Introduction

Summary Introduction Materials & Methods Results Discussion References

Histamine is an intercellular signal molecule that exerts its effect through H₁, H₂, and H₃ receptors (1). It is well known that human phagocytes possess H₂ receptors, which mediate activation of adenylyl cyclase with subsequent increases in cAMP and PKA activity (2). This in turn modulates different cellular processes such as proliferation and differentiation. Recently, other cAMP-independent pathways have been described for H₂ receptors, including regulation of the breakdown of phosphoinositides (3, 4), of intracellular Ca²⁺ levels (5, 6), and of phospholipase A₂ activity (7). The presence of H₁ and H₂ histamine receptors in U937 cells derived from the monocyte/macrophage lineage (8) has been reported (9, 10). H₂ receptor activation produces an increase in cAMP formation (9, 10), whereas the H₁ receptor seems to be coupled to enhancement of phosphoinositide turnover (9).

Certain subsets of early response genes code for transcription factors involved in nuclear signal transduction. For example, the c-fos gene is induced as an immediate early event by serum, growth factors, and phorbol ester (11). The Fos and Jun proteins are the components of the activator protein-1 transcription factor that binds to the consensus sequence TGA(C/G)TCA (TRE) and regulates the transcription of several genes (12). Although Jun is capable of binding to this sequence as a homodimer, the formation of the Jun/Fos heterodimer greatly enhances the ability of these proteins to stimulate transcription from activator protein-1 enhancer sequences (13). Another early response gene, c-myc, has long been implicated in the control of normal cell growth, whereas its deregulation is related to the development of neoplasia (14). Myc protein is a short-lived nuclear phosphoprotein that exhibits a dimerization domain structurally related to those described in a number of transcription factors (15).

Different agents such as PMA (16), interferon- (17), vitamin D₃ (18), and tumor necrosis factor- (19) can induce cellular differentiation in the U937 cell line. A decrease in c-myc and c-myb mRNA levels and an increase in c-fos and c-jun transcripts have been reported during this event (20, 21).

The aim of the current study was to investigate the effects of histamine and its agonists on the modulation of the c-fos and c-myc proto-oncogenes and their implication in U937 promonocytic cell differentiation. We demonstrate that histamine and H₂, but not H₁, agonists induce a transient increase in c-fos mRNA and protein levels through cAMP production without affecting cell differentiation or proliferation. In addition, these agents do not modulate c-myc expression.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Chemicals. Histamine dihydrochloride, PGE₂, IBMX, cAMP, H7, famotidine, pyrilamine, dbcAMP, PMA, and rhC5a were purchased from Sigma Chemical (St. Louis, MO). BM (Calbiochem, San Diego, CA), dimaprit (22), and 2-(3-trifluormethylphenyl)histamine (23) were kindly provided by Dr. W. Schunack (Freie Universität Berlin, Berlin, Germany) and Dr. A. Buschauer (Regensburg Universität, Regensburg, Germany). Random primer DNA labeling kit was from BioRad (Hercules, CA). [³H]cAMP and [³²P]dCTP were purchased from DuPont-New England Nuclear (Boston, MA). All other chemicals were of analytical grade.

Cell culture. The U937 cell line (American Type Culture Collection, Rockville, MD) was cultured in suspension at 37° in RPMI 1640 medium supplemented with 10% fetal calf serum and 50 µg/ml gentamicin in a 5% CO₂ humidified incubator. Cells were routinely passed every 3 days and seeded at a density of 2.5 × 10⁵ cells/ml.

Northern blots. The levels of c-fos and c-myc expression under various conditions were examined by Northern blot analysis. Total RNA was isolated as described previously (24), and 20-µg aliquots were electrophoresed in 1% agarose/0.8 M formaldehyde gels, transferred to ZetaProbe membranes (BioRad), and hybridized overnight at 45° in 0.125 M sodium chloride, 0.25 M sodium phosphate, pH 7.2, 5% SDS, 10% polyethylene glycol, 50% formamide, and >10⁶ cpm/ml random primer ³²P-labeled probe. Filters were washed and exposed to AGFA Curix RP1 films. Quantification of films was performed with an LKB scanner (Uppsala, Sweden). Results of specific mRNA hybridization were standardized to the "housekeeping" gene GAPDH and expressed as the ratio of specific mRNA to GAPDH mRNA.

Western blots. Treated and control cells (3 × 10⁶) were lysed in 300 µl of 50 mM Tris·HCl, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol, 10% glycerol, and 0.05% bromphenol blue and sonicated to shear DNA. Samples were then boiled for 5 min, and 20-µl samples were electrophoresed in 12% SDS-polyacrylamide gels and transferred to nitrocellulose paper. The residual binding sites were blocked with 5% nonfat dried milk in TBST (20 mM Tris·HCl, pH 7.6, 137 mM NaCl, 0.05% Tween-20), and membranes were incubated with 2 µg/ml concentration of the specific primary antibody in 3% nonfat dried milk in TBST. Rabbit sera against Myc, Jun, and Fos proteins were purchased from Santa Cruz Biochemicals (Santa Cruz, CA). All subsequent washes were performed in TBST. Reactivity was developed using an anti-rabbit polyclonal antibody linked to horseradish peroxidase and enhanced chemiluminescence reagents according to the manufacturer's instructions (Amersham International, Buckinghamshire, UK).

cAMP determination. Cells were stimulated with different concentrations of histamine, H₂ agonist (dimaprit), or H₁ agonist (2-(3-trifluormethylphenyl) histamine) in Hanks' balanced salt solution containing 1 mM IBMX. Reaction was terminated by 3-min centrifugation at 3000 × g followed by the addition of ethanol. Supernatants were centrifuged 10 min at 3000 × g, and the ethanol phase was dried and resuspended in 50 mM Tris·HCl buffer, pH 7.4. The cAMP content was determined by competition with [³H]cAMP for PKA, as described previously (9).

Desensitization experiments. Pretreatment of cells with dimaprit was performed in Hanks' solution at 37° in a humidified atmosphere containing 5% CO₂. Cells were exposed to 10 µM dimaprit (maximal response) for periods ranging from 1 min to 3 hr in the absence of IBMX, after which cells were washed and resuspended at a density of 10⁶ cells/ml in Hanks' solution containing 1 mM IBMX and exposed for 9 min to dimaprit to determine whether the histamine H₂ receptors could still generate a cAMP response.

Cell proliferation. Cells were seeded at 2 × 10⁵ cells/ml and incubated for diverse period of times (maximum, 6 days) with different doses of dimaprit, 2-(3-trifluormethylphenyl)histamine, dbcAMP, and forskolin. Cells were harvested, and their numbers were estimated using an hemocytometer chamber.

Cell differentiation. Control and U937 cells treated for 72 hr with 10 µM dimaprit or 0.4 mM dbcAMP were incubated 15 min at 37° with murine anti-CD14, anti-CD11b (25) antibodies, or the isotypic control. Cells were immunolabeled by the addition of a goat anti-murine IgG/fluorescein isothiocyanate conjugate. All of these reagents were purchased from DAKO (Carpinteria, CA). Labeled cells were analyzed in a FACStar plus flow cytometer (Becton Dickinson, San Jose, CA).

Chemotaxis assay. In vitro locomotion of control and treated U937 cells was assayed according to the micropore filter technique. Briefly, 10⁵ cells in 0.5 ml of RPMI 1640 supplemented with 0.5% human serum albumin were seeded onto the top compartment of the chemotactic chamber placed in a 24-well culture plate. The top and bottom compartments were separated by a PVP-free polycarbonate filter with a pore size of 5 µm. The bottom compartment was filled with 0.6 ml of control media alone or plus 5 nM rhC5a (26). Chambers were incubated for 1-3 hr at 37° in a 5% CO₂ atmosphere. Migrating cells were collected and counted by flow cytometry (Cytoron; Ortho Diagnosis, Raritan, NJ).

NBT reduction. Treated and control cells were mixed with equal volumes of 0.2% NBT and 200 nM PMA in phosphate-buffered saline. After 1 hr of incubation at 37°, the proportion of cells containing intracellular formazan deposits was determined on May-Grunwald-Giemsa-stained cytospin slide preparations.

