Loss of the MYC gene amplified in human HL-60 cells after treatment with inhibitors of poly(ADP-ribose) polymerase or with dimethyl sulfoxide.

In HL-60 cells, a human promyelocytic leukemia cell line, the human c-myc gene, designated MYC, is amplified about 16-fold. On differentiation of the HL-60 cells into granulocytes induced by several inhibitors of poly(ADP-ribose) polymerase [NAD+ poly(adenosine diphosphate D-ribose)ADP-D-ribosyltransferase, EC 2.4.2.30] including benzamide, nicotinamide, coumarin, and 4-hydroxyquinazoline or dimethyl sulfoxide, some MYC loss was observed. In contrast, benzoic acid, a noninhibitory analogue of benzamide, did not induce either granulocytic differentiation or loss of MYC. Loss of MYC seems to be associated with granulocytic differentiation because the time course of its loss was similar to that of appearance of nitroblue tetrazolium-positive cells, mature granulocytes, and its loss was not observed on differentiation of HL-60 cells into macrophages induced by phorbol 12-myristate 13-acetate or teleocidin. The loss of MYC is not the reason for the down regulation of MYC expression observed within 1 hr after addition of inducers, since the loss of MYC was not detected by 1-day treatment with inducers.     

Interferon gamma abrogates the differentiation block in v-myc-expressing U-937 monoblasts.

Extensive studies suggest a role for the myc protooncogene family in the control of cell proliferation and differentiation in vertebrates. Previously, deregulated expression of exogenous myc genes has been shown to inhibit induced differentiation in Friend erythroleukemia (MEL) cells and in human monoblastic U-937 cells. To examine the nature of the block of phorbol 12-myristate 13-acetate-induced differentiation in v-myc-expressing U-937 cells, we have studied the effect of other inducers utilizing signal pathways distinct from phorbol 12-myristate 13-acetate (i.e., 1 alpha, 25-dihydroxycholecalciferol and retinoic acid). We show that v-myc also inhibits differentiation associated with these inducers. However, the v-myc-associated block of phorbol 12-myristate 13-acetate-, 1 alpha,25-dihydroxycholecalciferol-, and retinoic acid-induced differentiation retinoic acid-induced differentiation can be overcome by adding interferon gamma as a costimulatory factor. Costimulation with interferon gamma restores terminal differentiation, as shown by acquisition of a macrophage phenotype and an irreversible growth arrest in the G0/G1 phase of the cell cycle, but induces only limited differentiation on its own. The differentiation is accomplished without altering the expression or nuclear localization of the v-myc protein. These results argue against the widely held view that down-regulation of myc expression is a general prerequisite for terminal differentiation of hematopoietic cells and suggests that interferon gamma induces a signal(s) that circumvents the v-myc activity.     

Coordinate viral induction of tumor necrosis factor alpha and interferon beta in human B cells and monocytes.

Human tumor necrosis factor alpha (TNF-alpha) gene expression can be induced primarily in cells of the monocyte/macrophage lineage by a variety of inducers, including lipopolysaccharide, phorbol esters such as phorbol 12-myristate 13-acetate, and virus or synthetic double-stranded RNA [poly(I).poly(C)]. In this paper we show that the TNF-alpha gene also responds to virus and phorbol 12-myristate 13-acetate in B lymphocytes and that virus is the most potent inducer of TNF-alpha mRNA in both monocyte and B-cell lines. In addition, we show that viral infection coinduces the expression of TNF-alpha and interferon beta mRNA and that viral induction of both genes is blocked by the kinase inhibitor 2-aminopurine. Inhibition of protein synthesis with cycloheximide had no effect on mRNA expression of the genes in one of three cell lines tested (U937) but blocked the viral induction of both genes in another (Namalwa). Thus, the regulatory factors required for mRNA induction of both genes are present prior to the addition of virus in U937 but not in Namalwa cells. However, in a third cell line (JY), cycloheximide blocked viral induction of the interferon beta gene but not the TNF-alpha gene. Taken together, these observations suggest that viral induction of TNF-alpha and interferon beta gene expression may involve overlapping pathways with both common and distinct regulatory factors.     

Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells.     

Decreased expression of transforming growth factor alpha during differentiation of human pancreatic cancer cells.

The relationship between cell differentiation and transforming growth factor alpha (TGF-alpha) expression in human pancreatic cancer cells was analyzed in Capan 1 cells. These cells differentiate either spontaneously or after butyrate treatment. During differentiation (spontaneous or butyrate induced), TGF-alpha messenger RNA (mRNA) levels decreased, whereas the TGF-beta 1 mRNA levels remained unchanged. TGF-alpha was present in cells as proTGF-alpha, which decreased after butyrate treatment. Secretion of TGF-alpha was not found. Under the two conditions of differentiation, the membrane-bound protein kinase C activity was also reduced. Conversely, long-term phorbol ester treatment increased both membrane-bound protein kinase C activity (260%) and TGF-alpha mRNA level (500%), a not significant increase of TGF-beta 1 mRNA was observed. However, phorbol 12-myristate-13-acetate did not induce TGF-alpha synthesis or secretion. These data suggest that expression of TGF-alpha can be reduced in cancer cells; they also suggest the existence of a relationship between TGF-alpha expression and cell differentiation. In addition, the protein kinase C-induced TGF-alpha mRNA level was not followed by the increase of TGF-alpha biosynthesis, suggesting a translational control. Finally, the expression of TGF-alpha and -beta 1 messengers appears to be differently regulated.     

Induction of human monocyte cell line U937 differentiation and CSF-1 production by phorbol ester.

The monoblast-like human histiocytic lymphoma cell line U937 can be induced by phorbol 12-myristate 13-acetate (PMA) at concentrations of 8-16 ng/ml to undergo differentiation. The induced cells show growth inhibition, morphological changes to monocyte-macrophage-like cells, and increases in nitroblue tetrazolium (NBT) and nonspecific esterase activities. The expression of surface markers MO1 and MO2 for macrophages is also elevated. The induced differentiation of U937 cells is accompanied by the appearance of colony-stimulating activity in the culture medium as assayed on mouse bone marrow cells. Gel filtration chromatography of the conditioned media shows a peak of colony-stimulating factor (CSF) activity with an apparent molecular weight of approximately 100 kd. Morphological analysis of the colonies reveals a predominant monocyte-macrophage population. This CSF activity can be neutralized by anti-human colony-stimulating factor 1 (CSF-1) antibody, and Western blot analysis shows a CSF-1 band. All these results strongly indicate that CSF produced by induced U937 cells is CSF-1. Northern blot analysis of RNA isolated from U937 cells demonstrated that CSF-1 gene expression started after PMA induction for 3 h and declined after 18 h. Protooncogenes c-fos and c-jun were expressed after half an hour of incubation with PMA.     

Expression of IMP dehydrogenase in differentiating HL-60 cells

Addition of mycophenolic acid to cultures of HL-60 cells results in a decreased cellular level of guanine nucleotides and the induction of cell differentiation. During the early stages of this induction, steady-state levels of cellular IMP dehydrogenase (IMPDH), messenger RNA (mRNA), and protein are increased, perhaps because of cellular compensation for the inhibition of IMPDH activity. The subsequent decrease in IMPH mRNA and protein levels after several days of treatment suggests a change in the control of IMPDH expression. In contrast to the pattern of increased IMPDH expression observed in the mycophenolic acid-treated cells, treatment of HL-60 cells with two other inducers of differentiation, namely retinoic acid and phorbol 12-myristate 13-acetate, resulted in stable or decreased levels of cellular IMPDH mRNA and protein. However, the kinetics of this expression were different. These results suggest that a number of factors influence the regulation of IMPDH expression during the induction of HL-60 cell differentiation, including the nature of the inducer. A decrease in the cellular IMPDH activity was observed for all of the inducers, suggesting that this decreased activity may be a determining factor in the acquisition of a mature phenotype in the HL-60 cells.     

