Reduced DNA repair during differentiation of a myogenic cell line

Repair synthesis induced by 4-nitroquinoline-1-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H] thymidine incorporation into unreplicated DNA. The level of repair synthesis was reuced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H] thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis, Both the initial rate and the overall incorporation of [3H] thymidine were found to be 50% lower in the myotubes. 4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were not longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single- strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.     

A human neuroblastoma cell line with an altered ornithine decarboxylase

A human neuroblastoma cell line (Paju) was resistant to 10 mM difluoromethylornithine, a concentration at which the growth of all mammalian cells normally stops. Ornithine decarboxylase from Paju was very resistant to inhibition by difluoromethylornithine in vitro (Ki = 10 microM compared to 0.5 microM for mouse kidney ornithine decarboxylase). After purification, apparently homogeneous Paju ornithine decarboxylase was inactivated with [3H]difluoromethylornithine and analyzed by polyacrylamide gel electrophoresis. Under denaturing conditions it was found to have an altered molecular structure, i.e. two nonidentical subunits of Mr = 55,000 and 60,000. Another unusual feature of Paju ornithine decarboxylase was its long half-life in vivo (T 1/2 = 8 h compared with 36 min in human HL-60 promyelocytic leukemia cells). The disappearance of immunoreactive protein was only slightly slower than the loss of catalytic activity. The long half-life of Paju ornithine decarboxylase was not shared by adenosylmethionine decarboxylase. Despite the altered structure of Paju ornithine decarboxylase, it was recognized by a specific antisera raised in rabbit against mouse kidney ornithine decarboxylase. The Paju karyotype did not contain double minute chromosomes or any large homogeneously staining region such as that seen in a mouse lymphoma cell mutant that is resistant to difluoromethylornithine and overproduces ornithine decarboxylase (McConlogue, L., and Coffino, P. (1983) J. Biol. Chem. 258, 12083-12086).     

Manipulation of medium conditions and differentiation in the rat myogenic cell line L6

Myoblasts of the L6 rat cell line were grown in Ham's F12 nutrient medium containing 10% fetal calf serum (F12 + FCS). Although the cells were confluent by 6 days in culture, fusion was not observed even if cultures were maintained for 10–14 days. At least 80% of the cells in such confluent unfused cultures were in the G₁ phase of the cell cycle and less than 5% of the cells in confluent cultures synthesized DNA during a 4-day period. The synthesis of muscle-specific proteins (α-actin, β-tropomyosin, and myosin light chains LC1_emb and LC2_F) was negligible when compared to fused cultures of L6 cells grown for a similar time in Dulbecco's medium with 10% FCS (DME + FCS). When the unfused cultures were shifted from F12 + FCS to DME + FCS, DNA synthesis could be demonstrated in more than 95% of the cells and fusion occurred, indicating that neither proliferative nor myogenic capacity had been irreversibly lost. Raising the levels of calcium, varying the serum concentration from 0 to 20%, or the addition of medium components (present in DME but reduced or absent in F12) all failed to induce fusion in the L6 cells grown in F12. However, L6 cells will fuse in mixtures of F12 + FCS and DME + FCS. Fusion will also occur if L6 cells are grown at clonal density in F12 + FCS supplemented with calcium. While it has not been possible to determine why F12 + FCS is nonpermissive for L6 cells in confluent mass cultures, the results demonstrate that prolonged residence in the G₁ phase of the cell cycle is not a sufficient condition for L6 myoblast differentiation to occur.     

Changes in adenyl cyclase specific activity during differentiation on an established myogenic cell line

The variation of specific activity of adenyl cyclase has been studied during differentiation of an established line of myoblast, strain L₆D and of a temperature sensitive developmental variant strain, H₆, derived from it. The specific activities of both basal and NaF stimulated adenyl cyclase were found to decrease 2 to 3 folds after fusion of myoblasts into myotubes in cultures of L₆D. Cultures of strain H₆ displayed the same decrease in specific activity of adenyl cyclase when grown at temperature which allows differentiation, while no decrease was observed at the temperature which does not allow cell fusion. These results indicare that the decrease in specific activity of adenyl cyclase is associated with cell fusion and reflects membrane changes ocurring during differentiation of myogenic cells.     

