BUdR, probability and cell variants: towards a molecular understanding of the decision to differentiate

The mechanism(s) by which the thymidine analogue 5-bromodeoxyuridine (BUdR) specifically inhibits the expression of differentiated functions is poorly understood, as are the ways in which cells regulate processes exhibiting probabilistic aspects. I have developed a theoretical model for the regulation of the decision of myogenic cells to differentiate that can explain both of the above phenomena. This model provided a strategy for isolating myoblast variants that had amplified the expression of the factors regulating the decision to differentiate. These myoblasts served as the source of mRNA for making and screening a cDNA library in order to isolate these factors. The successful cloning of these genes should represent a major advance in our understanding of the molecular basis for the major coordinated changes in gene expression that accompany cell differentiation.     

Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression

Skeletal muscle cells are a useful model for studying cell differentiation. Muscle cell differentiation is marked by myoblast proliferation followed by progressive fusion to form large multinucleated myotubes that synthesize muscle-specific proteins and contract spontaneously. The molecular analysis of myogenesis has advanced with the identification of several myogenic regulatory factors, including myod1, myd, and myogenin. These factors regulate each other's expression and that of muscle-specific proteins such as the acetylcholine receptor and acetylcholinesterase (AChE). In order to investigate the role of extracellular matrix (ECM) in myogenesis we have cultured myoblasts (C₂C₁₂) in the presence or absence of an exogenous ECM (Matrigel). In addition, we have induced differentiation of myoblasts in the presence or absence of Matrigel and/or chlorate, a specific inhibitor of proteoglycan sulfation. Our results indicated that the formation of fused myotubes and expression of AChE was stimulated by Matrigel. Treatment of myoblasts induced to differentiate with chlorate resulted in an inhibition of cell fusion and AChE activity. Chlorate treatment was also found to inhibit the deposition and assembly of ECM components such fibronectin and laminin. The expression of myogenin mRNA was observed when myoblasts were induced to differentiate, but was unaffected by the presence of Matrigel or by culture of the cells in the presence of chlorate. These results suggest that the expression of myogenin is independent of the presence of ECM, but that the presence of ECM is essential for the formation of myotubes and the expression of later muscle-specific gene products. © 1996 Wiley-Liss, Inc.     

Regulation of rat myosin light-chain synthesis in heterokaryons between 5-bromodeoxyuridine-blocked rat myoblasts and differentiated chick myocytes

Terminal cell differentiation in a variety of model systems is inhibited by the thymidine analogue 5-bromodeoxyuridine (BUdR). We investigated the mode of action of BUdR by forming heterokaryons between undifferentiated BUdR-blocked rat myoblasts and differentiated chick skeletal myocytes. We analyzed newly synthesized proteins on two- dimensional polyacrylamide gels. The induction of rat skeletal myosin light-chain synthesis was reduced fivefold, as compared with controls, when chick myocytes were fused to BUdR-blocked rat myoblasts. This indicates that plasma membrane effects cannot be the proximate cause for the inhibition of myogenesis by BUdR, since BUdR is able to block the effect of chick inducing factors even when a differentiated chick myocyte is in direct cytoplasmic continuity with the BUdR-blocked rat nucleus. The observation that chick cells required an 80% substitution of BUdR for thymidine to block myogenesis, whereas L6 rat myoblasts required only a 20% substitution led to a hypothesis involving a DNA- mediated action of BUdR. This model yielded three testable predictions: (a) putative chick inducing molecules should be present in limiting quantities, (b) exploiting gene-dosage effects to increase the quantity of putative chick inducing factors might overcome the inhibition produced in the rat myoblasts by a 35% BUdR for thymidine substitution, and (c) these gene-dosage effects should be abolished by increasing the level of BUdR substitution in the rat myoblast to 60-80%. All three of these predictions have been verified, providing strong indirect evidence that the inhibition of myogenesis produced by BUdR is a direct result of its incorporation into cellular DNA.     

Replication of mouse cytomegalovirus in thymidine kinase-deficient mouse cells.

In an attempt to determine whether mouse cytomegalovirus (MCMV) requires thymidine kinase (TK) for replication and whether it induces TK, TK-deficient mouse cells were isolated and used as host cells for MCMV. Mutant cells resistant to 200 μg/ml of 5-Bromodeoxyuridine (BUdR) were selected from SV40-transformed mouse cells, mks-A TU-7, by propagating the cells in the presence of varying concentrations of BUdR graded by serial 2-fold increments. The mutant cells, designated as TU-7 BU, showed a very low TK activity (less than 1/20 that of mks-A TU-7). Herpes simplex virus type 1 (HSV-1) replicated in starved as well as in unstarved TU-7 BU, whereas MCMV could replicate only in growing TU-7 BU and could not form plaques in monolayers of mks-A TU-7 or TU-7 BU. HSV-1 infection enhanced TK activity equally in both mks-A TU-7 and TU-7 BU. In contrast, TK activity of MCMV-infected mks-A TU-7 was lower than that of uninfected cells or cells inoculated with UV-inactivated virus. In addition, TK activity of the MCMV-infected TU-7 BU remained minimal without showing any increase. The replication of HSV-1 was completely inhibited in the presence of BUdR (10 μg/ml), whereas MCMV could replicate even in the presence of 50 μg/ml of BUdR. The results indicate that MCMV neither requires TK nor induces TK activity in the infected cells.     

