Objectives:

1. Radiation Targets 2: Cell Proliferation, Cell Death and SurvivalBill McBrideDept. Radiation OncologyDavid Geffen School MedicineUCLA, Los Angeles, Ca.wmcbride@mednet.ucla.edu

2. Objectives: • Know that senescence as well as cell death can lead to loss of reproductive colongenic cells and affect the outcome of RT • Be able to distinguish between interphase and mitotic (catastrophic) cell death following irradiation • Understand the physiologic, morphologic, and mechanistic differences between apoptosis, autophagy, and necrosis as deathstyles and how cells die in response to irradiation • Understand how survival pathways operate to affect cellular radiosensitivity and how these can be targeted for radiotherapeutic benefit. • Know the molecular basis for cell cycle arrest following IR and its importance in repair and carcinogenesis • Understand the importance of cell cycle kinetics, cell loss factors in tumor growth and regression • Recognize the importance of changes in these parameters during the course of a fractionated RT regimen

3. Intrinsic Radiosensitivity The outcome of radiation exposure depends on • The DNA lesions that are caused and their persistence • How cells and tissues ‘sense’ danger and respond by activating cell survival or death pathways

4. FRACTION OF CELLS SURVIVING 2 GY IN VITRO LYMPHOMA NEUROBLASTOMA MYELOMA SMALL CELL LUNG CANCER MEDULLOBLASTOMA BREAST CA SCC PANCREATIC CA COLORECTAL CA NON-SMALL CELL CA MELANOMA OSTEOSARCOMA GLIOBLASTOMA HYPERNEPHROMA 0.2 (0.08 - 0.37) 0.43 (0.14 - 0.75) 0.52 (0.2 - 0.86) Tumor cells vary dramatically in intrinsic radiosensitivity depending on their tissue of origin. The number of DNA lesions are the same but the outcome is different.

5. 20-40Gy Seminoma, Dysgerminoma, Acute Lymphocytic leukemia, Wilms’ tumor, Neuroblastoma 40-50Gy Hodgkin's, Lymphosarcoma, Seminoma, Histiocytic cell sarcoma, Skin ca. (basal and squamous cell) 50-60Gy Squamous cell ca. (cervix, head and neck), Breast ca., Ovarian ca.,Medulloblastoma, Retinoblastoma, Ewing's tumor 60-65Gy Larynx (<1 cm), breast cancer lumpectomy 70-75Gy Oral cavity (<2 cm, 2-4 cm), Oro-naso-laryngo-pharyngeal ca., Bladder ca., Cervix ca., Uterine ca., Ovarian ca., Lung ca. (<3 cm) >80Gy Head and neck ca. (~4 cm), Breast ca. (~5 cm), Glioblastomas, Osteogenic sarcomas (bone sarcomas), Melanomas, Soft tissue sarcomas (~5 cm), Thyroid Ca. (In Rubin P, et al, eds: Clinical Oncology: A Multidisciplinary Approach, edition 7, p 72. Saunders, 1993) Clinically, tumors show the same histological correlation with respect to sensitivity to RT.

6. Robert Hooke (1635-1703) was the first to use the term ‘cell’ in the 1665 Micrographia Not! Antony van Leeuwenhoek (1632-1723) - Made powerful lenses, discovered bacteria - father of microbiology Rudolph Virchow (1821-1902) - Recognized leukemia and mechanism of embolism - Developed theory that cells come from cells (“omnis cellula a cellula”) Walther Flemming (1843-1905) - identified chromatin and mitosis (Gk, thread) (“omnis nucleus a nucleo”) • Bergonie and Tribandeau. Action des rayou X sur le testicle Elect. Med.14, 779 • - radiosensitivity is related to cell proliferation

7. DSB repair, checkpoint arrest, and cell death are all part of the DNA damage response to DSBs. They function synergistically to dictate whether cells live or die following IR and to prevent development of chromosome instability. The relationship of repair, cell proliferation and cell death following IR has been the subject of many studies, primarily because, clinically, loss of reproductive, clonogenic cells following RT determines the outcome of cancer treatment.