A variant vascular endothelial cell line with altered growth characteristics.

A variant endothelial cell type was found to arise spontaneously from cultures of bovine aortal endothelial cells. This variant showed no contact inhibition and overgrew confluent cultures of wild-type endothelial cells. Unlike other reported variants of this cell type produced by chemical mutagenesis or by withdrawal of polypeptide growth factor, this variant retained the capacity to synthesis factor VIII antigen, but showed no alteration from wild-type in capacity to adsorb platelets. The variant also had an increased capacity to bind FITC-conjugated con A to its surface.     

Temperature-sensitive hamster cell line with altered membrane properties

The altered properties of a concanavalin A-resistant Chinese hamster ovary cell line with obvious temperature-sensitive growth properties is described. The variant cell line, C^R-7, was shown to have a higher efficiency of colony formation than the parental wild-type population after treatment with various concentrations of concanavalin A (ConA). The variant cells had the properties of a temperature-sensitive cell line as judged by growth studies performed on solid surfaces or in suspension culture at the permissive (34 °C) and non-permissive (39 °C) temperatures; by colony efficiency determinations performed at 34 °C and 39 °C; and by the altered ability to incorporate DNA, RNA, and protein precursors into acid-precipitable material at the non-permissive temperature. Evidence for changes in the membrane properties of C^R-7 cells included: a reduced agglutinability in the presence of ConA, an altered cellular morphology on solid surfaces, an enhanced sensitivity to the toxic effects of membrane-active agents, altered and temperature-sensitive adhesiveness properties, and a reduced ability to bind labelled ConA.     

Transgene expression in the QM myogenic cell line

We have isolated an avian muscle cell line (QM) which has the essential features of established mammalian muscle cell lines. The experiments reported here were undertaken to determine the suitability of QM cells for the introduction and analysis of cloned transgenes. The promoter of the cardiac troponin T (cTNT) gene has been previously shown to contain sequence elements which govern muscle-specific expression of the chloramphenicol acetyltransferase (CAT) gene in transiently transfected primary cell cultures. We show here that QM cells stably harboring cTNT promoter-CAT fusion genes up-regulate CAT expression in concert with myogenic differentiation, and that as few as 110 upstream nucleotides are sufficient for such differentiation-dependent regulation. In addition, both transient and stable transfection experiments demonstrate that differentiated QM cells possess trans-acting factors necessary for the expression of the skeletal alpha-actin promoter, despite the absence of mRNA or protein product from the endogenous sarcomeric actin genes in these cells. Finally, to follow the developmental potential of QM cells in vivo, we created a clone, QM2ADH, which constitutively expresses the histochemical marker transgene encoding Drosophila alcohol dehydrogenase. When surgically inserted into the limb buds of developing chick embryos, QM2ADH cells are incorporated into endogenous developing muscles, indicating that QM cells are capable of recognizing and responding to host cues governing muscle morphogenesis. Thus, QM cells are versatile as recipients of transgenes for the in vitro and in vivo analysis of molecular events in muscle development.     

Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C

The F3 molecule is a member of the immunoglobulin superfamily anchored to membranes by a glycane-phosphatidylinositol, and is predominantly expressed on subsets of axons of the central and peripheral nervous system. In a previous paper (Gennarini, G., P. Durbec, A. Boned, G. Rougon, and C. Goridis. 1991. Neuron. 6:595-606), we have established that F3 fulfills the operational definition of a cell adhesion molecule and that it stimulates neurite outgrowth when presented to sensory neurons as a surface component of transfected CHO cells. In the present study the question as to whether soluble forms of F3 would be functionally active was addressed in vitro on cultures of mouse dorsal root ganglion neurons. We observed that preparations enriched in soluble F3 had no effect on neuron attachment but enhanced neurite initiation and neurite outgrowth in a dose-dependent manner. By contrast, soluble NCAM-120 does not have any measurable effect on these phenomena. Addition of anti-F3 monovalent antibodies reduced the number of process-bearing neurons and the neuritic output per neuron to control values. Addition of cerebrospinal fluid, a natural source of soluble F3, also stimulated neurite extension, and this effect was partially blocked by anti-F3 antibodies. Our results suggest that the soluble forms of adhesive proteins with neurite outgrowth-promoting properties could act at a distance from their site of release in a way reminiscent of growth and trophic factors.     

