tretinoin and hexamethylene-bisacetamide

Topics: Acetamides; Animals; Antineoplastic Agents; Basophils; Carcinogens; Cell Differentiation; Chloroquine; DNA Methylation; Humans; Leukemia; Mice; Tetradecanoylphorbol Acetate; Tretinoin

Recently, a new therapeutic approach to leukemia has been developed by induction of differentiation of leukemic cells. To improve this therapy, various factors involved in in vivo induction of differentiation of leukemia cells should be clarified. Clinical studies showed that inducers of differentiation of leukemia cells including retinoic acid, hexamethylene bisacetamide and some chemotherapeutic drugs induced remission in patients with acute myelogenous leukemia. Clonal analysis of the matured granulocytes revealed that the effectiveness of these drugs was due to their ability to induce differentiation of the leukemic cells rather than their cytocidal activity. On the other hand, various types of agents have been found to induce in vitro differentiation of leukemia line cells and fresh human leukemic cells. To evaluate therapeutic efficacy of these differentiation-inducing agents, pre-clinical experiments using animal model and tests for in vivo inducibility of differentiation of the leukemia cells are essential. We are developing an experimental system to monitor in vivo induction of differentiation of murine leukemia cells inoculated into syngeneic mice.

Topics: Acetamides; Animals; Antineoplastic Agents; Cell Differentiation; DNA, Neoplasm; Flow Cytometry; Leukemia, Experimental; Leukemia, Promyelocytic, Acute; Mice; Polymorphism, Restriction Fragment Length; Translocation, Genetic; Tretinoin

Differentiation therapy focuses on the development and use of specific agents designed to selectively engage the process of terminal differentiation, leading to the eventual elimination of tumorigenic cells and rebalance of normal cellular homeostasis. Extensive in vitro study of the molecular mechanism involved during drug-induced maturation has allowed the realization and application of a differentiation-based therapy to the clinic. Rationalization of this mode of therapy has included the combined use of differentiation agents with low-dose chemotherapy to lessen adverse cytotoxicity and to enhance the efficacy of differentiation agents, allowing some success in their application to conditions resistant to conventional therapy. This review discusses some biological principles that underlie the concept of a differentiation therapy and compares the in vitro and in vivo effectiveness of the two differentiation agents, in particular retinoic acid (RA) and hexamethylene bisacetamide (HMBA). It also evaluates the prospects for differentiation therapy as an effective strategy in the treatment and management of malignancy.

Topics: Acetamides; Animals; Antineoplastic Agents; Cell Differentiation; Drug Therapy, Combination; Gene Expression; Humans; Neoplasms; Receptors, Retinoic Acid; Tretinoin

Topics: Acetamides; Cell Differentiation; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Growth Substances; Humans; Neoplasms, Germ Cell and Embryonal; Oncogene Proteins; Proto-Oncogene Proteins; Transforming Growth Factor alpha; Tretinoin

A prominent phenotypic abnormality of human acute myelogenous leukemia cells is the inability of the cells to differentiate to functional mature cells; instead, the cells are blocked at an early stage of development and remain in the proliferative pool and rapidly accumulate. Investigation of the induction of myeloid leukemic cell differentiation has made recent advances with the development of several human myelogenous leukemia cell lines. The lines provide models to study the biology of myeloid differentiation and to identify inducers of differentiation of myeloid leukemic blood cells. This review critically examines the inducers of leukemic cell differentiation and their potential therapeutic importance.

Topics: 3-Deazauridine; Acetamides; Alkaloids; Animals; Calcitriol; Cell Differentiation; Cell Line; Cells, Cultured; Dimethyl Sulfoxide; Glycoproteins; Granulocytes; Growth Inhibitors; Humans; Hypoxanthines; Interferons; Interleukin-6; Leukemia Inhibitory Factor; Leukemia, Myeloid, Acute; Lymphokines; Lyngbya Toxins; Macrophages; Mice; Phorbol Esters; Piperidones; Polyethylene Glycols; Tretinoin

CD44 is highly expressed in human acute myeloid leukemia (AML) cells. Some experiments had shown that it was possible to reverse differentiation blockage in AML cells by CD44 ligation with specific antibodies, indicating that CD44 was closely related to the differentiation of leukemia cells. The differentiation of acute promyelocytic leukemia cell line HL-60 cells could be induced by all trans-retinoic acid (ATRA) and hexamethylene bisacetamide (HMBA), but so far the mechanism was not demonstrated clearly. In the present study, we investigated whether ATRA or HMBA induced the growth arrest of HL-60 cells by down-regulating the expression of CD44. The results indicated that the proliferation of HL-60 cells was obviously inhibited and the differentiation was induced by both ATRA and HMBA. The decreased expression of CD44 and cyclin E mRNA, and the increased expression of p27 and p21 at mRNA levels were observed. Furthermore, there was a negative correlation between the expression of CD44 and p27. It was concluded that ATRA and HMBA played a role in the differentiation induction of HL-60 cells, which was mediated by the down-regulation of CD44, accompanied by down-regulation of cyclin E, and up-regulation of p27 and p21 mRNA.

Topics: Acetamides; Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Proliferation; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; HL-60 Cells; Humans; Hyaluronan Receptors; Leukemia, Promyelocytic, Acute; RNA, Messenger; Tretinoin; Up-Regulation

The HEXIM1 protein, in association with 7SK snRNA, binds and inhibits the kinase activity of P-TEFb (CDK9/cyclin T). P-TEFb activity is crucial for efficient transcription elongation of viral and cellular genes. HEXIM1 was originally isolated as a protein up-regulated by hexamethylene bisacetamide (HMBA), a prototypical inducer of differentiation. To determine the causative role of HEXIM1 during cell differentiation we analyzed the biochemical and functional consequences of HEXIM1 protein levels in several in vitro differentiation systems. We found that HEXIM1 mRNA and protein levels are up-regulated during differentiation of murine erythroleukemia cells upon treatment with HMBA or DMSO. Stimulation of HEXIM1 is not restricted to hematopoietic cells, as induction of phenotypic differentiation of neuroblastoma cells by retinoic acid results in up-regulation of HEXIM1. Moreover, ectopic expression of HEXIM1 causes growth inhibition and promotes neuronal differentiation. These findings highlight a crucial role of HEXIM1 protein during cell differentiation.

Topics: Acetamides; Animals; Cell Differentiation; Cell Line, Tumor; Dimethyl Sulfoxide; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Erythroblastic, Acute; Mice; Neuroblastoma; Positive Transcriptional Elongation Factor B; RNA-Binding Proteins; Transcription Factors; Transcription, Genetic; Tretinoin; U937 Cells

Following the differentiation of cultured stem cells is often reliant on the expression of genes and proteins that provide information on the developmental status of the cell or culture system. There are few molecules, however, that show definitive expression exclusively in a specific cell type. Moreover, the reliance on a small number of molecules that are not entirely accurate biomarkers of particular tissues can lead to misinterpretation in the characterization of the direction of cell differentiation. Here we describe the use of technology that examines the mass spectrum of proteins expressed in cultured cells as a means to identify the developmental status of stem cells and their derivatives in vitro. This approach is rapid and reproducible and it examines the expression of several different biomarkers simultaneously, providing a profile of protein expression that more accurately corresponds to a particular type of cell differentiation.