alpha7 integrin expressing human fetal myogenic progenitors have stem cell-like properties and are capable of osteogenic differentiation

During muscle development, precursor cells fuse to form myofibers. Following injury in adult muscle, quiescent satellite cells become activated to regenerate muscle in a fashion similar to fetal development. Recent studies indicate that murine skeletal myoblasts can differentiate along multiple cell lineages including the osteoblastic pathway. However, little is known about the multipotency of human myogenic cells. Here, we isolate myogenic precursor cells from human fetal and adult muscle by sorting for the laminin-binding alpha7 integrin and demonstrate their differentiation potential and alteration in adhesive behavior. The alpha7-positive human fetal progenitors were efficient at forming myotubes and a majority expressed known muscle markers including M-cadherin and c-Met, but were heterogeneous for desmin and MyoD expression. To test their pluripotent differentiation potential, enriched populations of alpha7-positive fetal cells were subjected to inductive protocols. Although the myoblasts appeared committed to a muscle lineage, they could be converted to differentiate along the osteoblastic pathway in the presence of BMP-2. Interestingly, osteogenic cells showed altered adhesion and migratory activity that reflected growth factor-induced changes in integrin expression. These results indicate that alpha7-expressing fetal myoblasts are capable of differentiation to osteoblast lineage with a coordinated switch in integrin profiles and may represent a mechanism that promotes homing and recruitment of myogenic stem cells for tissue repair and remodeling.     

Cultured myf5 null and myoD null muscle precursor cells display distinct growth defects

Myf-5 and MyoD are the two muscle regulatory factors expressed from the myoblast stage to maintain the identity and to promote the subsequent differentiation of muscle precursor cells. To get insight into their role we have studied the capacity to proliferate and to differentiate of myf-5 and myoD null myoblasts in primary cultures and in the subsequent passages. Our results indicate that myf-5 null myoblasts differ from wild type (wt) myoblasts in that they undergo precocious differentiation: they become myogenin- and troponin T-positive and fail to incorporate bromodeoxyuridine (BrdU) under culture conditions and at a time when wt cells are not yet differentiated and continue to proliferate. In primary cultures of myoD null cells, up to 60% of the cells were scored as myoblasts on the basis of the expression of myf-5. These myoD-deficient myoblasts, unlike myoD-expressing cells, were poorly differentiating and displayed a severe growth defect that led to their elimination from the cultures: within a few passages myoblasts were absent from myoD-deficient cultures, which mostly consisted of senescent cells. That a null mutation in either gene reduces the proliferative potential of cultured myoblasts raises the possibility that Myf-5 and MyoD serve proliferation of muscle precursor cells.     

5-Bromodeoxyuridine: A specific inhibitor of cytokinin-habituation in tobacco cell culture

Summary Cytokinin-habituated cells of Nicotiana tabacum L. cv. Havana 425 are able to grow in culture without added cytokinin. The thymidine analogue, 5-bromodeoxyuridine (BUdR), which selectively inhibits differentiation of animal cells, blocks expression of the cytokinin-habituated phenotype in culture. This effect is prevented by thymidine and is reversible. These findings suggest that habituation and animal cell differentiation have a common mechanism. BUdR provides a useful tool for investigating the metabolism of cell division factors and its regulation in higher plant cells.Abbreviation BUdR 5-bromodeoxyuridine - BU 5-bromouracil     

Hemoglobin synthesis in murine virus-induced leukemic cells in vitro. 3. Effects of 5-bromo-2'-deoxyuridine, dimethylformamide and dimethylsulfoxide

Erythroid differentiation of Friend leukemia cells is enhanced when the cells are grown for four days in the presence of dimethylsulfoxide (DMSO). Dimethylformamide (DMF) has a similar though less marked effect. 5-Bromo-2′-deoxyuridine (BUdR) (10^?5M) inhibits both DMF- and DMSO-stimulated differentiation. For maximum inhibition, BUdR must be present during the first two days of growth, during which time DNA synthesis is maximal. The addition of BUdR after the third day has no effect. Since BUdR is incorporated into DNA and thymidine prevents BUdR inhibition of DMSO-stimulated differentiation, it is likely that BUdR acts by virtue of its incorporation into DNA. Although BUdR alone had little effect upon cell multiplication, in combination with DMSO, cell growth was inhibited up to 40%. Since the BUdR-inhibition of the DMSO effect was approximately 70%, it is unlikely that its effect on differentiation is due to selective killing of those cells which are stimulated to differentiate.     

Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation

In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.     

The mitogen-activated protein kinase cascade promotes myoblast cell survival by stabilizing the cyclin-dependent kinase inhibitor,p21WAF1 protein

During myogenesis, proliferating myoblasts withdraw from the cell cycle and are either eliminated by programmed cell death or differentiate into mature myotubes. Previous studies indicate that mitogen-activated protein kinase (MAPK) activity is significantly induced with the onset of terminal differentiation of C2 myoblasts. We have investigated the part played by the MAPK pathway in the differentiation of C2 myoblasts. Specific activation of MAPK by expression of an active Raf1-estrogen receptor chimera protein reduced significantly the number of myoblasts undergoing programmed cell death in the differentiation medium. Activation of Raf1 prevented the proteolytic activation of the proapoptotic caspase 9-protein during differentiation. The antiapoptotic function of Raf1 correlated with accumulation of the p21WAF1 protein resulting from its increased stability. Antisense expression of p21 was used to determine whether the p21WAF1 protein mediated the antiapoptotic activity of Raf1. Reduction of p21WAF1 protein in muscle cells abolished the antiapoptotic activity of the MAPK pathway. We conclude that MAPK contributes to muscle differentiation by preventing apoptotic cell death of differentiating myoblasts and that this activity is mediated by stabilization of the p21WAF1 protein.     

Replicative aging down-regulates the myogenic regulatory factors in human myoblasts

Background information. Aging of human skeletal muscle results in a decline in muscle mass and force, and excessive turnover of muscle fibres, such as in muscular dystrophies, further increases this decline. Although it has been shown in rodents, by cross‐age transplantation of whole muscles, that the environment plays an important role in this process, the implication of proliferating aging of the muscle progenitors has been poorly investigated, particularly in humans, since the regulation of cell proliferation differs between rodents and humans. The myogenic differentiation of human myoblasts is regulated by the muscle‐specific regulatory factors. Cross‐talk between the muscle‐specific regulatory factors and the cell cycle regulators is essential for differentiation. The aim of the present study was to determine the effects of replicative senescence on the myogenic programme of human myoblasts. Results. We showed that senescent myoblasts, which could not re‐enter the cell cycle, are still able to differentiate and form multinucleated myotubes. However, these myotubes are significantly smaller. The expression of muscle‐specific regulatory factors and cell cycle regulators was analysed in proliferating myoblasts and compared with senescent cells. We have observed a delay and a decrease in the muscle‐specific regulatory factors and the cyclin‐dependent kinase inhibitor p57 during the early step of differentiation in senescent myoblasts, as well as an increase in the fibroblastic markers. Conclusions. Our results demonstrate that replicative senescence alters the expression of the factors triggering muscle differentiation in human myoblasts and could play a role in the regenerative defects observed in muscular diseases and during normal skeletal‐muscle aging.     

Transformation of avian myogenic cultures with myelocytomatosis virus strain 29

Summary Primary cultures of proliferating chick presumptive myoblasts were exposed of either to two RNA tumor viruses and shortly thereafter treated with 5-bromodeoxyuridine (BUdR) to suppress differentiation. The effect of a Rous sarcoma virus which was temperature-sensitive for transformation (tsRSV) has been characterized previously and was used as a reference for evaluating the effect of a myelocytomatosis virus (MC29) and its helper. Two subcultures following exposure, both infected cultures were extensively transformed as indicated by cell morphology. Relaxation of the BUdR block at this time resulted in cultures which still appeared transformed and did not contain myoblast or myotube-like cells or two of their molecular markers. In contrast, uninfected controls and tsRSV-infected cultures which were shifted-up to the nonpermissive temperature produced numerous spontaneously contracting myotubes. The results confirm previous evidence that infection of presumptive myoblasts by tsRSV at the premissive temperature preserves the extant state of differentiation of presumptive myoblasts and suggest, by analogy, that MC29-infection renders a similar effect.     

Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation

The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5- transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF- alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.     

Control of differentiation in heterokaryons and hybrids involving differentiation-defective myoblast variants

Clones of differentiation-defective myoblasts were isolated by selecting clones of L6 rat myoblasts that did not form myotubes under differentiation-stimulating conditions. Rat skeletal myosin light chain synthesis was induced in heterokaryons formed by fusing these defective myoblasts to differentiated chick skeletal myocytes. This indicates that the structural gene for this muscle protein was still responsive to chick inducing factors and that the defective myoblasts were not producing large quantities of molecules that dominantly suppressed the expression of differentiated functions. The regulation of the decision to differentiate was then examined in hybrids between differentiation- defective myoblasts and differentiation-competent myoblasts. Staining with antimyosin antibodies showed that the defective myoblasts and homotypic hybrids formed by fusing defective myoblasts to themselves could in fact differentiate, but did so more than a thousand times less frequently than the 64% differentiation achieved by competent L6 myoblasts or homotypic competent X competent L6 hybrids. Heterotypic hybrids between differentiation-defective myoblasts and competent L6 cells exhibited an intermediate behavior of approximately 1% differentiation. A theoretical model for the regulation of the commitment to terminal differentiation is proposed that could explain these results by invoking the need to achieve threshold levels of secondary inducing molecules in response to differentiation-stimulating conditions. This model helps explain many of the stochastic aspects of cell differentiation.