Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene
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
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
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