Identification of new genes related to the myogenic differentiation arrest of human rhabdomyosarcoma cells (original) (raw)
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Virchows Archiv. B, Cell pathology including molecular pathology, 1988
Three distinct subpopulations (A, B, C) derived from a dimethylbenzanthracene-induced rat rhabdomyosarcoma were established as permanent cell lines. Although the clonal nature of each of these subpopulations was confirmed by repeated recloning procedures, a striking intraclonal phenotypic heterogeneity was observed. By means of immunofluorescence microscopy and transmission electron microscopy, it could be shown that these subpopulations closely recapitulate stages of embryonic rhabdomyogenesis both in vitro and in vivo, but differ in their particular range of maximum differentiation. Embryonic rhabdomyogenesis is imitated most perfectly by subpopulation C, in which multinuclear myotubes are formed in vitro by fusion of mononuclear cells, and alpha-sarcomeric actin is expressed in the multinuclear cells and in a few mononuclear cells. After retransplantation in vivo, subpopulation C further proceeds in fine structural differentiation, now exhibiting myofibrils with a sarcomeric orga...
The Expression of c-Myb Correlates with the Levels of Rhabdomyosarcoma-specific Marker Myogenin
Scientific reports, 2015
The transcription factor c-Myb is required for modulation of progenitor cells in several tissues, including skeletal muscle and its upregulation is observed in many human malignancies. Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors with features of developing skeletal muscle. Several miRNAs are downregulated in RMS, including miR-150, a negative regulator of c-Myb expression. Using the C2C12 myoblast cell line, a cellular model of skeletal muscle differentiation, we showed that miR-150 controls c-Myb expression mainly at the level of translation. We hypothesized that a similar mechanism of c-Myb regulation operates in RMS tumors. We examined expression of c-Myb by immunohistochemistry and revealed c-Myb positivity in alveolar and embryonal tumors, the two most common subgroups of RMS. Furthermore, we showed direct correlation between c-Myb production and myogenin expression. Interestingly, high myogenin levels indicate poor prognosis in RMS patients. c-Myb co...
c-Myb regulates tumorigenic potential of embryonal rhabdomyosarcoma cells
Scientific Reports, 2019
Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors, the most common sub-types are embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma. Immunohistochemical analysis revealed c-Myb expression in both eRMS and aRMS. c-Myb has been reported to be often associated with malignant human cancers. We therefore investigated the c-Myb role in RMS using cellular models of RMS. Specific suppression of c-Myb by a lentiviral vector expressing doxycycline (Dox)-inducible c-Myb shRNA inhibited proliferation, colony formation, and migration of the eRMS cell line (RD), but not of the aRMS cell line (RH30). Upon c-Myb knockdown in eRMS cells, cells accumulated in G0/G1 phase, the invasive behaviour of cells was repressed, and elevated levels of myosin heavy chain, marker of muscle differentiation, was detected. Next, we used an RD-based xenograft model to investigate the role of c-Myb in eRMS tumorigenesis in vivo. We found that Dox administration did not result in efficient suppr...
Journal of Clinical Investigation, 1994
Skeletal myogenesis is regulated by a group of transcription factors (MyoD, myogenin, myf5, and myf6) that are "basic helix-loop-helix" proteins that bind to the promoters of muscle-specific genes and promote their expression. We have previously shown that after a mutation of Leu122 to Arg the DNA binding basic domain of MyoD confers c-myc-like functional characteristics to the protein. In this study we used singlestrand conformation polymorphism analysis to determine whether such mutations occur naturally in rhabdomyosarcomas. We have found that the basic domains of all the myogenic factors remain unaltered in rhabdomyosarcomas. Selection against such mutations may be the result of functional redundancy of these myogenic transcription factors.
RMZ: a new cell line from a human alveolar rhabdomyosarcoma. In vitro expression of embryonic myosin
1986
The RMZ cell line was established from a bone marrow metastasis of a human alveolar rhabdomyosarcoma. Since the beginning of the in vitro culture, RMZ cells showed a differentiation-related morphological heterogeneity: actively proliferating polygonal or spindle-shaped cells were observed along with a few multinucleated myotube-like structures and giant cells, frequently multinucleated. All these cell types were still present after over 40 passages. A set of clonal derivatives has been obtained from the second in vitro subculture. All the clones showed the same morphological heterogeneity of the parental cells, but differed from one another in the degree of differentiation.
Myogenic Differentiation of Human Rhabdomyosarcoma Cells Induced in Vitro by Antineoplastic Drugs1
The effect of various antineoplastic drugs (1-,8-D-arabinofuranosylcytosine, 5-azacytidine, cisplatin, dactinomycin, epirubicin, vincristine, and the activated metabolite of cyclophosphamide, mafosfamide) on cell differentiation in vitro was investigated using a human alveolar rhabdomyosarcoma cell clone, RMZ-RC2. These cells are able to differentiate spontaneously from small mononuclear proliferating elements to termi nal, extremely elongated multinuclear structures resembling myotubes; morphological differentiation is accompanied by the expression of myosins, in particular the embryonic isoform, which was used in this study as a specific marker of myogenic differentiation. The proportion of differentiated myosin-positive cells, which was around 10-15% in control cultures 10-15 days after seeding, was increased by some drug treatments up to 30-40%; the proportion of multinuclear elements was also in creased. 1-0-D-arabinofuranosylcytosine and 5-azacytidine were the most effective drugs, while dactinomycin had no effect; other molecules ranked in between. Since significant increments were usually observed after treatment with drug doses inhibiting cell growth, the kinetic behavior of the absolute number of myosin-positive cells or nuclei was analyzed to assess whether some effects could be due to a negative selection of proliferating, undifferentiated cells. This appeared to be the case for vincristine and epirubicin, while l-/3-D-arabinofuranosylcv tosine, 5-aza cytidine, and, to a lesser degree, mafosfamide and cisplatin actually seemed to increase differentiation ability.
