1. 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

2. CELL CYCLE AND CANCER, P53 Zoltan Balajthy Molecular Therapies- Lecture 13 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

3. 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 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.

4. CDK inhibitors Dihydrofolat reductase Thymidine kinase Thymidylate synthase DNA polymerase E2F: transcription factor E2F1 EGF: epidermal growth factor CDK: cyclin-dependent protein kinase Rb: retinoblastoma protein D1, A, E: Cyclin D1, A és E DNS replication machinery Start of S phase pozitív erősítés transzkripció leállítás Transcriptional Events in G1 Phase of Cell-cycle DNS replication machinery TÁMOP-4.1.2-08/1/A-2009-0011

5. Tumor Suppressor Genes, and Their Biochemical Functions NameChromosomal localizationsBiochemical function of missing protein p5317induces CDK inhibitor p21, induces GADD45 which induces DNS repair, induces apoptosis NF-117neurofibromine (activation of ras GTPase) Neurofibromatosis, type-1 WT- 1 (Wilms-tumor)11four Zn-finger transcription factor APC5induction of apoptosis, interacts with β-catenin adenoma polyposis coli P16 melanoma9inhibitor of cdk4 PTENP1 phosphatase deleted in prostate cancer BRCA117DNS repair breast cancer BRCA113DNS repair Breast cancer TÁMOP-4.1.2-08/1/A-2009-0011

6. 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) The Retinoblastoma Example 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

7. Tumor Suppressor Genes: Retinoblastoma and P53 TÁMOP-4.1.2-08/1/A-2009-0011

8. 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. Regulation Transcription Factor of p53 I. TÁMOP-4.1.2-08/1/A-2009-0011

9. Hmd2 p53 p p p p300 DNA damage ATM / ATR Chk2 Hmd2 gene expression Cell cycle arrest Cell death p53 destruction p53 stabilization and tetramerization ubiquitin Regulation Transcription Factor of p53 II. hdm2 p53 turnover in normal conditions TÁMOP-4.1.2-08/1/A-2009-0011

10. transcriptional activation domain Sequence-specific DNA-binding domain tetramerization domain CN 1 100 200300 393 nuclear export signal 1520 Ser 381382 Lys 372 Lys 383 Lys Hdm2 ubiquitination or p300 acetylation 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. Primary Structure of Transcription Factor p53 p p ATM/ATR Chk2 hdm2 TÁMOP-4.1.2-08/1/A-2009-0011

11. Cell stress Oncogene activity p53 Hdm2 Arf Nutlins Prima-1 CP-31398 Cell death Growth arrest Nutlins act by blocking interaction of Mdm2 with p53, therefore prevents its destruction leading to more of the the stable form of p53 Restoration of p53 Function in Tumor Cells III. TÁMOP-4.1.2-08/1/A-2009-0011

12. Restoration of p53 Function in Tumor Cells II. TÁMOP-4.1.2-08/1/A-2009-0011

14. 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 deathEmbryogenesis 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. ApoptosisMorphologic definition can be induced by non-physiologic agents does not always require active protein synthesis TÁMOP-4.1.2-08/1/A-2009-0011

15. 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

16. Morphology of Apoptosis Separation Condensation Fragmentation Phagocytosis Lysosomal digestion Residual body ‘HISTIOCYTE’ TÁMOP-4.1.2-08/1/A-2009-0011

17. Biochemical Pathways of Caspase Activation Dependent Cell Death TÁMOP-4.1.2-08/1/A-2009-0011

18. Killing Tumours by Induction of Apoptosis Untreated controlRadiotherapy (RT)Deathl ligand (TRAIL) Radiotherapy (RT) + Death ligand (TRAIL) Apoptosis staining Hystology staining TÁMOP-4.1.2-08/1/A-2009-0011