ROLE OF C-MYC IN ERYTHROID DIFFERENTIATION

We have been studying the role of the c-myc proto-oncogene on murine erythroleukemia cell differentiation. The expression of this gene changes dramatically following treatment with inducers of differentiation. Within 2 hours there is a 10-20 fold decline in the level of c-myc mRNA followed by a reexpression of the mRNA between 12-24 hours. The reexpression of c-myc mRNA occurs primarily in cells in the G1 phase of the cell cycle whereas in uninduced cells, c-myc mRNA is more equally distributed throughout the cell cycle. These changes may be important in the irreversible commitment of MEL cells to differentiate following inducer treatment. The primary objective of proposal 1 will be to examine the effect of an independently regulated c-myc gene on the irreversible commitment of MEL cells to differentiate following inducer treatment. MEL cells will be transfected with a recombinant plasmid I have constructed in which a murine c-myc gene has been placed under the control of a promoter from the metallothionein 1 gene. Following transfection, the recombinant c-myc plasmid will be regulated by the addition of heavy metals to the culture medium, independently from the endogenous c-myc gene. I will determine whether c-myc expression in the first 12 hours of inducer treatment affects the irreversible commitment of MEL cells to differentiate. Proposal 2 is designed to examine the transcriptional and post transcriptional regulation of the c-myc gene is uninduced and inducer treated MEL cells, by measuring in vitro transcription rates and mRNA stability. In proposal 3 the regulatory sequences involved in the regulation of c-myc gene expression will be determined by transfecting truncated versions of the c-myc gene into MEL cells and studying its expression following inducer treatment. Finally, the experiments in proposal 4 deal with the possibility that the cell cycle regulation of c-myc gene expression we have described, is a general phenomenon of terminally differentiating cells and not unique to the MEL cell system. In these experiments, I will study the cell cycle kinetics of c-myc gene expression in other differentiating systems. In the first 24 hours following inducer treatment, MEL cells are converted from cells with leukemogenic potential to cells committed to a program of terminal erythroid differentiation. Based on my previous work in this field, several experiments have been designed in which the molecular basis of this conversion will be investigated. Since leukemic cells exhibit a block in differentiation, an understanding of the genetic and molecular basis of this process may have clinical implications.