Differential effects of 3,5-T2 and T3 on the gill regeneration and metamorphosis of the Ambystoma mexicanum (axolotl).

Thyroid hormones (THs) regulate tissue remodeling processes during early- and post-embryonic stages in vertebrates. The Mexican axolotl (Ambystoma mexicanum) is a neotenic species that has lost the ability to undergo metamorphosis; however, it can be artificially induced by exogenous administration of thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3). Another TH derivative with demonstrative biological effects in fish and mammals is 3,5-diiodo-L-thyronine (3,5-T2). Because the effects of this bioactive TH remains unexplored in other vertebrates, we hypothesized that it could be biologically active in amphibians and, therefore, could induce metamorphosis in axolotl. We performed a 3,5-T2 treatment by immersion and observed that the secondary gills were retracted, similar to the onset stage phenotype; however, tissue regeneration was observed after treatment withdrawal. In contrast, T4 and T3 immersion equimolar treatments as well as a four-fold increase in 3,5-T2 concentration triggered complete metamorphosis. To identify the possible molecular mechanisms that could explain the contrasting reversible or irreversible effects of 3,5-T2 and T3 upon gill retraction, we performed a transcriptomic analysis of differential expression genes in the gills of control, 3,5-T2-treated, and T3-treated axolotls. We found that both THs modify gene expression patterns. T3 regulates 10 times more genes than 3,5-T2, suggesting that the latter has a lower affinity for TH receptors (TRs) or that these hormones could act through different TR isoforms. However, both TH treatments regulated different gene sets known to participate in tissue development and cell cycle processes. In conclusion, 3,5-T2 is a bioactive iodothyronine that promoted partial gill retraction but induced full metamorphosis in higher concentrations. Differential effects on gill retraction after 3,5,-T2 or T3 treatment could be explained by the activation of different clusters of genes related with apoptosis, regeneration, and proliferation; in addition, these effects could be initially mediated by TRs that are expressed in gills. This study showed, for the first time, the 3,5,-T2 bioactivity in a neotenic amphibian.

Insulin-like growth factor 1-coated sutures improve anastomotic healing in an experimental model of colitis.

BACKGROUND: : Exogenously applied insulin-like growth factor (rhIGF-1) may improve normal intestinal healing. This study examined the effect of rhIGF-1-coated sutures on anastomotic healing in experimental colitis. METHODS: : Acute colitis was induced in rats by dextran sodium sulphate (DSS). Inflammation was assessed by clinical Disease Activity Index (DAI), myeloperoxidase (MPO) measurement and histological examination. A distal colonic anastomosis was performed using sutures coated with rhIGF-1 dissolved in poly(D,L-lactide) (PDLLA) under general anaesthetic. Anastomotic healing was evaluated histologically, and by hydroxyproline measurement and bursting parameters after 1, 3 and 7 days, and compared with healthy, DSS and DSS + PDLLA controls. RESULTS: : DAI, MPO and histological inflammation scores were significantly increased in all animals treated with DSS. Bursting occurred less often within the anastomotic line on day 3 in the IGF group than in DSS controls (three versus eight of ten). On day 7, the IGF group had significantly increased histological healing scores (mean(s.e.m.) 12.5(0.7) versus 9.2(0.8) (P < 0.050)) and hydroxyproline content (4.6(0.3) versus 3.6(0.1) mg/g tissue; P < 0.050) compared with DSS controls. CONCLUSION: : IGF-1-coated sutures improve important aspects of anastomotic healing in rats with experimental colitis.

Cell-to-cell binding induced by different lectins.

The cell-to-cell binding induced by concanavalin A (Con A) and the lectins from wheatgerm, soybean, and waxbean has been analyzed by measuring the ability of single cells to bind to lectin-coated cells immobilized on nylon fibers. The cells used were lymphoma, myeloid leukemia, and normal fibroblast cells. With all lectins, cell-to-cell binding was inhibited if both cells were prefixed with glutaraldehyde. However, in most cases cell-to-cell binding was enhanced when only the lectin-coated cell was prefixed. With normal fibroblasts, treatment of either one or both cells with trypsin enhanced the cell-to-cell binding induced by Con A and the wheatgerm lectin. Neuraminidase, which increases the number of receptors for soybean agglutinin, increased cell-to-cell binding only if both cells were treated. Although cell-to-cell binding induced by the lectins from soybean and wheatgerm could be partially reversed by the appropriate competitive saccharide inhibitor, binding induced by Con A could not be reversed. The experiments indicate that cell-to-cell binding induced by a lectin can be prevented by an insufficient density of receptors for the lectin, insufficient receptor mobility, or induced clustering of receptors. These effects can explain the differences in cell-to-cell binding and agglutination observed with different cell types and lectins. They also suggest that cell-to-cell binding induced by different lectins with a variety of cell types is initiated by a mechanism involving the alignment of complementary receptors on the colliding cells for the formation of multiple cell-to-lectin-to-cell bridges.

