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Flow cytometric analysis of mammalian glial cultures treated with methotrexate
Author(s) -
Serrano Elba E.,
Schimke Robert T.
Publication year - 1990
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/glia.440030613
Subject(s) - dihydrofolate reductase , biology , methotrexate , cell culture , microbiology and biotechnology , somatic cell , antifolate , flow cytometry , transfection , cell , dna , gene , antimetabolite , biochemistry , genetics , immunology
Methotrexate (MTX) is an antineoplastic drug that acts by competitive inhibition of the enzyme dihydrofolate reductase (DHFR). MTX treatment of cultured cell lines leads to the emergence of resistant cell populations. Studies using stepwise selection procedures have demonstrated that MTX resistance conferred by overproduction of DHFR can be caused by DHFR gene amplification. We examined the effect of MTX on cells whose origin more closely approximates the in vivo condition by developing a culture system using dissociated brain tissue from 17–19 day old mouse embryos. At the first passage, cultures were divided into control and MTX groups. Cells were treated with the same or successively higher concentrations of MTX at each passage over a 3–4 month period. The first passage eliminated neurons and left a glial culture comprised of approximately 90% astrocytes. We used the Fluorescence Activated Cell Sorter in conjunction with fluorescent dyes to measure DHFR content, DNA content, size, and viability of glial cells following MTX treatment. MTX‐treated cells divided but grew more slowly and were larger than untreated cells. Stepwise selection in 30/60/90 nM or 60/120 nM MTX resulted in significant two‐ to threefold increases in fluorescence, and hence DHFR levels. Slot hybridizations assays demonstrated a threefold increase in DHFR gene copy number in the DNA from the 30/60/90 cultures. Thus, our findings were consistent with the results obtained from somatic cell lines, and lend support to the hypothesis that gene amplification may be a common mechanism for the acquisition of resistance in many types of cells. They also indicate that glial cells may be a specific target for cytotoxic effects of MTX on the central nervous system.