TPA-Induced Differentiation of Human Rhabdomyosarcoma Cells: Expression of the Myogenic Regulatory Factors

Istituto di Istologia ed Embriologia Generale, Fac. di Medicina, Università di Roma La Sapienza, Italy.
Experimental Cell Research (Impact Factor: 3.25). 10/1993; 208(1):209-17. DOI: 10.1006/excr.1993.1239
Source: PubMed


RD cells (a cell line derived from a human rhabdomyosarcoma) undergo a very limited myogenic differentiation despite the fact that they express several myogenic determination genes. Since we have previously shown (Aguanno et al., Cancer Res. 50, 3377, 1990) that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces myogenic differentiation in these cells, in this paper we investigate the mechanism by which TPA interferes with the expression and/or function of the myogenic determination genes. Northern blot analysis revealed that RD cells express the myf3 (the human analog of MyoD) and myf4 (the human analog of myogenin) transcripts, but not myf5 or myf6 transcripts. The myf3 and the myf4 gene products are correctly translated and accumulated in the nuclei as shown by immunofluorescence analysis. The tumor promoter (TPA) does not modify the pattern of expression of the myf factors while it induces the accumulation of muscle-specific transcripts, such as alpha-actin and fast myosin light chain 1, and their corresponding proteins. On the other hand, within 1 day of treatment, TPA inhibits the expression of the Id gene, which is a negative regulator of MyoD activity. However, while the TPA-induced inhibition of Id message accumulation correlates with differentiation, cell confluence also causes a reduction in Id message accumulation, without inducing differentiation. Under our experimental conditions, overexpression of any of the myf cDNAs in RD cells does induce spontaneous differentiation but enhances the effect of TPA treatment independently from the level of the expressed message. These data suggest that differentiation of RD cells is likely to depend upon the activity of complexes containing the various members of the MyoD family, which can be regulated by proteins affecting MyoD dimerization such as Id, but also by other mechanisms induced by TPA, such as phosphorylation.

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    • "There is evidence that RMS cells are susceptible to such therapeutic interventions. For example, some RMS cells differentiate upon treatment with retinoic acid (Crouch and Helman, 1991) or TPA (Aguanno et al., 1990; Bouche et al., 1993). Introduction of a constitutively active MKK6 induces myogenesis in RMS cells by reactivating the p38 MAPK that is normally required for muscle differentiation but is impaired in RMS cells (Puri et al., 2000). "
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    ABSTRACT: Rhabdomyosarcoma (RMS) is a morphologically and clinically heterogeneous group of malignant tumors that resemble developing skeletal muscle and is the most common soft-tissue sarcoma in children and adolescents. The most prominent sites involve head and neck structures (~40%), genito-urinary track (~25%), and extremities (~20%). Embryonal (ERMS) and alveolar (ARMS) are the two major RMS subtypes that are distinct in their morphology and genetic make-up. The prognosis for this cancer depends strongly on tumor size, location, staging, and child's age. In general, ERMS has a more favorable outcome, whereas the mortality rate remains high in patients with ARMS, because of its aggressive and metastatic nature. Over the past two decades, researchers have made concerted efforts to delineate genetic and epigenetic changes associated with RMS pathogenesis. These molecular signatures have presented golden opportunities to design targeted therapies for treating this aggressive cancer. This article highlights recent advances in understanding the molecular pathogenesis of RMS, and addresses promising research areas for further exploration.
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    • "Hereafter we employed different strategies to force the myogenic conversion of embryonal RD cells in order to analyze Neu2 expression. The first approach was to reactivate the p38 pathway, either via administration of the permeable molecule TPA (Bouché et al., 1993; Mauro et al., 2002), or via transient transfection of the constitutively activated MKK6EE kinase form (Puri et al., 2000). After TPA administration or MKK6EE transfection, the differentiation was monitored through the expression of Cav-3 and MyHC, which was significantly increased in terms of transcription (Fig. 2A and C) and protein levels (Fig. 2B and D) after 4 days compared to untreated cells. "
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    • "During the myogenic process of cultured cell lines, p21WAF1 expression is controlled by myogenic transcription factors such as MyoD [10,11]. In ERMS-derived RD cells with transcriptional inactive mutated p53, the myogenic transcription factors, MyoD and myogenin, are, despite being expressed, inactive [23,27]. Inactivation of p53 and myogenic transcription factors might explain the low level of p21WAF1 expression. "
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