Cell Cycle and Cancer , p53

1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011 ZoltanBalajthy MolecularTherapies-Lecture 13 CellCycle and Cancer, p53

3. 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

4. TÁMOP-4.1.2-08/1/A-2009-0011 Transcriptional Events in G1Phase 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

5. TÁMOP-4.1.2-08/1/A-2009-0011 Tumor Suppressor Genes, and Their Biochemical Functions Name Chromosomal localizations Biochemical function of missing protein p53 17 induces CDK inhibitor p21, induces GADD45 which induces DNS repair, induces apoptosis NF-1 17 neurofibromine (activation of ras GTPase) Neurofibromatosis, type-1 WT- 1 (Wilms-tumor) 11 four Zn-finger transcription factor APC 5 induction of apoptosis, interacts with β-catenin adenoma polyposis coli P16 melanoma 9 inhibitor of cdk4 PTEN P1 phosphatase deleted in prostate cancer BRCA1 17 DNS repair breast cancer BRCA1 13 DNS repair Breast cancer

6. TÁMOP-4.1.2-08/1/A-2009-0011 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. Theretinoblastoma 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)

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

8. TÁMOP-4.1.2-08/1/A-2009-0011 Regulation Transcription Factor of p53 I. The p53 transcription factor can either inducegrowth 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.

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

10. Primary Structure of Transcription Factor p53 ATM/ATR ubiquitination or Hdm2 acetylation p300 Chk2 Ser Lys Ser Lys Lys Lys 15 20 381 382 372 383 hdm2 nuclear export signal p p 1 100 200 300 393 N C tetramerization domain transcriptional activation domain Sequence-specific DNA-binding 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

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

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

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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 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 pointof 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. 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)

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

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

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

19. TÁMOP-4.1.2-08/1/A-2009-0011