Topics: Acetamides; Antigens, Surface; Antigens, Tumor-Associated, Carbohydrate; Biomarkers; Carcinoma, Embryonal; Cell Differentiation; Embryonal Carcinoma Stem Cells; Flow Cytometry; Gangliosides; Glycosphingolipids; Humans; Keratins; Neoplastic Stem Cells; Neurons; Peptides; Pluripotent Stem Cells; Proteoglycans; Proteome; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stage-Specific Embryonic Antigens; Tretinoin; Tubulin

In the course of examining the trafficking pathways of varicella-zoster virus (VZV) glycoproteins gE, gI, gH, and gB, we discovered that all four are synthesized within 4 to 6 h postinfection (hpi) in cultured cells. Thereafter, they travel via the trans-Golgi network to the outer cell membrane. When we carried out a similar analysis on VZV gC, we observed little gC biosynthesis in the first 72 hpi. Further examination disclosed that gC was present in the inocula of infected cells, but no new gC biosynthesis occurred during the first 24 to 48 h thereafter, during which time new synthesis of gE, gH, and major capsid protein was easily detectable. Similarly, delayed gC biosynthesis was confirmed with three different VZV strains and two different cell lines. Bioinformatics analyses disclosed the presence of PBX/HOX consensus binding domains in the promoter/enhancer regions of the genes for VZV gC and ORF4 protein (whose orthologs transactivate gC in other herpesviruses). Bioinformatics analysis also identified two HOXA9 activation regions on ORF4 protein. Treatment of infected cultures with chemicals known to induce the production of PBX/HOX transcription proteins, namely, hexamethylene bisacetamide (HMBA) and retinoic acid, led to more rapid gC biosynthesis. Immunoblotting demonstrated a fivefold increase in the HOXA9 protein after HMBA treatment. In summary, these results documented that gC was not produced during early VZV replication cycles, presumably related to a deficiency in the PBX/HOX transcription factors. Furthermore, these results explain the apparent spontaneous loss of VZV gC in some passaged viruses, as well as other anomalous gC results.

Topics: Acetamides; Cell Line; Giant Cells; Herpesvirus 3, Human; Humans; Microscopy, Electron, Scanning; Open Reading Frames; Promoter Regions, Genetic; Protein Binding; Protein Biosynthesis; Transcription Factors; Tretinoin; Up-Regulation; Viral Envelope Proteins

PML-RARalpha protein, the leukemogenic product of t(15,17) in acute promyelocytic leukemia, is cleaved into a truncated form termed deltaPML-RARalpha during all-trans retinoic acid (ATRA)-induced differentiation of NB4 cells. DeltaPML-RARalpha is not formed in ATRA differentiation resistant NB4 subclones. As(2)O(3) inhibits deltaPML-RARalpha formation and differentiation-induction when given in combination with ATRA. Treatment with hexamethylene bisacetamide (HMBA) combined with ATRA enhances ATRA-induced differentiation in ATRA-insensitive NB4-CI and arsenic-resistant NB4/As cells, and is associated with stabilization of PML-RARalpha protein and increased deltaPML-RARalpha formation. Unlike forced expression of PML-RARalpha, forced deltaPML-RARalpha expression based on an estimated deletion of the N-terminal PML portion does not repress RARE-tk-luc reporter activity mediated by endogenous retinoic acid receptors. The cleavage of PML-RARalpha is blocked by RARalpha antagonist Ro-41-5253 and cycloheximide and therefore requires a RARalpha transactivation-dependent pathway. Proteasome inhibitor MG-132 and caspase inhibitor Z-VAD-FMK do not block ATRA-induced PML-RARalpha cleavage and differentiation. These data suggest that (a) ATRA treatment induces PML-RARalpha cleavage by induction of unknown enzymes independent of proteasome- and caspase-mediated pathways; (b) deltaPML-RARalpha might function differently from both PML-RARalpha and RARalpha; (c) failure to cleave PML-RARalpha and form deltaPML-RARalpha after ATRA treatment may contribute to ATRA resistance in APL cells.

Topics: Acetamides; Antineoplastic Agents; Arsenic; Blotting, Western; Cell Differentiation; Cell Line; Cycloheximide; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Leukemia, Promyelocytic, Acute; Leupeptins; Luciferases; Multienzyme Complexes; Neoplasm Proteins; Oncogene Proteins, Fusion; Plasmids; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

The SOX genes comprise a family of transcriptional regulators implicated in the control of nervous system development. The developing brain is the major site of expression of many Sox genes. Sox2 and Sox3 genes are predominantly expressed in the immature, undifferentiated cells of the neural epithelium throughout the entire CNS. NTERA2 is a human embryonal carcinoma cell line that phenotypically represents undifferentiated, pluripotent embryonic stem cells. In the presence of retinoic acid, cells differentiate into mature neurons providing an in vitro model for studying human genes that promote and regulate neural differentiation. In this study it is shown for the first time that the retinoic acid-induced neuronal differentiation of NTERA2 cells is accompanied by down-regulation of SOX2 and up-regulation of SOX3 gene during early phases of induction. These data suggest that the effects of retinoic acid on neural differentiation of NTERA2 EC cells might be mediated by modulation of SOX2 and SOX3 gene expression.

Topics: Acetamides; Cell Differentiation; Cell Line; DNA-Binding Proteins; Gene Expression Regulation; High Mobility Group Proteins; HMGB Proteins; Humans; Neurons; Nuclear Proteins; SOXB1 Transcription Factors; Stem Cells; Transcription Factors; Tretinoin

All-trans retinoic acid (tRA)-induced differentiation in NB4 cells, a cell line derived from an acute promyelocytic leukemia patient with t(15;17) translocation, is markedly facilitated by sodium butyrate (NaB), a histone deacetylase inhibitor (HDACI), or by hexamethylene bisacetamide (HMBA), a non-HDACI tRA-differentiation inducer, as determined by nitroblue tetrazolium reduction. The tRA-induced expression of RIG-G, Bfl-1/A1, and p21(waf1) and, to a lesser extent, of CCAAT/enhancer binding protein-epsilon (C/EBPepsilon) are also enhanced by such combined treatments. Both responses are associated with a facilitated diminution of the leukemogenic PML-RARalpha protein and retained DeltaPML-RARalpha, a cleavage product. Treatment with tRA in tRA differentiation-resistant NB4 subclones R4 and MR-2 does not result in PML-RARalpha diminution and the tested gene expressions. Moreover, the addition of HMBA or NaB with tRA results in only minimal increase of differentiation in the tRA differentiation-resistant subclones. The increases in acetylated histone H3 (AcH3) and AcH4 in NaB-treated NB4, R4, and MR-2 cells are similar and do not correlate with the extent of differentiation induction when NaB and HMBA are given in combination with tRA. Arsenic trioxide (As2O3) treatment results in the total degradation of PML-RARalpha without increasing AcH3 or AcH4 or inducing differentiation in R4 cells. As2O3 in combination with tRA induces gene (Bfl-1/A1 and C/EBPepsilon) expression and partial differentiation. Both NaB and HMBA addition to As2O3-plus-tRA-treated R4 cells further enhances differentiation. These results suggest that elimination of the dominant negative PML-RARalpha protein is required prior to inhibition of histone deacetylase to fully overcome tRA-differentiation resistance in APL cells.

Topics: Acetamides; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Butyrates; Cell Differentiation; Drug Interactions; Drug Resistance, Neoplasm; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Oxides; Tretinoin; Tumor Cells, Cultured

We have analysed the surface antigen phenotype of a human embryonic stem (hES) cell line (H7) and the changes that occur upon differentiation induced by retinoic acid, hexamethylene bisacetamide and dimethylsulphoxide. The undifferentiated stem cells expressed Stage Specific Embryonic Antigen-3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-8 but not SSEA1. In these characteristics they closely resemble human embryonal carcinoma (EC) cells derived from testicular teratocarcinomas, and are distinct from murine EC and ES cells. The undifferentiated cells also expressed the liver/bone/kidney isozyme of alkaline phosphatase detected by antibody TRA-2-54, the class 1 major histocompatability antigens, HLA-ABC, and the human Thy1 antigen. Differentiation of hES cells was induced by retinoic acid, HMBA and DMSO with the appearance of various cell types including neurons and muscle cells. The surface antigens characteristically expressed by hES cells were down-regulated following induction of differentiation and other antigens appeared, notably several ganglioside glycolipids detected by antibodies VIN-IS-56 (GD3 and GD2), VIN-2PB-22 (GD2), A2B5 (GT3) and ME311 (9-O-acetyl-GD3). Whereas the expression of HLA was slightly down-regulated upon differentiation, its expression was strongly induced by interferon-y in both the undifferentiated and the differentiated cells, although the induction in the differentiated cultures was considerably stronger than in the stem cells. In all of these features the human ES cells, and their pattern of differentiation, resembled the pluripotent human EC cell line NTERA-2 although clearly the range of cells generated by the hES cells was considerably greater.