Effects of gender-specific adult bovine serum on myogenic satellite cell proliferation, differentiation and lipid accumulation

The study was performed to explore the effects of adult bovine male serum (MS), female serum (FS), and castrated male serum (C-MS) on myogenic satellite cells (MSCs) proliferation and differentiation into myotubes or into adipocyte-like cells (ALCs). MSC proliferation and differentiation was highest in the medium supplemented with MS, implying the important role of male steroid hormones. Myogenin and desmin were highly upregulated in cells cultured in MS-supplemented medium. In contrast, lipid accumulation in ALCs was highest in the medium supplemented with FS. Fatty acid transporter (FAT/CD36) was upregulated in FS-supplemented cultures. Detection of higher FAT/CD36 inducing fatty acids (arachidic acid and eicosapentaenoic acid) in FS compared with MS and C-MS suggests that these fatty acids may have influenced the enhanced formation of lipid droplets in ALCs. Effect of sex steroids on cell proliferation and cell growth of bovine MSCs and C2C12 cell in C-MS was greater than charcoal-dextran-treated fetal bovine serum (CDFBS). Concluding the above facts, the results indicate that each gender-specific bovine serum constitutes of different component, which leads to unique effects on cell behavior.     

Regulation of calcium binding proteins calreticulin and calsequestrin during differentiation in the myogenic cell line L6

In this report we defined the structural and temporal limits within which calreticulin and calsequestrin participate in the muscle cell phenotype, in the L6 model myogenic system. Calreticulin and calsequestrin are two Ca²⁺ binding proteins thought to participate in intracellular Ca²⁺ homeostasis. We show that calsequestrin protein and mRNA were expressed when L6 cells were induced to differentiate, during which time the level of expression of calreticulin protein did not change appreciably. Calreticulin mRNA levels, however, were constant throughout L6 cell differentiation except for slight decline in the mRNA levels at the very late stages of L6 differentiation (day 11–12). We also show that the two Ca²⁺ binding proteins are coexpressed in differentiated L6 cells. Based on its mobility in SDS-PAGE, L6 rat skeletal muscle cells in culture expressed cardiac isoform of calsequestrin. In the mature rat skeletal muscle, calreticulin and calsequestrin were localized to sarcoplasmic reticulum (SR). Calreticulin, but not calsequestrin, staining was also observed in the perinuclear region. These data suggest that expression of calreticulin and calsequestrin may be under different control during myogenesis in rat L6 cells in culture. © 1996 Wiley-Liss, Inc.     

Commitment,fusion and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis

L₆E₉ rat myoblasts derived from the L₆ cell line can be induced to differentiate to a very high percentage by manipulating the culture conditions. Under standard differentiating conditions, L₆E₉ cells divide an average of 2.5 times before differentiating and >99% of them incorporate ³H-TdR before fusing. By inhibiting DNA replication by a variety of means, data have been obtained which demonstrate that this DNa synthesis is not required to switch from growth to differentiation. After every cell division, L₆E₉ cells have the option either to fuse or to proliferate without intervening DNA synthesis.Cell cloning and DNA labeling experiments show a direct correlation between the time of culture in differentiating medium and a progressive loss of proliferative capacity of mononucleated L₆E₉ cells, demonstrating that these cells become irreversibly committed to differentiation and withdraw from the cell cycle prior to and not as a consequence of cell fusion. The commitment step occurs during the G1 phase prior to fusion. This G1 phase has a latent period during which no irreversible step toward differentiation occurs and the cells remain ambivalent toward growth or differentiation. Under proper conditions, this period is followed by an irreversible commitment toward differentiation and a loss of proliferative capacity. The kinetics of this commitment step strongly suggest that L₆E₉ cells become irreversibly committed in a stochastic manner. Once the cells have become committed, with or without DNA synthesis, they will fuse to form myotubes and biochemically differentiate in a deterministic fashion.The data presented are consistent with a stochastic model of differentiation for L₆E₉ cells and demonstrate that the switch from a proliferating to a differentiating genetic program can occur in the absence of DNA synthesis.     

