Presentation on theme: "Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill McBride Dept. Radiation Oncology David Geffen School Medicine UCLA, Los Angeles,"— Presentation transcript:
1Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill McBride Dept. Radiation Oncology David Geffen School Medicine UCLA, Los Angeles, Ca.Radiation Biology is study of the effects of radiation on living things. For the most part, this course deals with the effects of radiation doses of the magnitude of those used in radiation therapy.
2Objectives:Know that senescence as well as cell death can lead to loss of reproductive colongenic cells and affect the outcome of RTBe able to distinguish between interphase and mitotic (catastrophic) cell death following irradiationUnderstand the physiologic, morphologic, and mechanistic differences between apoptosis, autophagy, and necrosis as deathstyles and how cells die in response to irradiationUnderstand 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 carcinogenesisUnderstand the importance of cell cycle kinetics, cell loss factors in tumor growth and regressionRecognize the importance of changes in these parameters during the course of a fractionated RT regimen
3Intrinsic Radiosensitivity The outcome of radiation exposure depends onThe DNA lesions that are caused and their persistenceHow cells and tissues ‘sense’ danger and respond by activating cell survival or death pathways
4FRACTION OF CELLS SURVIVING 2 GY IN VITRO LYMPHOMANEUROBLASTOMAMYELOMASMALL CELL LUNG CANCERMEDULLOBLASTOMABREAST CASCCPANCREATIC CACOLORECTAL CANON-SMALL CELL CAMELANOMAOSTEOSARCOMAGLIOBLASTOMAHYPERNEPHROMA0.2 ( )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.
5Clinically, tumors show the same histological correlation 20-40Gy Seminoma, Dysgerminoma, Acute Lymphocytic leukemia, Wilms’ tumor, Neuroblastoma40-50Gy 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 tumor60-65Gy Larynx (<1 cm), breast cancer lumpectomy70-75Gy 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 correlationwith respect to sensitivity to RT.
6(“omnis cellula a cellula”) Robert Hooke ( ) was the first to use the term ‘cell’ in the 1665 MicrographiaNot!Antony van Leeuwenhoek ( ) - Made powerful lenses, discovered bacteria - father of microbiologyRudolph Virchow ( ) - Recognized leukemia and mechanism of embolism - Developed theory that cells come from cells(“omnis cellula a cellula”)Walther Flemming ( ) - 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
7DSB 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.
8Loss of Proliferative Ability can Occur in Different Ways Quiescence Senescence Terminal DeathDifferentiationIrreversible,non-physiological processIrreversible, physiologicalactive processCell cycle inhibition is a secondary effectApoptosisAutophagyNecrosisProperty of stem cellsReversible, physiological processApoptosis and differentiation is inhibitedHigh free radical scavenger levels(all with distinct, and common, gene patterns)IR is a pathological signal and can cause senescence
9Radiation-Induced Senescence Is particularly relevant to radiation fibrosis, but also occurs in cells other than fibroblasts.TGF-bp21ProliferativeProgenitorFibroblastPost-mitoticFibroblastStress-induced(Including radiation)Proliferation-inducedCancer-inducedCollagen production and fibrosisTumor progression
10Early 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 deathThis may take several days (as opposed to hours)It is due toChromosome lossFailure of spindle formation during cytokinesisEarly 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.
11RT RECURRENCE! 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
13Alternative Deathstyle Mechanisms Programmed cell death type 1: ApoptosisProgrammed cell death type 2: AutophagyPathological Death: NecrosisDeath is often an active process: cells decide to commit suicideDeath 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.
14Alternative Deathstyle Mechanisms Physiologic PathologicType 1: Apoptosis Type 1: ApoptosisType 2: Autophagy Type 2: AutophagyType 3: NecrosisType 1 and 2 are ProgrammedDeath is largely an active process: cells decide to commit suicideDeath 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.
15Physiologic Programmed Cell Death PCD is involved in:MorphogenesisTissue sculptingHomeostatic control of cell numbersPreventing autoimmunityPCD is immunologically “silent”“It is a myth to think death is just for the old. Death is there from the very beginning” Herman FeifelSelf-reactivelymphocytesIrradiationFingersGutTadpole Tailsproliferating cellsSex differentiationThis may be why proliferation often correlates with apoptotic indexCELL 88:350, 1997
16Pathologic Programmed Cell Death Self sacrifice by infected/damaged cellsSelf sacrifice by immune cells and other normal cells in the battle zoneCauses inflammationwound healingimmunity
17Programmed Cell Death Type I: Apoptosis Morphology Apoptosis is a tightly regulated “active” cell death process that is associated withCell and nuclear shrinkageNuclear fragmentation with formation of apoptotic bodiesBlebbing of cell membrane, but no early loss of membrane integrityDeletion of single cells in isolationLack 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
18Programmed 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 bp. This is called a “ladder.”-+Histones H2,H3,H4Nucleosome DNA Core(140 bp)DNA Spacer Region( bp)55 A110 AHISTONE H1Sites of endonuclease cleavage
19Detection 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 endonucleaseAn Apoptotic Index (AI) can be derived
20Apoptosis in Gut after IR Radiation-induced apoptosis occurs in normal tissues in specific sites and in cells that have a pro-apoptotic tendencyIn gut this is in the base of the cryptsSites ofapoptosis
21Programmed Cell Death Type 2: Autophagy Morphology A tightly regulated processA 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 condensationIn essence it is a defensive reaction that eventually can lead to cell death
22Pathological Cell Death Type 3: Necrosis Morphology Necrosis is a rapid non-physiological process associated withLoss of plasma membrane integrity and deregulated ion homeostasis.Swelling and bursting of cells as water entersGroups 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.
