Biomedical subjects

G C Yeh

Publications and source records attributed to G C Yeh.

At least 19 recordsLinked to original sources

PubMed1992

A new functional role for P-glycoprotein: efflux pump for benzo(alpha)pyrene in human breast cancer MCF-7 cells.

We propose that the cellular burden of certain carcinogens may be mitigated by P-glycoprotein (P-gp), the putative drug efflux pump. In a series of multidrug resistant human breast cancer MCF-7 cells with increasing P-gp expression we examined this hypothesis using benzo(alpha)pyrene, a widely distributed environmental and dietary carcinogen. We found that multidrug resistant cells were cross-resistant to benzo(alpha)pyrene and the rates of efflux for benzo(alpha)pyrene were higher in multidrug resistant cells than in wild type cells. Evidence supporting the involvement of P-gp included the inhibition of azidopine binding to P-gp benzo(alpha)pyrene and the inhibition of benzo(alpha)pyrene efflux by Adriamycin and verapamil. Our findings suggest that P-gp may play a role in the cellular defense to carcinogens. The expression of P-gp and the modulation of its function may affect the susceptibility of normal tissues to transformation by carcinogens.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines.

A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection. The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5-fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively. The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM). The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line. The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM. The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD-1 line. Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines. The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells. Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF-Ad10 (31-fold), and DLD-Ad (55-fold) cells. Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene. Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells. These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU.

Blotting, Southern↗

PubMed1991

Antimetabolites.

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Animals↗

PubMed1990

Evidence that zinc inhibits N-methyl-D-aspartate receptor-gated ion channel activation by noncompetitive antagonism of glycine binding.

Zinc noncompetitively antagonizes N-methyl-D-aspartate (NMDA) receptor-mediated responses in cultured neurons. We investigated the mechanism of this inhibition by examining the effect of zinc on ligand binding to three distinct sites on the NMDA receptor in rat hippocampal membranes. Zinc dose-dependently inhibited both the association and dissociation of the NMDA channel blocker [3H]N-(1-[thienyl]cyclohexyl)piperidine ([3H]TCP) but had no effect on steady state levels of [3H]TCP binding. This suggests that zinc inhibits the receptor-gated access of [3H]TCP to its site in the ion channel but has no effect on the binding site itself. Zinc inhibition of [3H]TCP association was not mediated by an action at the NMDA recognition site, because zinc had no effect on NMDA-displaceable L-[3H]glutamate binding. On the other hand, zinc dose-dependently inhibited [3H]glycine binding by a noncompetitive interaction. Stoichiometric analysis of equilibrium binding data indicated the presence of two [3H]glycine binding sites/[3H]TCP binding site. Comparison of the potencies of zinc in inhibiting glycine-dependent [3H]TCP association and [3H]glycine binding suggests that blockade of only one of the two glycine sites is sufficient to prevent [3H]TCP association. We hypothesize that synaptically released zinc inhibits NMDA receptor-mediated responses by binding to a site on the receptor/channel complex, reducing glycine binding, and thereby decreasing what would otherwise be a tonically present action of endogenous extracellular glycine.

Animals↗

PubMed1990

Antimetabolites.

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Animals↗

PubMed1989

Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH.

Pyrroline-5-carboxylate reductase catalyzes the final step in proline synthesis by NAD(P)H-dependent reduction of pyrroline-5-carboxylate. We have purified and characterized this enzyme from human erythrocytes. Purification to homogeneity (approximately 600,000-fold) was accomplished by sonication, ultracentrifugation, 2',5'-ADP-Sepharose affinity chromatography, and DEAE-Sephacel ion exchange chromatography. The enzyme runs as a single band of 30,000 Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sizing chromatography under nondenaturating conditions demonstrates activity in the 300,000-350,000 Mr range, suggesting that the native enzyme exists as a 10- to 12-mer. The purified enzyme exhibits kinetic characteristics similar to those previously described for whole red cell homogenates. The Vmax is 10-fold higher and the Km for pyrroline-5-carboxylate is 7-fold higher with NADH versus NADPH as cofactor. The affinity for NADPH is 15-fold higher than that for NADH. Erythrocyte pyrroline-5-carboxylate reductase is competitively inhibited by NADP+. Unlike the enzyme from some other sources, erythrocyte pyrroline-5-carboxylate reductase is not inhibited by proline or ATP. Double label studies using [14C]pyrroline-5-carboxylate and [3H]exNADPH in the presence of both NADH and NADPH were performed to determine the preferred source of reducing equivalents. In the presence of physiologic concentrations of pyrroline-5-carboxylate and both pyridine nucleotides, all of the reducing equivalents came from NADPH. We suggest that, in some cell types including human erythrocytes, a physiologic function of pyrroline-5-carboxylate reductase is the generation of NADP+.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1989

N-methyl-D-aspartate receptor plasticity in kindling: quantitative and qualitative alterations in the N-methyl-D-aspartate receptor-channel complex.

