Browsing by Subject "Cell cycle"

Now showing 1 - 10 of 26
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    A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response-Dependent Survival of Quiescent Cancer Cells
    (American Association for Cancer Research, 2023) Calvo, Veronica; Zheng, Wei; Adam-Artigues, Anna; Staschke, Kirk A.; Huang, Xin; Cheung, Julie F.; Nobre, Ana Rita; Fujisawa, Sho; Liu, David; Fumagalli, Maria; Surguladze, David; Stokes, Michael E.; Nowacek, Ari; Mulvihill, Mark; Farias, Eduardo F.; Aguirre-Ghiso, Julio A.; Biochemistry and Molecular Biology, School of Medicine
    Purpose: The integrated stress response (ISR) kinase PERK serves as a survival factor for both proliferative and dormant cancer cells. We aim to validate PERK inhibition as a new strategy to specifically eliminate solitary disseminated cancer cells (DCC) in secondary sites that eventually reawake and originate metastasis. Experimental design: A novel clinical-grade PERK inhibitor (HC4) was tested in mouse syngeneic and PDX models that present quiescent/dormant DCCs or growth-arrested cancer cells in micro-metastatic lesions that upregulate ISR. Results: HC4 significantly blocks metastasis, by killing quiescent/slow-cycling ISRhigh, but not proliferative ISRlow DCCs. HC4 blocked expansion of established micro-metastasis that contained ISRhigh slow-cycling cells. Single-cell gene expression profiling and imaging revealed that a significant proportion of solitary DCCs in lungs were indeed dormant and displayed an unresolved ER stress as revealed by high expression of a PERK-regulated signature. In human breast cancer metastasis biopsies, GADD34 expression (PERK-regulated gene) and quiescence were positively correlated. HC4 effectively eradicated dormant bone marrow DCCs, which usually persist after rounds of therapies. Importantly, treatment with CDK4/6 inhibitors (to force a quiescent state) followed by HC4 further reduced metastatic burden. In HNSCC and HER2+ cancers HC4 caused cell death in dormant DCCs. In HER2+ tumors, PERK inhibition caused killing by reducing HER2 activity because of sub-optimal HER2 trafficking and phosphorylation in response to EGF. Conclusions: Our data identify PERK as a unique vulnerability in quiescent or slow-cycling ISRhigh DCCs. The use of PERK inhibitors may allow targeting of pre-existing or therapy-induced growth arrested "persister" cells that escape anti-proliferative therapies.
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    Analysis of the cytotoxic activity of carboplatin and gemcitabine combination
    (The International Institute of Anticancer Research, 2010-11) Wang, Sisi; Zhang, Hongyong; Evans, Christopher; Pan, Chong-Xian; Department of Pathology and Laboratory Medicine, IU School of Medicine
    Aim To determine if the drug doses and administration schedules of carboplatin and gemcitabine combination affect antitumor effects. Materials and Methods The inhibition of cell viability was measured by MTT assay. Median effect analysis was conducted to determine the cytotoxicity activity of carboplatin and gemcitabine combination. Cell cycle changes were analyzed by flow cytometry. Results Synergism was observed when the bladder cancer cell line 5637 cells were treated with gemcitabine followed by carboplatin or concurrent carboplatin/gemcitabine. In contrast, moderate antagonism was observed when cells were treated with carboplatin followed by gemcitabine. Cell cycle analysis showed that the combined effect of these two drugs was cell cycle disturbance. Conclusions Different doses and administration schedules affect the anti-tumor effect of carboplatin/gemcitabine combination that may have clinical significance in the treatment for bladder cancer.
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    Apicoplast Dynamics During Plasmodium Cell Cycle
    (Frontiers Media, 2022-04-29) Elaagip, Arwa; Absalon, Sabrina; Florentin, Anat; Pharmacology and Toxicology, School of Medicine
    The deadly malaria parasite, Plasmodium falciparum, contains a unique subcellular organelle termed the apicoplast, which is a clinically-proven antimalarial drug target. The apicoplast is a plastid with essential metabolic functions that evolved via secondary endosymbiosis. As an ancient endosymbiont, the apicoplast retained its own genome and it must be inherited by daughter cells during cell division. During the asexual replication of P. falciparum inside human red blood cells, both the parasite, and the apicoplast inside it, undergo massive morphological changes, including DNA replication and division. The apicoplast is an integral part of the cell and thus its development is tightly synchronized with the cell cycle. At the same time, certain aspects of its dynamics are independent of nuclear division, representing a degree of autonomy in organelle biogenesis. Here, we review the different aspects of organelle dynamics during P. falciparum intraerythrocytic replication, summarize our current understanding of these processes, and describe the many open questions in this area of parasite basic cell biology.
