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W L Yang

Publications and source records attributed to W L Yang.

At least 37 records · Page 2Linked to original sources
PubMed2000

Telomere shortening by cisplatin in yeast nucleotide excision repair mutant.

Telomeres are unique DNA tandem repeats that form the ends of eukaryotic chromosomes to protect the chromosomes from degradation and illegitimate recombination. In yeast, loss of telomere may be compensated for through the acquisition of new telomere by RAD52-mediated or RAD52-independent recombinational repair. In this report, the effects of cis-dichlorodiammine-platinum (II) (cisplatin) on telomere length and the role of nucleotide excision repair in telomere maintenance were examined in the yeast Saccharomyces cerevisiae. We showed that the SSL2 (RAD25) DNA repair yeast mutant exhibited a gradual shortening of the telomere in the presence of cisplatin. Further telomere shortening was prevented upon the withdrawal of cisplatin. Complementation of the mutant with the wild-type SSL2 (RAD25) gene abolished the cisplatin-induced telomere degradation. These results suggest that telomeres are susceptible to cisplatin-induced intrastrand crosslinks and that Ssl2 (Rad25) or the nucleotide excision repair pathway may play a critical role in the repair and the maintenance of telomere integrity.

Cell Survival↗
PubMed2000

Cholinergic receptor up-regulates COX-2 expression and prostaglandin E(2) production in colon cancer cells.

The M(3) muscarinic cholinergic receptor has important physiological functions on normal colonic cells. It is frequently expressed on human colon cancer cells and is biologically active. Although it is mitogenic in certain cell models, the importance of this receptor on colon carcinogenesis is unknown. In the present study we have determined expression of the M(3) receptor on human colon cancer tissue compared with matched normal tissue and examined the downstream effect of receptor activation in the HT-29 human colon carcinoma cell line. Using reverse transcription-PCR, M(3) receptor RNA expression was detected in all matched colon carcinoma and normal specimens from eight patients. Five of the eight (62%) patients showed an up to 8-fold greater level of M(3) receptor expression in cancer compared with the matched normal tissue. Exposure of HT-29 cells to carbachol, a stable receptor agonist, results in a 10-fold increase in cyclooxygenase-2 (COX-2) protein. This induction of COX-2 protein was dose dependent and was inhibited by the cholinergic receptor antagonist N-methylscopolamine (NMS). Carbachol caused a dose-dependent increase in prostaglandin E(2) (PGE(2)), the main product of cyclooxygenase activity. The maximum stimulatory effect (40-fold increase) was noted with 1mM carbachol. The increase in PGE(2) was completely abolished by NMS and by the COX-2 selective inhibitor NS398. This suggests that the M(3) receptor mediates PGE(2) production by a mechanism involving COX-2. As COX-2 and PGE(2) are known promoters of gastrointestinal cancer, these data suggest that M(3) receptor activation may facilitate progression of colon carcinoma, in part by a COX-2-mediated cellular mechanism.

Carbachol↗
PubMed1999

Transcriptional activation of the cyclin-dependent kinase inhibitor p21 by PML/RARalpha.

Acute promyelocytic leukemia (APL) is a result of clonal expansion of hematopoietic precursors blocked at the promyelocytic stage and is associated with a t(15;17) chromosomal translocation and the expression of the PML/RARalpha fusion protein. Treatment of APL cells with retinoic acid (RA) leads to complete remission by inducing growth arrest and differentiation of these cells into granulocytes. The cyclin-dependent kinase inhibitor p21WAF1/CIP1 may be involved in terminal differentiation associated growth arrest. We showed in this study that PML/RARalpha increased the transcription of p21WAF1/CIP1 gene and the activation was further induced by RA treatment. Deletion analysis revealed a region upstream of the p21WAF1/CIP1 promoter that is required for transactivation by PML/RARalpha. Transient transfection of PML/RARalpha in cells increased the endogenous p21WAF1/CIP1 protein levels. These results suggest that the induction of APL cells differentiation by RA may be a result of the activation of p21WAF1/CIP1 by PML/RARalpha.

Cell Extracts↗
PubMed1999

Human colon cancer cell proliferation mediated by the M3 muscarinic cholinergic receptor.

