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C-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor
M C Bondurant1, T Yamashita, K Muta
1Department of Medicine, Department of Veterans Affairs Medical Center, Nashville, Tennessee, USA.
Abstract:
The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-Myc and resultant depression of proliferation.
Insights
Erythropoietin (EPO) regulates c-myc expression in differentiating erythroblasts, impacting cell proliferation but not differentiation. Maintaining c-myc expression is crucial for erythroid cell development.
Area of Science:
- Hematology
- Molecular Biology
- Cellular Biology
Background:
- Erythropoiesis is a complex process involving precise regulation of gene expression.
- The c-myc proto-oncogene plays a critical role in cell proliferation and differentiation.
- Understanding c-myc regulation during erythroid differentiation is key to deciphering hematopoietic stem cell biology.
Purpose of the Study:
- To investigate the role of c-myc expression during murine and human erythroid differentiation.
- To determine the regulatory effects of erythropoietin (EPO) and other growth factors on c-myc in erythroblasts.
- To elucidate the functional significance of c-myc in erythroid cell proliferation and differentiation.
Main Methods:
- Analysis of c-myc mRNA and protein levels in murine (FVA cells) and human (CFU-E) erythroblasts during in vitro differentiation.
- Treatment with EPO, insulin-like growth factor 1 (IGF-1), and c-kit ligand (SCF).
- Application of antisense oligonucleotides targeting c-myc to assess its functional role.
Main Results:
- EPO is required to maintain c-myc expression in FVA cells; EPO stimulation rapidly increases c-myc mRNA and protein.
- c-myc mRNA kinetics and EPO response in human CFU-E parallel murine cells.
- IGF-1 and SCF additively increase c-myc mRNA with EPO, suggesting distinct signaling pathways.
- Antisense inhibition of c-myc suppressed proliferation but did not induce differentiation or apoptosis in human CFU-E.
Conclusions:
- EPO signaling is a primary regulator of c-myc expression during erythroid differentiation.
- c-myc plays a crucial role in regulating erythroblast proliferation.
- Erythroid differentiation is dependent on signals beyond c-myc-mediated proliferation control.