Receptor mobility and the binding of cells to lectin-coated fibers.

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation

Formation of bone tissue in culture from isolated bone cells.

A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.

The molecular control of blood cell development.

The establishment of a cell culture system for the clonal development of blood cells has made it possible to identify the proteins that regulate the growth and differentiation of different blood cell lineages and to discover the molecular basis of normal and abnormal cell development in blood forming tissues. A model system with myeloid blood cells has shown that (i) normal blood cells require different proteins to induce cell multiplication (growth inducers) and cell differentiation (differentiation inducers), (ii) there is a hierarchy of growth inducers as cells become more restricted in their developmental program, and (iii) a cascade of interactions between proteins determines the correct balance between immature and mature cells in normal blood cell development. Gene cloning has shown that there is a family of different genes for these proteins. Normal protein regulators of blood cell development can control the abnormal growth of certain types of leukemic cells and suppress malignancy by inducing differentiation to mature nondividing cells. Chromosome abnormalities that give rise to malignancy in these leukemic cells can be bypassed and their effects nullified by inducing differentiation, which stops cells from multiplying. These blood cell regulatory proteins are active in culture and in the body, and they can be used clinically to correct defects in blood cell development.

Epigenetics and the plasticity of differentiation in normal and cancer stem cells.

Embryonic stem cells are characterized by their differentiation to all cell types during embryogenesis. In adult life, different tissues also have somatic stem cells, called adult stem cells, which in specific niches can undergo multipotent differentiation. The use of these adult stem cells has considerable therapeutic potential for the regeneration of damaged tissues. In both embryonic and adult stem cells, differentiation is controlled by epigenetic mechanisms, and the plasticity of differentiation in these cells is associated with transcription accessibility for genes expressed in different normal tissues. Abnormalities in genetic and/or epigenetic controls can lead to development of cancer, which is maintained by self-renewing cancer stem cells. Although the genetic abnormalities produce defects in growth and differentiation in cancer stem cells, these cells have not always lost the ability to undergo differentiation through epigenetic changes that by-pass the genomic abnormalities, thus creating the basis for differentiation therapy. Like normal stem cells, cancer stem cells can show plasticity for differentiation. This plasticity of cancer stem cells is also associated with transcription accessibility for genes that are normally expressed in different tissues, including tissues other than those from which the cancers originated. This broad transcription accessibility can also contribute to the behavior of cancer cells by overexpressing genes that promote cell viability, growth and metastasis.

Autoregulation of interleukin 6 and granulocyte-macrophage colony-stimulating factor in the differentiation of myeloid leukemic cells.

Induction of differentiation in one type of clone of mouse myeloid leukemic cells by mouse or human interleukin 6 (IL-6) and in another type of clone by mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to be associated with induction of IL-6 and GM-CSF mRNA and protein. The results indicated that IL-6 and GM-CSF could positively autoregulate their own gene expression during myeloid cell differentiation. It is suggested that this autoregulation may serve to enhance and prolong the signal induced by these proteins in cells transiently exposed to IL-6 or GM-CSF.

Control of hematopoietic cell growth regulators during mouse fetal development.

Gene expression for the four different growth-regulatory proteins for cells of the myeloid hematopoietic cell lineages was analyzed in mouse fetal and extraembryonic tissues at various stages of development. The macrophage growth inducer MGI-1M (colony-stimulating factor 1) was the only myeloid hematopoietic growth regulator detected as both mRNA and bioactive protein during fetal development. This regulator was produced predominantly in extraembryonic tissues, and the production of hematopoietic growth regulators in embryogenesis was regulated by transcriptional and posttranscriptional controls.

The oncogenic potential of an activated Hox-2.4 homeobox gene in mouse fibroblasts.

