Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Glia. 1990;3(6):539-49.

Flow cytometric analysis of mammalian glial cultures treated with methotrexate.

Author information

  • 1Department of Biological Sciences, Stanford University, California 94305.

Abstract

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.

PMID:
2148556
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Loading ...
    Write to the Help Desk