Browsing by Subject "Cell cycle -- Regulation"
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Item Author Correction: REST regulates the cell cycle for cardiac development and regeneration(Springer Nature, 2018-01-12) Zhang, Donghong; Wang, Yidong; Lu, Pengfei; Wang, Ping; Yuan, Xinchun; Yan, Jianyun; Cai, Chenleng; Chang, Ching-Pin; Zheng, Deyou; Wu, Bingruo; Zhou, Bin; Medicine, School of MedicineDespite the importance of cardiomyocyte proliferation in cardiac development and regeneration, the mechanisms that promote cardiomyocyte cell cycle remain incompletely understood. RE1 silencing transcription factor (REST) is a transcriptional repressor of neuronal genes. Here we show that REST also regulates the cardiomyocyte cell cycle. REST binds and represses the cell cycle inhibitor gene p21 and is required for mouse cardiac development and regeneration. Rest deletion de-represses p21 and inhibits the cardiomyocyte cell cycle and proliferation in embryonic or regenerating hearts. By contrast, REST overexpression in cultured cardiomyocytes represses p21 and increases proliferation. We further show that p21 knockout rescues cardiomyocyte cell cycle and proliferation defects resulting from Rest deletion. Our study reveals a REST-p21 regulatory axis as a mechanism for cell cycle progression in cardiomyocytes, which might be exploited therapeutically to enhance cardiac regeneration.Item Characterization of the MDM2 binding regions of ribosomal protein L5(2010-07-20T16:29:38Z) Plummer, Kevin D.; Lu, Hua; Goebl, Mark, 1958-; Hurley, Thomas D., 1961-The MDM2-p53 feedback loop is a well-characterized pathway. p53 is a transcription factor and regulates the transcriptional expression of genes that encode proteins responsible for cellular senescence, cell cycle arrest, apoptosis, and DNA repair. Various cellular stresses can result in p53 activation, including hypoxia, DNA damage by agents such as UV or IR, oncogenic signaling, nucleotide depletion and nucleolar stress from perturbation of ribosomal biogenesis. Under normal conditions, MDM2’s role in the pathway is to inhibit p53 function by directly binding to this protein and facilitating its ubiquitylation and 26S proteasome-mediated degradation. Under stressful cellular conditions, certain proteins interact with and rescue MDM2’s inhibition of p53. For example, upon exposure to small amounts of Actinomycin D, rRNA transcript synthesis is stalled resulting in the release of various ribosomal proteins including RPL5, RPL11 and RPL23; each of which has been shown to bind MDM2 within its central acidic domain and inhibit its ability to destabilize p53. Although the RPL5 binding region of MDM2 have been mapped in prior investigations, the MDM2-binding region(s) of RPL5 have yet to be characterized. By employing RPL5 deletion mutagenesis and in vitro GST-fusion protein-protein association assays with purified proteins, this dissertation attempts to elucidate those regions of RPL5 that may interact with MDM2. Normalizing RPL5-WT to 1.00, our study reveals that the basic N and C-terminals of RPL5 appear to bind with MDM2 while RPL5’s central region displays negligible binding to the central acidic domain of MDM2. Also, the possible meanings of these RPL5 MDM2 binding domains are discussed along with their utilization in potential future applications.Item Differential role of PI-3Kinase p85 (α & β) regulatory subunits in mast cell development(2011-08) Krishnan, Subha; Kapur, Reuben; Wek, Ronald C.; Quilliam, Lawrence; Mooney, Sean D.Stem cell factor (SCF) mediated c-Kit signaling, and downstream activation of Phosphatidylinositol-3 Kinase (PI-3K) is critical for multiple biological effects mediated by mast cells. Mast cells express multiple regulatory subunits of PI-3Kinase, including p85α, p85β, p50α and p55α. In the present study, we have examined the relationship between p85α and p85β subunit in mast cell development and show that loss of p85α in mast cell progenitors impairs their growth, maturation and survival whereas loss of p85β enhances this process. To further delineate the mechanism (s) by which p85α provides specificity to mast cell biology, we compared the amino acid sequences between p85α and p85β subunits. The two isoforms share significant