DOCSLIB.ORG

The Cyclin D1-Dependent Kinase Associates with the Pre-Replication Complex and Modulates RB⅐MCM7 Binding*

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Cyclin D1-Dependent Kinase Associates with the Pre-Replication Complex and Modulates RB⅐MCM7 Binding* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 11, Issue of March 14, pp. 9754–9760, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Cyclin D1-dependent Kinase Associates with the Pre-replication Complex and Modulates RB⅐MCM7 Binding* Received for publication, November 27, 2002, and in revised form, December 23, 2002 Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212088200 Andrew B. Gladden and J. Alan Diehl‡ From the Leonard and Madlyn Abramson Family Cancer Research Institute, Department of Cancer Biology, Abramson Family Cancer Center of the University of Pennsylvania, Philadelphia, Pennsylvania 19104 The capacity of the cyclin D-dependent kinase to pro- Following ORC binding, CDC6 (5) and Cdt1 (6) associate and ⅐ mote G1 progression through modulation of RB E2F is function to recruit the minichromosome maintenance proteins well documented. We now demonstrate that the cyclin (MCM), a group of highly conserved proteins essential for the D1/CDK4 kinase binds to components of the MCM com- initiation of DNA synthesis (7–9). The MCM complex is com- Downloaded from plex. MCM7 and MCM3 were identified as cyclin D1- posed of six distinct, but related polypeptides, MCM2, MCM3, binding proteins. Catalytically active cyclin D1/CDK4 MCM4, MCM5, MCM6, and MCM7 (10), that form a hexameric complexes were incorporated into chromatin-bound helicase the activity of which is essential for initiation and protein complexes with the same kinetics as MCM7 and elongation of the replication fork (11). Origin firing at the G1/S MCM3, where they associated specifically with MCM7. boundary is also highly regulated, but the precise regulatory Although the cyclin D1-dependent kinase did not phos- mechanisms remain unclear. Both cyclin E/CDK2 and a dis- www.jbc.org phorylate MCM7, active cyclin D1/CDK4, but not cyclin tinct kinase composed of Cdc7 and Dbf4 function to promote S E/CDK2, did catalyze the dissociation of an RB⅐MCM7 phase entry, perhaps through direct phosphorylation of MCM complex. Finally, expression of an active D1/CDK4 ki- subunits (12–16). nase but not cyclin E/CDK2 promoted the removal of RB from chromatin-bound protein complexes. Our data sug- Although current data support the notion that the phospho- at University of Pennsylvania Library on September 24, 2008 gest that D1/CDK4 complexes play a direct role in alter- rylation status of pre-RC subunits determines activity, it is also ing an inhibitory RB⅐MCM7 complex possibly allowing becoming clear that interactions with cell cycle inhibitors also for setting of the origin in preparation for DNA contribute. The RB tumor suppressor protein functions as the replication. primary block that prevents premature activation of cell cycle entry and thus premature S phase entry. Although the cell cycle inhibitory activity of RB is most thoroughly understood in External stimuli activate cellular signaling cascades, terms of its ability to directly repress E2F-dependent transcrip- thereby promoting the induction of cellular activities needed to tion (17), RB also directly associates with the pre-RC through initiate DNA replication. During the G1 phase, growth factor- MCM7 (18) and perhaps subunits of ORC (19). initiated signals promote the accumulation and assembly of the Given that RB associates with components of the pre-RC and D-type cyclins (D1, D2, D3) with their cognate cyclin-dependent that RB is the only known substrate of cyclin D1, we considered 1 kinase (CDK4 or 6). The active holoenzyme promotes G1 phase the possibility that cyclin D1 might target components of the progression and S phase entry by virtue of its ability to phos- DNA replication machinery that are inhibited by RB. Using phorylate the retinoblastoma protein (RB) and titrate the CDK cyclin D1 