8. Loss of Proliferative Ability can Occur in Different Ways • Quiescence Senescence Terminal Death • Differentiation Irreversible, non-physiological process Irreversible, physiological active process Cell cycle inhibition is a secondary effect Apoptosis Autophagy Necrosis Property of stem cells Reversible, physiological process Apoptosis and differentiation is inhibited High free radical scavenger levels (all with distinct, and common, gene patterns) IR is a pathological signal and can cause senescence

9. Radiation-Induced Senescence Is particularly relevant to radiation fibrosis, but also occurs in cells other than fibroblasts. TGF-b p21 Proliferative Progenitor Fibroblast Post-mitotic Fibroblast Stress-induced (Including radiation) Proliferation-induced Cancer-induced Collagen production and fibrosis Tumor progression

10. Early Observations on Cell Death after Irradiation • Radiobiologists like Puck and Marcus (1956) showed that most reproductive cells die a mitotic death, also known as mitotic catastrophe, after IR. • It may take several cell divisions, the number depending on the radiation dose. • After 2 Gy, it may average 2-3 cell divisions before death • This may take several days (as opposed to hours) • It is due to • Chromosome loss • Failure of spindle formation during cytokinesis • Early radiobiologists also discovered that a few cells of specific types die by interphase death (without dividing) • This is generally more rapid than mitotic death, occurring 4-24hrs after irradiation.

11. Lethal Sectoring in Mitotic Death RT RECURRENCE! The fear of death is the most unjustified of all fears, for there's no risk of an accident for someone who's dead. Albert Einstein

12. Courtesy: Randi Syljuasen Control Cells Irradiated Cells Irradiated - Nuclei Stained Control - Nuclei Stained

13. Alternative Deathstyle Mechanisms Programmed cell death type 1: Apoptosis Programmed cell death type 2: Autophagy Pathological Death: Necrosis • Death is often an active process: cells decide to commit suicide • Death pathways prevent carcinogenesis and mutations in them are associated with cancer. They provide potential tumor-specific targets for therapeutic intervention. • Death pathways, and mutations in them, affect intrinsic cellular radiosensitivity. They provide potential tumor-specific targets for radiosensitization.

14. Alternative Deathstyle Mechanisms Physiologic Pathologic Type 1: Apoptosis Type 1: Apoptosis Type 2: Autophagy Type 2: Autophagy Type 3: Necrosis • Type 1 and 2 are Programmed • Death is largely an active process: cells decide to commit suicide • Death pathways prevent carcinogenesis and mutations in molecules in these pathways are associated with cancer. They provide potential tumor-specific targets for therapeutic intervention. • The same death pathways and mutations affect intrinsic cellular radiosensitivity. They provide potential tumor-specific targets for radiosensitization.

15. Fingers Gut Tadpole Tails proliferating cells Irradiation Self-reactive lymphocytes Physiologic Programmed Cell Death • PCD is involved in: • Morphogenesis • Tissue sculpting • Homeostatic control of cell numbers • Preventing autoimmunity • PCD is immunologically “silent” • “It is a myth to think death is just for the old. Death is there from the very beginning” Herman Feifel Sex differentiation This may be why proliferation often correlates with apoptotic index CELL 88:350, 1997

16. Pathologic Programmed Cell Death • Self sacrifice by infected/damaged cells • Self sacrifice by immune cells and other normal cells in the battle zone • Causes inflammation • wound healing • immunity

17. Programmed Cell Death Type I: Apoptosis Morphology • Apoptosisis a tightly regulated “active” cell death process that is associated with • Cell and nuclear shrinkage • Nuclear fragmentation with formation of apoptotic bodies • Blebbing of cell membrane, but no early loss of membrane integrity • Deletion of single cells in isolation • Lack of an inflammatory response and phagocytosis by local cells (a silent death!) The word comes from  - from and  - falling. “Like leaves on trees the race of man is found, now green in youth, now withering on the ground”The Iliad of Homer. Book vi. Line 181

