Involvement of phosphatidylserine externalization in the down-regulation of c-myb expression in differentiating C2C12 cells

Abstract Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression.     

Constitutive versus cell cycle regulation of c-myb mRNA expression correlates with developmental stages in murine B lymphoid tumors.

The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.     

c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.     

Activation of c-myb expression by phytohemagglutinin stimulation in normal human T lymphocytes.

The expression of c-myb in normal human T lymphocytes directly derived from a normal subject and not adapted to continuous growth in culture was found to be markedly increased after phytohemagglutinin stimulation. In the same cells, the expression of c-myc mRNA is a much earlier event compared with the appearance of c-myb mRNA, which takes place soon after that of histone H3 mRNA. The increase in c-myb expression was not due to a particular T-lymphocyte subset, as shown by in situ hybridization assays.     

Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene.

During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-induced differentiation of these transfectants was inhibited in proportion to the level of exogenous c-myb mRNA expression. By adding or removing ZnCl2 at different times during the induction process, it was possible to show that up-regulation of exogenous c-myb limited to the first 2 days of induction had little or no effect on differentiation. In contrast, continuous expression of exogenous c-myb beginning at any time during the period of induction blocked further differentiation. These results suggest that during HMBA induction of MEL cells, the early down-regulation of c-myb mRNA is not necessary for terminal differentiation, whereas the down-regulation of c-myb at a later time is necessary.     

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.     

c-myb protein expression is a late event during T-lymphocyte activation.

The expression of p80c-myb was examined during the activation of resting human T lymphocytes. Before activation, no detectable p80c-myb was present. Synthesis of p80c-myb was observed only after initiation of the S phase of the cell cycle.     

Sublocalization of c-myb to 6q21----q23 by in situ hybridization and c-myb expression in a human teratocarcinoma with 6q rearrangements

We have sublocalized the human proto-oncogene c-myb by applying two different techniques: in situ hybridization of metaphase spreads and chromosome spot hybridization of flow-sorted chromosomes. For this we used a teratocarcinoma cell line carrying specific chromosome translocations involving the two chromosomes 6 and one chromosome 11. The distribution of the c-myb gene copies on the different translocation chromosomes revealed that c-myb is located in the region 6q21----q23. Because of the close proximity of the c-myb locus to the chromosomal breakpoints in the teratocarcinoma, we investigated whether c-myb was implicated in the development of this tumor. No rearrangement, deletion, or amplification of the gene was detected in the teratocarcinoma cells. Furthermore, the level of c-myb expression was comparable to that of other cell lines of nonhematopoietic origin. These results suggest that c-myb was not affected by the translocation and played no significant role in the development of this teratocarcinoma.