Quantitation of dihydrofolate reductase in individual parental and methotrexate-resistant murine cells. Use of a fluorescence activated cell sorter.

A fluorescent labeling technique has been devised to quantitate dihydrofolate reductase levels in individual cells and has been used to examine the heterogeneity in dihydrofolate reductase levels within various populations of methotrexate-resistant and parental cells. Precise quantitation of cellular dihydrofolate reductase levels in parental and methotrexate-resistant cells (having up to several hundred times the level of dihydrofolate reductase present in parental cells) is made possible by saturating the enzyme with a fluorescein derivative of methotrexate and then using a fluorescence-activated cell sorter to analyze the degree of fluorescence emission from individual cells. Saturation of the cellular dihydrofolate reductase, specificity of the binding, and the linear relationship between dihydrofolate reductase specific activity and the mean fluorescence emission per cell in various populations of methotrexate-resistant and parental cell lines validate the use of this method as an index of cellular dihydrofolate reductase levels. The fluorescent labeling technique has been used to study the variations in dihydrofolate reductase levels within the population of methotrexate-resistant cells as well as in resistant cells undergoing reversion to methotrexate sensitivity during growth in the absence of methotrexate. The methotrexate-resistant line was composed of a complex mixture of cells with varying levels of fluorescence. That population has been fractionated on the basis of cellular fluorescence; the subpopulations obtained contain dihydrofolate reductase specific activities and rates of dihydrofolate reductase synthesis proportional to the mean fluorescence emission/cell of that subpopulation. A revertant population, generated by growth of the methotrexate-resistant population in the absence of methotrexate for 400 cell doublings, contains ‘I-fold greater amounts of dihydrofolate reductase than the sensitive, parental line and does not represent a mixture of sensitive and resistant cells but rather a homogeneous population of cells with dihydrofolate reductase levels 7-fold greater than those of the sensitive cell line. These investigations are the first to illustrate the use of a fluorescent probe directed at an intracellular protein to analyze and sort living populations of cells. This technique should be of general