Topics: Acetamides; Animals; Antigens, Surface; Cell Culture Techniques; Cell Differentiation; Coculture Techniques; Dimethyl Sulfoxide; Embryo, Mammalian; Humans; Interferon-gamma; Mice; Stem Cells; Tretinoin

TUMOR necrosis factor-alpha (TNF-alpha) plays an important role in the pathogenesis of rheumatoid arthritis. The present study was to evaluate the effects of lipopolysaccharide (LPS), phytomitogens and cytodifferentiation agents on cytotoxicity of TNF-alpha secreted by adherent human mononuclear cells (AMC). TNF-alpha cytotoxicity in LPS-treated, phytomitogen-treated, and cytodifferentiation agent-treated AMC supernatants were analyzed by the L929 bioassay system. Our results showed that LPS could induce homogeneous TNF-alpha production by AMC whereas, in addition to TNF-alpha, phytomitogens could also induce other TNF-like factors. Neither methotrexate, retinoic acid nor sodium butyrate can inhibit TNF-alpha cytotoxicity, while hexamethylene bisacetamide could not only inhibit TNF-alpha cytotoxicity but also TNF-alpha inducing ability of LPS to AMC.

Topics: Acetamides; Animals; Antineoplastic Agents; Biological Assay; Butyrates; Cell Adhesion; Cell Differentiation; Cell Line; Cytotoxicity Tests, Immunologic; Dose-Response Relationship, Immunologic; Humans; Immunosuppressive Agents; Lectins; Leukocytes, Mononuclear; Lipopolysaccharides; Methotrexate; Mice; Recombinant Proteins; Tretinoin; Tumor Necrosis Factor-alpha

Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 showed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation pattern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90alpha or -beta isoform via the gene transfer method. On the other hand, the overexpression of HSP90beta alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiologically critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.

Topics: Acetamides; Blotting, Western; Cell Differentiation; Cell Line; Down-Regulation; Embryonal Carcinoma Stem Cells; Flow Cytometry; Hot Temperature; HSP90 Heat-Shock Proteins; Humans; Neoplastic Stem Cells; Tretinoin

Cytodifferentiation therapy by all-trans-retinoic acid (RA) for acute promyelocytic leukemia patients is encouraging in spite of several limitations preventing better clinical outcomes. Most patients in complete remission induced by RA experience relapse and resist further treatment with RA. This resistance primarily is due to a systemic self-induced catabolism of RA, which interferes with the maintenance of effective plasma levels of RA. In this report we explored the possibility that treatment with combinations of RA and other differentiation agents may induce differentiation at lower RA concentrations, which in turn may produce diminished levels of resistance. We found that although n-butyric acid (BA), tributyrin (TB) (a prodrug of BA), or hexamethylene bisacetamide (HMBA) were inactive as sole agents they potentiated RA-induced differentiation of human acute promyelocytic NB4 cells. A measure of the effectiveness of these combinations was that the concentrations of RA in combination with BA and HMBA inducing half-maximal differentiation were 20- to 40-fold lower than those needed with RA alone. Furthermore, the concentrations of BA and HMBA in these combinations were at achievable plasma levels. Therefore, these combinations may have clinical utility for treatment of a variety of malignancies that are sensitive to RA alone.

Topics: Acetamides; Antineoplastic Agents; Butyrates; Butyric Acid; Cell Differentiation; Drug Synergism; Humans; Leukemia, Promyelocytic, Acute; Tretinoin; Triglycerides; Tumor Cells, Cultured

The Wnt gene family encodes a series of conserved glycoproteins that regulate pattern formation during embryogenesis, in a variety of tissues including the nervous system. As with other genes that control embryonic cell differentiation, members of the Wnt family have also been implicated in tumourigenesis. To search for Wnt genes involved in human teratocarcinomas, with a possible role in human embryogenesis, we used RT-PCR primed with degenerate oligonucleotides to analyse mRNA from differentiating cultures of the pluripotent human embryonal carcinoma (EC) cell line NTERA-2. NTERA-2 EC cells differentiate into neurons and other cell types when induced with retinoic acid. Wnt gene expression was not detected in the undifferentiated EC cells, but Wnt-related PCR fragments were amplified from differentiating cultures, 4-14 days after induction with retinoic acid. The RT-PCR products were composed primarily of DNA fragments corresponding to the recently identified human Wnt-13 gene. No other Wnt-related genes were identified. Northern analysis confirmed induction of Wnt-13 as a 2.4 kb mRNA during the early phases of retinoic acid-induced differentiation, and during differentiation along a non-neural pathway induced by hexamethylene bisacetamide (HMBA), but not in the terminally differentiated neurons. Wnt-13 remained expressed in non-neural differentiated NTERA-2 cells, even several weeks after the induction of differentiation. The time course of induction, its induction by HMBA, and its persistence in differentiated cells indicate that Wnt-13 expression is not dependent upon direct activation by retinoic acid. Wnt-13 was not detected, or only detected at low levels, in other human EC cells. However, it was found to be expressed at a high level in one malignant teratoma cell line, 577MF, that does not exhibit an EC phenotype although it was derived from a testicular teratocarcinoma. At least two members of the human frizzled gene family, thought to encode receptors for Wnt proteins, were also expressed in the NTERA-2 cells, suggesting the presence of a mechanism by which endogenously expressed Wnt-13 could modulate the histogenesis of teratocarcinomas by mediating interactions between sub-populations of differentiating EC cells. We note that Wnt-13 maps to chromosome 1p13, a region reported to be subject to relatively frequent loss of heterozygosity in germ cell tumours. Further analysis indicated that 465 bp of the published Wnt-13 sequence, within the pred

Topics: Acetamides; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Drosophila Proteins; Frizzled Receptors; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Molecular Sequence Data; Neoplasms, Germ Cell and Embryonal; Neurons; Receptors, G-Protein-Coupled; Sequence Homology, Nucleic Acid; Teratocarcinoma; Testicular Neoplasms; Tretinoin; Wnt Proteins

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

Phorbol 12-myristate 13-acetate (PMA), N,N'-hexamethylenebisacetamide (HMBA) and retinoic acid induce cell differentiation in U-937 promonocytic cells. This report examines the effects of these agents on DNA topoisomerase I activity. A decrease in enzyme activity could be detected as early as 30 min after treatment with all three differentiating compounds and lasted at least 48 h. No alteration in the levels of DNA topoisomerase I transcript or protein was observed during these treatments. The results might be explained by post-translational events that render DNA topoisomerase type I less active.

Topics: Acetamides; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; DNA Topoisomerases, Type I; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia, Myeloid; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The hyaluronan-rich matrix that surrounds many tumours and facilitates tumour cell growth and invasion is thought to be predominantly synthesized by normal stromal cells stimulated by tumour cell-derived factors. This study examines the possibility that the production of tumour cell-derived factors that stimulate fibroblast glycosaminoglycan (GAG) synthesis may be blocked by exposure to differentiation-inducing agents such as retinoic acid. We have demonstrated that Hs294T, C8161 and A375 human melanoma cell lines release factors into their medium that stimulate normal fibroblast GAG synthesis. Exposure of these melanoma cells to retinoic acid failed to mediate any significant reduction in growth over a 7-day period. Retinoic acid failed to block the tumour cell production of GAG-stimulating activities and even enhanced the activities produced by the C8161 cell line, particularly at low retinoic acid concentrations (48% stimulation at 10(-9) M retinoic acid; P < 0.02). Addition of retinoic acid directly to fibroblast cultures exposed to fibroblast-conditioned medium resulted in an inhibition of GAG synthesis with a 33% inhibition observed at 10(-5) M. Addition of retinoic acid to fibroblast cultures exposed to the tumour cell-conditioned medium failed to inhibit the stimulation of GAG synthesis. Other differentiation-inducing agents, such as hexamethylene-bis-acetamide and butyrate, also failed to block the production of tumour cell-derived GAG-stimulating activities. These results demonstrate that retinoic acid and other differentiation-inducing agents fail to inhibit melanoma cell production of fibroblast GAG synthesis-stimulating factors or their action upon fibroblasts.

