Alteration of myoblast phenotype by dimethyl sulfoxide.

Application of dimethyl sulfoxide to proliferating L8 myoblasts (an established cell line of rat skeletal muscle) for 72 hr completely prevented fusion and induction of creatine phosphokinase (EC 2.7.3.2) activity (an indicator of muscle differentiation). The growth pattern changed from the usual sheets of randomly oriented cells to flattened, whorled monolayers of elongated fibroblast-like cells. By electron microscopy, rough endoplasmic reticulum increased and extracellular material appeared that had the morphologic and staining characteristics of collagen. After 120 hr in dimethyl sulfoxide-containing medium, the cells secreted about 6 times more collagen than untreated controls. Dimethyl sulfoxide was ineffective when applied to L8 cells just prior to fusion, and effects of dimethyl sulfoxide were not readily reversible unless treated cells were subcultured at low density.     

Metalloendoprotease inhibitors that block fusion also prevent biochemical differentiation in L6 myoblasts.

The effect of metalloendoprotease inhibitors on the biochemical differentiation of the rat skeletal muscle line, L6, was investigated. Confluent unfused L6 cells exposed briefly to 1,10-phenanthroline, a chelator of divalent metal cations, or continuously to dipeptide amide metalloendoprotease substrates that are blocked at the NH2-terminals, N-carbobenzyloxyserylleucyl amide and N-carbobenzyloxyglycylleucyl amide, did not fuse or express creatine kinase, myosin heavy chain, or alpha-actin. These effects were reversible and dose-dependent. Exposure to N-carbobenzyloxylglycylglycyl amide, which is not a metalloendoprotease inhibitor, had no effect. As the differentiation in a culture progressed, 1,10-phenanthroline became less effective in blocking the accumulation of creatine kinase and myosin heavy chain. Exposure of partially fused cultures to N-carbobenzyloxyserylleucyl amide prevented any further accumulation of muscle-specific proteins. In confluent cultures where cell division was blocked before the onset of differentiation, N-carbobenzyloxyserylleucyl amide still prevented fusion and the induction of creatine kinase. This indicates that these inhibitors do not act by interfering with the cell cycle. Experiments that measured DNA synthesis rates, plating efficiencies, and the effects of sequential dipeptide and dimethyl sulfoxide treatments indicate that L6 myoblasts do not irreversibly withdraw from the cell cycle when exposed to N-carbobenzyloxyserylleucyl amide. These results are consistent with the role of a metalloendoprotease in initiating the terminal differentiation of cultured muscle cells.     

Human--rat muscle somatic cell hybrids form myotubes and express human muscle gene products.

Somatic cell hybrids have been prepared at high frequency between the rat muscle cell line L6 and human fetal muscle cells. The hybrid cells express several human gene products including an antigen, 12E7, controlled by the human X chromosome, Thy-1, and several human isoenzymes. In addition, one clone (37-11) expresses a human muscle-specific surface antigen (5.1 H11) and, under appropriate conditions, can be induced to form myotubes. Upon myotube formation, this clone switches on the production of the human muscle-specific creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) M subunit. This is an example of developmental regulation of human muscle-specific genes in somatic cell hybrids.     

Antiretroviral protease inhibitors prevent l6 muscle cell fusion by reducing calpain activity

The antiretroviral protease inhibitors indinavir (IDV) and ritonavir (RTV) are used in highly active antiretroviral therapies (HAART). Side effects from long-term HAART therapy include loss of muscle mass. Myoblasts when cultured in media low in growth factors withdraw from the cell cycle, express muscle-specific differentiation inducers and proteins, and fuse to form myotubes. The neutral protease, calpain, is required for myotube formation and RTV decreased calpain activity in vitro. We found lower calpain activity, but not protein, in homogenates of RTV-treated L6 cells than in control cultures. Importantly, L6 and C2C12 myoblasts did not form myotubes when cultured with 10 or 20 microM IDV or RTV. Control and drug-related L6 myoblasts showed identical decreases in proliferating cell nuclear antigens expression indicating proliferation arrest. Similarly, muscle differentiation inducers MyoD and myogenin and their downstream target, myosin heavy chain, were expressed at similar levels in control and drug-treated cells. Thus, whereas muscle differentiation was unaffected by protease inhibitors, calpain activity was reduced and myotube formation prevented. We conclude that RTV and IDV reduced myotube formation by reducing calpain activity. Our data suggest that protease inhibitors included in HAART might be directly involved in muscle wasting by reducing muscle remodeling.     

