tretinoin and Lymphoma--Large-B-Cell--Diffuse

The same progress in the recent therapeutic strategy for older adults with hematological malignancies has also been seen in younger adults. The standard initial therapy for elderly acute promylocytic leukemia is the combination with all-trans retinoic acid and anthracyclines. For other acute myeloid leukemias (AML), many trials of combination chemotherapy have not improved the outcome of elderly patients. Gemtuzumab ozogamicin,which is an immunoconjugate binding to CD 33 on the surface of AML blasts, has produced good results for elderly patients in either monotherapy or in combination with conventional chemotherapeutic drugs. One of the BCR-ABL tyrosine kinase inhibitors, imatinib mesylate, is active for elderly Philadelphia-positive leukemia including acute lymphoblastic leukemia and chronic myeloid leukemia. In the treatment of elderly diffuse large B cell lymphoma, combination of rituximab and cyclophosphamide+doxorubicin+vincristine+prednisone (CHOP) has become the therapy of choice based upon a Groupe d'Etude des Lymphomes de l'Adulte (GELA) trial even though there are some other trials for elderly patients such as dose-dense CHOP therapy. For follicular lymphoma, combination therapies of rituximab and cytotoxic drugs such as R-CHOP and R-CVP are also considered as promising therapies. For the management of multiple myeloma, high-dose chemotherapy, mainly melphalan with autologous stem cell transplantation, has become the standard treatment even for elderly patients less than 65 years of age.

Topics: Aged; Aminoglycosides; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Cyclophosphamide; Doxorubicin; Drug Administration Schedule; Gemtuzumab; Hematologic Neoplasms; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Middle Aged; Multiple Myeloma; Prednisone; Rituximab; Survival Rate; Tretinoin; Vincristine

Topics: Animals; Cell Differentiation; Cholecalciferol; Cytokines; Humans; Lymphoma, Large B-Cell, Diffuse; Macrophages; Models, Biological; Monocytes; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The aim of this study was to develop an intravenous delivery of all-trans retinoic acid (ATRA) for the treatment of cancer. Two kinds of liposomes composed of dipalmitoylphosphatidylcholine and cholesterol with sterylglucoside (SG) mixture (SG liposomes) or without SG (non-SG liposomes) were prepared. A limited amount of ATRA was incorporated into liposomes approximately 3mol%. ATRA-loaded SG liposomes were more stable at 4 degrees C with light protection for 24 days than non-SG liposomes; maintaining 83.1% ATRA and the average diameter 198.5 nm (36 days), and in 80% rat serum for 120 min. SG seems to increase the ATRA-loaded efficiency in liposomes and stability of liposomes compared with cholesterol. The mean survival time of mice given SG liposomes (18.2 days) indicated a greater antitumor activity than saline (14.1 days) (P<0.001) without change of mean body weight. It is concluded that the current ATRA-loaded SG liposomes are potentially applicable for hepatic metastasis of M5076 tumor.

Topics: Animals; Antineoplastic Agents; Cholestenes; Chromatography, High Pressure Liquid; Drug Carriers; Drug Compounding; Drug Stability; Female; Injections, Intravenous; Liposomes; Liver Neoplasms, Experimental; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Inbred C57BL; Particle Size; Tretinoin

Retinoic acid (RA), a potent inducer of cell differentiation, and N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide), a potent inducer of apoptosis, are well known as anticancer agents that are administered orally to patients for leukemia, breast and prostate cancer, respectively. However, it has not been studied whether both retinoids are effective on metastatic cancer. In mice implanted with M5076 cells, murine reticulum cell sarcoma survival times were prolonged by i.v. treatment of RA and 4-HPR entrapped in liposomes containing soybean-derived sterylglucoside mixture (SG), which accumulates in liver. In contrast, free RA and 4-HPR were inactive. These results indicate that RA and 4-HPR in SG-liposomes exhibit anticancer efficacy on metastatic cancers, and may have great potential for clinical use in the treatment of various cancers.

Topics: Animals; Cell Line, Tumor; Cholestenes; Drug Evaluation, Preclinical; Female; Fenretinide; Liposomes; Liver Neoplasms, Experimental; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Inbred C57BL; Tretinoin; Xenograft Model Antitumor Assays

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cyclophosphamide; Cytarabine; Doxorubicin; Etoposide; Female; Humans; Idarubicin; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Non-Hodgkin; Neoplasm Proteins; Neoplasms, Second Primary; Oncogene Proteins, Fusion; Piperazines; Prednisolone; Remission Induction; Tretinoin; Vincristine

Association of the ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) with liposomes (ELL-12) reduces acute toxicity while maintaining or enhancing anticancer activity in experimental tumor models. ELL-12 has been shown to induce apoptosis by a cytochrome-c-dependent caspase-mediated pathway, which results in proteolytic cleavage of poly(ADP-ribose) polymerase and lamins, but the antitumor effects of ET-18-OCH3 or ELL-12 could result from tumor cell differentiation or activation. Here we compared the effects of ET-18-OCH3 and ELL-12 on the expression of cell-surface proteins associated with cell differentiation and/or activation in U-937 cells. Phorbol 12-myristate 13-acetate and all-trans-retinoic acid, which induce differentiation in U-937 cells, up-regulated CD11b (MAC1 alpha-integrin) and CD82 and down-regulated CD71 (transferrin receptor) in a time- and dose-dependent manner. In contrast, ET-18-OCH3 and ELL-12 up-regulated both CD71 and CD11b and did not have any effect on expression of CD82 in U-937 cells, suggesting that the ELL-12 may activate these cells rather than induce differentiation. Further evidence of activation was that ET-18-OCH3 and ELL-12 strongly induced tumor necrosis factor alpha production by U-937 cells.

Topics: Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Antineoplastic Agents; Cell Differentiation; Drug Carriers; Drug Synergism; Humans; Kangai-1 Protein; Liposomes; Lymphoma, Large B-Cell, Diffuse; Macrophage-1 Antigen; Membrane Glycoproteins; Phagocytosis; Phospholipid Ethers; Proto-Oncogene Proteins; Receptors, Cell Surface; Receptors, Transferrin; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Necrosis Factor-alpha; U937 Cells

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1,25-dihydroxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide synthase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2.-) but also nitric oxide (.NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2.- determined. Conversely, superoxide dismutase (SOD) scavenged the O2.- released and increased the .NO-derived nitrite concentration detected. These data suggested a possible interaction between O2.- and .NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abrogated by SOD and partially inhibited by aminoguanidine or L-NMMA. Since the iNOS inhibitors did not directly scavenge O2.-, LDCL determination in the presence or absence of SOD and/or iNOS inhibitors demonstrated a concomitant production of O2.- and .NO. These radicals induced the formation of a .NO-derived product(s), probably peroxynitrite (ONOO-), which was required to elicit maximal LDCL. Finally, LDCL measurement provided a convenient tool to characterize iNOS triggering and demonstrated an interaction between NADPH oxidase and iNOS products in human macrophagic cells phagocytizing opsonized-zymosan. These findings show that in activated macrophages, iNOS activity can be involved in LDCL and support the debated hypothesis of iNOS participation to the microbicidal activity of human macrophages.