Data analysis. EC₅₀ values and doses required to reach maximal response were obtained by analyzing the concentration-response curves (see Figs. 2 and 6). Statistical analysis was carried out with Student's t test or one-way analysis of variance. A Bonferroni post hoc test for multiple pairwise comparisons was performed when significant differences were detected. Values of p < 0.05 were considered to indicate statistically significant differences.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent effects of histamine, H1 agonist, and H2 agonist on c-fos mRNA levels. U937 cells were incubated 1 hr with increasing concentrations of histamine (HIST), 2-(3 trifluormethylphenyl)histamine (H1), or dimaprit (H2). Total cellular RNA (20 µg) was hybridized to 32P-labeled c-fos and GAPDH probes. A, autoradiograms. B, quantification of autoradiograms. Arbitrary units represent the ratio of c-fos mRNA to GAPDH mRNA. Data are representative of at least three independent experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Concentration-response curves for cAMP production. U937 cells were incubated with increasing concentrations of histamine (Hist), 2-(3-trifluormethylphenyl)histamine (H1), or dimaprit (H2) for 9 min at 37° in Hanks' medium supplemented with 1 mM IBMX, and cAMP levels were determined. Data are the mean ± standard error of triplicate assays. Similar results were obtained in at least four independent experiments.

	Results

Summary Introduction Materials & Methods Results Discussion References

Time-dependent induction of c-fos expression by histamine and H₂ agonist. U937 cells treated with 10 µM histamine or H₂ agonist showed a remarkable transient increase in c-fos levels after 30 min (Fig. 1, top). Quantitative analysis of this assay clearly showed maximal levels of c-fos mRNA after 60 min of treatment. These mRNA levels consequently diminished, reaching values close to the basal level at 4 hr of treatment (Fig. 1, bottom). Identical experiments carried out in the presence of 10 µM H₁ agonist showed no effects on the levels of c-fos mRNA. The levels of the control GAPDH transcript were essentially constant throughout the assay.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Time-dependent effects of histamine, H1 agonist, and H2 agonist on c-fos mRNA levels. U937 cells were cultured in the presence of histamine (HIST), 2-(3-trifluormethylphenyl)histamine (H1), or dimaprit (H2) (10 µM each) for the indicated periods of time. Total cellular RNA (20 µg) was hybridized to 32P-labeled c-fos and GAPDH probes. A, autoradiograms. B, quantification of autoradiograms. Arbitrary units represent the ratio of c-fos mRNA to GAPDH mRNA. Data are representative of at least three independent experiments.

Dose-response analysis of the effects of histamine and the H₁ and H₂ agonists on the quantitative levels of c-fos mRNA. Concentration-response curves were performed after 1-hr incubation in the presence of histamine or the H₂ agonist. Both agents induced dose-dependent increases in the levels of c-fos mRNA (Fig. 2, top). EC₅₀ values and doses required to obtain maximal response were obtained by densitometric analysis of the autoradiograms. The EC₅₀ values for histamine and dimaprit were 1.4 ± 0.3 and 0.6 ± 0.1 µM, respectively, whereas the maximal response was obtained with doses of >10 µM for both reagents (Fig. 2, bottom). Again, no change was observed in cells treated with the H₁ agonist.

The increase in c-fos expression is induced specifically via the H₂ receptor. A combined treatment with the agonist and antagonists showed that only H₂ receptors were directly linked to the increases in c-fos levels. As shown in Fig. 3, the effect of histamine was completely blocked by famotidine, an H₂ antagonist, whereas no changes were seen with pyrilamine, an H₁ antagonist. These observations were reinforced by the fact that famotidine, but not pyrilamine, abrogates the increase in c-fos induced by the H₂ agonist. Neither pyrilamine nor famotidine was able to alter c-fos expression alone.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Effects of H1 and H2 antagonists on histamine-induced c-fos expression. U937 cells were pretreated 5 min with 10 µM pyrilamine (PYR) or 10 µM famotidine (FAM). Cultures were continued 1 hr in the presence of histamine (HIST), 2-(3-trifluormethylphenyl)histamine (H1), or dimaprit (H2) (10 µM each). Total cellular RNA (20 µg) was hybridized with 32P-labeled c-fos and GAPDH probes. Data are representative of at least three independent experiments.

The stimulatory effect of dimaprit on c-fos mRNA levels is mediated by PKA activation. To investigate whether protein phosphorylation is involved in the mechanism of c-fos modulation, a number of assays were carried out in which H7, a PKA/PKC inhibitor (27), was included before the addition of histamine or H₂ agonist to U937 in culture. The results shown in Fig. 4 demonstrate that H7 dramatically blocked the increase in c-fos mRNA levels evoked by histamine or dimaprit. In addition, dbcAMP, a PKA activator, induced an increase in c-fos transcript levels that could be blocked by H7. BM, a specific PKC inhibitor (28), did not modify c-fos expression induced by histamine or dimaprit but instead completely abolished the increase in c-fos mRNA evoked by PMA, a well-known PKC activator (29). These results suggest that protein phosphorylation mediated by PKA is part of the mechanism involved in histamine-induced c-fos gene expression.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Effects of H7 and bisindolylmaleimide on c-fos expression. U937 cells were pretreated 20 min with 20 µM H7 or 0.5 µM BM. Cultures were continued 1 hr in the presence of 10 µM histamine (HIST), 10 µM dimaprit (H2), 0.4 mM dbcAMP, or 10 nM PMA. Total cellular RNA (20 µg) was hybridized with 32P-labeled c-fos and GAPDH probes. Data are representative of at least three independent experiments.