Kindling is an animal model of epilepsy and neuronal plasticity produced by periodic electrical stimulation of the brain. Electrophysiologic studies indicate that this phenomenon is associated with increased participation of N-methyl-D-aspartate (NMDA) receptors in excitatory synaptic transmission. Biochemical studies suggest that a change intrinsic to the NMDA receptor-channel complex may contribute to the increase in NMDA receptor-mediated synaptic transmission. We tested this idea by measuring the binding of 3-[(+)-2-(carboxypiperazin-4-yl)][1,2-3H]propyl-1-phosphonic acid ([3H]CPP), [3H]glycine, and tritiated N-[(1-thienyl)cyclohexyl]piperidine [( 3H]TCP) to rat hippocampal membranes. In this preparation these ligands are selective for the NMDA receptor, the strychnine-insensitive glycine receptor, and the NMDA receptor-gated ion channel, respectively. Kindling increased the density of CPP, glycine, and TCP binding sites in hippocampal membranes by 47%, 42%, and 25%, respectively. No significant changes were detected in the affinity of these binding sites. Surprisingly, alterations in the glycine binding site were detected in animals sacrificed 1 month but not 1 day after the final kindling stimulation. Thus, delayed upregulation of the NMDA receptor-channel complex may be one molecular mechanism that maintains the long-lasting hyperexcitability of hippocampal neurons in kindled animals.

Animals↗

PubMed1989

Glycine regulation of the N-methyl-D-aspartate receptor-gated ion channel in hippocampal membranes.

The N-methyl-D-aspartate receptor-gated ion channel (NMDA channel) is regulated by glycine. To examine the interaction of glycine and NMDA receptor ligands on NMDA channel function, we used a biochemical marker of channel opening, [3H]N-(1-[thienyl]cyclohexyl)piperidine (TCP). We quantified [3H]glycine,L-[3H]glutamate, and TCP binding in an identical membrane preparation. This allowed direct comparison of NMDA and glycine receptor occupancy and channel activation. Glycine increased the association and dissociation rates of NMDA-dependent TCP binding to hippocampal membranes, without altering the Kd or Bmax for TCP binding. Structurally similar amino acids mimicked the action of glycine, with D-isomers being more potent than L-isomers. The potency of glycine in regulating TCP binding matched that for displacing [3H]glycine. Glycine stimulation of TCP binding required the presence of NMDA agonists and was inhibited by the NMDA antagonist D-2-amino-5-phosphonovaleric acid. Glycine stimulation of NMDA-dependent TCP binding was not associated with an increase in agonist binding to the NMDA receptor. Likewise, NMDA stimulation of glycine-dependent TCP binding was not associated with an increase in the binding of glycine to the glycine receptor. These findings permit the following conclusions: 1) glycine stimulates TCP binding solely by increasing the access of TCP to its site in the NMDA channel; 2) TCP binding can be used to quantify glycine regulation of the NMDA channel; 3) a stereospecific glycine receptor, as part of the NMDA receptor-channel complex, regulates NMDA-evoked channel opening by a mechanism not involving increased agonist binding to the NMDA receptor. Thus, it appears that the mechanism of glycine and NMDA receptor regulation of the NMDA channel is analogous to that of a two-key lock; both receptors, by independent and mutually required mechanisms, alter channel conformation to allow ion passage.

Allosteric Regulation↗

PubMed1988

Stimulation of phosphoribosyl pyrophosphate and purine nucleotide production by pyrroline 5-carboxylate in human erythrocytes.