We have demonstrated previously cell surface receptors for gastrointestinal peptides on 10 human colon cancer cell lines. Because most of the cells studied bind muscarinic cholinergic agonists, we undertook the determination of the cholinergic receptor subtype expressed by human colon cancer cells, as well as the biological function of these receptors, and more specifically, the effect on cell proliferation. We used radiolabeled ligand binding, PCR, calcium mobilization, and cellular proliferation studies. The present study demonstrates a muscarinic cholinergic receptor having two classes of binding site for carbamylcholine. Analysis demonstrated 2499+/-153 binding sites/cell, of which 75% had a high affinity for carbamylcholine (Kd 55 microM), and 25% had a low affinity (Kd 0.33 mM). N-Methylscopolamine, a receptor antagonist, recognized only one binding site having high affinity (Kd 0.20 nM). The number of muscarinic cholinergic binding sites/cell found on colon cancer cells is 50% of the number of receptors found on guinea pig chief cells in physiological conditions. Specific cholinergic receptor antagonists inhibit binding in the following order of potency: N-methylscopolamine > 4-DAMP >> pirenzipine > AF-DX116. This order of potency pharmacologically classifies the receptor as an M3 subtype. Receptor expression, studied by reverse transcription-PCR, correlates with the binding data. Specifically, cell lines that exhibit binding, abundantly expressed the M3 receptor subtype, whereas cell lines that do not exhibit binding for muscarinic cholinergic agonists did not abundantly express the M3 receptor. Agonist activation of the M3 receptor on these cells resulted in intracellular calcium mobilization. The dose-response curve of calcium mobilization suggests that there are spare receptors on these cells. Signal transduction can be inhibited by receptor antagonists in the same order of potency in which the binding is inhibited. Exogenous agonist added to the cells in culture induces significant cell proliferation. These results demonstrate a muscarinic cholinergic receptor of the M3 subtype on human colon cancer cells. This receptor induces intracellular calcium mobilization and mediates cell proliferation. The data suggest that there are spare receptors present, and that there may be enhanced intracellular signal activation in response to receptor binding.

Animals↗
PubMed1998

Novel function of the regulatory subunit of protein kinase A: regulation of cytochrome c oxidase activity and cytochrome c release.

There have been speculations that the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) may have other functions. A recent study has shown that the catalytic (C) subunit of PKA may be regulated in a cAMP- and R subunit-independent manner. However, evidence linking a function to the R subunit apart from inhibiting the C subunit has been elusive. In this report, interaction cloning experiments showed that the RIalpha subunit association with the cytochrome c oxidase subunit Vb (CoxVb) is cAMP-sensitive. Interaction was detected with a GST-RIalpha fusion protein as well as by coimmunoprecipitation. Transient treatment with cAMP-elevating agents inhibited cytochrome c oxidase in Chinese hamster ovary (CHO) cells with a concomitant decrease in cytochrome c levels in the mitochondria and an increase in its release into the cytosol. Furthermore, mutant cells harboring a defective RIalpha show increased cytochrome c oxidase activity and also constitutively lower levels of cytochrome c in comparison to either the wild-type cells or the C subunit mutant. These results suggest a novel mechanism of cAMP signaling through the interaction of RIalpha with CoxVb thereby regulating cytochrome c oxidase activity as well as the cytochrome c levels.

Animals↗
PubMed1998

The requirement of yeast Ssl2 (Rad25) for the repair of cisplatin-damaged DNA.

Cisplatin is one of the most widely used anticancer agents. Cisplatin-induced cytotoxicity results from its ability to form cisplatin-DNA adducts within the cellular genome which can inhibit the transcription of genes and the replication of DNA. Cisplatin-adducts are primarily removed by the nucleotide excision repair (NER) pathway. The SSL2 (RAD25) gene of Saccharomyces cerevisiae, a homolog of the XPB (ERCC3) gene in humans, is involved in the nucleotide excision repair of UV-damaged DNA and is also required for cell viability. However, the role of Ssl2 (Rad25) in cisplatin sensitivity has not been examined. In this study, we have demonstrated that a yeast strain carrying the mutant allele SSL2-XP, a truncated form of SSL2 (RAD25) at the carboxyl terminus to mimic the human XPB (ERCC3) mutation, has increased cellular sensitivity to cisplatin in comparison to wild type cells. Analysis by host cell reactivation (HCR) assay further shows that Ssl2 (Rad25) is required for the repair of cisplatin-damaged DNA.

Antineoplastic Agents↗
PubMed1998

Nucleotide-dependent tetramerization of CTP synthetase from Saccharomyces cerevisiae.

