DOCSLIB.ORG

Host Cell Factors in Ovarian Cancer Influencing Efficacy of Oncolytic Adenovirus Dl922-947

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Host Cell Factors in Ovarian Cancer Influencing Efficacy of Oncolytic Adenovirus Dl922-947 Host Cell Factors in Ovarian Cancer Influencing Efficacy of Oncolytic Adenovirus dl922-947 Magdalena Barbara Flak A thesis submitted to the University of London (Faculty of Science) for the degree of Doctor of Philosophy February 2010 Centre for Molecular Oncology and Imaging Institute of Cancer Barts and the London School of Medicine and Dentistry Charterhouse Square, London EC1M 6BQ 1 I, Magdalena Barbara Flak, hereby confirm that the work presented in this thesis is original and is my own. Work undertaken by others in contribution to this thesis has been clearly indicated. Where information has been derived from other sources, this has been acknowledged and appropriately referenced. Signed ………………………………... 2 Acknowledgments First of all, I would like to thank my supervisor Prof Iain McNeish. He has given me the opportunity to work on this exciting project. But more importantly, Iain, your expertise, encouragment and genuine support have guided me throughout my PhD and allowed me to grow as a scientist and as a person. I owe my deepest gratitude to Jes, without whom I may not have come this far. Your support, neverending patience and love have carried me through the most frustrating, difficult times. Having you as a companion to celebrate the great moments made them all the more precious. You have always believed in me, and I am forever in your debt. I would like to thank my parents. They were always, always there to listen, to share disappointments and successes. You have instilled in me the awe and curiosity of all things around me. You have dedicated your strength to letting me follow my ambitions. This thesis would not have been possible without you. Thomas, I thank you for brotherly supervision of my first “lab experiments” at home, mixing coloured water in test tubes, and for my first microscope. In short, for supporting a dream and putting things into perspective. Z całego serca dziękuję moim kochanym Babcią i Dziadką za pomyślność i modlitwe. Za miejsce w waszych sercach. Many friends have supported me on my path to a PhD, for which I cannot thank enough. Anke has been patient, wise and wonderful from day one. I am grateful to Maren, Mary, Manuel and Charmaine for their great support. I thank Prof B. Volk for encouragment and inspiration. Heartfelt gratitude goes to my wonderful colleagues and friends Kyra, Carin, AM, Sarah, Michael, Lynda, Katrina, Michelle and Claire, for lab wisdom, for listening to my wingeing, for laughter, midnight meetings and chocolate. You have made the past years cheerful and enjoyable. Never forget: Doppelt gemoppelt haelt besser. I would like to thank Katrina, Claire, Sarah and Claude in particular, for contributing valuable work to this thesis. I am truly grateful for your genious heads and hands making these experiments happen. It was an honour and a pleasure to work with all the great people that make the Centre for Molecular Ongology & Imaging. I am very thankful for generous help and interesting discussions. I have met some great people and friends amongst you. I thank all those, whose names do not appear here, despite their contribution to this PhD. Please forgive me. 3 Abstract Adenoviral gene therapy holds great potential for cancer treatment, but is limited by a lack of efficient vectors. dl922-947, an E1A CR2-deleted adenovirus, replicates selectively within and lyses cancer cells. It is believed that its selectivity depends upon abnormalities in the cell cycle regulatory Rb pathway and subsequent G1/S checkpoint, observed in 90% of human cancers. The cytotoxic efficacy of dl922-947 is greater than that of wild-type adenovirus and dl1520 (Onyx-015). Nevertheless, sensitivity to dl922- 947 varies widely among ovarian cancer cell lines, despite similar infectivity. My work aimed to identify host cell factors that influence cytotoxicity and which could be potential biomarkers in clinical trials. Surprisingly, comparison of ovarian cancer lines indicated that cytotoxicity correlated poorly with infectivity, replication and virion production. Immunoblotting suggested correlation between sensitivity to dl922-947 and overexpression of p21, p27, Cyclin D, cdk4 and p16. Subsequent experiments confirmed a role for p21 in dl922-947 cytotoxic activity. In vitro and in vivo, Hct116 p21+/+ cells were significantly more sensitive to dl922-947 than matched p21-/- cells. p21 knock-down by siRNA in TOV21G and IGROV-1 cells reduced dl922-947 cytotoxicity, whilst re-expression in ACP-WAF1 cells increased activity. p21 expression was also greater in sensitive transformed TOSE cells compared to resistant normal IOSE25 cells. Results suggest that p21 promotes dl922-947 activity by stabilising Cyclin D thus promoting cell cycle progression. Comparative microarray analysis in TOSE1, 4 and IOSE25 cells and in MRC5 and MRC5-VA cells suggested determinants of dl922-947 activity beyond the Rb pathway, which may also prove valuable biomarkers. Moreover, pathways and processes emerged, correlating with sensitivity, and meriting future investigation. Together, my results suggest that a cellular environment conducive for dl922-947 function includes mediators of proliferative capacity, amongst which p21 plays a role in enhancing activity of the virus. 