18. - + Programmed Cell Death Type I: Apoptosis Molecular Hallmarks During apoptosis, endonucleases are induced that cleave between nucleosomes. On agarose gel electrophoresis, the DNA separates into fragments with sizes that are multiples of 180-200 bp. This is called a “ladder.” Histones H2,H3,H4 Nucleosome DNA Core (140 bp) DNA Spacer Region (60-100 bp) 55 A 110 A HISTONE H1 Sites of endonuclease cleavage

19. Detection of Apoptosis - TUNEL Assay • Apoptosis can be visualized in tissue sections using terminal deoxynucleotidyl transferase (TdT) to add fluorescein-labeled (dUTP) nucleotides onto 3’-OH ends of DNA that result from the action of the apoptotic endonuclease • An Apoptotic Index (AI) can be derived

20. Apoptosis in Gut after IR • Radiation-induced apoptosis occurs in normal tissues in specific sites and in cells that have a pro-apoptotic tendency • In gut this is in the base of the crypts Sites of apoptosis

21. Programmed Cell Death Type 2: Autophagy Morphology Autophagy • A tightly regulated process • A response to nutrient and growth factor deprivation, but is also seen in physiologic processes, eg morphogenesis. • Organelles and other cell components are sequestered in autophagosomes that fuse with lysosomes (self-digestion) • Increased endocytosis, vacuolation, membrane blebbing, nuclear condensation • In essence it is a defensive reaction that eventually can lead to cell death

22. Necrosisis a rapid non-physiological process associated with Loss of plasma membrane integrity and deregulated ion homeostasis. Swelling and bursting of cells as water enters Groups of cells, rather than single cells, are affected. DNA forms a random “smear” on agarose gel. There is no pattern to its fragmentation. Associated with inflammation. Pathological Cell Death Type 3: NecrosisMorphology

23. Triggers for Cell Death • Type 1 - Apoptosis: • Extrinsic triggering of “death” receptors (some TNFR family members) • Intrinsic DNA damage response pathway • Alterations in mitochondria membrane permeability • Type 2 - Autophagy: • Removal of growth/survival factor signaling. Often called “death by neglect.”Cells have to receive the appropriate stimuli from their environment to survive, if not they die often by autophagy. Death is the default pathway of life! Cells in the wrong microenvironment die of “homelessness” (anoikis), a form of death by neglect. • The PI3K/Akt/mTOR pathway is activated by growth factors allowing increased expression of transporters for glucose, amino acids, etc. Akt increases glycolysis. mTOR drives protein translation rates. • Type 3 - Necrosis: • Extrinsic activation of immune cells leads to release of cytotoxins - perforins, etc. that cause necrosis

24. What Deathstyles are Associated with Radiation-Induced Death? Any of them • Mitotic death after irradiation can be by any molecular mechanism • Interphase death after irradiation is by rapid apoptosis • Prominent in lymphocytes, spermatogonia, oligodendrocytes, salivary gland • Occurs in many tumors and tissues, normally in specific sites • Cells that are most sensitive to radiation considered to have a pro-apoptotic phenotype

25. How do cells commit suicide? • Apoptosis is Mediated by Caspases - “Roads to Ruin” • The morphological and biochemical hall-marks of apoptosis are the result of cascadic activation of members of a family of pro-enzyme proteases called Caspases by • Extrinsic pathway through Tumor Necrosis Factor Receptor (TNFR) family members, which activates caspase 8 • Intrinsic pathway through cytochrome c leaking from mitochondria, which activates caspase 9. • Irrespective of the apoptotic death signal, all caspases converge to activate a terminal Caspase 3-dependent pathway

26. Executioner Caspases • Executioner caspases cleave >40 substrates (including each other) leading to the morphological features of apoptosis • Blocking these caspases does not generally prevent radiation-induced cell death - by then it is too late! Caspase 3 Caspase 7 Caspase 6 Lamin A Actin DNA-PKcs PARP iCAD - CAD CAD Cell Shrinkage DNA Fragmentation DNA Repair ICAD (inhibitor of caspase activated DNase) DNA-PK (DNA protein kinase) PARP (poly-ADP-ribose polymerase)