The nucleotide-dependent tetramerization of purified native URA7-encoded CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) from the yeast Saccharomyces cerevisiae was characterized. CTP synthetase existed as a dimer in the absence of ATP and UTP. In the presence of saturating concentrations of ATP and UTP, the CTP synthetase protein existed as a tetramer. Increasing concentrations of ATP and UTP caused a dose-dependent conversion of the dimeric species to a tetramer. The kinetics of enzyme tetramerization correlates with the kinetics of enzyme activity. The tetramerization of CTP synthetase was dependent on UTP and Mg2+ ions. ATP facilitated the UTP-dependent tetramerization of CTP synthetase by a mechanism that involved the ATP-dependent phosphorylation of UTP catalyzed by the enzyme. The glutaminase reaction that is catalyzed by the enzyme was not required for enzyme tetramerization. CTP, a potent inhibitor of CTP synthetase activity, did not inhibit the ATP/UTP-dependent tetramerization of the enzyme. Phosphorylation of the purified native CTP synthetase with protein kinase A and protein kinase C facilitated the nucleotide-dependent tetramerization. Dephosphorylation of native CTP synthetase with alkaline phosphatase prevented the nucleotide-dependent tetramerization of the enzyme. This correlated with the inactivation of CTP synthetase activity. Rephosphorylation of the dephosphorylated enzyme with protein kinase A and protein kinase C resulted in a partial restoration of the nucleotide-dependent tetramerization of the enzyme. This tetramerization correlated with the partial restoration of CTP synthetase activity. Taken together, these results indicated that enzyme tetramerization was required for CTP synthetase activity and that enzyme phosphorylation played an important role in the tetramerization and regulation of the enzyme.

Adenylyl Imidodiphosphate↗
PubMed1998

Cisplatin resistance in cyclic AMP-dependent protein kinase mutants.

The emergence of cisplatin resistance poses a major problem to the successful treatment of a variety of human malignancies. Therefore, understanding the molecular mechanisms that underlie cisplatin resistance could significantly improve the clinical efficacy of this cytotoxic agent. Various studies have described that cellular sensitivity to cisplatin can be influenced by several signal transduction pathways. In this review, we examine the role of the cyclic AMP-dependent protein kinase (PKA) in the modulation of drug resistance in cancer. By a somatic mutant genetic approach, the role of PKA in the development of resistance to chemotherapeutic agents has been investigated. A series of mutants with decreased PKA activity was examined for their sensitivity to cisplatin. PKA mutants with defective regulatory (RIalpha) subunits, but not altered catalytic (C) subunits, exhibit increased resistance to cisplatin, as well as other DNA-damaging agents. Furthermore, since RIalpha subunit mutants show enhanced DNA repair we, therefore, hypothesize that functional inactivation of PKA may result in increased recognition and repair of cisplatin lesions. Alternatively, it seems likely that mutation of the RIalpha subunit may affect cellular sensitivity to various anticancer drugs, suggesting that the RIalpha subunit may have other physiological functions in addition to inhibiting the kinase activity of the C subunit. Therefore, exploitation of cyclic AMP levels or functional alteration of the R subunit may potentiate the cytotoxicity of chemotherapeutic agents and circumvent drug resistance in cancer. More importantly, the altered pattern and mechanism of drug resistance may offer the opportunity to investigate novel regulatory functions of the RIalpha subunit of PKA.

Antineoplastic Agents↗
PubMed1998

Paraquat-induced cell death in PC12 cells.

Paraquat was taken up by PC12 cells in a carrier-mediated, saturable manner. When PC12 cells were permeabilized with digitonin (50 microg/ml) lipid peroxidation was observed after paraquat treatment in the presence of NADPH and chelated iron. The fact that lipid peroxidation preceded the appearance of LDH release provides positive evidence that lipid peroxidation may be one of the important factors leading to cytotoxicity of cells. Furthermore, the fact that addition of superoxide dismutase, catalase and promethazine efficiently blocked the malondialdehyde formation and attenuated the cell death indicated the involvement of reactive oxygen radicals in mediating the cytotoxicity induced by paraquat. Taken together the results present in vitro evidence that neurotoxicity of paraquat may be a consequence of cellular lipid peroxidation, which leads to cell death and may have great implications in assessing the risk of exposure to paraquat in Parkinson's disease.

Animals↗
PubMed1998

The effects of ethnicity on disease patterns in 472 Orientals with systemic lupus erythematosus.

