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D J Fernandes

Publications and source records attributed to D J Fernandes.

At least 37 records · Page 2Linked to original sources
PubMed1995

GTP depletion induced by IMP dehydrogenase inhibitors blocks RNA-primed DNA synthesis.

Inhibitors of IMP dehydrogenase (EC 1.2.1.14), including mizoribine (Bredinin) and mycophenolic acid, have significant antitumor and immunosuppressive activities. Studies were aimed at determining the mechanism by which intracellular GTP depletion induced by these agents results in inhibition of DNA synthesis. Incubation of human CEM leukemia cells for 2 hr with IC50 concentrations of either mizoribine (4 microM) or mycophenolic acid (0.5 microM) reduced cellular GTP levels an average of 68% or 58%, respectively, compared with the levels in control cells. Under similar conditions, mizoribine and mycophenolic acid decreased the amount of [3H]adenosine incorporated into primer RNA by 75% and 70%, respectively, relative to the untreated controls, but had no significant effect on total RNA synthesis. Repletion of the guanine nucleotide pools by coincubation of CEM cells with guanosine plus 8-aminoguanosine prevented both the inhibition of primer RNA synthesis and the inhibition of tumor cell growth induced by these agents. Additional studies demonstrated that GTP depletion alone was capable of directly inducing inhibition of primer RNA synthesis. Primer RNA synthesis was inhibited an average of 84% in whole-cell lysates that lacked GTP but contained all remaining ribo- and deoxyribonucleoside triphosphates. On an M13 DNA template, RNA-primed DNA synthesis catalyzed by the purified complex of DNA primase (EC 2.7.7.6) and DNA polymerase alpha (EC 2.7.7.7) was decreased an average of 70% in the absence of GTP, compared with synthesis in the presence of 0.5 mM GTP. These results provide evidence that mizoribine and mycophenolic acid inhibit DNA replication by inducing GTP depletion, which suppresses the synthesis of RNA-primed DNA intermediates.

Adenosine Triphosphate↗
PubMed1994

Subcellular distribution of the alpha and beta topoisomerase II-DNA complexes stabilized by VM-26.

Studies were done to determine (a) the subcellular distribution of the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II, and (b) the extent to which each isozyme forms complexes with DNA in tumor cells incubated with and without VM-26. Western blotting revealed that topoisomerase II beta was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5-100 microM VM-26 for 30 min protected the beta isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase II beta detected in nuclei of CEM cells incubated for 30 min with 25 microM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase II beta in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase II beta from degradation during cell fractionation. The stabilization of topoisomerase II beta by VM-26 allowed subsequent studies of the subcellular distribution of the topoisomerase II isozymes. Both isozymes were detected in the nonmatrix (high salt-soluble) fraction of nuclei from CEM cells, but only topoisomerase II alpha was present in the nuclear matrix. VM-26 stabilized binding of the alpha and beta topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase II alpha to matrix DNA. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function, and that both the alpha and beta isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26 is a useful way to stabilize topoisomerase II beta during cell fractionation.

Amsacrine↗
PubMed1993

Primer RNA chain termination induced by 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate. A mechanism of DNA synthesis inhibition.

The studies described herein were aimed at defining the mechanism by which 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (FaraATP), the active intracellular metabolite of fludarabine phosphate, inhibits the synthesis of primer RNA and RNA-primed DNA by the polymerase alpha-primase complex. Incubation of the purified DNA polymerase alpha-primase complex with a poly(dT) template, 500 microM ATP, and increasing concentrations of FaraATP from 2.5 to 50 microM resulted in the progressive accumulation of smaller oligoribonucleotides (2-6 nucleotides) at the expense of the full-length products of DNA primase (7-10 nucleotides). Comparison of the kcat/KM values for incorporation of FaraATP and ATP into oligoribonucleotides revealed that DNA primase incorporated FaraATP 30-fold more efficiently than ATP. FaraAMP was present exclusively at the 3'-termini of the growing primer RNA chains, which prevented further elongation of the primers by DNA primase (primer RNA chain termination). At all FaraATP concentrations tested, inhibition of RNA-primed DNA synthesis was accompanied by primer chain termination. In contrast, DNA polymerase alpha added FaraATP onto full-length primer RNAs about 8-fold less efficiently than dATP, and the incorporation of FaraAMP at the 3'-termini of the primers did not prevent further elongation of these primers by DNA polymerase alpha. These results indicate that primer RNA chain termination is the major effect responsible for the inhibition of RNA-primed DNA synthesis by fludarabine phosphate.