Development of a clonal myogenic cell line with unusual biochemical properties.

The morphological, ultrastructural, biochemical and electrophysiological properties of B104-F, a clonal cell line derived from a nitrosoethylurea-induced neoplasm in a rat, were studied as a function of the growth phase of the culture. Cells in exponentially growing cultures are mononucleate and produce action potentials when stimulated electrically. Stationary phase cultures contain three types of cells: cells of the first type are mononucleate and have long processes containing microfilaments and many parallel microtubules; cells of the second type are mononucleate but contain no microtubules and few microfilaments; and cells of the third type have ultrastructural features typical of multinucleate, striated myotubes. Multinucleate cells generate action potentials with both sodium and calcium components and are depolarized by acetylcholine. The acetylcholine response is blocked by d-tubocurarine. The specific activity of creatine phosphokinase is nine times higher in stationary phase cultures than in exponentially growing ones while the myokinase specific activity is unchanged. The gamma-aminobutyric acid content of the cells is 3.5- to 26-fold higher in stationary phase than in exponentially growing cultures, depending on the degree of fusion of the culture. The properties of B104-F are discussed in relation to the properties of developing skeletal muscle and of central nervous system cell lines.     

Expression of Transthyretin during bovine myogenic satellite cell differentiation

Adult myogenesis responsible for the maintenance and repair of muscle tissue is mainly under the control of myogenic regulatory factors (MRFs) and a few other genes. Transthyretin gene (TTR), codes for a carrier protein for thyroxin (T₄) and retinol binding protein bound with retinol in blood plasma, plays a critical role during the early stages of myogenesis. Herein, we investigated the relationship of TTR with other muscle-specific genes and report their expression in muscle satellite cells (MSCs), and increased messenger RNA (mRNA) and protein expression of TTR during MSCs differentiation. Silencing of TTR resulted in decreased myotube formation and decreased expression of myosin light chain (MYL2), myosin heavy chain 3 (MYH3), matrix gla protein (MGP), and voltage-dependent L type calcium channel (Cav1.1) genes. Increased mRNA expression observed in TTR and other myogenic genes with the addition of T₄ decreased significantly following TTR knockdown, indicating the critical role of TTR in T₄ transportation. Similarly, decreased expression of MGP and Cav1.1 following TTR knockdown signifies the dual role of TTR in controlling muscle myogenesis via regulation of T₄ and calcium channel. Our computational and experimental evidences indicate that TTR has a relationship with MRFs and may act on calcium channel and related genes.     

