UMLS:C0019204 - FACTA Search

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Query: UMLS:C0019204 (hepatocellular carcinoma)
71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamine-dependent carbamyl phosphate synthetase [EC 2.7.2.9] was purified 1,300-fold from rat ascites hepatoma cells (AH 13) as a multienzyme complex with aspartate transcarbamylase[EC 2.1.3.2] and dihydroorotase[EC 3.5.2.3], using dimethyl sulfoxide, glycerol, and dithiothreitol as stabilizers. The purified complex was essentially homogeneous on agarose-acrylamide composite gel electrophoresis and analytical ultracentrifugation. Its molecular weight was estimated to be about 870,000 by sedimentation equilibrium studies. After alkylation with iodoacetamide or reduction with 0.6% dithiothreitol at 100 degrees, the complex gave a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate in a position corresponding to a molecular weight of 210,000. These results indicate that the complex consists of four subunits of similar size.
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PMID:Purification of homogeneous glutamine-dependent carbamyl phosphate synthetase from ascites hepatoma cells as a complex with aspartate transcarbamylase and dihydroorotase. 17 92

Carbamoyl-phosphate synthetase II [EC 6.3.5.5] of rat ascites hepatoma cells (AH 13), the first and regulatory enzyme of de novo pyrimidine nucleotide biosynthesis, exists as a multienzyme complex (molecular weight, 870,000) with aspartate carbamoyltransferase [EC 2.1.3.2] and dihydroorotase [EC 3.5.2.3] (Mori, M. & Tatibana, M. (1975) J. Biochem. 78, 239-242). The purified complex was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase [EC 2.7.1.37] of rabbit skeletal muscle. The incorporation of 32Pi was 2.2 mol/mol of the complex. The phosphorylation was completely inhibited by the inhibitor protein of the cAMP-dependent protein kinase. Among the substrates and effectors of the enzyme complex tested, only MgUTP, an allosteric inhibitor of carbamoyl-phosphate synthetase II, strongly inhibited the phosphorylation; this inhibition was due probably to the competition of MgUTP with y inhibited by the inhibitor protein of the cAMP-dependent protein kinase. Among the substrates and effectors of the enzyme complex tested, only MgUTP, an allosteric inhibitor of carbamoyl-phosphate synthetase II, strongly inhibited the phosphorylation; this inhibition was due probably to the competition of MgUTP with y inhibited by the inhibitor protein of the cAMP-dependent protein kinase. Among the substrates and effectors of the enzyme complex tested, only MgUTP, an allosteric inhibitor of carbamoyl-phosphate synthetase II, strongly inhibited the phosphorylation; this inhibition was due probably to the competition of MgUTP with the substrate MgATP for the protein kinase. The complex that was phosphorylated by cAMP-dependent protein kinase was dephosphorylated by phosphoprotein phosphatase [EC 3.1.3.16] of rat skeletal muscle. The complex was also phosphorylated by cAMP-independent protein kinase activity present in the extract of AH 13 cells and dephosphorylated by phosphoprotein phosphatase activity of the same origin. These results suggest that the complex is subject to phosphorylation and dephosphorylation in the living cells. Phosphorylation of the complex by cAMP-dependent protein kinase was associated only with a slight change, albeit definite, in the activity of carbamoyl-phosphate synthetase II under the assay conditions. Thus, the physiological significance of phosphorylation-dephosphorylation remains to be further studied.
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PMID:Phosphorylation and dephosphorylation of carbamoyl-phosphate synthetase II complex of rat ascites hepatoma cells. 611 55

