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Temporal Mapping of the Differentiation Pathway of the Murine Erythroleukemia Cell¹

Departments of Microbiology and Immunology [K. E., A. G., L. W.] and Biochemistry [K. E., W. L. C.], The University of Western Ontario, London, Ontario, N6A 5C1, Canada

These studies are concerned with "mapping" the temporal order of the precommitment events in the differentiation pathway of the Friend erythroleukemia cell. We have used a single-block procedure in which a differentiation-specific inhibitor of a temperature-sensitive (ts) differentiation-defective mutation was used to block the differentiation program. Later, the block was removed, differentiation was allowed to proceed, and the time required to reach a reference marker was monitored. These studies have indicated that the mutations tsC2GP1 and tsB5A, the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide, and the glucocorticoid hormone dexamethasone blocked functions which are required just prior to commitment. We have also used a double-block procedure involving two consecutive restrictive conditions, which suggests that the 3-aminobenzamide- and tsC2GP1-blocked functions constitute a part of a sequentially ordered pathway leading to terminal differentiation. The convergence of the 3-aminobenzamide, dexamethasone, and ts mutational blocks just prior to commitment suggests that the blocked functions may be part of a major control mechanism for commitment.

In these studies, we have introduced a cytochalasin B-based assay to monitor commitment. The use of cytochalasin B permits a direct assay for commitment and obviates the need for colony-forming assays using semisolid medium, which have inherent problems such as efficiency of plating.

¹ Supported by the Medical Research Council of Canada (Grant MA-10239) and the National Cancer Institute of Canada (Grant 2127).

² To whom requests for reprints should be addressed.

Received 9/13/89. Accepted 1/ 8/91.