Topics: Acetamides; Adult; Antineoplastic Agents; Butyrates; Butyric Acid; Cell Count; Cell Differentiation; Cells, Cultured; Culture Media, Conditioned; Female; Fibroblasts; Glycosaminoglycans; Humans; Male; Melanoma; Middle Aged; Skin Neoplasms; Tretinoin

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

The effects of induced differentiation on telomerase activity were examined in human acute promyelocytic leukemic (NB4) and human embryonal carcinoma (NTERA-2) cells exposed to all-trans-retinoic acid or hexamethylene bisacetamide. Retinoic acid treatment of NB4 and NTERA-2 cells, and hexamethylene bisacetamide treatment of NTERA-2 cells caused a decline in telomerase activity in differentiation-sensitive but not in resistant clones of these cell lines. Changes in telomerase activity as measured by the PCR-based telomeric repeat amplification protocol assay were noted by 24-72 h of exposure to the inducer, suggesting that its regulation may precede terminal differentiation. The degree of telomerase activity decline was greater in NB4 cells than in NTERA-2 cells, probably reflecting in part a more mature state of NB4 cells after 5 days of exposure to the inducer. Mixing of protein extracts from treated and untreated cells did not suggest the presence of diffusible telomerase inhibitors. Expression of the RNA component of telomerase was also examined in NB4 cells, and its decline correlated with the reduced telomerase activity measured by the telomeric repeat amplification protocol assay during induced differentiation of these tumor cells. Taken together, these findings indicate that telomerase is a regulated enzyme system during induced human tumor cell differentiation, showing an inverse relationship between the degree of differentiation and telomerase activity. These models will be be useful to study the regulation and role of telomerase during induced differentiation of human tumor cells.

Topics: Acetamides; Cell Differentiation; Embryonal Carcinoma Stem Cells; Humans; Leukemia, Promyelocytic, Acute; Neoplastic Stem Cells; Telomerase; Tretinoin; Tumor Cells, Cultured

The retinoids exert potent growth and differentiation effects on normal and neoplastic cells through two families of nuclear receptors. These are the retinoic acid receptors (RAR alpha, RAR beta, RAR gamma) and the retinoid-X receptors (RXR alpha, RXR beta, RXR gamma). All-trans retinoic acid (RA) induces terminal neuronal differentiation and represses tumorigenicity of the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1). Hexamethylene bisacetamide (HMBA) induces a phenotype distinct from RA-treated NT2/D1 cells. This study reports the derivation and characterization of RA- and HMBA-resistant NT2/D1 clones. Nine RA-resistant (NT2/D1-R1 through NT2/D1-R9) and one HMBA-resistant (NT2/D1-H1) clones were derived after mutagen treatment of NT2/D1 cells and selection in RA or HMBA. NT2/D1-R cells were cross-resistant to 9-cis retinoic acid (9-cis RA), a ligand activating the RAR and RXR pathways, but retained maturation response to HMBA. A representative RA-resistant clone, NT2/D1-R1, overcame the antitumorigenic actions of RA as assessed in athymic mice. NT2/D1-H1 cells were dually resistant to RA and 9-cis RA. All these retinoid resistant cells exhibit deregulated expression of RAR gamma but not RAR alpha or RAR beta. Southern analysis using RAR gamma probes shows no apparent structural differences in genomic DNA between NT2/D1 cells and the RA-resistant subclones. Pulsed-field gel electrophoresis (PFGE) with RAR gamma probes demonstrated an Mlu-I restriction fragment length polymorphism, but no other structural abnormalities in these cells or a panel of germ cell tumor (GCT) cell lines. Full-length RAR gamma 1 coding region cDNAs were cloned from NT2/D1 and NT2/D1-R1 cells and these sequences were identical, suggesting RA resistance in these cells is due to altered regulation of RAR gamma. These differentiation-resistant cells are useful to study RAR gamma target genes or mechanisms engaged by these differentiation inducing agents in human embryonal carcinomas.

Topics: Acetamides; Animals; Antineoplastic Agents; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Clone Cells; Drug Resistance, Neoplasm; Germinoma; Humans; Immunophenotyping; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

The alterations in the mucociliary unit in the course of chronic inflammation of the upper respiratory tract correspond to morphologic anomalies of the respiratory epithelium and induce cuboidal and squamous metaplasia. While the squamous pattern is most probably irreversible, it is still not clear whether it is possible to restore ciliogenesis in cuboidal metaplasia. In the present study, the action of different inductors of differentiation was evaluated in vitro in isolated cells and explants from human nasal metaplastic epithelium. Polar/apolar compounds induced secretory activity, whereas retinoic acid was able to induce ciliogenesis in some cases. Therefore, the cuboidal metaplastic condition appears to be reversible, and two distinct pathways of differentiation, secretory and ciliogenetic, are identifiable.

Topics: Acetamides; Cell Differentiation; Cells, Cultured; Chronic Disease; Cilia; Cryoprotective Agents; Dimethyl Sulfoxide; Epithelium; Hematinics; Humans; Keratolytic Agents; Metaplasia; Microscopy, Electron; Microscopy, Electron, Scanning; Nasal Cavity; Nasopharyngitis; Tretinoin

We observed the enhancing effect of dimethylsulfoxide (DMSO) on infection of human T cells with human immunodeficiency virus type 1 (HIV-1). Similar enhancing effects were also found in related polar chemicals such as dimethylformamide, hexamethylenebisacetamide, sodium butyrate and retinoic acid. In acute infection of the human MT-4 T cell line, DMSO at a concentration of 1% reduced the amounts of HIV-1 required to establish similar infection by one log. Furthermore, infection of peripheral blood lymphocytes with HIV-1 was also augmented several times by DMSO. HIV-1 production from persistently infected human T cell lines, but not monocytic cell lines, was enhanced by DMSO and related polar chemicals. DMSO enhanced transcription of HIV-1 RNA in persistently infected T cell lines, and the enhancing effect of DMSO on HIV-1 production was inhibited by staurosporine, a protein kinase inhibitor. These findings suggested that DMSO enhanced HIV-1 infection of T cells mainly at the step of transcription of viral RNA.

Topics: Acetamides; Butyrates; Butyric Acid; Cell Line; Dimethyl Sulfoxide; Dimethylformamide; HIV-1; Humans; T-Lymphocytes; Tretinoin; Virus Replication

Osteogenic protein-1 (OP-1) is a member of the transforming growth factor-beta super family closely related to the bone morphogenetic proteins and also known as bone morphogenetic protein-7. Other members of this family of growth factors influence cell differentiation as well as cell growth in a number of systems. The Drosophila homolog encoded by the decapentaplegic locus is involved in dorsal-ventral pattern formation during embryogenesis, whereas the expression of several bone morphogenetic proteins including OP-1 is developmentally regulated in mammalian embryos.. The effect of recombinant human OP-1 on the proliferation and differentiation of an established pluripotent human embryonal carcinoma (EC) cell line, NTERA2, and three nullipotent human EC cell lines, 2102Ep, 833KE and TERA-1, was investigated. These cells were grown under reduced serum conditions, and differentiation was monitored by morphology and expression of marker antigens.. OP-1 inhibited proliferation of NTERA2 and induced their differentiation, marked by changes in cellular morphology, the loss of EC cell antigens (SSEA3, SSEA4, the liver isozyme of alkaline phosphatase), and the appearance of new antigens, notably SSEA1 and class 1 major histocompatibility complex antigens. These changes were irreversible and did not involve significant cell degeneration or cell death. The OP-1-induced differentiation of NTERA2 appeared distinct from that induced by either retinoic acid or hexamethylene bisacetamide. Nevertheless, OP-1 did induce the homeobox gene, HOXA1. By contrast, OP-1 elicited only a limited and partial response from the nullipotent EC cell lines.. Our results suggest that pluripotent human EC cells differentiate in response to OP-1 and that this factor can modulate the differentiation induced by retinoic acid. Like other members of the transforming growth factor-beta super family, OP-1 might play an inductive role in the early embryo. The results also suggest a possible therapeutic value for OP-1 in the treatment of some germ cell tumors.