Recent studies have shown that pyrroline 5-carboxylate, the intermediate in the interconversions of proline, ornithine, and glutamate, can regulate the metabolism of erythrocytes. We now report that the formation of 5-phosphoribosyl 1-pyrophosphate (PP-Rib-P) was markedly stimulated by pyrroline 5-carboxylate in intact red cells. The production of PP-Rib-P is an important point of regulation in nucleotide metabolism. We found that pyrroline 5-carboxylate increased glucose metabolism through the oxidative arm of the pentose shunt, ribose 5-phosphate formation, and PP-Rib-P production and subsequently augmented purine nucleotide production through the salvage pathway in erythrocytes. We now report that pyrroline 5-carboxylate markedly stimulated the net synthesis of inosine monophosphate from hypoxanthine in intact human red cells so that the pool of inosine monophosphate became 20-30% of the total pool of purine nucleotides. Inosine monophosphate has been considered to be a "mobile pool" of purines, i.e. a reservoir from which peripheral tissues can be supplied; the effect of pyrroline 5-carboxylate on the inosine monophosphate pool may be a mechanism for regulating the function of erythrocytes in purine delivery.

Adenine Nucleotides↗

PubMed1988

Antimetabolites.

The mechanisms of action of MTX and 5-FU have been further elucidated. Such studies will be important for the design of drug combinations and for the development of novel antifolate and fluoropyrimidine analogs. A greater understanding of MTX and ara-C transport and drug levels required to optimize transport may also aid in these endeavors. Pharmacokinetic parameters have been found to be predictors of relapse in children with acute leukemia, particularly with respect to MTX, 6-MP and ara-C. The intracellular terminal half-life of ara-C was correlated with remission duration in AML. Assay systems aimed at uncovering response predictors through biochemical analysis of patient tumor samples are being developed, including an interesting use of NMR spectroscopy to study the pharmacokinetics of fluorine-19-labeled 5-FU in vivo. Such an approach may yield valuable information on 5-FU anabolism in tumors in situ. A high frequency of resistance to MTX apparently may be generated within a single cell cycle by transient exposures to DNA synthesis inhibitors. The resistance may be based on either target enzyme amplification or altered membrane transport. These important studies provided bases for the rapid emergence of clinical resistance. Further, the multidrug-resistant phenotype appears to be a much broader based phenomenon as MTX resistance was found to be a frequent event in cells selected for multidrug resistance. A variety of novel approaches have been proposed to overcome antimetabolite resistance and to improve the selectivity of these agents, including the use of guanosine nucleotides, leucovorin and allopurines as biochemical modulators of 5-FU. Efficient techniques for the transfection of resistant DHFR into tissues using retroviruses have been reported. These studies serve as starting point for the ultimate development of more effective strategies for the treatment of human malignancies.

Antimetabolites, Antineoplastic↗

PubMed1987

Adriamycin resistance in human tumor cells associated with marked alteration in the regulation of the hexose monophosphate shunt and its response to oxidant stress.

We found that Adriamycin increased the pentose phosphate shunt activity in both Adriamycin-sensitive (WT) and Adriamycin-resistant (ADRR) human breast cancer MCF-7 cells. In contrast, hydrogen peroxide and cumene hydroperoxide markedly stimulated pentose-shunt activity in ADRR but only moderately increased the activity in WT cells. Furthermore, the altered oxidation-reduction regulation is associated with changes intrinsic to the key enzymes of the pentose-shunt pathway, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase and with glutathione peroxidase. We found the Vmax values for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were 50- and 4-fold lower, respectively, in ADRR than WT cells and the Kms of NADP+ were 10-fold lower in ADRR than WT. The activity of glutathione reductase in ADRR is 42% of that in WT. In spite of these changes, the response of the cells to both hydrogen peroxide and organic peroxide is not limited by either the capacity of the pentose shunt or glutathione reductase, but is determined by the activity of glutathione peroxidase and a glutathione transferase which possess peroxidase activity. The kinetic properties of the glucose-6-phosphate dehydrogenase in ADRR may, however, seriously limit the activity of cytochrome P-450 reductase, a major enzyme of Adriamycin conversion to a free radical.

Benzene Derivatives↗

PubMed1987

Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as a principal mode of metabolic inhibition by methotrexate.

