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The spacer region of pRb2/p130 was reported to be able to inhibit the kinase activity of Cdk2. The region responsible for the inhibitory effect was further narrowed down to a 39-amino-acid sequence, which was named as Spa310. In this dissertation, the anti-cancer functions and mechanisms of Spa310 were studied. The synthesized Spa310 peptide was able to inhibit the kinase activities of Cdk2/Cyclin E/A complexes. In vitro kinase assays showed the inhibition occurred in a dose-dependent manner. The half maximal inhibition concentration of the Spa310 in the kinase assay was 1.67mM. In addition, it has been shown that Spa310 peptide is able to inhibit the kinase activities of both Cdk2/Cyclin E and Cdk2/Cyclin A. Intra-cellular distribution study using fluorescein-labeled Spa310 peptide showed that Spa310 was able to localize to the nuclei of A549 cancer cells. Some data indicated the endoplasmic reticulum might play a role in transporting Spa310 peptide from cytoplasm to the nucleus. At high concentration, the treatment of Spa310 peptide was able to arrest cells at the G0/G1 phase of the cell cycle and reduce the growth of xenografted tumors in nude mice. Further studies indicated Spa310 peptide is not a specific inhibitor for Cdk2/Cyclin E/A. It is also able to inhibit the kinase activities of Cdk1/Cyclin B, Cdk4/Cyclin D and Cdk9/Cyclin T/K. Result of a binding assay using GST-Spa310 and in vitro transcribed/translated Cdk2 did not support a direct binding between Spa310 and Cdk2. Additionally, GST-Spa310 was unable to bind to the in vitro transcribed/translated Cyclin E. At first, co-immunoprecipitation experiments indicated a weak binding between Spa310 peptide and Cdk2. However, later this weak binding was proven to be unspecific and only occurred when the concentration of Spa310 peptide was high. Thus, the hypothesized mechanism of the inhibitory effect of Spa310 was not supported. After noticing three classic Cdk phosphorylation sites present in Spa310, it was proven that Spa310 is a substrate for Cdk1, 2, 4 and 9. Results of kinase assays supported the inhibitory effect of Spa310 on the different Cyclin-dependent kinases was resulted from a substrate-competitive mechanism. Although the data generated from this study does not support Spa310 is a potent peptide inhibitor for the Cdks, knowledge gained from and the approach used in this research can be applied to design and develop more potent and specific Cdk2 peptide inhibitors, which have their potentials to work as powerful anti-cancer reagents. / Biology

The DREAM complex assembles during G0/G1 when RB-like protein p130 recruits E2F4, DP1, and a core complex of five MuvB proteins to repress genes involved in cell cycle progression. In S-phase, the MuvB core dissociates from p130 and binds to BMYB transcription factor. Binding of the MuvB core to p130 requires phosphorylation of its subunit LIN52 at S28 residue by DYRK1A protein kinase. However, little is known about how the MuvB core interacts with p130 to form the DREAM complex, and how these interactions are manipulated throughout the cell cycle. In collaboration with Dr. Seth Rubin, we characterized the structural basis for DREAM assembly, and found that the LxSxExL sequence in LIN52 directly interacts with the LxCxE binding cleft within the pocket domain of p130. Furthermore, immunoprecipitation and proliferation assays revealed that mutating the LIN52 LxSxExL sequence to mimic the canonical LxCxE motif found in viral oncoproteins reduces cellular proliferation and stabilizes the DREAM complex in the presence of viral proteins. We addressed how the DREAM complex is disassembled upon cell cycle entry and found that CDK phosphorylation of p130 inactivates the DREAM complex by displacing p130 from the MuvB core. Under certain conditions, we found that BMYB and p130 simultaneously bind the MuvB core, while overexpression of BMYB disrupts DREAM assembly. Together, our study provides insight into the structural mechanisms of DREAM assembly and function, which can help identify novel approaches to halt tumor cell proliferation or dormancy.

