Showing papers on "Cellular differentiation published in 1986"

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells.

Abstract: In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."

Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells

Abstract: Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.

Stimulation of neuronal acetylcholine receptors induces rapid gene transcription

Abstract: Cholinergic agonists rapidly and transiently induced transcription of the c-fos protooncogene and one or more actin genes in neuronally differentiated PC12 cells. Transcription was activated within minutes after stimulation of the nicotinic acetylcholine receptor and required an influx of extracellular Ca2+ ions through voltage-sensitive calcium channels. Nicotine activation proceeded by a different pathway from activation by nerve growth factor, whose stimulation of these genes is independent of extracellular Ca2+ ions. These findings suggest that neurotransmitters may rapidly activate specific gene transcription in nondividing neuronally differentiated cells. They also suggest a functional role for neurotransmitter induction of c-fos and actin expression in the nervous system.

Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes.

Abstract: The growth and differentiation of B cells to immunoglobulin (Ig)-secreting cells is regulated by a variety of soluble factors. This study presents data that support a role for transforming growth factor (TGF)-beta in this regulatory process. B lymphocytes were shown to have high-affinity receptors for TGF-beta that were increased fivefold to sixfold after in vitro activation. The addition of picogram quantities of TGF-beta to B cell cultures suppressed factor-dependent, interleukin 2 (IL 2) B cell proliferation and markedly suppressed factor-dependent (IL 2 or B cell differentiation factor) B cell Ig secretion. In contrast, the constitutive IgG production by an Epstein Barr virus-transformed B cell line was not modified by the presence of TGF-beta in culture. This cell line was found to lack high-affinity TGF-beta receptors. The degree of inhibition of B cell proliferation observed in in vitro cultures was found to be dependent not only on the concentration of TGF-beta added but also on the concentration of the growth stimulatory substance (IL 2) present. By increasing the IL 2 concentrations in culture, the inhibition of proliferation induced by TGF-beta could be partially overcome. In contrast, the inhibition of Ig secretion induced by TGF-beta could not be overcome by a higher concentration of stimulatory factor, demonstrating that the suppression of B cell differentiation by TGF-beta is not due solely to its effects on proliferation. Furthermore, it was demonstrated that B lymphocytes secrete TGF-beta. Unactivated tonsillar B cells had detectable amounts of TGF-beta mRNA on Northern blot analysis, and B cell activation with Staphylococcus aureus Cowan (SAC) resulted in a twofold to threefold increase in TGF-beta mRNA. Supernatants conditioned by unactivated B cells had small amounts of TGF-beta, SAC activation of the B cells resulted in a sixfold to sevenfold increase in the amount of TGF-beta present in the supernatants. Thus, B lymphocytes synthesize and secrete TGF-beta and express receptors for TGF-beta. The addition of exogenous TGF-beta to cultures of stimulated B cells inhibits subsequent proliferation and Ig secretion. TGF-beta may function as an autocrine growth inhibitor that limits B lymphocyte proliferation and ultimate differentiation.

Tau protein function in living cells.

Abstract: Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis.

A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells.

Abstract: The c-myc gene product is a nuclear protein1,2 expressed in a wide variety of cell types3. It has been implicated in the control of normal cell growth as well as transformation4–7, but its exact function is unknown. When the human promyelocytic leukaemia cell line HL60 is treated with retinoic acid, the cells differentiate into granulocytes, and there is a reduction in steady state c-myc RNA of more than 10-fold8. Nuclear runoff assays show that this reduction is caused by a corresponding decrease in the transcription of exon 2. However, only a minor decrease in exon 1 transcription is observed upon differentiation. In undifferentiated HL60 cells there is an approximately 3-fold molar excess of exon 1 transcription over exon 2, and this excess increases to about 15-fold in differentiated cells. This observation suggests that a major component of c-myc transcriptional down-regulation in HL60 cells is at the level of elongation rather than at the level of initiation. The position of the elongation block was mapped to the region of the boundary between exon 1 and intron 1. During HL60 differentiation, a DNase I hypersensitive site in the chromatin about 300 bases downstream of the 5′ end of of intron 1 increases in intensity relative to other sites, possibly reflecting events associated with the termination of transcription. Our runoff analysis also revealed transcription of both strands immediately upstream of exon 1 in HL60 cells. The sense strand transcription of this region produces a novel c-myc RNA which initiates several hundred bases upstream of the previously defined promoters and is found in a variety of cell types.

Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein.

Abstract: Formation of syncytia, with progression to cell death, is a characteristic feature of in vitro cultures of susceptible cells infected with human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV)1–3. Viral antigen-positive multi-nucleated giant cells have also been observed in histological sections from infected individuals4,5. In vitro, formation of these multinucleated giant cells occurs through cell fusion which is dependent on cell-surface expression of the differentiation antigen CD4 (ref. 1). Utilizing a recombinant vaccinia virus containing the gene for the envelope glycoprotein of HTLV-III/LAV6, we demonstrate that cell-surface expression of this protein, in the absence of other HTLV-III/LAV structural or regulatory proteins, is sufficient to induce CD4-dependent cell fusion, leading to cell death, one of the characteristic manifestations of AIDS (acquired immune deficiency syndrome) virus cytopathology. This process may contribute to the loss of CD4+ T cells seen in AIDS.

Type beta transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells.

Abstract: Type beta transforming growth factor (TGF-beta) was shown to be the serum factor responsible for inducing normal human bronchial epithelial (NHBE) cells to undergo squamous differentiation. NHBE cells were shown to have high-affinity receptors for TGF-beta. TGF-beta induced the following markers of terminal squamous differentiation in NHBE cells: (i) increase in Ca ionophore-induced formation of crosslinked envelopes; (ii) increase in extracellular activity of plasminogen activator; (iii) irreversible inhibition of DNA synthesis; (iv) decrease in clonal growth rate; and (v) increase in cell surface area. The IgG fraction of anti-TGF-beta antiserum prevented both the inhibition of DNA synthesis and the induction of differentiation by either TGF-beta or whole blood-derived serum. Therefore, TGF-beta is the primary differentiation-inducing factor in serum for NHBE cells. In contrast, TGF-beta did not inhibit DNA synthesis of human lung carcinoma cells even though the cells possess comparable numbers of TGF-beta receptors with similar affinities for the factor. Epinephrine antagonized the TGF-beta-induced inhibition of DNA synthesis and squamous differentiation of NHBE cells. Although epinephrine increased the cyclic AMP levels in NHBE cells, TGF-beta did not alter the intracellular level in NHBE cells in either the presence or absence of epinephrine. Therefore, epinephrine and TGF-beta appear to affect different intracellular pathways that control growth and differentiation processes of NHBE cells.

Transgenesis by means of blastocyst-derived embryonic stem cell lines

Abstract: This study demonstrates that blastocyst-derived embryonic stem cells (ES cells) can be used as a vehicle for transgenesis. The method is nearly as efficient as other methods, and the introduced neomycin phosphotransferase (neo) gene is stably transmitted through several generations with no apparent loss in G418 resistance. An important factor contributing to the efficiency of this process is the rigorous selection, before blastocyst injection, of genetically transformed cells for in vitro developmental pluripotency. One of the advantages of the ES cell route to transgenesis is that it provides investigators with the opportunity to screen for the desired genetic alterations before reintroducing the ES cells into the animal.

Type beta transforming growth factor is an inhibitor of myogenic differentiation.

Abstract: We have investigated the effect of type beta transforming growth factor (TGF-beta) on the differentiation of skeletal muscle myoblasts. TGF-beta potently (ID50 approximately 10 pM) prevents established cell lines and primary cultures of rat and chicken embryo myoblasts from fusing into multinucleated myotubes. Inhibition of morphological differentiation by TGF-beta correlates with inhibition of the expression of muscle-specific mRNAs and proteins, strong induction of extracellular matrix type I collagen and fibronectin, and a marked tendency of the treated myoblasts to aggregate into densely multilayered arrays or clusters. Myogenic differentiation can resume after removal of TGF-beta from the medium. Examination of the time of action of TGF-beta shows that myoblasts stochastically reach a point beyond which they become insensitive to the inhibitory action of TGF-beta. This resistance of committed myoblasts to the inhibitory action of TGF-beta is not associated with any measurable change in the number or affinity of TGF-beta receptors in those cells. The results indicate that TGF-beta is a potent inhibitor of myogenesis and may regulate muscle development in vivo.