Topics: Acetamides; Antineoplastic Agents; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Cell Differentiation; Cell Division; Gene Expression; Genes, Homeobox; Humans; Proteins; Recombinant Proteins; RNA; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

Topics: Acetamides; Butyrates; Cell Differentiation; Cholecalciferol; Cytarabine; Drug Synergism; Humans; In Vitro Techniques; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured

The multipotent human teratocarcinoma (TC) cell NTera-2 clone D1 (abbreviated NT2/D1) differentiates into a neuronal lineage after retinoic acid (RA) treatment and a distinct phenotype after hexamethylene bisacetamide (HMBA) treatment. We previously reported that RA treatment of NT2/D1 cells reduces cellular cloning efficiency and nude mouse tumorigenicity. This accompanied a loss of mRNA expression of transforming growth factor-alpha (TGF-alpha) and the fibroblast growth factor kFGF, also known as hst-1 (abbreviated hst-1/kFGF). This study extends prior work by reporting that the distinct phenotype induced by HMBA also decreases cloning efficiency, tumorigenicity, and TGF-alpha and hst-1/kFGF mRNA expression in NT2/D1 cells. These RNA findings were confirmed by measurements of growth factor protein in the conditioned media of inducer-treated and untreated NT2/D1 cells. In two established human TC lines refractory to the actions of RA, N2102ep and Tera-1, RA fails to decrease expression of either growth factor despite induction of its nuclear receptor, RAR-beta. However, HMBA induces morphologic maturation and down-regulation of these growth factors in N2102ep cells. This indicates that the loss of TGF-alpha and hst-1/kFGF expression serves as a new marker of differentiation in human TCs. To explore the effects of these growth factors on growth and differentiation of NT2/D1 cells, TGF-alpha or hst-1/kFGF protein was added following inducer treatment or no treatment. Neither growth factor blocked immunophenotypic differentiation, but both promoted the growth of uninduced NT2/D1 cells in cloning assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetamides; Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Line; Clone Cells; Drug Resistance; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Expression; Humans; Proto-Oncogene Proteins; Teratocarcinoma; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

The undifferentiated Y-79 retinoblastoma cell line can be induced by specific agents to express characteristics of mature retinal cells. In the present study, attached Y-79 cell cultures were treated with hexamethylene bis-acetamide (HMBA) and other differentiating agents and examined for "neuronal" and other properties. Immunocytochemical staining was performed with antibodies against neuron- and retina-specific antigens, [synaptophysin, interphotoreceptor retinoid-binding protein (IRBP), neural cell adhesion molecule (N-CAM), and rod- and cone-specific transducin (TR alpha and TC alpha)] and microtubule-associated protein (MAP-1) and tubulin. Enhanced expression of tubulin was observed with cAMP treatment in FBS media. Expression of N-CAM was observed in all groups. Morphological differentiation was pronounced with HMBA and butyrate treatment, with HMBA inducing increased tubulin expression after 2 weeks of treatment. Expression of TR alpha was minimal under all culture conditions, whereas TC alpha was ubiquitously expressed. This supports the concept that Y-79 retinoblastoma is predominantly of cone neuronal origin and that, surprisingly, immunocytochemical differentiation is not correlated with the marked morphological changes induced by the major differentiating agents used.

Topics: Acetamides; Antineoplastic Agents; Butyrates; Cell Adhesion Molecules, Neuronal; Cell Transformation, Neoplastic; Cyclic AMP; Eye Neoplasms; Eye Proteins; Humans; Immunohistochemistry; Microtubule-Associated Proteins; Regression Analysis; Retinoblastoma; Retinol-Binding Proteins; Synaptophysin; Transducin; Tretinoin; Tubulin; Tumor Cells, Cultured

SV40 large T oncoprotein-transformed murine mesenchymal 3T3 T stem cells (CSV3 cells) can be induced to growth arrest and then differentiate into adipocytes. When differentiation occurs, SV40 T oncoprotein expression is repressed (Estervig et al., J Virol 63:2718, 1989). To determine if repression of T oncoprotein expression can also be induced pharmacologically, the effect of a variety of agents that have been reported to effect differentiation in various cell types but not in 3T3 T or CSV3 cells was tested. This rationale suggests that if any of these agents repress T oncoprotein expression in CSV3 cells, then the results would establish that repression of T oncoprotein expression can be mediated by mechanisms independent of overt differentiation. The results show that dimethylsulfoxide (DMSO) is the only agent tested that represses T oncoprotein expression in CSV3 cells. Repression occurs in a dosage-dependent manner within 24-96 hours after exposure to DMSO. The effect of DMSO on T oncoprotein expression is mediated by posttranslational mechanisms that decrease the stability of the T oncoprotein. DMSO-induced repression of T oncoprotein expression is also associated with reversion of the transformed phenotype in CSV3 cells as demonstrated by the loss of responsiveness to a specific transformation-associated mitogen. These data support the conclusion that the pharmacological repression of T oncoprotein expression represents a form of cancer suppressor activity that can be mediated by a distinct molecular mechanism.

Topics: Acetamides; Animals; Antigens, Polyomavirus Transforming; Azacitidine; Blotting, Northern; Blotting, Western; Butyrates; Butyric Acid; Cell Line, Transformed; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Insulin; Interferons; Mice; Phenotype; Stem Cells; T-Lymphocytes; Tretinoin

We have investigated the effect of all-trans Retinoic acid, and of substances (Hemine and Hexamethylene bisacetamide) which interfere with "in vitro" differentiation of mesenchyme derived cell lineages on the expression of specific markers of hyperthrophy in "in vitro" differentiating chick embryo chondrocytes. (Castagnola P., et al., 1986). Continuous treatment of chondrogenic cells in conditions allowing differentiation "in vitro" with Retinoic acid resulted in persistence of type I collagen synthesis and in lack of type X collagen and Ch 21 protein expression. Hemin treated cells secreted a reduced amount of type X collagen. HMBA treatment inhibited type X collagen expression and caused reduction of the ratio between type II collagen and Ch 21 synthesized. The data indicate an independent regulation of these markers during chondrocyte differentiation.

Topics: Acetamides; Animals; Avian Proteins; Carrier Proteins; Cartilage; Cell Differentiation; Chick Embryo; Collagen; Fatty Acid-Binding Proteins; Gene Expression Regulation; Hemin; Lipocalins; Tretinoin

All-trans-retinoic acid, hexamethylene bisacetamide and 5-azacytidine are inducers of granulocytic differentiation of HL-60 human myeloid leukaemic cells, which eventually leads to inhibition of cell proliferation. The effect of graded concentrations of all-trans-retinoic acid (RA) (1 nM-1 microM), hexamethylene bisacetamide (HMBA) (0.5-4 mM) and/or 5-azacytidine (5azaC) (1 nM-1 mM), alone and in combination with each other on colony formation and growth of HL-60 cells was studied in agar capillary clonogenic micro assays in order to identify new potential therapeutic regimens for elderly patients with acute myeloid leukaemia. ED90 concentrations, inducing 90% inhibition of colony formation for RA, HMBA and 5azaC, were 128 nM, 2.7 mM and 40 microM, respectively. The drug interactions between these differentiating agents were analysed by Berenbaum's general algebraic solution. The combinations: RA + HMBA, 5azaC + HMBA and RA + 5azaC were significantly synergistic in inhibiting HL-60 colony formation. Their interaction indices were 0.62, 0.83, and 0.97, respectively, at a specific effect level of 15%. The addition of 1 mM HMBA to 100 nM 5azaC- and 1 nM RA-treated cultures significantly increased the colony-formation inhibition from only 2.6% and 7.0% to 46.4%, and 43.1%, respectively. Also, HMBA showed marked synergism with RA and 5azaC in inhibiting colony growth. The interaction indices (I) of HMBA + RA and HMBA + 5azaC were 0.013 and 0.009, respectively, at the same specific level of 15%. Moreover, the triple combination of RA + HMBA + 5azaC showed synergism in inhibiting both the colony formation (I = 0.7) and colony growth (I = 0.4) at the same specific level of 15%. Since RA, HMBA and 5azaC were effective when administered alone in phase I clinical trials of myeloid leukaemic patients, their synergistic combinations could provide shorter and less toxic courses of treatment in elderly myeloid leukaemic patients. I is less than 1, = 1 or greater than 1 in synergistic, additive or antagonistic interactions, respectively.