27. Radiation-Induced Apoptosis Members of TNFR family with Death Domains (TNFR1, Fas, TRAIL) DNA Damage INITIATORS Sphingomyelin Ceramide FADD JNK P38 MAPK ATM x Activation of Pro-caspase 8 EFFECTORS Bax Mitochondria p53 JNK - jun kinase ATM - mutated in ataxia telangiectasia FADD - Fas activated death domain Apaf - apoptosis activating factor Cytochrome c Caspase 8 Pro-caspase 9 Caspase 9 Apaf-1 Apoptosome Complex TERMINAL PHASE Caspase 3, 6, 7

28. The decision to commit apoptosis is determined by an internal “rheostat” within the cell i.e. cells have a pro-apoptotic or anti-apoptotic phenotype • Radiation increases the AI, but does not change a cell from an anti-apoptotic to pro-apoptotic phenotype • Apoptotic cells reappear between radiation fractions “There is only one serious philosophical problem. It is suicide. To judge whether life is, or is not, worth living” Albert Camus

29. Why don’t all cells die by apoptosis after RTx? • Mitochondrial Control: Members of the Bcl-2 family (B cell lymphoma oncogene) localize in the outer membrane of the mitochondria • Bcl-2 is the prototypical inhibitor of apoptosis • Bax is from the same family and activates apoptosis • The balance of pro-apototic (bax) to anti-apoptotic (Bcl-2) factors control the “leakiness” of the membranes. • Survival pathways: These affect intrinsic and extrinsic apoptotic and autophagic pathways and alter the rheostat away from cell death and towards radioresistancy - acting often through the Bcl-2 family. Major survival pathways are • phosphoinositol kinase 3 (PI3K) • nuclear factor kappa B (NF-B) • Cancer is associated with mutations in cell death/survival pathways, as is radioresistance, and these are targets for theraputic intervention

30. Control Over Radiation-Induced Apoptosis DNA Damage Stress Members of TNFR family With Death Domains INITIATORS Sphingomyelin Ceramide JNK P38 MAPK ATM FADD x NF-kB IAPs EFFECTORS p53 Bax Bcl-2/Bcl-xl Mitochondria Cytochrome c Caspase 8 Caspase 9 Apaf-1 Apoptosome Complex IAP - inhibitors of apoptosis FLIP - FLICE (procaspase 8) inhibitory protein TERMINAL PHASE Caspase 3, 6, 7

31. “Survival Pathways” Growth Factors, Cytokines, Proliferative Signals Sphingomyelin Ceramide TNFR2 TNFR1 PI 3-kinase PDK1 AKT Bad Ras Raf Proliferation ERK Metabolic Pathway P90 RSK NFkB mTOR Inhibitors of Apoptosis (IAPs) Bcl-2/Bcl-XL caspases Context is everything - “Location, location, location” Survival

32. Clinical Significance of Cell Death • Intrinsic cellular radiosensitivity is determined in part by the balance of the signals transducing cell death or survival pathways • Clinical RT response is superior in tumors with pathways primed for an active form of cell death, but the relationship between AI (or BAX/Bcl-2) and local tumor control or patient survival after RT are controversial, perhaps because excessive cell death often correlates with high cell proliferation or because multiple pathways to cell death are possible • Apoptosis may affect the clinical response of normal tissues to RT e.g. serous cells - “dry mouth” • In general, RT increases the A.I. only in cells with a pro-apoptotic phenotype and apoptotic cells reappear between fractions of RT • Enhancing PCD in a proportion of cells does not necessarily affect the shape of the clonogenic survival curves following radiation - this depends on the response of the surviving cells