Stress protein systems of mammalian cells.

Abstract: Living organisms are known to react to a heat stress by the selective induction in the synthesis of several polypeptides. In this review we list the major stress proteins of mammalian cells that are induced by heat shock and other environments and categorize these proteins into specific subgroups: the major heat shock proteins, the glucose-regulated proteins, and the low-molecular-weight heat shock proteins. Characteristics of the localization and expression of proteins in each of these subgroups are presented. Specifically, the nuclear/nucleolar locale of certain of the major heat shock proteins is considered with respect to their association with RNA and the recovery of cells after a heat exposure. The induction of these major heat shock proteins and the repression of the glucose-regulated proteins as a result of reoxygenation of anoxic cells or by the addition of glucose to glucose-deprived cultures is described. Changes in the expression of these protein systems during embryogenesis and differentiation in mammalian and nonmammalian systems is summarized, and the protective role that some of these proteins appear to play in protecting the animal against the lethal effects of a severe heat treatment and against teratogenesis is critically examined.

Long-term cultures of HTLV-III--infected T cells: a model of cytopathology of T-cell depletion in AIDS.

Abstract: Long-term cultures were established of HTLV-III-infected T4 cells from patients with the acquired immune deficiency syndrome (AIDS) and of T4 cells from normal donors after infection of the cells in vitro. By initially reducing the number of cells per milliliter of culture medium it was possible to grow the infected cells for 50 to 60 days. As with uninfected T cells, immunologic activation of the HTLV-III-infected cells with phytohemagglutinin led to patterns of gene expression typical of T-cell differentiation, such as production of interleukin-2 and expression of interleukin-2 receptors, but in the infected cells immunologic activation also led to expression of HTLV-III, which was followed by cell death. The results revealed a cytopathogenic mechanism that may account for T4 cell depletion in AIDS patients and suggest how repeated antigenic stimulation by infectious agents, such as malaria in Africa, or by allogeneic blood or semen, may be important determinants of the latency period in AIDS.

Constitutive c- myc oncogene expression blocks mouse erythroleukaemia cell differentiation but not commitment

Abstract: Mouse erythroleukaemia cells (also called Friend cells) can be isolated from the spleen of certain strains of mice that have been infected with the Friend virus complex1. The cells resemble pro-erythroblasts and, when exposed to dimethyl sulphoxide (DMSO) or a variety of other chemicals, can be induced to undergo a programme of differentiation which closely resembles the final stages of normal erythropoiesis2. This includes the cessation of proliferation and large increases in the production of messenger RNA for both α- and β-globin. In addition, DMSO induces a rapid (<2h) decrease in c-myc mRNA levels3,4. The c-myc oncogene is expressed in the majority of proliferating normal cells5 and altered expression of the gene has been implicated in the genesis of a wide variety of tumours6–9. To study the influence of oncogene activation on differentiation, we have transfected viral-promoter-driven c-myc genes into mouse erythroleukaemia cells. Constitutive c-myc expression was found to block DMSO-induced differentiation.

Differential expression of myc family genes during murine development

Abstract: The myc family of cellular oncogenes contains three known members. The N-myc and c-myc genes have 5'-noncoding exons, strikingly homologous coding regions, and display similar oncogenic potential in an in vitro transformation assay. The L-myc gene is less well characterized, but shows homology to N-myc and c-myc (ref. 6; also see below). c-myc is expressed in most dividing cells, and deregulated expression of this gene has been implicated in the development of many classes of tumours. In contrast, expression of N-myc has been found only in a restricted set of tumours, most of which show neural characteristics; these include human neuroblastoma, retinoblastoma and small cell lung carcinoma (SCLC). L-myc expression has so far been found only in SCLC. Activated N-myc and L-myc expression has been implicated in oncogenesis; for example, although N-myc expression has been found in all neuroblastomas tested, activated (greatly increased) N-myc expression, resulting from gene amplification, is correlated with progression of the tumour. We now report that high-level expression of N- and L-myc is very restricted with respect to tissue and stage in the developing mouse, while that of c-myc is more generalized. Furthermore, we demonstrate that N-myc is not simply a neuroectoderm-specific gene; both N- and L-myc seem to be involved in the early stages of multiple differentiation pathways. Our findings suggest that differential myc gene expression has a role in mammalian development and that the normal expression patterns of these genes generally predict the types of tumours in which they are expressed or activated.

