Abstract
Backgroundp21WAF1, implicated in the cell cycle control of both normal and malignant cells, can be induced by p53-dependent and independent mechanisms. In some cells, MEKs/ERKs regulate p21WAF1 transcriptionally, while in others they also affect the post-transcriptional processes. In myogenic differentiation, p21WAF1 expression is also controlled by the myogenic transcription factor MyoD. We have previously demonstrated that the embryonal rhabdomyosarcoma cell line undergoes growth arrest and myogenic differentiation following treatments with TPA and the MEK inhibitor U0126, which respectively activate and inhibit the ERK pathway.In this paper we attempt to clarify the mechanism of ERK-mediated and ERK-independent growth arrest and myogenic differentiation of embryonal and alveolar rhabdomyosarcoma cell lines, particularly as regards the expression of the cell cycle inhibitor p21WAF1.Resultsp21WAF1 expression and growth arrest are induced in both embryonal (RD) and alveolar (RH30) rhabdomyosarcoma cell lines following TPA or MEK/ERK inhibitor (U0126) treatments, whereas myogenic differentiation is induced in RD cells alone. Furthermore, the TPA-mediated post-transcriptional mechanism of p21WAF1-enhanced expression in RD cells is due to activation of the MEK/ERK pathway, as shown by transfections with constitutively active MEK1 or MEK2, which induces p21WAF1 expression, and with ERK1 and ERK2 siRNA, which prevents p21WAF1 expression. By contrast, U0126-mediated p21WAF1 expression is controlled transcriptionally by the p38 pathway. Similarly, myogenin and MyoD expression is induced both by U0126 and TPA and is prevented by p38 inhibition. Although MyoD and myogenin depletion by siRNA prevents U0126-mediated p21WAF1 expression, the over-expression of these two transcription factors is insufficient to induce p21WAF1. These data suggest that the transcriptional mechanism of p21WAF1 expression in RD cells is rescued when MEK/ERK inhibition relieves the functions of myogenic transcription factors. Notably, the forced expression of p21WAF1 in RD cells causes growth arrest and the reversion of anchorage-independent growth.ConclusionOur data provide evidence of the key role played by the MEK/ERK pathway in the growth arrest of Rhabdomyosarcoma cells. The results of this study suggest that the targeting of MEK/ERKs to rescue p21WAF1 expression and myogenic transcription factor functions leads to the reversal of the Rhabdomyosarcoma phenotype.
Highlights
Permanent withdrawal from the cell cycle is a crucial event during terminal differentiation
We demonstrated that PKC-α-mediated MAPK (ERKs, JNKs and p38) activation is responsible for orchestrating growth arrest and myogenic differentiation induced by the phorbol ester TPA [30]
Sustained post-transcriptional and transient transcriptional p21WAF1 expression respectively after ERK pathway activation and down-regulation In order to identify the molecular mechanism of G1 arrest following ERK activation and MEK/ERK inhibition in RD cells, we first determined the pattern of G1/S cyclins, CDKs and CDK inhibitor proteins after TPA and U0126 treatments
Summary
Permanent withdrawal from the cell cycle is a crucial event during terminal differentiation. Dysfunction of either cell cycle control or differentiation machinery is responsible for deregulated growth and transformed phenotype [1]. Control of G1/S transition is regulated by a set of specific CDK and cyclin complexes, sequentially expressed, activated and degraded to ensure both entry and progress in the cell cycle [2]. P21WAF1, a cyclin-dependent kinase inhibitor (CKI), which inhibits all cyclin/CDK complexes, those in the G1 phase, has been found to be associated with the growth arrest of both normal and malignant cells [4]. Of genetic alterations which define the embryonal [22,23] and the alveolar subtype [24] These different subtypes share molecular changes, including disruption of the p53 pathway through mutation or MDM2 amplification, and deregulation of imprinted genes at the chromosome region 11p15.5 [22,25]
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