Cytometry of Cyclin Proteins
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Cyclin D1/Cyclin-Dependent Kinase 4 Interacts with Filamin a and Affects the Migration and Invasion Potential of Breast Cancer Cells
Published OnlineFirst February 28, 2010; DOI: 10.1158/0008-5472.CAN-08-1108 Tumor and Stem Cell Biology Cancer Research Cyclin D1/Cyclin-Dependent Kinase 4 Interacts with Filamin A and Affects the Migration and Invasion Potential of Breast Cancer Cells Zhijiu Zhong, Wen-Shuz Yeow, Chunhua Zou, Richard Wassell, Chenguang Wang, Richard G. Pestell, Judy N. Quong, and Andrew A. Quong Abstract Cyclin D1 belongs to a family of proteins that regulate progression through the G1-S phase of the cell cycle by binding to cyclin-dependent kinase (cdk)-4 to phosphorylate the retinoblastoma protein and release E2F transcription factors for progression through cell cycle. Several cancers, including breast, colon, and prostate, overexpress the cyclin D1 gene. However, the correlation of cyclin D1 overexpression with E2F target gene regulation or of cdk-dependent cyclin D1 activity with tumor development has not been identified. This suggests that the role of cyclin D1 in oncogenesis may be independent of its function as a cell cycle regulator. One such function is the role of cyclin D1 in cell adhesion and motility. Filamin A (FLNa), a member of the actin-binding filamin protein family, regulates signaling events involved in cell motility and invasion. FLNa has also been associated with a variety of cancers including lung cancer, prostate cancer, melanoma, human bladder cancer, and neuroblastoma. We hypothesized that elevated cyclin D1 facilitates motility in the invasive MDA-MB-231 breast cancer cell line. We show that MDA-MB-231 motility is affected by disturbing cyclin D1 levels or cyclin D1-cdk4/6 kinase activity. -
Introduction to Flow Cytometry Principles Data Analysis Protocols Troubleshooting
Flow Cytometry ipl.qxd 11/12/06 11:14 Page i Introduction to Flow Cytometry Principles Data analysis Protocols Troubleshooting By Misha Rahman, Ph.D. Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc. Your first choice for antibodies! Flow Cytometry ipl.qxd 11/12/06 11:14 Page ii Flow Cytometry ipl.qxd 11/12/06 11:14 Page iii Introduction to Flow Cytometry Principles Data analysis Protocols Troubleshooting By Misha Rahman, Ph.D. Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc. Flow Cytometry ipl.qxd 11/12/06 11:14 Page 2 Preface How can I explain what flow cytometry is to someone that knows nothing about it? Well, imagine it to be a lot like visiting a supermarket. You choose the goods you want and take them to the cashier. Usually you have to pile them onto a conveyor. The clerk picks up one item at a time and interrogates it with a laser to read the barcode. Once identified and if sense prevails, similar goods are collected together e.g. fruit and vegetables go into one shopping bag and household goods into another. Now picture in your mind the whole process automated; replace shopping with biological cells; and substitute the barcode with cellular markers – welcome to the world of flow cytometry and cell sorting! We aim to give you a basic overview of all the important facets of flow cytometry without delving too deeply into the complex mathematics and physics behind it all. For that there are other books (some recommended at the back). -
Cyclin-Dependent Kinase 5 Decreases in Gastric Cancer and Its
Published OnlineFirst January 21, 2015; DOI: 10.1158/1078-0432.CCR-14-1950 Biology of Human Tumors Clinical Cancer Research Cyclin-Dependent Kinase 5 Decreases in Gastric Cancer and Its Nuclear Accumulation Suppresses Gastric Tumorigenesis Longlong Cao1,2, Jiechao Zhou2, Junrong Zhang1,2, Sijin Wu3, Xintao Yang1,2, Xin Zhao2, Huifang Li2, Ming Luo1, Qian Yu1, Guangtan Lin1, Huizhong Lin1, Jianwei Xie1, Ping Li1, Xiaoqing Hu3, Chaohui Zheng1, Guojun Bu2, Yun-wu Zhang2,4, Huaxi Xu2,4,5, Yongliang Yang3, Changming Huang1, and Jie Zhang2,4 Abstract Purpose: As a cyclin-independent atypical CDK, the role of correlated with the severity of gastric cancer based on tumor CDK5 in regulating cell proliferation in gastric cancer remains and lymph node metastasis and patient 5-year