Adenosine Triphosphate↗
PubMed1992

Radiotherapy and verrucous carcinoma of the oral cavity. A study of 107 cases.

One hundred and seven cases of oral verrucous carcinoma treated primarily with radiotherapy at Kasturba Hospital, Manipal, India between 1977 and 1987 were analysed concerning location within the oral cavity, clinical extent, and effectiveness of radiotherapy. The most common site was the buccal mucosa followed by the buccogingival sulcus. Only 13.2% of the patients presented with T1 or T2 tumours and 32.7% had clinically negative nodes. Biopsy had to be repeated more than once in 22 patients to get confirmation of invasive carcinoma. The 5-year survival rate was 35% for stage III and 26% for stage IV. The treatment results with radiotherapy were comparable with those for ordinary squamous cell carcinoma of the oral cavity. It is felt that the treatment policy for verrucous carcinoma can be the same as for ordinary squamous cell carcinoma. In order to prevent delay in diagnosis and treatment, proper cooperation between the treating oncologist and the pathologist is essential.

Adult↗
PubMed1991

Inhibition of primer RNA formation in CCRF-CEM leukemia cells by fludarabine triphosphate.

The effects of fludarabine triphosphate (Fara-ATP), 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer RNA and DNA synthesis in human CCRF-CEM leukemia cells were investigated. RNA-primed Okazaki fragment synthesis was monitored by first incubating whole cell lysates for 10 min in the presence or absence of the compound and then following the incorporation of [alpha-32P]ATP and [3H]dTTP into the primer RNA and DNA portions, respectively, of the Okazaki fragments. In whole cell lysates the degree of DNA synthesis inhibition induced by Fara-ATP was directly related to the extent of primer RNA synthesis inhibition over the entire range of Fara-ATP concentrations tested (10-50 microM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25-200 microM) and aphidicolin (0.5-5 micrograms/ml) that inhibited DNA synthesis. The primer RNA recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or aphidicolin was of normal length, predominately 11 nucleotides. Fara-ATP was a more potent inhibitor of the polydeoxythymidylate primase activity than of the DNA polymerase alpha/delta activities present in the 100,000 x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive inhibitor of DNA primase with respect to ATP [50% inhibitory concentration, 2.3 +/- 0.3 (SD) microM, Ki = 6.1 +/- 0.3 (SE) microM] and the Km(ATP)/Ki (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases alpha/delta activities on calf thymus DNA were 43 +/- 1.6 (SD) microM and greater than 100 microM with respect to dATP and dTTP. The effects of ara-CTP and aphidicolin on these enzymes were opposite those seen with Fara-ATP, since 50% inhibitory concentrations of either ara-CTP or aphidicolin for DNA polymerases alpha/delta did not inhibit polydeoxythymidylate primase activity. The results provide evidence that fludarabine phosphate blocks DNA synthesis in CCRF-CEM cells through inhibition of primer RNA formation. In contrast, the accumulation of primer RNA and RNA-primed Okazaki fragments that is induced by ara-CTP and aphidicolin could lead to the rereplication and amplification of chromosomal DNA segments.

Aphidicolin↗
PubMed1991

Antimetabolites.

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Antimetabolites, Antineoplastic↗
PubMed1991

Nuclear matrix targets for anticancer agents.

The nuclear matrix of eukaryotic cells comprises a dynamic framework on which DNA is organized into discrete functional units of replication and transcription. There is growing evidence that matrix-associated DNA and proteins are direct targets of a wide range of clinically active anticancer agents. DNA associated with matrix-bound replication and transcription sites has a relatively open conformation and is preferentially damaged by ionizing radiation and certain alkylating agents. Fludarabine phosphate, a purine antimetabolite, inhibits DNA replication by blocking the synthesis of matrix-associated primer RNA and RNA-primed Okazaki fragments. VM-26 and m-AMSA appear to interact specifically with nuclear matrix topoisomerase II, and one mechanism of cellular resistance to these agents is associated with depletion of the matrix enzyme. Studies of the interactions of anticancer agents with targets in the nuclear matrix should provide further insight into the mechanisms by which these agents exert their therapeutic effects.