The homeobox gene Hox-2.4 is transcriptionally activated in cells of the mouse myeloid leukemia WEHI-3B. The constitutive Hox-2.4 expression in WEHI-3B cells is due to insertion of a transposable element belonging to the family of intracisternal A particles. In this study, we demonstrated the oncogenic potential of this activated homeobox gene. NIH 3T3 fibroblast clones bearing the activated Hox-2.4 gene produced fibrosarcomas in nude mice.

Differential transcriptome regulation by 3,5-T2 and 3',3,5-T3 in brain and liver uncovers novel roles for thyroid hormones in tilapia.

Although 3,5,3'-triiodothyronine (T3) is considered to be the primary bioactive thyroid hormone (TH) due to its high affinity for TH nuclear receptors (TRs), new data suggest that 3,5-diiodothyronine (T2) can also regulate transcriptional networks. To determine the functional relevance of these bioactive THs, RNA-seq analysis was conducted in the cerebellum, thalamus-pituitary and liver of tilapia treated with equimolar doses of T2 or T3. We identified a total of 169, 154 and 2863 genes that were TH-responsive (FDR < 0.05) in the tilapia cerebellum, thalamus-pituitary and liver, respectively. Among these, 130, 96 and 349 genes were uniquely regulated by T3, whereas 22, 40 and 929 were exclusively regulated by T2 under our experimental paradigm. The expression profiles in response to TH treatment were tissue-specific, and the diversity of regulated genes also resulted in a variety of different pathways being affected by T2 and T3. T2 regulated gene networks associated with cell signalling and transcriptional pathways, while T3 regulated pathways related to cell signalling, the immune system, and lipid metabolism. Overall, the present work highlights the relevance of T2 as a key bioactive hormone, and reveals some of the different functional strategies that underpin TH pleiotropy.

Enhancement of hormone action by a phorbol ester and anti-tubulin alkaloids involves different mechanisms.

The tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhanced 1-isoproterenol and prostaglandin E1 stimulated cyclic AMP formation in clones of mouse myeloid leukemic cells. The enhancement was found up to 3h after TPA treatment and had disappeared after 24h, indicating its reversibility. The effect of TPA was not inhibited by removal of extracellular Ca2+ or pre-treatment with the calcium ionophore A23187. This enhancement by TPA seems to involve a different pathway than enhancement of response to the same hormones after treatment with the anti-tubulin alkaloids colchicine or vinblastine, since a myeloid leukemic cell mutant clone that was non-responsive to the anti-tubulin alkaloids responded to TPA. Furthermore, combined treatment of colchicine-sensitive cells with TPA and colchicine showed an additive stimulating effect. The enhancement of cell response to hormones by TPA was found in myeloid leukemic cell clones that either were or were not induced to differentiate after treatment with TPA. This suggests that enhancement of the effect of these and possibly other hormones by TPA may be an initial step of TPA action, but that this enhancement is not sufficient to induce the wide repertoire of TPA effects including induction of differentiation.

A highly specific aminotripeptidase of rat brain cytosol. Substrate specificity and effects of inhibitors.

An aminopeptidase preferentially hydrolyzing Leu- or Ala-Gly-Gly was purified from rat brain cytosol and detailed studies have been performed on its substrate specificity and the effects of inhibitors. The enzyme was devoid of di- and oligopeptidase contamination. Biologically active tripeptides such as Met-Leu-Tyr (chemotactic factor), Gly-His-Lys (liver growth factor) and Thr-Val-leu central nervous system tripeptide) were hydrolyzed at rates 0.05-0.15-times that of Leu-Gly-Gly. Melanostatin (Pro-Leu-GlyNH2) did not serve as a substrate. Substrates bearing N-terminal charged groups, or ones with proline in positions 2 or 3, or those with D-amino acid in positions 1 or 2, or with C-terminal CONH2, were poorly hydrolyzed or did not act as substrates, providing information on subsites involved in enzyme catalysis. The enzyme was inhibited competitively by bestatin (Ki 10-7 M) and by Captopril (2.5.10-7 M, D-3-thio-2-methylpropanyl proline) and by low concentrations of Zn2+ or PCMB, and at higher concentrations by TPCK and PMSF. Inhibition was observed for the chemotactic factor (I50 13 microM) and for the central nervous system tripeptide (195 microM). The enhanced action of Captopril was attributed to the presence of the -SH and -CH3 groups, since inhibition was shared by di- and tripeptides with proline in positions 2 and 3. The specificity pattern of brain enzyme was different from that reported for kidney and intestine.