Topics: Calcitriol; Cell Differentiation; Cell-Free System; Free Radical Scavengers; Guanidines; Humans; Immunoglobulin E; Immunoglobulin G; Leukemia, Monocytic, Acute; Luminescent Measurements; Luminol; Lymphoma, Large B-Cell, Diffuse; Macrophage Activation; Macrophages; Neoplastic Stem Cells; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; omega-N-Methylarginine; Opsonin Proteins; Phagocytosis; Superoxide Dismutase; Superoxides; Tretinoin; Tumor Cells, Cultured; Xanthine; Xanthine Oxidase; Zymosan

The EVI-1 gene encodes a Zn finger, DNA binding protein previously detected in some acute myelogenous leukemias (AML) and myelodysplasias (MDS), but not in normal marrow or cord blood cells. Experimental studies suggest EVI-1 blocks cellular differentiation by binding to GATA-1 or other specific DNA sequences controlling gene expression, and may be involved in the pathogenesis of some AMLs. To further define potential roles for EVI-1 in leukemia pathogenesis, we studied its regulation in acute promyelocytic leukemias (APL). Seven of 11 APL cases expressed EVI-1 RNA detected by RNA PCR at diagnosis, and expression was detected in two additional cases after treatment with all-trans retinoic acid (ATRA). Two of four cases studied at relapse also expressed EVI-1 RNA. To investigate regulation of EVI-1 expression in APL, we examined its expression in the NB4 APL cell line. NB4 cells did not express EVI-1 under basal conditions, but expressed EVI-1 after ATRA-induced differentiation. When NB4 cells were exposed to ATRA and transferred to cultures with N,N'-hexamethylene-bis-acetamide (HMBA), differentiation occurred but EVI-1 RNA was not detected, indicating that EVI-1 expression was not required for terminal, NB4 differentiation. ATRA-resistant NB4 cells were obtained by continuous culture in gradually increasing concentrations of ATRA. These cells did not express markers of differentiation but continued to express EVI-1 for several weeks even after ATRA withdrawal. To assess whether expression of the APL PML-RAR alpha fusion gene alone was sufficient for ATRA induction of EVI-1, the PML-RAR alpha gene cDNA was expressed in U937 histiocytic lymphoma cells. ATRA treatment of PML-RAR alpha-transfected or control U937 cells did not induce EVI-1 expression. In conclusion, this study demonstrates the EVI-1 gene is consistently expressed in APL cells either constitutively or after ATRA treatment. ATRA represents the first biologically active agent shown to specifically regulate EVI-1 expression in blood cells. In contrast to previous studies in AML and MDS, the pattern of EVI-1 expression suggests it may facilitate rather than inhibit myeloid differentiation during ATRA treatment. However, effects of EVI-1 expression are likely to be complex, and expression in ATRA-resistant APL cells may indicate multiple roles for this gene.

Topics: Acetamides; Alitretinoin; Cell Differentiation; DNA-Binding Proteins; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Isotretinoin; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; MDS1 and EVI1 Complex Locus Protein; Neoplasm Proteins; Oncogene Proteins, Fusion; Proto-Oncogenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Zinc Fingers

Retinoids and vitamin D (VD) cooperate to induce the differentiation and inhibit the proliferation of human myelomonocytic leukemia cells. Two classes of retinoids receptors, the RARs and RXRs, respectively, can mediate these effects. RXR forms heterodimers with a variety of nuclear receptors, including RAR and the VD receptor. We have previously found that VD treatment increases RXR alpha levels in myelomonocytic leukemia cells. By immunoanalysis, we observed in the present work that the RAR alpha protein is expressed in proliferating U937, HL-60 and THP-1 human leukemia cells and that VD treatment induces alterations of its electrophoretic pattern, although with large differences between cell lines. In the three cell lines, 9-cis RA, an agonist of both RARs and RXRs, cooperated with VD more efficiently than all-trans RA and RAR-specific synthetic ligands, thus suggesting an involvement of both RAR and RXR pathways in cell differentiation. Using U937 cells as a model, we delineated the relative contributions of RAR and RXR by assessing the effects of receptor-selective synthetic retinoids. The synergy between VD and all-trans RA or RAR-specific agonists (TTNPB and Ro 40-6055) was abrogated by a RAR alpha-specific antagonist (Ro 41-5253), confirming an involvement of RAR alpha. However, the cooperation between VD and 9-cis RA, although reduced, was not suppressed by the antagonist, suggesting also an involvement of the RXR pathway. The role of RXR as a ligand-activated receptor was confirmed using RXR-specific agonists (CD2608 and LGD1069), which also proved able to cooperate with VD. Finally, while each synthetic agonist alone was significantly less potent than 9-cis RA, combinations of the RAR and RXR selective agonists TTNPB and LGD1069 appeared to be as effective as the pan agonist 9-cis-RA. These results confirm that various retinoids can cooperate with VD and demonstrate that, at a whole cell level, optimal effects require the activation of both RAR and RXR receptors.

Topics: Alitretinoin; Animals; Benzoates; Bexarotene; Cell Differentiation; Chromans; COS Cells; HL-60 Cells; Humans; Leukemia, Monocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Molecular Structure; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Signal Transduction; Tetrahydronaphthalenes; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Vitamin D

The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regulation of the cell cycle, and in mediating cell differentiation. RB is necessary for hematopoiesis in mice, and aberrant RB-expression is associated with the progress and prognosis of leukemia. We have used antisense oligonucleotides, established clones stably expressing an antisense RB construct, and also established clones over expressing the retinoblastoma protein (pRb) to study the role of RB expression in monocytic differentiation induced by all-trans retinoic acid (ATRA) or 1-alpha-25-dihyroxycholecalciferol (Vit D3) in the monoblastic cell line U-937 and erythroid differentiation induced by transforming growth factor beta1 (TGFbeta1) and hemin in the erythroleukemic cell line K562. A reduction in pRb production in antisense RB-transfected U-937 clones was shown. Antisense oligonucleotides as well as expression of the antisense RB construct suppressed differentiation responses to ATRA or Vit D3, as judged by the capability to reduce nitro blue tetrazolium, by the appearance of monocyte-related cell surface antigens and by morphologic criteria. K562 cells showed decreased differentiation response to TGFbeta1, but not to hemin, when incubated with antisense oligonucleotides. U-937 antisense RB-transfected cells were also suppressed in their ability to upregulate levels of hypophosphorylated pRb when induced to differentiate. Although U-937 cells incubated with antisense oligonucleotides and clones expressing the antisense RB construct were hampered in their ability to differentiate on incubation with ATRA or Vit D3, the induced G0/G1-accumulation was similar to differentiating control cells treated with ATRA or Vit D3. Intriguingly, U-937 clones overexpressing RB were also inhibited in their differentiation response to ATRA or Vit D3 but not inhibited in their ability to respond with G0/G1 accumulation when induced with these substances. The results indicate that pRb plays a role in induced differentiation of U-937 cells as well as K562 cells involving mechanisms that, at least partially, are distinct from those inducing G1 accumulation.