fatality rate. unknown. Nuclear localization of CDK5 was found to be significantly Experimental Design: Expression of CDK5 in gastric tumor decreased in tumor tissues and gastric cancer cell lines, and paired adjacent noncancerous tissues from 437 patients was whereas exogenously expression of nucleus-targeted CDK5 measured by Western blotting, immunohistochemistry, and real- inhibited the proliferation and xenograft implantation of time PCR. The subcellular translocation of CDK5 was monitored gastric cancer cells. Treatment with the small molecule during gastric cancer cell proliferation. The role of nuclear CDK5 NS-0011, which increases CDK5 accumulation in the nucleus, in gastric cancer tumorigenic proliferation and ex vivo xenografts suppressed both cancer cell proliferation and xenograft was explored. Furthermore, by screening for compounds in the tumorigenesis. PubChem database that disrupt CDK5 association with its nu- Conclusions: Our results suggest that low CDK5 expression is clear export facilitator, we identified a small molecular (NS-0011) associated with poor overall survival in patients with gastric that inhibits gastric cancer cell growth. -
Cyclin-Dependent Kinases and P53 Pathways Are Activated Independently and Mediate Bax Activation in Neurons After DNA Damage
The Journal of Neuroscience, July 15, 2001, 21(14):5017–5026 Cyclin-Dependent Kinases and P53 Pathways Are Activated Independently and Mediate Bax Activation in Neurons after DNA Damage Erick J. Morris,1 Elizabeth Keramaris,2 Hardy J. Rideout,3 Ruth S. Slack,2 Nicholas J. Dyson,1 Leonidas Stefanis,3 and David S. Park2 1Massachusetts General Hospital Cancer Center, Laboratory of Molecular Oncology, Charlestown, Massachusetts 02129, 2Neuroscience Research Institute, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada, and 3Columbia University, New York, New York 10032 DNA damage has been implicated as one important initiator of ization, and DNA binding that result from DNA damage are not cell death in neuropathological conditions such as stroke. Ac- affected by the inhibition of CDK activity. Conversely, no de- cordingly, it is important to understand the signaling processes crease in retinoblastoma protein (pRb) phosphorylation was that control neuronal death induced by this stimulus. Previous observed in p53-deficient neurons that were treated with camp- evidence has shown that the death of embryonic cortical neu- tothecin. However, either p53 deficiency or the inhibition of rons treated with the DNA-damaging agent camptothecin is CDK activity alone inhibited Bax translocation, cytochrome c dependent on the tumor suppressor p53 and cyclin-dependent release, and caspase-3-like activation. Taken together, our re- kinase (CDK) activity and that the inhibition of either pathway sults indicate that p53 and CDK are activated independently alone leads to enhanced and prolonged survival. We presently and then act in concert to control Bax-mediated apoptosis. show that p53 and CDKs are activated independently on par- allel pathways. -
The Cryoelectron Microscopy Structure of the Human CDK-Activating Kinase
The cryoelectron microscopy structure of the human CDK-activating kinase Basil J. Grebera,b,1,2, Juan M. Perez-Bertoldic, Kif Limd, Anthony T. Iavaronee, Daniel B. Tosoa, and Eva Nogalesa,b,d,f,2 aCalifornia Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720; bMolecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; cBiophysics Graduate Group, University of California, Berkeley, CA 94720; dDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; eQB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, CA 94720; and fHoward Hughes Medical Institute, University of California, Berkeley, CA 94720 Edited by Seth A. Darst, Rockefeller University, New York, NY, and approved August 4, 2020 (received for review May 14, 2020) The human CDK-activating kinase (CAK), a complex composed of phosphoryl transfer (11). However, in addition to cyclin binding, cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical full activation of cell cycle CDKs requires phosphorylation of the regulator of transcription initiation and the cell cycle. It acts by T-loop (9, 12). In animal cells, these activating phosphorylations phosphorylating the C-terminal heptapeptide repeat domain of are carried out by CDK7 (13, 14), itself a cyclin-dependent ki- the RNA polymerase II (Pol II) subunit RPB1, which is an important nase whose activity depends on cyclin H (14). regulatory event in transcription initiation by Pol II, and it phos- In human and other metazoan cells, regulation of transcription phorylates the regulatory T-loop of CDKs that control cell cycle initiation by phosphorylation of the Pol II-CTD and phosphor- progression. -