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    Cardiac Troponin I-interacting Kinase impacts cardiomyocyte S-phase activity but not cardiomyocyte proliferation
    (American Heart Association, 2023) Reuter, Sean P.; Soonpaa, Mark H.; Field, Dorothy; Simpson, Ed; Rubart-von der Lohe, Michael; Lee, Han Kyu; Sridhar, Arthi; Ware, Stephanie M.; Green, Nick; Li, Xiaochun; Ofner, Susan; Marchuk, Douglas A.; Wollert, Kai C.; Field, Loren J.; Pediatrics, School of Medicine
    Background: Identifying genetic variants that affect the level of cell cycle reentry and establishing the degree of cell cycle progression in those variants could help guide development of therapeutic interventions aimed at effecting cardiac regeneration. We observed that C57Bl6/NCR (B6N) mice have a marked increase in cardiomyocyte S-phase activity after permanent coronary artery ligation compared with infarcted DBA/2J (D2J) mice. Methods: Cardiomyocyte cell cycle activity after infarction was monitored in D2J, (D2J×B6N)-F1, and (D2J×B6N)-F1×D2J backcross mice by means of bromodeoxyuridine or 5-ethynyl-2'-deoxyuridine incorporation using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus analysis, fine scale genetic mapping, whole exome sequencing, and RNA sequencing analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype. Results: (D2J×B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar compared with D2J mice (0.798±0.09% versus 0.056±0.004%; P<0.001). Quantitative trait locus analysis of (D2J×B6N)-F1×D2J backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of chromosome 3 (logarithm of the odds score=6.38; P<0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, Tnni3k (troponin I-interacting kinase) is expressed in B6N hearts but not in D2J hearts. Transgenic expression of TNNI3K in a D2J genetic background results in elevated cardiomyocyte S-phase activity after injury. Cardiomyocyte S-phase activity in both Tnni3k-expressing and Tnni3k-nonexpressing mice results in the formation of polyploid nuclei. Conclusions: These data indicate that Tnni3k expression increases the level of cardiomyocyte S-phase activity after injury.
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    Cell-Cycle-Based Strategies to Drive Myocardial Repair
    (Springer, 2009-04-02) Zhu, Wuqiang; Hassink, Rutger J.; Rubart, Michael; Field, Loren J.; Pediatrics, School of Medicine
    Cardiomyocytes exhibit robust proliferative activity during development. After birth, cardiomyocyte proliferation is markedly reduced. Consequently, regenerative growth in the postnatal heart via cardiomyocyte proliferation (and, by inference, proliferation of stem-cell-derived cardiomyocytes) is limited and often insufficient to affect repair following injury. Here, we review studies wherein cardiomyocyte cell cycle proliferation was induced via targeted expression of cyclin D2 in postnatal hearts. Cyclin D2 expression resulted in a greater than 500-fold increase in cell cycle activity in transgenic mice as compared to their nontransgenic siblings. Induced cell cycle activity resulted in infarct regression and concomitant improvement in cardiac hemodynamics following coronary artery occlusion. These studies support the notion that cell-cycle-based strategies can be exploited to drive myocardial repair following injury.
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    Circadian clock core component Bmal1 dictates cell cycle rhythm of proliferating hepatocytes during liver regeneration
    (American Physiological Society, 2021) Jiang, Huaizhou; Garcia, Veronica; Yanum, Jennifer Abla; Lee, Joonyong; Dai, Guoli; Biology, School of Science
    After partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH on Bmal1+/+ and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, Bmal1+/+ hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, in Bmal1hep-/- hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 h, and the third such wave was lost. Following PH, Bmal1+/+ hepatocytes underwent three major waves of mitosis, whereas Bmal1hep-/- hepatocytes fully abolished mitotic oscillation. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied by suppressed expression peaks of a group of cell cycle components and regulators and dysregulated activation patterns of mitogenic signaling molecules c-Met and epidermal growth factor receptor. Moreover, Bmal1+/+ hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this phenomenon was largely inhibited in Bmal1hep-/- hepatocytes. In addition, during late stages of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated liver tissue. Collectively, we demonstrated that during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advancement, and largely governs fluctuations in fat metabolism in replicating hepatocytes, as well as eventually determines the redox state of regenerated livers. NEW & NOTEWORTHY: We demonstrated that Bmal1 centrally controls the synchronicity and rhythmicity of the cell cycle and lipid accumulation in replicating hepatocytes during liver regeneration. Bmal1 plays these roles, at least in part, by ensuring formation of the expression peaks of cell cycle components and regulators, as well as the timing and levels of activation of mitogenic signaling molecules.