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells.

Phospholipase A2 activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A2 activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-3H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7-14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E1 did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A2 activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A2, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A2 activity was also related to previously found differences in the ability of the clones to develop desensitization to beta-adrenergic hormones or prostaglandin E1.

Characterization of macrophage- and granulocyte-inducing proteins for normal and leukemic myeloid cells produced by the Krebs ascites tumor.

Medium from serum-free cultures of Krebs ascites tumor cells contains two macrophage and granulocyte inducing (MGI) activities that can act on the myeloid precursors of these hematopoietic cells. One activity, MGI-1, induced the formation of macrophage and granulocyte colonies from normal myeloid precursors. The second activity, MGI-2, induced macrophage and granulocyte differentiation in myeloid leukemic cells that no longer required MGI-1 for colony formation. The medium contained one species of MGI-1 and two species of MGI-2. One species of MGI-2, MGI-2A, copurified through five stages of purification with MGI-1, but separated from the other MGI-2 species, MGI-2B, at an early stage in purification. MGI-1, MGI-2A and MGI-2B were purified 1490, 1140 and 678-fold, respectively. When bands with biological activity gel from non-denaturing polyacrylamide gels were run on SDS-polyacrylamide gel electrophoresis, MGI-1 and MGI-2A activities were associated with similar Mr and each activity showed two bands, one of 23 000 and the other 25 000. MGI-2B activity showed one band with a Mr of 45 000. Secretion did not appear to involve glycosylation, none of the species bound to concanavalin A, soybean agglutinin, or wheat germ agglutinin agarose columns and they did not appear to contain carbohydrates. The assays for MGI-1 and MGI-2 activities were not affected by adding protease inhibitors. But MGI-2 was more readily destroyed by treatment with proteases and was more labile at high temperature and low pH than MGI-1. It is suggested that the level of cellular proteases may play a role in regulating the relative amounts of MGI-1 and MGI-2 that are present in vivo.

Ion metabolism in a Halobacterium. I. Influence of age of culture on intracellular concentrations.

Work is described on the changes in cell ions during growth of cultures of a species of Halobacterium isolated from the Dead Sea. Cell K concentration fell from 5.5 to 3.8 moles per kg cell water during the logarithmic phase of growth and maintained the latter value during the stationary phase (initial medium concentration, 7 mM). Cell Na and Cl followed a complex series of roughly parallel changes. The logarithmic phase ion concentrations were: Na, 1.0-2.3 moles/kg cell water; Cl, 2.3-3.7 moles/kg cell water. The final stationary phase values were: Na, 0.5 moles/kg cell water; Cl, 2.3-2.9 moles/kg cell water (medium NaCl concentration, 3.9 Molal). It is suggested that most of the K(+) is bound within the cytoplasm.

Control of in vivo differentiation of myeloid leukemic cells.

The differentiation of leukemic cells in vivo can be a useful approach to therapy. In vivo differentiation of myeloid leukemic cells was studied in intraperitoneally implanted diffusion chambers, containing different soluble antigens. The presence of these antigens in the chambers induced differentiation of myeloid leukemic cells and this was inhibited in immune-deficient mice. Transfer of normal spleen cells enriched for T-lymphocytes or antigen-specific helper T lymphocyte cell lines to mice in which differentiation of leukemic cells was inhibited, restored in vivo differentiation of the leukemic cells. Antigen-specific helper T cells produce myeloid regulatory proteins and can accumulate at a site that contains the specific antigen. It is suggested that migration in response to antigen of helper T cells producing regulatory proteins may play an important role in inducing in vivo differentiation of leukemic cells. We have identified a class of myeloid leukemic cells that can be induced to differentiate in vitro by incubation with pure MGI-1GM (GM-CSF) or IL-3, but not with MGI-1G (G-CSF). Experiments with pure recombinant proteins have shown that MGI-1GM and IL-3, but not MGI-1G, can also induce these myeloid leukemic cells to differentiate in vivo. These results and our previous studies on the myeloid cell differentiation-inducing protein MGI-2, demonstrate the potential use of normal hematopoietic regulatory proteins not only in regulation of normal hematopoiesis, but also in the treatment of myeloid leukemia by in vivo induction of terminal cell differentiation.