Topics: Animals; Antigens, Differentiation; Antigens, Neoplasm; Calcitriol; Cell Differentiation; G1 Phase; Gene Expression Regulation, Leukemic; Genes, Retinoblastoma; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoma, Large B-Cell, Diffuse; Mice; Monocytes; Neoplasm Proteins; Oligonucleotides, Antisense; Retinoblastoma Protein; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Retinoids have been shown to modulate cell growth and differentiation in a variety of human tumor cell types, but their effects on B-cell non-Hodgkin's lymphomas (NHL-B) have not been explored. In this study, all-trans retinoic acid (ATRA) in the free form and liposome-encapsulated form (L-ATRA) were used to determine effects on fresh NHL-B patient cells as well as cell lines recently established from both HIV-negative and -positive NHL-B patient biopsies. Both ATRA and L-ATRA were found to inhibit cell proliferation in NHL-B cells. However, L-ATRA was found to be superior to free ATRA in inhibiting cell proliferation of NHL-B cells and resulted in greater than 90% cell growth inhibition in a dose-dependent manner. In addition, L-ATRA also induced high levels of apoptosis in NHL-B cells in vitro. To delineate the apoptotic pathways involved, the expression of the apoptosis suppressor oncogene bcl-2 was evaluated in different NHL-B cells with and without the t(14;18) chromosomal translocation. After L-ATRA exposure, more than a 50% reduction in the expression of bcl-2 protein was observed. bcl-2 message levels were also down-regulated in the L-ATRA-sensitive NHL-B cells. Bax protein levels were analyzed and found to be up-regulated in L-ATRA-sensitive NHL-B cells. Similar results were observed in sensitive AIDS/lymphoma cell lines. Experiments using an RAR-alpha antagonist (RO 41-5253) showed that both the proliferation inhibition and apoptosis induced by L-ATRA could be blocked in NHL-B cells. The findings of the present study indicate that L-ATRA may possess therapeutic potential in blocking cell proliferation, inducing apoptosis, and

Topics: Apoptosis; bcl-2-Associated X Protein; Benzoates; Blotting, Western; Cell Division; Chromans; Dosage Forms; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Liposomes; Lymphoma, AIDS-Related; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Microscopy, Electron; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin; Tumor Cells, Cultured

Although retinoic acid (RA) has been known for many years to be a modulating agent that plays a role in generating both granulocytes and monocytes, the molecular mechanism underlying this role has not been defined in the monoblast lineage. In particular, the part played by the retinoid X receptors (RXRs), which are members of the steroid/thyroid hormone nuclear receptor family, has not been explored. In this study, therefore, the human monoblastic leukemia cell line U937 has been used as a model system to investigate the role of one of the RXRs, RXR-alpha, in monoblast differentiation. RXR-alpha mRNA was present in untreated U937 cells, and levels increased after induction of differentiation with phorbol ester. The same was found for RXR-beta mRNA. Using plasmids containing sense or antisense RXR-alpha sequences under the control of an inducible promoter, we generated stably transfected cell lines which expressed either increased or decreased levels of RXR-alpha, respectively. The sense cell lines (U alpha S and its clonal derivative alpha G2S) showed increased sensitivity to RA, while the antisense cell lines (U alpha A and its clonal derivative alpha B5A) showed decreased sensitivity to RA, as demonstrated by growth inhibition and by regulation of an RA-responsive reporter gene. Both U alpha A and alpha B5A also failed to respond to another modulating agent, 1 alpha,25-dihydroxycholecalciferol (DHCC), but only U alpha S and not alpha G2S showed an enhanced response to DHCC. The combination of RA and DHCC together inhibited growth of both sense and antisense cell lines. In addition, alpha G2S exhibited increased expression of CD11b and CD54, while alpha B5A cells showed increased expression of CD102, suggesting that RXR-alpha has a role in regulating expression of cell adhesion molecules in U937 cells. These results demonstrate that RXR-alpha has a role in mediating growth inhibition and cell adhesion during myelomonocytic differentiation, and suggest that different species of heterodimers involving RXR-alpha may control the acquisition of different features of mature monocyte/macrophage function.

Topics: Antigens, CD; Antineoplastic Agents; Calcitriol; Cell Adhesion Molecules; Cell Differentiation; DNA-Binding Proteins; DNA, Antisense; Gene Expression Regulation, Neoplastic; Humans; Intercellular Adhesion Molecule-1; Lymphoma, Large B-Cell, Diffuse; Macrophage-1 Antigen; Monocytes; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vitamin D

Defensins are microbicidal peptides and the principal constituents of neutrophil primary granules. They are presumed to play a prominent role in innate host defenses. We examined defensin mRNA levels during drug-induced differentiation of the promyelocytic leukemia cell line, HL-60. Transcription was restricted to promyelocyte, myelocyte, and very early metamyelocyte stages of the granulocytic pathway. Complete downregulation occurred during late granulocytic maturation or early during phorbol ester-promoted differentiation along the monocyte/macrophage lineage. Retinoic acid (RA) was the strongest inducer of defensin mRNA accumulation, even at doses too low to effect morphologic changes; the initial (first 48 hours), gradual increase resulted from transcriptional activation and was enhanced by granulocyte colony-stimulating factor. In contrast, addition of hybrid polar compounds led to a transient, drug-specific downregulation within the same time period, apparently by means of selectively induced, biphasic degradation of transcripts. Subsequent increase in transcript levels was faster and more pronounced with hexamethylene bisacetamide, relative to dimethyl sulfoxide (DMSO). DMSO-promoted effects were strikingly different in serum-free medium or in the presence of the tyrosine kinase inhibitor, genistein. Under these conditions, and although differentiation was unaffected, early defensin mRNA downregulation was final. The effect did not occur with RA and expression of other myeloid-specific genes was also unchanged. Addition of selected cytokines caused a similar "dip," only at earlier times and uncoupled from differentiation. Tumor necrosis factor-alpha markedly induced defensin levels after 2 days in previously untreated HL-60 cells, but inhibited expression in RA-differentiated cells. These results begin to detail a complex regulation of defensin mRNA synthesis with both spatial and temporal control elements, and a unique modulation by chemical agents, cytokines, and serum-factors.

Topics: Acetamides; Biomarkers; Blood Proteins; Cell Differentiation; Cycloheximide; Cytokines; Dactinomycin; Defensins; Dimethyl Sulfoxide; Dimethylformamide; DNA, Complementary; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; Genistein; Granulocyte Colony-Stimulating Factor; Granulocytes; HL-60 Cells; Humans; Interferon-gamma; Isoflavones; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Lymphocytes; Lymphoma, Large B-Cell, Diffuse; Neoplasm Proteins; Recombinant Proteins; RNA Processing, Post-Transcriptional; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We have recently found that all-trans retinoic acid (ATRA) upregulates thrombomodulin (TM) and downregulates tissue factor (TF) expression in acute myelogenous leukemia (AML) M3 cells (NB4) and acute monoblastic leukemia cells (U937) (Koyama et al, Blood 84:3001, 1994). We have further investigated the effects of ATRA on leukemic cells freshly isolated from patients at diagnosis. Increase of TM antigen was documented in all AML cells: M0 (n = 1), M2 (n = 5), M3 (n = 3), M4 (n = 3), M5 (n = 3), and M6 (n = 1). Decrease of TF antigen was observed in 4 M2, 1 M4, and all M3 and M5 patients. However, no TM and TF antigens were detected in all chronic lymphocytic leukemia cells (n = 3) with or without ATRA treatment. Changes of TM and TF antigen levels were associated with those of TM and TF cofactor levels on the cell surface. A stereoisomer of RA, 9-cis RA, is a high-affinity ligand for the RA receptors (RARs) and the retinoid X receptors, although ATRA and another isomer, 13-cis RA, solely bind to RARs. We have also studied the effects of 9-cis RA and 13-cis RA on the expressions of TM and TF in NB4 and U937 cells. A relatively wide range of 9-cis RA concentrations (0.01 to 1 mumol/L) compared with ATRA was optimal for prolongation of normal plasma-based recalcification time (reduction of cell surface TF activity), decrease of TF antigen, and increase of TM antigen on the surface and in the lysates of NB4 and U937 cells. Western blot analysis under nonreducing conditions showed that both ATRA and 9-cis RA markedly induced the prominent band at 75 kD of TM and reduced the band at 45 kD of TF. Northern blot analysis has shown similar changes of mRNA levels, which indicates that RAs regulate TM and TF expression in leukemic cells at transcriptional levels. Anticoagulant effects of ATRA, ie, upregulation of TM expression and downregulation of TF expression, are applied not only to established cell lines of specific subtypes (M3 and M5) but also to more universal AML (most cases of M3 and M5 and a part of the other types of AML) cells freshly isolated from patients. 9-cis RA may be more effective than ATRA as an inducer of differentiation of AML M3 cells and as an anticoagulant agent for patients with certain types of AML as well.