structural homology in the two SH2 domains, but show significant differences in the N-terminal SH3 domain as well as the BCR homology domain. To determine whether the c-Kit induced reduction in growth of mast cells is contributed via the N-terminal SH3 or the BCR homology domain, we cloned and expressed the shorter splice variant p50α, and various truncated mutant versions of p85α in p85α deficient mast cells. We demonstrate both invitro and invivo that while the SH3 and the BH domains of p85 are dispensable for mast cell maturation; they are essential for normal growth and survival. In contrary to existing dogma on redundant functional role of PI-3K regulatory subunits, this study proves that p85α and p85β regulatory subunits of PI-3K have unique roles in mast cell development. We prove that p85α deficiency impairs the expression of multiple growth, survival and maturation related genes whereas p85β deficiency inhibits c-Kit receptor internalization and degradation. This novel finding on negative role of p85β in mast cell development has significant clinical implication, as this knowledge could be used to develop treatments for mast-cell-associated leukemia and mastocytosis.Item Effects of Altering Cell Proliferation on Hematopoietic Stem and Progenitor Cell Function(2011-06-14) Rohrabaugh, Sara L.; Broxmeyer, Hal E.; Pelus, Louis; Roman, Ann; Yoder, Mervin C.Cell cycle checkpoints guarantee movement through the cell cycle in an appropriate manner. The spindle assembly checkpoint (SAC) ensures the proper segregation of chromosomes into daughter cells during mitosis. Mitotic arrest deficiency 2 (Mad2), a member of the mitotic checkpoint proteins, appears to be crucial for generating the wait anaphase signal to prevent onset of anaphase. We first studied the SAC in hematopoietic stem cells (HSC) to ensure that it was functional. Our previous studies found that prolonged SAC activation was uncoupled from apoptosis initiation in mouse and human embryonic stem cells (ESC). We found that upon treatment with a microtubule-destabilizing agent, HSC arrested in M-phase and subsequently initiated apoptosis. Thus unlike ESC, HSC exhibit coupling of prolonged SAC activation with apoptosis. We studied the effects of Mad2+/- on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2-haploinsufficient (Mad2+/-) mice. We found that Mad2+/- HPCs were protected from the cytotoxic effects of cytarabine (Ara-C), a cycle specific agent, consistent with Mad2+/- HPCs being in a slow or non-cycling state. Mad2 haploinsufficiency did not affect recovery of functional HPC after treatment with cyclophosphamide or high sub-lethal dose irradiation, both non-cycle specific agents. There were no differences in immunophenotype defined HSCs in Mad2+/- and Mad2+/+ mice, data confirmed by functional HSC competitive repopulation assays. To better understand the role of Mad2 in HPC, E3330, a cytostatic agent, was used to assess the redox function of Ape1/Ref-1, and colony formation in vitro was examined under normoxic and lowered O2 tension. Mad2+/- HPCs were less responsive to E3330 than Mad2+/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2+/- HPCs did not exhibit enhanced growth in lowered oxygen tension, in contrast to Mad2+/+ HPCs. Our studies have unexpectedly found that Mad2 haploinsufficiency is protective from the cytotoxic effects of a cycle specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.Item Expression and Function of the PRL Family of Protein Tyrosine Phosphatase(2012-12) Dumaual, Carmen Michelle; Stauffacher, Cynthia; Randall, Stephen Karl, 1953-; Malkova, Anna; Sandusky, George Earl, 1945-The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.Item The Fanconi anemia signaling network regulates the mitotic spindle assembly checkpoint(2014) Enzor, Rikki S.; Clapp, D. Wade; Broxmeyer, Hal E.; Haneline, Laura S.; Srour, Edward F.Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.Item mTOR regulates Aurora A via enhancing protein stability(2013-12) Fan, Li; Quilliam, Lawrence; Atkinson, Simon; Goebl, Mark G.; Harrington, Maureen A.; Wek, Ronald C.Mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis. Dysregulation of mTOR signaling occurs in many human cancers and its inhibition causes arrest at the G1 cell cycle stage. However, mTOR’s impact on mitosis (M-phase) is less clear. Here, suppressing mTOR activity impacted the G2-M transition and reduced levels of M-phase kinase, Aurora A. mTOR inhibitors did not affect Aurora A mRNA levels. However, translational reporter constructs showed that mRNA containing a short, simple 5’-untranslated region (UTR), rather than a complex structure, is more responsive to mTOR inhibition. mTOR inhibitors decreased Aurora A protein amount whereas blocking proteasomal degradation rescues this phenomenon, revealing that mTOR affects Aurora A protein stability. Inhibition of protein phosphatase, PP2A, a known mTOR substrate and Aurora A partner, restored mTOR-mediated Aurora A abundance. Finally, a non-phosphorylatable Aurora A mutant was more sensitive to destruction in the presence of mTOR inhibitor. These data strongly support the notion that mTOR controls Aurora A destruction by inactivating PP2A and elevating the phosphorylation level of Ser51 in the “activation-box” of Aurora A, which dictates its sensitivity to proteasomal degradation. In summary, this study is the first to demonstrate that mTOR signaling regulates Aurora-A protein expression and stability and provides a better understanding of how mTOR regulates mitotic kinase expression and coordinates cell cycle progression. The involvement of mTOR signaling in the regulation of cell migration by its upstream activator, Rheb, was also examined. Knockdown of Rheb was found to promote F-actin reorganization and was associated with Rac1 activation and increased migration of glioma cells. Suppression of Rheb promoted platelet-derived growth factor receptor (PDGFR) expression. Pharmacological inhibition of PDGFR blocked these events. Therefore, Rheb appears to suppress tumor cell migration by inhibiting expression of growth factor receptors that in turn drive Rac1-mediate actin polymerization.Item The role of acid sphingomyelinase in autophagy(2014-07-11) Justice, Matthew Jose; Petrache, Irina; Blum, Janice Sherry, 1957-; Wek, Ronald C.Autophagy is a conserved cellular process that involves sequestration and degradation of cytosolic contents. The cell can engulf autophagic cargo (lipids, long-lived proteins, protein aggregates, and pathogens) through a double bound membrane called an autophagosome that fuses with a lysosome where hydrolases then degrade these contents. This process is one of the main defenses against starvation and is imperative for newborns at birth. Research on this process has increased exponentially in the last decade since its discovery almost a half a century ago. It has been found that autophagy is an important process in many diseases, continues to be at the forefront of research, and is clearly not fully understood. Our preliminary cell culture data in endothelial and epithelial cells show that a blockade of the de novo ceramide synthesis pathway, during treatment with an autophagy stimulus (cigarette smoke extract exposure), does not result in any reduction in autophagy or autophagic flux. Conversely, when acid sphingomyelinase (ASM) is pharmacologically inhibited, which prevents the generation of ceramide from sphingomyelin in an acidic environment, a profound increase in autophagy is observed. In this work, we hypothesize that (ASM) is an endogenous inhibitor of autophagy. ASM has two forms, a secreted form and a lysosomal form. N-terminal processing in the Golgi determines its cellular fate. In the lysosomal form, the phosphodiesterase is bound in the lysosomal membrane. The pharmacological inhibition mechanism is to release ASM from the membrane and allow other hydrolases to actively degrade the enzyme which, in turn, decreases the activity of ASM. This suggests that either the activity of ASM is a regulator of autophagy or that the presence of ASM, activity aside, is required for the lysosomal nutrient sensing machinery (LYNUS) to function properly. Here, we show that ASM is, in fact, an endogenous inhibitor of autophagy in vitro. The phosphorylation status of P70 S6k, a downstream effector of mammalian target of rapamycin (mTOR), which is part of the LYNUS, shows that dissociation of ASM from the membrane regulates mTOR and disturbs the LYNUS in such a manner as to signal autophagy.