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Title: Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene

Abstract

Methotrexate (MTX), a folate analog which inhibits the enzyme dihydrofolate reductase (DHFR), is an effective antineoplastic drug. However, MTX-induced myelosuppression limits the effectiveness of this agent. Selective induction of MTX resistance in bone marrow stem cells, prior to treatment with MTX, might prevent this toxicity and improve the therapeutic index of the drug. In these studies drug resistance was transferred to mouse and human bone marrow stem cells by retroviral expression vectors containing coding sequences of a mutant DHFR with a decreased affinity for MTX. Three retroviral expression vectors were analyzed. The CIS DR vector contained the mutant DHFR gene inserted into the replication-defective amphotropic 4070 virus, Cistor. The other vectors contained the mutant DHFR inserted into either the env region (SDHT1) or gag-pol region (SDHT2) of a replication-defective spleen focus-forming virus. All three constructs induced approximately a 200-fold resistance to MTX when transfected into NIH3T3 cells. Amphotropic infectious retroviruses were obtained by transfecting the mutant DHFR vectors into a packaging cell line, which supplied the gag, pol, and env proteins for virus production. Virus titers of 4.5 x 10/sup 3/ colony-forming units (CFU)/ml (CIS DR), 1.5 x 10/sup 4/ CFU/ml (SDHT2), and 5 x 10/sup 5/ CFU/ml (SDHT1) weremore » measured by the transfer of MTX resistance to NIH3T3 cells. The amphotropic SDHT1 virus efficiently induced MTX resistance in cells of several species, including mouse NIH3T3 cells (5 x 10/sup 5/ CFU/ml), monkey CV1 cells (4 x 10/sup 3/ CFU/ml), and human MCF-7 cells (6 x 10/sup 4/ CFU/ml). When cocultured with SDHT1 virus-producing cells, both mouse and human bone marrow cells could be infected and rendered resistant to MTX. Mouse cytotoxic T lymphocytes and mouse helper T lymphocytes can also be made resistant to MTX.« less

Authors:
Publication Date:
Research Org.:
American Univ., Washington, DC
OSTI Identifier:
7138109
Resource Type:
Thesis/Dissertation
Resource Relation:
Other Information: Thesis (Ph. D.)
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 59 BASIC BIOLOGICAL SCIENCES; METHOTREXATE; SIDE EFFECTS; TOXICITY; MUTANTS; GENE REGULATION; OXIDOREDUCTASES; RECOMBINANT DNA; ANTIGENS; BIOCHEMICAL REACTION KINETICS; BIOLOGICAL RADIATION EFFECTS; BONE MARROW; CELL KILLING; DEOXYURIDINE; DNA; ENZYME INHIBITORS; FOLIC ACID; LYMPHOCYTES; MICE; MONKEYS; RESPONSE MODIFYING FACTORS; STEM CELLS; THERAPY; TRACER TECHNIQUES; TRITIUM COMPOUNDS; VIRUSES; AMINO ACIDS; ANIMAL CELLS; ANIMAL TISSUES; ANIMALS; ANTIMETABOLITES; AROMATICS; AZAARENES; AZINES; BIOLOGICAL EFFECTS; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY; BODY FLUIDS; CARBOXYLIC ACIDS; CONNECTIVE TISSUE CELLS; DRUGS; ENZYMES; HEMATINICS; HEMATOLOGIC AGENTS; HEMATOPOIETIC SYSTEM; HETEROCYCLIC COMPOUNDS; HYDROXY COMPOUNDS; ISOTOPE APPLICATIONS; KINETICS; LABELLED COMPOUNDS; LEUKOCYTES; MAMMALS; MATERIALS; MICROORGANISMS; NUCLEIC ACIDS; NUCLEOSIDES; NUCLEOTIDES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PARASITES; PRIMATES; PTERIDINES; PYRIMIDINES; RADIATION EFFECTS; REACTION KINETICS; RIBOSIDES; RODENTS; SOMATIC CELLS; TISSUES; URACILS; VERTEBRATES; VITAMIN B GROUP; VITAMINS; 560300* - Chemicals Metabolism & Toxicology; 550401 - Genetics- Tracer Techniques; 560120 - Radiation Effects on Biochemicals, Cells, & Tissue Culture

Citation Formats

Ricciardone, M D. Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene. United States: N. p., 1986. Web.
Ricciardone, M D. Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene. United States.
Ricciardone, M D. Wed . "Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene". United States.
@article{osti_7138109,
title = {Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene},
author = {Ricciardone, M D},
abstractNote = {Methotrexate (MTX), a folate analog which inhibits the enzyme dihydrofolate reductase (DHFR), is an effective antineoplastic drug. However, MTX-induced myelosuppression limits the effectiveness of this agent. Selective induction of MTX resistance in bone marrow stem cells, prior to treatment with MTX, might prevent this toxicity and improve the therapeutic index of the drug. In these studies drug resistance was transferred to mouse and human bone marrow stem cells by retroviral expression vectors containing coding sequences of a mutant DHFR with a decreased affinity for MTX. Three retroviral expression vectors were analyzed. The CIS DR vector contained the mutant DHFR gene inserted into the replication-defective amphotropic 4070 virus, Cistor. The other vectors contained the mutant DHFR inserted into either the env region (SDHT1) or gag-pol region (SDHT2) of a replication-defective spleen focus-forming virus. All three constructs induced approximately a 200-fold resistance to MTX when transfected into NIH3T3 cells. Amphotropic infectious retroviruses were obtained by transfecting the mutant DHFR vectors into a packaging cell line, which supplied the gag, pol, and env proteins for virus production. Virus titers of 4.5 x 10/sup 3/ colony-forming units (CFU)/ml (CIS DR), 1.5 x 10/sup 4/ CFU/ml (SDHT2), and 5 x 10/sup 5/ CFU/ml (SDHT1) were measured by the transfer of MTX resistance to NIH3T3 cells. The amphotropic SDHT1 virus efficiently induced MTX resistance in cells of several species, including mouse NIH3T3 cells (5 x 10/sup 5/ CFU/ml), monkey CV1 cells (4 x 10/sup 3/ CFU/ml), and human MCF-7 cells (6 x 10/sup 4/ CFU/ml). When cocultured with SDHT1 virus-producing cells, both mouse and human bone marrow cells could be infected and rendered resistant to MTX. Mouse cytotoxic T lymphocytes and mouse helper T lymphocytes can also be made resistant to MTX.},
doi = {},
url = {https://www.osti.gov/biblio/7138109}, journal = {},
number = ,
volume = ,
place = {United States},
year = {1986},
month = {1}
}

Thesis/Dissertation:
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