Carbamoyl-phosphate synthetase II of higher animals, the first enzyme of de novo pyrimidine biosynthesis, forms a multienzyme complex with aspartate carbamoyltransferase and dihydroorotase, the second and third enzymes of the pathway. The hypothesis that the complex serves to channel carbamoyl-phosphate, synthesized by the first enzyme of the complex, to the second enzyme was tested using a highly purified complex preparation from Yoshida ascites hepatoma cells (AH 13). Experimentally, aspartate carbamoyltransferase in the complex was allowed to compete with exogenously added ornithine carbamoyltransferase, another carbamoyl-phosphate-utilizing enzyme, for carbamoyl-phosphate which was either synthesized endogenously or added exogenously. The ratios of amounts of the two enzymic products, carbamoyl-aspartate and citrulline, were compared. In the absence of enzyme stabilizers dimethyl sulfoxide or glycerol, a slight channeling of the intermediate in the complex was observed. The further addition of 5-phosphoribosyl 1-pyrophosphate, MgUTP (positive and negative allosteric effectors of carbamoyl-phosphate synthetase II), 30% (v/v) dimethyl sulfoxide or 30% (w/v) glycerol did not affect the extent of channeling. It was slightly increased in the presence of 7.5% (v/v) dimethyl sulfoxide plus 2.5% (w/v) glycerol. Any shift of the assay temperature, pH or concentration of MgATP or of the enzyme complex resulted in little further increase in the extent of channeling. Even when a larger amount of the enzyme complex was used to approximate physiological conditions, there was no increase in the extent of channeling either without or with allosteric effectors. MgUTP even abolished channeling under these conditions. These results indicate that carbamoyl-phosphate can be channeled in the multienzyme complex of AH 13 cells, but the extent of channeling is very small, contrary to expectation.
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PMID:Studies on channeling of carbamoyl-phosphate in the multienzyme complex that initiates pyrimidine biosynthesis in rat ascites hepatoma cells. 613 83

On the basis of our observation of the increased specific activities of glutamine-utilizing enzymes in purine and pyrimidine metabolism in hepatoma 3924A, and because the concentration of glutamine is ten times lower in the hepatomas than in the liver, the biochemical pharmacology of the anti-glutamine agent, acivicin, was examined. (1) Acivicin competitively inhibited the activities of amidophosphoribosyl-transferase, CTP synthetase and carbamoyl-phosphate synthetase II from extracts of liver and hepatoma 3924A. (2) In addition to the competitive inhibition exerted by acivicin, evidence was obtained that this drug also irreversibly inactivated in vitro the glutamine-utilizing enzymes. It is particularly relevant for the selectivity of acivicin that the activity of aspartate carbamoyltransferase, an enzyme present in the same complex as carbamoyl-phosphate synthetase II, was not affected by the anti-glutamine agent. (3) Acivicin in vivo brought down the activities of glutamine-utilizing enzymes in a period of 10 min to 1 hr after injection. CTP synthetase activity declined to less than 10% of that observed in the uninjected rats. The decreases were not reversible by various in vitro methods, but in vivo the activities returned to normal range in 72 hr. (4) The activity of aspartate carbamoyltransferase, which exists as a multi-enzyme complex with synthetase II, was not altered by acivicin injection. Similar results were observed in transplantable sarcoma in the rat. (5) The acivicin-induced decrease in enzymic activities could not be restored by purification of the enzymes. (6) In vitro studies indicated that addition of acivicin to liver or hepatoma extracts or purified enzymes rapidly decreased enzymic activities; the activities could not be restored. These results are consistent with an interpretation that acivicin acts either as a tight-binding inhibitor or as an inactivator through alkylation of the enzymes of glutamine utilization. (7) Acivicin in combination with actinomycin provided a synergistic kill of hepatoma cells in tissue culture and also inhibited the growth of transplantable solid hepatoma 3924A in the rat. (8) The synergistic biological results of combination chemotherapy with acivicin and actinomycin can be accounted for by the action of acivicin in inhibiting GMP and CTP synthetases, resulting in a decrease in GTP and CTP content, and by the actinomycin-caused inhibition of RNA polymerase in selectively blocking the utilization of GTP and CTP.
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PMID:Multi-enzyme-targeted chemotherapy by acivicin and actinomycin. 618 Jun 9