33. The pathways that govern cell death/survival also govern radioresistance and radiosensitivity!!!!! • Manipulation of apoptotic pathways genetically, or with drugs, can affect clonogenic cell survival • Survival pathways are appropriate targets for tumor radiosensitization • EGFR • Iressa, Tarceva, C225, Farnesyl Transferase Inhibitors • NF-kB • COX-2 inhibitors • Survival pathways form appropriate targets for normal tissue radioprotection • Keratinocyte growth factor (KGF) in bone marrow transplant patients

34. Volume 354:567-578 February 9, 2006 • Radiotherapy plus Cetuximab for Squamous-Cell Carcinoma of the Head and Neck • James A. Bonner, M.D., Paul M. Harari, M.D., Jordi Giralt, M.D., Nozar Azarnia, Ph.D., Dong M. Shin, M.D., Roger B. Cohen, M.D., Christopher U. Jones, M.D., Ranjan Sur, M.D., Ph.D., David Raben, M.D., Jacek Jassem, M.D., Ph.D., Roger Ove, M.D., Ph.D., Merrill S. Kies, M.D., Jose Baselga, M.D., Hagop Youssoufian, M.D., Nadia Amellal, M.D., Eric K. Rowinsky, M.D., and K. Kian Ang, M.D., Ph.D. • The median duration of locoregional control was 24.4 months among patients treated with cetuximab plus radiotherapy and 14.9 months among those given radiotherapy alone ….. • the median duration of overall survival was 49.0 months among patients treated with combined therapy and 29.3 months among those treated with radiotherapy alone ….. • Radiotherapy plus cetuximab significantly prolonged progression-free survival … With the exception of acneiform rash and infusion reactions, the incidence of grade 3 or greater toxic effects, including mucositis, did not differ significantly between the two groups.

35. Cell Proliferation and Cell Death: Two Sides of the Same Coin?

36. Timeframe of Cellular LifeThe Cell Cycle • Under the microscope, Flemming identified cells in mitosis (M) and in interphase - i.e 2 cell cycle phases • Howard & Pelc, 1951 & 1953, - bean root cells in interphase incorporate 32P for DNA synthesis (S phase) and there is a time gap (G2) before the beginning of cell division (M) and there is another gap (G1) between M and S to complete the cell cycle - i.e. 4cell cycle phases • Taylor et al., 1957 looked at tritiated thymidine uptake (in S) and measured the time it takes for labeled cells to enter M (= time in G2), and the other cell cycle kinetic parameters • More recently, bromodeoxyuridine detected by fluorescent antibody is used to label cells (in S) and measure cell cycle kinetics by flow cytometry or U.V. microscopy

37. Labeling Index … .. … .. … .. … .. … .. … .. … .. … .. … .. Mitotic Index Flash label with 3H-TdR or BdUR for 20 mins Fix and stain If 3H-TdR labeled If BdUR labeled AR film mitosis *Anti-BdUR Mitotic Index (M.I.) = l TM/TC Autoradiography • U.V. microscopy … .. … .. … .. … .. … .. … .. … .. … .. … .. … .. … .. … .. … .. Where  is a correction factor for cell division, about 0.69 Labeling Index (L.I.) = l TS/TC

38. Frequency of Labeled Mitosis Technique (FLM) • By counting the number of mitoses that are labeled at various times after 3H-thymidine incorporation, the time taken for a cell to traverse a specific cell cycle phase, and the cell cycle time, can be estimated • But, it is easier to use BUdR and flow cytometry

39. From FLM to FACS Label cells with dye and use a laser to excite it. Collect output by photomultiplier tubes. E.g. DNA can be labeled by propidium iodide (P.I.) PM tubes LASER Cells in fine stream

40. 4n 4n M G2 G1 2n S 2n +  n Flow Cytometry for DNA Quantity 1. label DNA with propidium iodide (fluorescent dye) 2. measure light output by flow cytometry 3. analyze DNA histograms G1 # cells G2M S 2n 4n degree of fluorescence

