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www.radbiol.ucla.edu Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill McBride Dept. Radiation Oncology David Geffen School Medicine.

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Presentation on theme: "www.radbiol.ucla.edu Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill McBride Dept. Radiation Oncology David Geffen School Medicine."— Presentation transcript:

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2 Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill McBride Dept. Radiation Oncology David Geffen School Medicine UCLA, Los Angeles, Ca.

3 Objectives: Know that senescence as well as cell death can lead to loss of reproductive colongenic cells and affect the outcome of RTKnow 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 irradiationBe 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 irradiationUnderstand 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.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 carcinogenesisKnow 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 regressionUnderstand 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 regimenRecognize the importance of changes in these parameters during the course of a fractionated RT regimen

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

5 FRACTION OF CELLS SURVIVING 2 GY IN VITRO LYMPHOMANEUROBLASTOMAMYELOMA SMALL CELL LUNG CANCER MEDULLOBLASTOMA BREAST CA SCC PANCREATIC CA COLORECTAL CA NON-SMALL CELL CA MELANOMAOSTEOSARCOMAGLIOBLASTOMAHYPERNEPHROMA 0.2 ( ) 0.43 ( ) 0.52 ( ) 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.

6 Gy 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.

7 Not! Robert Hooke ( ) was the first to use the term ‘cell’ in the 1665 Micrographia Antony van Leeuwenhoek ( ) - Made powerful lenses, discovered bacteria - father of microbiology Rudolph Virchow ( ) - Recognized leukemia and mechanism of embolism - Developed theory that cells come from cells (“omnis cellula a cellula”) (“omnis cellula a cellula”) Walther Flemming ( ) - identified chromatin and mitosis (Gk, thread) (“omnis nucleus a nucleo”) 1906 Bergonie and Tribandeau. Action des rayou X sur le testicle 1906 Bergonie and Tribandeau. Action des rayou X sur le testicle Elect. Med.14, radiosensitivity is related to cell proliferation

8 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. 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.

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

10 Stress-induced (Including radiation) Proliferation-inducedCancer-induced ProliferativeProgenitorFibroblast Post-mitoticFibroblast TGF-  Radiation-Induced Senescence p21 Collagen production and fibrosis Tumor progression Is particularly relevant to radiation fibrosis, but also occurs in cells other than fibroblasts.

11 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.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 lossChromosome loss Failure of spindle formation during cytokinesisFailure of spindle formation during cytokinesis Early radiobiologists also discovered that a few cells of specific types die by interphase death (without dividing)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.

12 RECURRENCE! RT Lethal Sectoring in Mitotic Death 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

13 Control Cells Control - Nuclei Stained Irradiated Cells Irradiated - Nuclei Stained Courtesy: Randi Syljuasen

14 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 suicideDeath 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 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.Death pathways, and mutations in them, affect intrinsic cellular radiosensitivity. They provide potential tumor-specific targets for radiosensitization.

15 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 ProgrammedType 1 and 2 are Programmed Death is largely an active process: cells decide to commit suicideDeath 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.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.The same death pathways and mutations affect intrinsic cellular radiosensitivity. They provide potential tumor-specific targets for radiosensitization.

16 Physiologic Programmed Cell Death Sex differentiation PCD is involved in: MorphogenesisMorphogenesis Tissue sculptingTissue sculpting Homeostatic control of cell numbersHomeostatic control of cell numbers Preventing autoimmunityPreventing autoimmunity PCD is immunologically “silent”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 Self-reactivelymphocytes Irradiation Fingers Gut Tadpole Tails proliferating cells This may be why proliferation often correlates with apoptotic index CELL 88:350, 1997

17 Pathologic Programmed Cell Death Self sacrifice by infected/damaged cellsSelf sacrifice by infected/damaged cells Self sacrifice by immune cells and other normal cells in the battle zoneSelf sacrifice by immune cells and other normal cells in the battle zone Causes inflammationCauses inflammation –wound healing –immunity

18 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 Programmed Cell Death Type I: Apoptosis Morphology Apoptosis is 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!)