OBJECTIVE: To determine the effects of ethnicity on disease manifestations in Oriental patients with systemic lupus erythematosus (SLE) and to describe the risk of developing renal or central nervous system (CNS) involvement with time. METHODS: A retrospective study of 472 patients with SLE seen at the only Rheumatology Unit in Singapore. The effect of ethnicity on selected disease manifestations at diagnosis was assessed after adjusting for demographic variables using multiple logistic regression. The probability of developing selected disease manifestations with time was determined using the Kaplan-Meier product limit method. RESULTS: At diagnosis, Malays had a higher risk of renal or CNS involvement than Chinese (OR 2.26, 95% CI 1.21 to 4.21, and OR 3.07, 95% CI 1.01 to 9.34, respectively), and Indians a lower risk of malar rash and a higher risk of oral ulcers than Chinese (OR 0.30, 95% CI 0.13 to 0.68, and OR 2.90, 95% CI 1.45 to 7.34, respectively). The prevalence of renal or CNS involvement in the entire cohort increased with time, reaching 75.6% (95% CI 66.1% to 85.0%) and 16.7% (95% CI 11.7% to 21.6%), respectively, after 18 years of disease. CONCLUSION: Ethnicity influenced disease manifestations at diagnosis in this cohort of Oriental patients with SLE. Renal or CNS involvement developed in previously unaffected patients up to 18 years after diagnosis, highlighting the need for continued vigilance in patients with lupus.

Adolescent↗
PubMed1997

Modulation of muscle creatine kinase promoter activity by the inducible orphan nuclear receptor TIS1.

TIS1, an inducible orphan nuclear receptor, was originally isolated as a tumour-promoter-inducible gene in mouse 3T3 cells and later shown to be induced by growth factors and other extracellular stimuli. We show here that TIS1 mRNA was expressed in proliferating C2C12 mouse skeletal muscle cells out that the level of TIS1 expression increased during muscle differentiation. Overexpression of TIS1 transactivated muscle creatine kinase (MCK) reporter genes containing as little as 80 bp of the proximal 5' flanking region. In contrast, a promoterless TIS1 construct and a frameshift mutant TIS1 construct were unable to transactivate the MCK reporter gene. Moreover, the effect exerted by TIS1 appeared to be selective for the MCK promoter. Treatment of C2C12 cells with forskolin, which is known to induce TIS1 expression, also stimulated MCK reporter gene activity. Interestingly, in vitro translated TIS1 protein failed to bind to the MCK promoter region, suggesting that the transactivation effect of TIS1 may be mediated without direct interaction of the protein with the MCK promoter DNA. Collectively, these results suggest that changing levels of TIS1 may help to modulate the expression of MCK, and perhaps other muscle-specific genes, in response to physiological changes.

3T3 Cells↗
PubMed1996

Phosphorylation and regulation of CTP synthetase from Saccharomyces cerevisiae by protein kinase A.

The phosphorylation and regulation of the URA7-encoded CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae by cAMP-dependent protein kinase (protein kinase A) were examined. Protein kinase A is the principal mediator of signals transmitted through the RAS/cAMP pathway in S. cerevisiae. The results of labeling experiments indicated that the phosphorylation of CTP synthetase was mediated by the RAS/cAMP pathway in vivo. In vitro, protein kinase A phosphorylated CTP synthetase at a serine residue with a stoichiometry consistent with one phosphorylation site per CTP synthetase subunit. Protein kinase A activity was dose- and time-dependent using CTP synthetase as a substrate. The dependence of protein kinase A activity on CTP synthetase was cooperative (n = 1.8) and the Km value for CTP synthetase was 73 nM. Phosphorylation of CTP synthetase with protein kinase A resulted in the stimulation (190%) of activity. The mechanism of this stimulation included an increase in the Vmax of the reaction with respect to UTP and ATP, a decrease in the Km for ATP, and a decrease in the cooperative kinetic behavior of the enzyme. Phosphorylated CTP synthetase was less sensitive to product inhibition by CTP. Protein kinase C also phosphorylates and activates CTP synthetase. Phosphorylation of CTP synthetase with protein kinases A and C together resulted in an increase in CTP synthetase activity that was slightly greater than that obtained when the enzyme was phosphorylated with either protein kinase alone.

Adenosine Triphosphate↗
PubMed1996

Metabotropic glutamate receptor 5 is a disulfide-linked dimer.

The sequences of the metabotropic glutamate receptors (mGluRs) show little homology with other members of the G protein-coupled receptor family and exhibit several distinctive features, including a large N-terminal extracellular domain with 17 cysteines in conserved positions. Here we demonstrate that mGluR5, as well as other mGluRs, behave as species approximately twice as large as expected from their sequence, but reducing conditions cause a decrease to the predicted molecular mass. Co-immunoprecipitation experiments using wild type and epitope-tagged receptors demonstrate that this is due to specific, disulfide-dependent dimerization of the receptor. The intermolecular disulfide that mediates dimerization occurs in the extracellular domain, within about 17 kDa from the N terminus.