We have investigated the role of dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo purine synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Previous studies have shown that cytotoxic concentrations of MTX that inhibit dihydrofolate reductase produce only minimal depletion of the reduced folate cofactor, 10-formyltetrahydrofolate, required for purine synthesis. At the same time, de novo purine synthesis is totally inhibited. In these studies, we show that 10 microM MTX causes inhibition of purine synthesis at the step of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase, as reflected in a 2-3-fold expansion of the intracellular AICAR pool. The inhibition of purine synthesis coincides with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of AICAR transformylase. When the generation of H2PteGlu is blocked by pretreatment with 50 microM 5-fluorodeoxyuridine (FdUrd), an inhibitor of thymidylate synthase, MTX no longer causes inhibition of purine synthesis. Intermediate levels of H2PteGlu produced in the presence of lower (0.1-10 microM) concentrations of FdUrd led to proportional inhibition of purine biosynthesis, and the exogenous addition of H2PteGlu to breast cells in culture re-established the block in purine synthesis in the presence of FdUrd and MTX. The early phases of inhibition of purine biosynthesis could be ascribed only to H2PteGlu accumulation. MTX polyglutamates, also known to inhibit AICAR transformylase, were present in breast cells only after 6 h of incubation with the parent compounds and were not formed in cells preincubated with FdUrd. The lipid-soluble antifolate trimetrexate, which does not form polyglutamates, produced modest 10-formyltetrahydrofolate depletion, but caused marked H2PteGlu accumulation and a parallel inhibition of purine biosynthesis. This evidence leads to the conclusion that MTX and the lipid-soluble analog trimetrexate cause inhibition of purine biosynthesis through the accumulation of H2PteGlu behind the blocked dihydrofolate reductase reaction.

Acyltransferases↗

PubMed1987

Antimetabolites.

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Animals↗

PubMed1985

Unique coronary vasodilator induction by leukotriene D4.

Coronary blood flow (CBF) and myocardial contractility decrease markedly in response to intracoronary administration of leukotriene D4 (LTD4). With steady infusion, however, both CBF and contractility escape, approaching preinfusion values despite ongoing LTD4 administration. To clarify the mechanism of this escape, we reinfused plasma from the coronary vein draining the myocardial area receiving LTD4. Introducing this plasma into a coronary artery caused a marked rise in coronary flow for the duration of the plasma infusion. Coronary flow reduction with vasopressin or mechanical occlusion matching that caused by LTD4 failed to elicit vasodilator production. Thus a unique coronary vasodilator factor is induced by LTD4. Whole blood or platelet-rich plasma incubated with LTD4 in vitro produced the same pattern of coronary dilation on intracoronary infusion; LTD4 incubation with platelet-poor plasma failed to elicit a vasodilation. The vasodilator factor is stable and is not potassium, a prostaglandin, catecholamine, histamine, serotonin, adenosine, adenosine diphosphate, or platelet-activating factor. Production of this leukotriene-induced vasodilator factor may account for the escape from LTD4-induced coronary constriction.

Adenosine↗

PubMed1984

The effect of pyrroline-5-carboxylic acid on nucleotide metabolism in erythrocytes from normal and glucose-6-phosphate dehydrogenase-deficient subjects.

Pyrroline-5-carboxylate, the intermediate in the interconversion of proline, ornithine, and glutamate, increases 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) and purine nucleotide formation in intact human erythrocytes. We proposed that: 1) pyrroline-5-carboxylate is converted to proline by pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH, 2) NADP+ augments glucose-6-phosphate dehydrogenase activity, and 3) production of ribose-5-phosphate via the pentose shunt is increased. Since glucose-6-phosphate dehydrogenase plays a central role in this proposed mechanism, we examined the responsiveness of glucose-6-phosphate dehydrogenase-deficient erythrocytes to pyrroline-5-carboxylate. We compared erythrocytes from four Sardinian glucose-6-phosphate dehydrogenase-deficient subjects and four Sardinian normal controls. Without pyrroline-5-carboxylate treatment, the levels of pentose shunt activity, PP-ribose-P, and inosine monophosphate were comparable in the two populations. However, the response to pyrroline-5-carboxylate in erythrocytes from normal and glucose-6-phosphate dehydrogenase-deficient subjects was markedly different. In normal erythrocytes, pyrroline-5-carboxylate treatment increased pentose shunt activity 600%, PP-ribose-P formation 250%, and the incorporation of hypoxanthine into inosine monophosphate 260%. In contrast, pyrroline-5-carboxylate had no effect on glucose-6-phosphate dehydrogenase-deficient erythrocytes. These findings strongly support our proposed mechanism for the pyrroline-5-carboxylate effect on nucleotides. Furthermore, the markedly different capacities for nucleotide synthesis in the two populations with pyrroline-5-carboxylate treatment suggest a role for pyrroline-5-carboxylate-mediated modulation of nucleotide metabolism in normal cells.

