Reactivation and Dedifferentiation of Differentiated Murine Erythroleukemic Cell Nuclei

A murine erythroleukemic cell line, 745 A4-TG, deficient in hypoxanthine-guanine-phosphoribosyl transferase, can be induced with 3 mM hexamethylene bisacetamide to yield at least 50% of cells undergoing irreversible erythroid differentiation and finally losing capacity for cell divisions. The effects of such induced differentiation of 745 A4-TG on its ability to form viable and proliferating hybrids when fused with 3T3 1T22 fibroblasts were investigated. We found that when the induced 745 A4-TG cells were used, more continuously proliferating hybrids were obtained than could be accounted for by the residual uninduced cells which remained in these induced preparations. This suggests that some of the induced 745 A4-TG cells, when fused with 3T3 1T22 reverted from the induced phenotype of a limited capacity for cell proliferation to an uninduced state of continuous proliferation. This observation was further confirmed with the use of fully differentiated 745 A4-TG cells, which were obtained after selection with a bromodeoxyuridine suicide treatment to eliminate the uninduced and the partially differentiated cells in the preparations. When these selected, fully differentiated cells, as characterized by their lack of proliferation capacity and thymidine kinase activity, were fused with 3T3 1T22 (also deficient in thymidine kinase), it was found that not only were viable hybrid colonies obtained in a selection medium, which precluded the proliferation of either parental cells, but these hybrids continued to proliferate for more than two months in selection medium. These data thus confirmed that some fully differentiated erythroleukemic nucleus components in the hybrids were reactivated to regain capacity for cell proliferation and to dedifferentiate to synthesize thymidine kinase for survival in the selection medium. The lack of hemoglobin synthesis by these hybrids also indicates dedifferention of these murine erythroleukemic components in the hybrids.