Alkylating Agents↗
PubMed1990

Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA.

CEM leukemia cells selected for resistance to VM-26 (CEM/VM-1) are cross-resistant to various other DNA topoisomerase II inhibitors but not to Vinca alkaloids. Since DNA topoisomerase II is a major protein of the nuclear matrix, we asked if alterations in nuclear matrix topoisomerase II might be important in this form of multidrug resistance. Pretreatment of drug-sensitive CEM cells for 2 h with either 5 microM VM-26 or 3 microM m-AMSA reduced the specific activity of newly replicated DNA on the nuclear matrix by 75 and 50%, respectively, relative to that of the bulk DNA. However, neither VM-26 nor m-AMSA affected the relative specific activity of nascent DNA isolated from the nuclear matrices of drug-resistant CEM/VM-1 cells. The decatenating and unknotting activities of DNA topoisomerase II were 6- and 7-fold lower, respectively, in the nuclear matrix preparations from the CEM/VM-1 cells compared to parental CEM cells. Western blot analysis revealed that the amount of immunoreactive topoisomerase II in the nuclear matrices of the CEM/VM-1 cells was decreased 3.2-fold relative to that in CEM cells, but there was no significant difference in the amount of enzyme present in the nonmatrix (1.5 M salt soluble) fractions of nuclei from these cell lines. Increasing the NaCl concentration used in the matrix isolation procedure from 0.2 to 1.8 M resulted in a progressive decrease in the specific activity of topoisomerase II in matrices of CEM/VM-1 but not CEM cells, which suggested that the association of the enzyme with the matrix is altered in the resistant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amsacrine↗
PubMed1990

Synthesis and distribution of primer RNA in nuclei of CCRF-CEM leukemia cells.

The distribution of primer RNA and RNA-primed nascent DNA in nuclei of CCRF-CEM leukemia cells was examined, and the primer RNA purified from the nuclear matrices of these cells was characterized. RNA-primed nascent DNA was radiolabeled by incubating whole-cell lysates with [alpha-32P]ATP and [3H]dTTP in the presence of approximately physiological concentrations of the remaining ribo- and deoxyribonucleoside triphosphates. The primer RNA was purified by cesium chloride density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. Nuclear subfractionation studies revealed that at least 94% of the primer RNA and RNA-primed nascent DNA were located within the insoluble matrix fraction of the nucleus. The predominant primer RNA isolated from the nuclear matrix was 8-10 nucleotides in length, and several lines of evidence indicated that this oligoribonucleotide was the functional primer RNA. Essentially all of the matrix primer RNA was covalently linked to the newly replicated DNA as demonstrated by its buoyant density in cesium chloride gradients, phosphate-transfer analysis, and sensitivity to DNase I. Analysis of 32P transfer from [alpha-32P]dTTP revealed a random distribution of ribonucleotides at the 3'-end of the primer RNA. Data obtained from mixing experiments indicated that the association of RNA-primed nascent DNA with the nuclear matrix was not the result of aggregation of these fragments with the nuclear matrix. No significant amount of either primer RNA, RNA-primed nascent DNA, or phosphate transfer was detected in the high-salt-soluble (nonmatrix) fraction of the nucleus, although the nonmatrix fraction contained most of the newly replicated DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

Proliferation-dependent cytotoxicity of methotrexate in murine L5178Y leukemia.

The basis for the proliferation-dependent cytotoxicity of methotrexate has been investigated in mice bearing the L5178Y ascites leukemia. Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase tumor cells accumulated 8-fold higher levels of methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing tumor cells were observed in the cellular levels of unmetabolized methotrexate. Intestinal mucosa and bone marrow from non-tumor-bearing mice resembled slowly growing tumor cells and had markedly lower levels of methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of methotrexate polyglutamates in the logarithmically growing tumor cells was consistent with an increased synthesis of methotrexate polyglutamates in these cells. The enhanced methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular methotrexate transport, transmembrane efflux of methotrexate, or hydrolysis of methotrexate polyglutamates. Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells. Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased methotrexate polyglutamate accumulation and thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of methotrexate for tumors with a high growth fraction is a consequence of the rapid rates of both cellular methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by thymidylate synthase.