Erythrocytes↗

PubMed1983

Pyrroline-5-carboxylate stimulates the conversion of purine antimetabolites to their nucleotide forms by a redox-dependent mechanism.

The activation of purine antimetabolites to their respective nucleotides is a step critical to their effectiveness as chemotherapeutic agents. Erythrocytes, with their relatively simple purine metabolism, are useful as a model for identifying mechanisms which enhance this 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP)-dependent activation. We previously showed that pyrroline-5-carboxylate, a physiologic intermediate in the interconversions of proline, ornithine, and glutamate, markedly stimulated the pentose phosphate pathway, increased the formation of P-Rib-PP, and increased purine incorporation into nucleotides. We now report that the events initiated by pyrroline-5-carboxylate markedly increased the activation of 6-thiohypoxanthine, 6-thioguanine, and azathioprine to their respective nucleotides in intact human erythrocytes. The mechanism of this effect was directly demonstrated in studies using the conversion of hypoxanthine to inosine monophosphate as a model for pyrroline-5-carboxylate-mediated stimulation of P-Rib-PP-dependent nucleotide formation. Since the P-Rib-PP-dependent activation of these chemotherapeutic agents may be important to their clinical effectiveness, the events initiated by pyrroline-5-carboxylate may provide new insight into the nature of tumor sensitivity and resistance to these agents.

Azathioprine↗

PubMed1982

Transfer of 1-pyrroline-5-carboxylate as oxidizing potential from hepatocytes to erythrocytes.

The interconversions of proline and 1-pyrroline-5-carboxylate form an intercellular cycle that is the basis of a metabolic interaction between hepatocytes and erythrocytes. The cycle transfers oxidizing potential from hepatocytes to erythrocytes, which stimulates pentose phosphate pathway in erythrocytes. This interaction depends on the differential metabolism of proline and 1-pyrroline-5-carboxylate in erythrocytes and hepatocytes and consists of the following: in hepatocytes proline oxidase converts proline into 1-pyrroline-5-carboxylate, which is released into the medium and taken up by erythrocytes; erythrocyte 1-pyrroline-5-carboxylate reductase converts 1-pyrroline-5-carboxylate into proline and concomitantly generates NADP+; the generated oxidizing potential drives glucose metabolism through the pentose phosphate pathway in erythrocytes; finally, erythrocytes release proline into the medium, enabling it to re-enter hepatocytes and repeat the cycle. The increased activity of the pentose phosphate pathway in erythrocytes may enhance the production of 5-phosphoribosyl pyrophosphate, a necessary moiety for the processing of purines.

Animals↗

PubMed1982

Stimulation of the hexosemonophosphate-pentose pathway by pyrroline-5-carboxylate in cultured cells.

delta 1-Pyrroline-5-carboxylic acid, an intermediate in the interconversions of proline, ornithine, and glutamate, is a potent stimulator of glucose oxidation through the hexosemonophosphate-pentose pathway. The effect is observed in cultured human fibroblasts, Chinese hamster ovary cells (CHO-K1), and rabbit kidney cells (LLC-RK1). In human fibroblasts, the magnitude of the stimulation of the hexosemonophosphate-pentose pathway is dependent on the concentration of added pyrroline-5-carboxylate and the effect is observed over a wide range of glucose concentrations. The mechanism of the effect is related to the generation of oxidizing potential in the form of NADP+ by pyrroline-5-carboxylate reductase concomitant with the conversion of pyrroline-5-carboxylate to proline. In LLC-RK1 cells, a cell line unique in having proline oxidase activity, proline also stimulated hexosemonophosphate-pentose pathway activity. Although pyrroline-5-carboxylate markedly stimulated the hexosemonophosphate-pentose pathway, it has no effect on glucose metabolism in the Embden-Meyerhof pathway or the tricarboxylic acid cycle. Since the hexosemonophosphate-pentose pathway is a source of ribose-5-phosphate, the precursor of phosphoribosyl pyrophosphate, the effect of pyrroline-5-carboxylate on the hexosemonophosphate-pentose pathway may link amino acid and nucleic acid metabolism.

Animals↗