Functional interactions between bile acids, all-trans retinoic acid, and 1,25-dihydroxy-vitamin D3 on monocytic differentiation and myeloblastin gene down-regulation in HL60 and THP-1 human leukemia cells.

Bile acids were shown previously to inhibit proliferation and to induce monocytic differentiation in HL60 human acute promyelocytic leukemia cells (A. Zimber et al., Int. J. Cancer, 59: 71-77, 1994). In this report, we hypothesized that bile acids may exert a positive cooperativity with two known inducers of leukemic cell differentiation, all-trans retinoic acid and 1,25(OH)2-vitamin D3. Our results provide evidence that bile acids induced the monocytic differentiation of HL60 and THP-1 human leukemia cells exposed to ineffective concentrations of these inducers. The protein kinase C (PKC) inhibitors H-7 (10 and 20 microM) and staurosporine (5 and 20 nM) modulated the effects of bile acids on HL60 cell differentiation. Most interestingly, bile acids are shown herein to down-regulate the expression of the serine protease myeloblastin gene involved in the differentiation of myeloid hematopoietic cells. In agreement with the recent identification of nuclear receptors for bile acids, our data suggest that functional interactions between nuclear bile acid signaling pathways, PKC, and nuclear receptors for retinoic acid and vitamin D3 are involved in the down-regulation of the myeloblastin gene and the induction of cell differentiation in human leukemic cells.

Inhibition of proliferation and induction of monocytic differentiation on HL60 human promyelocytic leukemia cells treated with bile acids in vitro.

We have tested the effect of several bile acids on the proliferation and differentiation of the HL60 human promyelocytic leukemia cell line in vitro. Deoxycholate, chenodeoxycholate and lithocholic acid caused dose-dependent inhibition of cell proliferation and induction of differentiation along the monocyte/macrophage pathway as determined by morphology, NBT test, non-specific esterase, and staining by monoclonal antibodies against specific cell-surface antigens. Optimal effects were obtained at 100, 75, and 60 microM of the 3 bile acids respectively. Cell-cycle flow-cytometric analysis showed that a substantial fraction of HL60 cells accumulated at the G0/G1 transition. Protein-kinase-C inhibitors such as sphinganine and H-7 inhibited the differentiation-inducing effect of bile acids, suggesting a possible role for PKC in this regulation. When bile acids were combined with non-effective concentrations of all-trans retinoic acid, enhancement of the monocytic differentiation of THP-1 human leukemia cells was observed. Our findings demonstrate induction of tumor-cell differentiation by bile acids, compounds that present minimal undesirable effects in humans.

Histamine H2 receptors and histidine decarboxylase in normal and leukemic human monocytes and macrophages.

Spontaneous and all-trans-retinoic acid (RA)-induced differentiation of normal human monocytes and of leukemic THP-1 monocytes into macrophages resulted in a progressive loss of adenosine 3',5'-cyclic monophosphate production induced by histamine via typical H2 receptors (H2R). In THP-1 cells and in HL-60 human acute myelocytic leukemia cells, RA treatment increased the abundance of the 4.5-kb messenger RNA of the H2R gene fourfold, suggesting transcriptional control by a RA response element. Scatchard plots of [3H]tiotidine binding indicated the expression of H2R with similar affinity and binding capacity in THP-1 monocytes and macrophages, while the conversion of normal monocytes into macrophages decreased H2R density from 91.8 to 43.1 fmol/mg protein, with no change in affinity (Kd = 9.9 to 11.2 nM). In THP-1 macrophages, histamine inhibited 4 beta-phorbol 12-myristate 13-acetate (PMA)-induced H2O2 formation via the activation of H2 receptors. Expression of the H2R gene, histamine accumulation, and histidine decarboxylase activity were also demonstrated in normal human monocytes/macrophages and peripheral lymphocytes. Histamine and H2R may therefore affect, via intracrine, autocrine, and paracrine pathways, various immune and inflammatory responses of the lymphoid and myeloid progenitors and lineages in the bone marrow and peripheral tissues.

Gs alpha availability to cholera toxin-catalysed ADP-ribosylation is decreased in membranes of retinoic acid-treated leukemic cell lines HL-60 and THP-1. A posttranslational effect.

