COMPUTERIZED IMAGE-ANALYSIS QUANTITATION OF HETEROGENEITY OF RESPONSEIN K562 HUMAN LEUKEMIA-CELLS AFTER METHOTREXATE EXPOSURE

Methotrexate (MTX) induces a concentration-dependent accumulation of c ells in S-phase, an increase in average cell size and RNA and protein content ('unbalanced growth') and finally induces cell death. Even aft er high-dose MTX treatment, however, some cells escape the drug effect and this heterogeneity in response may play a major role in therapeut ic outcome. We investigated heterogeneity in the response of K562 huma n leukemia cells, which exhibited population characteristics of unbala nced growth when treated with MTX, including a concentration-dependent increase in average-cell size and a block in S-phase of the cell cycl e 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 heter ogeneity in both untreated K562 cells and their response to MTX treatm ent, as well as distinctive features of the response. Colonies of untr eated 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 ov er a 2-fold range of doubling times for individual cells. In addition, there was heterogeneity in the size of individual untreated K562 cell s. Following MTX treatment, the iCFA detected shifts in the growth rat e that were not detected in suspension culture and also drug-induced i ncreased heterogeneity in growth rates at relatively nontoxic MTX conc entrations. More importantly, the iCFA demonstrated that at a MTX conc entration where no cells reproduced sufficiently to reach the threshol d necessary to be counted as a colony, continued slow, logarithmic gro wth of a number of individual cells was observed. Thus, in addition to cytotoxicity, growth slow-down was a major effect exhibited in K562 c ells treated with MTX. There was no correlation between MTX-induced un balanced growth (as indicated by an increase in individual cell size) and the proliferative capacity of each cell. At cytotoxic MTX concentr ations, there were individual cells that showed dramatically increased cell size but continued to proliferate, while other cells did not enl arge, but were still killed. These data show directly for the first ti me that the increase in cell size following MTX treatment is not likel y to be the primary mechanism of cell kill. Similar changes were obser ved with gamma-fluoromethotrexate (FMTX), an MTX analog which is defic ient in polyglutamylation. Since FMTX caused similar effects to MTX, w e 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 a pproaches to arrest the growth of cells escaping primary drug effects.