A single injection of the anti-glutamine drug, acivicin (NSC 163501), in tumor-bearing rats in 30 min decreased the activities of amidophosphoribosyltransferase, carbamoyl-phosphate synthetase II and CTP synthetase to 56, 50, and 7% of those of the controls. By 1 hr the activities were down to 32, 13 and 3% and they remained low for 12 hr, after which they slowly returned towards normal range in 72 hr. The decline of the activity of CTP synthetase (a loss of 80% in 10 min) was the most rapid, and the activity only returned to 60% of the controls by 3 days after the acivicin injection. In the hepatoma the concentrations of ATP and UTP changed little, but those of GTP and CTP rapidly decreased, reaching at the lowest point 32 and 2%, respectively, of control values 2 hr after acivicin; concentrations started to rise at 12 hr, reaching normal levels by 48 hr. The drop in enzyme activities preceded the decline in the pools of GTP and CTP. The behavior of enzyme activities and nucleotide concentrations in the host liver had a pattern similar to that in the hepatoma; however, the changes were less extensive than those in the tumor. The differential response between tumor and liver is attributed, in part at least, to the tissue L-glutamine concentration which in the hepatoma (0.5 mM) was 9 times lower than in the liver (4.5mM). The selectivity of acivicin action in inhibiting glutamine-utilizing enzymes is also demonstrated by the lack of effect on aspartate carbamoyltransferase, an enzymic activity which resides in the same complex as that of carbamoyl-phosphate synthetase II. The rapid decline in the activities of glutamine-utilizing enzymes is attributed to an inactivation of the enzymes by acivicin which functions as an active sitedirected affinity analog of L-glutamine. The rapid modulation of the enzymic phenotype and ribonucleotide concentrations by acivicin provides a useful tool for elucidating the role of enzymic and nucleotide imbalance in the commitment of cancer cells to replication and in the targeting of anticancer chemotherapy.
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PMID:Rapid in vivo inactivation by acivicin of CTP synthetase, carbamoyl-phosphate synthetase II, and amidophosphoribosyltransferase in hepatoma. 707 46

The antitumor drug acivicin, L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, in vivo irreversibly inactivated carbamoyl-phosphate synthetase II(glutamine-dependent)(EC 6.3.5.5), the first and rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, in transplantable rat hepatoma and host liver. With two injections of 0.5 mg acivicin per 100 g body weight to one group and two injections of 5 mg to another group, enzyme activity decreased to 20 and 1% in hepatoma and to 99 and 31% in liver respectively. Aspartate carbamoyltransferase (EC 2.1.3.2) activity was not affected. Acivicin in vitro selectively inactivated glutamine-dependent activity of the synthetase II from the hepatoma and liver, with an inactivation constant (Kinact) of 90 microM and a minimum inactivation half-time (T) of 0.7 min. The inactivation velocity with 10 microM acivicin was 5.0-fold stimulated by 2 mM MgATP and 18.4-fold by 2 mM MgATP plus 16.7 mM bicarbonate. MgATP at 0.5 mM caused half-maximum stimulation of the inactivation velocity. Under in vitro conditions, L-glutamine (1 mM) protected the enzyme from inactivation by 10 microM acivicin. The synthetase activity was protected in vitro by 6 mM concentrations for glycine (84%), L-glutamate (59%) and L-aspartate (51%) and by 0.5 mM UTP (35%) from inactivation by 20 microM acivicin. The results are compatible with the suggestion that acivicin is an active site-directed affinity analog of L-glutamine.
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PMID:In vivo inactivation by acivicin of carbamoyl-phosphate synthetase II in rat hepatoma. 708 74

d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [(14)C]Uridine labelling of hepatoma cells indicated a division of [(14)C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmolxh(-1)x(kg AS-30D or liver wet wt.)(-1). As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of (14)CO(2) into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5'-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37 degrees C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue.
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PMID:Uridylate trapping, induction of UTP deficiency, and stimulation of pyrimidine synthesis de novo by D-galactosone. 712 88

