Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

Probability that the commitment of murine erythroleukemia cell differentiation is determined by the c-myc level

During the commitment of mouse erythroleukemia cell differentiation, c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the commitment of these cells, we have introduced the rat c-myc gene driven by inducible, heterologous (human metallothionein IIA) gene promoter into murine erythroleukemia cells and we have examined the ability of the transformed cells to undergo commitment to terminal differentiation. The induction of the exogenous c-myc gene expression inhibited the commitment of these cells. Time-dependent inhibition of the commitment was observed with the addition of zinc at an appropriate time after the induction with dimethyl sulfoxide. The result clearly indicated that late decline, not early decline, is required for the commitment. By examining the transformants expressing the exogenous c-myc mRNA at different levels, and the induction of the exogenous c-myc mRNA by varying the concentration of zinc, we demonstrated that the commitment may be determined by a stoichiometric amount of c-myc in the defined period. The data also suggest that the probability value for the commitment process occurring in a stochastic manner is well-correlated with the amount of c-myc mRNA.     

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.     

Microarray profiling of genes differentially expressed during erythroid differentiation of murine erythroleukemia cells

Murine erythroleukemia (MEL) cells are widely used to study erythroid differentiation thanks to their ability to terminally differentiate in vitro in response to chemical induction. At the molecular level, not much is known of their terminal differentiation apart from activation of adult-type globin gene expression. We examined changes in gene expression during the terminal differentiation of these cells using microarray-based technology. We identified 180 genes whose expression changed significantly during differentiation. The microarray data were analyzed by hierarchical and k-means clustering and confirmed by semi-quantitative RT-PCR. We identified several genes including H1f0, Bnip3, Mgl2, ST7L, and Cbll1 that could be useful markers for erythropoiesis. These genetic markers should be a valuable resource both as potential regulators in functional studies of erythroid differentiation, and as straightforward cell type markers.     

Dissection of the immediate early response of myeloid leukemia cells to terminal differentiation and growth inhibitory stimuli

To better understand the immediate early genetic response of myeloid cells to terminal differentiation and growth inhibitory stimuli, complementary DNA clones of myeloid differentiation primary response (MyD) genes have recently been isolated. In this study, a set of known (junB, c-jun, ICAM-1, H1(0), and H3.3 histone variants) and novel (MyD88, MyD116) MyD genes were used as immediate early molecular markers to further dissect the primary genetic response of myeloid cells to various differentiation and growth inhibitory stimuli. Expression of all of these MyD genes was highly induced in autonomously replicating differentiation inducible M1D+ myeloblasts following induction of terminal differentiation and growth inhibition by interleukin 6. Expression of all MyD genes except MyD88 was induced upon inhibition of M1D+ cell growth and induction of early, but not late, differentiation markers by interleukin 1 and lipopolysaccharide. In sharp contrast, only expression of H1(0) and H3.3 histone variants was increased following inhibition of M1D+ cell growth by interferon beta or gamma, which did not induce any differentiation associated properties. No increase in the expression of any of these MyD genes was seen in a clone of WEHI-3B D- myelomonocytic cells following stimulation with interleukin 6, which neither induced it for differentiation nor inhibited its growth. 12-O-Tetradecanoylphorbol-13-acetate, known to be a potent inducer of jun expression in many cell types, failed to induce high or stable expression of junB and c-jun in M1D+ cells, where it did not induce differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histone deacetylase inhibitors induce apoptosis in peripheral blood lymphocytes along with histone H4 acetylation and the expression of the linker histone variant, H1 degrees

The results of this study show that H1 degrees can be induced by sodium butyrate and trichostatin A in peripheral blood lymphocytes, a cell system which does not normally express this linker histone variant. Moreover, this induced expression was found to be correlated in a dose-dependent manner with the concomitant induction of apoptosis and increased levels of histone H4 acetylation. Sodium butyrate and trichostatin A, both inhibitors of histone deacetylases, are known to induce terminal differentiation and at the same time the induction of the linker histone variant, H1 degrees, in a number of tissue/cell systems. Moreover, aside from induced expression by histone deacetylase inhibitors, H1 degrees gene expression has also been tightly associated with the process of terminal differentiation in many physiological tissue/cell systems. The concomitant induction of H1 degrees expression along with apoptosis and histone acetylation in the same cell system has not been previously reported. Histone acetylation is known to be involved in chromatin remodelling events. Such events also occur during apoptosis. The association of H1 degrees gene expression with apoptosis, and not with differentiation in these cells, leads to more general implications as to a potential functional role of H1 degrees during chromatin remodelling.     

A transfected L-myc gene can substitute for c-myc in blocking murine erythroleukemia differentiation.

We investigated the ability of the proto-oncogene L-myc to substitute for c-myc in blocking murine erythroleukemia differentiation. Murine erythroleukemia cells (line C19) were transfected with recombinant plasmids containing genomic and cDNA fragments of the L-myc gene driven by a Moloney murine leukemia virus long terminal repeat. Clones expressing constitutive high levels of L-myc failed to differentiate in response to the chemical inducer N,N'-hexamethylene bisacetamide (HMBA). The block to differentiation correlated with the level of L-myc expression. Furthermore, transfected clones grown in the presence of inducer for an extended period of time showed an increased level of L-myc expression. These results suggest that functional domains of the c-myc gene involved in differentiation are located in the discrete regions of homology between the c- and L-myc genes.     

Molecular probes for the development and plasticity of neural crest derivatives

We have isolated cDNA clones for several mRNAs expressed in sympathetic neurons but not in adrenal chromaffin cells, two neural crest derivatives thought to share a common precursor. The tissue specificity, developmental expression, and hormonal regulation of these genes have been characterized using Northern blot and in situ hybridization analysis. We find that these mRNAs are independently regulated in development rather than synchronously induced. Our evidence also implicates Nerve Growth Factor (NGF) in the induction of one of these genes in postmigratory crest cells. Two of these genes become induced in mature chromaffin cells, which express a neuronal morphology in response to NGF. These results support the idea that the phenotypic plasticity of neural crest derivatives reflects a common precursor, the multipotentiality of which is sustained through terminal differentiation.     

Isolation and characterization of a mouse fully replication-dependent H1 gene within a genomic cluster of core histone genes

We have used an oligonucleotide complementary to a sequence coding for the conserved central globular domain of H1s to screen a mouse genomic library for H1 genes. We then used a series of universal histone oligonucleotides to identify five different H1 genes which were linked to core histone genes. We characterized one of the H1 genes which was linked to an H2a, an H2b, an H3, and an H4 histone gene. This characterization involved: 1) sequencing of the coding region of the gene and several hundred base pairs of flanking region. 2) Comparison of this sequence to other H1 sequences from other organisms. This sequence analysis clearly showed that the gene coded for an H1 and identified H1 consensus sequences in the 5'- and 3'-flanking region. 3) Mapping of the 5'- and 3'-ends of the mRNA complementary to this gene by S1 nuclease analysis. 4) Identifying this gene and an adjacent H3 gene as being of the fully replication-dependent expression class, by measuring changes in the steady state levels of their mRNAs in the presence of hydroxyurea and during differentiation of murine erythroleukemia cells.     

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.     

Increased Ser-10 phosphorylation of histone H3 in mitogen-stimulated and oncogene-transformed mouse fibroblasts.

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.