41. BrdUrd green BrdUrd green BrdUrd green G1 G1 G1 s s s G2/M DNA red G2/M DNA P.I. red DNA P.I red G2/M Cell Cycle Kinetic Analysis by Flow Cytometry P.I. (DNA - red) combined with Bromodeoxyuridine uptake followed by staining with fluorescently labeled anti-BrdUrd (green) Time

42. Cell Cycle M phase 0.5-1 hr G2 phase 1-2 hrs G0 quiescent S phase DNA synthesis 6-8 hrs G1 phase variable length If all cells in a population are dividing Mitotic Index (M.I.) = lTm / Tc Labeling Index (L.I.) = lTs /Tc Where l is a correction for uneven cell numbers due to mitosis (0.69)

43. Cell Cycle Synchronisation The best estimates of kinetics come from use of cells synchronized in a specific cell cycle phase • Mitotic cells can be shaken off from some cell lines - M phase cells • Serum deprivation - G1 phase cells • Hydroxyurea synchronizes cells at the G1/S transition

44. 1 .1 G1 S G2 M .01 Cell Cycle and Radiosensitivity Variations in sensitivity and in cell cycle arrest after irradiation could be important in radiation therapy, because fractionated irradiation can lead to sensitization by reassortment. The oxygen enhancement ratio (OER) does not vary much with the phase of the cell cycle. High LET responses are less affected by cell cycle phase than low LET radiation responses. S.F. LATE S EARLY S G1 PHASE G2/M PHASE 0 0 4 8 12 16 20 Dose (Gy) Increasing radioresistance

45. Cell Cycle Arrest • Cells have “checkpoints” where they “proof-read” DNA for damage before continuing to cycle. This ensures faithful chromosome replication and maintains genomic integrity. • Irradiation causes cells to arrest at these checkpoints • Cells tend to arrest at • G1 - especially if they have wt p53. This may lead to apoptosis • Intra S phase - initiation and elongation stages of DNA replication are affected by p53 independent mechanisms • G2 - most cells arrest here - allows chromatid repair prior to segregation in M • M phase - block in anaphase until all sister chromatids have aligned properly on the spindle - Monitors spindle integrity for cytokinesis

46. Cell Cycle Arrest DNA Damage Dependent Checkpoints • Irradiated (7Gy) • P.I stain at 9hr wild-type irradiated Decrease in S Increase in G2M i.e. G1 and G2M arrest P53 or ATM deficient irradiated loss of G1/S checkpoint and only G2M arrest

47. What Drives Cell Cycle Progression? Growth factors are required for G0 through G1 to S (and cell survival) • To activate resting cells to enter G1 • To allow cells to pass through G1 phase • To gain competence to progress into S phase The growth factors that are required vary with the cell type. For example, for fibroblasts: • PDGF (platelet derived GF) activates cells • EGF (epidermal GF) and insulin act as competence factors to progress into S phase • IGF (insulin GF) promotes progression into S Cycling is growth factor independent through S, G2, M

48. Molecular Mechanism of Cell Cycle Progression Progression through each checkpoint requires: • Retinoblastoma (Rb) tumor suppressor gene family • especially G1-S transition • Regulatory Factors • Cyclinsthat are synthesized at the appropriate time for each phase and then degraded to coordinate cell cycle progression. Growth factors induce cyclin expression in G1. • Cyclin Dependent Kinases (CDK)are activated by cyclins and phosphorylate targets required for the next cell cycle phase • Regulators of CDKs • Inhibitory kinases • Activated phosphatases • Non-kinase inhibitors

49. Retinoblastoma Protein pRb • Cyclin D/cdk4/6 and cyclin E/cdk2 phosphorylate Rb, which is essential for cell cycle progression into S • Phosphorylation of Rb releases E2F, which it normally is bound to. E2F is a transcription factor for 20-30 genes that are required for S phase gene expression. • pRB mutation often leads to cancer.

50. Cyclins • Have no intrinsic enzymatic activity • Cyclins A to J have been identified (no I) • Synthesized and degraded during each cell cycle phase • Bind and activate cdks