The retinoblastoma family of phosphoproteins consisting of the retinoblastoma protein (pRB) and the two structurally related proteins p130 and p107 play an important role in the negative regulation of cell cycle progression. Hypophosphorylated pocket proteins interact with the different members of the E2F family and repress the transcription of E2F-dependent genes and consequently suppress cell cycle progression through the G0/G1 transition and the restriction point in G1. Mitogenic stimulation results in sequential activation of cyclin/CDK complexes in mid to late G1, leading to subsequent hyperphosphorylation at multiple Ser/Thr sites of pocket proteins triggering dissociation of pocket protein/E2F complexes. This disruption leads to de-repression of many E2F dependent genes whose products are essential for cell cycle progression. The traditional view has been that pocket proteins continue to be hyperphosphorylated through the S and G2 phases and following cyclin/CDK inactivation during mitotic exit become dephosphorylated by action of PP1. However, our lab observed that upon treatment of asynchronously growing cells with the CDK inhibitor Flavopiridol or CHX, pocket proteins, are rapidly dephosphorylated correlating with the inactivation of G1/CDKs and down regulation of D-type cyclins, respectively. Pocket protein dephosphorylation was prevented by pre-treating these cells with phosphtase inhibitors at a concentration selective for PP2A, implicating PP2A or PP2A-like serine/threonine phosphatase in this iii process. The involvement of PP2A on pocket protein dephosphorylation was further strengthened by the observation that SV40 small t antigen (ST) delays/prevents p107 dephosphorylation. Moreover, a physical association between PP2A/C and p130/p107 was observed throughout the cell cycle that was not affected by CHX treatment, strongly suggesting that CHX-induced dephosphorylation is not the result of increased pocket protein targeting by PP2A, but rather that a dynamic equilibrium between CDKs and PP2A is shifted to dephosphorylation when CDK activity is compromised. This dynamic equilibrium operates throughout the cell cycle. PP2A is a trimeric enzyme complex consisting of a catalytic C, a structural A and substrate specific B subunit. There are four families of regulatory B subunits designated B, B’, B’’ and B’’’, each with several members encoded by genes with multiple splice variants that mediate substrate specificity and subcellular localization. It has been reported recently that in excess of 200 functional distinct PP2A holoenzymes can assemble with distinct specificities. Therefore, to gain insight into the mechanisms that regulate the steady state phosphorylation of pocket proteins throughout the cell cycle, it was essential to identify the specific holoenzyme complexes involved. To this end, it was identified that a PP2A trimeric holoenzyme containing B55α specifically targets and dephosphorylates p107/p130 both in vitro and in mammalian cells. B55α associates directly with the spacer of p107 and this interaction seems to be indirectly enhanced by the C-terminus of p107. The decreased association of p107 with PP2A/C of the B55α/PP2A holoenzyme complex upon treatment with ST further confirmed the role of B55α in mediating p107-PP2A/C interaction. Our data also revealed an interaction between B55α and p130, but not pRb, which appears to prefer a PR70, suggesting selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes. In accordance with this, recombinant purified B55α dephosphorylates p107 in vitro. Limited ectopic expression of B55α but not other subunits, result in ST sensitive dephosphorylation of p107 and p130 in cells. Further shRNA mediated knockdown of B55α results in hyperphosphorylation of p107 and p130. This suggests that the cellular levels of B55α are critical in modulating the phosphorylation status of p107/p130 rather than just catalyzing the dephosphorylation of these proteins when the activity of CDKs is compromised. Since ST disrupts the B55α/PP2A holoenzyme complex by binding to the PP2A-A-C dimer and leads to hyperphosphorylation of pocket proteins it is conceivable that ST mediates its effects on cell proliferation at least in part, via inactivation of the PP2A holoenzymes that activates pocket proteins. Given the sensitivity of p107 phosphorylation to the cellular levels of B55α, future analyses should ascertain if deregulation of B55α leads to hyperphosphorylation of pocket proteins and abnormal cell cycle progression. / Molecular Biology and Genetics

High expression of B-Myb (encoded by MYBL2), an oncogenic transcription factor, is associated with cell cycle deregulation and poor prognosis in several cancers, including ovarian cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adaptor for assembly of both the DREAM and MMB complexes, by a mechanism that requires the S28 phosphorylation site in LIN52. To validate our cellular findings, we determined the effect of B-Myb levels on DREAM target gene expression in HGSOC tissue samples and corresponding patient outcomes. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification and establish the rationale for targeting B-Myb to restore cell cycle control.

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.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. HPVs are divided into high-risk (HR) or low-risk (LR) types based on their oncogenic potential. HPVs 16 and 18 are considered HR types and can cause cervical cancer. HPVs 6 and 11 are classified as LR and are associated with condyloma acuminata (genital warts). Viral proteins of both HR and LR HPVs must be able to facilitate a replication competent environment. The E7 proteins of LR and HR HPVs are responsible for maintenance of S-phase activity in infected cells. HR E7 proteins target all pRb family members (pRb, p107 and p130) for degradation. LR E7 does not target pRb or p107 for degradation, but does target p130 for degradation. Immunohistochemistry experiments on HPV 6 infected patient biopsies of condyloma acuminata showed that detection of p130 was decreased in the presence of the whole HPV 6 genome. Further, the effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Experiments were performed using human foreskin keratinocytes transduced with HPV 6 E7, HPV 16 E7 or parental vector. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the viii presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. Experiments were also conducted to detect E7-binding partners. Cyclin C and cullin 5 were identified as proteins capable of binding to both HPV 6 E7 and HPV 16 E7. Preliminary experiments showed that decreasing protein levels of p600, a binding partner of both HPV 6 E7 and HPV 16 E7, by RNA interference might affect p130 stability. Elucidating the mechanisms of p130 degradation may identify potential targets for preventing degradation of p130 and allowing restoration of cell cycle control.

