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B A Moroson

Publications and source records attributed to B A Moroson.

33 records · Page 2Linked to original sources
PubMed1985

Methotrexate resistant cells as targets for selective chemotherapy.

Strategies that are selective for eradicating methotrexate resistant cells are described. These strategies have been developed based on knowledge of the mechanism of drug resistance encountered in experimental systems and in the clinic. Drug resistance to methotrexate in experimental tumors is commonly due to either gene amplification (dihydrofolate reductase) or to impaired transport of methotrexate. While no effective drugs or methods to prevent gene amplification have been described, the concept of developing "pro drugs", i.e. a drug that is selectively reduced by dihydrofolate reductase to an inhibitor of another critical folate enzyme (thymidylate synthase, methionine synthetase, folylpolyglutamate synthetase) remains worthwhile. Second generation antifolates such as trimetrexate which are effective vs methotrexate transport resistant cells have already been developed and are in clinical trial.

Animals↗
PubMed1985

Molecular and karyological analysis of methotrexate-resistant and -sensitive human leukemic CCRF-CEM cells.

A methotrexate-resistant subline, CCRF-CEM/R1, was selected stepwise from the human leukemic lymphoblast T-cell line, CCRF-CEM, and maintained in 0.2 microM methotrexate. The development of resistance to methotrexate (75-fold) was associated with a 20-fold increase of dihydrofolate reductase activity. The affinity of dihydrofolate reductase from the resistant cells for methotrexate did not vary significantly as compared to the enzyme from the parent cells. Southern blot analysis of DNA from parent and CCRF-CEM/R1 cells demonstrated amplification of the dihydrofolate reductase gene in the resistant line. Quantitative dot-blot DNA hybridization demonstrated the presence of about 18 reductase gene copies in the R1 cells. The human dihydrofolate reductase gene contained at least 4 intervening sequences and was about 30 kilobases in size. Northern blot analysis demonstrated an increase in dihydrofolate reductase messenger RNA species, the predominant message was 3.8 kilobases. Cytogenetic analysis of CCRF-CEM/R1 cells revealed an elongated marker chromosome containing a homogeneous staining region not present in the parent line. This chromosome appeared to be derived from chromosome 21.

Base Sequence↗
PubMed1985

Cytotoxic effects of folate antagonists against methotrexate-resistant human leukemic lymphoblast CCRF-CEM cell lines.

A human T-lymphoblast cell line, CCRF-CEM/R1, resistant to methotrexate by virtue of increased dihydrofolate reductase activity, was grown in stepwise increasing concentrations of methotrexate. This additional selection pressure resulted in a cell line, CCRF-CEM/R2, resistant to methotrexate by virtue of both an elevation of dihydrofolate reductase activity and a marked decrease in methotrexate transport. The R1 and R2 cells were approximately 70- and 350-fold more resistant to methotrexate than were the parent cells. The effects of three folate antagonists were studied on these cell lines and also on CCRF-CEM/R3 cells, characterized by impaired methotrexate transport but normal levels of dihydrofolate reductase. The elevated reductase subline CCRF-CEM/R1 was cross-resistant to triazinate [Baker's antifol, NSC 139105; ethanesulfonic acid compounded with alpha-(2-chloro-4-[4,6-diamino-2,2-dimethyl-S-triazine-1-(2H)-yl] phenoxyl)-N,N-dimethyl-m-toluamide (1:1)] and trimetrexate (NSC 249008, JB-11, TMQ; 2,4-diamino-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline), two nonclassical folate antagonists. In contrast, the transport defective subline, CCRF-CEM/R3 was not cross-resistant to these two compounds. In cells resistant to MTX by virtue of both mechanisms, CCRF-CEM/R2, triazinate, and trimetrexate were partially cross-resistant. All three methotrexate-resistant sublines showed minor cross-resistance to isoaminohydroxyquinazoline (IAHQ, NSC 289517; 5,8-dideazaisopteroylglutamate), a folate antagonist inhibitor of thymidylate synthase. These data demonstrate that methotrexate-resistant tumor cells may be effectively inhibited by antifolates with different route of entry into cells or with different enzyme targets.

Antineoplastic Agents↗
PubMed1984

Synthesis and evaluation of 2,4-diaminoquinazoline antifolates with activity against methotrexate-resistant human tumor cells.

In an attempt to find potent antifolates with selectivity against tumor cells with intrinsic or acquired resistance to methotrexate, eleven nonclassical 2,4-diaminoquinazoline antifolates were synthesized and tested as inhibitors of dihydrofolate reductase from L5178Y cells. Several compounds appeared to be good enzyme inhibitors, with I50 values around 1 nM. Two of the compounds were also good inhibitors of cell growth in vitro. One of these (PKC-32, 9-(2,4-diamino-5-methylquinazoline-6-methylene)aminophenanthren e) appeared to be 100-fold more potent than methotrexate as an inhibitor of growth of a methotrexate-resistant cell line with impaired transport for methotrexate. PKC-32 and PKC-155 were also tested against mouse tumors in vivo. PKC-32 was modestly active in vivo as compared with methotrexate. This drug may be a useful agent in the treatment of methotrexate-resistant tumors.

Animals↗
PubMed1983

Amplification and organization of dihydrofolate reductase genes in a human leukemic cell line, K-562, resistant to methotrexate.

A subline of human leukemia cells (K-562), highly resistant to methotrexate, was developed by stepwise selection in the presence of increasing concentrations of this drug. The ED50 of these resistant cells was 1 mM compared to 10 nM for the parental line. Comparison of certain folate-requiring enzymes from crude extracts of the parent and resistant cells showed a 240-fold elevation of dihydrofolate reductase activity in the resistant cells with no significant increase in the levels of the other enzymes. Purified dihydrofolate reductase from the resistant cells had the same physical and kinetic properties as the enzyme from the sensitive cells. Southern blot analysis showed a marked increase in the number of dihydrofolate reductase genes in the resistant line. The genomic organization of the human dihydrofolate reductase gene was determined by hybridization with specific cDNA sequences from a human cDNA to DNA fragments from K-562 cells generated by restriction endonucleases. The human dihydrofolate reductase gene contained at least four intervening sequences and was approximately 30 kb in size. Northern blot studies demonstrated an increase of dihydrofolate reductase mRNA species; the predominant message was 3.8 kb. Karyotype analysis revealed three elongated marker chromosomes, derived from chromosomes 5, 6, and 19 which contained homogeneous staining regions, which were not present in the parent cell line.

Cell Line↗
PubMed1983

Gene amplification and altered enzymes as mechanisms for the development of drug resistance.

Two known mechanisms by which neoplastic cells may become resistant to chemotherapeutic agents are reviewed, using methotrexate (MTX) resistance as a model. These mechanisms are an increased level of target enzyme, found in several instances to be a consequence of gene amplification, or an altered target enzyme or receptor, less capable of binding the drug. An example of MTX resistance due to low-level gene amplification in leukemia cells from an MTX-resistant patient is described. Strategies for selectively eradicating these resistant cell populations may be formulated based on the mechanism by which these cells became drug-resistant.

Animals↗
PubMed1982

Pharmacology and toxicity of a potent "nonclassical" 2,4-diamino quinazoline folate antagonist, trimetrexate, in normal dogs.

The pharmacology of trimetrexate (JB-11, NSC 249008, 2,4-diamino-5-methyl-5-[(3,4,5-trimethoxyanilino)methyl]quinazoline), an antitumor agent effective against several mouse tumors, was studied in normal dogs. A high-performance liquid chromatographic technique with electrochemical detection, dihydrofolate reductase inhibition assay, and 14C-labeled drug were used to measure plasma disappearance, tissue distribution, excretion, and metabolism of the drug at doses from 0.5 to 6 mg/kg. Doses of 2 mg/kg were well tolerated without toxicity. Higher doses (3 to 6 mg/kg) produced mainly intestinal toxicity without significant hematological or liver abnormalities. The 6-mg/kg dose caused severe bloody diarrhea. After administration of 3 mg/kg, plasma concentrations of trimetrexate were 1 microM and were equal to or greater than 0.1 microM at 1 and 24 hr, respectively. The predominant pharmacokinetics of trimetrexate plasma disappearance was an elimination phase with a t1/2 of 3.5 hr. Concentrations in the cerebrospinal fluid were 2 to 5% of that in plasma and were maximum within 1 to 2 hr after i.v. administration. Highest tissue concentrations of drug were measured in liver and kidney; lowest were found in brain and lung. A dose equivalent to 3 mg/kg in humans (on a sq m basis) should produce adequate plasma concentrations (greater than 0.1 microM) for therapeutic effects.

Administration, Oral↗
PubMed1981

The role of methotrexate and dihydrofolate polyglutamates in the enhancement of fluorouracil action by methotrexate.

The effect of the methotrexate (MTX)-5-fluorouracil (FUra) combination on L1210 cell viability was highly sequence dependent in that a 1.4 log (25-fold) increase in cytotoxicity was observed when the MTX preceded FUra, as compared to the reverse sequence. The formation of ternary complexes of thymidylate synthetase has been examined as a basis for the interaction of FUra and MTX in L1210 cells. L1210 cells converted 39% of the total intracellular MTX into MTX-poly-gamma-glutamates within 4 hours of a 1 microM MTX exposure. MTX-diglutamate (2,4-diamino,N 10-methylpteroyl-diglutamate) and MTX-triglutamate were the predominant metabolites. In contrast to the ternary complexes formed with MTX and MTX-diglutamate, the 5-fluorodeoxy-uridylate (FdUMP)-7,8-dihydropteroylpentaglutamate-enzyme and the FdUMP-5,10-methylenetetrahydropteroylpentaglutamate-enzyme complexes were stable to polyacrylamide gel electrophoresis under nondenaturing conditions. MTX-diglutamate enhanced the extent of tight-binding inhibition of thymidylate synthetase activity by FdUMP in the presence of saturating 5,10-methylenetetrahydropteroylpentaglutamate, suggesting that MTX-diglutamate did not antagonize the formation of FdUMP-5,10-methylenetetrahydropteroylpentaglutamate enzyme complex. We propose that the sequence-dependent effect of MTX plus FUra on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase, and consequently a trapping of intracellular folates as dihydrofolate polyglutamates that could increase the extent of FdUMP binding to thymidylate synthetase.