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

20 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 endonucleaseApoptosis 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 derivedAn Apoptotic Index (AI) can be derived

21 Apoptosis in Gut after IR Sites of apoptosis Radiation-induced apoptosis occurs in normal tissues in specific sites and in cells that have a pro- apoptotic tendencyRadiation-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 cryptsIn gut this is in the base of the crypts

22 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

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

24 Triggers for Cell Death Type 1 - Apoptosis: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: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. 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. 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:Type 3 - Necrosis: –Extrinsic activation of immune cells leads to release of cytotoxins - perforins, etc. that cause necrosis

25 What Deathstyles are Associated with Radiation-Induced Death? Any of them Mitotic death after irradiation can be by any molecular mechanismMitotic death after irradiation can be by any molecular mechanism Interphase death after irradiation is by rapid apoptosisInterphase 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 phenotypeCells that are most sensitive to radiation considered to have a pro-apoptotic phenotype

26 How do cells commit suicide? Apoptosis is Mediated by Caspases - “Roads to Ruin”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 byThe 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 pathwayIrrespective of the apoptotic death signal, all caspases converge to activate a terminal Caspase 3-dependent pathway

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

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

29 The decision to commit apoptosis is determined by an internal “rheostat” within the cell i.e. cells have a pro-apoptotic or anti- apoptotic phenotypeThe 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 phenotypeRadiation increases the AI, but does not change a cell from an anti-apoptotic to pro-apoptotic phenotype Apoptotic cells reappear between radiation fractionsApoptotic 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

30 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 mitochondriaMitochondrial 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 areSurvival 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 interventionCancer is associated with mutations in cell death/survival pathways, as is radioresistance, and these are targets for theraputic intervention

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

32 Ras Raf ERK P90 RSK Survival PI 3-kinase PDK1AKTBad TNFR1 NF  B TNFR2 “Survival Pathways” Bcl-2/Bcl-XL Growth Factors, Cytokines, Proliferative Signals SphingomyelinCeramide Inhibitors of Apoptosis (IAPs) caspases Context is everything - “Location, location, location” Proliferation mTOR MetabolicPathway

33 Clinical Significance of Cell Death Intrinsic cellular radiosensitivity is determined in part by the balance of the signals transducing cell death or survival pathwaysIntrinsic 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 possibleClinical 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”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 RTIn 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 cellsEnhancing 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

34 The pathways that govern cell death/survival also govern radioresistance and radiosensitivity!!!!!The pathways that govern cell death/survival also govern radioresistance and radiosensitivity!!!!! Manipulation of apoptotic pathways genetically, or with drugs, can affect clonogenic cell survivalManipulation of apoptotic pathways genetically, or with drugs, can affect clonogenic cell survival Survival pathways are appropriate targets for tumor radiosensitizationSurvival pathways are appropriate targets for tumor radiosensitization EGFREGFR Iressa, Tarceva, C225, Farnesyl Transferase InhibitorsIressa, Tarceva, C225, Farnesyl Transferase Inhibitors NF-  BNF-  B COX-2 inhibitorsCOX-2 inhibitors Survival pathways form appropriate targets for normal tissue radioprotectionSurvival pathways form appropriate targets for normal tissue radioprotection Keratinocyte growth factor (KGF) in bone marrow transplant patientsKeratinocyte growth factor (KGF) in bone marrow transplant patients

35 Volume 354: February 9, 2006Volume 354: February 9, 2006 Radiotherapy plus Cetuximab for Squamous-Cell Carcinoma of the Head and NeckRadiotherapy 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.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 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 ….. 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. 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.