Induction of interleukin 2 receptor gene expression by p40x encoded by human T-cell leukemia virus type 1.

Abstract: Human T-cell leukemia virus type 1 (HTLV-1) is an etiologic agent of adult T-cell leukemia (ATL). A viral product, p40x, encoded by the pX sequence of HTLV-1 is a trans-acting transcriptional activator of the long terminal repeat (LTR) and has been suspected of involvement in leukemogenesis, activating the cellular genes. The cellular interleukin-2 (IL-2) and its receptor (IL-2R), the latter of which is expressed on ATL leukemic cells, were shown to be transiently induced by transfection of plasmid pMTPX expressing pX in two T-cell lines, Jurkat and HSB-2, but not in other human T- or B-cell lines. The cell type specificity of IL-2R induction by pX expression was the same as that by phytohaemagglutinin/phorbol ester activation, indicating the requirement for some specific cellular factors or a certain state of cellular differentiation. Induction of IL-2 and IL-2R at mRNA level was also demonstrated in transfected cells. Transfections with mutants of pMTPX in which the open reading frames for p40x, p27x-III and p21x-III were inactivated indicated that p40x alone was sufficient for induction of the IL-2R in inducible cells. This induction of the IL-2R by p40x of HTLV-1 may contribute to preferential proliferation of HTLV-1 infected cells at an early stage of ATL development and eventually increase the number of putative target cells for malignant transformation.

Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50.

Abstract: Two human B-cell differentiation antigens, Bp35 and Bp50, apparently play distinct roles as signal receptors in B-cell activation. Monoclonal antibodies (mAbs) to either Bp35 or Bp50 deliver positive signals to B cells that stimulate their transition through the cell cycle. mAb to Bp35, like anti-immunoglobulin antibodies, functions principally to activate resting B cells to become competent to enter the G1 phase of the cell cycle. In contrast, mAb to Bp50, a 50-kDa polypeptide expressed on all B cells, functions to stimulate activated B cells to traverse the cell cycle. mAb to Bp35, like anti-immunoglobulin antibodies, activates tonsillar B cells and induces low levels of B-cell proliferation. In contrast, anti-Bp50 mAb alone neither activates B cells nor induces B cells to proliferate but, together with anti-Bp35 or anti-immunoglobulin, augments B-cell proliferation. In this respect the action of anti-Bp50 antibody resembles the activity of B-cell growth factor(s) (BCGF). As little as 0.05 microgram of anti-Bp50 per ml is needed to augment proliferation and, like BCGF, anti-Bp50 is effective even when added 12-24 hr after B cells are activated with anti-immunoglobulin or anti-Bp35. Without additional exogenous signals, anti-Bp35 and anti-Bp50 together induce strong proliferation of purified resting B cells. These results suggest that the Bp35 and Bp50 surface molecules function in the regulatory control of B-cell activation and progression through the cell cycle.

Differentiation of mammalian embryonic gonad

Abstract: Formation and differentiation of a gonad depend on finely controlled interactions between germ cells and various types of somatic cells. These interactions already begin when the germ cells start migrating toward the gonadal ridge. Reaching the presumptive gonadal area on the mesonephros, the germ cells join with the mesonephric-derived cells. These mesonephric cells are probably the precursors of the steroid-producing cells. A crucial event for gonadal function is the enclosure of germ cells and somatic cells in specific germ cell compartments. Survival and differentiation of the germ cells depend on this separation. Differentiation of the steroid-producing cells depends in turn on remaining outside the cell compartments. The mechanisms directing the gonad to develop into a testis or an ovary are still obscure, but specific gene products from the sex chromosomes probably play a basic role in gonadal sex differentiation.