Induction of c-fos and c-myc mRNA by epidermal growth factor or calcium ionophore is cAMP dependent.

Phorbol esters activate protein kinase C and induce expression of the c-fos and c-myc protooncogenes in density-arrested BALB/c 3T3 (A31) cells; in contrast, epidermal growth factor (EGF) does not activate protein kinase C and is a poor inducer of c-fos and c-myc in these confluent cells. We show that, when A31 cells were subconfluent and made quiescent by serum deprivation, the phorbol ester phorbol 12-myristate 13-acetate induced c-fos and c-myc mRNA poorly, whereas EGF was a better inducer. Another platelet-derived growth factor-inducible gene, JE, did not show this differential regulation by phorbol 12-myristate 13-acetate and EGF. The ability of EGF to induce protooncogene mRNA was associated with elevated levels of intracellular cAMP. First, serum-deprived cells maintained cAMP at about 2-fold higher level than density-arrested cells. Second, induction was greatly enhanced by cholera toxin and 3-isobutyl-1-methylxanthine, which increased intracellular cAMP 3- to 10-fold. The calcium ionophore A23187 mimicked EGF in that it elevated c-fos and c-myc mRNA when administered with cholera toxin and isobutylmethylxanthine. Neither cholera toxin and isobutyl-methylxanthine nor A23187 appreciably induced these mRNAs when used alone. Our results suggest that c-fos and c-myc expression can be regulated by an EGF-directed pathway that utilizes calcium and cAMP as cooperating cytoplasmic messengers.     

Contributions of p53 and PMA to g-irradiation induced apoptosis in Jurkat cells

Several mutations prevent the expression of p53 in the human lymphoblastoid T cell line Jurkat. Restoration of p53 in Jurkat cells had no effect on the cell growth but markedly increased the amount of apoptosis induced by g-irradiation. Inhibition of RNA synthesis using 5,6-dichlorobenimidizole riboside had little effect on apoptosis induced by irradiation in the presence of p53 and did not affect the p53-independent apoptotic pathway. Expression of p53 also had no effect on the expression levels of proteins such as Fas, GADD45, Bax, Bcl-2, Bcl-x_L or p53 induced proteins (PIGS) in resting cells or after irradiation. Activation of protein kinase C by phorbol 12-myristate 13-acetate produced an almost complete inhibition of p53-independent apoptosis following irradiation, whereas no significant effect was observed on the rate of p53-induced apoptosis. Although phorbol 12-myristate 13-acetate strongly induced p21 and stabilised p53 in the resting transfected Jurkat cells, neither apoptosis nor cell arrest was observed. In summary, this work shows that p53 enhances the radiosensitivity of Jurkat cells through an apoptotic process that is triggered by irradiation and is largely independent of RNA synthesis and protein kinase C activation. Apoptosis in p53- negative Jurkat cells is strongly inhibited by PMA indicating that the pathway triggered by p53 may be distinct from apoptotic pathways used in its absence.     

Phorbol ester-induced terminal differentiation is inhibited in human U-937 monoblastic cells expressing a v-myc oncogene.

Induction of differentiation of the human monoblastic cell line U-937 in vitro by several physiologic and nonphysiologic inducers is accompanied by a rapid decrease in expression of MYC, the endogenous human myc protooncogene. To investigate whether this reduction is a prerequisite for terminal differentiation, we introduced a constitutively expressed v-myc gene into U-937 cells. The results show that constitutive expression of an avian v-myc oncogene does not interfere with phorbol 12-myristate 13-acetate-induced differentiation of U-937 cells early after stimulation. However, after 24 hr the differentiation process is reversed, as judged by a full recovery of the proliferative capacity and reexpression of the immature phenotype, within the next 2-4 days. We conclude that the terminal stage of macrophage differentiation is inhibited in U-937 cells constitutively expressing a v-myc oncogene.