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

37 Timeframe of Cellular Life The Cell Cycle Under the microscope, Flemming identified cells in mitosis (M) and in interphase - i.e 2 cell cycle phasesUnder 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 32 P 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. 4 cell cycle phasesHoward & Pelc, 1951 & 1953, - bean root cells in interphase incorporate 32 P 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. 4 cell 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 parametersTaylor 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. microscopyMore 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

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

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

40 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.) LASER Cells in fine stream PM tubes

41 Flow Cytometry for DNA Quantity 1. label DNA with propidium iodide (fluorescent dye) (fluorescent dye) 2. measure light output by flow cytometry 3. analyze DNA histograms G1 S G2 M 2n 2n +  n 4n4n2n 4n # cells degree of fluorescence G1 S G2M

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

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

44 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 cellsMitotic cells can be shaken off from some cell lines - M phase cells Serum deprivation - G1 phase cellsSerum deprivation - G1 phase cells Hydroxyurea synchronizes cells at the G1/S transitionHydroxyurea synchronizes cells at the G1/S transition

45 Cell Cycle and Radiosensitivity S.F Dose (Gy) LATE S EARLY S G1 PHASE G2/M PHASE 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. G1 S G2 M Increasing radioresistance

46 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.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 checkpointsIrradiation causes cells to arrest at these checkpoints Cells tend to arrest atCells tend to arrest at G1 - especially if they have wt p53. This may lead to apoptosisG1 - 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 mechanismsIntra 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 MG2 - most cells arrest here - allows chromatid repair prior to segregation in M M phase - b lock in anaphase until all sister chromatids have aligned properly on the spindle - Monitors spindle integrity for cytokinesisM phase - b lock in anaphase until all sister chromatids have aligned properly on the spindle - Monitors spindle integrity for cytokinesis

47 Irradiated (7Gy) Irradiated (7Gy) P.I stain at 9hr 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 Cell Cycle Arrest DNA Damage Dependent Checkpoints

48 What Drives Cell Cycle Progression? Growth factors are required for G0 through G1 to S (and cell survival) To activate resting cells to enter G1To activate resting cells to enter G1 To allow cells to pass through G1 phaseTo allow cells to pass through G1 phase To gain competence to progress into S phaseTo 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 cellsPDGF (platelet derived GF) activates cells EGF (epidermal GF) and insulin act as competence factors to progress into S phaseEGF (epidermal GF) and insulin act as competence factors to progress into S phase IGF (insulin GF) promotes progression into SIGF (insulin GF) promotes progression into S Cycling is growth factor independent through S, G2, M

49 Molecular Mechanism of Cell Cycle Progression Progression through each checkpoint requires: Retinoblastoma (Rb) tumor suppressor gene familyRetinoblastoma (Rb) tumor suppressor gene family especially G1-S transitionespecially G1-S transition Regulatory FactorsRegulatory Factors Cyclins that are synthesized at the appropriate time for each phase and then degraded to coordinate cell cycle progression. Growth factors induce cyclin expression in G1.Cyclins that 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 phaseCyclin Dependent Kinases (CDK) are activated by cyclins and phosphorylate targets required for the next cell cycle phase Regulators of CDKsRegulators of CDKs 1.Inhibitory kinases 2.Activated phosphatases 3.Non-kinase inhibitors

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

51 Cyclins Have no intrinsic enzymatic activityHave no intrinsic enzymatic activity Cyclins A to J have been identified (no I)Cyclins A to J have been identified (no I) Synthesized and degraded during each cell cycle phaseSynthesized and degraded during each cell cycle phase Bind and activate cdksBind and activate cdks

52 Cyclin Dependent Kinases Cyclins bind and activate Cdks, whichCyclins bind and activate Cdks, which –Are serine/threonine kinases with multiple substrates e.g. pRb, p53, E2F, etc. that they activate/inactivatee.g. pRb, p53, E2F, etc. that they activate/inactivate –Have regulatory domains E.g. inhibitory and activating phosphatesE.g. inhibitory and activating phosphates –Are present throughout cell cycle –To move cells from G0 to G1 to S Cyclin D activates cdks 4/6 andCyclin D activates cdks 4/6 and Cyclin E activates cdk2Cyclin E activates cdk2 P P cdk Inhibitory phosphate activating phosphate Cyclin kinase site

