Cytokine-dependent granulocytic differentiation. Regulation of proliferative and differentiative responses in a murine progenitor cell line

Human granulocyte colony stimulating factor (G-CSF) can support the survival and short term proliferation of the interleukin 3 (IL 3)-dependent diploid murine hemopoietic progenitor cell line 32D C13. After 8 days in the presence of 30 U/ml of G-CSF and in the absence of IL 3, the great majority of 32D C13 cells becomes positive for myeloperoxidase (a marker that appears at the promyelocytic stage of the granulocytic lineage) and progressively differentiates into lactoferrin-containing neutrophilic granulocytes. Myeloperoxidase mRNA rapidly increases after 24 to 48 hr of treatment with G-CSF, peaks at day 6 and is no longer detectable at day 9 and 12, paralleling the appearance of myeloperoxidase-positive promyelocytes and myelocytes in the culture. After 12 days, 100% of the cells terminally differentiate, and clonogenic assays in IL 3-containing semisolid media indicate that the whole population has irreversibly lost proliferative capability. By using varying concentrations of both murine IL 3 and recombinant human G-CSF, the cultures develop an heterogeneous population of cells representing all the differentiation stages of the myeloid lineage, and the relative ratios of immature proliferating precursors and terminally differentiated cells present in the cultures can be modulated by modifying the concentrations of IL 3 or recombinant human G-CSF. Isobolic curves indicate that IL 3 and G-CSF have an antagonistic effect on the proliferation of 32D C13 cells. Thus, these cells represent a simplified in vitro model of normal granulocytic differentiation whose extent may be modulated completely in the presence of serum by two well-defined growth and differentiation factors: IL 3 and G-CSF.     

The Promyelocytic Leukemia Zinc Finger Protein Affects Myeloid Cell Growth, Differentiation, and Apoptosis

The promyelocytic leukemia zinc finger (PLZF) gene, which is disrupted in therapy-resistant, t(11;17)(q23;q21)-associated acute promyelocytic leukemia (APL), is expressed in immature hematopoietic cells and is down-regulated during differentiation. To determine the role of PLZF in myeloid development, we engineered expression of PLZF in murine 32Dcl3 cells. Expression of PLZF had a dramatic growth-suppressive effect accompanied by accumulation of cells in the G₀/G₁ compartment of the cell cycle and an increased incidence of apoptosis. PLZF-expressing pools also secreted a growth-inhibitory factor, which could explain the severe growth suppression of PLZF-expressing pools that occurred despite the fact that only half of the cells expressed high levels of PLZF. PLZF overexpression inhibited myeloid differentiation of 32Dcl3 cells in response to granulocyte and granulocyte-macrophage colony-stimulating factors. Furthermore, cells that expressed PLZF appeared immature as demonstrated by morphology, increased expression of Sca-1, and decreased expression of Gr-1. These findings suggest that PLZF is an important regulator of cell growth, death, and differentiation. Disruption of PLZF function associated with t(11;17) may be a critical event leading to APL.     

Expression profiles of micro RNA in proliferating and differentiating 32D murine myeloid cells

32D cells are murine myeloid cells that grow indefinitely in Interleukin-3 (IL-3). In these cells, the type 1 insulin-like growth factor (IGF-I) and granulocytic-colony stimulating factor (G-CSF) induce differentiation to granulocytes. 32D cells do not express insulin receptor substrate-1 (IRS-1) or IRS-2, docking proteins of the IGF-I receptor. Ectopic expression of IRS-1 in these cells inhibits differentiation, the cells become IL-3 independent and IGF-1 dependent and can form tumors in mice. 32D and 32D-derived cells offer a good model in which to study the expression profiles of Micro Rna (miR) related to sustained proliferation or differentiation. We present here the data obtained with miR micro-arrays and identify the miR that are regulated by IGF-1 or G-CSF and are associated with either differentiation or indefinite cell proliferation of 32D murine myeloid cells.     

Molecular mechanisms associated with the regulation of apoptosis by the two alternatively spliced products of c-Myb

The c-myb proto-oncogene encodes two alternatively spliced mRNAs, which in turn code for proteins of 75 kDa and 89 kDa. It is at present unclear whether the two isoforms of c-Myb perform identical functions or whether they mediate different biological effects. To assess their role in apoptotic death of hematopoietic cells, we expressed the two isoforms of c-Myb in the murine myeloid cell lines 32Dcl3 and FDCP1. Our results show that while ectopic overexpression of p75 c-Myb results in the acceleration of cell death, similar overexpression of p89 c-Myb results in the protection of cells from apoptotic death. An analysis of gene expression changes with mouse cDNA expression arrays revealed that while p75 c-Myb blocked the expression of glutathione S-transferase micro mRNA, p89 c-Myb greatly enhanced the expression of this gene. These results were further confirmed by Northern blot analysis. Ectopic overexpression of the glutathione S-transferase micro gene in 32Dcl3 cells resulted in protection of cells from interleukin-3 withdrawal-induced cell death similar to that seen with the ectopic overexpression of p89 c-Myb. These results suggest that the two isoforms of c-Myb differentially regulate apoptotic death of myeloid cells through differential regulation of glutathione S-transferase micro gene expression.     

24p3 in differentiation of myeloid cells

24p3 is a secreted lipocalin that has been variously related to apoptosis, proliferation, and the neutrophil lineage of blood cells. We have investigated the expression of 24p3 mRNA and protein in myeloid cell lines induced to differentiate by insulin-like growth factor 1 (IGF-1) and the granulocytic-colony simulating factor (G-CSF). Both these growth factors, which cause myeloid cells to differentiate into granulocytes, induced a marked increase in the expression of both 24p3 protein and mRNA. The mRNA especially appeared early after the cells were induced with either IGF-1 or G-CSF, at a time when the cells were still proliferating and are morphologically undifferentiated. 24p3 can be considered an early marker of granulocytic differentiation.     

Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1

To study the nature of genes that are induced during the apoptotic death of myeloid precursor cells, we performed representational difference analysis (RDA) using 32Dcl3 myeloblastic cells that were deprived of IL-3 for 24h. We have isolated a novel cDNA (g1-related protein, G1RP) that is homologous to g1, a Drosophila melanogaster zinc-finger protein that is expressed in the mesoderm. Northern blot analysis using RNAs derived from 32Dcl3 cells that have been grown in the absence of IL-3 demonstrates that the G1RP message is upregulated in these cells following the removal of IL-3, suggesting that this gene may regulate growth factor withdrawal-induced apoptosis of myeloid precursor cells.     

Expression of the Evi-1 zinc finger gene in 32Dc13 myeloid cells blocks granulocytic differentiation in response to granulocyte colony-stimulating factor.

Expression of the Evi-1 gene is frequently activated in murine myeloid leukemias by retroviral insertions immediately 5' or 90 kb 5' of the gene. The Evi-1 gene product is a nuclear, DNA-binding zinc finger protein of 145 kDa. On the basis of the properties of the myeloid cell lines in which the Evi-1 gene is activated, it has been hypothesized that its expression blocks normal differentiation. To explore this proposed role, we have constructed a retrovirus vector containing the gene and examined its effects on an interleukin-3-dependent myeloid cell line that differentiates in response to granulocyte colony-stimulating factor (G-CSF). Expression of the Evi-1 gene in these cells did not alter the normal growth factor requirements of the cells. However, expression of the Evi-1 gene blocked the ability of the cells to express myeloperoxidase and to terminally differentiate to granulocytes in response to G-CSF. This effect was not due to altered expression of the G-CSF receptor or to changes in the initial responses of the cells to G-CSF. These results support the hypothesis that the inappropriate expression of the Evi-1 gene in myeloid cells interferes with the ability of the cells to terminally differentiate.     

Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis.

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.     

Downregulation of the upstream binding factor1 by glycogen synthase kinase3beta in myeloid cells induced to differentiate

The upstream binding factor 1 (UBF1), one of the proteins that regulate the activity of RNA polymerase I, is downregulated in 32D myeloid cells induced to differentiate into granulocytes, either by the type 1 insulin-like growth factor (IGF-1) or the granulocytic colony stimulating factor (G-CSF). Downregulation of UBF1 is largely due to protein degradation, while mRNA levels are not affected. Inhibition of UBF1 degradation by lithium chloride (LiCl)and lactacystin suggest a role of glycogen synthase kinase beta (GSK3beta) in a proteasome-dependent degradation of UBF. GSK3beta phosphorylates in vitro and in vivo the UBF protein, which has five putative motifs for phosphorylation by GSK3beta. Elimination and/or mutations of these motifs stabilize the UBF1 protein even in cells induced to differentiate. Conversely, a stably transfected, constitutively active GSK3beta accelerates the downregulation of UBF1. We show further that activation of the differentiating protein C/EPBalpha in 32D cells transformed by the oncogenic BCR/ABL protein causes downregulation of UBF1. Finally, inhibition of differentiation of myeloid cells by a dominant negative mutant of Stat3 stabilizes the UBF1 protein, while rapamycin-induced differentiation of myeloid cells downregulates UBF1 levels. Taken together, our results indicate that the induction of granulocytic differentiation in 32D murine myeloid cells causes the degradation of UBF1, via GSK3beta and the proteasome pathway.