Methotrexate (MTX) induces a concentration-dependent accumulation of cells in S-phase, an increase in average cell size and RNA and protein content ('unbalanced growth') and finally induces cell death. Even after high-dose MTX treatment, however, some cells escape the drug effect and this heterogeneity in response may play a major role in therapeutic outcome. We investigated heterogeneity in the response of K562 human leukemia cells, which exhibited population characteristics of unbalanced growth when treated with MTX, including a concentration-dependent increase in average-cell size and a block in S-phase of the cell cycle that correlated with growth inhibition. To investigate for the first time the response to MTX at the individual cell level, an individual colony formation assay (iCFA) was used. The iCFA revealed marked heterogeneity in both untreated K562 cells and their response to MTX treatment, as well as distinctive features of the response. Colonies of untreated K562 cells grew logarithmically with modal doubling times of 21.9 h (compared to 18.8+/-1.1 h in suspension culture), but there was over a 2-fold range of doubling times for individual cells. In addition, there was heterogeneity in the size of individual untreated K562 cells. Following MTX treatment, the iCFA detected shifts in the growth rate that were not detected in suspension culture and also drug-induced increased heterogeneity in growth rates at relatively nontoxic MTX concentrations. More importantly, the iCFA demonstrated that at a MTX concentration where no cells reproduced sufficiently to reach the threshold necessary to be counted as a colony, continued slow, logarithmic growth of a number of individual cells was observed. Thus, in addition to cytotoxicity, growth slow-down was a major effect exhibited in K562 cells treated with MTX. There was no correlation between MTX-induced unbalanced growth (as indicated by an increase in individual cell size) and the proliferative capacity of each cell. At cytotoxic MTX concentrations, there were individual cells that showed dramatically increased cell size but continued to proliferate, while other cells did not enlarge, but were still killed. These data show directly for the first time that the increase in cell size following MTX treatment is not likely to be the primary mechanism of cell kill. Similar changes were observed with gamma-fluoromethotrexate (FMTX), an MTX analog which is deficient in polyglutamylation. Since FMTX caused similar effects to MTX, we conclude that under these conditions poly(gamma-glutamate) synthesis is not the most significant factor in producing the observed effects in K562 cells. These results point out the utility of this approach as an alternative to the plating efficiency assay in order to identify approaches to arrest the growth of cells escaping primary drug effects.

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