Animals↗
PubMed1988

Effects of antileukemia agents on nuclear matrix-bound DNA replication in CCRF-CEM leukemia cells.

The effects of various antileukemic agents on DNA replication associated with the nuclear matrix were investigated in CCRF-CEM leukemia cells. Residual nuclear matrices were prepared by sequential treatment of nuclei with 1.5 M NaCl, DNase I, and Triton X-100 and contained 1-5, 10, and 37% of the total nuclear DNA, protein, and phospholipid, respectively. In control cells pulse-labeled for 45 s with [3H]thymidine, the specific activity of nascent DNA was four-fold greater in the nuclear matrix fraction relative to the specific activity of the high salt-soluble (nonmatrix) DNA fraction. Pulse-labeling and reconstitution experiments indicated that this enrichment of newly replicated DNA on the nuclear matrix did not result from aggregation of nascent DNA with the matrix. A 2-h incubation of tumor cells with either 0.1 microM teniposide (VM-26), 0.2 microM VM-26, or 0.5 microM amsacrine (m-AMSA) reduced the relative specific activity of nascent DNA on the nuclear matrix by 59, 61, and 54%, respectively, compared to control cells. In contrast hydroxyurea and cytosine arabinoside, at concentrations that markedly inhibited total nuclear DNA synthesis, did not decrease the relative specific activity of newly replicated DNA on the matrix. The results provide evidence that the antiproliferative effects of the DNA topoisomerase II inhibitors, VM-26 and m-AMSA, are localized on the nuclear matrix of CCRF-CEM leukemia cells.

Amsacrine↗
PubMed1987

L-asparaginase-induced modulation of methotrexate polyglutamylation in murine leukemia L5178Y.

The modulation of methotrexate polyglutamylation by L-asparaginase has been examined in mice bearing sublines of leukemia L5178Y that have different sensitivities to asparaginase. A single i.p. injection of 200 IU/kg of asparaginase completely inhibited ascites tumor cell growth in the parental L5178Y/S+ tumor for 120 h compared to 72 and 30 h in the L5178Y/S and L5178Y/S+/- sublines, respectively. Similarly, DNA and protein synthesis were completely inhibited by asparaginase for 96 h in L5178Y/S+ cells, but only for 72 and 24 h in L5178Y/S and L5178Y/S+/- cells. In each tumor the temporal patterns of depletion and recovery of S-phase cells were similar to the patterns of suppression and recovery of DNA and protein synthesis observed in that tumor. When methotrexate was administered at either 96 or 24 h after asparaginase during the asparaginase-induced S-phase nadirs of L5178Y/S+ and L5178Y/S+/- cells, respectively, subsequent methotrexate polyglutamylation was inhibited 83 and 92% compared to tumor cells exposed to methotrexate only. Recovery of methotrexate polyglutamylation in both tumors following L-asparaginase pretreatment coincided in time with the return in the fraction of S-phase cells towards the pretreatment values. The inhibition of methotrexate polyglutamate accumulation by asparaginase was associated with decreased retention of methotrexate in tumor cells. In contrast, asparaginase had no significant effect on methotrexate polyglutamate accumulation and methotrexate retention when administered after methotrexate. These data indicated that the asparaginase-induced modulation of methotrexate polyglutamylation in mice was directly related to the time course of inhibition and recovery of tumor cell proliferation by asparaginase, and thus varied with the intrinsic sensitivity of the individual tumor to the enzyme.

Animals↗
PubMed1986

Effect of liposomes sensitized with methotrexate-gamma-dimyristoylphosphatidylethanolamine on cells that are resistant to methotrexate.