Ischemic stroke occurs as a result of a lack or severe reduction of blood supply to the brain. Presently therapeutic interventions are limited and there is a need to develop new and efficacious stroke treatments. To this end, a great deal of research effort has been devoted to studying the potential molecular mechanisms involved in ischemic neuronal death. Correlative evidence demonstrated a paradoxical activation of the cell cycle machinery in ischemic neurons. The levels and activity of key cell cycle regulators including cyclin D1, Cdk2 and Cdk4 are upregulated following ischemic insults. However, the functional relevance of these various signals following ischemic injury was unclear. Accordingly, the research described in this thesis address the functional relevance of the activation of the cell cycle machinery in ischemic neuronal death. The data indicate that the inhibition of Cdk4 protects neurons from ischemia-induced delayed death, whereas abrogation of Cdk5 activity prevents excitotoxicity-induced damage in vitro and in vivo. Examination of upstream activators of mitotic-Cdks showed that Cdc25A is a critical mediator of delayed ischemic neuronal death. Investigation of the potential molecular mechanism by which cell cycle regulators induced neuronal death revealed perturbations in the levels and activity of key downstream targets of Cdk4. The retinoblastoma protein family members, pRb and p130 are increasingly phosphorylated following ischemic stresses. Importantly, p130 and E2F4 proteins are drastically reduced following ischemic insults. Additionally, E2F1 association with promoters of pro-apoptotic genes are induced while that of E2F4 is reduced. These changes appear to be important determinants in ischemic neuronal death. Cumulatively, the data supports the activation of the cell cycle machinery as a pathogenic signal contributing to ischemic neuronal death. The development of neuroprotectant strategies for stroke has been hampered in part by its complex pathophysiology. Previous research indicated that flavopiridol, a general CDK-inhibitor, is unable to provide sustained neuroprotection beyond one week following cerebral ischemia. The potential benefit of combining flavopiridol with another neuroprotectant, minocycline, was explored. The data indicate that while this approach provided histological protection 10 weeks after insult, the protected neurons are not functional due to progressive dendritic degeneration. This evidence indicates that targeting cell cycle pathways in stroke while important must be combined with other therapeutic modalities to fully treat stroke-induced damage.

Ischemic stroke occurs as a result of a lack or severe reduction of blood supply to the brain. Presently therapeutic interventions are limited and there is a need to develop new and efficacious stroke treatments. To this end, a great deal of research effort has been devoted to studying the potential molecular mechanisms involved in ischemic neuronal death. Correlative evidence demonstrated a paradoxical activation of the cell cycle machinery in ischemic neurons. The levels and activity of key cell cycle regulators including cyclin D1, Cdk2 and Cdk4 are upregulated following ischemic insults. However, the functional relevance of these various signals following ischemic injury was unclear. Accordingly, the research described in this thesis address the functional relevance of the activation of the cell cycle machinery in ischemic neuronal death. The data indicate that the inhibition of Cdk4 protects neurons from ischemia-induced delayed death, whereas abrogation of Cdk5 activity prevents excitotoxicity-induced damage in vitro and in vivo. Examination of upstream activators of mitotic-Cdks showed that Cdc25A is a critical mediator of delayed ischemic neuronal death. Investigation of the potential molecular mechanism by which cell cycle regulators induced neuronal death revealed perturbations in the levels and activity of key downstream targets of Cdk4. The retinoblastoma protein family members, pRb and p130 are increasingly phosphorylated following ischemic stresses. Importantly, p130 and E2F4 proteins are drastically reduced following ischemic insults. Additionally, E2F1 association with promoters of pro-apoptotic genes are induced while that of E2F4 is reduced. These changes appear to be important determinants in ischemic neuronal death. Cumulatively, the data supports the activation of the cell cycle machinery as a pathogenic signal contributing to ischemic neuronal death. The development of neuroprotectant strategies for stroke has been hampered in part by its complex pathophysiology. Previous research indicated that flavopiridol, a general CDK-inhibitor, is unable to provide sustained neuroprotection beyond one week following cerebral ischemia. The potential benefit of combining flavopiridol with another neuroprotectant, minocycline, was explored. The data indicate that while this approach provided histological protection 10 weeks after insult, the protected neurons are not functional due to progressive dendritic degeneration. This evidence indicates that targeting cell cycle pathways in stroke while important must be combined with other therapeutic modalities to fully treat stroke-induced damage.