Genomic stability was investigated in Chinese hamster ovary (CHO) and human hepatocellular carcinoma HepG2 cells selected for growth in the presence of cytotoxic concentrations of N-(phosphonacetyl-L-asparate) (PALA). In CHO cells selected with 9 x LD50 PALA the carbamyl p-synthetase, aspartate transcarbamylase and dihydroorotase (CAD) gene complex was amplified two-fold while in HepG2 cells selected at comparable PALA concentrations a 7- to 10-fold increase in the CAD gene was observed. Concomitant with amplification of the CAD gene were increases in CAD mRNA and protein expression in both CHO and HepG2 cells. In long-term cultures of HepG2 cells the CAD gene underwent spontaneous amplification (5-fold) in the absence of PALA treatment with increasing passage number. In an attempt to define proteins and/or family of proteins that may either directly or indirectly influence DNA amplification potential through a mechanism of enhanced genomic instability, immobilized pH gradient-two-dimensional polyacrylamide gel electrophoresis (IPG 2-D PAGE) analysis of silver-stained nuclear cytoplasmic polypeptides concomitant with PALA resistance and CAD amplification was performed. Analysis of silver-stained polypeptides from 3 x LD50 PALA-selected CHO and HepG2 cells revealed no significant alterations in polypeptide expression. In CHO cells selected at 5 x and 7 x PALA LD50, and HepG2 cells selected at 5 x and 9 x PALA LD50, one subset of 4-8 polypeptides (pl: pI 7.2-7.6/36-38 kDa) were increased 2- to 3-fold in both 5 x and 7 x- and 5 x and 9 x LD50 PALA-selected CHO and HepG2, respectively, while five relatively neutral-to-basic, low M(r) polypeptides (p2: 18/7.30; p3: 16/7.00; p4: 14/7.00; p5: 14/7.40; and p6: 13.5/7.00) were markedly increased in CHO cells selected at 7 x LD50 PALA. In addition to these PALA-associated increases, four polypeptides (p7a: pI 6.50/40 kDa; p7b: 6.55/40; p7c: 6.60/40; and p7d: 6.65/40) were significantly increased in high-passage (p159) HepG2 cells undergoing spontaneous CAD gene amplification in the absence of PALA exposure. In CHO cells, polypeptides p7 a, b, d were increased while the expression of p7c (pI 6.60/40 kDa) was unaltered in 7 x LD50-treated CHO cells. Although neither the identity nor biological function of polypeptides 1-7 is known, a proposed mechanism involving interaction with certain growth regulatory proteins such as p53 for mediating genomic instability is given.
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PMID:Protein alterations associated with gene amplification in cultured human and rodent cells. 885 14

Argininosuccinate synthetase (ASS), a key enzyme to synthesize arginine is down regulated in many tumors including hepatocellular carcinoma (HCC). Similar to previous reports, we have found the decrease in ASS expression in poorly differentiated HCC. These ASS(-) tumors are auxotrophic for arginine. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine, has shown activity in these tumors, but the antitumor effect is not robust and hence combination treatment is needed. Herein, we have elucidated the effectiveness of ADI-PEG20 combined with 5-Fluorouracil (5-FU) in ASS(-)HCC by targeting urea cycle and pyrimidine metabolism using four HCC cell lines as model. SNU398 and SNU387 express very low levels of ASS or ASS(-) while Huh-1, and HepG2 express high ASS similar to normal cells. Our results showed that the augmented cytotoxic effect of combination treatment only occurs in SNU398 and SNU387, and not in HepG2 and Huh-1 (ASS(+)) cells, and is partly due to reduced anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP), myeloid leukemia cell differentiation protein (Mcl-1) and B-cell lymphoma-2 (Bcl-2). Importantly, lack of ASS also influences essential enzymes in pyrimidine synthesis (carbamoyl-phosphate synthetase2, aspartate transcarbamylase and dihydrooratase (CAD) and thymidylate synthase (TS)) and malate dehydrogenase-1 (MDH-1) in TCA cycle. ADI-PEG20 treatment decreased these enzymes and made them more vulnerable to 5-FU. Transfection of ASS restored these enzymes and abolished the sensitivity to ADI-PEG20 and combination treatment. Overall, our data suggest that ASS influences multiple enzymes involved in 5-FU sensitivity. Combining ADI-PEG20 and 5-FU may be effective to treat ASS(-)hepatoma and warrants further clinical investigation.
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PMID:The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma. 2858 70