The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this thesis I have shown that in the absence of pRb, differentiating primary myoblasts fused to form short myotubes that never twitched and degenerated via a non-apoptotic mechanism. The shortened myotubes exhibited an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation and reduced ATP production. Bcl-2 and autophagy inhibitors restored mitochondrial function and rescued muscle degeneration, leading to twitching myotubes that expressed normal levels of muscle-specific proteins and eventually exited the cell-cycle. A hypoxia-induced glycolytic switch also rescued the myogenic defect after chronic or acute inactivation of Rb in a HIF-1-dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program.
I next tested the effect of retinoblastoma protein family members – p107 and p130 – on skeletal myogenesis in the absence of Rb. Chronic or acute inactivation of Rb plus p130 or Rb plus p107 increased myoblast cell death and reduced myotube formation, yet expression of Bcl-2, treatment with autophagy antagonist or exposure to hypoxia extended myotube survival, leading to long, contracting myotubes that appeared indistinguishable from control myotubes. Triple mutations in Rb family genes further accelerated cell death and led to elongated myocytes or myotubes containing two nuclei, some of which survived and twitched under hypoxia. Whereas nuclei in Rb-/- myotubes were unable to stably exit the cell-cycle, myotubes lacking both p107/p130 became permanently post-mitotic, suggesting that pRb, but not p107 or p130 may be lost in cancer because of the unique requirement for cell-cycle exit during terminal differentiation. This thesis demonstrates that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and reveal a novel link between pRb and cell metabolism.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/29688 |
| Date | 30 August 2011 |
| Creators | Ciavarra, Giovanni |
| Contributors | Zacksenhaus, Eldad |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis, Video |
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