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Mouse P19 embryonal carcinoma cells undergo cardiogenesis in response to high density and DMSO. We have derived a clonal subline which undergoes cardiogenesis in response to high density, but without requiring exposure to DMSO. The new subline retains the capacity to differentiate into skeletal muscle and neuronal cells in response to DMSO and retinoic acid. However, upon aggregation, these Oct 4-positive cells, termed P19-SI because they “self-induce” cardiac muscle, exhibit increased mRNAs encoding the mesodermal factor Brachyury, cardiac transcription factors Nkx 2.5 and GATA 4, the transcriptional repressor Msx-1, and cytokines Wnt 3a, Noggin and BMP 4. Exposure of aggregated P19-SI cells to BMP 4, a known inducer of cardiogenesis, accelerates cardiogenesis, as determined by rhythmic beating and myosin staining. However, cardiogenesis is severely inhibited when P19-SI cells are aggregated in the presence of BMP 4. These results demonstrate that cell-cell interaction is required before P19-SI cells can undergo a cardiogenic response to BMP 4. A concurrent increase in the expression of Msx-1 suggests one possible process underlying the inhibition of cardiogenesis. The phenotype of P19-SI cells offers an opportunity to explore new aspects of cardiac induction.

Transcriptional regulatory element X (Trex) is a positive control site within the Muscle creatine kinase (MCK) enhancer. Cell culture and transgenic studies indicate that the Trex site is important for MCK expression in skeletal and cardiac muscle. After selectively enriching for the Trex-binding factor (TrexBF) using magnetic beads coupled to oligonucleotides containing either wild-type or mutant Trex sites, quantitative proteomics was used to identify TrexBF as Six4, a homeodomain transcription factor of the Six/sine oculis family, from a background of approximately 900 copurifying proteins. Using gel shift assays and Six-specific antisera, we demonstrated that Six4 is TrexBF in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle, while Six5 is the major TrexBF in adult mouse heart. In cotransfection studies, Six4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. Our results are consistent with Six4 being a key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle. The Trex/MEF3 composite sequence ([C/A]ACC[C/T]GA) allowed us to identify novel putative Six-binding sites in six other muscle genes. Our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification.

The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF-2) or interfere with complex formation between FGF-2 and its high-affinity FGF receptor-1 (FGFR1), yet both native SPARC and a peptide derived from the C-terminal high-affinity Ca(2+)-binding region of protein significantly inhibit ligand-induced autophosphorylation of FGFR1 (>80%), activation of mitogen-activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF-2 (>80%). We also report that in the presence of FGF-2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca(2+)-binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)-deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1-mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell-surface, low-affinity FGF-2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand-binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf-MAPK pathway. SPARC thus modulates the mitogenic effect of FGF-2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade.

MM14 murine myoblast cells remain in an undifferentiated and proliferative state if they are maintained in the continuous presence of basic fibroblast growth factor (FGF-2) and serum factors, but terminally differentiate into myocytes and myotubes if deprived of FGF-2 during G1 of the cell cycle. We find that 2-aminopurine (2-AP) induces a reversible, rapid, and profound alteration in the morphology of proliferating MM14 cells to a polarized shape which is similar to that observed during normally induced differentiation. This change requires neither a differentiation signal norde novoprotein synthesis. In contrast, we do not observe a morphological change in response to 2-AP in nonmyogenic cell lines. The morphological alteration of MM14 cells in response to 2-AP requires reorganization of the microtubule and actin cytoskeletons, in common with changes during normal differentiation. Additionally, we show that cytoskeletal rearrangements that occur during both normal differentiation and in response to 2-AP require the activity of the small GTPase Rho. Differentiation defective MM14 cells (DD-1 cells), which lack MyoD, also undergo a profound morphological alteration in the presence of 2-AP. These results indicate that morphological changes consistent with myoblast differentiation are regulated by a pathway independent of MyoD transcriptional control.

Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries--a phenomenon referred to as "spontaneous angiogenesis in vitro". We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to identify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.

β-galactosidase (β-gal) is among the most frequently used markers for studying a wide variety of biological mechanisms, e.g., gene expression, cell migration, stem cell conversion to different cell types, and gene silencing. Many of these studies require the histochemical detection of relative β-gal levels in tissue cross-sections mounted onto glass slides and visualized by microscopy. This is particularly useful for the analysis of promoter activity in skeletal muscle tissue since the β-gal levels can vary dramatically between different anatomical muscles and myofiber types. The differences in promoter activity can be due to a myofiber's developmental history, innervation, response to normal or experimental physiological signals, and its disease state. It is thus important to identify the individual fiber types within muscle cross-sections and to correlate these with transgene expression signals. Here, we provide a detailed description of how to process and analyze muscle tissues to determine the fiber-type composition and β-gal transgene expression within cryosections.

β-galactosidase (β-gal) is among the most frequently used markers for studying a wide variety of biological mechanisms, e.g., gene expression, cell migration, stem cell conversion to different cell types, and gene silencing. Many of these studies require the histochemical detection of relative β-gal levels in tissue cross-sections mounted onto glass slides and visualized by microscopy. This is particularly useful for the analysis of promoter activity in skeletal muscle tissue since the β-gal levels can vary dramatically between different anatomical muscles and myofiber types. The differences in promoter activity can be due to a myofiber's developmental history, innervation, response to normal or experimental physiological signals, and its disease state. It is thus important to identify the individual fiber types within muscle cross-sections and to correlate these with transgene expression signals. Here, we provide a detailed description of how to process and analyze muscle tissues to determine the fiber-type composition and β-gal transgene expression within cryosections.

BackgroundHundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood.ResultsModulatory region 1 (MR1) is a 1-kb regulatory region withinMCKintron 1 that is highly active in terminally differentiating skeletal myocytesin vitro. AMCKsmall intronic enhancer (MCK-SIE) containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bpMCK5'-enhancer, but theMCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and theMCK5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kbMCKgenomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical forMCKexpression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively), but is not required for expression in fast-twitch muscle fibers (types IIb and IId).ConclusionsIn this study, we discovered that MR1 is critical forMCKexpression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls forMCKexpression in different skeletal muscle fiber types.

The in vitro replicative potential of human diploid fibroblasts can be increased by polypeptide growth factors such as epidermal growth factor (EGF). Also, the cycle time of EGF-stimulated cells is, on average, decreased and their mitotic cell volume is reduced. Therefore, the regulation of cell size by the duration of the cell cycle may be one process which determines replicative potential. The growth response to a continuous presence of EGF, however, appears to be limited by eventual desensitization to the growth factor.

We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase α activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase α activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.

Interactions between Leydig cells and the extracellular matrix (ECM) within the interstitial compartment of the mammalian testis have not been characterized. We have examined the influence of ECM on adult mouse Leydig cells by culturing the cells on different ECM substrates. Leydig cells adhere weakly to hydrated gels of type I collagen (including those supplemented with collagen types IV, V, or VIII), or to air-dried films of collagen types I, V, or VIII. In contrast, the cells attach firmly to substrates of purified type IV collagen, fibronectin, or laminin. Leydig cells also attach rapidly and adhere strongly to gelled basement membrane matrix derived from the murine Englebreth-Holm-Swarm sarcoma (Matrigel). Leydig cells assume spherical shapes and form aggregates on thick (1.5-mm) layers of Matrigel; however, on thin (0.1-mm) layers, networks of cell clusters linked by cords of elongated cells are formed within 48 h. Similar networks are formed on thick layers of Matrigel that are supplemented with type I collagen. On substrates with high ratios of collagen I to Matrigel or on untreated tissue culture plastic, Leydig cells flatten and do not aggregate. On substrates that induce rounded shapes, proliferation is inhibited and the cells maintain the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase for as long as 2 wk. Under conditions where Leydig cells are flattened, they divide and cease expressing the enzyme. Proliferating Leydig cells also exhibit elevated levels of mRNA for SPARC (Secreted Protein, Acidic and Rich in Cysteine), a Ca2(+)-binding glycoprotein associated with changes in cell shape that accompany morphogenesis and tissue remodeling. Our results indicate that the shape, association, proliferation, and expression of gene products by Leydig cells can be significantly affected in vitro by altering the composition of the extracellular substratum.

Gene therapy for Duchenne muscular dystrophy (DMD) will require sustained expression of therapeutic dystrophins in striated muscles. Lentiviral vectors have a relatively large transgene carrying capacity and can integrate into nondividing cells. We therefore explored the use of lentiviral vectors for transferring genes into mouse skeletal muscle cells. These vectors successfully transferred a minidystrophin expression cassette into mdx muscles, and minidystrophin expression persisted and prevented subsequent muscle fiber degeneration for at least 6 months. However, only low to moderate levels of skeletal muscle transduction could be obtained by intramuscular injection of the highest currently available lentiviral doses. Using cultured cells, the lentiviral vectors effectively transduced proliferating and terminally differentiated muscle cells, indicating that cell cycling is not essential for transduction of myogenic cells. We further showed that lentiviral vectors efficiently transduced both primary myoblasts and multipotent adult progenitor cells (MAPCs) in vitro, and the cells persistently expressed transgenes without any obvious toxicity. When mdx primary myoblasts were genetically modified with minidystrophin vectors and transplanted into mdx skeletal muscles, significant numbers of dystrophin-expressing myofibers formed. Finally, we showed that a short, highly active CK6 regulatory cassette directed muscle-specific activity in the context of the lentiviral vectors. The ability of lentiviral vectors to transduce myogenic progenitors using a minidystrophin cassette regulated by a muscle-specific promoter suggests that this system could be useful for ex vivo gene therapy of muscular dystrophy.