Topics: Anticoagulants; Base Sequence; Cell Separation; Cysteine Endopeptidases; Flow Cytometry; Gene Expression Regulation, Leukemic; Humans; Isotretinoin; Leukemia; Leukemia, Monocytic, Acute; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Molecular Sequence Data; Neoplasm Proteins; Neoplastic Stem Cells; Receptors, Retinoic Acid; Thrombomodulin; Thromboplastin; Tretinoin; Tumor Cells, Cultured

Tretinoin tocoferil is an alpha-tocopherol ester of all-trans retinoic acid (RA) and safely used in the treatment of skin ulcer. Tretinoin tocoferil inhibited proliferation of human promyelocytic leukemia HL-60 cells and induced granulocytic differentiation of the cells, but less than RA. alpha-Tocopherol did not affect differentiation of HL-60 cells, but at high concentrations enhanced its nitroblue tetrazolium (NBT)-reducing activity and expression of surface antigen CD11b, which are markers of myelomonocytic differentiation induced by RA. Tretinoin tocoferil increased NBT reduction in HL-60 cells treated with RA. It also enhanced the differentiation of HL-60 cells induced by dimethyl sulfoxide, phorbol-12-myristate 13-acetate or 1alpha,25-dihydroxyvitamin D3 (VD3). In combination with a low concentration of VD3, it induced the NBT-reducing activity of human monoblastic U937 cells very effectively. Moreover, it enhanced the differentiation of human myelomonocytic ML-1, THP-1, P39/TSU, and P31/FUJ cells induced by VD3. In combination with VD3, it synergistically inhibited the proliferation of HL-60, U937, ML-1, THP-1, P39/TSU, and P31/FUJ cells and decreased the effective concentration of VD3 to a 10(-10) mol/L level. Because tretinoin tocoferil was reported to induce neither retinoid-related toxicity nor teratogenicity, the therapeutic advantage of the use of it in treatment of myelomonocytic leukemia is suggested.

Topics: Antineoplastic Agents; Calcitriol; Cell Differentiation; Cell Division; Dimethyl Sulfoxide; Drug Combinations; Drug Screening Assays, Antitumor; Drug Synergism; Growth Inhibitors; HL-60 Cells; Humans; Leukemia, Monocytic, Acute; Leukemia, Myelomonocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Neoplastic Stem Cells; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Vitamin E

All-trans-retinoic acid (ATRA) is the drug of choice in the treatment of acute promyelocytic leukemia (APL). ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes. However, the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood. The NB4 cell line, which is derived from the bone marrow of a patient with APL during relapse, can be used as a model system to study the growth and differentiation of APL cells. Because interferon (IFN) regulatory factors (IRF-1 and IRF-2) and other IFN-inducible gene products regulate cell growth, we analyzed the effects of ATRA on the expression of these genes. We show that ATRA directly activates IRF-1 gene expression, followed by activation of IRF-2 and 2'-5' oligoadenylate synthetase (OAS) gene expression with slower kinetics. In addition to NB4 cells, ATRA also activated IRF-1 gene expression in HL-60, U937, and THP-1 cells, which all respond to ATRA by growth inhibition. A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells. ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription (STAT) activation pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. The ATRA-induced expression of IRF-1, an activator of transcription and repressor of transformation, may be one of the molecular mechanisms of ATRA-induced growth inhibition, and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells.

Topics: 2',5'-Oligoadenylate Synthetase; Base Sequence; Cell Differentiation; DNA; DNA-Binding Proteins; Drug Synergism; Enzyme Induction; Gene Expression Regulation; HL-60 Cells; Humans; Interferon Regulatory Factor-1; Interferon Regulatory Factor-2; Interferon-gamma; Leukemia, Monocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Macrophages; Molecular Sequence Data; Monocytes; NF-kappa B; Phosphoproteins; Repressor Proteins; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.

Topics: Cell Differentiation; Cycloheximide; Endopeptidases; Humans; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Neoplasm Proteins; Oncogene Proteins, Fusion; Protein Synthesis Inhibitors; Ribosomes; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

We have previously demonstrated that human promyelocytic HL-60 cells express transforming growth factor-alpha (TGF-alpha) during granulocytic differentiation. The present experiments were carried out in order to determine whether cells differentiated towards monocytes/macrophages will analogously express the TGF-alpha proto-oncogene product. HL-60 cells were induced to differentiate with 1 microM 1,alpha 25-dihydroxycholecalciferol (vitamin D3), and the human monocytoid cell line, U-937, was induced with 1 microM retinoic acid (RA), 0.1 microM vitamin D3, or 0.16 microM phorbol-12-myristate-13-acetate (PMA), ie experimental protocols known to induce monocyte/macrophage differentiation in these cells. In HL-60 cells, lacking constitutive TGF-alpha mRNA, vitamin D3 caused expression of the TGF-alpha gene and protein as demonstrated by Northern blot analysis and enzyme-linked immunoabsorbant assay (ELISA). In U-937 cells, showing constitutive TGF-alpha expression, RA but not vitamin D3 or PMA, caused marked increase in TGF-alpha mRNA (approximately 5-fold) and protein (approximately 3-fold) levels. In both cell lines the increase in TGF-alpha mRNA was evident within 24 h and continued throughout the observation period. Thus, it is established that differentiation of human leukemia cells towards monocytes/macrophages may be accompanied by TGF-alpha gene and protein expression in vitro. This is in conformity with the observed ability of mature activated macrophages to produce TGF-alpha.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Calcitriol; Cell Differentiation; Gene Expression; Humans; In Vitro Techniques; Integrin alphaXbeta2; Leukemia, Promyelocytic, Acute; Lipopolysaccharide Receptors; Lymphoma, Large B-Cell, Diffuse; Monocytes; Proto-Oncogene Mas; RNA, Messenger; RNA, Neoplasm; Time Factors; Transforming Growth Factor alpha; Tretinoin

Because retinoids are known to modulate the growth and differentiation effects of tumor necrosis factor (TNF), we investigated the effect of all-trans-retinoic acid (RA) on the cell surface expression of TNF receptors in human histiocytic lymphoma U-937 cells. RA decreased the specific binding of 125I-labeled TNF to these cells in a dose- and time-dependent manner. The maximal decrease occurred when cells were treated with 1 mumol/L RA for 24 hours at 37 degrees C. Scatchard analysis of the binding indicated that the decrease by RA was caused by a decrease in receptor number and not by a decrease in affinity. The downmodulation of TNF receptors was also confirmed by covalent receptor-ligand cross-linking studies. Receptor-mediated internalization of the ligand was also found to be decreased on treatment of cells with RA. Northern blot analysis also indicated a decrease in the transcript of the receptor. By using antibodies specific to either the p60 or p80 form of the TNF receptor, we found that both receptors were downregulated by RA. RA treatment also decreased TNF receptors on acute monocytic leukemia cell line THP-1. Other analogues of RA, specifically 9-cis-RA, (E)-4-[2-(5,6,7,8- tetrahydro-2-naphthalenyl)-1-propenyl]-benzoic acid (TTNPB), and 3-methyl-TTNPB, which differ in their specificity towards different RA receptors, were also active in downregulating TNF receptors. 3-Methyl-TTNPB, which is more specific for the RXR form of the RA receptor, was found to be most potent. The downregulation of TNF receptors by RA correlated with the downmodulation of the antiproliferative effects of TNF against U-937 cells. Overall, our results indicate that RA downmodulates both the p60 and p80 form of the TNF receptor on cells of myeloid origin, which correlates with the cellular response.