4 A scientist in his laboratory is not a mere technician: He is also a child confronting natural phenomena that impress him as though they were fairy tales. Marie Curie 5 Contents 1 Introduction ................................................................................................ 18 1.1 Ovarian Cancer .................................................................................... 19 1.2 Stratified Medicine ............................................................................... 24 1.3 Gene Therapy ...................................................................................... 25 1.3.1 Oncolytic gene therapy.................................................................. 27 1.3.2 Oncolytic viruses in cancer therapy ............................................... 28 1.3.3 Deletion mutants for cancer therapy ............................................. 30 1.4 Adenoviridae ........................................................................................ 33 1.4.1 Structure and Genome .................................................................. 34 1.4.2 Early genes ................................................................................... 35 1.4.3 Life cycle of Adenovirus ................................................................ 39 1.5 Oncolytic adenoviruses ........................................................................ 43 1.5.1 Second Generation Adenoviral Vectors ........................................ 44 1.5.2 dl922-947 ...................................................................................... 46 1.5.3 dl922-947 in cancer cells ............................................................... 46 1.5.4 Immunogenicity of Adenoviral vectors ........................................... 47 1.5.5 Further limitations of dl922-947 and other adenoviruses .............. 48 1.5.6 Future of virus-based therapy in cancer ........................................ 49 1.6 The Rb pathway and cell cycle control ................................................ 51 1.6.1 Rb pathway and cell cycle regulation ............................................ 51 1.6.2 Rb pathway and cancer ................................................................. 64 1.6.3 E2F and the Rb pathway in ovarian cancer ................................... 66 1.7 Aims of this study ................................................................................. 68 2 Materials and Methods .............................................................................. 69 2.1 Cell lines .............................................................................................. 70 2.1.1 Human ovarian cancer cell lines ................................................... 70 2.1.2 Other human cell lines................................................................... 70 2.2 Adenoviral mutants .............................................................................. 70 2.3 Virus amplification ................................................................................ 71 2.4 Viral particle count ............................................................................... 72 2.5 Viral titration by TCID50 assay .............................................................. 72 2.6 Virion detection in serum samples by TCID50 ...................................... 73 2.7 Sensitivity of cells to adenoviral cytotoxicity ........................................ 73 2.8 Infectability with adenoviral vectors ..................................................... 74 2.8.1 Flow cytometry .............................................................................. 74 6 2.8.2 Quantitative PCR .......................................................................... 74 2.9 Protein expression ............................................................................... 78 2.9.1 Protein concentration evaluation ................................................... 78 2.9.2 Western blot analysis (Immunoblotting) ........................................ 78 2.9.3 Densitometric analysis .................................................................. 80 2.10 Viral replication ................................................................................. 81 2.10.1 TCID50 assays ..............................................................................
Load more

Recommended publications
  • The Transcription Factor Hey and Nuclear Lamins Specify and Maintain
    RESEARCH ARTICLE The transcription factor Hey and nuclear lamins specify and maintain cell identity Naama Flint Brodsly1†, Eliya Bitman-Lotan1†, Olga Boico1, Adi Shafat1, Maria Monastirioti2, Manfred Gessler3, Christos Delidakis2, Hector Rincon-Arano4, Amir Orian1* 1Rappaport Research Institute and Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; 2Institute of Molecular Biology and Biotechnology (IMBB), Foundation for Research and Technology - Hellas (FORTH), Heraklion, Greece; 3Biocenter of Developmental Biochemistry, University of Wu¨ rzburg, Wu¨ rzburg, Germany; 4Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States Abstract The inability of differentiated cells to maintain their identity is a hallmark of age- related diseases. We found that the transcription factor Hey supervises the identity of differentiated enterocytes (ECs) in the adult Drosophila midgut. Lineage tracing established that Hey-deficient ECs are unable to maintain their unique nuclear organization and identity. To supervise cell identity, Hey determines the expression of nuclear lamins, switching from a stem-cell lamin configuration to a differentiated lamin configuration. Moreover, continued Hey expression is required to conserve large-scale nuclear organization. During aging, Hey levels decline, and EC identity and gut homeostasis are impaired, including pathological reprograming and compromised gut integrity. These phenotypes are highly similar to those observed upon acute targeting of Hey or perturbation of lamin expression in ECs in young adults. Indeed, aging phenotypes were suppressed by continued expression of Hey in ECs, suggesting that a Hey-lamin network *For correspondence: safeguards nuclear organization and differentiated cell identity. [email protected] DOI: https://doi.org/10.7554/eLife.44745.001 †These authors contributed equally to this work Competing interests: The authors declare that no Introduction competing interests exist.
    [Show full text]
  • 2012 Annual Report
    Memorial Sloan-Kettering Cancer Center 2012 ANNUAL REPORT A SHARED VISION A SINGULAR MISSION Nurse practitioner Naomi Cazeau, of the Adult Bone Marrow Transplant Service. PING CHI PHYSICIAN-SCIENTIST 10 STEPHEN SOLOMON ALEXANDER RUDENSKY INTERVENTIONAL IMMUNOLOGIST RADIOLOGIST 16 12 VIVIANE TABAR The clinicians and scientists of NEUROSURGEON Memorial Sloan-Kettering share a vision and 18 a singular mission — to conquer cancer. STEPHEN LONG STRUCTURAL BIOLOGIST They are experts united against a 20 SIMON POWELL complex disease. Each type of cancer R ADIATION ONCOLOGIST 24 ETHEL LAW is different, each tumor is unique. Set free NURSE PRACTITIONER in surroundings that invite the sharing of 26 ideas and resources, they attack the CHRISTINA LESLIE complexity of cancer from every angle COMPUTATIONAL BIOLOGIST and every discipline. 34 SCOTT ARMSTRONG PEDIATRIC ONCOLOGIST 30 TO JORGE REIS-FILHO EXPERIMENTAL PATHOLOGIST CONQUER 38 CANCER 04 Letter from the Chairman and the President A complete version of this report — 42 Statistical Profile which includes lists of our donors, 44 Financial Summary doctors, and scientists — 46 Boards of Overseers and Managers is available on our website at 49 The Campaign for Memorial Sloan-Kettering www.mskcc.org/annualreport. 4 5 Letter from the Chairman In 2012 the leadership of Memorial Sloan-Kettering endorsed Douglas A. Warner III These programmatic investments require leadership and and the President a $2.2 billion investment in a clinical expansion that will set vision. Our new Physician-in-Chief, José Baselga, joined the stage for a changing care paradigm into the next decade us on January 1, 2013. An internationally recognized and beyond.
    [Show full text]
  • POTENTIAL ROLE of MUC1 by Dahlia M. Besmer a Dissertatio
    THE DEVELOPMENT OF NOVEL THERAPEUTICS IN PANCREATIC AND BREAST CANCERS: POTENTIAL ROLE OF MUC1 by Dahlia M. Besmer A dissertation submitted to the faculty of The University of North Carolina at Charlotte in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Charlotte 2013 Approved by: _____________________________ Dr. Pinku Mukherjee _____________________________ Dr. Valery Grdzelishvili _____________________________ Dr. Mark Clemens _____________________________ Dr. Didier Dréau _____________________________ Dr. Craig Ogle ii © 2013 Dahlia M. Besmer ALL RIGHTS RESERVED iii ABSTRACT DAHLIA MARIE BESMER. Development of novel therapeutics in pancreatic and breast cancers: potential role of MUC1. (Under the direction of DR. PINKU MUKHERJEE) Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer-related deaths in the US, and breast cancer (BC) contributes to ~40,000 deaths annually. The development of novel therapeutic agents for improving patient outcome is of paramount importance. Importantly, MUC1 is a mucin glycoprotein expressed on the apical surface of normal glandular epithelia but is over expressed and aberrantly glycosylated in >80% of human PDA and in >90% of BC. In the present study, we first utilize a model of PDA that is Muc1-null in order to elucidate the oncogenic role of MUC1. We show that lack of Muc1 significantly decreased proliferation, invasion, and mitotic rates both in vivo and in vitro. Next, we evaluated the anticancer efficacy of oncolytic virus (OV) therapy that utilizes viruses to kill tumor cells. The oncolytic potential of vesicular stomatitis virus (VSV) was analyzed in a panel of human PDA cell lines in vitro and in vivo in immune compromised mice.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8.450,106 B2 Kaur Et Al
    USOO845O106B2 (12) United States Patent (10) Patent No.: US 8.450,106 B2 Kaur et al. (45) Date of Patent: May 28, 2013 (54) ONCOLYTIC VIRUS 2009.0117644 A1 5/2009 Yazaki et al. 2009/0215147 A1 8/2009 Zhang et al. (75) Inventors: Balveen Kaur, Dublin, OH (US); 2009, 0220460 A1 9, 2009 Coffin Antonio Chiocca, Powell, OH (US); 2010.0086522 A1 4/2010 Bell et al. Yoshinaga Saeki, Toyama (JP) FOREIGN PATENT DOCUMENTS WO 2008/O12695 A2 1, 2008 (73) Assignee: The Ohio State University Research Foundation, Columbus, OH (US) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Abdallah, B.M. etal...TheUse of Mesenchymal (Skeletal) StemCells patent is extended or adjusted under 35 for Treatment of Degenerative Diseases: Current Status and Future U.S.C. 154(b) by 410 days. Prespectives, Journal of Cellular Physiology, 2009, pp. 9-12, 218. Aboody, K.S. et al., Neural stem cells display extensive tropism for (21) Appl. No.: 12/697,891 pathology in adult brain: Evidence from intracranial gliomas, PNAS, Nov. 7, 2000, pp. 12846-12851,97(23). Aboody, K.S. et al., Stem and progenitor cell-mediated tumor selec (22) Filed: Feb. 1, 2010 tive gene therapy, Gene Therapy, 2008, pp. 739-752, 15. (65) Prior Publication Data Abordo-Adesida, E. et al., Stability of Lentiviral Vector-Mediated Transgene Expression in the Brain in the Presence of Systemic US 2010/0272686 A1 Oct. 28, 2010 Antivector Immune Responses, Hum Gene Ther. 2005, pp. 741-751, 16(6). Related U.S. Application Data Abremski, K. et al., Bacteriophage P1 Site-specific Recombination, The Journal of Biological Chemistry, Feb.
    [Show full text]
  • ASBMB President Urges Support for NSF Page 2 Experimental Smallpox
    MAY 2004 www.asbmb.org Constituent Society of FASEB AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY ALSO IN THIS ISSUE ASBMB President Urges Support for NSF Page 2 Experimental Smallpox Vaccine Tested Page 9 See You in Boston! “A“A MolecularMolecular ExplorationExploration ofof thethe Cell”Cell” ASBMB Annual Meeting and 8th IUBMB Conference JuneJune 12-16,12-16, 20042004 Boston, Massachusetts IUBMB/ASBMB 2004 “A Molecular Exploration of the Cell” June 12 – 16 Boston, MA American Society for Biochemistry and Molecular Biology Annual Meeting and 8th IUBMB Conference l Biology ■ Mole ■ Chemica cular Recog atics nition ■ nform Ce Bioi llula and r Bi ics och om em te ist Pro Opening Lecture ry First Annual Herbert Tabor/Journal of Biological Chemistry Lectureship Robert J. Lefkowitz, HHMI, Duke University Medical Center Organized by: John D. Scott, HHMI, Vollum Institute; Alexandra C. Newton, UCSD; Julio Celis, Danish Cancer Society, and the 2004 ASBMB Program Planning Committee Cellular Organization and Dynamics Regulation of Gene Expression and Organizer: Harald A. Stenmark, Norwegian Rad. Hosp. Chromosome Transactions Organizer: Joan W. Conaway, Stowers Inst. for Med. Res. Genomics, Proteomics and Bioinformatics Organizers: Charlie Boone, Univ. of Toronto and Signaling Pathways in Disease Michael Snyder, Yale Univ. Organizers: Alexandra Newton, UCSD and John D. Scott, HHMI, Vollum Inst. Integration of Signaling Mechanisms Organizer: Kjetil Tasken, Univ. of Oslo, Norway The Future of Education and Professional Development in the Molecular Life Sciences Molecular and Cellular Biology of Lipids Organizer: J. Ellis Bell, Univ. of Richmond Organizer: Dennis Vance, Univ. of Alberta Molecular Recognition and Catalysis For further information: Organizer: Jack E.
    [Show full text]
  • Advances and Potential Pitfalls of Oncolytic Viruses Expressing Immunomodulatory Transgene Therapy for Malignant Gliomas Qing Zhang1,2,3 and Fusheng Liu1,2,3
    Zhang and Liu Cell Death and Disease (2020) 11:485 https://doi.org/10.1038/s41419-020-2696-5 Cell Death & Disease REVIEW ARTICLE Open Access Advances and potential pitfalls of oncolytic viruses expressing immunomodulatory transgene therapy for malignant gliomas Qing Zhang1,2,3 and Fusheng Liu1,2,3 Abstract Glioblastoma (GBM) is an immunosuppressive, lethal brain tumor. Despite advances in molecular understanding and therapies, the clinical benefits have remained limited, and the life expectancy of patients with GBM has only been extended to ~15 months. Currently, genetically modified oncolytic viruses (OV) that express immunomodulatory transgenes constitute a research hot spot in the field of glioma treatment. An oncolytic virus is designed to selectively target, infect, and replicate in tumor cells while sparing normal tissues. Moreover, many studies have shown therapeutic advantages, and recent clinical trials have demonstrated the safety and efficacy of their usage. However, the therapeutic efficacy of oncolytic viruses alone is limited, while oncolytic viruses expressing immunomodulatory transgenes are more potent inducers of immunity and enhance immune cell-mediated antitumor immune responses in GBM. An increasing number of basic studies on oncolytic viruses encoding immunomodulatory transgene therapy for malignant gliomas have yielded beneficial outcomes. Oncolytic viruses that are armed with immunomodulatory transgenes remain promising as a therapy against malignant gliomas and will undoubtedly provide new insights into possible clinical uses or strategies. In this review, we summarize the research advances related to oncolytic viruses that express immunomodulatory transgenes, as well as potential treatment pitfalls in patients with malignant gliomas. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Facts Open questions 1.
    [Show full text]
  • Use of Replicating Oncolytic Adenoviruses in Combination Therapy for Cancer
    Vol. 10, 5299–5312, August 15, 2004 Clinical Cancer Research 5299 Review Use of Replicating Oncolytic Adenoviruses in Combination Therapy for Cancer Roland L. Chu,1,2 Dawn E. Post,1 therapy and radiation damage to their DNA. This confers resist- Fadlo R. Khuri,1 and Erwin G. Van Meir1 ance to treatment through clonal expansion of genetically re- sistant tumor cells. Much effort has been directed toward finding 1Laboratory of Molecular Neuro-Oncology, Departments of Neurosurgery, Hematology/Oncology, and Winship Cancer Institute, alternate pathways that would complement therapeutic induc- and 2AFLAC Cancer and Blood Disorders Center, Children’s tion of apoptosis, overcome multidrug resistance, and ultimately Healthcare of Atlanta, Emory University, Atlanta, Georgia improve overall cure rates. Although the dream of developing a single curative drug per cancer type drives the field, the reality is that combination therapy will be the most effective way of ABSTRACT improving survival. Oncolytic virotherapy is the use of genetically engi- The use of viruses that specifically kill tumor cells while neered viruses that specifically target and destroy tumor sparing normal cells, known as oncolytic virotherapy, has re- cells via their cytolytic replication cycle. Viral-mediated tu- emerged over the past 7 years (1, 2). Viruses have evolved to mor destruction is propagated through infection of nearby maximize their ability to enter cells, use the machinery of the tumor cells by the newly released progeny. Each cycle host cell to replicate and package their own genome and lyse the should amplify the number of oncolytic viruses available for cells to release their progeny and propagate the viral replicative infection.
    [Show full text]
  • Oncolytic Herpes Simplex Virus-Based Therapies for Cancer
    cells Review Oncolytic Herpes Simplex Virus-Based Therapies for Cancer Norah Aldrak 1 , Sarah Alsaab 1,2, Aliyah Algethami 1, Deepak Bhere 3,4,5 , Hiroaki Wakimoto 3,4,5,6 , Khalid Shah 3,4,5,7, Mohammad N. Alomary 1,2,* and Nada Zaidan 1,2,* 1 Center of Excellence for Biomedicine, Joint Centers of Excellence Program, King Abdulaziz City for Science and Technology, P.O. Box 6086, Riyadh 11451, Saudi Arabia; [email protected] (N.A.); [email protected] (S.A.); [email protected] (A.A.) 2 National Center for Biotechnology, Life Science and Environmental Research Institute, King Abdulaziz City for Science and Technology, P.O. Box 6086, Riyadh 11451, Saudi Arabia 3 Center for Stem Cell Therapeutics and Imaging (CSTI), Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; [email protected] (D.B.); [email protected] (H.W.); [email protected] (K.S.) 4 Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 5 BWH Center of Excellence for Biomedicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 6 Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA 7 Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA * Correspondence: [email protected] (M.N.A.); [email protected] (N.Z.) Abstract: With the increased worldwide burden of cancer, including aggressive and resistant cancers, oncolytic virotherapy has emerged as a viable therapeutic option. Oncolytic herpes simplex virus (oHSV) can be genetically engineered to target cancer cells while sparing normal cells.
    [Show full text]
  • Genomic DNA Damage and ATR-Chk1 Signaling Determine Oncolytic Adenoviral Efficacy in Human Ovarian Cancer Cells Claire M
    Research article Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells Claire M. Connell,1,2 Atsushi Shibata,2 Laura A. Tookman,1 Kyra M. Archibald,1 Magdalena B. Flak,1 Katrina J. Pirlo,1 Michelle Lockley,1 Sally P. Wheatley,2 and Iain A. McNeish1 1Centre for Molecular Oncology and Imaging, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom. 2Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom. Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being devel- oped as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sen- sitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3- related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells.
    [Show full text]
  • Meeting Product Development Challenges in Manufacturing Clinical Grade Oncolytic Adenoviruses
    Oncogene (2005) 24, 7792–7801 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Meeting product development challenges in manufacturing clinical grade oncolytic adenoviruses Peter K Working*,1, Andy Lin1 and Flavia Borellini1 1Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, CA 94080, USA Oncolytic adenoviruses have been considered for use as issue, is possible because the expression of the adeno- anticancer therapy for decades, and numerous means of virus genome is a tightly regulated cascade, which can be conferring tumor selectivity have been developed. As with considered as divided into early (E) and late (L) phases. any new therapy, the trip from the laboratory bench to the The E1 region gene products are critical for efficient clinic has revealed a number of significant development expression of essentially all the other regions of the hurdles. Viral therapies are subject to specific regulations adenovirus genome, such that controlling the expression and must meet a variety of well-defined criteria for purity, of the E1 region will effectively control adenoviral potency, stability, and product characterization prior to replication. Oncolytic adenoviruses that utilize this their use in the clinic. Published regulatory guidelines, approach include CG7870 (Yu et al., 2001), which although developed specifically for biotechnology-derived utilizes prostate-specific transcription regulatory ele- products, are applicable to the production of oncolytic ments to control E1 and target prostate cancer, and adenoviruses and other cell-based products, and they CG0070 (Bristol et al., 2003), which utilizes the human should be consulted early during development. Most E2F-1 promoter cloned in place of the endogenous E1a importantly, both the manufacturing process and the promoter to target the diverse group of cancers that development of characterization and release assays should have a defective retinoblastoma (Rb) pathway.
    [Show full text]
  • Combining Oncolytic Virotherapy with P53 Tumor Suppressor Gene Therapy
    Review Combining Oncolytic Virotherapy with p53 Tumor Suppressor Gene Therapy Christian Bressy,1 Eric Hastie,2 and Valery Z. Grdzelishvili1 1Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA; 2Department of Biology, Duke University, Durham, NC 27708, USA Oncolytic virus (OV) therapy utilizes replication-competent ing and killing cancer cells (oncotoxicity); (3) able to stimulate an viruses to kill cancer cells, leaving non-malignant cells un- adaptive immune response against cancer cells; and (4) resistant to harmed. With the first U.S. Food and Drug Administration- premature clearance by the immune system during treatment. approved OV, dozens of clinical trials ongoing, and an abun- Despite encouraging results, OV monotherapy based exclusively on dance of translational research in the field, OV therapy is virus replication-induced oncolysis often does not demonstrate all poised to be one of the leading treatments for cancer. A number of these desired qualities, especially when tested against virus-resis- of recombinant OVs expressing a transgene for p53 (TP53) or tant malignancies. Today’s hurdles facing OV therapies remain the – another p53 family member (TP63 or TP73) were engineered same as those described in early6 8 and recent reviews.9,10 Several with the goal of generating more potent OVs that function methods have been developed to increase the anti-cancer activities synergistically with host immunity and/or other therapies to of OVs. Most commonly, OVs are engineered to express an exoge- reduce or eliminate tumor burden. Such transgenes have nous transgene with anti-tumor activity and/or combined with proven effective at improving OV therapies, and basic research standard treatments like radiotherapy or chemotherapy, or with has shown mechanisms of p53-mediated enhancement of OV small-molecule inhibitors of virus-host interactions.
    [Show full text]
  • Review the Oncolytic Virotherapy Treatment Platform for Cancer
    Cancer Gene Therapy (2002) 9, 1062 – 1067 D 2002 Nature Publishing Group All rights reserved 0929-1903/02 $25.00 www.nature.com/cgt Review The oncolytic virotherapy treatment platform for cancer: Unique biological and biosafety points to consider Richard Vile,1 Dale Ando,2 and David Kirn3,4 1Molecular Medicine Program, Mayo Clinic, Rochester, Minnesota, USA; 2Cell Genesys Corp., Foster City, California, USA; 3Department of Pharmacology, Oxford University Medical School, Oxford, UK; and 4Kirn Oncology Consulting, San Francisco, California, USA. The field of replication-selective oncolytic viruses (virotherapy) has exploded over the last 10 years. As with many novel therapeutic approaches, initial overexuberance has been tempered by clinical trial results with first-generation agents. Although a number of significant hurdles to this approach have now been identified, novel solutions have been proposed and improvements are being made at a furious rate. This article seeks to initiate a discussion of these hurdles, approaches to overcome them, and unique safety and regulatory issues to consider. Cancer Gene Therapy (2002) 9, 1062 – 1067 doi:10.1038/sj.cgt.7700548 Keywords: oncolytic; virotherapy; experimental therapeutics; cancer ew cancer treatments are needed. These agents must limitation of levels of delivery to cancer cells in a solid tumor Nhave novel mechanisms of action and thereby lack mass. Over 10 different virotherapy agents have entered, or cross-resistance with currently available treatments. Viruses will soon be entering, clinical trials; one such adenovirus have evolved to infect, replicate in, and kill human cells (dl1520) has entered a Phase III clinical trial in recurrent through diverse mechanisms. Clinicians treated hundreds of head and neck carcinoma.
    [Show full text]