A model experimental system for investigating myogenic induction signals has been devised with mouse P19 embryonal carcinoma cells. When cocultured with pieces of chick neural tube, aggregated P19 cells are induced to become skeletal muscle. The most potent inducing activity is localized to the dorsal neural tube. Less activity was found in the ventral neural tube, notochord, ectoderm, and lateral plate mesoderm, and none was detected in the neural retina. These results suggest that P19 cells may be a useful model system for investigating the mechanisms underlying induction of somite myogenesis.

We have previously reported that the DNA polymerase alpha activity/unit cellular protein is decreased in late-passage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase alpha and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase alpha activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase alpha activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via "dilution" of factors essential for cellular replication.

We previously reported a model of clonal attenuation which assumes three classes of cells: small highly replicative cells; intermediate size cells of limited replicative potential and large non-dividing cells. Computer simulations carried out with the model lead to predictions of how the relative proportion of each cell type varies throughout the in vitro replicative life span of a mass population. These predictions appear to be broadly confirmed by independent data recently reported by another laboratory.

Evidence that exogenous hyaluronate (HA) binds to the surface of muscle fibroblast cultures was obtained by incubating confluent fibroblasts with 14C-HA purified from fibroblast cell surfaces. Surface-bound 14C-HA was operationally defined as material resistant to six saline washes and solubilized by brief trypsinization. All of the surface-bound radioactivity remains as authentic HA. Exposure of fibroblasts to 100 μg/ml cold HA caused a nearly 3-fold ‘reduction’ in incorporation of isotopic precursors into glycosaminoglycan (GAG); but when intracellular 14C precursors to GAG were quantitated, the entire ‘reduction’ could be accounted for by decreased precursor uptake. Exposure to exogenous HA altered the distribution of newly synthesized GAG by stimulating an increase in total GAG secreted to the medium at the expense of that bound to the culture surface. Qualitatively, the cell surface ratio of 14C-HA: 14C-sulfated GAG (SGAG) of HA-treated cells is about 2.5 times greater than that of untreated cells and the medium ratio is correspondingly reversed. This is primarily the result of stimulated 14C-SGAG release to the culture medium. Addition of cold HA to prelabeled cultures also stimulates the selective turnover of SGAG from the culture surface. Thus, exposure to HA alters the fibroblast surface by accumulation of exogenous HA as well as by stimulation of SGAG turnover.

Radioactive species in commercial preparations of [14C]glucosamine bind to macromolecular components in horse, fetal calf and human sera, as well as to bovine serum albumin. The binding appears to be non-enzymatic and is stable through extensive dialysis and acid precipitation. On the basis of competition and chromatographic experiments glucosamine appears to be the major reactive species. When chick muscle fibroblasts are grown in serum-containing medium with [14C]glucosamine, at least some of their radioactive cell surface components as well as radioactive components in the overlying conditioned medium, display chromatographic properties which are similar to those detected in complete medium which has been exposed to [14C]glucosamine in the absence of cells. Preliminary results with [3H]fucose indicate that it also binds to acid-precipitable medium proteins in the absence of cells. The possible relevance of such “anomalously labeled” serum components to the study of cell surface and conditioned medium glycoproteins is discussed.

It is shown by computer simulation that an established commitment model of clonal attenuation can account for clone size distribution data obtained from three vertebrate species — chick, hamster and human — from two evolutionarily divergent classes. The different in vitro replicative lifespans of each cell strain can be explained simply by differences in cell kinetics. These results suggest that the process of clonal attenuation is qualitatively similar in fibroblasts from all vertebrate species.

Volcanic ash was collected immediately after the eruption of Mount St. Helens on May 18, 1980. This ash was extracted with water. The elemental composition of the extracted portion was determined by atomic absorption spectrometry. The aqueous extract was applied at high concentrations (up to 37.5 μg/ml) to non-confluent mixed cultures of mouse lung cells. Even after treatment for up to 10 days, cell number was typically unaffected by the ash extract. Cell viability was also unaltered, and no grossly observable changes were noted in the cells by light muscopy. We conclude that the water-soluble portion of the ash we tested does not markedly affect growth of the cells most at risk, those of the lung.