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    Correlation Analysis of Histopathology and Proteogenomics Data for Breast Cancer
    (American Society for Biochemistry and Molecular Biology, 2019-08-09) Zhan, Xiaohui; Cheng, Jun; Huang, Zhi; Han, Zhi; Helm, Bryan; Liu, Xiaowen; Zhang, Jie; Wang, Tian-Fu; Ni, Dong; Huang, Kun; Medicine, School of Medicine
    Tumors are heterogeneous tissues with different types of cells such as cancer cells, fibroblasts, and lymphocytes. Although the morphological features of tumors are critical for cancer diagnosis and prognosis, the underlying molecular events and genes for tumor morphology are far from being clear. With the advancement in computational pathology and accumulation of large amount of cancer samples with matched molecular and histopathology data, researchers can carry out integrative analysis to investigate this issue. In this study, we systematically examine the relationships between morphological features and various molecular data in breast cancers. Specifically, we identified 73 breast cancer patients from the TCGA and CPTAC projects matched whole slide images, RNA-seq, and proteomic data. By calculating 100 different morphological features and correlating them with the transcriptomic and proteomic data, we inferred four major biological processes associated with various interpretable morphological features. These processes include metabolism, cell cycle, immune response, and extracellular matrix development, which are all hallmarks of cancers and the associated morphological features are related to area, density, and shapes of epithelial cells, fibroblasts, and lymphocytes. In addition, protein specific biological processes were inferred solely from proteomic data, suggesting the importance of proteomic data in obtaining a holistic understanding of the molecular basis for tumor tissue morphology. Furthermore, survival analysis yielded specific morphological features related to patient prognosis, which have a strong association with important molecular events based on our analysis. Overall, our study demonstrated the power for integrating multiple types of biological data for cancer samples in generating new hypothesis as well as identifying potential biomarkers predicting patient outcome. Future work includes causal analysis to identify key regulators for cancer tissue development and validating the findings using more independent data sets.
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    Evaluation of ATM Kinase Inhibitor KU-55933 as Potential Anti-Toxoplasma gondii Agent
    (Frontiers, 2019-02-13) Munera López, Jonathan; Ganuza, Agustina; Bogado, Silvina S.; Muñoz, Daniela; Ruiz, Diego M.; Sullivan, William J., Jr.; Vanagas, Laura; Angel, Sergio O.; Pharmacology and Toxicology, School of Medicine
    Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933.
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    FANCA maintains genomic stability through regulating BUBR1 acetylation
    (2017-08) Abdul Sater, Zahi Abass; Nalepa, Grzegorz; Clapp, Wade; Goebl, Mark; Wek, Ronald
    Fanconi Anemia (FA), a chromosomal instability syndrome, is characterized by bone marrow failure, genetic malformations, and predisposition to malignancies like acute myeloid leukemia (AML) and solid tumors. FA is caused by germline bi-allelic mutations in one of 21 known FA pathway genes and somatic mutations in FA genes are also found in a variety of sporadic cancers. Recently, numerous reports have discovered that the protective function of the FA pathway extends beyond its canonical role in regulation of DNA repair in interphase. In particular, the FA pathway has been shown to function in essential mitotic processes including spindle assembly checkpoint (SAC), cytokinesis, and centrosome maintenance. Understanding of the mechanistic origins of genomic instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. To this end, using a micronucleus assay, we showed that both interphase DNA damage and mitotic errors contribute to genomic instability in FA ex vivo and in vivo. Functional studies of primary FA patient cells coupled with super-resolution microscopy revealed that FANCA is important for centrosome dependent spindle assembly supporting the protective role of FA pathway in mitotic processes. Furthermore, we dissected the interactions between the FA pathway and cellular kinase networks by employing a synthetic lethality sh-RNA screen targeting all human kinases. We mapped kinases that were synthetically lethal upon loss of FANCA, particularly those involved in highly conserved signal transduction pathways governing proliferation and cell cycle homeostasis. We mechanistically show that loss of FANCA, the most abundant FA subtype, results in in premature degradation of the mitotic kinase BUBR1 and faster mitotic exit. We further demonstrate that FANCA is important for PCAF-dependent acetylation of BUBR1 to prevent its premature degradation. Our results deepen our understanding of the molecular functions of the FA pathway in mitosis and uncover a mechanistic connection between FANCA and SAC phosphosignaling networks. These findings support the notion that further weakening the SAC through targeting kinases like BUBR1 in FA-deficient cancers may prove to be a rational therapeutic strategy.
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    Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness
    (Cold Spring Harbor Laboratory, 2023-02-24) Vanagas, Laura; Muñoz, Daniela; Cristaldi, Constanza; Ganuza, Agustina; Nájera, Rosario; Bonardi, Mabel C.; Turowski, Valeria R.; Guzman, Fanny; Deng, Bin; Kim, Kami; Sullivan, William J., Jr.; Angel, Sergio O.; Pharmacology and Toxicology, School of Medicine
    Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by “reader” proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N-terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis.