Showing papers on "Cellular differentiation published in 1987"

Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production

Abstract: Gamma interferon (IFN-gamma) and B cell stimulatory factor-1 (BSF-1), also known as interleukin-4, are T cell-derived lymphokines that have potent effects on B cell proliferation and differentiation. They are often secreted by distinct T cell clones. It is now shown that IFN-gamma stimulates the expression of immunoglobulin (Ig) of the IgG2a isotype and inhibits the production of IgG3, IgG1, IgG2b, and IgE. By contrast, BSF-1 has powerful effects in promoting switching to the expression of IgG1 and IgE but markedly inhibits IgM, IgG3, IgG2a, and IgG2b. These results indicate that BSF-1 and IFN-gamma as well as the T cells that produce them may act as reciprocal regulatory agents in the determination of Ig isotype responses. The effects of IFN-gamma and BSF-1 on isotype expression are independent.

A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor

Abstract: Nerve growth factor (NGF) is a trophic agent that promotes the outgrowth of nerve fibers from sympathetic and sensory ganglia. The neuronal differentiation stimulated by this hormone was examined in the NGF-responsive cell line PC12. Differential hybridization was used to screen a complementary DNA library constructed from PC12 cells treated with NGF and cycloheximide. One of the complementary DNA clones that was rapidly induced by NGF was found to have a nucleotide sequence that predicts a 54-kilodalton protein with homology to transcriptional regulatory proteins. This clone, NGFI-A, contains three tandemly repeated copies of the 28- to 30-amino acid "zinc finger" domain present in Xenopus laevis TFIIIA and other DNA-binding proteins. It also contains another highly conserved unit of eight amino acids that is repeated at least 11 times. The NGFI-A gene is expressed at relatively high levels in the brain, lung, and superior cervical ganglion of the adult rat.

Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer.

Abstract: We describe a cell-lineage marking system applicable to the vertebrate nervous system. The basis of the technique is gene transfer using the retroviral vector system. We used Escherichia coli beta-galactosidase as a marker gene and demonstrate a high level of expression of this marker from the viral long terminal repeat promoter, with simultaneous expression of the Tn5 neo gene from the simian virus 40 early promoter. This expression has allowed us to detect individual infected cells histochemically. We applied this marking technique to the study of lineage relationships in the developing vertebrate nervous system, both in vivo and in culture. In the rat retina, we injected virus in vivo and histochemically identified clones of marked neural cells. In addition, we used this virus to infect cultures of rat cerebral cortex and have analyzed the clonal relationships of morphologically different neural cell types. The host range of the marking system extends to avian as well as mammalian species. Thus, this system should have broad applicability as a means of gene transfer and expression in the nervous system.

The role of depolarization in the survival and differentiation of cerebellar granule cells in culture

Abstract: Cultures greatly enriched in granule cells from early postnatal cerebellum (P8) were grown in a medium containing fetal calf serum. Under the conditions used, nerve cells died, usually within a week, unless the K+ concentration in the medium was greater than or equal to 20 mM. The requirement for elevated [K+]e was manifested by about 3 d in vitro, and after this time continuous exposure to high [K+]e was essential for the survival of the granule cells. The initial morphological and biochemical maturation of the granule cells was similar in the presence and the absence of elevated [K+]e, suggesting that the dependence on depolarizing conditions develops in parallel with the expression of the differentiated characteristics of the cells. The positive effect of elevated [K+]e on granule cell survival was not influenced by preventing bioelectric activity in the cultures with TTX and xylocaine. On the other hand, depolarization-induced transmembrane Ca2+ flux was essential in securing the maintenance of the granule cells. Depolarized nerve cells were compromised when Ca2+ entry was blocked by elevated Mg2+, EGTA, or organic Ca2+ antagonists, while dihydropyridine Ca2+ agonists [BAY K 8644, (+)-(S)-202 79 1 and CGP 28392] were potent agents preventing nerve cell loss in the presence of 15 mM [K+]e, which was ineffective on its own. Calmodulin inhibitors (1 microM trifluoperazine or calmidazolium) blocked the beneficial effect of K+-induced depolarization on granule cells. The comparison of the timing of the differentiation and innervation of the postmitotic granule cells in vivo with the development of the K+ dependence in vitro would indicate that depolarization of the granule neurons in culture mimics the influence of the physiological stimulation in vivo through excitatory amino acid receptors, including N-methyl-D-aspartate receptors, involving Ca2+ entry and the activation of a Ca2+/calmodulin- dependent protein kinase.

A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor- β family

Abstract: The decapentaplegic gene complex (DPP-C) has been implicated in several events in pattern formation during Drosophila development. During embryogenesis, the DPP-C participates in the establishment of dorsal-ventral specification. Later, it is required for the correct morphogenesis of the imaginal disks, which will form much of the adult epidermis. We have undertaken a molecular analysis of the DPP-C to determine what role it plays in positional information. It appears to be a large genetic unit (greater than 40 kilobases (kb] consisting mostly of cis-regulatory information controlling the expression of a set of overlapping transcripts that differ at their 5' ends, but share the bulk of their transcribed sequences. Here, we describe the sequence analysis of two complementary DNAs comprising 4.0 kb of a 4.5-kb transcript. The C-terminus of the protein thereby deduced exhibits strong sequence homology (25-38% amino-acid identity) to the C-termini of a class of mammalian proteins that includes transforming growth factor-beta (TGF-beta), inhibin and Mullerian inhibiting substance (MIS). These proteins act on target cells to produce a variety of responses, such as stimulation or inhibition of cell division or differentiation. The homology suggests that the DPP-C protein contributes to correct morphogenesis as a secreted factor involved in the differential regulation of cell growth. This is the first report of a member of the TGF-beta gene family in a non-mammalian organism, and indicates that one or more members of this gene family existed before arthropod and vertebrate lineages diverged.

The molecular basis of blood diseases

Abstract: PART I: Hematopoiesis Stem Cell Biology Hemopoietic Growth Factors and Receptors Transcriptional Factors that Regulate Lineage Decisions Growth Factor Signaling PART II: Red Cells Hemoglobin Switching The Thalassemias Human Hemoglobins: Sickle Cell Hemoglobin and Other Mutants The Erythrocyte Membrane and Cytoskeleton: Structure, Function, and Disorders Red Blood Cell Antigenes Molecular Basis of Iron Metabolism PART III: Lymphopoiesis Gene rearrangements in Lymhoic Cells Lymphoiesis Antigen Processing and T-Cell Effector Mechanisms PART IV: Myelopoiesis/White Blood Cells Integrins, Selectins, and LAD Chemokines Genetic Disorders of Phagocyte Function Paroxymal Nocturnal Hemoglobins PART V: Hemostasis Vitamin K-Dependent Proteins The Protein C Anticoagulant System Regulation of Blood Coagulation by Protease Inhibitors Hemophilia A, Hemophilia B, and von Willenbrand Disease The Molecular Basis of Fibrin Fibrinilysis and the Control of Hemostasis Platelets PART VI: Molecular Oncology Molecular Aspects of Oncogenesis Mechanisms of Leukemogenesis PART VII: Viruses The Molecular and Biological Properties of the Human Immunodeficiency Virus Viral Pathogenesis of Hematological Disorders B19 Parvoviruses HTLVs Herpesviruses Gene Therapy of Hemopoietic Diseases

Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment

Abstract: Haematopoietic progenitor cells proliferate and mature in semi-solid media when stimulated by exogenous haematopoietic cell growth factors (HCGFs) such as granulocyte-macrophage colony-stimulating factor (GM-CSF)1,2. They also proliferate in association with marrow-derived stromal cells3,4 although biologically active amounts of HCGFs cannot be detected in stromal culture supernatants5. It is possible that HCGFs are synthesized in small amounts by stromal cells but remain bound to the stromal cells and/or their extracellular matrix (ECM). This interpretation accords with haematopoietic progenitor cell proliferation in close association with stromal layers in long-term cultures6. Glycosaminoglycans (GAGs) are found in the ECM produced by stromal cells27,8. They are prime candidates for selectively retaining HCGFs in the stromal layer9; they influence embryonic morphogenesis and cyto-differentiation10 and they may regulate haematopoiesis11–13. We now report that granulocyte-macrophage colony-stimulating activity can be eluted from cultured stromal layers and that exogenous GM-CSF binds to GAGs from bone marrow stromal ECM. Selective Compartmentalization of HCGFs in this manner may be an important function of the marrow microenvironment and may be involved in haematopoietic cell regulation.

Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF).

Abstract: Leukaemia inhibitory factor (LIF) can induce macrophage differentiation in M1 murine myeloid leukaemic cells and suppress their proliferation in vitro. It does not stimulate the proliferation of normal progenitor cells and is apparently distinct from known colony-stimulating factors. We have used oligo-nucleotides complementary to partial amino acid sequence of LIF to isolate a LIF clone from a T lymphocyte cDNA library. When this cDNA was coupled to a yeast expression vector (YEpsec1) and introduced into yeast cells, a molecule with the biological properties characteristic of native LIF was secreted into the growth medium. The amino acid sequence of LIF established it to be a unique molecular entity, distinct from the other known haemopoietic growth factors. Since LIF is encoded by a unique gene, two biochemically separable forms of LIF probably represent post-transcriptional or posttranslational variants of the same gene product. In contrast to several other haemopoietic regulators, the 0.8- to 1-kb LIF mRNA was expressed constitutively in two murine T lymphocyte cell lines examined, and its abundance was not enhanced by stimulation with concanavalin A. Cloning, sequencing and expressing LIF has resolved several discrepancies in the literature concerning the identity of factors capable of inducing differentiation of murine myeloid leukaemic cells in vitro.

Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation.

Abstract: The Pgp-1 glycoprotein was identified on a minor (27%) subset of peripheral Lyt-2+ or L3T4+ T cells. In contrast, mature medullary-type thymocytes (Lyt-2+ L3T4-, Lyt-2- L3T4+) were nearly devoid of cells expressing detectable surface Pgp-1. The appearance of peripheral Pgp-1- T cells was found to be thymus dependent, as demonstrated by the diminished proportion of Pgp-1- T cells after thymectomy and their virtual absence in athymic nude mice. The subsequent acquisition of surface Pgp-1 was found to be a stable differentiation event occurring concomitantly with primary antigenic stimulation; selected Pgp-1- mature T cells from thymus or periphery acquired constitutive expression of Pgp-1 after stimulation in vitro with alloantigen or mitogens. These observations were extended by studies in vivo showing that immunization with various antigens augmented the percentage of Pgp-1+ spleen cells within the Lyt-2+ subset. Furthermore, the frequencies of antigen-specific CTLp, after immunization by any of three different antigens tested, were greatly enriched in the Pgp-1+ compared with the Pgp-1- subpopulations. Peritoneal exudate Lyt-2+ cells, after a localized allograft rejection, demonstrated a particularly prominent Pgp-1+ subpopulation (78%) that contained virtually all the allospecific cytolytic activity. A model consistent with all of these data proposes that mature thymocytes lacking surface Pgp-1 upon emigration to the periphery acquire its expression at the time of primary antigenic stimulation. Hence, expression of Pgp-1 among peripheral T cells is an important differentiation marker for identifying antigen-stimulated memory T cells.

Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor.

Abstract: Analysis of MM14 mouse myoblasts demonstrates that terminal differentiation is repressed by pure preparations of both acidic and basic fibroblast growth factor (FGF). Basic FGF is approximately 30-fold more potent than acidic FGF and it exhibits half maximal activity in clonal assays at 0.03 ng/ml (2 pM). FGF repression occurs only during the G1 phase of the cell cycle by a mechanism that appears to be independent of ongoing cell proliferation. When exponentially growing myoblasts are deprived of FGF, cells become postmitotic within 2-3 h, express muscle-specific proteins within 6-7 h, and commence fusion within 12-14 h. Although expression of these three terminal differentiation phenotypes occurs at different times, all are initiated by a single regulatory "commitment" event in G1. The entire population commits to terminal differentiation within 12.5 h of FGF removal as all cells complete the cell cycle and move into G1. Differentiation does not require a new round of DNA synthesis. Comparison of MM14 behavior with other myoblast types suggests a general model for skeletal muscle development in which specific growth factors serve the dual role of stimulating myoblast proliferation and directly repressing terminal differentiation.

A new T-cell receptor gene located within the alpha locus and expressed early in T-cell differentiation

Abstract: A new T-cell receptor gene lies just 5' to the JαCα coding regions. Its placement in this location suggests a novel mechanism for the regulation of expression of one T-cell receptor polypeptide to another during ontogeny. Rearrangement of this locus occurs very early in thymic differentiation and its RNA expression parallels that of the γ-chain in thymic subpopulations, making this a possible candidate for the recently described δ-chain of the T-cell receptor.

The Influence of Extracellular Matrix on Gene Expression: Is Structure the Message?

Abstract: The study of the regulation of gene expression in cultured cells, particularly in epithelial cells, has been both hampered and facilitated by the loss of function that accompanies culture on traditional plastic substrata. Initially, investigations of differentiated function were thwarted by the inadequacy of tissue culture methods developed to support growth of mesenchymal cells. However, with the recognition that the unit of function in higher organisms is larger than the cell itself, and that gene expression is dependent upon cell interactions with hormones, substrata and other cells, came the understanding that the epithelial cell phenotype is profoundly influenced by the extracellular environment. In the last decade research on epithelial cells has centred on culture conditions that recreate the appropriate environment for function with very promising and important results. The investigations into the modulation of phenotype in culture produced not only a better model, but also contributed to a better understanding of the regulation of normal function. Using cultured mammary gland epithelial cells as a primary model of these interactions, our studies of gene expression are based on three premises. (1) That the extracellular matrix (ECM) on which the cells sit is an extension of the cells and an active participant in the regulation of cellular function; i.e. the ECM is an 'informational' entity in the sense that it receives, imparts and integrates structural and functional signals. (2) That ECM-induced functional differentiation in the mammary gland is mediated through changes in cell shape, i.e. that the structure is in large part 'the message' required to maintain differentiated gene expression. (3) That the unit of function includes the cell plus its extracellular matrix; in a larger context, the unit is the organ itself. These tenets and the data presented below are consistent with a model of 'Dynamic Reciprocity', where the ECM is postulated to exert an influence on gene expression via transmembrane proteins and cytoskeletal components. In turn, cytoskeletal association with polyribosomes affects mRNA stability and rates of protein synthesis, while its interaction with the nuclear matrix could affect mRNA processing and, possibly, rates of transcription.

Multicellular spheroids. A review on cellular aggregates in cancer research.

Abstract: Cellular aggregates have been used in developmental biology and in experimental cancer research for several decades. Spherical aggregates of malignant cells, i.e. multicellular tumor spheroids, may serve as in vitro models of tumor microregions and of an early, avascular stage of tumor growth. The similarities between the original tumor and the respective spheroids include volume growth kinetics, cellular heterogeneity, e.g. the induction of proliferation gradients and quiescence, as well as differentiation characteristics, such as the development of specific histological structures or the expression of antigens. Research using cell aggregates has been focussed on mechanisms involved in the control of proliferation, invasion and metastasis. Immunological studies with spheroids have resulted in the characterization of defense cells which are responsible for specific host-versus-tumor reactions. The vast majority of investigations on spheroids concerns the simulation of therapy with regard to various treatment modalities, combination treatments and systematic analyses of using various endpoints in predictive assays. Only a few pathophysiological studies on the interrelationship among tumor-specific micromilieu, cellular metabolism, proliferative status, and cellular viability have been undertaken with the spheroid model up to now. Since these studies are indicative of a large influence of the cellular microenvironment on basic biological properties of cancer cells, investigations of these epigenetic mechanisms should be intensified in future research on cell aggregates. Similarly, the molecular basis of the biological peculiarities found in malignant cells grown as three-dimensional aggregates has to be investigated more intensively.