The development of insulin receptors and responsiveness is an early marker of differentiation in the muscle cell line L6

After reaching confluence, mononucleated L6 myoblasts fuse into multinucleated contracting myotubes. This process is accompanied by the synthesis of characteristic skeletal muscle proteins, such as myosin heavy chain and the MM isoenzyme of creatine kinase. We have studied the development of insulin receptors and insulin responsiveness during differentiation in the L6 cells. Insulin was bound to high affinity receptors in both myoblasts and differentiated myotubes. The binding showed characteristics typical for insulin binding in other cell types, including high affinity, appropriate specificity, an upwardly concave Scatchard plot, and down-regulation. In the logarithmic growth phase, the myoblasts exhibited a low level of insulin binding, but on initiation of cell fusion, the resulting myotubes progressively developed a 2-fold increase in specific [125I]iodoinsulin binding as a result of a 2-fold increase in receptor number. The increase in insulin binding was an early differentiation event, preceding the accumulation of creatine kinase by 24 h. The development of insulin binding during differentiation correlated closely with an increased ability of the hormone to stimulate maximal 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake at physiological concentrations. The L6 cells are a useful model for studying the binding and effects of physiological insulin concentrations in skeletal muscle before and after differentiation.     

Temperature-Sensitive Variants of an Established Myoblast Line

Upon reaching confluency, mononucleated myoblasts fuse into multinucleated myotubes and concomitantly accumulate various characteristic muscle proteins, including myosin, actin, and several enzymes. We have approached the problem of determining the relationship between morphological and biochemical differentiation of muscle cells by isolating a series of temperature-sensitive clones from the established myoblast line, L(6).Twelve phenotypically variant clones were isolated from mutagenized populations of myoblasts. These fell into five classes, distinguishing conditional growth variants from conditional developmental variants. The phenotype of these strains, at least for the more extensively studied ones, was stable for more than 80 generations.Synthesis of characteristic proteins such as myosin, glycogen phosphorylase (EC 2.4.1.1), and phosphocreatine kinase (EC 2.7.3.2) has been studied in two conditional developmental mutants. One mutant, E(3), fuses into myotubes at 37 degrees but not at 40 degrees ; the other, H(6), does not fuse into myotubes at 37 degrees but does so at 40 degrees . At permissive temperatures the enzymes accumulated in mutant cells with the same time course as in the parent cell line. Myosin accumulated in strain E(3) but not in strain H(6). At nonpermissive temperatures neither fusion into myotubes nor accumulation of any of the proteins occured in the cells of these two variant lines.     

Is the regulation of the expression of MMTV proviruses in various cell types linked to glucocorticoid receptors and/or the structure of chromatin?

The expression of the mouse mammary tumor virus (MMTV) was studied in undifferentiated embryonal carcinoma cells (EC), in partially differentiated myoblasts derived from EC cells and in fully differentiated myotubes. Whereas no MMTV RNA could be detected in EC cells, relatively large amounts of tumor virus RNA were found in myoblasts. The MMTV RNA level was reduced in myotubes derived from myoblastic differenciation. DNaseI sensibility of MMTV DNA in chromatin allows us to show conformational changes in EC cell myoblasts and GR mammary cell chromatin which could be associated with MMTV RNA synthesis. The glucocorticoid hormones dexamethasone which stimulates the MMTV RNA synthesis in differentiated mammary cells did not affect this synthesis in myoblasts. By contrast, the reduced synthesis of MMTV RNA in myotubes was overcome with dexamethasone. The differential MMTV RNA synthesis is not related to different amount of receptor and its hormonal regulation is not associated with a conformational change of chromatin.     