53 Activating Phosphatases CDC25 Removes Phosphate from Tyr-15 –CDC25A = cyclin E/CDK2 = G1/S specific –CDC25B = cyclin A/CDK2 = S-phase exit –CDC25C = cyclin B/CDK1 = G2/M specific

54 Cyclin D CDK 4/6 Cyclin E CDK 2 Responsible for pRb phosphorylation Cyclin A CDK 1/2 Cyclin B CDK 1 Cyclin A CDK 1/2 Early - mid G1 Cyclin D CDK 4/6 Responsible for pRb phosphorylation cdk1 phosphorylates substrates leads to Nuclar envelope breakdownNuclar envelope breakdown Chromosome separationChromosome separation Spindle assemblySpindle assembly Chromosome condensationChromosome condensation Cyclosome (APC) pRb dephosphorylation G0 quiescent

55 Cyclin Kinase Inhibitors PhaseComplexesInhibitors G1 cyclin D-CDK4, 6p16 (INK 4a), p19 ARF (INK 4a) p15 (INK4b) G1/S cyclin E-CDK2, 3p21 CIP1, p27 KIP1 S cyclin A-CDK2p21, p57 G2/M cyclin B-CDK1 p21 p53 is a transcription factor for p21, which is why it is involved in cell cycle arrest after IR Inhibitors (CKIs) belong to 2 families INK4 and KIP/CIP INK4 and KIP/CIP Generally compete with cyclins for CDKs

56 ATM MRN complex NHEJ 53BP1 MDC1 MRN BRCA1  H2AX mediators p53CHK2 CDC25A phosphorylation p21 CYCLIN E CDK2 CYCLIN E CDK2 P-thr14/tyr15 p21 G1/S Arrest Sensescence/transient CDC25A degradation rapid slow transactivation ATR 53BP1 MDC1 MRN BRCA1 CHK1 CDC25A phosphorylation CYCLIN A/E CDK2 S Phase Arrest DSB SSB/Base damage Replication stress, UV, MMC, hypoxia Stalled Replication Fork HR ATM ATR 53BP1 MDC1 MRN BRCA1 CHK2CHK1 CDC25C phosphorylation and nuclear export CYCLIN B CDK! P-thr14/tyr15 P G2/M Arrest DSB Resection MDM2 sensors transducers effectors

57 If p53 or any other molecule governing cell cycle arrest is mutated, genetic instability results as well as more rapid cell cycle progression. If p53 or any other molecule governing cell cycle arrest is mutated, genetic instability results as well as more rapid cell cycle progression. Cyclins, cdks, cdkis and other molecules involved in cell cycle progression are frequently mutated or have altered expression in cancer Cyclins, cdks, cdkis and other molecules involved in cell cycle progression are frequently mutated or have altered expression in cancer e.g. cyclin D amplification and/or p16 deletion or silencing and/or p53 mutation in Head and Neck Cae.g. cyclin D amplification and/or p16 deletion or silencing and/or p53 mutation in Head and Neck Ca Cell Cycle in Cancer

58 Growth Factor/Cytokine Receptor Proliferation Cell death Oncogenes RasRafMAPK PI3K NF-  B SurvivalSignals Cancer

59 DNA damage response ATM, ATR, MRN P53, Chk1, Chk2 Initial damage ROS Cell cycle arrest Cell death /survival DNA repair JNK P38 MAPK NF-kB Tissue recovery /lesion formation Cell death /survival Cell proliferation Immediate early gene response AU-rich control: TNF- , IL1 , IL-2, IL-3, GM- CSF, IL-6, IL-8, IL-12, IFN  / , VEGF, PDGFB, NGF, IGFR, DR5, COX-2 Proteasome inhibition Mitochondrial damage Activation of EGFR, TGF- , etc Inflammatory Cytokines and Growth Factors P21, Bax, caspase 8,etc.