Two forms of transforming growth factor-β distinguished by multipotential haematopoietic progenitor cells

Abstract: Type-β transforming growth factors (TGF-βs) are polypeptides that act hormonally to control proliferation and differentiation of many cell types1,2. Two distinct homodimeric TGF-β polypeptides, TGF-β1 and TGF-β2 have been identified which show ˜70% amino-acid sequence similarity3,4. Despite their structural differences, TGF-β1 and TGF-β2 are equally potent at inhibiting epithelial cell proliferation and adipogenic differentiation3. The recent immunohistochemical localization of high levels of TGF-β in the bone marrow and haematopoietic progenitors of the fetal liver5 has raised the possibility that TGF-βs might be involved in the regulation of haematopoiesis. Here we show that TGF-β1, but not TGF-β2, is a potent inhibitor of haematopoietic progenitor cell proliferation. TGF-β1 inhibited colony formation by murine factor-dependent haematopoietic progenitor cells in response to interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF), as well as colony formation by marrow progenitor cells responding to CSF-1 (M-CSF). The progenitor cell lines examined were ˜100-fold more sensitive to TGF-β1 than TGF-β2, and displayed type-I TGF-β receptors with affinity ˜20-fold higher for TGF-β1 than TGF-β2. These results identify TGF-β1 as a novel regulator of haematopoiesis that acts through type-I TGF-β receptors to modulate proliferation of progenitor cells in response to haematopoietic growth factors.

Polypeptide Growth Factors: Roles in Normal and Abnormal Cell Growth

Abstract: An increasing number of polypeptide growth factors have been identified that regulate not only cell proliferation but an extraordinary range of cell activities, including matrix protein deposition and resolution, the maintenance of cell viability, cell differentiation, inflammation, and tissue repair. Normal cells appear to require growth factors for proliferation and for maintenance of viability. Cells that secrete a polypeptide growth factor have an advantage in growth. These factors can act either externally through cell surface receptors or perhaps internally during the transport of receptors and growth factors through the ER and Golgi, causing autocrine stimulation of cell growth. Depending on the cell type, growth factors can also be potent inhibitors of cell growth rather than stimulating growth, and the effects can depend on the presence or absence of other growth factors. Platelet-derived growth factor has been shown to be nearly identical to the product of the v-sis gene of the simian sarcoma virus, which appears to cause cell transformation through its interactions with the PDGF receptor activating the tyrosine kinase activity of the PDGF receptor. Similarly, two proto-oncogenes, c-erbB and c-fms, encode growth factor receptors. The EGF receptor activity of the v-erb oncogene product appears to be constitutively activated without the need for growth factor, perhaps because of the truncation at the amino terminus deleting the EGF binding domain. The induction of the myc and the fos proteins by growth factor stimulation of quiescent cells, as well as the potential for the p21 product of the ras oncogene to act as an intermediate in transducing adrenergic signals, provide direct evidence that these pathways are important for stimulation of cell growth. Cells transformed by the v-sis oncogene always appear to bear PDGF cell surface receptors, which suggests that this oncogene has a specific requirement of the PDGF receptor for transformation. In contrast, cells transformed by the v-erbB and v-fms oncogenes are not stimulated by EGF or by CSF-1. Thus it seems likely that the tyrosine kinase activity of the corresponding receptor is ubiquitously expressed in these cases. Major questions remain unanswered. In particular, what are the mechanisms by which growth factors initiate pathways leading to DNA synthesis? What are the physiological substrates of the growth factor receptor tyrosine kinase? Considerable effort also is needed to further define the cellular specificity of the different growth factors, particularly within intact tissues, and to determine how the various growth factors interact.(ABSTRACT TRUNCATED AT 400 WORDS)

Human B-cell differentiation factor defined by an anti-peptide antibody and its possible role in autoantibody production

Abstract: The partial amino acid sequence of the NH2 terminus of a factor named human B-cell differentiation factor or B-cell stimulatory factor 2 (BSF-2) has been determined. Antibodies raised against the synthetic peptide corresponding to residues 1-13 of the NH2-terminal sequence specifically react with BSF-2 generated by a T-cell line and by phytohemagglutinin-stimulated normal T cells. Furthermore, the antipeptide antibodies react with a BSF-2-like factor produced by cardiac myxoma as well as uterine cervical carcinoma cells. The results show that BSF-2 functions in vivo as well and suggest that the constitutive production of BSF-2 may be involved in autoantibody production, since patients with cardiac myxoma and uterine carcinoma showed autoantibody production.