Amino Acid Sequence↗
PubMed1996

Regulation of yeast CTP synthetase activity by protein kinase C.

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purified CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this activation involved an increase in the apparent Vmax of the reaction and an increase in the enzyme's affinity for ATP. In vitro phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to product inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity.

Adenosine Triphosphate↗
PubMed1995

Differential biochemical regulation of the URA7- and URA8-encoded CTP synthetases from Saccharomyces cerevisiae.

The URA7- and URA8-encoded CTP synthetases (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming) are functionally overlapping enzymes responsible for the biosynthesis of CTP in the yeast Saccharomyces cerevisiae. URA8-encoded CTP synthetase was purified to apparent homogeneity by ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Q-Sepharose, Affi-Gel Blue, Mono Q, and Superose 6. The subunit molecular mass (67 kDa) of purified URA8-encoded CTP synthetase was in good agreement with the predicted size of the URA8 gene product. Antibodies raised against a fusion protein constructed from the coding sequences of the URA8 gene and expressed in Escherichia coli reacted with purified URA8-encoded CTP synthetase. Native URA8-encoded CTP synthetase existed as a dimer which oligomerized to a tetramer in the presence of its substrates UTP and ATP. Maximum URA8-encoded CTP synthetase activity was dependent on Mg2+ ions (Ka = 2.4 mM) and 2-mercaptoethanol at the pH optimum of 7.5. The enzyme followed saturation kinetics toward UTP (Km = 74 microM), ATP (Km = 22 microM), and glutamine (Km = 0.14 mM). GTP stimulated (Ka = 26 microM) URA8-encoded CTP synthetase activity 12-fold. CTP potently inhibited (IC50 = 85 microM) URA8-encoded CTP synthetase activity and, in addition, caused the dependence of activity toward UTP to become cooperative. The URA8-encoded CTP synthetase and the previously purified URA7-encoded CTP synthetase differed significantly with respect to several biochemical properties including turnover number, pH optimum, substrate dependences, and sensitivity to inhibition by CTP. The URA7-encoded CTP synthetase mRNA was 2-fold more abundant when compared with URA8-encoded CTP synthetase mRNA. Both CTP synthetase isoforms were maximally expressed in the exponential phase of growth.

Base Sequence↗
PubMed1995

Phosphorylation of CTP synthetase from Saccharomyces cerevisiae by protein kinase C.

Phosphorylation of CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae protein kinase C was examined. Using pure CTP by synthetase as a substrate, protein kinase C activity was dose- and time-dependent and required calcium, diacylglycerol, and phosphatidylserine for full activation. Protein kinase C activity was also dependent on the concentration of CTP synthetase. Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro whereas the enzyme was primarily phosphorylated on serine residues in vivo. Phosphopeptide mapping analysis of CTP synthetase phosphorylated in vitro and in vivo indicated that the enzyme was phosphorylated on more than one site. Most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from CTP synthetase phosphorylated by protein kinase C in vitro. The stoichiometry of the phosphorylation of native CTP synthetase was 0.4 mol of phosphate/mol of enzyme whereas the stoichiometry of the phosphorylation of alkaline phosphatase-treated CTP synthetase was 2.2 mol of phosphate/mol of enzyme. This indicated that CTP synthetase was purified in a phosphorylated state. Phosphorylation of CTP synthetase resulted in a 3-fold activation in enzyme activity whereas alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold decrease in enzyme activity. Overall, the results reported here were consistent with the conclusion that CTP synthetase was regulated by protein kinase C phosphorylation.

Adenosine Triphosphate↗
PubMed1995

Signal-transduction-pathway-specific desensitization of expression of orphan nuclear receptor TIS1.

Induction of many primary-response genes by a variety of stimuli occurs in a transient manner. The precise mechanism responsible for these transient kinetics is not completely understood. We report here that the orphan nuclear receptor, TIS1, which is a potent sequence-specific transcription factor, was transiently induced by the adrenergic agonist isoprenaline in the C2C12 skeletal-muscle cell line. Moreover, we showed that the rapid decline in mRNA level after peak induction was due in part to a specific desensitization of the isoprenaline-mediated induction pathway. Desensitization of the induction response presumably occurred at the level of the receptor, as agents that either bypass the adrenergic receptor or activate alternative signalling pathways were able to induce TIS1 expression in the desensitized cells. However, stimulation by agents that directly activate intracellular enzymes also resulted in the signal-transduction-pathway-specific desensitization of TIS1 inducibility. Our results suggest that the pathway-specific nature of the desensitization process may be important for directing an integrated response to multiple physiological stimuli.

Adrenergic beta-Agonists↗