Cell Cycle Arrest and DNA Endoreduplication Following P21waf1/Cip1 Expression
Oncogene (1998) 17, 1691 ± 1703 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression Stewart Bates, Kevin M Ryan, Andrew C Phillips and Karen H Vousden ABL Basic Research Program, NCI-FCRDC, Building 560, Room 22-96, West 7th Street, Frederick, Maryland 21702-1201, USA p21Waf1/Cip1 is a major transcriptional target of p53 and there are an increasing number of regulatory mechan- has been shown to be one of the principal mediators of isms that serve to inhibit cdk activity. Primary amongst the p53 induced G1 cell cycle arrest. We show that in these is the expression of cdk inhibitors (CDKIs) that addition to the G1 block, p21Waf1/Cip1 can also contribute can bind to and inhibit cyclin/cdk complexes (Sherr to a delay in G2 and expression of p21Waf1/Cip1 gives rise and Roberts, 1996). These fall into two classes, the to cell cycle pro®les essentially indistinguishable from INK family which bind to cdk4 and cdk6 and inhibit those obtained following p53 expression. Arrest of cells complex formation with D-cyclins, and the p21Waf1/Cip1 in G2 likely re¯ects an inability to induce cyclin B1/cdc2 family that bind to cyclin/cdk complexes and inhibit kinase activity in the presence of p21Waf1/Cip1, although the kinase activity. inecient association of p21Waf1/Cip1 and cyclin B1 The p21Waf1/Cip1 family of CDKIs include p21Waf1/Cip1, suggests that the mechanism of inhibition is indirect. p27 and p57 and are broad range inhibitors of cyclin/ Cells released from an S-phase block were not retarded cdk complexes (Gu et al., 1993; Harper et al., 1993; in their ability to progress through S-phase by the Xiong et al., 1993; Firpo et al., 1994; Polyak et al., presence of p21Waf1/Cip1, despite ecient inhibition of 1994; Toyoshima and Hunter, 1994; Lee et al., 1995; cyclin E, A and B1 dependent kinase activity, suggesting Matsuoka et al., 1995). -
Phosphatidylinositol-3-Kinase Related Kinases (Pikks) in Radiation-Induced Dna Damage
Mil. Med. Sci. Lett. (Voj. Zdrav. Listy) 2012, vol. 81(4), p. 177-187 ISSN 0372-7025 DOI: 10.31482/mmsl.2012.025 REVIEW ARTICLE PHOSPHATIDYLINOSITOL-3-KINASE RELATED KINASES (PIKKS) IN RADIATION-INDUCED DNA DAMAGE Ales Tichy 1, Kamila Durisova 1, Eva Novotna 1, Lenka Zarybnicka 1, Jirina Vavrova 1, Jaroslav Pejchal 2, Zuzana Sinkorova 1 1 Department of Radiobiology, Faculty of Health Sciences in Hradec Králové, University of Defence in Brno, Czech Republic 2 Centrum of Advanced Studies, Faculty of Health Sciences in Hradec Králové, University of Defence in Brno, Czech Republic. Received 5 th September 2012. Revised 27 th November 2012. Published 7 th December 2012. Summary This review describes a drug target for cancer therapy, family of phosphatidylinositol-3 kinase related kinases (PIKKs), and it gives a comprehensive review of recent information. Besides general information about phosphatidylinositol-3 kinase superfamily, it characterizes a DNA-damage response pathway since it is monitored by PIKKs. Key words: PIKKs; ATM; ATR; DNA-PK; Ionising radiation; DNA-repair ABBREVIATIONS therapy and radiation play a pivotal role. Since cancer is one of the leading causes of death worldwide, it is DSB - double stand breaks, reasonable to invest time and resources in the enligh - IR - ionising radiation, tening of mechanisms, which underlie radio-resis - p53 - TP53 tumour suppressors, tance. PI - phosphatidylinositol. The aim of this review is to describe the family INTRODUCTION of phosphatidyinositol 3-kinases (PI3K) and its func - tional subgroup - phosphatidylinositol-3-kinase rela - An efficient cancer treatment means to restore ted kinases (PIKKs) and their relation to repairing of controlled tissue growth via interfering with cell sig - radiation-induced DNA damage. -