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis.

Abstract: The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.

Glucocorticoids and Insulin Promote the Differentiation of Human Adipocyte Precursor Cells into Fat Cells

Abstract: To study the in vitro differentiation of human adipocyte precursor cells and its regulation by hormones, primary cultures of stromal vascular cells of human adipose tissue were established. A 30- to 70-fold increase in the number of developing fat cells was achieved by the addition of cortisol or related corticosteroids in the presence of insulin. Either of the two hormones alone was ineffective. The stimulatory action of cortisol was dose dependent and occurred at physiological concentrations. The results suggest that glucocorticoids may play a role in the development of human hyperplastic obesity by stimulating the formation of adipocytes from precursor cells.

Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation.

Abstract: A clone HT29-18 has been isolated from the parent cell line HT-29, which derived from a human colon adenocarcinoma (Fogh, J., and G. Trempe, 1975, Human Tumor Cells in Vitro, J. Fogh, editor, Plenum Publishing Corp., New York, 115-141). This clone is able to differentiate as the parent cell line does. Differentiation occurs when glucose is replaced by galactose in the culture medium (Pinto, M., M.D. Appay, P. Simon-Assman, G. Chevalier, N. Dracopoli, J. Fogh, and A. Zweibaum, 1982, Biol. Cell., 44:193-196). We demonstrate here that the differentiated cloned population HT29-18/gal is heterogenous: although 90% of the cells show morphological characteristics of "absorptive cells", only 20-30% of them display sucrase-isomaltase in their apical microvillar membranes. About 10% of the entire cell population consists of cells containing mucous granules similar to intestinal goblet cells. We have isolated two subclones, HT29-18-C1 and HT29-18-N2, from the differentiated HT29-18/gal cells. HT29-18-C1 cells show morphological characteristics of polarized absorptive cells, when growing either in glucose- or in galactose-containing media, but the sucrase-isomaltase is not expressed in the cells grown in glucose-containing medium. The clone HT29-18-N2 is also polarized in both culture conditions and is similar to globlet cells in vivo. It grows as a monolayer, exhibits tight junctions, and contains numerous mucous granules whose exocytosis can be triggered by carbachol, a parasympathomimetic drug. We conclude that the clone HT29-18 first isolated was a multipotent cell population from which we isolated several subclones that differentiate either as absorptive (HT29-18-C1) or as mucous (HT29-18-N2) cells. In contrast to the parent HT-29 cell line, the subclones retain most of their differentiated properties in glucose-containing medium.

Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.

Abstract: When human epidermal cells were seeded on floating rafts of collagen and fibroblasts, they stratified at the air-liquid interface. The suprabasal cells synthesized the large type II (K1) and type I (K10/K11) keratins characteristic of terminal differentiation in skin. At earlier times in culture, expression of the large type II keratins appeared to precede the expression of their type I partners. At later times, all suprabasal cells expressed both types, suggesting that the accumulation of a critical level of K1 keratin may be a necessary stimulus for K10 and K11 expression. Expression of the terminal differentiation-specific keratins was completely suppressed by adding retinoic acid to the culture medium, or by submerging the cultures in normal medium. In submerged cultures, removal of vitamin A by delipidization of the serum restored the keratinization process. In contrast, calcium and transforming growth factor-beta did not influence the expression of the large keratins in keratinocytes grown in the presence of retinoids, even though they are known to induce certain morphological features of terminal differentiation. Retinoic acid in the raft medium not only suppressed the expression of the large keratins, but, in addition, induced the synthesis of two new keratins not normally expressed in epidermis in vivo. Immunofluorescence localized one of these keratins, K19, to a few isolated cells of the stratifying culture. In contrast, the other keratin, K13, appeared uniformly in a few outer layers of the culture. Interestingly, K13 expression correlated well with the gradient of retinoid-mediated disruptions of intercellular interactions in the culture. These data suggest that K13 induction may in some way relate to the reduction in either the number or the strength of desmosomal contacts between suprabasal cells of stratified squamous epithelial tissues.