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

Thesis/Dissertation · Wed Jan 01 04:00:00 EST 1986

OSTI ID:7138109

Ricciardone, M D

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.

• Research Organization: American Univ., Washington, DC
• OSTI ID: 7138109
• Country of Publication: United States
• Language: English

Related Subjects

550401 -- Genetics-- Tracer Techniques
560120 -- Radiation Effects on Biochemicals
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560300* -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACIDS
ANIMAL CELLS
ANIMAL TISSUES
ANIMALS
ANTIGENS
ANTIMETABOLITES
AROMATICS
AZAARENES
AZINES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
BIOLOGICAL MATERIALS
BIOLOGICAL RADIATION EFFECTS
BLOOD
BLOOD CELLS
BODY
BODY FLUIDS
BONE MARROW
CARBOXYLIC ACIDS
CELL KILLING
CONNECTIVE TISSUE CELLS
DEOXYURIDINE
DNA
DRUGS
ENZYME INHIBITORS
ENZYMES
FOLIC ACID
GENE REGULATION
HEMATINICS
HEMATOLOGIC AGENTS
HEMATOPOIETIC SYSTEM
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LEUKOCYTES
LYMPHOCYTES
MAMMALS
MATERIALS
METHOTREXATE
MICE
MICROORGANISMS
MONKEYS
MUTANTS
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
OXIDOREDUCTASES
PARASITES
PRIMATES
PTERIDINES
PYRIMIDINES
RADIATION EFFECTS
REACTION KINETICS
RECOMBINANT DNA
RESPONSE MODIFYING FACTORS
RIBOSIDES
RODENTS
SIDE EFFECTS
SOMATIC CELLS
STEM CELLS
THERAPY
TISSUES
TOXICITY
TRACER TECHNIQUES
TRITIUM COMPOUNDS
URACILS
VERTEBRATES
VIRUSES
VITAMIN B GROUP
VITAMINS