Topics: Acetamides; Aged; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Cell Differentiation; Cell Division; Drug Synergism; Humans; Leukemia, Myeloid, Acute; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay

Differentiation inducing agents in double and triple combinations can induce differentiation in primary culture of more than 80% of blast cells from some AML patients. In the present study, the interactions between these differentiating agents have been analysed using Berenbaum's general algebraic solution and three new, potentially clinically useful synergistic combinations: have been identified all trans retinoic acid (RA) + hexamethylene bisacetamide (HMBA), cytosine arabinoside (Ara-C) + HMBA and RA + Ara-C + HMBA. A measure of the effectiveness of these combinations was that the doses of Ara-C and HMBA required to induce 50% differentiation were decreased about 10-fold and 5-fold, respectively, in combination with 1 microM RA. The new synergistic combinations are important not only to limit toxicity but also because multiple drug combinations may better overcome the inherent molecular heterogeneity of the differentiation defect in AML patients. They warrant clinical trial in AML patients who are either unsuitable for or are unresponsive to conventional cytotoxic chemotherapy.

Topics: Acetamides; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cytarabine; Drug Synergism; Humans; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured

cDNA libraries have been generated from Nulli-SCCl murine embryonal carcinoma (EC) cells untreated or treated for 24 h with all-trans retinoic acid (RA) or hexamethylenebisacetamide (HMBA), two chemically unrelated inducers of differentiation of EC cells. The libraries were screened for gene sequences whose expression was differentially regulated by one or both compounds. Of 20,000 cDNA clones screened, only 12 showed reproducible quantitative differences. One of the latter clones (pH 34) has been studied in detail. pH 34 cDNA hybridizes with a polyadenylated RNA (650 nucleotides) which is abundant in untreated Nulli-SCCl EC cells but whose steady-state levels decrease within 6 h of exposure to HMBA, reaching a minimum at 24 h. RA has a less-marked effect on this mRNA. Addition of inducers to the cells in fresh medium produces an early (15 min) transient increase in pH 34 mRNA levels. Nuclear run-on experiments are consistent with the view that the decrease in pH 34 mRNA is due to post-transcriptional events. Subclones of pH 34 in pGEM-4 were used to synthesize mRNA which could be translated in vitro into a 14-kDa protein. DNA sequencing of the pH 34 cDNA revealed that it is 607 bp in length with a single open reading frame capable of encoding a protein of 118 amino acids. Primer extension experiments revealed that the insert contains the full 5' sequence. Comparison with known sequences failed to reveal significant homology with previously sequenced proteins.

Topics: Acetamides; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cell Differentiation; Cell Nucleus; DNA, Neoplasm; Gene Expression; Gene Library; Mice; Molecular Sequence Data; Protein Biosynthesis; Restriction Mapping; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

Retinoic acid, hexamethylene bisacetamide, sodium butyrate, and dimethylsulfoxide, four compounds which modulate phenotypic expression in a variety of neoplastic cell lines, all inhibited the induction of tyrosinase activity and melanogenesis by the combination of melanocyte-stimulating hormone and isobutylmethyxanthine in Cloudman S91 melanoma cells. Results were the same in assays of whole cells or in extracts made from them. Only retinoic acid, however, was effective at inhibiting the activation of dopachrome isomerase, another regulatory enzyme in melanogenesis. Despite inhibiting the effects of melanocyte-stimulating hormone (MSH) and isobutylmethylxanthine on tyrosinase activity, all of the agents tested increased the binding of MSH to intact cells. Ultrastructural analysis of treated cells following DOPA cytochemistry revealed that both retinoic acid and hexamethylene bisacetamide arrested melanosomal maturation at stage I-II. Retinoic acid resulted in a derangement of melanosomal structure. The specificity of these agents for preventing the induction of melanogenesis makes them powerful tools for the dissection of this complex cellular process.

Topics: 1-Methyl-3-isobutylxanthine; Acetamides; Animals; Butyrates; Butyric Acid; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Indolequinones; Indoles; Melanins; Melanocyte-Stimulating Hormones; Melanocytes; Melanoma, Experimental; Mice; Microscopy, Electron; Monophenol Monooxygenase; Phenotype; Quinones; Tretinoin; Tumor Cells, Cultured

Alterations in the pattern of gene expression were studied during differentiation of the human embryonal carcinoma (EC) cell line NEC14. NEC14 cells can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The efficiency of DNA transfection of undifferentiated and differentiated NEC14 cells was compared by measuring the activities of endogenous and exogenously introduced promoters for the beta-actin gene and heat shock protein 70 gene. The results indicated that the efficiency was not significantly different in cells of these two states. Under the conditions used, all the viral enhancer-promoters tested showed very little or no activity in undifferentiated cells, but activities of SV40, BKV, adenovirus and RSV enhancers were greatly increased after differentiation. Activities of these viral enhancers in differentiated cells were completely repressed by cotransfection with the adenovirus E1A gene. An E1A-inducible promoter of the adenovirus E2 gene showed stronger activity in differentiated than in undifferentiated cells, and was not activated efficiently by cotransfection with the E1A gene in either undifferentiated or differentiated cells. These results indicate that factor(s) regulating activities of various enhancer-promoters in NEC14 cells is or are different from E1A-like factor(s) present in mouse EC F9 cells.

Topics: Acetamides; Adenovirus Early Proteins; Cell Transformation, Neoplastic; Chloramphenicol O-Acetyltransferase; DNA, Neoplasm; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasms, Germ Cell and Embryonal; Oncogene Proteins, Viral; Testicular Neoplasms; Transfection; Tretinoin; Tumor Cells, Cultured

The effects of the differentiation inducing agents (DIAS), sodium butyrate (NaBu), retinoic acid (RA), dimethylformamide (DMF), hexamethylene bisacetamide (HMBA), forskolin, and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the growth, morphology, and estrogen receptor (ER) content and epithelial membrane antigen (EMA) expression on a serumless human breast cancer cell line (MCF-7) were compared. All these agents reversibly caused a concentration-dependent growth inhibition in monolayers and markedly reduced colony-forming efficiency in soft agar. A twofold increase in doubling time was obtained with RA (1 microM), but cell replication ceased with NaBu (1 mM), forskolin (50 microM), DMF (1%), HMBA (5 mM), and TPA (8 nM). Total growth arrest induced by these last compounds was preceded by an accumulation of cells in G0/G1 phase observed at 24 h by flow cytometry and accompanied by a change in cell morphology as seen by light and electronic microscopy. An increase in cell volume and the presence of lipid droplets was noted in treated cells that were spread out, as compared with controls. The acquisition of a more mature phenotype was confirmed by an increased expression of EMA monitored by flow cytometry. A specific reduction in the number of ER without any constant dissociation (Kd) modification was also observed after treatment with the 5 DIAs. No modification of morphological or biochemical characteristics, including EMA expression and ER binding, were observed for RA (1 microM)-treated cells. All these results suggest that induction of a more differentiated phenotype is associated with a block in G1 cell cycle phase, resulting in total growth arrest. Apparently, RA (1 microM)-treated cells did not fulfill these criteria, since only a slight accumulation in G1 and a slowed growth rate were evaluated.

Topics: Acetamides; Antigens, Neoplasm; Breast Neoplasms; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Colforsin; Dimethylformamide; Fluorescent Antibody Technique; Humans; Membrane Glycoproteins; Mucin-1; Receptors, Estrogen; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Differentiation induction therapy provides an alternative therapeutic approach for patients with acute myeloid leukaemia (AML) who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + 6-thioguanine (6-Th) + hexamethylene bisacetamide (HMBA) on differentiation of blasts from 24 AML patients was studied. Nonadherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in 24-well tissue culture plates containing RPMI-1640 culture medium with 20% fetal calf serum and 10% 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 1.5 X 10(-6) M 6-thioguanine and/or 2 mM hexamethylene bisacetamide for six days at 37 degrees C in a humidified incubator under 5% CO2. Morphological, cytochemical and functional differentiation into mature cells were induced in blasts from 22 out of the 24 AML patients following exposure to the triple combination of 10(-6) M RA + 1.5 X 10(-6) M 6-Th + 2 mM HMBA in primary culture. These effective results justify a clinical trial of such triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.

