Lineage-specific expression of c-fos and c-fms in human hematopoietic cells: Discrepancies with the in vitro differentiation of leukemia cells

In vitro differentiation studies using the bipotential human leukemia cell line, HL60, have indicated that high levels of expression of two proto-oncogenes, c-fos and c-fms, are restricted to the myelomonocytic lineage. No such expression has been detected in induced granulocytic cells. In striking contrast to these observations, we found that c-fos mRNA levels are very high in purified human granulocytes, but barely detectable in blood monocytes and tissue macrophages. Human granulocytes contain, however, relatively low levels of c-fos protein, indicating that c-fos mRNA is inefficiently translated or that the protein is rapidly degraded in these cells. In closer agreement with the in vitro results, the level of the expression of c-fms is high in purified blood monocytes and undetectable in granulocytes. We found, however, that the evolution of monocytes into tissue macrophages is accompanied by a significant decrease in c-fms expression, suggesting that the function of c-fms is restricted to specific stages of monocytic differentiation. Our observations also show that results obtained using in vitro differentiation systems have to be regarded with caution, since they may not reflect the in vivo situation.     

Temporal relationships between induced changes in c-myc mRNA abundance, proliferation, and differentiation in HL60 cells

Changes in the relative abundances of c-myc mRNA have been related to changes in other parameters of differentiation (histochemical, clonogenic) during the course of the differentiation of HL60 cells to monocytes/macrophages or to granulocytes. Induction of differentiation to monocytes/macrophages was marked by a rapid rate of appearance of committed cells (80 to 90% in 24 hours) and a concomitant rapid loss of c-myc mRNA. Induction of granulocytic differentiation resulted in a much slower rate of appearance of committed cells (50% in 48 hours), and a much faster rate of loss of c-myc mRNA (tenfold in 1 hour). These data are consistent with there being a direct link between down-regulation of the expression of c-myc and the onset of proliferation arrest and monocytic differentiation, but show there is no such association of c-myc mRNA abundance and proliferation or differentiation during the maturation of HL60 granulocytes.     

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Potentiate c-fos Expression Induced by Glutamate in Cultured Cortical Neurons

Abstract: Previous reports have demonstrated that glutamate stimulates c- fos mRNA expression in primary cultures of mouse cerebral cortical neurons. We show here that vasoactive intestinal peptide (VIP) induces c- fos mRNA expression; however, this effect of VIP is completely inhibited by the noncompetitive NMDA receptor antagonist MK-801, therefore indicating that VIP stimulates c- fos expression in a glutamate-dependent manner. A similar effect was observed with pituitary adenylate cyclase-activating polypeptide27 (PACAP27). At the intracellular level, coactivation of protein kinases A and C mediates the glutamate-dependent stimulation of c- fos expression evoked by VIP, because either H-89 or staurosporin inhibits the effect of VIP as well as that of glutamate. These results point to a "biochemical AND gate" mechanism, which implies the obligatory activation of both protein kinases A and C in the transduction of c- fos expression. In summary, this article provides evidence that VIP and PACAP27 potentiate the effect of glutamate, the principal effector on c- fos expression, suggesting that both peptides can increase the "throughput" or "strength" of glutamate-containing circuits in the cerebral cortex.     

Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation

The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, [125I]-labeled human transferrin and a [32P]-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (Ka) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 X 10(8) M-1, and the number of receptors per cell was 4.4 +/- 1.2 X 10(4). In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, greater than 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.     

Regulation of c-fos Expression by Convulsants and Hexachlorocyclohexane Isomers in Primary Cultures of Cortical Neurons

Abstract: Primary cortical cultures were used to study the effects of four convulsants on c- fos expression. Approximately 30% of the neurons in these cultures displayed c- fos nuclear immunostaining under basal conditions. The addition of tetrodotoxin, nifedipine, or δ-hexachlorocyclohexane produced a significant decrease in c- fos basal values. Lindane (γ-hexachlorocyclohexane), Bay K 8644, pentylenetetrazole, and picrotoxinin produced a significant increase in c- fos immunoreactivity and in c- fos mRNA expression. Treatment of cells with tetrodotoxin before administration of the convulsant agents lowered c- fos staining below basal levels. In contrast, δ-hexachlorocyclohexane or nifedipine failed to block only the picrotoxinin-induced increase. The differential pattern of expression shown by c- fos after these treatments suggests various mechanisms of action for the compounds studied. The results obtained with δ-hexachlorocyclohexane and nifedipine suggest that picrotoxinin activates c- fos expression by calcium-requiring intracellular signaling pathways that are different from those activated by Bay K 8644, pentylenetetrazole, or γ-hexachlorocyclohexane, which, at least in part, act via L-type calcium channels.     