Topics: Down-Regulation; Humans; Lymphoma, Large B-Cell, Diffuse; Receptor Aggregation; Receptors, Tumor Necrosis Factor; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Among the nuclear hormone receptors, the retinoid X receptors (RXRs) play a central role through their ability to heterodimerize with other members of this family of transcription factors, including retinoic acid (RA) and vitamin D (VD3) receptors. We have previously found that all-trans retinoic acid and 1 alpha, 25-dihydroxyvitamin D3 cooperate to induce monocytic differentiation of U937 human leukemic cells. Here the expression of RXR alpha protein in myelomonocytic cells was studied by immunodetection using polyclonal antibodies. RXR alpha was detected upon exposure of cells to VD3 and higher levels were found in cells treated by combinations of RA and VD3 under conditions where both agents synergized for inducing monocytic properties.

Topics: Animals; Blotting, Western; Calcitriol; Cell Differentiation; Cell Division; Cell Line; Cell Nucleus; Chlorocebus aethiops; DNA, Neoplasm; Drug Synergism; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Thymidine; Time Factors; Transcription Factors; Transfection; Tretinoin; Tritium; Tumor Cells, Cultured

The activation of human immunodeficiency virus type 1 (HIV-1) expression in latently infected cells by exogenous agents is believed to be important in the progression of AIDS. Most factors that are known to activate HIV-1 gene expression increase the binding of NF-kappa B or NF-kappa B-like transcription factors to the HIV-1 core enhancer region. In this report, we demonstrate that retinoic acid (RA) treatment of promonocytic U937 cells stimulates expression from the simian immunodeficiency virus (SIVmac) long terminal repeat (LTR). Furthermore, RA and phorbol 12-myristate 13-acetate (PMA) synergistically stimulated both SIVmac and HIV-1 LTRs to levels of expression comparable to that achieved by the viral transactivator Tat. The cis-acting elements required for a response to RA and PMA cotreatment are located between nucleotides -50 and +1 of SIVmac and between nucleotides -83 and +80 of HIV-1. Thus, the synergistic stimulation induced by RA and PMA is NF-kappa B independent. Analysis of deletion mutants of the SIVmac LTR demonstrates that RA and PMA stimulation cooperates with NF-kappa B and Sp1. An SIVmac LTR-reporter gene construct [pLTR(-50/+466)CAT] lacking NF-kappa B and Sp1 binding sites was not activated by Tat in untreated cells but was activated in cells that were cotreated with RA and PMA. Furthermore, gel retardation assays demonstrated that RA treatment causes a change in the pattern of a cellular factor(s) which binds to the -50 through +1 region of the SIVmac LTR. These data suggest that RA induces a PMA-activatable cellular factor that cooperates with NF-kappa B, Sp1, or Tat to stimulate LTR-directed transcription.

Topics: Base Sequence; Binding Sites; Cell Line; Drug Synergism; Gene Expression Regulation, Viral; Genes, fos; HIV-1; Humans; Lymphoma, Large B-Cell, Diffuse; Molecular Sequence Data; NF-kappa B; Oligodeoxyribonucleotides; Plasmids; Repetitive Sequences, Nucleic Acid; RNA, Messenger; RNA, Viral; Simian Immunodeficiency Virus; TATA Box; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Ferritin is an ubiquitous protein that has been shown to regulate cell differentiation in several experimental systems. In this study we have investigated the expression of ferritin genes encoding the heavy (H) and light (L) chains in t'B U937 cell line, induced to differentiate to macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA) or 1-beta-D-arabinofuranosylcytosine (Ara-C). An increase in the level of H ferritin mRNA was detected in U937 cells that had been incubated with Ara-C. Treatment of U937 cells with Actinomycin D suggested that the H ferritin mRNA increase was mediated by post-transcriptional mechanisms. The L ferritin mRNA level increased only following stimulation of U937 cells with RA. Immunophenotypic and cytochemical analyses showed that Ara-C was the strongest inducer of the macrophagic differentiation of U937 cells. These results suggest that the increase of H ferritin mRNA expression may represent a sensitive marker of myeloid cells differentiating along the monocyte-macrophage lineage.

Topics: Antigens, CD; Cell Differentiation; Cell Line; Cytarabine; Ferritins; Gene Expression; HLA-DR Antigens; Humans; Lymphoma, Large B-Cell, Diffuse; Macromolecular Substances; Macrophages; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The individual and combined effects of heat shock, all-trans retinoic acid and 1,25-dihydroxyvitamin D3 on inhibition of cell growth and initiation of differentiation were investigated on U937 human leukemia cells. Incubation of U937 cells at 43 degrees C for 1 h did not affect cell viability but induced a reduction of cell growth and the emergence of a differentiated phenotype, characterized by the acquisition of chemiluminescent responses to various oxidative burst inducers and by the capacity to produce IL-6 in response to bacterial lipopolysaccharide. Heat shock alone, therefore, appears to be an efficient inducer of cell differentiation. In addition, heat shock primed the cells to respond more efficiently to the action of retinoic acid and vitamin D, and amplified the phenotypic changes initiated by pretreatment of U937 cells with these agents.

Topics: Calcitriol; Cell Differentiation; Cell Division; Hot Temperature; Humans; Kinetics; Leukemia; Luminescent Measurements; Lymphoma, Large B-Cell, Diffuse; Monocytes; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Tumor Cells, Cultured; Zymosan

The human histiocytic lymphoma cell line U937 can be induced to differentiate along the monocyte/macrophage pathway by either phorbol myristate acetate (PMA) or by the combination of retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (VD). U937 cells treated with either PMA or RA/VD were able to phagocytose Salmonella typhimurium in the presence of non-immune human serum. However, only cells differentiated by RA/VD were capable of developing an oxidative metabolic burst in response to infection. Since the oxidative burst is considered to be a potent antimicrobial mechanism, we investigated its effect on S. typhimurium. The oxidative burst failed to affect either the viability or the multiplication of S. typhimurium suggesting that it plays only a minor role in the host defence against S. typhimurium.

