Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2020, College of Sciences and Health Professions

The formation of functional skeletal muscle is the consequence of both the differentiation and apoptosis of skeletal myoblasts. Ex vivo culture of skeletal myoblasts provides a tractable model for the study of these two coordinately regulated processes. We have previously reported that 23A2 myoblasts stably expressing a dominant negative Death Receptor 5 (A2:dnDR5 myoblasts) exhibit decreased basal mRNA and protein expression of the master muscle regulatory transcription factor MyoD. This decrease at the mRNA level is not a consequence of altered stability. Binding of the transcription factor SRF to a non-canonical CArG box within a serum response element (SRE) in the distal regulatory region (DRR) of the MyoD gene is required for basal MyoD expression. Herein, we report that A2:dnDR5 myoblasts exhibit a decrease in the amount of SRF bound at this CArG box. Additionally, in A2:dnDR5 myoblasts, we observe a decrease in the phosphorylation indicative of activation of SRF as well as a decrease in the phosphorylation indicative of activation of the mitogen-activated protein kinase p38, which is known to activate SRF. Pharmacological inhibition of p38, or of caspase-3, in parental 23A2 myoblasts mimics the decreased activation of SRF and p38, the decreased binding of SRF to the MyoD CArG box, and the decreased levels of MyoD mRNA and protein detected in the A2:dnDR5 myoblasts. Taken together, these results suggest that basal signaling through DR5 to caspase 3 leads to the activation of p38 and subsequently SRF to maintain basal expression of MyoD.

Committee: Crystal Weyman (Advisor) Subjects: Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2009, College of Science

We have previously reported that the level of MyoD expression correlates with the level of apoptosis that occurs in a subpopulation of skeletal myoblasts induced to differentiate by serum withdrawal. Herein we document that MyoD expression dictates the apoptotic threshold in myoblasts and fibroblasts in response to a variety of apoptotic stimuli. Specifically, re-expression of MyoD inskeletal myoblasts rendered defective for both differentiation and apoptosis by the expression of oncogenic Ras restores their ability to undergo both differentiation and apoptosis in response to serum withdrawal. Further, using a fibroblast cell line expressing an estrogen receptor:MyoD fusion protein, we have determined that addition of estrogen sensitizes these fibroblasts to apoptosis induced by serum withdrawal, or by treatment with etoposide or thapsigargin. RNAi mediated silencing of MyoD in either 23A2 or C2C12 myoblasts renders these cells resistant to apoptosis induced by serum withdrawal, or by treatment with etoposide or thapsigargin. Finally, MyoD mediated regulation of the apoptotic response to these various stimuli correlates with the level of induction of the pro-apoptotic Bcl-2 family member PUMA.

Committee: Crystal Weyman PhD (Advisor); Anton Komar PhD (Committee Member); Barsanjit Mazumder PhD (Committee Member); Martha Cathcart PhD (Committee Member); Alexandru Almasan (Other); Andrew Resnick PhD (Other) Subjects: Cellular Biology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2011, Biochemistry

Rhabdomyosarcoma (RMS) is one of the most common soft-tissue sarcomas and is a highly aggressive, malignant solid tumor that primarily affects children and young adults. RMS is thought to arise as a consequence of regulatory disruption of the differentiation program of the skeletal muscle cells. Current knowledge of the molecular mechanisms responsible for this disruption in RMS tumors, however, is limited. The most aggressive form of this muscle cancer is alveolar rhabdomyosarcoma (ARMS), which has a poor prognosis and a high frequency of metastasis. Current aggressive chemotherapeutic approaches have improved the outcome of ARMS treatment; however, the cure rate for metastatic ARMS is still only 20% to 30%. Previously, our laboratory has reported that histone methyltransferase Suv39h (mouse homologue of human SUV39H)-mediated epigenetic mechanism controls the growth and differentiation of murine skeletal muscle progenitor cells. We demonstrated that Suv39h blocks MyoD, which acts as a key transcriptional regulator of the muscle differentiation program. In our present study, we have found increased expression of SUV39H in ARMS cells when they are cultured under differentiation-permissible conditions. Moreover, SUV39H-depleted ARMS cells showed MyoD-mediated transcriptional activation, MyoD-dependent growth arrest, reduced anchorage-independent growth, replacement of a repressive mark with an active mark on the muscle-specific gene promoter, and induction of differentiation-associated gene expression. These results suggest that SUV39H overexpression blocks myogenic differentiation program of ARMS cells. Altogether, our results from the current study indicate that SUV39H negatively regulates MyoD in ARMS cells in the failure of muscle differentiation. Based on these results on ARMS cells, we aimed to isolate the pharmacological compound(s) that target the SUV39H-associated mechanism and restore the differentiation program in ARMS cells. To achieve this aim, we gener (open full item for complete abstract)

Committee: Asoke Mal PhD (Advisor); Andrei Gudkov PhD (Advisor); Ed Stavnezer PhD (Committee Chair); Hung-Ying Kao PhD (Committee Member); David Danielpour PhD (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology; Oncology; Pharmaceuticals

Doctor of Philosophy, Case Western Reserve University, 2009, Biochemistry

Ski is the most studied member of a family of proteins all sharing a conserved Dachshund homology domain. It has been implicated in oncogenic transformation, myogenic conversion of avian embryo fibroblasts and also many aspects of vertebrate development, especially myogenesis. Ski-/- mice exhibit severe defects in skeletal muscle and die at birth, yet little is know about either the underlying mechanisms or the role of Ski in adult muscle regeneration. In these studies, I used Ski knockout mice and C2C12 myoblast cultures to address these issues, respectively. Detailed analysis of Ski-/- embryos revealed dramatically reduced hypaxial muscles but less affected epaxial muscles. The reduced number of myogenic regulatory factor positive cells in Ski-/- mice suggested an insufficient myogenic cell pool to support muscle formation. However, both the dermomyotomal hypaxial progenitors and myotomal epaxial progenitors formed and committed to myogenic fate appropriately. The hypaxial muscle defect in Ski-/- mice was not caused by abnormal proliferation, terminal differentiation or apoptosis of the myogenic cells either, but due to impaired migration of embryonic hypaxial progenitors. Surprisingly, the normal distribution of fetal/postnatal myogenic progenitors in Ski-/- mice suggested different effects of Ski on the behaviors of embryonic and fetal/postnatal myogenic progenitors. In addition, although not affecting the terminal differentiation of embryonic myogenic cells, Ski was necessary for that of adult satellite-cell derived C2C12 myoblasts as evidenced by impaired myotube formation and reduced induction of genes essential for myogenic differentiation in the absence of Ski. This function was mainly mediated by Ski's ability to form a complex with Six1 and Eya3 and activate Myog transcription through a MEF3 site. It is important in the future to further study mechanisms underlying the contrasting effects of Ski on embryonic, fetal and adult muscle development, to investi (open full item for complete abstract)

Committee: David Samols PhD (Committee Chair); Ed Stavnezer PhD (Advisor); Clemencia Colmenares PhD (Committee Member); Nikki Harter PhD (Committee Member); Lynn Landmesser PhD (Committee Member) Subjects: Biomedical Research