Role for Cyclin D1 in UVC-Induced and P53-Mediated Apoptosis
Cell Death and Differentiation (1999) 6, 565 ± 569 ã 1999 Stockton Press All rights reserved 13509047/99 $12.00 http://www.stockton-press.co.uk/cdd Role for cyclin D1 in UVC-induced and p53-mediated apoptosis Hirofumi Hiyama1 and Steven A. Reeves*,1 irradiation of human fibroblasts is mediated by the p53- induced cyclin/cdk inhibitor, p21.3 Cell cycle progression 1 Molecular Neuro-Oncology, Neuroscience Center, Neurosurgical Services, through the G1/S boundary is controlled by G1 cyclins, Massachusetts General Hospital and Harvard Medical School, Boston, including D and E type cyclins and their cyclin-dependent Massachusetts 02129, USA kinases (cdk), whose phosphorylation of the retinoblastoma * corresponding author: Steven A. Reeves, Molecular Neuro-Oncology, gene product (pRB) causes a dissociation of the pRB and Neuroscience Center, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA. tel.: (617) 726-5510; fax: (617) 726-5079; E2F-1 interaction, and subsequent activation of E2F- e-mail: [email protected] mediated transcription. As an universal inhibitor of cdks, p21 can inhibit phosphorylation of pRB and allow for G1 arrest.4 Recent studies have shown that overexpression of cyclin Received 13.10.98; revised 19.03.99; accepted 23.03.99 D1 in serum-starved cell types can induce apoptosis.5 Edited by T. Cotter Interestingly, the induction of the cell death program was found to be associated with an increase in cyclin D1- dependent kinase activity.6 Moreover, in cells that contain Abstract wild-type p53, the overexpression of E2F-1 leads to S- 7 DNA damaging agents such as ultraviolet (UV) induce cell phase entry and p53-dependent apoptosis. -
Comparison of Count Reproducibility, Accuracy, and Time to Results
Comparison of Count Reproducibility, Accuracy, and Time to Results between a Hemocytometer and the TC20™ Automated Cell Counter Tech Frank Hsiung, Tom McCollum, Eli Hefner, and Teresa Rubio Note Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA Cell Counting Bulletin 6003 Introduction Bead counts were performed by sequentially loading For over 100 years the hemocytometer has been used by and counting the same chamber of a Bright-Line glass cell biologists to quantitate cells. It was first developed for the hemocytometer (Hausser Scientific). This was repeated ten quantitation of blood cells but became a popular and effective times. The number of beads was recorded for all nine tool for counting a variety of cell types, particles, and even 1 x 1 mm grids. small organisms. Currently, hemocytometers, armed with Flow Cytometry improved Neubauer grids, are a mainstay of cell biology labs. Flow cytometry was performed using a BD FACSCalibur flow Despite its longevity and versatility, hemocytometer counting cytometer (BD Biosciences) and CountBright counting beads suffers from a variety of shortcomings. These shortcomings (Life Technologies Corporation). Medium containing 50,000 include, but are not limited to, a lack of statistical robustness at CountBright beads was combined one-to-one with 250 µl low sample concentration, poor counts due to device misuse, of cells in suspension, yielding a final solution containing and subjectivity of counts among users, in addition to a time- 100 beads/µl. This solution was run through the flow consuming and tedious operation. In recent years automated cytometer until 10,000 events were collected in the gate cell counting has become an attractive alternative to manual previously defined as appropriate for non-doublet beads in hemocytometer–based cell counting, offering more reliable the FSC x SSC channel. -
The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression
International Journal of Molecular Sciences Review The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression Tingting Zou and Zhenghong Lin * School of Life Sciences, Chongqing University, Chongqing 401331, China; [email protected] * Correspondence: [email protected] Abstract: The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), which activities are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins, CDK inhibitors (CKIs), other kinases and phosphatases. Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell cycle progression via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor or abnormally high accumulation of oncoproteins often results in deregulation of cell proliferation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the regulation of UPS machinery of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell cycle transition, cancer treatment, and the development of anti-cancer drugs. Keywords: cell cycle regulation; CDKs; cyclins; CKIs; UPS; E3 ubiquitin ligases; Deubiquitinases (DUBs) Citation: Zou, T.; Lin, Z. The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression. 1. Introduction Int. J. Mol. Sci. 2021, 22, 5754. https://doi.org/10.3390/ijms22115754 The cell cycle is a ubiquitous, complex, and highly regulated process that is involved in the sequential events during which a cell duplicates its genetic materials, grows, and di- Academic Editors: Kwang-Hyun Bae vides into two daughter cells. -
Requirement for Cyclin D3 in Germinal Center Formation and Function
Cell Research (2010) :1-16. © 2010 IBCB, SIBS, CAS All rights reserved 1001-0602/10 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr Requirement for cyclin D3 in germinal center formation and function Jonathan U Peled1, J Jessica Yu1, Jeganathan Venkatesh2, Enguang Bi1, B Belinda Ding1, 5, Melissa Krupski-Downs1, Rita Shaknovich3, Piotr Sicinski4, Betty Diamond2, Matthew D Scharff1, B Hilda Ye1 1Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; 2The Center for Autoimmune and Musculoskeletal Disease, The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA; 3Depart- ments of Medicine and Pathology, Weill Cornell College of Medicine, New York, NY 10021, USA; 4Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA Germinal centers (GC) of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these pro- cesses, little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins constitute an im- portant component of the cell cycle engine that enables the cells to respond to physiological changes. Cell type- and developmental stage-specific roles of D-type cyclins have been described but the cyclin D requirement during GC reaction has not been addressed. In this study, we report that cyclin D3 is largely dispensable for proliferation and Ig class switching of in vitro activated B cells. -
The Expression of P21/WAF-1 and Cyclin B1 Mediate Mitotic Delay in X-Irradiated Fibroblasts
ANTICANCER RESEARCH 25: 1123-1130 (2005) The Expression of p21/WAF-1 and Cyclin B1 Mediate Mitotic Delay in x-Irradiated Fibroblasts MICKAEL J. CARIVEAU1,2, CHARLES J. KOVACS2, RON R. ALLISON2, ROBERTA M. JOHNKE2, GERHARD W. KALMUS3 and MARK EVANS2 1Department of Physics, East Carolina University, Greenville NC, 27858; 2Department of Radiation Oncology, The Brody School of Medicine, East Carolina University, Greenville NC, 27858; 3Department of Biology, East Carolina University, Greenville NC, 27858, U.S.A. Abstract. Background: To better understand the relationship Initiation and maintenance of cell cycle arrest is regulated between mitotic delay and the disruption of cyclin B1 and p21 in by a group of proteins known as the cyclins which function x-irradiated fibroblasts, studies were carried out to establish by coupling to their corresponding cyclin dependent kinases correlations between the downregulation of cyclin B1 by the cyclin (CDK) (6-9). Of particular interest to cell cycle arrest in kinase inhibitor (CKI) p21 and the induction of mitotic delay in response to DNA damage is the G2/M damage checkpoint the NIH3T3 fibroblast. Materials and Methods: Cell cycle kinetics which is regulated by cyclin B1/CDK1 and initiated by DNA were used to analyze mitotic delay in irradiated NIH3T3 cells and damage activation of the cyclin kinase inhibitor p21(10-14). immunocytochemistry incorporated to assess the expression of The inhibitory potential of p21 is well established; however, cyclin B1 and p21, following 2 or 4Gy x-irradiation. Results and there is still much confusion about the role of p21 in the Discussion: Results indicate a dose dependent increase in mitotic irradiated cell (15, 16).