as bait for a yeast two-hybrid screen, we identified inhibitors p27Kip1 and p21Cip1 (1, 2). Initial phosphorylation of MCM7 as a cyclin D1 binding partner. We demonstrate that RB and titration of the CDK inhibitors expedites the activation cyclin D1 mediates binding of the cyclin D1/CDK4 complex to of the cyclin E/CDK2 kinase, thereby completing RB phospho- MCM7 in mammalian cells. We demonstrate that the cyclin rylation and inactivation prior to initiation of DNA replication. D1/CDK4 kinase is incorporated into chromatin-bound protein Initiation of DNA replication at the G1/S phase boundary is complexes, with the same kinetics as MCM7 and that the cyclin meticulously regulated to ensure the cell has assembled the D1/CDK4 kinase specifically dissociates RB⅐MCM7 complexes, appropriate machinery needed for high fidelity genome dupli- thereby facilitating establishment of the pre-RC. cation. Prior to S phase entry, pre-replication complexes (pre- EXPERIMENTAL PROCEDURES RC) form at replication origins during G1 phase. Origins are Yeast Two-hybrid Screen—Yeast strain Y190 (his3 leu2 trp1), which first marked by the origin recognition complex (ORC), which is carries GAL4-dependent lacZ and HIS3 genes, was transformed with composed of six subunits (ORC1–6) (3) and serves as a landing pAS2D1, and TRPϩ transformants were examined for lacZ and HIS3 pad for recruitment of the DNA replication machinery (4). expression via a ␤-galactosidase assay and growth on media lacking histidine. A lacZϪ HIS3Ϫ TRP1ϩ clone was transformed with a HEK293 cDNA library (Clontech) fused to the GAL4 activation domain in plas- * This work was supported by National Institutes of Health Grant mid pGAD-GH, a LEU2 yeast plasmid. Transformants were replica CA93237 (to J. A. D.). The costs of publication of this article were plated onto indicator plates to score for growth in media lacking histi- defrayed in part by the payment of page charges. This article must dine and ␤-galactosidase activity. This selection resulted in 51 positive therefore be hereby marked “advertisement” in accordance with 18 colonies. Further analysis revealed that the cDNAs encoded by each of U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. E-mail: adiehl@ the 51 colonies interacted with GAL4-D1 and not other bait constructs mail.med.upenn.edu. containing GAL4 fusions with p53 or large T antigen. 1 The abbreviations used are: CDK, cyclin-dependent kinase; MEF, Cell Culture Conditions and Transfections—NIH-3T3 cells and murine embryonic fibroblast; MCM, minichromosome maintenance; RBϪ/Ϫ mouse embryonic fibroblasts (MEFs) were maintained in Dul- ORC, origin recognition complex; RB, retinoblastoma protein; Pipes, becco’s modified Eagle’s medium containing glutamine supplemented 1,4-piperazinediethanesulfonic acid; pre-RC, pre-replication complex; with antibiotics (BioWhittaker) and 10% fetal calf serum (BioWhit- HA, hemagglutinin; IP, immunoprecipitation; HRP, horseradish perox- taker). Derivatives of NIH-3T3 cells engineered to overexpress Flag- idase; GST, glutathione S-transferase. tagged cyclin D1 and Flag-tagged cyclin D1-T286A have been described 9754 This paper is available on line at http://www.jbc.org Cyclin D1/CDK4-dependent Dissociation of a RB⅐MCM7 Complex 9755 previously (20). Insect Sf9 cells were grown in Grace’s medium supple- mented with 10% heat-inactivated fetal calf serum. Procedures for manipulation of baculoviruses have been described previously (21). Transient expression of plasmids encoding HA-tagged MCM7, Flag- tagged cyclin D1, CDK4, RB, CDK4/K35M, Myc-tagged cyclin E, and CDK2 was achieved by using LipofectAMINE Plus (Invitrogen) accord- ing to the instructions from the manufacturer. Purification of Active Cyclin-CDK Complexes—Following infection of insect Sf9 cells with the indicated viral supernatants, cells were lysed in Tween 20 buffer (50 mM HEPES (pH 8), 150 mM NaCl, 2.5 mM EGTA, 1mM EDTA, 0.1% Tween 20) and cleared by sedimentation for 10 min. Flag-D1 complexes were purified by affinity chromatography using