Topics: Cell Differentiation; Cholecalciferol; Humans; Lymphoma, Large B-Cell, Diffuse; Phagocytosis; Respiratory Burst; Salmonella typhimurium; Tretinoin; Tumor Cells, Cultured

We examined the effects of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), and its synthetic analogue, 22-oxa-1,25-(OH)2D3, on differentiation of U937 cells by studying the cellular growth, surface marker expression and cytosolic free Ca2+ concentration ([Ca2+]i). RA inhibited cellular growth but did not induce expression of Mo2 (CD14), a monocyte/macrophage specific surface marker. To the contrary, 1,25-(OH)2D3 did not inhibit cellular growth, but increased CD 14-positive cells. Simultaneous addition of 1,25-(OH)2D3 and RA had no additive effect on cellular growth inhibition or CD14 expression. With regard to [Ca2+]i, however, 5 days' incubation with either of them increased the basal [Ca2+]i level and induced U937 cells to respond to formyl-methionyl-leucyl-phenylalanine (FMLP). When the cells were incubated with both 10(-6) M RA and 10(-8) M 1,25-(OH)2D3, basal [Ca2+]i was higher and FMLP caused a greater increase in [Ca2+]i than when only RA or 1,25-(OH)2D3 was added. These data suggest that RA and 1,25-(OH)2D3 induce monocytoid differentiation in U937 cells through different pathways and act synergistically in the differentiation process. The 22-oxa-1,25-(OH)2D3 induced CD14 expression, basal [Ca2+]i increase and [Ca2+]i response to FMLP, but did not cause cellular growth inhibition in U937 cells, and in these points, 22-oxa-1,25-(OH)2D3 exhibited no significantly different effects from 1,25-(OH)2D3. Thus, 22-oxa-1,25-(OH)2D3 has the same potent activity as 1,25-(OH)2D3 in inducing differentiation of U937 cells.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Calcitriol; Calcium; Cell Differentiation; Cytosol; Drug Synergism; Flow Cytometry; Humans; Lipopolysaccharide Receptors; Lymphoma, Large B-Cell, Diffuse; Tretinoin; Tumor Cells, Cultured

Activated monocytes play an important role as producers of IL-6 during inflammation and immune response. We show that during monocytic differentiation of U-937 cells, induced by phorbolester (PMA), IL-6 mRNA expression was transiently up-regulated and IL-6 protein was secreted into the medium. In contrast, differentiation induced by VitD3 or Retinoic acid (RA) did not lead to an increase in the IL-6 expression. Thus, IL-6 expression does not seem to be associated with monocyte differentiation per se. However, U-937 cells terminally differentiated by VitD3, rapidly responded to bacterial lipopolysaccharide (LPS) induced activation by IL-6 expression and secretion. In cells, differentiated by PMA, the IL-6 expression was super-induced after activation by interferon-gamma (IFN-gamma) and LPS. The capacity of U-937 cells to respond to LPS activation by IL-6 expression was associated with the expression of CD14 and some serum components(s) were a prerequisite for a successful LPS induction. The IL-6R expression was down-regulated during monocytic differentiation of U-937 cells. In the terminally differentiated U-937 cells, the expression of IL-6R could be induced after activation by IFN-gamma and to a lesser extent by LPS, suggesting a mechanism by which activation positively regulates the response to IL-6 in macrophages.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Calcitriol; Cell Differentiation; Cell Line; Gene Expression; Genes, myc; Humans; Interferon-gamma; Interleukin-6; Lipopolysaccharide Receptors; Lipopolysaccharides; Lymphoma, Large B-Cell, Diffuse; Monocytes; Receptors, Immunologic; Receptors, Interleukin-6; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Necrosis Factor-alpha

The uptake of colloidal gold particles by human monocytes was studied by electron microscopy, with special emphasis on changes in this uptake during the differentiation and maturation of these cells. The way in which leukemic cells of childhood acute non-lymphocytic leukemia (ANLL) can function in this gold uptake was also examined. In monocytes, microendocytosis was temperature-dependent; colloidal gold uptake increased as temperatures rose from 4 degrees C to 37 degrees C. It appeared that gold particles first adhered to the cell surface membrane, were then incorporated into the cytoplasmic vesicles, and then were transported into the granules. Original HL-60 cells and retinoic acid (RA)-treated HL-60 cells, which were differentiating and maturing along the granulocyte lineage, did not ingest colloidal gold particles, but 1,25(OH)2D3-treated HL-60 cells showed colloidal gold uptake during their differentiation and maturation along the monocyte lineage: 68.6% of the cells contained gold particles. Gold uptake was demonstrated in 27.3% of original U937 cells; the percentage increased to 70.3% when they were induced to mature by RA. In 15 specimens of childhood ANLL, none of the M1, M2 or M3 cells showed colloidal gold uptake, whereas 76-97% of M4 and M5 cells showed this uptake. These findings indicate that colloidal gold uptake is a marker of monocyte differentiation and maturation and can provide additional information for ANLL cytology.

Topics: Adolescent; Biomarkers; Calcitriol; Cell Differentiation; Child; Child, Preschool; Endocytosis; Female; Gold; Humans; Infant; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Leukemia, Promyelocytic, Acute; Leukocytes, Mononuclear; Lymphoma, Large B-Cell, Diffuse; Male; Microscopy, Electron; Neoplastic Stem Cells; Tretinoin; Tumor Cells, Cultured

We report the selection and characterization of a U-937 subline which is capable of long-term growth in serum-free medium and can be induced to differentiate. The subline (U-937-1SF) can be maintained in standard RPMI-1640 medium supplemented by antibiotics only. As compared to the serum-dependent U-937 parental cell line, U-937-1SF produced lower amounts of lysozyme and elastase and had a decreased surface expression of complement receptor 1 (CD35) and myeloid antigens CDw17 and CD38. Apart from these alterations, the U-937-1SF cells appear to be morphologically, cytogenetically and phenotypically similar to the parental U-937 clone-1 cells. The capacity of U-937 clone-1 cells to undergo phorbol myristic acid (PMA)-, vitamin D3 (VitD3)- and retinoic-acid (RA)-induced differentiation was retained in the U-937-1SF cells as evidenced by the induced growth arrest, development of a monocyte/macrophage morphology and increased expression of differentiation-associated antigens, e.g. CD11b, CD11c, CD14 and CD18. The growth-inhibitory response to cytokines involved in the activation and differentiation of monocytes, IFN-gamma, TNF-alpha, IL-1 beta, IL-6 and GM-CSF, was normal. Our results suggest that the U-937-1SF subline can be used as a serum-free model system for studies on various aspects of monocyte differentiation.

Topics: Animals; Antigens, CD; Antigens, Neoplasm; Cell Differentiation; Culture Media, Serum-Free; Cytokines; Gene Expression; Hematopoiesis; Humans; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Nude; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Vitamin D

The retinoids: all-transretinoic acid (tretinoin), 13-cis retinoic acid (isotretinoin) and the aromatic retinoids etretinate and acitretin have a preventive and therapeutic effect on chemically-induced tumours. Clinically, retinoids have shown variable effectiveness in therapy and/or prevention of oncological diseases of skin, head and neck, lung, bladder, vulva and bone marrow. With a few exceptions, monotherapy with retinoids has not been satisfactory. Similarly, monotherapy with interferon alpha has been used successfully only for some specific indications. Retinoids have a marked differentiation-inducing effect which may contribute to their therapeutic effect. Experiments were carried out in transformed cell lines to test the combination of retinoids with interferon alpha and other cytokines on differentiation. In HL-60 cells, an acute promyelocytic leukaemia cell line, induction of differentiation was determined by induction of an oxidative burst potential. Retinoids showed the following order of activity: tretinoin greater than isotretinoin greater than acitretin. Cytokines had no differentiation-inducing effect by themselves. However, the addition of the following cytokines to retinoids potentiated the retinoid-induced differentiation: IFN alpha, IFN beta, IFN gamma, TNF alpha, G-CSF, IL-1 alpha and IL-4. In experiments with HL-60 or other cell lines, the pattern of differentiation-induction was always dependent on the particular retinoid/cytokine combination. IFN alpha provoked a marked potentiation of retinoid-induced differentiation. The combination of the antiproliferative and differentiation-inducing effect of the retinoids together with the antiproliferative, immunostimulatory and differentiation-potentiating effects of IFN alpha suggests that this combination might be a particularly promising treatment for neoplastic diseases.