Characterization of a New Human Embryonal Rhabdomyosarcoma Cell Line, RMS-GR
Cancer Science, 1998
A human tumor cell line designated RMS-GR was established from an embryonal rhabdomyosarcoma. The monolayer cells were polygonal, round or spindle-shaped. The RMS-GR cell line became stable with a doubling time of 42 h. Tumorigenicity of the cells was confirmed by heterotransplantion into nude mice. Electron microscopic images showed typical cytoplasmic inclusion of aggregated intermediate filaments and myofibril-like thin filaments. The expression of desmin, vimentin, actin and human myoglobin was recognized by cytofluorometric analyses, and a large fraction of CK-MM and small fractions of CK-BB and MCK-1 isoenzymes were found. Chromosomal analysis showed that the modal chromosome number was consistently near triploid with structural abnormalities mostly involving chromosomes 1, 3 and 8, and additional unidentified markers. No alteration of chromosome 2 was observed. The RMS-GR cell line may provide a system to identify genes which are involved in the pathogenic mechanism of rhabdomyosarcomas, and to investigate the modulation of myogenic differentiation.
Oncogene, 2003
Rhabdomyosarcomas (RMSs) are one of the most common solid tumor of childhood. Rhabdomyosarcoma (RMS) cells fail to both complete the skeletal muscle differentiation program and irreversibly exit the cell cycle as a consequence of an active repression exerted on the muscle-promoting factor MyoD. Myostatin is a negative regulator of normal muscle growth, we have thus studied its possible role in RMS cells. Here, we present evidence that overexpression of myostatin is a common feature of RMS since both subtypes of RMS (embryonal RD and alveolar Rh30 cells) express high levels of myostatin when compared to nontumoral skeletal muscle cells. Interestingly, we found that inactivation of myostatin through overexpression of antisense myostatin or of follistatin (a myostatin antagonist) constructs enhanced differentiation of RD cells. In addition, RD and Rh30 cells treated with blocking antimyostatin antibodies progress into the myogenic terminal differentiation program. Finally, our results suggest that high levels of myostatin could impair MyoD function in RMS cells. These results show that an autocrine myostatin loop contributes to maintain RMS cells in an undifferentiating stage and suggest that new therapeutic approaches could be exploited for the treatment of RMS based on inactivation of myostatin protein.
PLoS ONE, 2013
Dlk1, a member of the Epidermal Growth Factor family, is expressed in multiple tissues during development, and has been detected in carcinomas and neuroendocrine tumors. Dlk1 is paternally expressed and belongs to a group of imprinted genes associated with rhabdomyosarcomas but not with other primitive childhood tumors to date. Here, we investigate the possible roles of Dlk1 in skeletal muscle tumor formation. We analyzed tumors of different mesenchymal origin for expression of Dlk1 and various myogenic markers and found that Dlk1 was present consistently in myogenic tumors. The coincident observation of Dlk1 with a highly proliferative state in myogenic tumors led us to subsequently investigate the involvement of Dlk1 in the control of the adult myogenic programme. We performed an injury study in Dlk1 transgenic mice, ectopically expressing ovine Dlk1 (membrane bound C2 variant) under control of the myosin light chain promotor, and detected an early, enhanced formation of myotubes in Dlk1 transgenic mice. We then stably transfected the mouse myoblast cell line, C2C12, with full-length Dlk1 (soluble A variant) and detected an inhibition of myotube formation, which could be reversed by adding Dlk1 antibody to the culture supernatant. These results suggest that Dlk1 is involved in controlling the myogenic programme and that the various splice forms may exert different effects. Interestingly, both in the Dlk1 transgenic mice and the DLK1-C2C12 cells, we detected reduced myostatin expression, suggesting that the effect of Dlk1 on the myogenic programme might involve the myostatin signaling pathway. In support of a relationship between Dlk1 and myostatin we detected reciprocal expression of these two transcripts during different cell cycle stages of human myoblasts. Together our results suggest that Dlk1 is a candidate marker for skeletal muscle tumors and might be involved directly in skeletal muscle tumor formation through a modulatory effect on the myogenic programme. Citation: Jørgensen LH, Sellathurai J, Davis EE, Thedchanamoorthy T, Al-Bader RWA, et al. (2013) Delta-Like 1 Homolog (Dlk1): A Marker for Rhabdomyosarcomas Implicated in Skeletal Muscle Regeneration. PLoS ONE 8(4): e60692.