This study compares the ability of methotrexate and liposomes, in which the drug is anchored to the lipid bilayers via methotrexate-gamma-dimyristoylphosphatidylethanolamine, to inhibit proliferation of human leukemic cells (CEM/O) and cells derived from this line that are resistant to methotrexate because of either a defective transport system (CEM/MTX cells) or elevated levels of dihydrofolate reductase (CEM/R1 cells). Whereas CEM/O and CEM/MTX cells show a 120-fold difference in their susceptibility to methotrexate (as measured by the incorporation of tritiated deoxyuridine into DNA), both lines are equally sensitive to the liposomes. In contrast, proliferation of CEM/MTX cells is not inhibited significantly by methotrexate-gamma-glycerophosphorylethanolamine (MTX-gamma-glyceroPE), the water-soluble analog of MTX-gamma-DMPE. Both the ability of the liposomes to circumvent the transport defect, and the inability of MTX-gamma-glyceroPE to do so, were anticipated on the basis of previous experiments which show that thiamine pyrophosphate could antagonize inhibition of mouse 3T3 and L1210 cell proliferation by methotrexate and MTX-gamma-glyceroPE, but not inhibition by liposomes. Human cells (CEM/O) behave similarly. The present experiments also suggest that liposomes prepared with MTX-gamma-DMPE can partially reverse the methotrexate resistance of CEM/R1 cells that is due to overproduction of the target enzyme.

Cell Division↗
PubMed1986

Dissociation of thymidylate biosynthesis from DNA biosynthesis by 5-fluoro-2'-deoxyuridine and 5,8-dideazaisofolic acid.

The effects of 5-fluoro-2'-deoxyuridine (FdUrd) and 5,8-dideazaisofolic acid on the coordination of thymidylate synthase activity and DNA synthesis were examined in human CCRF-CEM leukemic cells following a continuous exposure to these agents. In logarithmically growing control tumor cells, the rate of in situ thymidylate synthase activity equaled the rate of DNA synthesis. However, in tumor cells incubated with growth-inhibitory concentrations of either FdUrd or 5,8-dideazaisofolic acid for 48 h, the rate of thymidylate synthase activity was between 15- and 17-fold greater than the rate of DNA synthesis. The loss in tumor cell viability of FdUrd-treated cells was temporally related to this prolonged dissociation of thymidylate biosynthesis from DNA biosynthesis. The dissociation of thymidylate from DNA biosynthesis in cells incubated with FdUrd was not closely related to thymidylate depletion. The intracellular concentrations and activities of thymidylate synthase were comparable in tumor cells incubated for 24 or 48 h with either a growth-inhibitory or non-growth-inhibitory concentration of FdUrd, indicating no direct relationship among these parameters. Indirect thymidylate depletion induced by the combination of 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, hypoxanthine, and glycine inhibited in situ thymidylate synthase activity and DNA synthesis to an equal extent. In addition, the intracellular concentrations of all four deoxyribonucleoside 5'-triphosphates in tumor cells incubated with FdUrd for 48 h were between 1.3- and 3.1-fold greater than their respective concentrations in control cells, reflecting their decreased utilization in DNA synthesis in FdUrd-treated cells. These data indicated that inhibition of CCRF-CEM cell growth and DNA synthesis following a continuous exposure to cytostatic concentrations of either FdUrd or 5,8-dideazaisofolic acid resulted primarily from interference with thymidylate incorporation into DNA, and not simple blockade of thymidylate synthase.

DNA↗
PubMed1985

Resistance of CCRF-CEM cloned sublines to 5-fluorodeoxyuridine associated with enhanced phosphatase activities.

Resistance of human CCRF-CEM leukemic cells in tissue culture to 5-fluoro-2'-deoxyuridine (FdUrd) has been examined following a single drug exposure (FS sublines). In two FS sublines generated by soft agar cloning of FdUrd sensitive cells in the presence of 10 nM FdUrd, the level of drug resistance was maintained at 22- to 30-fold following 1 month growth in the absence of FdUrd. Characteristic of the FS sublines was a decreased accumulation and retention of free intracellular 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) averaging 3% of FdUrd sensitive cells, a more rapid rate of disappearance of free FdUMP and FdUMP-bound thymidylate synthase (EC 2.1.1.45, 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase), and enhanced alkaline and acid phosphatase activities. There was no significant difference in the number of nucleoside transport sites per cell among the FS sublines and FdUrd-sensitive cells, indicating that the decreased accumulation of FdUMP in the resistant sublines was not the result of impaired FdUrd transport across the plasma membrane. The more rapid turnover of FdUMP-bound TMP synthase observed in the FS sublines was neither accompanied by a decreased stability of the TMP synthase-FdUMP-5,10-methylenetetrahydrofolate ternary complex, nor an enhanced rate of degradation of FdUrd to the less potent agent, 5-fluorouracil. In addition, the growth rates of the two FS sublines were similar to that of FdUrd sensitive cells in medium containing hypoxanthine, methotrexate, and thymidine, indicating that there was no depletion of thymidine kinase (EC 2.7.1.21, ATP : thymidine-5'-phosphotransferase) in the FS sublines. Therefore, we propose that enhanced activities of acid and alkaline phosphatases, which influence the intracellular accumulation and retention of FdUMP, are important determinants of stable FdUrd resistance in CCRF-CEM cells.