Phenotypic and functional characteristics of macrophage-like cells differentiated in pro-inflammatory cytokine-containing cultures

Previous studies have demonstrated an infiltration of monocytes and increased levels of IL-1beta and TNF-alpha in some chronic inflammatory tissues. Interleukin-1beta and TNF-alpha are capable of protecting monocytes from spontaneous apoptosis and thus maintain their viability in vitro. To study the possible effects of these cytokines on the differentiation and function of recruited monocytes, a model has been developed in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of IL-1beta or TNF-alpha. Monocytes cultured with IL-1beta and TNF-alpha underwent substantial changes in morphology, similar to those observed in monocytes undergoing differentiation into macrophages. The cultured cells increased in size and vacuolization and their content of acid phosphates increased 10-fold. Although they exhibited the morphological characteristics of macrophages, monocytes matured in the cytokines differed functionally from those cultured in serum in a lower expression of HLA-DR, lower ability for triggering the proliferation of allogeneic lymphocytes, higher expression of mannose receptor and greater production of superoxide and TNF-alpha. This data suggests that IL-1beta and TNF-alpha direct monocyte differentiation into macrophages with a reduced antigen-presenting and an increased pro-inflammatory factor-releasing phenotype. Elevated levels of IL-1beta and TNF-alpha in the inflammatory tissues may therefore not only prolong the survival of recruited monocytes, but maintain them in an inflammatory state.     

Interferon α selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed constitutively in cells of the myeloid lineage, appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes. The human myeloid leukemia cell lines, HL-60, U937, and THP-1, express similar levels of immunochemically detectable MNDA. Although, the level of MNDA mRNA in primary monocytes is very low it was up-regulated at 6 h following the addition of interferon α. The effect of interferon α on the MNDA mRNA is also observed in the cell lines HL-60, U937, and THP-1. The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon α and other agents including interferon γ, endotoxin, poly (I) · poly (C), and FMLP. The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents. Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level. This reduced level of mRNA could then be elevated with subsequent interferon α treatment. The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed. The ability of interferon α to up-regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes. Granulocyte differentiation induced by retinoic acid treatment of HL-60 cells did not alter the MNDA mRNA level which was also unchanged following subsequent treatment with interferon α. The lack of interferon α effects on retinoic acid treated HL-60 cells is consistent with its inability to influence MNDA mRNA level in primary granulocytes.     

Oncogene expression in a medullary thyroid carcinoma

We report the expression of Ha-ras, fos, c-myc and N-myc mRNA in a human medullary carcinoma of the thyroid gland, both in primary tumor and lymph node metastasis, as demonstrated by in situ hybridization and Northern blot analysis. A significant difference in the oncogene expression in the primary tumor and the metastasis was not observed. Tumor tissue revealed a significant overexpression of Ha-ras, c-myc and N-myc mRNA as compared to the normal thyroid gland. The amount of fos mRNA expression in non tumorous thyroid gland did not significantly differ from tumor tissue, sis, fms and abl mRNA expression was not detectable in tumor tissue and non tumorous thyroid gland. We conclude, that the (over)expression of the oncogenes Ha-ras, c-myc and N-myc may be associated with initiation and progression of medullary thyroid carcinoma. Similar studies on additional cases of human medullary thyroid carcinoma will be necessary to reveal further information.     

Production and characterization of a monoclonal antibody (A1-3) that binds selectively to activated monocytes and inhibits monocyte procoagulant activity

We describe the generation and characterization of a new monoclonal antibody, A1-3, which possesses two unique properties. First, A1-3 binds selectively to stimulated human monocytes. Secondly, A1-3 inhibits the procoagulant activity expressed by stimulated monocytes and by human brain tissue factor. Unstimulated human peripheral blood cells (granulocytes, lymphocytes, monocytes, red blood cells, and platelets), prepared in the absence of detectable endotoxin, express no procoagulant activity and fail to bind A1-3. Stimulation of peripheral blood monocytes. alveolar macrophages, or the monocyte-like cell line U937, however, results in the expression of procoagulant activity and the binding of A1-3. The surface antigen recognized by A1-3 was recovered from endotoxin-stimulated human monocyte vesicles by immune precipitation and demonstrated an apparent m.w. of approximately 52,000. It is proposed that the monoclonal antibody A1-3 detects a differentiation antigen on human monocytes that is expressed in response to stimuli for monocyte activation.