Expression of smooth muscle-specific alpha-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation.

Abstract: The relationship between growth and cytodifferentiation was studied in cultured rat aortic smooth muscle cells (SMCs) using expression of the smooth muscle (SM)-specific isoactins (Vanderkerckhove, J., and K. Weber, 1979, Differentiation, 14:123-133) as a marker for differentiation in these cells. Isoactin expression was evaluated by: (a) measurements of fractional isoactin content and synthesis ([35S]methionine incorporation) by densitometric evaluation of two-dimensional isoelectric focusing sodium dodecyl sulfate gels, and (b) immunocytological examination using SM-specific isoactin antibodies. Results showed the following: (a) Loss of alpha-SM isoactin was not a prerequisite for initiation of cellular proliferation in primary cultures of rat aortic SMCs. (b) alpha-SM isoactin synthesis and content were low in subconfluent log phase growth cells but increased nearly threefold in density-arrested postconfluent cells. Conversely, beta-nonmuscle actin synthesis and content were higher in rapidly dividing subconfluent cultures than in quiescent postconfluent cultures. These changes were observed in primary and subpassaged cultures. (c) alpha-SM actin synthesis was increased by growth arrest of sparse cultures in serum-free medium (SFM; Libby, P., and K. V. O'Brien, 1983, J. Cell. Physiol., 115:217-223) but reached levels equivalent to density-arrested cells only after extended periods in SFM (i.e., greater than 5 d). (d) SFM did not further augment alpha-SM actin synthesis in postconfluent SMC cultures. (e) Serum stimulation of cells that had been growth-arrested in SFM resulted in a dramatic decrease in alpha-SM actin synthesis that preceded the onset of cellular proliferation. These findings demonstrate that cultured vascular SMCs undergo differential expression of isoactins in relation to their growth state and indicate that growth arrest promotes cytodifferentiation in these cells.

Oncostatin M: a growth regulator produced by differentiated histiocytic lymphoma cells.

Abstract: A polypeptide termed oncostatin M, which inhibits the replication of A375 melanoma and other human tumor cells, but not normal human fibroblasts, has been isolated from serum-free supernatants of U-937 histiocytic lymphoma cells that have been induced to differentiate into macrophage-like cells following treatment with the phorbol ester phorbol 12-myristate 13-acetate. No such growth inhibitory activity is detected in the supernatant of untreated U-937 cells, indicating that the protein is induced or increased in expression in the phorbol ester-induced differentiated cells. Oncostatin M is stable between pH 2 and 11 and after heating for 1 hr at 56 degrees C but is not stable at 90 degrees C. Purification of oncostatin M has been achieved by gel chromatography and reversed-phase HPLC, using sequentially acetonitrile and n-propanol in the presence of aqueous trifluoroacetic acid. The apparent molecular weight of oncostatin M is approximately 18,000, as determined by gel chromatography, and 28,000, as determined by polyacrylamide gel electrophoresis. The amino-terminal amino acid sequence of the purified polypeptide has been determined. No substantial sequence homology between oncostatin M and other proteins was found, including other tumor cell inhibitory proteins produced by mononuclear cells. Oncostatin M, therefore, appears to represent a distinct cell growth regulator.

Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21

Abstract: The protein products (p21) of the ras cellular proto-oncogenes are thought to transduce membrane signals necessary for the induction of cell division. However, there is uncertainty as to the precise role of ras p21 in mediating ligand-membrane receptor signals leading to cell differentiation. Treatment of rat phaeochromocytoma cells (PC12) with nerve growth factor (NGF) results in the induction of a number of phenotypic characteristics of sympathetic neurones, including cessation of cell division and outgrowth of neuronal processes (neurites). Here we report that microinjection of antibody to ras p21 into PC12 cells inhibited neurite formation and resulted in temporary regression of partially extended neurites, an effect which was observed up to 36 h after initiation of NGF treatment. Neurite formation induced by cyclic AMP was unaffected by injection of anti-p21 antibody. These results indicate that p21 is involved in the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in hormone-mediated cellular responses distinct from cell proliferation.