Erythroid differentiation of K562 cells: mixed colonies as an index of delayed expression of commitment

K562 cells demonstrate commitment, defined as the clonal expression of a differentiated phenotype coupled with a limitation in proliferation. Upon exposure to certain agents, K562 cells are induced to synthesize hemoglobin, detectable by benzidine staining. If plated in semisolid medium, they produce benzidine-positive colonies, benzidine-negative colonies, and mixed colonies, the latter containing both positive and negative cells. To test whether or not mixed colonies represent a delay in the expression of commitment, we conducted two types of experiments. The first type showed that, following inducer removal, a delay in plating causes not only a decline in the number of mixed colonies, but also a rise in the proportion of negative colonies, with no change in the proportion of positive colonies. To explain this result, we propose that a plating delay can conceal a negative cell producing a positive cell if that cell division has occurred before plating. Instead of one mixed colony, one observes one positive colony and one other colony, either negative or mixed (depending on subsequent negative-to-positive events). Thus delay does not change the proportion of positive colonies, presumably because they breed true. But delay causes an increase in negative colonies to balance the decrease in mixed colonies due to concealment of negative-to-positive events and provides evidence that the converse, positive-to-negative events, do not occur. The second type of experiment utilized cordycepin, which inhibits commitment. We predicted that, if mixed colonies represent a delay in the expression of commitment, the addition of cordycepin to cells already exposed to thymidine should increase the percentage of mixed colonies. We found that cordycepin does indeed preferentially increase the proportion of mixed colonies. These two types of experiments provide evidence that mixed colonies represent a delay in expression of commitment. Such an inducible system, in which the commitment event and its expression can be separated in time by a generation or more, may provide an opportunity to more fully characterize the commitment process.