Human foreskin fibroblast-like cells were separated on the basis of DNA content and cell size by fluorescence-activated cell sorting. Subpopulations of "large" or "small" cells with the same (G1) DNA content were clonally expanded and found to contain predominantly nondividing or highly proliferative cells, respectively. From the rate of clonal growth, we deduce that small cells divide faster than large cells. Intermediate-sized cells were found to yield primarily smaller ("attenuated") clones. The clonal data can be incorporated into a previously reported kinetic model of clonal attenuation. This version of the model postulates that small "stem" cells yield larger daughters which have only a limited proliferative potential. We also postulate that a progressive increase in cell size can account for the decreasing concentration of DNA polymerase alpha, which has been reported in older cultures.

Analysis of glycosaminoglycan (GAG) synthesis by high density and clonal cultures of chick myoblasts and fibroblasts, and by cultures enriched for myotubes, indicates that all culture types produce hyaluronic acid (HA) and sulfated polysaccharides (S-GAG). Of these cell types the myotube synthesizes the most HA-rich complement of polysaccharides and has the most HA-rich cell surface. It appears that this is the result of decreased S-GAG synthesis rather than increased HA synthesis. Myotube cell surface HA is qualitatively different from HA released to the medium; cell surface HA is predominantly a single high molecular weight species, whereas HA isolated from the medium comprises a heterogeneous mixture of lower molecular weight HA. Since HA molecules of similar size classes can be generated by treating high molecular weight HA with hyaluronidase, an endogenous hyaluronidase could be involved. Alternatively, myotube-enriched cultures may synthesize and secrete HA polymers of several intermediate lengths.

In kinetic studies with human diploid fibroblast-like (HDFL) cells carried out in heterokaryons and in monokaryons, we have observed a first-order relationship between the level or concentration of DNA polymerase alpha and the rate of initiation of new rounds of DNA synthesis. Because cell size is inversely proportional to the concentration of DNA polymerase alpha and presumably other replication factors, it is inversely related to the initiation of new rounds of DNA synthesis. An inverse relationship between cell size and clonogenic activity was also observed in both unsorted HDFL cells and in HDFL cells sorted on the basis of size. Experimental enlargement of cells by serum deprivation at low density resulted in changes in colony-forming ability that would be predicted by these studies. A causal relationship between the observed increase in cell size with advancing passage level and the loss of proliferative activity is suggested by these studies; in addition, cell size may be a useful biophysical marker for cellular aging.

SPARC (Secreted Protein Acidic and Rich in Cysteine) is a Ca+2-binding glycoprotein that is differentially associated with morphogenesis, remodeling, cellular migration, and proliferation. We show here that exogenous SPARC, added to cells in culture, was associated with profound changes in cell shape, caused rapid, partial detachment of a confluent monolayer, and inhibited spreading of newly plated cells. Bovine aortic endothelial cells, exposed to 2-40 micrograms SPARC/ml per 2 x 10(6) cells, exhibited a rounded morphology in a dose-dependent manner but remained attached to plastic or collagen-coated surfaces. These round cells synthesized protein, uniformly excluded trypan blue, and grew in aggregates after replating in media without SPARC. SPARC caused rounding of bovine endothelial cells, fibroblasts, and smooth muscle cells; however, the cell lines F9, PYS-2, and 3T3 were not affected. The activity of native SPARC was inhibited by heat denaturation and prior incubation with anti-SPARC IgG. The effect of SPARC on endothelial cells appeared to be independent of the rounding phenomenon produced by the peptide GRGDSP. Immunofluorescence localization of SPARC on endothelial cells showed preferential distribution at the leading edges of membranous extensions. SPARC bound Ca+2 in both amino- and carboxyl-terminal (EF-hand) domains and required this cation for maintenance of native structure. Solid-phase binding assays indicated a preferential affinity of native SPARC for several proteins comprising the extracellular matrix, including types III and V collagen, and thrombospondin. This binding was saturable, Ca+2 dependent, and inhibited by anti-SPARC IgG. Endothelial cells also failed to spread on a substrate of native type III collagen complexed with SPARC. We propose that SPARC is an extracellular modulator of Ca+2 and cation-sensitive proteins or proteinases, which facilitates changes in cellular shape and disengagement of cells from the extracellular matrix.

Multicellular aggregates of tumorigenic mouse mammary epithelial cells contain a hyaluronate-rich matrix, both at the aggregate periphery and within the growing spheroid. It is proposed that the establishment of a hyaluronaterich matrix is essential to spheroid growth in vitro, and that the spheroid is a good model system for analysis of this aspect of early tumor development.