Isolation and characterization of an avian myogenic cell line

Myogenic cell lines have proven extremely valuable for studying myogenesis in vitro. Although a number of mammalian muscle cell lines have been isolated, attempts to produce cell lines from other classes of animals have met with only limited success. We report here the isolation and characterization of seven avian myogenic cell lines (QM1-4 and QM6-8), derived from the quail fibrosarcoma cell line QT6. A differentiation incompetent QM cell derivative was also isolated (QM5DI). The major features of QM cell differentiation in vitro closely resemble those of their mammalian counterparts. Mononucleated QM cells replicate in medium containing high concentrations of serum components. Upon switching to medium containing low serum components, cells withdraw from the cell cycle and fuse to form elongated multinucleated myotubes. Cultures typically obtain fusion indices of 43-49%. Northern blot and immunoblot analyses demonstrate that each differentiated QM cell line expresses a wide variety of genes encoding muscle specific proteins: desmin, cardiac troponin T, skeletal troponin T, cardiac troponin C, skeletal troponin I, alpha-tropomyosin, muscle creatine kinase, myosin light chain 2, and a ventricular isoform of myosin heavy chain. While all QM lines analyzed to date express at least some myosin light chain 2, only one line, QM7, expresses this gene at high levels. Surprisingly, none of the QM lines reported here express any known form of alpha-actin. The absence of sarcomeric actin expression may explain the absence of myofibrils in QM myotubes. These novel features of muscle gene expression in QM cells may prove useful for studying the role of specific muscle proteins during myogenesis. More importantly, however, the isolation of QM cell lines indicates that it may be feasible to isolate other avian myogenic cell lines with general utility for the study of muscle development.     

Protein synthesis and secretion in a myogenic cell line

Myogenesis in a clonal myoblast cell line is accompanied by an increase in the specific activities of creatine phosphokinase and myokinase and in the rates of synthesis and accumulation of myosin heavy chain. Exponentially dividing myoblasts synthesize myosin heavy chain at a rate of about 1% of their rate of total protein synthesis; this rate increases 7-fold during the differentiation process. Both myoblasts and myotubes secrete a minimum of 12-soluble proteins. Although there is a quantitative change in the rates of appearance of five of these proteins during myogenesis, no qualitative changes in the profile of the secreted proteins are detected. Three of the secreted proteins share several properties of soluble collagen molecules. Basal laminae and polymerized collagen fibrils are associated with myotubes, but not with exponentially dividing myoblasts.     

GREG cells, a dysferlin-deficient myogenic mouse cell line

The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (~200kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.     

Iron and copper requirements for proliferation and differentiation of a human promyelocytic leukemia cell line (HL-60)

Trace mineral deficiencies tend to have profound effects on the integrity of formed blood elements. Anemia and neutropenia are commonly seen in copper (Cu) deficiency. We therefore developed a serum-free medium to examine the trace mineral requirements, in particular iron and Cu, for proliferation and retinoic acid (RA)-induced differentiation of HL-60 cells. This defined medium (DFM) was composed of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with insulin and human apo-transferrin (each at 5 μg/ml) and 1.4 μM FeSO₄. The iron concentration range for optimal cellular proliferation was narrow (2–3 μM). HL-60 cells could be maintained in DFM for 15 passages with a doubling time of 38–40 hr. The Cu content of IMDM was very low. Thus, by the fourth passage in DFM, the activity of cuproenzymes (cytochrome c oxidase, CCO; and copperzinc superoxide dismutase, CuZnSOD) began to decline. Supplementation of DFM with CuSO₄ (50 nM) restored enzyme activities. Treatment of cells with a Cu chelator (tetrathiomolybdate, 1 μM) rapidly reduced the activities of both CCO and CuZnSOD. Over the Cu concentration range examined (5–350 nM), Cu supplementation had little effect on HL-60 proliferation. Cell retained the ability to differentiate along the granulocytic pathway when treated with RA, but seemed to be less sensitive to the inducing agent except at the highest concentration tested (1 μM). This decreased sensitivity to RA did not seem to be related to the Cu status of the cells but rather to the absence of a component of serum. Indeed, cells grown in DFM regained their sensitivity to RA when allowed to differentiate in IMDM with 5% serum. These data indicate that the processes of growth and terminal differentiation in HL-60 cells are not greatly influenced by Cu. Thus, it seems likely that the insult resulting in neutropenia which is associated with Cu deficiency may occur earlier than the promyelocytic stage. However, the possibility that the mechanisms contributing to neutropenia may be unrelated to primary defects in the biochemistry of neutrophil maturation cannot be ruled out. © 1995 Wiley-Liss, Inc.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.