Erythropoietin-specific cell cycle progression in erythroid subclones of the interleukin-3-dependent cell line 32D

Recently, a variety of growth factor-dependent subclones of the murine interleukin-3 (IL-S)-dependent cell line 32D have been isolated. These subclones include those dependent for growth on erythropoietin (Epo) (32D Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) (32D GM), or granulocyte colony-stimulating factor (G-CSF) (32D G). 32D Epo1.1 is a revenant of 32D Epo and is capable of growing in IL-3. These cell lines express the differentiation program appropriate to the specific growth factor and depend on the growth factors not only for proliferation but also for survival. To determine how the signal for proliferation is triggered by various growth factors, we examined the DNA histograms and the expression of cell cycle-specific genes in the different cell lines. The cell cyclespecrfic genes analyzed were myc (early G₁), myb (late G₁), and the structural genes for the calcium-binding protein 2A9 (middle G₁) and histone H3 (G₁-S boundary). The DNA histogram analysis of cells in the logarithmic phase of growth showed that approximately 40% of 32D, 32D GM, 32D G, and 32D Epo1.1 (growing in IL-3) were cells with a 2N DNA content (and therefore in G₀/G₁), and another 40% have a DNA content intermediate between 2N and 4N (in S phase) In contrast, 32D Epo and 32D Epo1.1 (growing in Epo) had fewer cells in the G₀/G₁ phase of the cell cycle compared with the number of cells that were in the S phase (19% to 31 % v 69% to 78%, respectively). Because all the cell lines have comparable doubling times (15 to 18 hours), the cell distribution among the phases of the cell cycle is proportional to the length of the phase. Therefore, cells growing in IL-3 (32D and 32D Epo1.1 ), GM-CSF (32D GM), or G-CSF (32D G) progress along the cycle in a manner typical of previously reported nontransformed cell lines. In contrast, cells growing in Epo (32D Epo or 32D Epo1.1) spend relatively less time in G₀/G₁ and correspondingly more time in S. These data were confirmed by the analysis of the tritiated thymidine (³H-TdR) suicide rate and of the expression of cell cycle-specific genes. The 32D and 32D Epo1.1 cells growing in IL-3 had a suicide rate of ≃50%, whereas the suicide rate of 32D Epo and 32D Epo1.1 growing in Epo was higher than 75%. All the cell lines expressed comparable levels of myc and myb, whereas 32D Epo expressed undetectable levels of 2A9 and increased levels of histone H3. These results indicate that cells may progress along the cell cycle according to different pathways depending on which growth factor triggers the process. The prolonged S phase induced by Epo and described here could provide a cellular mechanism for the high ³H-TdR suicide rate previously reported for normal murine and human Epo-dependent erythroid progenitors.