Topics: Cell Differentiation; Cytokines; Drug Synergism; Humans; Interferons; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Recombinant Proteins; Retinoids; Tretinoin; Tumor Cells, Cultured

The human-derived leukemia cell lines HL-60 and U937 are known to differentiate into more mature phagocytic cells in the presence of retinoic acid or 1,25-dihydroxyvitamin D3. We studied the effects of combinations of these two agents on cell growth and differentiation. These treatments were found to increase inhibition of cell proliferation. A dramatic enhancement of functional properties was observed in U937, but not HL-60 cells exposed to combinations of the two inducers. We investigated the conditions required to obtain the highest synergistic effects on the differentiation of U937 cells. These effects were found to be highly dose-dependent. We found that synergism required the simultaneous presence of both inducers and did not occur upon sequential exposure to each agent used separately.

Topics: Calcitriol; Cell Differentiation; Cell Division; Drug Synergism; Humans; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Monocytes; Tretinoin; Tumor Cells, Cultured

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.

Topics: Animals; Humans; Leishmania; Leishmania donovani; Leishmania tropica; Leukemia, Myeloid; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Inbred BALB C; Monocytes; Tretinoin; Tumor Cells, Cultured

Human monoblastoid cell line U-937 was induced by recombinant or leukocyte human interferon-alpha, retinoic acid, by their combination, or by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), to express some differentiation-associated markers and characteristics. Induced biochemical alterations were studied with the aid of two-dimensional electrophoretic analysis (isoelectrofocusing/SDS-PAGE) of 125I-lactoperoxidase radioiodinated cell surface proteins, 35S-methionine metabolically radiolabeled cell proteins and 32P orthophosphate radiolabeled cell phosphoproteins. Alteration of immunophenotype markers was studied by continuous flow immunocytofluorometry with a panel of monoclonal antibodies directed to leukocyte antigens, cell proliferation, DNA, RNA, and protein synthesis by radioactive precursor incorporation techniques. Furthermore, cytochemical markers, cell adherence and nitroblue tetrazolium (NBT) reduction were utilized to follow the induction of differentiation-associated characteristics. Differential alterations of some immunophenotype markers induced by diverse inducers were observed. Major induced alterations included down-regulation of CD4 (induced by TPA, and to a lesser extent by IFN-alpha), TPA-induced decrease of cell surface expression of transferrin receptor (unmodified by IFN-alpha) and IFN-alpha induced increase of antigen density (fluorescence intensity) of MHC class I antigen. Marked retinoic acid and interferon-alpha induced increase in membrane expression of antigen(s) detected by monoclonal antibodies BraC6 and BraC8, elicited with healthy donor's granulocytes, was also observed.

Topics: Antigens, Surface; CD4 Antigens; Cell Division; Flow Cytometry; Fluorescent Antibody Technique; Histocytochemistry; HLA-DR Antigens; Humans; Interferon Type I; Isoelectric Focusing; Lymphoma, Large B-Cell, Diffuse; Peptide Mapping; Phenotype; Phorbol Esters; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

We have studied the expression of vimentin in the human histiocytic lymphoma cell line U937, induced to differentiate along the monocyte/macrophage pathway. Normal monocytes possess a network of vimentin intermediate filaments (IFs) at all stages of maturation. The undifferentiated U937 leukemia cells contain very low amounts of vimentin, but express a conspicuous IF network when exposed to phorbol myristate acetate. In parallel, they acquire functional properties typical of cells of the monocyte lineage. These concomitant variations suggest that vimentin IFs could play a role in the process of differentiation. However, we observed that all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 confer monocyte-like properties upon U937 cells without inducing vimentin expression. We obtained increased phenotypic changes, yet in the absence of a vimentin network, by combining the effects of both inducers. These results show that vimentin expression is not crucial for the acquisition of some of the functions characteristic of the monocyte/macrophage lineage.

Topics: Calcitriol; Cell Transformation, Neoplastic; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Intermediate Filaments; Lymphoma, Large B-Cell, Diffuse; Phagocytosis; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Vimentin

The effect of retinoic acid (RA) on the level of interferon (IFN)-induced 2-5 oligoadenylate (2-5A) synthetase activity was examined in human histiocytic lymphoma U937 cells and WISH cells** in order to ascertain the role of this polymerase in interaction between IFNs and RA. Cultures containing both IFNs (1-100 U/ml) and RA (0.1-10 microM) consistently had higher levels of enzyme activity than corresponding cells treated with IFN alone and this was true for all three types of IFNs in both cell lines. The potentiating effect of RA was dose- and time-dependent and under optimal conditions, the induction of the synthetase was synergistic between IFN-beta (10-100 U/ml) and RA (0.1-10 microM). Furthermore, pretreatment (but not posttreatment) with RA followed by subsequent treatment with IFNs preferentially induced higher levels of enzyme activity in U937 cells but not in WISH cells. In addition, our results indicated that the modulating effect of RA on IFNs did not involve interaction at the receptor level and the level of enhancement of 2-5A synthetase activity was not in parallel with either cell-growth arrest or promotion of differentiation. Lastly, the present study raises the possibility that interactions between IFNs and RA, in either a synergistic or antagonistic manner, may be mediated through amplification of the 2-5A system.

Topics: 2',5'-Oligoadenylate Synthetase; Epithelium; Humans; Infant, Newborn; Interferons; Lymphoma, Large B-Cell, Diffuse; Tretinoin; Tumor Cells, Cultured

A monoblastic cell line U-937 (clone 4), was induced to differentiate along the monocytoid lineage by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), and vitamin D3 (VD3). By immunochemical and morphological criteria the cells were found to differentiate into macrophage-like cells in the presence of all three inducers. The expression of proteoglycans was investigated in control cultures and in cells differentiated in the presence of both TPA, RA, and VD3. The cells were labeled with [35S]sulfate and cell and medium-associated 35S-macromolecules were either solubilized in sodium dodecyl sulfate or subjected to proteolytic digestion. By use of chondroitinase ABC digestions and deaminative cleavage at pH 1.5 it was demonstrated that all cell cultures incorporated [35S]sulfate exclusively into chondroitin sulfate proteoglycan (CSPG). The expression of CSPG was found to decrease with differentiation to 60% in the presence of TPA, 67% in RA, and 40% in VD3 of control cultures on a cellular basis. The CSPG synthesized was consistently recovered from the medium fractions, whereas free glycosaminoglycan (GAG) chains were found in the cell fraction in all the cell cultures. GAG chains from both control and TPA-, RA-, and VD3-induced cultures were found to be exclusively of the chondroitin 4-sulfate type. However, the CSPGs from RA- and VD3-treated cells were found to differ in molecular size from those of control and TPA-induced cultures, as judged by Sepharose CL-6B gel chromatography. This difference in macromolecular properties following the induced differentiation of the monoblastic cells into macrophage-like cells was found to reside in expression of CSPGs (in the presence of RA and VD3) with smaller GAG chains. Control cells and TPA-induced cells synthesized CSPGs with GAG chains of approximate Mr of 30,000, contrasted by approximate Mr of 17,000 and 16,000 in RA- and VD3-induced cells, respectively. Accordingly, all three agents used in this study were found to induce differentiation of the U-937-4 cells and a decrease in the expression of CSPG, but only RA and VD3 were found to influence the structure of the proteoglycans synthesized.

