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Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Zoltan Balajthy Molecular Therapies- Lecture 13 CELL CYCLE AND CANCER, P53 TÁMOP-4.1.2-08/1/A-2009-0011 Learning objectives of chapter 12 and 13 . The purpose of this chapter is to describe the processes and regulations of both cell cycle and cell death, explain the unregulated cell division, and to point out the therapeutic intervention in cancer at molecular levels. Topics in chapter 13. 13.1. Tumor suppressor genes, and their biochemical functions The retinoblastoma protein Primary structure of transcription factor p53 and its regulation Restoration of p53 function in tumor cells 13.2. Natural Cell Death Common elements of the three forms of natural cell death Biochemical pathways of caspase activation dependent cell death Killing tumours by induction of apoptosis TÁMOP-4.1.2-08/1/A-2009-0011 Transcriptional Events in G1 Phase of Cell-cycle CDK inhibitors Start of S phase DNS replication machinery pozitív erősítés Dihydrofolat reductase Thymidine kinase Thymidylate synthase DNA polymerase CDK inhibitors E2F: transcription factor E2F1 EGF: epidermal growth factor CDK: cyclin-dependent protein kinase Rb: retinoblastoma protein D1, A, E: Cyclin D1, A és E transzkripció leállítás DNS replication machinery CDK inhibitors TÁMOP-4.1.2-08/1/A-2009-0011 Tumor Suppressor Genes, and Their Biochemical Functions Name Chromosomal localizationsBiochemical function of missing protein p53 17 induces CDK inhibitor p21, induces GADD45 which induces DNS repair, induces apoptosis NF-1 17 Neurofibromatosis, type-1 neurofibromine (activation of ras GTPase) WT- 1 (Wilms-tumor) 11 four Zn-finger transcription factor APC adenoma polyposis coli 5 induction of apoptosis, interacts with β-catenin P16 melanoma 9 inhibitor of cdk4 PTEN deleted in prostate cancer P1 phosphatase BRCA1 breast cancer 17 DNS repair BRCA1 Breast cancer 13 DNS repair TÁMOP-4.1.2-08/1/A-2009-0011 The Retinoblastoma Example The retinoblastoma gene 180 kb, 27 exon 4.7 kb mRNA 105 kD fehérje Deletion was observed in this gene when observed from isolated tumor cells. The frequency of deletions in this genes corresponded to the rate of occurrence of of this tumor. From neuroblastoma tumor cells only damaged or mutated forms of this gene could be isolated. Re-introducing the cloned Rb gene into the tumor cells led to their normal proliferation (loss of tumor forming Potential) Some part of chromosome 13 were very often missing when it was isolated from neuroblastoma tumors. From the corresponding part in normal chromosome 13 the neuroblastoma gene could be cloned and characterized. TÁMOP-4.1.2-08/1/A-2009-0011 Tumor Suppressor Genes: Retinoblastoma and P53 Rb Blockade of cell divisin Damage to DNA E2F G1 CDK active p p Rb p P53 p TF E2F Ac 1. Halts cell cycle at G1 csheckpoit 2. Activates DNA repair system Ac S phase / G2 Initiates transcription of p21 p21 Mithosis p21 Cell Division Blocks cell cycle at G1 checkpoin G1 CDK active Initiates transcription of repair enzymes DNA repair Prevents DNA replication TÁMOP-4.1.2-08/1/A-2009-0011 Regulation Transcription Factor of p53 I. DNA Damage, UV Oncogenic Stimulation Hypoxia Hypoglycemia (cyclins, c-Myc, E2F-1, Ras) BRCA1 (ATM, DNA-PK, ATR) p19ARF The p53 transcription factor can either induce growth arrest or apoptosis in response to a variety of cellular stresses including exposure to DNA damaging agents, hypoxia and inappropriate proliferative signals. DNA damaging agents and UV irradiation stabilize p53 through phosphorylation of p53 at its N-terminal and activate its DNA binding through dephosphorylation and acetylation of its C-terminal region. Hypoxia and hypoglycemia stabilize p53 through both phosphorylation dependent and independent mechanisms. Inappropriate oncogene stimulation leads to p53 stabilization through the p19ARF pathway. Binding of hdm2 to p53 inhibits its transactivation activity and leads to its degradation. ARF overexpression leads to p53 stabilization by binding to hdm2 and preventing the hdm2 mediated p53 inhibition and degradation. Disruption of hdm2 and p53 interactions appears to be critical for the stabilization of p53. Stabilized and activated p53 can then transactivate its target genes. HIF-1 h d m 2 ? p p53 h d m 2 p53 acetylation dephosphorylation Stable Degraded p53 p53 p53 p53 p53 Stable and Active Transcriptional repression Transcription of target genes IAP survivin PUMA NOXA APAF-1 P21(WAF1) GADD45 14-3-3 PIG3 BAX FAS/APO1 KILLER/DR5 cell death proteins TÁMOP-4.1.2-08/1/A-2009-0011 Regulation Transcription Factor of p53 II. DNA damage hdm2 ubiquitin ATM / ATR Chk2 p300 p300 Hmd2 p53 Hmd2 p p53 p p p53 Hmd2 p53 destruction p53 stabilization and tetramerization gene expression p53 turnover in normal conditions Cell cycle arrest Cell death TÁMOP-4.1.2-08/1/A-2009-0011 Primary Structure of Transcription Factor p53 ATM/ATR p p Hdm2 p300 Chk2 Ser Ser 15 20 ubiquitination or acetylation Lys Lys Lys Lys 372 381 hdm2 1 382 383 nuclear export signal 100 200 300 393 N transcriptional activation domain C Sequence-specific DNA-binding domain tetramerization domain p53 is of central importance in the response to DNA damage and other cellular stresses, and its activation can cause the death of the cell. It is therefore subject to an unusually large array of regulatory modifications that ensure it is present and active only when necessary. Most of these modifications increase its concentration or its intrinsic gene regulatory activity, or both, when DNA damage occurs Mutation of the gene for ATM in humans results in the disease ataxia telangiectasia, which is characterized by, among other things, a greatly reduced ability to repair radiation-induced double-strand breaks – and an increased risk of developing cancer. ATM is recruited to sites of double-strand break formation, where it phosphorylates effector molecules that carry out the damage response. TÁMOP-4.1.2-08/1/A-2009-0011 Restoration of p53 Function in Tumor Cells III. Cell stress Oncogene activity Prima-1 CP-31398 Cell death Hdm2 p53 Growth arrest Arf Nutlins Nutlins act by blocking interaction of Mdm2 with p53 , therefore prevents its destruction leading to more of the the stable form of p53 TÁMOP-4.1.2-08/1/A-2009-0011 Restoration of p53 Function in Tumor Cells II. DNA Damage, UV Oncogenic Stimulation Hypoxia Hypoglycemia (cyclins, c-Myc, E2F-1, Ras) BRCA1 (ATM, DNA-PK, ATR) p19ARF HIF-1 ? The p53 transcription factor p h acts as a tumor suppressor Oncogenic d by inducing growth arrest or mutations m p53 2 apoptosis in response to a variety of cellular stresses Nutlin therapy including DNA damage, hypoxia and inappropriate p53 acetylation dephosphorylation proliferative signals. Stable Gene therapy Stabilizing molecules h d m 2 Degraded p53 p53 p53 p53 p53 Stable and Active Transcriptional repression Transcription of target genes IAP survivin PUMA NOXA APAF-1 P21(WAF1) GADD45 14-3-3 PIG3 KILLER/DR5 FAS/APO1 BAX cell death proteins TÁMOP-4.1.2-08/1/A-2009-0011 TÁMOP-4.1.2-08/1/A-2009-0011 13.2. Natural Cell Death is a physiologic phenomenon occurring continuously in living tissues to remove cells without any function (morphogenesis, duplicate structures, sexual dimorphism), which are produced in excess (e.g. in bone marrow), which develop improperly (e.g. part of lymphocytes), which completed their function (endometrium, tissue turnover), which are potentially dangerous (e.g. autoreactive T cells, neutrophil granulocytes). Forms of natural cell death a. Programmed cell death Embryogenesis functional, developmental definition; predictable in space and time; requires active protein synthesis b. Specialized forms of cell death tissue-specific terminal differentiation; the death program is suspended at one point of the death pathway; the partial death forms serve specific tissue functions; requires active protein (e.g., red blood cell, platelets, lens, cornification) c. Apoptosis Morphologic definition can be induced by non-physiologic agents does not always require active protein synthesis TÁMOP-4.1.2-08/1/A-2009-0011 Common Elements of The Three Forms of Natural Cell Death elimination of the nucleus DNA degradation by endonucleases acting at internucleosomal sites activation and/or induction of protein cross-linker transglutaminases Activation of specific proteases there is no leakage of intracellular macromolecules effective phagocytosis with the help of integrin receptors (except cornification and lens epithelial cells) TÁMOP-4.1.2-08/1/A-2009-0011 Morphology of Apoptosis Condensation Separation Fragmentation Phagocytosis Residual body Lysosomal digestion ‘HISTIOCYTE’ TÁMOP-4.1.2-08/1/A-2009-0011 Biochemical Pathways of Caspase Activation Dependent Cell Death Mitochondrion Plasma membrane Truncated Bid Bcl-2, … Bid Cytochrome c Procaspase-3 Procaspase-8 ATP Bax, Noxa, Puma, … Apaf-1 Procaspase-9 Caspase-8 p53 Mdm2 Apoptosome Caspase-3 XIAP Diablo/SMAC Cell death Cytoplasma Nucleus Prot e deat olysis o h su bstra f tes DNA cleavage ICAD CAD DNA-PK, ATM DNA damage TÁMOP-4.1.2-08/1/A-2009-0011 Hystology staining Apoptosis staining Killing Tumours by Induction of Apoptosis Untreated control Radiotherapy (RT) Deathl ligand (TRAIL) Radiotherapy (RT) + Death ligand (TRAIL) TÁMOP-4.1.2-08/1/A-2009-0011