Topics: Acetamides; Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Drug Screening Assays, Antitumor; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Thioguanine; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid (RA), sodium n-butyrate (NaB), hexamethylene bisacetamide (HMBA), and dimethyl sulfoxide (DMSO) induce differentiation of the human acute myeloid leukemia cell line HL60. In the clinic, RA, NaB, or HMBA induce complete or partial remissions. However, the achievement and maintenance of effective plasma concentrations and toxicity have been problems. These difficulties led us to study the interaction of RA with these inducers. We found that combinations of RA with either NaB, HMBA, or DMSO synergistically induced terminal differentiation of HL60. A measure of the effectiveness of these combinations was that the doses of NaB, HMBA, and DMSO required alone to induce half-maximal differentiation were decreased about 4-fold in combination with normal plasma concentrations of about 30 nM RA. RA or NaB alone did not enhance the growth of HL60 cells. In contrast, HMBA or DMSO alone increased growth of HL60 cells even at concentrations that did not induce differentiation. The addition of RA reduced the promotion of growth and increased the extent of terminal differentiation seen with HMBA and DMSO alone. These data suggest that treatment of some malignancies with combinations of RA with HMBA or NaB may maintain differentiation-inducing effects and decrease the problems associated with the achievement and maintenance of effective plasma concentrations as single agents.

Topics: Acetamides; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Humans; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured

We have studied the expression of nine homeobox genes from Hox 1, Hox 2, Hox 3 and Hox 5 clusters in human embryonal carcinoma (EC) cell lines analyzed as both stem cells and after exposure to the differentiation-inducing agents retinoic acid (RA), hexamethylenebisacetamide (HMBA) and bromodeoxyuridine (BUdR). None of the homeobox genes was expressed in stem cells, whereas all were activated, although with different kinetics, in cultures of the pluripotent EC cell line NTERA-2, clone D1 (NT2/D1), following differentiation induced by RA. At least some homeobox genes were stably expressed in differentiated cells several weeks after removal of RA from the culture medium. However, the length of initial exposure to RA is a critical factor in achieving stable gene expression, and differs among the different sets of genes and, at least in one case, among different transcripts from the same gene. No homeobox gene expression was detected in NT2/D1 cells induced to differentiate with HMBA or BUdR. Also, no expression was detectable in xenograft tumors generated by NT2/D1 cells in nude mice, even though tumors of this type contain mostly differentiated cells. Other human EC lines tested, i.e., 833KE, 2102Ep or 1156QE, did not differentiate in response to RA and did not express homeobox genes. No expression was detectable in xenograft tumors of 833KE and 2102Ep, containing essentially EC cells. These data indicate that homeobox-gene activation specifically accompanies RA-induced differentiation of NT2/D1 cells, thereby providing an excellent model for studying the molecular basis of homeobox-gene regulation and the possible role of the homeobox in cell differentiation.

Topics: Acetamides; Animals; Antigens, Surface; Bromodeoxyuridine; Cell Differentiation; Cell Line; Gene Expression Regulation; Genes, Homeobox; Humans; Mice; Stem Cells; Time Factors; Transcriptional Activation; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

The status of the T cell receptor beta and gamma genes in natural killer (NK) cells was investigated in two patients with a marked expansion of CD2+, CD3- NK cells. Both genes were found to be in the germline state. The T alpha and complete T beta gene transcripts were not detected, but a 1.0-kilobase T beta gene transcript could be demonstrated at low levels in freshly isolated cells and at a much higher level in interleukin-2 (IL-2)-cultured cells. The transcript coding for the delta chain of the CD3 complex was also absent. These cells were cultured in IL-2 with or without the addition of the differentiation-inducing agents: retinoic acid, N,N-hexamethylene bisacetamide, and sodium butyrate. The cultured cells retained their NK activity except in culture with sodium butyrate at greater than or equal to 1 mmol/L. Expression of CD3 or other T cell surface markers by the NK cells was not observed in these cultures. Either CD2+, CD3- NK cells are derived from a non-T lineage, or they have diverged from the T cell lineage earlier than the stage of T gamma gene rearrangement and CD3 delta chain expression; they are refractory to many induction signals in undergoing further T cell differentiation.

Topics: Acetamides; Antigens, Differentiation, T-Lymphocyte; Antigens, Surface; Butyrates; Butyric Acid; CD3 Complex; Cell Differentiation; Cells, Cultured; Cytotoxicity, Immunologic; Humans; Interleukin-2; Lymphocytes; Lymphoproliferative Disorders; Phenotype; Receptors, Antigen, T-Cell; Tretinoin

In this study we followed the effects of various differentiating agents on the expression of carcinoembryonic antigen (CEA) released into the medium by a colon carcinoma cell line HT-29. Butyric acid 1 mM markedly increased the level of CEA (12-fold in comparison to control levels). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) 50 ng/ml and 5-azacytidine 4 x 10(-6) M increased the amount of CEA, 2- and 1.5-fold respectively. On the other hand retinoic acid 10(-5) M, N methyl-formamide 1% and N,N hexamethylene bisacetamide 2.5 mM decreased CEA 2-, 4- and 3-fold respectively. Our results emphasize that various differentiating agents affect CEA levels differently. Thus changes in CEA levels appear not to be reliable as a marker of a more differentiated phenotype.

Topics: Acetamides; Azacitidine; Butyrates; Butyric Acid; Carcinoembryonic Antigen; Cell Differentiation; Colonic Neoplasms; Drug Synergism; Formamides; Humans; Tetradecanoylphorbol Acetate; Tretinoin

Genomic and cDNA clones of the mouse Hox2.3 gene have been isolated. Expression of this gene was characterized in differentiating embryonal carcinoma (EC) and embryonic stem (ES) cells, and in the 13.5-day embryo. Hox2.3 is expressed at a very low level, if at all, in undifferentiated ES and EC cells. As previously reported for the Hox1.1 and Hox2.1 genes, differentiation of pluripotent stem cells induced by a nonchemical method is not accompanied by strong accumulation of Hox2.3 transcripts. Treatment of the stem cells with a chemical inducer like retinoic acid (RA), and also hexamethylenebisacetamide (HMBA), or 5-bromo-2'-deoxyuridine (BUdR), simultaneously accelerates differentiation and stimulates accumulation of Hox2.3 mRNA to high levels. Addition of RA several days after the cells have been induced to differentiate by a nonchemical method induces Hox2.3-transcript accumulation as well. For comparison, expression of the En-1 gene, which contains a homeobox belonging to a different class from that of the Antennapedia-related Hox1.1, Hox2.1, and Hox2.3 genes, was analyzed. The En-1 gene was found also to be sensitive to this regulation by chemical inducers of differentiation. It was observed that treatment of undifferentiated EC cells with the inhibitor of protein synthesis cycloheximide resulted in slight accumulation of Hox2.3 mRNA, suggesting the involvement of a short-lived protein in keeping the level of homeobox-gene transcription low in EC cells. The highest level of Hox2.3 transcripts in 13.5-day embryos in vivo was observed in the spinal cord. Comparison with the expression pattern of three other homeobox genes revealed overlapping gradients of mRNA along the longitudinal brain-spinal-cord axis. An important question is that of the molecular basis for such a spatially restricted accumulation of homeobox transcripts. Hox2.3 is expressed at a much lower level in rat and mouse embryonic midbrain than in spinal cord in vivo. We have shown that addition of RA to primary cultures of cells from rat embryo mesencephalon leads to strong accumulation of Hox2.3 mRNA. A possible interpretation is that RA mimics one or more spatially restricted effectors, accounting for the local accumulation of Hox2.3 transcripts in the embryonic central nervous system. Control of Hox2.3 gene expression in vivo may obey some similar mechanisms as in chemically stimulated EC and ES cells in vitro.

