Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate

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Abstract
A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 .mu.M MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 .mu.M MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting establihed that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division. Induced differentiation also resulted in a decrease of DHFR mRNA levels of up to 100-fold relative to uninduced matched control cells. These decreases of DHFR mRNA content were readily apparent to uninduced matched control cells. These decreases of DHFR mRNA content were readily apparent by 48 h of exposure to inducer. We have thus established a series of MELC variants in which the expression, i.e., downregulation, of a nonstructural housekeeping gene can be studied during induced terminal erythroid differentiation.

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