M2-agarose (Sigma) and eluted with FLAG peptide (5 ␮g/ml) (Sigma) FIG.1. Cyclin D1 binds to the COOH terminus of MCM7. A, predicted amino acid sequence, corresponding to residues 587–719, of solubilized in kinase buffer (50 mM HEPES (pH 8), 10 mM MgCl , 2.5 2 the MCM7 cDNA identified in a cyclin D1 yeast two-hybrid screen. B, mM EGTA, 1 mM dithiothreitol, 20 ␮M ATP, 10 mM ␤-glycerol phos- cyclin D1 binds specifically to MCM7 in vitro. MCM2–7 were labeled phate, 0.1 mM NaVO ,and1mM NaF). Cyclin E/His-CDK2 complexes 3 with [35S]methionine by in vitro transcription and translation. Labeled were purified using TALON metal affinity resin (Clontech) and eluted proteins were mixed with Sf9 lysates programmed with Flag-D1 and with 150 mM imidazole solubilized in His-CDK2 kinase buffer (50 mM immunoprecipitated with control rabbit serum (NRS) or with the M2 ␮ HEPES (pH 8), 10 mM MgCl2,1mM dithiothreitol, 20 M ATP, 10 mM monoclonal antibody. Ten percent of the MCM proteins mixed with ␤ Downloaded from -glycerol phosphate, 0.1 mM NaVO3,and1mM NaF). Purified com- Flag-D1 were resolved by SDS-PAGE (lower panel). MCM proteins were plexes were either used immediately or stored at 4 °C. visualized by autoradiography. Chromatin-binding Assays—Cells harvested by trypsinization were lysed in CSKϩ buffer (10 mM Pipes (pH 7.0), 100 mM NaCl, 300 mM Myc-tagged cyclin E was visualized with the 9E10 monoclonal antibody sucrose, 3 mM MgCl , 0.5% Triton X-100, 1 mM ATP, 0.1 mM phenyl- 2 followed by biotinylated anti-mouse and subsequently streptavidin methylsulfonyl fluoride, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 ␮g/ml Texas Red. MCM7 was visualized using a polyclonal rabbit anti-MCM7 pepstatin, and 25 mM ␤-glycerol phosphate). Lysates were incubated on (PharMingen) followed by fluorescein isothiocyanate-conjugated anti- ice for 20 min and then subjected to low speed centrifugation (1000 ϫ g) www.jbc.org rabbit (Amersham Biosciences). RB was visualized using a polyclonal for 5 min. The soluble fraction was removed, and the chromatin pellet rabbit anti-RB (Santa Cruz Biotechnologies, sc-50-G) followed by fluo- was washed one time with DNase I buffer and then digested with rescein isothiocyanate-conjugated anti-rabbit.
Load more

Recommended publications
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • Cyclin D1 Amplification Is Independent of P16 Inactivation in Head And
    Oncogene (1999) 18, 3541 ± 3545 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Cyclin D1 ampli®cation is independent of p16 inactivation in head and neck squamous cell carcinoma K Okami1,3, AL Reed1, P Cairns1, WM Koch1, WH Westra2, S Wehage1, J Jen1 and D Sidransky*,1 1Department of OtolaryngologyÐHead & Neck Surgery, Division of Head and Neck Cancer Research, Johns Hopkins University School of Medicine, 818 Ross Research Building, 720 Rutland Avenue, Baltimore, Maryland, MD 21205-2196, USA; 2Department of Pathology, 7181 Meyer Building, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, Maryland, MD 21287, USA; 3Department of Otolaryngology, Yamaguchi University, Ube 755, Japan Progression through the G1 phase of the cell cycle is kinase (cdk) 4 complexes. This cdk4 phosphorylation mediated by phosphorylation of the retinoblastoma activity is negatively regulated by the tumor suppressor protein (pRb) resulting in the release of essential gene p16 (a CDK inhibitor), and positively by cyclin transcription factors such as E2F-1. The phosphorylation D1. Each individual component of this p16-cyclin D1- of pRb is regulated positively by cyclin D1/CDK4 and Rb pathway is commonly targeted in various malig- negatively by CDK inhibitors, such as p16 (CDKN2/ nancies (Weinberg, 1995; Sherr, 1996). We previously MTS-1/INK4A). The p16 /cyclin D1/Rb pathway plays reported a high frequency (83%) of p16 inactivation in a critical role in tumorigenesis and many tumor types human HNSCC (Reed et al., 1996) and less frequent display a high frequency of inactivation of at least one inactivation (13%) of pRb (Yoo et al., 1994).
    [Show full text]
  • Immunohistochemical Evaluation of P63 and Cyclin D1 in Oral Squamous Cell Carcinoma and Leukoplakia
    https://doi.org/10.5125/jkaoms.2017.43.5.324 ORIGINAL ARTICLE pISSN 2234-7550·eISSN 2234-5930 Immunohistochemical evaluation of p63 and cyclin D1 in oral squamous cell carcinoma and leukoplakia Sunit B. Patel1, Bhari S. Manjunatha2, Vandana Shah3, Nishit Soni4, Rakesh Sutariya5 1Department of Oral Pathology, Ahmedabad Dental College, Ahmedabad, India, 2Department of Oral Biology, Basic Dental Sciences, Faculty of Dentistry, Al-Huwaiyah, Taif University, Taif, Kingdom of Saudi Arabia, 3Department of Oral Pathology, K.M.Shah Dental College, Vadodara, 4Department of Oral Pathology, Karnavati School of Dentistry, Gandhinagar, 5Department of Oral Pathology, Vaidik Dental College, Daman, India Abstract (J Korean Assoc Oral Maxillofac Surg 2017;43:324-330) Objectives: There are only a limited number of studies on cyclin D1 and p63 expression in oral squamous cell carcinoma (OSCC) and leukoplakia. This study compared cyclin D1 and p63 expression in leukoplakia and OSCC to investigate the possible correlation of both markers with grade of dys- plasia and histological grade of OSCC. Materials and Methods: The study included a total of 60 cases, of which 30 were diagnosed with OSCC and 30 with leukoplakia, that were evalu- ated immunohistochemically for p63 and cyclin D1 expression. Protein expression was correlated based on grades of dysplasia and OSCC. Results: Out of 30 cases of OSCC, 23 cases (76.7%) were cyclin D1 positive and 30 cases (100%) were p63 positive. Out of 30 cases of leukoplakia, 21 cases (70.0%) were cyclin D1 positive and 30 (100%) were p63 positive (P<0.05). Conclusion: The overall expression of cyclin D1 and p63 correlated with tumor differentiation, and increases were correlated with poor histological grades, from well-differentiated to poorly-differentiated SCC.
    [Show full text]
  • Cyclin D1 Degradation Is Sufficient to Induce G1 Cell Cycle Arrest Despite Constitutive Expression of Cyclin E2 in Ovarian Cancer Cells
    Published OnlineFirst July 28, 2009; DOI: 10.1158/0008-5472.CAN-09-0913 Experimental Therapeutics, Molecular Targets, and Chemical Biology Cyclin D1 Degradation Is Sufficient to Induce G1 Cell Cycle Arrest despite Constitutive Expression of Cyclin E2 in Ovarian Cancer Cells Chioniso Patience Masamha1 and Doris Mangiaracina Benbrook1,2 Departments of 1Biochemistry and Molecular Biology and 2Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma Abstract All cancers are characterized by abnormalities in apoptosis and differentiation and altered cell proliferation (4). Cancer cells often D- and E-type cyclins mediate G1-S phase cell cycle progres- sion through activation of specific cyclin-dependent kinases have a selective growth advantage due to deregulation of cell cycle (cdk) that phosphorylate the retinoblastoma protein (pRb), proteins, causing aberrant growth signaling that drives tumor thereby alleviating repression of E2F-DP transactivation of development (1, 5). Exit of cells from quiescence and cell cycle S-phase genes. Cyclin D1 is often overexpressed in a variety of progression is induced by sequential activation of cyclin-dependent cancers and is associated with tumorigenesis and metastasis. kinases (cdk) by cyclins. Once the cell progresses through late G1 into the Sphase, it is irrevocably committed to DNA replication Loss of cyclin D can cause G1 arrest in some cells, but in other cellular contexts, the downstream cyclin E protein can and cell division (6). Deregulation of G1 to S-phase transition is implicated in the pathogenesis of most human cancers, including substitute for cyclin D and facilitate G1-S progression. The objective of this study was to determine if a flexible ovarian cancer (7).
    [Show full text]
  • Upregulation of CDKN2A and Suppression of Cyclin D1 Gene
    European Journal of Endocrinology (2010) 163 523–529 ISSN 0804-4643 CLINICAL STUDY Upregulation of CDKN2A and suppression of cyclin D1 gene expressions in ACTH-secreting pituitary adenomas Yuji Tani, Naoko Inoshita1, Toru Sugiyama, Masako Kato, Shozo Yamada2, Masayoshi Shichiri3 and Yukio Hirata Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan, Departments of 1Pathology and 2Hypothalamic and Pituitary Surgery, Toranomon Hospital, Tokyo 105-8470, Japan and 3Department of Endocrinology, Diabetes and Metabolism, School of Medicine, Kitasato University, Kanagawa 252-0375, Japan (Correspondence should be addressed to Y Hirata; Email: [email protected]) Abstract Objective: Cushing’s disease (CD) is usually caused by ACTH-secreting pituitary microadenomas, while silent corticotroph adenomas (SCA) are macroadenomas without Cushingoid features. However, the molecular mechanism(s) underlying their different tumor growth remains unknown. The aim of the current study was to evaluate and compare the gene expression profile of cell cycle regulators and cell growth-related transcription factors in CD, SCA, and non-functioning adenomas (NFA). Design and methods: Tumor tissue specimens resected from 43 pituitary tumors were studied: CD (nZ10), SCA (nZ11), and NFA (nZ22). The absolute transcript numbers of the following genes were quantified with real-time quantitative PCR assays: CDKN2A (or p16INK4a), cyclin family (A1, B1, D1, and E1), E2F1, RB1, BUB1, BUBR1, ETS1, and ETS2. Protein expressions of p16 and cyclin D1 were semi-quantitatively evaluated by immunohistochemical study. Results and conclusion: CDKN2A gene expression was about fourfold greater in CD than in SCA and NFA.
    [Show full text]
  • Cytometry of Cyclin Proteins
    Reprinted with permission of Cytometry Part A, John Wiley and Sons, Inc. Cytometry of Cyclin Proteins Zbigniew Darzynkiewicz, Jianping Gong, Gloria Juan, Barbara Ardelt, and Frank Traganos The Cancer Research Institute, New York Medical College, Valhalla, New York Received for publication January 22, 1996; accepted March 11, 1996 Cyclins are key components of the cell cycle pro- gests that the partner kinase CDK4 (which upon ac- gression machinery. They activate their partner cy- tivation by D-type cyclins phosphorylates pRB com- clin-dependent kinases (CDKs) and possibly target mitting the cell to enter S) is perpetually active them to respective substrate proteins within the throughout the cell cycle in these tumor lines. Ex- cell. CDK-mediated phosphorylation of specsc sets pression of cyclin D also may serve to discriminate of proteins drives the cell through particular phases Go vs. GI cells and, as an activation marker, to iden- or checkpoints of the cell cycle. During unper- tify the mitogenically stimulated cells entering the turbed growth of normal cells, the timing of expres- cell cycle. Differences in cyclin expression make it sion of several cyclins is discontinuous, occurring possible to discrirmna* te between cells having the at discrete and well-defined periods of the cell cy- same DNA content but residing at different phases cle. Immunocytochemical detection of cyclins in such as in G2vs. M or G,/M of a lower DNA ploidy vs. relation to cell cycle position (DNA content) by GI cells of a higher ploidy. The expression of cyclins multiparameter flow cytometry has provided a new D, E, A and B1 provides new cell cycle landmarks approach to cell cycle studies.
    [Show full text]
  • Cyclin D1 and P16 Alterations in Advanced Premalignant Lesions of the Upper Aerodigestive Tract: Role in Response to Chemoprevention and Cancer Development1
    Vol. 7, 3127–3134, October 2001 Clinical Cancer Research 3127 Cyclin D1 and p16 Alterations in Advanced Premalignant Lesions of the Upper Aerodigestive Tract: Role in Response to Chemoprevention and Cancer Development1 Vassiliki A. Papadimitrakopoulou,2,3 Julie Izzo,2 tified in two cases. Cyclin D1 protein dysregulation at last Li Mao, Jamie Keck, David Hamilton, follow-up alone and in combination with p16 loss was asso- Dong Moon Shin, Adel El-Naggar, ciated with histological progression and cancer development (P < 0.01). Petra den Hollander, Diane Liu, Conclusions: Additional study of these alterations in a Walter N. Hittelman, and Waun K. Hong larger sample and exploration of the upstream signaling Departments of Thoracic/Head and Neck Medical Oncology (V. A. P., partners of these cell cycle regulators in vivo is warranted to J. K., D. H., D. M. S., W. K. H.), Experimental Therapeutics (J. I., identify cancer risk profiles that would be meaningful tar- P. D. H., W. H.), Pathology (A. E. N.) and Biostatistics (D. L.), The gets for chemopreventive intervention. University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 INTRODUCTION Although tobacco and its derivatives are considered to be ABSTRACT leading causes for HNSCC4 development, their underlying ge- Purpose: To better understand the role of G1-S transi- netic targets are not yet completely clarified. Cell cycle dys- tion regulator abnormalities in the pathogenesis of advanced regulation is intimately linked to carcinogenesis. In the past premalignant lesions of the upper aerodigestive tract and decade, scientists have uncovered several important hints to how the biological effects of chemoprevention, we studied biop- mechanisms that control the cell cycle play central roles in both sies obtained sequentially from participants in a prospective the development and the prevention of cancer (1).
    [Show full text]
  • Role of P16/MTS1, Cyclin D1 and RB in Primary Oral Cancer and Oral
    British Journal of Cancer (1999) 80(1/2), 79–86 © 1999 Cancer Research Campaign Article no. bjoc.1998.0325 Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines M Sartor1, H Steingrimsdottir1, F Elamin1, J Gäken2, S Warnakulasuriya1, M Partridge3, N Thakker4, NW Johnson1 and M Tavassoli1 Oral Oncology Group, Departments of 1Oral Medicine and Pathology and 2Molecular Medicine, 3Molecular Oncology Group, Department of Oral and Maxillofacial Surgery, The Rayne Institute, King’s College School of Medicine and Dentistry, 125 Coldharbour Lane, London SE5 9NU, UK; 4Department of Medical Genetics, University of Manchester, St Mary’s Hospital, Manchester M13 0JH, UK Summary One of the most important components of G1 checkpoint is the retinoblastoma protein (pRB110). The activity of pRB is regulated by its phosphorylation, which is mediated by genes such as cyclin D1 and p16/MTS1. All three genes have been shown to be commonly altered in human malignancies. We have screened a panel of 26 oral squamous cell carcinomas (OSCC), nine premalignant and three normal oral tissue samples as well as eight established OSCC cell lines for mutations in the p16/MTS1 gene. The expression of p16/MTS1, cyclin D1 and pRB110 was also studied in the same panel. We have found p16/MTS1 gene alterations in 5/26 (19%) primary tumours and 6/8 (75%) cell lines. Two primary tumours and five OSCC cell lines had p16/MTS1 point mutations and another three primary and one OSCC cell line contained partial gene deletions. Six of seven p16/MTS1 point mutations resulted in termination codons and the remaining mutation caused a frameshift.
    [Show full text]
  • Cyclin D2 and Cyclin D3 Play Opposite Roles in Mouse Skin Carcinogenesis
    Oncogene (2007) 26, 1723–1730 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Cyclin D2 and cyclin D3 play opposite roles in mouse skin carcinogenesis P Rojas1,4, MB Cadenas2,4, P-C Lin3, F Benavides1, CJ Conti1 and ML Rodriguez-Puebla2 1Department of Carcinogenesis, Science Park Research Division, MD Anderson Cancer Center, Smithville, TX, USA; 2Center for Comparative Medicine and Translational Research, Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA and 3Department of Molecular and Cellular Oncology, MD Anderson Cancer Center, Houston, TX, USA D-type cyclins are components ofthe cell-cycle engine a family of key cell-cycle regulators, as they are the that link cell signaling pathways and passage throughout regulatory subunits of the CDK4 and CDK6, which G1 phase. We previously described the effects of over- phosphorylate pRb family of proteins that are critical expression cyclin D1, D2 or D3 in mouse epidermis and substrates for cell-cycle progression (Matsushine et al., tumor development. We now asked whether cyclin D2 1994; Meyerson and Harlow, 1994; Sherr and Roberts, and/or cyclin D3 play a relevant role in ras-dependent 1999; Farkas et al., 2002; Leng et al., 2002). Initially, tumorigenesis. Here, we described the effect of cyclin D3 several reports assigned redundant roles to the three and cyclin D2 overexpression in mouse skin tumor members of D-type cyclins, but in the last few years, it development. Notably, overexpression ofcyclin D3 results has become evident that each member is differentially in reduced tumor development and malignant progression expressed in various tissues playing specific roles (Sherr, to squamous cell carcinomas (SCC).
    [Show full text]
  • The Interplay of Proteins Encoded by CDKN1A, CDKN1B, CDKN2A and TP53 in the Prognosis of Breast Cancer
    Research Article Clinics in Oncology Published: 02 Aug, 2019 The Interplay of Proteins Encoded by CDKN1A, CDKN1B, CDKN2A and TP53 in the Prognosis of Breast Cancer Valentina Robila1*, Bryce S Hatfield1, Nitai Mukhopadhyay2 and Michael O Idowu1 1Department of Pathology, VCU School of Medicine, USA 2Department of Biostatistics, VCU School of Medicine, USA Abstract The use of palbociclib has increased the spotlight on cell cycle regulators, especially on the proteins encoded by CDKN2A (p16), CDKN1A (p21), CDKN1B (p27), cyclin D1 and TP53 (p53). Cell cycle regulators and cyclin-dependent kinases show significant promise as new breast cancer biomarkers. Expression patterns of small sets of proteins in breast carcinoma were reported, and their overall role in breast cancer progression was investigated, with mixed results. The goal of our study was to simultaneously evaluate multiple cell cycle regulators in breast carcinoma. The protein expression was assessed on Tissue Microarrays (TMA) and the intensity and percentage of positive tumor cells were quantified. Statistical analysis was performed to establish patterns of protein associations, as well as correlation with prognostic factors, including metastases/recurrence and overall survival. Non-triple negative carcinoma showed significantly increased expression of cyclin D1, marginally increased expression of p21, 27, and decreased expression of p16, p53. This is in contrast to triple negative carcinoma which expressed high levels of p16 and p53. Statistically significant dual proteins associations were demonstrated, including direct correlations of expression of cyclin D1 with p21 or 27, and indirect correlations with p16 and p53. Cyclin D1 (p=0.0001) and p21 (p=0.0049) expression imparted a favorable outcome on development of metastases and/or survival, while p16 (p=0.0048) had a negative impact, particularly on non-triple negative carcinoma.
    [Show full text]
  • Full Text (PDF)
    654 Vol. 1, 654–664, July 2003 Molecular Cancer Research Expression of Cyclin-Dependent Kinase 6, but not Cyclin-Dependent Kinase 4, Alters Morphology of Cultured Mouse Astrocytes Karen K. Ericson, David Krull, Peter Slomiany, and Martha J. Grossel Department of Biology, Connecticut College, New London, CT Abstract mechanism involving the accumulation of the p53 and p130 Disruption of the pRb pathway is a common mechanism growth-suppressing proteins (6), and activation of cdk6 in tumor formation. The D-cyclin-associated kinases, precedes cdk4 activation in T cells (7, 8). Also, differences in cyclin-dependent kinase (cdk) 4 and cdk6, are important subcellular localization of cdk4 and cdk6 and in the timing of regulators of the G1-S phase transition and are elevated nuclear localization have been noted in several cell types by in several types of cancers, including gliomas. To several researchers (9–12). Data from tumor studies also investigate potential functional differences in these suggest differences in these kinases. Cdk4, and not cdk6, is kinases, mouse astrocytes were taken from chimeric specifically targeted in melanoma (13, 14) while cdk6 activity mice and propagated in tissue culture. These multipolar has been found to be elevated in squamous cell carcinomas (15, tissue-culture astrocytes were infected with viruses 16) and neuroblastomas (17) without alteration of cdk4 activity. expressing either cdk4 or cdk6. Interestingly, expression Cumulatively, these data suggest that cdk4 and cdk6 have of cdk6 resulted in a distinct and rapid morphology unique functions that may be cell-type specific, temporally change from multipolar to bipolar. This change was not regulated, or developmentally distinct.
    [Show full text]