c-myc is not affected by histamine. As indicated in Fig. 5, U937 cells express the c-myc gene. This expression was studied after the addition of 10 µM concentration of histamine, H₁ agonist, or H₂ agonist. During different periods of time (1-48 hr), no variations in the expression of this oncogene could be observed. Prolonged treatments of 6 days gave similar results (data not shown).

View larger version (79K): [in this window] [in a new window]   Fig. 5.   Kinetics analysis of the effects of histamine, H1 agonist, or H2 agonist treatment on c-myc mRNA levels. U937 cells were cultured in the presence of histamine (HIST), 2-(3-trifluormethylphenyl)histamine (H1), or dimaprit (H2) (10 µM each) for the indicated periods of time. Total cellular RNA (20 µg) was hybridized to 32P-labeled c-myc and GAPDH probes. Data are representative of at least three independent experiments.

Histamine and H₂ agonist induce cAMP levels in U937 cells. Histamine and dimaprit produced a dose-dependent increase in cAMP levels, with a maximum accumulations of ~100-fold on the basal value for histamine and >50-fold for dimaprit, with EC₅₀ values of 1.8 ± 0.2 and 0.7 ± 0.2 µM, respectively (Fig. 6). The EC₅₀ values for histamine and the H₂ agonist that produced increase in cAMP levels were found to coincide with the EC₅₀ values that induce c-fos expression (Figs. 2, bottom, and 6).

c-fos and c-myc levels are regulated by dbcAMP. U937 cells in culture were supplemented for various period of times with 0.4 mM dbcAMP, after which both c-fos and c-myc mRNA levels were examined. As indicated in Fig. 7, dbcAMP remarkably increased c-fos mRNA level within the first hour, and the level remain elevated for 12 hr after treatment. This sustained effect was not observed using histamine or the H₂ agonist. Under similar conditions, the levels of c-myc mRNA were markedly reduced within 1 hr of treatment with dbcAMP.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Effect of dbcAMP treatment on c-fos and c-myc mRNA levels. U937 cells were cultured in the presence of 0.4 mM dbcAMP for the indicated periods of time. Total cellular RNA (20 µg) was hybridized to 32P-labeled c-myc and GAPDH or to c-fos and GAPDH probes. Data are representative of at least three independent experiments.

Levels of Fos and Myc proteins in U937 cells treated with H₂ histamine agonist and dbcAMP. Kinetic analysis of Fos and Myc protein content was carried out on cells treated with dimaprit or dbcAMP. Translation was found to follow a pattern that paralleled the corresponding oncogene mRNA levels. Dimaprit produced a transient increase in Fos protein with a maximal response after 2 hr, which remained higher than controls for 6 hr and progressively decreased to basal concentrations after 12- 24 hr. In contrast, levels of this protein remain constantly elevated in cells exposed 24 hr to dbcAMP (Fig. 8). Myc protein levels were not affected by dimaprit in cells treated 24 hr but showed a remarkable and progressive decrease when cultured in the presence of dbcAMP (Fig. 8). Analysis of Jun protein levels showed a behavior similar to the one found for Fos in cells treated with dimaprit or dbcAMP (Fig. 8).

View larger version (56K): [in this window] [in a new window]   Fig. 8.   Time-dependent effects of dimaprit and dibutyryl cAMP on Fos, Myc, and Jun protein levels. U937 cells were incubated with 10 µM dimaprit or 0.4 mM dbcAMP for the indicated periods of time before harvest and lysis as described in Materials and Methods. Samples were electrophoresed in 12% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with polyclonal purified rabbit serum against Fos, Myc, and Jun. Data are representative of at least three independent experiments.

Histamine does not induce changes in cell proliferation. Fig. 9 illustrate the results on U937 cell proliferation after 3 days of treatment with histamine, H₁ or H₂ agonist, or dbcAMP at the indicated concentrations. Neither histamine nor its agonists were able to inhibit cell division. In contrast, dbcAMP inhibited U937 cell proliferation. This effect was dose dependent, exhibiting an EC₅₀ value of 116 ± 12 µM. Forskolin showed a similar inhibitory effect with an EC₅₀ value of 19 ± 6 µM.

View larger version (53K): [in this window] [in a new window]   Fig. 9.   Effects of histamine, H1 and H2 agonists, and dbcAMP in U937 cell proliferation. U937 cells were cultured with histamine (Hist), 2-(3-trifluormethylphenyl)histamine (H1), dimaprit (H2), or dbcAMP and treated for 3 days. Cells were harvested, and an aliquot was counted using an hemocytometer chamber. Data are the mean ± standard error of assay quadruplicates. Similar results were obtained in at least three independent experiments.

H₂ agonist has no effect on cell differentiation. Essentially all cells remained functionally undifferentiated after 3 days of treatment with dimaprit (Table 1). As expected, cell differentiation takes place in the presence of dbcAMP. This was judged by the ability of dbcAMP to promote reduction of NBT, to increase migration rates of cells challenged with the chemoattractant rhC5a, and to induce the expression of CD14 and CD11b myeloid differentiation cell surface antigens (Table 1). Forskolin, an inducer of the PKA pathway, showed similar results to dbcAMP (data not shown).

Histamine H₂ receptor desensitization. The susceptibility of the histamine H₂ receptor to desensitization was further investigated in this cell line. Results showed a dramatic reduction in cAMP production by the H₂ agonist when U937 cells were pretreated with dimaprit. In fact, the agonist could no longer induce a response when cells were pretreated for 2 hr with 10 µM concentration of the H₂ agonist. Half-maximal desensitization was observed at 20 ± 3 min after cell preexposure (Fig. 10). To establish whether desensitization was restricted to the histamine H₂ receptor, PGE₂-mediated cAMP induction was examined in cells pretreated with dimaprit. PGE₂ increased cAMP production in the U937 cell line for 2 hr. Identical assays carried out in the presence of forskolin showed similar results (Fig. 10, inset).

View larger version (27K): [in this window] [in a new window]   Fig. 10.   Dimaprit induces desensitization of the histamine H2 receptor. U937 cells were preincubated for different period of times with 10 µM dimaprit, further washed, and stimulated with dimaprit in the presence of 1 mM IBMX; cAMP production was determined as described in Materials and Methods. Inset, Cells pretreated with dimaprit for 0, 30, and 240 min were stimulated with 1 µM PGE2 or 10 µM forskolin (FORSK), and cAMP production was determined. Data are shown as the percentage of cAMP production in response to dimaprit in function of the basal level in nonpretreated cells and are the mean ± standard error of assay triplicates. Similar results were obtained in at least three independent experiments.

	Discussion

Summary Introduction Materials & Methods Results Discussion References

The current study reports the effects of histamine on the levels of two nuclear proto-oncogenes, c-fos and c-myc, in U937 cells. Results indicate that histamine and dimaprit induce a transient increase in the levels of c-fos that is specifically mediated through H₂ receptors.

The presence of this type of receptor in U937 cells was reported in a study by Gespach et al. (10), in which H₂ receptors exhibit a classic pharmacological pattern described in the right atrium of guinea pig. In addition, the cAMP response to H₂ receptor stimulus obtained in the current study was concordant with data reported in the literature (1).

The demonstration that the EC₅₀ values for cAMP and c-fos expression were similar suggests that this increase is modulated via cAMP-PKA. Studies performed in the presence of H7 showed dramatic blocking of the effects of histamine and its agonist on the expression of this proto-oncogene.

Previous studies performed by our group demonstrated that histamine modulates the turnover of phosphoinositides in U937 cells in a dose-dependent manner through H₁ receptors (9). In addition, the regulation of PKC activity by the activation of this pathway via H₁ receptors has been described (1). Furthermore, the existence of diverse PKC isoenzymes with activity for distinct substrates in vitro has revealed differences that may indicate an even more pronounced specificity toward natural substrates under physiological conditions (29). Therefore, unlike PMA, possibly histamine or H₁ agonists activate PKC isoforms that are unable to stimulate c-fos transcription via PKC. The fact that the H₁ agonist did not induce changes in c-fos mRNA levels supports the idea that the effect of histamine at this level is independent of PKC. In addition, pretreatment with bisindolylmaleimide, a selective PKC inhibitor, did not alter the expression of the c-fos proto-oncogene increased by either histamine or dimaprit. This allows us to conclude that the intracellular increase in cAMP through H₂ receptors, together with the increase in PKA activity, might be responsible for the increased levels of c-fos mRNA. This is supported by the fact that dbcAMP, a direct activator of PKA, also increased c-fos expression and can be blocked by H7.