Topics: Acetamides; Animals; Brain; Bromodeoxyuridine; Cell Differentiation; Cells, Cultured; Cloning, Molecular; DNA; DNA Restriction Enzymes; Embryo, Mammalian; Genes, Homeobox; Mice; Spinal Cord; Transcription, Genetic; Tretinoin

The human promyelocytic leukemia cell line HL60 differentiates to either granulocytes or monocytes/macrophages when induced with various chemicals and lymphokines. Retinoic acid (RA) induces HL60 to differentiate to granulocyte-like cells. However, HL60/MRI cells, derived from a transplantable HL60 tumor established in athymic nude mice, differentiate to monocytoid cells when cultured with RA in vitro. HL60/MRI induced with RA are monocytes based on morphology and the expression of markers and functions specific for monocytes such as: the OKM5 monocyte-specific antigen, nonspecific esterase activity, and adhesiveness. HL60/MRI is much more sensitive to RA than is HL60. Thus, the RA concentrations that induce 50% differentiation are 0.41 nM for HL60/MRI and 37 nM for HL60, and maximum differentiation occurs at 2 days for HL60/MRI and at 4 days for HL60. While RA induces HL60/MRI to monocytoid cells, other inducers of granulocytic differentiation of HL60, such as dimethyl sulfoxide and hexamethylene bisacetamide, induce HL60/MRI to granulocytes. Furthermore, 12-O-tetradecanoylphorbol-13-acetate induces both HL60 and HL60/MRI to macrophage-like cells. The isozyme phenotypes of HL60/MRI and HL60 are identical. Cytogenetic analysis of HL60/MRI indicates that many of its normal chromosomes are triploid and that it has five abnormal chromosome markers, M1-M5, three of which, M1-M3, are seen also in HL60. This unique cell line, HL60/MRI, may be useful for studying the event(s) triggering differentiation of myelomonocytic cells and the mechanism of action of RA.

Topics: Acetamides; Animals; Antigens, Surface; Cell Differentiation; Cell Line; Chromosome Aberrations; Dimethyl Sulfoxide; Humans; Isoenzymes; Leukemia, Myeloid, Acute; Mice; Mice, Inbred BALB C; Monocytes; Neoplasm Transplantation; Tetradecanoylphorbol Acetate; Tretinoin

Retinoic acid induces the differentiation of NTERA-2 cl. D1 human embryonal carcinoma (EC) cells into neurons, cells permissive for the replication of human cytomegalovirus (HCMV), and other cell types that cannot as yet be classified but are distinguishable from the stem cells. We tested several additional agents for their ability to induce the differentiation of these EC cells. No differentiation was induced by butyrate, cyclic AMP, cytosine arabinoside, the tumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA), or the chemotherapeutic agent cis-diaminedichloroplatinum, although morphological changes were detected at the highest concentrations of these agents that permitted cell survival. However, retinal, retinol, 5-bromouracil 2'deoxyribose (BUdR), 5-iodouracil 2'deoxyribose (IUdR), hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), and dimethylsulfoxide (DMSO) all induced some neuronal differentiation, but to a lesser extent than retinoic acid. Also, BUdR, IUdR, HMBA, and DMA induced the appearance of many cells permissive for the replication of HCMV. Differentiation was, in all cases, accompanied by the loss of SSEA-3, a globoseries glycolipid antigen characteristically expressed by human EC cells. However, another glycolipid antigen, A2B5, which appears in 60%-80% of differentiated cells 7 days following retinoic acid induction, was detected in less than 20% of the cells induced by the other agents studied. This implies that the HCMV-permissive cells induced by retinoic acid are not identical to those induced by BUdR, IUdR, and DMA.

Topics: Acetamides; Bromodeoxyuridine; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Cisplatin; Clone Cells; Cyclic AMP; Cytarabine; Dimethyl Sulfoxide; Humans; Idoxuridine; Kinetics; Neurons; Teratoma; Tetradecanoylphorbol Acetate; Tretinoin

During treatment of HL-60 myeloid leukemia cells in culture with polar solvents or retinoic acid at a concentration inducing terminal maturation in 90-95% of the cells, there is a rapid decline (within 2 h) in the Vmax for influx of the folate analogue, [3H]methotrexate. Following 24 h of exposure to these agents, there is no effect on growth, but influx Vmax is reduced by 70%. After 7 days of exposure, influx Vmax is reduced 90-95%. A similar time course was seen for the reduction in intracellular levels of dihydrofolate reductase, a marker of cellular proliferation. Both the extent of terminal maturation (as determined by the extent of nitro blue tetrazolium reduction) and decrease in influx showed the same dependence on the concentration of inducer. In contrast to the effect seen on influx Vmax, both influx Km and mediated efflux of [3H]methotrexate remained unchanged in HL-60 cells exposed to inducers of maturation. Finally, evidence is presented for the coupling of this alteration on [3H]methotrexate influx with commitment of HL-60 cells to terminal maturation. This evidence shows that the effect on folate analogue influx precedes commitment and documents the irreversible nature of the reduction in influx once the majority of the cells exposed to inducer were committed to the process of maturation. The possible relevance of these results to the process of neoplastic transformation is discussed.

Topics: Acetamides; Biological Transport, Active; Cell Differentiation; Cell Line; Cells, Cultured; Folic Acid; Humans; Kinetics; Leukemia, Myeloid, Acute; Mathematics; Methotrexate; Tretinoin

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.

Topics: Acetamides; Animals; Antigens, Surface; Carrier Proteins; Cell Differentiation; Cell Line; Embryonal Carcinoma Stem Cells; Glycolipids; Hybrid Cells; Lewis X Antigen; Male; Mice; Mutation; Neoplastic Stem Cells; Plasminogen Activators; Receptors, Retinoic Acid; Stem Cells; Teratoma; Tretinoin

In monolayer cultures, embryonal carcinoma (EC) cells from the cell line Nulli-SCCl can be induced to differentiate at high efficiency by exposure to either retinoic acid or hexamethylenebisacetamide. Depending on which inducing agent is used, two distinct differentiated phenotypes result. These phenotypes resemble two of the earliest differentiated derivatives of the cells of the inner cell mass of a mouse blastocyst, i.e., parietal and visceral endoderm. Both differ from EC cells as well as from each other on the basis of their morphology, antigenic expression, secretion of plasminogen activator, and protein-synthetic profiles.

Topics: Acetamides; Animals; Antigens, Neoplasm; Blastocyst; Cell Differentiation; Cell Line; Mice; Phenotype; Teratoma; Tretinoin

We have generated by mutagenesis eight differentiation-defective sublines from three murine embryonal carcinoma (EC) cell lines. These mutants grossly resemble parental cells in the absence of inducers of differentiation. Based upon response to retinoic acid (RA) or hexamethylenebisacetamide (HMBA), the mutants can be grouped into three types: (a) RA-selected cells that lack cellular RA binding protein (cRABP) activity and fail to differentiate in response to RA or HMBA; (b) RA- or HMBA-selected cells that possess cRABP but differentiate poorly, if at all, in the presence of RA or HMBA; and (c) cells originally selected for lack of response to HMBA but which retain cRABP and the ability to differentiate in response to RA.

Topics: Acetamides; Animals; Carrier Proteins; Cell Differentiation; Cell Line; Mice; Mutation; Plasminogen Activators; Receptors, Retinoic Acid; Teratoma; Tretinoin

The human leukemic cell lines HL60 and K562, were induced to differentiate terminally by chemical agents. The isoenzyme patterns of lactate dehydrogenase (LD) in the cells before and after differentiation were determined electrophoretically on agarose gels. In general, treatment of the leukemic cells with inducers of differentiation resulted in a quantitative shift of the isoenzyme pattern towards anodic or cathodic forms. This was correlated with the conversion of the chemically treated cells to morphologically more normal cells, as verified by light microscopy and/or synthesis of hemoglobin. The LD isoenzyme patterns of the chemically differentiated cells were: (a) characteristic for the particular cell type obtained rather than for the nature of the inducer used; and (b) not similar to those of normally differentiated cells of the corresponding lineage, indicating that incomplete differentiation had occurred.

Topics: Acetamides; Butyrates; Cell Differentiation; Cell Line; Cell Survival; Diamines; Dimethyl Sulfoxide; Erythropoiesis; Granulocytes; Hematopoiesis; Humans; Isoenzymes; L-Lactate Dehydrogenase; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Tetradecanoylphorbol Acetate; Tretinoin