Autocrine nerve growth factor is essential for cell survival and viral maturation in HHV-8-infected primary effusion lymphoma cells

High levels of nerve growth factor (NGF) are found in sera from individuals infected with human herpesvirus 8 (HHV-8). BC-1 and BCBL-1 cells are primary effusion lymphoma-derived B-cell lines; BC-1 cells are infected by HHV-8 and the Epstein-Barr virus (EBV), and BCBL-1 cells are infected only by HHV-8. Both cells express NGF receptors and produce NGF, whereas RAMOS cells (a B-cell line that is negative for HHV-8 and EBV) express NGF receptors but do not produce detectable NGF. Neutralization of endogenous NGF results in cell growth inhibition and apoptosis in BCBL-1 cells and, to a minor extent, in BC-1 cells. When the HHV-8 lytic cycle is induced in BCBL-1 cells by tetradecanoyl phorbol acetate (TPA), an initial reduction of endogenous NGF production is observed, and many cells undergo apoptosis. However, at 48 hours, TPA-treated cells produce significantly more NGF than untreated controls, and a subsequent recovery of cell viability is observed. Consistent with this finding, the addition of exogenous NGF or anti-NGF antibodies to TPA-treated cells reduces or increases, respectively, the rate of apoptosis in response to TPA. Finally, electron microscopy of TPA-treated BCBL-1 cells shows that the addition of exogenous NGF increases the number of cells producing and releasing complete virions as compared with the controls (25% versus 5%). On the contrary, NGF neutralization leads to the production of defective viral progeny in about 2% of cells. These data indicate that NGF is essential for both cell survival and virus maturation in HHV-8-infected cell lines. (Blood. 2000;95:2905-2912)     

GLUT4 facilitates insulin stimulation and cAMP-mediated inhibition of glucose transport.

The glucose transporter isoform GLUT4 is found only in cells that exhibit insulin-sensitive glucose transport. To investigate the function of this transporter, L6 myoblasts were stably transfected with GLUT4 cDNA. GLUT4 underwent insulin-dependent movement to the cell surface in myoblasts overexpressing the transporter. One cell line (243-6) expressed sufficient levels of the GLUT4 protein to study insulin-dependent glucose transport. Unlike wild-type L6 cells, 243-6 myoblasts exhibited two features that are characteristic of differentiated muscle fibers and adipocytes in vivo: a large insulin-stimulated component of glucose transport and inhibition of this stimulated component by cAMP. Relative to normal L6 cells, 243-6 cells responded to insulin or insulin-like growth factor 1 with a 5-fold larger increase in 2-deoxy[3H]glucose uptake. N6,O2'-Dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) did not inhibit transport in normal L6 myoblasts, which express only GLUT1, but inhibited IGF-1/insulin-stimulated transport by 50% in 243-6 cells. The effect of cAMP was investigated further by using Chinese hamster ovary cells transiently expressing GLUT1 and GLUT4. Bt2cAMP inhibited glucose transport only in Chinese hamster ovary cells expressing GLUT4. These results indicate that cAMP-mediated inhibition of glucose transport is dependent on expression of the GLUT4 isozyme.     

DNA-transformed murine teratocarcinoma cells: regulation of expression of simian virus 40 tumor antigen in stem versus differentiated cells.

Thymidine kinase-deficient (TK-; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21)F9 teratocarcinoma stem cells have been transformed with a recombinant plasmid genome consisting of the pBR322 genome linked to a herpes simplex virus type 1 thymidine kinase gene (HSV-1 tk) and a simian virus 40 (SV40) genome. A clonal line of stem cells was obtained that contains only one copy of plasmid DNA, which is integrated into murine chromosomal DNA through a site on the pBRR322 genome. The HSV-1 tk gene, which is adjacent to the SV40 genome, is expressed in stem cells, whereas SV40 gene expression is not detectable. If differentiation of these stem cells is induced, the differentiated cells express SV40 early gene products. Thus, we have constructed a stem cell which contains a set of genes (SV40), the expression of which is regulated differently in stem and differentiated cells. This cell line could be used to determine the mechanism of suppression of expression of these genes in stem cells.     

Transformation of human skeletal muscle cells by simian virus 40.

