to MCMV in Western blot analysis. Antibodies to gH were generated in mice immunized with the GST-gH fusion protein SK and shown to react with an 87-kDa polypeptide present in virion particles which was conserved in MCMV isolates obtained from diverse locations. They also recognized the gH protein in MCMV-infected cells, as well as gH expressed in Chinese Hamster Ovary cells. The antibodies to gH had a significant ELISA titer but no neutralizing activity in vitro. The antibody response to the GST-gH fusion protein did not modify the level of MCMV replication in the spleens of mice.
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PMID:Expression of the glycoprotein H of murine cytomegalovirus and identification of an N-terminal antibody-binding region. 752 76

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.
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PMID:Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV. 753 46

In a retrospective study the expression of the resistance proteins P-170 glycoprotein (P-170), glutathione S-transferase pi (GST-pi), thymidylate-synthase (TS), dihydrofolate reductase (DHFR) and metallothionein (MT) was investigated in 111 patients with newly diagnosed acute lymphoblastic leukemia (ALL) using the streptavidin-biotin-peroxidase complex method. The expression of the resistance proteins was found in following frequency: P-170 in 39 (35%), GST-pi in 54 (49%), TS in 46 (42%), DHFR in 21 (20%) and MT in 30 (33%) cases of the investigated patients. Patients with overexpression of P-170 or GST-pi had a significant lower probability of remaining in continuous first remission (P < 0.05 for P-170 and P < 0.01 for GST-pi). The expression of TS and DHFR had no prognostic significance on the probability of first remission. Patients with MT-overexpression showed only a tendency for a lower probability of continuous first remission. Coexpression of P-170 and GST-pi was observed in leukemias of 22 patients (21%) and 38 patients (37%) showed no evidence for the expression of both markers. Combining P-170 and GST-pi improved the prognostic value. The expression of the resistance proteins was independent of age, sex, FAB-type, immunological subtype and of the initial peripheral blast cell count. The multivariate analysis indicated that only the expression of P-170 was an independent unfavorable prognostic factor for children with initial ALL. The reason for this was an minor correlation of P-170 and GST-pi (P = 0.01).
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PMID:[Expression and clinical significance of resistance proteins in initial acute lymphatic leukemia (ALL) in childhood]. 756 59


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