Topics: Cell Differentiation; Cholecalciferol; Chondroitin; Chondroitin Sulfates; Glycosaminoglycans; Humans; Lymphoma, Large B-Cell, Diffuse; Monocytes; Proteoglycans; Tetradecanoylphorbol Acetate; Tretinoin

Differentiation-inducing agents have recently been applied clinically in various myeloproliferative diseases, based on their in vitro ability to induce differentiation in myeloid and monocytic cell lines. In this study we compared the abilities of two agents, dibutyryl cyclic AMP (DBcAMP) and retinoic acid (RA) to induce monocytic functions in the human histiocytic lymphoma cell line U937. Both agents produced similar induction of phagocytosis and reduction of nitroblue tetrazolium (NBT). Other monocytic functions, including accumulation of lysozyme, spontaneous and directed migration toward zymosan-activated serum (ZAS), the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and leukotriene B4 (LTB4) were induced by DBcAMP, while RA induced migration toward LTB4 only. Simultaneous treatment with both inducers proved synergistic only with respect to phagocytosis. NBT reduction and migration toward FMLP and LTB4 were unchanged, while migration toward ZAS and lysozyme accumulation were suppressed by the addition of RA. The results suggest that the inducer affects the expression of cellular functions and characteristics specific to a particular lineage. In addition, the data presented indicate that the U937 cell line may serve as an excellent model system for the study of the regulation and mechanisms of chemotactic receptor expression in monocytes.

Topics: Bucladesine; Cell Differentiation; Cell Line; Cell Movement; Chemotaxis, Leukocyte; Humans; L-Lactate Dehydrogenase; Lymphoma, Large B-Cell, Diffuse; Monocytes; Muramidase; Phagocytosis; Tretinoin

Phorbol esters stimulate differentiation of certain human leukemic cell lines. Although activation of protein kinase C may mediate certain effects of phorbol esters, controversy exists as to the role of protein kinase C activation in phorbol ester-induced differentiation. Retinoic acid modulates responses to phorbol esters in several cell types. Retinoic acid has also been found to alter protein kinase C-dependent phosphorylation in leukemic cells. We correlated the effects of retinoic acid on protein kinase C-dependent phosphorylation and differentiation stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester, in the human monoblastoid U937 cell line. At concentrations less than 1 nM, which were 100-fold less than those directly stimulating differentiation, retinoic acid potentiated TPA-induced differentiation of the U937 cell as assessed by enhanced adherence to plastic and acquisition of nonspecific esterase activity. TPA-stimulated decreases in cellular proliferation were not affected by retinoic acid treatment. Without altering the sensitivity to TPA, retinoic acid increased the maximal response to this agent. Retinoic acid enhanced TPA-stimulated phosphorylation of a Mr 48,000 substrate in intact 32P-labeled U937 cells and also increased the protein kinase C-dependent phosphorylation of a similar Mr 48,000 substrate and a Mr 80,000 substrate in cellular extracts. In cellular extracts the retinoic acid-induced enhancement of protein kinase C-dependent phosphorylation was predominantly localized to the cytosolic fraction. Increases in protein kinase C-dependent phosphorylation were evident within a 12-h exposure to 1 nM retinoic acid and were observed at retinoic concentrations of 0.01 to 1 nM. A retinoic acid-induced increase in the protein kinase C-dependent phosphorylation of an exogenous substrate, histone, was observed following diethylaminoethyl extraction of cytosol, but not a solubilized particulate fraction. The conditions of retinoic acid treatment increasing protein kinase C activity and enhancing protein kinase C-dependent phosphorylation of endogenous substrates were similar to those conditions potentiating phorbol ester-induced differentiation. Thus, the retinoic acid-induced amplification of phorbol ester signal transduction at the level of protein kinase C activation could mediate the effects of this vitamin on phorbol ester-induced differentiation.

Topics: Cell Adhesion; Cell Differentiation; Cell Line; Enzyme Activation; Humans; Lymphoma, Large B-Cell, Diffuse; Molecular Weight; Peptide Mapping; Phorbol Esters; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin; Vitamin A

Functional and specific histamine H2 receptors were characterized in human peripheral monocytes and in U-937 cells, before and after retinoic acid--induced differentiation into monocyte/macrophage-like cells. The relative potencies of histamine and the H1, H2 receptor agonists and antagonists studied are remarkably similar in U-937 cells and U-937 monocytes. There is no change in histamine concentration and activity of the enzymes forming and degrading histamine during monocytic-like differentiation. The results raise the possibility that histamine H2 receptors might be involved in pathophysiological regulations (proliferation/differentiation and biological function) of normal and leukemic monocytes.

Topics: Adult; Cell Differentiation; Cell Line; Cyclic AMP; Histamine; Humans; Leukemia, Myeloid, Acute; Lymphoma, Large B-Cell, Diffuse; Middle Aged; Monocytes; Receptors, Histamine; Receptors, Histamine H2; Tretinoin

The monoblast-like human histiocytic lymphoma cell line, U-937, is induced to differentiate into monocyte-like cells by incubation with 0.1 to 1.0 microM retinoic acid (RA). These induced cells are phagocytic, reduce nitroblue tetrazolium, and show an increased hexose monophosphate shunt activity, consistent with monocyte-like cells. Prostaglandin E, cholera toxin, or dibutyryl cyclic adenosine 3':5'-monophosphate, all inactive alone, increased markedly the extent of RA-induced differentiation of U-937. These data suggest that the intracellular level of cyclic adenosine 3':5'-monophosphate is of importance for expression of the RA-induced effect. Responsiveness to RA seems to be limited to those leukemic myeloid cells blocked at a relatively late stage of maturation, like the promyelocytic HL-60 and the monoblast-like U-937 cell lines.

Topics: Bucladesine; Cell Differentiation; Cell Division; Cell Line; Cyclic AMP; Dinoprostone; Humans; Kinetics; Lymphoma, Large B-Cell, Diffuse; Prostaglandins E; Tretinoin

The human cell lines HL-60 (acute promyelocytic leukemia) and U-937 (histiocytic monoblast-like lymphoma) differentiate to functionally mature cells by incubation with retinoic acid (RA). This differentiation is potentiated by agents known to increase intracellular cyclic adenosine 3':5'-monophosphate (cAMP) levels. The present study shows that these cells can be primed for differentiation by treatment for approximately one day with RA followed by exposure to a cAMP-inducing agent. The reverse sequence was ineffective. Thus, HL-60 could be primed by incubation for less than 20 hr with 10 nM RA to respond by differentiation to the addition of 10 nM prostaglandin E2 or 1 nM cholera toxin, whereas 10 nM RA alone was almost inactive. RA-primed HL-60 also responded with differentiation to a concentration of T-lymphocyte-derived differentiation-inducing factor which alone was inactive. U-937 primed by incubation for 24 hr with 100 nM RA responded to cAMP-inducing agents and differentiation-inducing factor, which alone were inactive on this cell line. Priming of these cell lines does not depend on the normal rate of protein synthesis, as it occurs even better in the presence of a concentration of cycloheximide that inhibits growth completely, suggesting that a decrease in synthesis of some protein(s) favors RA-induced differentiation. Cycloheximide alone produced some priming of HL-60 but not of U-937. HL-60, but not U-937, primed with RA responded to adenosine triphosphate and other nucleoside triphosphates, consistent with the notion that modulation of the RA effect may be mediated through protein kinase activity at the plasma membrane. The finding that myeloid cell lines, like HL-60 and U-937, blocked at a late stage of maturation can be primed by RA to respond to cAMP-inducing agents and differentiation-inducing factor may improve our understanding of the arrest in maturation typical of some forms of leukemia.

Topics: Cell Differentiation; Cell Line; Cholera Toxin; Cyclic AMP; Cycloheximide; Dinoprostone; Humans; Insulin; Kinetics; Lymphoma, Large B-Cell, Diffuse; Prostaglandins E; T-Lymphocytes; Tretinoin