Selective regulation by hydrocortisone of induction of in vivo differentiation of myeloid leukemic cells with granulocyte-macrophage colony-stimulating factor, interleukin 6 and interleukin 1 alpha.

Clones of myeloid leukemic cells can differ in their ability to be induced to differentiate in vitro by different cytokines. Using such leukemic clones, we studied the regulation by hydrocortisone of induction of in vivo differentiation by injection of recombinant interleukin 6 (IL-6), interleukin 1 alpha (IL-1 alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Injection of IL-6 and IL-1 alpha induced in vivo differentiation of leukemic cells that were induced to differentiate by these cytokines in vitro, but not of leukemic cells that were not susceptible to these cytokines in vitro. In contrast, injection of GM-CSF induced in vivo differentiation both in leukemic cells that were susceptible or not susceptible to GM-CSF in vitro. The effect of GM-CSF, but not of IL-6 or IL-1 alpha, on inducing differentiation in vivo was inhibited by pretreatment with hydrocortisone. In leukemic cells that were not induced to differentiate with GM-CSF in vitro, this inhibition of differentiation by pretreatment with hydrocortisone was greater than inhibition of differentiation obtained by pretreatment with cyclophosphamide or irradiation or the use of nude mice. After hydrocortisone pretreatment, the number of peritoneal cells and their ability to produce GM-CSF and IL-6 were suppressed. It is suggested that hydrocortisone can inhibit the effect of an injected cytokine such as GM-CSF on induction of in vivo differentiation of leukemic cells by inhibiting the ability of host cells to produce cytokines to which the leukemic cells are susceptible.

Interferon-gamma inhibits apoptosis induced by wild-type p53, cytotoxic anti-cancer agents and viability factor deprivation in myeloid cells.

Different hematopoietic cytokines including colony-stimulating factors and interleukins can inhibit apoptotic cell death induced in myeloid cells by the tumor-suppressor gene wild-type 53 and a variety of cytotoxic anti-cancer agents. In this study we identity interferon-gamma as an anti-apoptotic cytokine for myeloid cells in which apoptosis was induced by wild-type p53, cytotoxic anti-cancer agents or viability factor deprivation. The inhibition of wild-type p53-mediated apoptosis in myeloid leukemic cells by interferon-gamma was not associated with downregulated expression of wild-type p53 or the p53-induced cyclin-dependent kinase inhibitor gene WAF-1, or with upregulated expression of the apoptosis-inhibiting gene bcl-2. Interferon-gamma also inhibited induction of apoptosis by a p53-independent pathway. Interferon-gamma inhibited apoptotic cell death caused by withdrawal of viability factors in normal myeloid precursor cells, the interleukin 3-dependent 32D cell line and differentiating myeloid leukemic cells. Interferon-alpha/beta did not inhibit apoptotic cell death in any of these systems. The results indicate that although interferon-gamma can inhibit cell multiplication and differentiation in myeloid cells, it shares with other hematopoietic cytokines the ability to protect normal and leukemic myeloid cells from induction of apoptosis.

Control of apoptosis in hematopoiesis and leukemia by cytokines, tumor suppressor and oncogenes.

Hematopoietic cells require certain cytokines including colony-stimulating factors and interleukins to maintain viability. Without these cytokines the program of apoptotic cell death is activated. Cells from many myeloid leukemias require cytokines for viability, and apoptosis is also activated in these leukemic cells after cytokine withdrawal resulting in reduced leukemogenicity. The same cytokines protect normal and leukemic cells from induction of apoptosis by irradiation and cytotoxic chemotherapeutic compounds. This suggests that decreasing the levels of viability inducing cytokines may increase the effectiveness of cytotoxic anti-cancer therapy. The susceptibility of normal and cancer cells to induction of apoptosis is also regulated by the balance between apoptosis-inducing genes such as the tumor suppressor wild-type p53, and c-myc and bax, and apoptosis-suppressing genes such as the oncogene mutant p53, and bcl-2 and bcl-XL. Cell susceptibility to induction of apoptosis in leukemic cells could be enhanced by increased expression of apoptosis-inducing genes and/or decreased expression of apoptosis-suppressing genes. Modulation of expression of apoptosis-regulating genes should thus also be useful for improvement of anti-cancer therapy.