The induction of c-fos by cAMP has been demonstrated in numerous systems. The characteristics of this control are partly cell-type specific, as illustrated by the very high and stable transcription levels of c-fos in cAMP-stimulated macrophages and a weaker and transient effect in other cells (30). Enhanced c-fos expression associated with an increase in the intracellular concentration of cAMP have been described in systems in which this cyclic nucleotide can induce cellular proliferation [e.g., Swiss 3T3 cells (31) and dog thyroid cells (32)].

Contrarily to the response of c-fos, we did not detect modifications in the expression of the c-myc proto-oncogene in cells treated with histamine or its agonists. These results differ from those described for other hematopoietic cell lines in which intracellular increases in cAMP by histamine induced a pronounced down-regulation of c-myc expression (3, 33). However, using a membrane-permeable analogue of cAMP, dbcAMP, we have shown an increase in c-fos transcript concomitant with a down-regulation of c-myc expression. Similar results concerning c-myc expression were previously reported in HL-60 cells (34). It is not clear whether butyrate, a putative hydrolytic product of dbcAMP, contributes to U937 cell differentiation. Exogenously added sodium butyrate does not affect c-fos mRNA expression (35) or induce morphological or functional alterations characteristic of differentiated phagocytes (36). Consequently, further studies are needed to clarify this issue. However, using forskolin to induce accumulation of intracellular cAMP, we observed in U937 cells the appearance of several characteristics of differentiated cells, disregarding any participation of butyrate (data not shown).

Because gene expression regulation in general is not restricted to mRNA levels alone (37), the effects of H₂ agonist and dbcAMP on Fos, Jun, and Myc protein expression were also studied. Dimaprit transiently elevated Fos levels, whereas dbcAMP produced increases for longer periods of times. In addition, we observed that the levels of Jun protein, the other component of the activator protein-1 complex, in cells treated with H₂ agonist or dbcAMP showed similar patterns. A great deal of circumstantial evidence suggests that control of Myc function may be important during differentiation. Heightened Myc expression in murine erythroleukemia cells and monocytes can block differentiation (38). It appears that the transient elevation in cAMP induced by H₂ agonist is insufficient to down-modulate Myc protein expression over the long term, whereas in cells incubated in the presence of dbcAMP, we clearly observed a significant and sustained reduction in Myc protein levels.

In many G protein-coupled systems, such as histamine receptors, prolonged exposure to agonists leads to a state of reduced responsiveness to these agents, a process termed "desensitization" (39). In this regard, we show that the desensitization of H₂ receptors in U937 cells exhibits a half-life of 20 min and that by 120 min this process is complete. Furthermore, desensitization proved to be homologous because cells pretreated with dimaprit showed an intact cAMP response to both PGE₂ and forskolin. This is in agreement with the transient increase in c-fos expression and supports the hypothesis that this effect is mediated by cAMP. On the other hand, this rapid desensitization followed by a decrease in cAMP levels could be the basis for the lack of response in c-myc expression. In other cellular systems in which the desensitization of the H₂ receptor is retarded, histamine and its agonists down-regulate the levels of c-myc gene expression (40).

We demonstrate here that histamine is incapable of inhibiting U937 cell proliferation despite the noticeable though transient increase in cAMP levels, whereas an important inhibition by dbcAMP could be observed. Accordingly, we have shown that forskolin and dbcAMP, but not dimaprit, induced a significant proportion of cells to acquire the ability to (i) produce superoxide anion, (ii) express cell surface maturation markers, and (iii) increase chemotaxis. Taken together, these results indicate that the binding of agonist to H₂ histamine receptors triggers an early specific response characterized by an increase in cAMP levels, but their rapid desensitization makes this response insufficient to induce differentiation of U937 cells. It could be possible that in promonocytic U937 cells, but not in normal progenitors, desensitization via H₂ receptors plays a significant role in reducing their capacity to reach terminal cell differentiation. Supporting this idea, although not rigorously related to this cell hierarchy, histamine has been reported to cooperate with granulocyte-colony stimulating factor in the induction of normal cell differentiation (40). Therefore, a better understanding of this issue could make possible new avenues in therapeutic intervention.