Animals↗
PubMed1980

Effect of [3H]methotrexate impurities on apparent transport of methotrexate by a sensitive and resistant L1210 cell line.

Transport of methotrexate by L1210 sensitive and resistant cell lines was studied using [3H]methotrexate and methotrexate. The intracellular and cellular efflux of drug was analyzed by radioligand binding assay and high performance liquid chromatography. It was shown that the initial, rapid uptake of [3H]methotrexate was not methotrexate but rather [3H]p-aminobenzoylglutamate, even when the [3H]methotrexate was greater than 97% homogeneous. In the mutant cell line, the impurity could account for all of the apparent methotrexate uptake at 1 to 10 micro M extracellular drug. Similarly, the rapid efflux of [3H]methotrexate was also shown to be all, or in part, 3H-impurities, in both the mutant and sensitive cell lines. These results do not conflict with the currently accepted model of a carrier-mediated process for reduced folate and antifolate transport but do suggest that the quantitative interpretation of the early time points of transport experiments be more critically evaluated, especially when mutant cell lines are being analyzed.

4-Aminobenzoic Acid↗
PubMed1980

Regulation of thymidylate synthetase in mouse leukemia cells (L1210).

L1210 mouse leukemia cells were synchronized by isoleucine deprivation followed by hydroxyurea treatment. Changes of thymidylate synthetase activity both in cell extracts and in the intact cells (in situ) were followed throughout the cell cycle. The enzyme activity assayed in situ, by measuring tritium released from [5-3H]deoxyuridine, changed markedly in relation to the DNA synthesis pattern, whereas that assayed in cell extracts remained unchanged. In the S phase of the cell cycle, activity of the enzyme was much higher as measured in situ than as measured in cell extracts. Hydroxyurea (1 mM) inhibited DNA synthesis in exponentially growing L1210 cells and was found to inhibit thymidylate synthetase activity in situ. However, the enzyme activity in cell extracts was not inhibited by the drug. In cells with DNA synthesis inhibited by hydroxyurea, the pattern of change of in situ activity with time suggested product inhibition. Results of isotope-dilution analysis of the release of tritium from [5-3H]deoxyuridine in the intact cells indicated that thymidine added to the medium caused strong inhibition of thymidylate synthetase, the most likely limiting step in this pathway. These data allow the suggestion that, during cell growth, thymidylate synthetase activity is regulated by thymine nucleotide(s), and is dependent on the rate of DNA synthesis as well as the availability of the salvage pathway substrate, thymidine.

Animals↗
PubMed1979

Enhancement of antitumor activity of 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine and Baker's antifol (triazinate) with carboxypeptidase G1.

Carboxypeptidase G1 (CPDG1), an enzyme that degrades folates but not the nonclassical folate antagonists triazinate (TZT, Baker's antifol) and 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP), enhanced the antineoplastic activity of these antifolates. In 6-day cell culture experiments with Walker 256 carcinosarcoma, CPDG1 at levels up to 0.54 unit/ml showed very little inhibitory effect on growth. In the presence of 10(-7) M DDMP, the grown of Walker 256 cells was similar to that of controls, but in combination with CPDG1 (0.1 unit/ml) 80% growth inhibition was observed. TZT at a concentration of 10(-8) M did not affect the growth of Walker 256 cells. The combination of 10(-8) M TZT with CPDG1 (0.1 unit/ml), however, strongly inhibited cell growth. In experiments with rats bearing Walker 256 carcinosarcoma, the administration of CPDG1 (800 units/kg/day) from Day 1 to Day 6 resulted in no significant increase in life span. Administration of TZT in doses up to 0.05 mg/kg on Day 1 or that of DDMP up to 15 mg/kg on Days 1, 3, and 5 had no antitumor effects as measured by survival of the animals. However, CPDG1 (800 units/kg/day) from Day 1 to Day 6 in combination with TZT (0.05 mg/kg on Day 1) or DDMP (15 mg/kg on Days 1, 3, and 5) resulted in increases of 50 and 30%, respectively, in the survival of the tumor-bearing animals. These results demonstrate that CPDG1 enhances the antitumor effects of TZT or DDMP.

Animals↗