60 Loss of Proliferative Ability can Occur in Different Ways Quiescence Senescence Terminal Death Differentiation Property of stem cells Reversible, physiological process Apoptosis and differentiation is inhibited High free radical scavenger levels Irreversible, physiological active process Cell cycle inhibition is a secondary effect Irreversible, non-physiological process ApoptosisAutophagyNecrosis

61 Tissue Kinetics Kinetics in tumors or normal tissues depend upon Cell cycleCell cycle Growth fraction (G.F.)Growth fraction (G.F.) G.F. is the proportion of proliferating cellsG.F. is the proportion of proliferating cells G.F. = P / (P + Q) where P = proliferating cells and Q = non- proliferating cells (quiescent/senescent/differentiated cells)G.F. = P / (P + Q) where P = proliferating cells and Q = non- proliferating cells (quiescent/senescent/differentiated cells) Cell loss factorCell loss factor Cell Loss Factor  is due to death or loss of cellsCell Loss Factor  is due to death or loss of cells If  = 0, Td = Tpot where Td is the actual volume doubling time and Tpot is potential volume doubling timeIf  = 0, Td = Tpot where Td is the actual volume doubling time and Tpot is potential volume doubling time  = 1 - Tpot / Td  = 1 - Tpot / Td if G.F. = 1 then Tpot = Tc = Ts / L.I.if G.F. = 1 then Tpot = Tc = Ts / L.I. Under steady state conditions, a constant cell number is maintained by the balance between cell proliferation and cell loss i.e.  = 1.0. In tumors and embryos,  < 1.0Under steady state conditions, a constant cell number is maintained by the balance between cell proliferation and cell loss i.e.  = 1.0. In tumors and embryos,  < 1.0

62 Tumor Kinetics Tc Cell cycle time G.F. Growth fraction Tpot Pot. doubling time Td Actual doubling time  Cell loss factor Human SCC 36 hrs days 60 days 0.9 Rate of tumor growth, and the rate of tumor regression, are determined largely by the cell loss factor! VARIES GREATLY WITH TUMOR (36hr x 4) (1-6/60)

63 Tumor Regression The rate of tumor growth and regression is determined byThe rate of tumor growth and regression is determined by rate of cell loss ( rate of cell loss (  G.F.G.F. cell cycle kineticscell cycle kinetics Slow growing tumors may regress rapidlySlow growing tumors may regress rapidly Rapidly growing tumors are expected to regress and regrow rapidlyRapidly growing tumors are expected to regress and regrow rapidly Slow regression is not an indication of treatment failureSlow regression is not an indication of treatment failure The rate of tumor regression after Tx is not, in general, prognosticThe rate of tumor regression after Tx is not, in general, prognostic

64 Tumor Regeneration Rat rhabdomyosarcoma Hermans and Barendsen, 1969 Tumors can regenerate at the same time as they regress! ControlIrradiated Surviving clonogens measured in vitro Growth delay Time Relative tumor volume X-rays

65 EVIDENCE FOR ACCELERATED REPOPULATION IN TUMORS Time to tumor recurrence after therapy is shorter than than would be expected from the original growth rateTime to tumor recurrence after therapy is shorter than than would be expected from the original growth rate Split-course radiation therapy often gives poor resultsSplit-course radiation therapy often gives poor results Protraction of treatment time often results in poor resultsProtraction of treatment time often results in poor results Accelerated treatment has been shown to be of benefit in some circumstances.Accelerated treatment has been shown to be of benefit in some circumstances.

66 Accelerated Tumor Repopulation T2 and T3 SCC head and neck ( excluding nasopharynx and vocal cord). TCD 50 values are consistent with onset of repopulation at 4 weeks followed by accelerated repopulation with a 3-4 day doubling time, implying a loss in dose of about 0.6 Gy/dy Withers et al, 1988 T2 T3 local control no local control

67 Accelerated Tumor Repopulation Onset may be about day 21. Repopulation may not be constant and may increase from 0.6 Gy / day around week 3-4 to even 1.6 – 1.8 Gy / day around week 6-7 and thereafter.

68 Accelerated repopulation in human tumors provided the rationale for accelerated fractionation protocols


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