Retinoic acid (RA) induces HL-60 and THP-1 leukemic cell lines to differentiate into granulocyte-like and monocyte-like cells. Limited data are available concerning the effects of RA on components of the cyclic AMP pathway in human myeloid leukemic cells. We showed previously a decrease in adenylate cyclase activity in the presence of histamine, prostaglandin E1 and forskolin in RA-treated HL-60 cells as compared to untreated cells. We examined the elements of the signal transduction pathway utilized by RA in the human myeloid cell line HL-60 and the human monocytic cell line THP-1. We therefore studied the effect of RA on the activity of the stimulatory G-protein (Gs). We demonstrate that addition of RA to two human myeloid leukemia cell lines, HL-60 and THP-1, does not induce a reduction of the 2 subunit of Gs (Gs alpha) RNA or Gs alpha protein in the plasma membrane but leads to a rapid decrease in the cholera toxin (CTX)-catalysed ADP-ribosylation of Gs alpha. In addition, this effect seems to be specific to RA, since there was no modification in Gs alpha ADP-ribosylation in the membranes of cells treated with dimethyl sulfoxide (DMSO), another inducer of differentiation in HL-60 cells.

Retinoic acid inhibits the expression of cytidine deaminase linked to the differentiation of the human leukemic cell line HL-60.

The activity of cytidine deaminase markedly increases during the differentiation of HL-60 cells induced by dimethylsulfoxide or 1,25-dihydroxy vitamin D3, but does not increase when the inducer is retinoic acid. Here it is demonstrated that retinoic acid inhibits the increase in cytidine deaminase activity elicited by the other two inducers. This inhibitory effect of retinoic acid (i) was not the result of a direct action on the enzymatic activity; (ii) was correlated with the differentiating effect of retinoic acid, as indicated by the similar time-course and dose-dependence of both effects, and by additional studies with various retinoids and with an HL-60 variant resistant to retinoic acid-induced differentiation; (iii) required the continued presence of the drug for more than 24 h, and could not be reversed after 48 h; (iv) was manifest, after a lag-time of 24 h, at whatever time retinoic acid was added during the 5 days of treatment of the cells with the differentiation inducers; and (v) was prevented by the addition of the protein synthesis inhibitor cycloheximide. These data indicate that retinoic acid negatively regulates the expression of cytidine deaminase in HL-60 cells, and suggest that this effect is mediated by a protein, the synthesis of which should be controlled by the nuclear receptor of retinoic acid.

The differentiating agent, retinoic acid, causes an early inhibition of inositol lipid-specific phospholipase C activity in HL-60 cells.

Retinoic acid, a derivative of vitamin A, is shown to inhibit the levels of inositol phosphates and diacylglycerol by 25-30% when added to intact HL-60 cells at concentrations which induce differentiation. The onset of inhibition occurs after 10 min and reaches a maximum at 45 min. To study the mechanism and the site of action of retinoic acid, the activity of the phosphatidylinositol bisphosphate-specific phospholipase C was studied in cells permeabilized with streptolysin O and in membrane preparations. Phospholipase C activity was stimulated either via the guanine nucleotide regulatory protein (G-protein) or directly by Ca2+. Retinoic acid treatment, in a time- and concentration-dependent manner, led to a decrease in phospholipase C activity when stimulated with either GTP gamma S or NaF, both of which activate the enzyme via the G-protein. By contrast, it had no effect on the enzyme activity when stimulated with Ca2+ alone. This indicates that retinoic acid interferes with the coupling of the G-protein and phospholipase C. A relationship between the inhibition of phospholipase C activity and the induction of differentiation by retinoic acid was investigated. Only a small inhibition of GTP gamma S-stimulated phospholipase C activity was observed when an analogue of retinoic acid, etretine or Ro10-1670, with low differentiating activity, was used. Moreover, no inhibition of the GTP gamma S-stimulated phospholipase C activity was observed in an HL-60 sub-line resistant to retinoic acid. These results suggest that phospholipase C inhibition is an important step in the induction of differentiation.

Characterization, in some human breast cancer cell lines, of gastrin-releasing peptide-like receptors which are absent in normal breast epithelial cells.

The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.

Interactions between the human monocytic leukaemia THP-1 cell line and Old and New World species of Leishmania.

The human promyelocytic THP-1 cell line has been found to support the growth of Leishmania parasites. THP-1 cells, differentiated with retinoic acid, cease replication while remaining in suspension. 72 +/- 8% of THP-1 cells became infected after inoculation with promastigotes of several Old and New World Leishmania species. The resulting amastigotes (19 +/- 5 per infected cell) were easy to harvest, capable of reinfecting cultures of normal human cells and, in the case of L. major and L. infantum, caused specific lesions in BALB/c mice. This culture system should facilitate biochemical and immunological studies on amastigotes and be of use in screening anti-parasite drugs.

In vitro effects of retinoic acid.

Retinoids, synthetic and natural analogues of vitamin A, play fundamental roles both in directing the spatial organization of cells during the development of vertebrate limbs and in the maintenance of growth and differentiation of many adult tissues. They also block the phenotypic expression of cancer in vitro; inhibit growth and induce differentiation in many animal and human malignant cell types. They have proved beneficial in skin diseases, cancer prevention and in acute promyelocytic leukemia.

HLA-DR and DQ antigens in chronic lymphocytic leukemia: dissociation of expression revealed by cell surface, protein, and mRNA studies.

We studied peripheral blood lymphocytes (PBL) of eight B chronic lymphocytic leukemia (CLL) patients for the expression of the human leucocyte antigens, HLA-DR and HLA-DQ. Cell surface expression of HLA class II epitopes was analyzed by fluorescent activated cell sorter (FACS) using three monomorphic anti-HLA class II monoclonal antibodies (mAb) specific for DR (D1.12) and DQ (TU22, L2) and a polymorphic anti-DQ (G2A5). The DR and DQ molecules were characterized by two-dimensional gel electrophoresis (2D-PAGE) of the specific immunoprecipitates from biosynthetically labeled cells. DR, DQ specific probes were used to characterize the class II transcripts of the corresponding genes. The data obtained with immunofluorescence disclosed two distinct patterns of HLA class II expression leading to two cell surface phenotypes: (DR+DQ+) and (DR+DQ-). In all cases the cells expressed normal amounts of HLA-DR gene products in terms of mRNA. DR cell surface determinants were present in more than 80% of cells in every sample. By 2-D gel analysis DR proteins disclosed the normal classical pattern associating the alpha, beta, and invariant chain gamma with normal level of biosynthesis for alpha and gamma but decreased biosynthesis for one of the beta gene products. Moreover, the three chains demonstrated defect in glycosylation process. In half of the cases studied the cells lacked DQ molecules at the cell surface. DQ alpha and DQ beta transcripts were detected in all cases, although the amount was extremely low in one case. DQ proteins were variable in the DQ+ phenotype and absent in the DQ- one. Interestingly, TPA and rIFN gamma treatment could restore normal glycosylation process of the DR isotype and increase biosynthesis of DQ alpha and beta chains. Those combined results support the view that transcriptional, post-transcriptional, and posttranslational mechanisms underlie the heterogeneity of class II expression observed in CLL. Moreover, 2-D gel analysis may be an invaluable tool for the analysis of the biosynthetic process of class II molecules.

[Large-scale production of amastigotes by a human monoblastoid cell line]. / Production massive d'amastigotes par une lignée de cellules monoblastoides humanines.

In this study, a human monoblastoid cell line (TPH-1) was tested in vitro for the production of Leishmania amastigotes. The number of TPH1 cells increased with time and 6 days after promastigote infection the percentage of infected cells was around 45%. Pre-treatment of TPH1 cells with retinoic acid induced the cells to differentiate into unreplicating macrophage-like cells. Ninety per cent was parasitized 6 days after promastigote infection; the number of amastigotes quintuplied during this period of time; this result was irrespective of the Leishmania species used for experiments. Viable and infective parasites were obtained from treated and nontreated cells. TPH1 cells merit further consideration for research concerning new molecules active against Leishmania.

Single-cell analysis of the intracellular pH and its regulation during the monocytic differentiation of U937 human leukemic cells.

Monocytic differentiation of U937 cells induced by retinoic acid is accompanied by a 0.2-pH-unit cell alkalinisation. The effect of retinoic acid on intracellular pH (pHi) develops slowly and it precedes the differentiation of the cells by 24 h. Heterogeneity in cellular pHi values was assessed using flow cytometry. It was higher at the differentiated stage than at the undifferentiated stage. It was reduced under conditions of clamped pHi values. Two membrane mechanisms allow U937 cells to recover from an intracellular acidosis. These are the Na+/H+ exchange system and a Na+-dependent HCO3-/Cl- exchange system. The increase in the pHi observed after monocytic differentiation resulted from a twofold increase in the maximum activity of the Na+/H+ exchange system with no change in the activity of the bicarbonate-dependent system. The properties of interaction of the Na+/H+ exchanger of U937 cells with Na+, Li+, amiloride and its derivatives were defined and appeared to be unique to human leukemic cells.

An increase in intracellular pH is a general response of promonocytic cells to differentiating agents.

Intracellular pH (pHi) was measured in HL60 and U937 cells before and after differentiation into monocyte-macrophage like cells. 12-O-Tetradecanoyl phorbol-13-acetate (PMA), butyrate, interferon, retinoic acid and 1,25-dihydroxyvitamin D3 all increased pHi. The increases elicited were rapid with PMA, much slower with retinoic acid and interferon and still slower with 1,25-dihydroxyvitamin D3. Increases in pHi are due to an activation of the Na+/H+ exchange system. High pHi values are unlikely to serve as an early intracellular signal for initiating monocytic differentiation.

Fatty acid composition of HL-60 cells is modified upon proliferation arrest and differentiation.

The human leukemic cell line HL-60 undergoes differentiation to granulocytic-like cells in response to dimethyl sulfoxide (DMSO) or retinoic acid (RA). This differentiation is accompanied by an arrest in cell proliferation. Studies have implicated alterations in the phospholipid fatty acid (FA) composition as a result of HL-60 differentiation. However, changes in FA's are also known to occur during the arrest of cellular proliferation. Using a highly efficient capillary gas-liquid chromatography technique, the phospholipid FA composition of HL-60 and of DMSO-resistant and RA-resistant HL-60 subclones was determined in proliferating cells, in density-arrested cells, and in terminally differentiated cells. The same specific modifications in some of the FAs of the three cell lines were observed when proliferation was inhibited by cell density; 16:0 and 18:2n-6 were decreased and 22:6n-3 increased. Moreover, 16 and 18 dimetylacetals were both increased when proliferation was decreased, indicating modifications in plasmalogen contents. Granulocytic differentiation of HL-60 cells and of its subclones with DMSO and/or RA provoked modifications in phospholipid FAs different from that found in density-arrested, undifferentiated cells such as decreases in monoenoic FAs of 16 and 18 carbons as well as an increase in arachidonic acid, the major polyunsaturated FA. The biological significance of these changes upon arrest of proliferation and differentiation are discussed. These results indicate that, when arrest of proliferation accompanies differentiation, these two phenomena can be responsible for different changes and, whenever possible, they have to be considered separately in order to know which modifications are effectively due to differentiation itself.

Discrepancy between in vitro and in vivo passaged U-937 human leukemic cells: tumorigenicity and sensitivity to differentiating drugs.

The treatment of nude mice bearing tumors of transplanted human leukemic cells with drugs known to induce differentiation of the same leukemic cells in vitro does not always affect tumor yield, tumor cell differentiation or nude mice survival. We have transplanted human monoblastic leukemic cells of the U-937 cell line into newborn Swiss nu/nu mice. Priming with cyclophosphamide, followed by subcutaneous injections of at least 10 x 10(6) cells allowed us to obtain solid tumors. The cytology, HLA phenotype and in vitro proliferation characteristics of the U-937 tumor cells were conserved. However, these tumor cells were more tumorigenic when reinjected into nude mice and showed a modified response to differentiation induction. A decreased capacity to differentiate with retinoic acid (RA) and a resistance to 1-beta-D arabinofuranosyl cytosine (Ara-C) and 1-25 dihydroxy vitamin D3 (1-25 (OH)2 D3) were noted in three tumor cell lines tested. With regard to the latter, the resistance was not due to a modification of the number of cell receptors. The study shows that though in vivo transplantation of human leukemic cells in nude mice may lead to a selection of resistant cells, systematic checking of in vitro differentiation characteristics of the tumor cells permits the nude mouse model to be maintained for the in vivo screening of new differentiating agents.

Properties of the Na+-dependent Cl-/HCO3- exchange system in U937 human leukemic cells.

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl- influx and efflux experiments. Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na+ which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl- and increased by removal of external Cl-. 36Cl- efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl- at a cell pHi of 6.3. Finally a 36Cl- uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (K0.5 = 10 microM) and bicarbonate (K0.5 = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+-independent Cl-/HCO3- exchange system could be found. The Na+-dependent Cl-/HCO3- exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO3-/Cl- exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.

PSL, a nuclear cell-cycle associated antigen is increased during retinoic acid-induced differentiation of HL-60 cells.

PSL(p55) is a nuclear 55kD antigen present in various mammalian cell systems, which has been first identified by use of human autoimmune antibodies (Barque et al. 1983, EMBO J. 2, 743). It has been shown to be associated with interphase chromatine and to be synthesized in during the S phase of the cell cycle. In this work, we have analysed the status of PSL in promyelocytic HL-60 human cells in exponential or stationary growth, or undergoing granulocytic differentiation in presence of Retinoic acid. By use of 2-dimensional electrophoresis, PSL was found to be composed of two acidic proteins designated p55A and p55B. Unexpectedly, estimated 10-20 fold higher amounts of each species were found in cells treated for 5 days with 10(-6)M Retinoic acid, than in asynchronously growing cells or resting cells. Moreover, the p55A protein was phosphorylated during the process. On the basis of these results, PSL appears to be involved in some steps of the granulocytic differentiation process.

Differential expression of c-myc and c-fos oncogenes during the differentiation of human leukemic cells by Ara-C.

1-B-D-arabinofuranosyl cytosine (Ara-C) is, at very low concentrations, an inducer of monocytic differentiation of human leukemic cells. By what mechanisms this differentiation is obtained and how the monocytic pathway is determined, is not yet understood. We studied the effect of Ara-C on the RNA transcript levels of two c-oncogenes often associated to cell proliferation and differentiation. In the monoblastic U-937 cell line, where Ara-C has a maximum (100%) monocytic differentiation, RNA transcript level of c-myc is not detectable, whereas c-fos levels are rapidly increased. On the other hand, in the bipotential promyelocytic HL-60 cells, Ara-C induces only 40% monocytic differentiation after 7 days; this is associated to a weaker decrement of c-myc expression; c-fos is however greatly induced 3 log after 2 days treatment, but before monocytic phenotype is observed. Ara-C, at low concentrations, may, by arresting cell proliferation, restore the leukemic's cell proliferation/differentiation equilibrium. The rapid inducement of c-fos expression may allow to foresee a rapid prescreening of Ara-C sensitive-blasts.

1,25-Dihydroxycholecalciferol induces an increase in PGE1- and forskolin-stimulated cyclic-AMP production in T47D human breast cancer cell line.

The effect of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], the active form of vitamin D3, on cell growth, clonogenicity, and cyclic adenosine monophosphate (cAMP) production was examined in human breast cancer cell line T47D. 1,25(OH)2D3 markedly inhibited proliferation of T47D cells in a time- and concentration-dependent manner. 1,25(OH)2D3 5 X 10(-7) reduced to 70% [3H]thymidine incorporation into DNA. Specific high affinity nuclear receptors for 1,25(OH)2D3 were present in this cell line. The cAMP produced by T47D cells was measured during 10 min stimulation by effectors (prostaglandin E1 or forskolin). Without effector, T47D cells produced similar amounts of cAMP in control and 1,25(OH)2D3-treated cells. After 3 days in the presence of 1,25(OH)2D3, cAMP production was significantly increased compared to control cells when stimulated by 10(-4) M prostaglandin E1 or 5 X 10(-7) M forskolin (3.2- and 2.4-fold increase, respectively). This cAMP increase was concentration dependent within the same range that inhibited cell growth and clonogenicity. These results suggest that 1,25(OH)2D3 may indirectly affect cAMP production by modulating the target cell response to stimulatory agents of cAMP production.