Molecular studies of the biochemical alterations involved in human myopathies have been restricted because of the finite life-span and slow growth rate of cultures derived from primary tissue. Because the tumor virus simian virus 40 (SV40) can alter both the growth properties and longevity of human cells, we have infected skeletal muscle cultures derived from four biopsies with a small-plaque variant of SV40 and analyzed the biological and biochemical properties of cloned myoblast derivatives. At early times after infection, myoblasts fused normally into multinucleated myotubes, and both unfused and fused cells contained SV40 tumor antigen (T antigen). After six to eight subcultures after infection, the ability of myoblasts to fuse diminished, and clonal cell lines were generated with increased growth rates and saturation densities. Transformed cultures also lost contact inhibition of growth and became anchorage independent. Unlike untransformed myoblasts, SV40-transformed clones did not undergo an increase in creatine kinase activity or a transition of creatine kinase isoenzymes from the BB form to the muscle-specific MM form. Analysis of the pattern of SV40 DNA integration by Southern blotting hybridization analysis in two cloned SV40-transformed myoblast cell lines (KJ-SV40 and PK-SV40) indicated that KJ-SV40 contained at least one site of SV40 DNA integration into chromosomal DNA and PK-SV40 contained at least three sites of SV40 DNA covalently linked to cellular DNA. Cell lysates and growth medium from PK-SV40 transformants contained infectious small-plaque variant SV40, whereas KJ-SV40 did not contain or produce detectable virus. These studies demonstrate that human myoblasts can be immortalized by SV40. This procedure may prove useful for generating large quantities of genetically deficient human cells for biochemical and molecular analysis.     

Inhibitory effects of extracellular adenosine triphosphate on growth of esophageal carcinoma cells

AIM: To study the growth inhibitory effects of ATP on TE-13 human squamous esophageal carcinoma cells in vitro. METHODS: MTT assay was used to determine the inhibition of proliferation of ATP or adenosine (ADO) on TE-13 cell line. The morphological changes of TE-13 cells induced by ATP or ADO were observed under fluorescence light microscope by acridine orange (A0)/ethidium bromide (EB) double stained cells. The internucleosomal fragmentation of genomic DNA was detected by agarose gel electrophoresis. The apoptotic rate and cell cycle after treatment with ATP or ADO were determined by flow cytometry. RESULTS: ATP and ADO produced inhibitory effects on TE-13 cells at the concentration between 0.01 and 1.0 mmol/L. The IC50 of TE-13 cells exposed to ATP or ADO for 48 and 72 h was 0.71 or 1.05, and 0.21 or 0.19 mmol/L, respectively. The distribution of cell cycle phase and proliferation index (PI) value of TE-13 cells changed, when being exposed to ATP or ADO at the concentrations of 0.01, 0.1, and 1 mmol/L for 48 h. ATP and ADO inhibited the cell proliferation by changing the distribution of cell cycle phase via either G0/G1 phase (ATP or ADO, 1 mmol/L) or S phase (ATP, 0.1 mmol/L) arrest. Under light microscope, the tumor cells exposed to 0.3 mmol/L ATP or ADO displayed morphological changes of apoptosis. A ladder-like pattern of DNA fragmentation was obtained from TE-13 cells treated with 0.1-1 mmol/L ATP or ADO in agarose gel electrophoresis. ATP and ADO induced apoptosis of TE-13 cells in a dose-dependent manner at the concentration between 0.03 and 1 mmol/L. The maximum apoptotic rate of TE-13 cells exposed to ATP or ADO for 48 h was 16.63% or 16.9%, respectively. CONCLUSION: ATP and ADO inhibit cell proliferation, arrest cell cycle, and induce apoptosis of TE-13 cell line.     

Extracellular creatine regulates creatine transport in rat and human muscle cells.

Muscle cells do not synthesize creatine; they take up exogenous creatine by specific Na+-dependent plasma membrane transporters. We found that extracellular creatine regulates the level of expression of these creatine transporters in L6 rat muscle cells. L6 myoblasts maintained for 24 hr in medium containing 1 mM creatine exhibited 1/3rd of the creatine transport activity of cells maintained for 24 hr in medium without creatine. Down-regulation of creatine transport was partially reversed when creatine-fed L6 cells were incubated for 24 hr in medium lacking creatine. Down-regulation of creatine transport occurred independently of amino acid and glucose transport. Furthermore, the down-regulation of creatine transporters by extracellular creatine was slowed by inhibitors of protein synthesis. These results suggest that creatine induces the expression of a protein that functionally inactivates the creatine transporters. Regulation of creatine transport by extracellular creatine also was observed in L6 myotubes and in cultures of human myoblasts and myotubes. Hence, the activity of creatine transport represents another site for the regulation of creatine homeostasis.     

戊型肝炎病毒ORF3融合蛋白对L02细胞增殖和凋亡的影响

目的检测ORF3融合蛋白对L02细胞增殖与凋亡的影响。方法将重组质粒ORF3-pEGFP和ORF3-pXF2RH脂质体法转染L02细胞,用MTT比色法测定细胞增殖情况,用流式细胞仪碘化丙啶染色法检测细胞周期和细胞凋亡情况。结果转染重组质粒的L02细胞自第4d开始,明显增殖(P<0.05);L02细胞凋亡率由转染前的1.41%分别下降为0.83%和0.88%,差异有统计学意义(P<0.05)。结论戊型肝炎ORF3融合蛋白可减少L02细胞凋亡,而使L02增殖速度增加(P<0.05)。     

An activated c-Ha-ras allele blocks the induction of muscle-specific genes whose expression is contingent on mitogen withdrawal.

During myogenesis, induction of muscle-specific genes is subject to negative control by polypeptide mitogens and type-beta transforming growth factor. Since transduction of growth factor signals may require proteins encoded by cellular ras oncogenes, we have tested whether a mutationally altered Harvey ras expression vector, by itself, can prevent establishment of a differentiated phenotype in BC3H1 mouse myoblasts. Transfection with the valine-12 allele of the human Harvey ras gene, under the control of its own promoter, was sufficient to prevent the induction of both muscle creatine kinase activity and the nicotinic acetylcholine receptor following mitogen withdrawal but did not inhibit withdrawal from the cell cycle. The loss of creatine kinase activity resulted from a corresponding block to induction of muscle creatine kinase mRNA. Similarly, mitogen withdrawal elicited little or no alpha-actin mRNA in ras-transfected cells. These results suggest that an activated ras allele can inhibit myogenesis through a mechanism independent of cell proliferation and can preclude activation of genes whose up-regulation normally accompanies mitogen withdrawal.     

Induction of Endogenous Virus and of Thymidine Kinase by Bromodeoxyuridine in Cell Cultures Transformed by Friend Virus

Thymidine kinase positive (TK(+)) N type cell lines that had been transformed by spleen focus-forming virus were established by transformation with NB tropic Friend virus complex. Thymidine kinase deficient (TK(-)) cell clones were isolated. Some of these cell clones release 1000- to 100,000-fold reduced amounts of Friend virus complex as compared to the TK(+) parental cell clone. TK(-) clones were grown in medium without BrdUrd. Some of these TK(-) clones can be induced to release endogenous helper virus and transforming spleen focus-forming virus on reexposure to 10(-6)-10(-4) M BrdUrd. The induced Friend virus complex is of N host range as expected with induced endogenous virus in N-type cells. Before the induction of the endogenous virus spleen focus-forming virus complex, an induction of thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) activity is observed. The latter is possibly a prerequisite for the induction of endogenous virus in TK(-) cells. Induction of thymidine kinase activity and of endogenous virus is transient and always correlated. The role of BrdUrd and another thymidine analogue, azidothymidine, in interfering with C-type virus release in virus positive cells is discussed. Azidothymidine is unable to induce endogenous virus. Induction of endogenous virus by BrdUrd and inhibition of virus release in virus positive cells is apparently not caused by the same mechanism.     

Regulation of energy metabolism by creatine in cardiac and skeletal muscle cells in culture

Cardiac and skeletal muscle cells grown in culture were used in experiments designed to test the regulatory function of creatine. An increased concentration of intracellular phosphorylcreatine resulted from the addition of creatine to the growth media and the addition of metabolic inhibitors prevented the new high steady state concentration without depletion of ATP. With 1-fluoro-2, 4-dinitrobenzene the high concentration of phosphorylcreatine remained unchanged, while ATP was depleted. The use of the inhibitors provided additional evidence for creatine as the effector molecule in a feedback regulation of energy production, and for phosphorylcreatine in the regulation of energy charge at the contractile site. The effect of creatine on the fusion of myoblasts into myotubes supported its role in the control of cellular functions other than those involved in the energy metabolism. The correlation of the growth of muscle with increased muscular activity might be mediated through creatine, the end product of muscle activity. Creatine, synthesized in tissues other than muscle, might be involved in the coordination of muscle development throughout the body. Furthermore, it is suggested that the metabolic regulatory function of creatine is limited to the cells which do not synthesize creatine de novo, while in the tissues, such as the liver and kidney which have the mechanism for de novo synthesis of creatine, the absence of mitochondrial CPK precludes metabolic regulation of ～ P synthesis by creatine.