Acid Phosphatase↗
PubMed1984

A method for the determination of total, free, and 5-fluorodeoxyuridylate-bound thymidylate synthase in cell extracts.

A radiochemical assay for thymidylate synthase (EC 2.1.1.45, dTMP synthase), which permits the accurate determination of total, free, and 5-fluoro-2'-deoxyuridylate (FdUMP)-bound enzyme in cells exposed to the 5-fluoropyrimidine anticancer agents, is described. The total intracellular concentrations of dTMP synthase (free plus FdUMP-bound enzyme) in extracts from CCRF-CEM leukemic cells incubated with 5-fluoro-2'-deoxyuridine were determined following dissociation of the covalent dTMP synthase-5,10-methylenetetrahydrofolate-FdUMP ternary complex in the presence of the substrate, 2'-deoxyuridine-5'-monophosphate. The addition of substrate prevented reformation of the ternary complex during the dissociation procedure, and allowed complete recovery of FdUMP binding sites in cells exposed to a high concentration of 5-fluoro-2'-deoxyuridine. After removal of the substrate by charcoal adsorption, the concentration of total FdUMP binding sites was determined by titration of the enzyme with a saturating concentration of [6-3H]FdUMP and 5,10-methylenetetrahydrofolate. The concentration of FdUMP-bound dTMP synthase was then calculated as the difference between the total and free (without prior ternary complex disruption) enzyme values. The high sensitivity of this assay coupled with its ability to accurately quantitate both free and FdUMP-bound dTMP synthase in cells exposed to a wide range of fluoropyrimidine concentrations should make it useful for a variety of experimental and clinical studies.

Binding Sites↗
PubMed1983

Effects of 5,8-dideazaisopteroylglutamate and its possible tri-gamma-glutamyl metabolite (5,8-dideazaisoPteGlu3) on colon adenocarcinoma, and the folate dependent enzymes thymidylate synthase and dihydrofolate reductase.

A series of 2-amino-4-hydroxy-quinazolines was synthesized and evaluated as inhibitors of colon adenocarcinoma and the folate-dependent enzymes, thymidylate synthase and dihydrofolate reductase. Of the quinazolines tested, 5,8-dideazaisopteroylglutamate, (IAHQ), when administered at 85 mg/kg on days 2 and 10 after tumor implantation delayed the growth of colon tumor No. 38, and resulted in 6 of 20 tumor-free animals at 90 days. In contrast, methotrexate had no effect on the growth of colon tumor No. 38 at maximally tolerated doses. IAHQ was also active against human colon adenocarcinoma cells (HCT-8) in tissue culture, requiring a concentration of 5 X 10(-7) M to inhibit cell growth 50% after 72 hours continuous exposure. Since IAHQ was an effective substrate for folylpolyglutamate synthetase, we examined the effects of IAHQ and its possible tri-gamma-glutamyl metabolite, 5,8-dideazaisoPteGLu3, on thymidylate synthase and dihydrofolate reductase. Neither IAHQ nor 5,8-dideazaisoPteGlu3 stimulated significant binding of 5-fluorodeoxyuridylate to thymidylate synthase. This was consistent with the observation that IAHQ antagonized the killing of HCT-8 cells by 5-fluorouracil. 5,8 DideazaisoPteGlu3 bound more tightly to thymidylate synthase than dihydrofolate reductase as indicated by Kis of 0.09 and 0.7 microM when deoxyuridylate and dihydropteroylglutamate, respectively, were the variable substrates. Inhibition studies also revealed that binding of IAHQ and 5,8-dideazaisoPteGlu3 to thymidylate synthase is promoted and not antagonized by deoxyuridylate. The data suggests that the biochemical basis for the antitumor effects of IAHQ is the intracellular conversion of IAHQ to poly-gamma-glutamyl metabolites, which inhibit thymidylate synthase via formation of an inhibitor-deoxyuridylate-enzyme complex.

Adenocarcinoma↗