23Triggers for Cell Death Type 1 - Apoptosis:Extrinsic triggering of “death” receptors (some TNFR family members)Intrinsic DNA damage response pathwayAlterations in mitochondria membrane permeabilityType 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
24What Deathstyles are Associated with Radiation-Induced Death? Any of themMitotic death after irradiation can be by any molecular mechanismInterphase death after irradiation is by rapid apoptosisProminent in lymphocytes, spermatogonia, oligodendrocytes, salivary glandOccurs in many tumors and tissues, normally in specific sitesCells that are most sensitive to radiation considered to have a pro-apoptotic phenotype
25How 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 byExtrinsic pathway through Tumor Necrosis Factor Receptor (TNFR) family members, which activates caspase 8Intrinsic 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
26Executioner Caspases Caspase 3 Caspase 7 Caspase 6 Executioner caspases cleave >40 substrates (including each other) leading to the morphological features of apoptosisBlocking these caspases does not generally prevent radiation-induced cell death - by then it is too late!Caspase 3Caspase 6Caspase 7Lamin AActiniCAD - CADDNA-PKcs PARPCADCellShrinkageDNAFragmentationDNARepairICAD (inhibitor of caspase activated DNase)DNA-PK (DNA protein kinase)PARP (poly-ADP-ribose polymerase)
27Radiation-Induced Apoptosis DNA DamageMembers of TNFR familywith Death Domains(TNFR1, Fas, TRAIL)INITIATORSSphingomyelinCeramideFADDJNKP38 MAPKATMxActivation ofPro-caspase 8EFFECTORSp53BaxMitochondriaJNK - jun kinaseATM - mutated in ataxia telangiectasiaFADD - Fas activated death domainApaf - apoptosis activating factorCytochrome cCaspase 8Pro-caspase 9Caspase 9Apaf-1Apoptosome ComplexTERMINAL PHASECaspase 3, 6, 7
28Apoptotic cells reappear between radiation fractions The decision to commit apoptosis is determined by an internal “rheostat” within the cell i.e. cells have a pro-apoptotic or anti-apoptotic phenotypeRadiation increases the AI, but does not change a cell from an anti-apoptotic to pro-apoptotic phenotypeApoptotic 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
29Why 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 mitochondriaBcl-2 is the prototypical inhibitor of apoptosisBax is from the same family and activates apoptosisThe 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 arephosphoinositol 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
30Control Over Radiation-Induced Apoptosis DNA DamageStressMembers of TNFR familyWith Death DomainsINITIATORSSphingomyelinCeramideJNKP38 MAPKATMFADDxNF-kBIAPsEFFECTORSp53BaxBcl-2/Bcl-xlMitochondriaCytochrome cCaspase 8Caspase 9Apaf-1Apoptosome ComplexIAP - inhibitors of apoptosisFLIP - FLICE (procaspase 8) inhibitory proteinTERMINAL PHASECaspase 3, 6, 7
31“Survival Pathways” Survival Growth Factors, Cytokines, Proliferative SignalsTNFR2TNFR1SphingomyelinCeramidePI 3-kinasePDK1AKTBadRasRafProliferationERKMetabolicPathwayP90 RSKNFkBmTORInhibitors of Apoptosis (IAPs)Bcl-2/Bcl-XLcaspasesContext is everything -“Location, location, location”Survival
32Clinical Significance of Cell Death Intrinsic cellular radiosensitivity is determined in part by the balance of the signals transducing cell death or survival pathwaysClinical 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 possibleApoptosis 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 RTEnhancing 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
33Survival pathways are appropriate targets for tumor radiosensitization The pathways that govern cell death/survival also govern radioresistance and radiosensitivity!!!!!Manipulation of apoptotic pathways genetically, or with drugs, can affect clonogenic cell survivalSurvival pathways are appropriate targets for tumor radiosensitizationEGFRIressa, Tarceva, C225, Farnesyl Transferase InhibitorsNF-kBCOX-2 inhibitorsSurvival pathways form appropriate targets for normal tissue radioprotectionKeratinocyte growth factor (KGF) in bone marrow transplant patients
34Volume 354: February 9, 2006Radiotherapy plus Cetuximab for Squamous-Cell Carcinoma of the Head and NeckJames 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.
35Cell Proliferation and Cell Death: Two Sides of the Same Coin?
36Timeframe of Cellular Life The Cell Cycle Under the microscope, Flemming identified cells in mitosis (M) and in interphase - i.e 2 cell cycle phasesHoward & 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. 4 cell cycle phasesTaylor 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 parametersMore 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
37Labeling Index (L.I.) = l TS/TC Mitotic IndexLabeling IndexFlash label with3H-TdR or BdUR for 20 minsFix and stainIf 3H-TdR labeledIf BdUR labeledAR filmmitosis*Anti-BdURMitotic Index (M.I.)= l TM/TCU.V. microscopyAutoradiography…....…..……....……..…....……..…....…..…..…..…..…..…Where is a correction factor for cell division, about 0.69Labeling Index (L.I.) = l TS/TC
38Frequency 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 estimatedBut, it is easier to use BUdR and flow cytometry
39From FLM to FACS PM tubes LASER Cells in fine stream 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 tubesLASERCells in fine stream
40Flow Cytometry for DNA Quantity 1. label DNA with propidium iodide(fluorescent dye)2. measure light output by flow cytometry3. analyze DNA histogramsG1SG2M2n2n + n4nG1# cellsG2MS2n4ndegree of fluorescence
41Cell Cycle Kinetic Analysis by Flow Cytometry P.I. (DNA - red) combined with Bromodeoxyuridine uptake followed by staining with fluorescently labeled anti-BrdUrd (green)G1sG2/MBrdUrdgreenDNAredG1sG2/MBrdUrdgreenDNAP.I. redG1sG2/MBrdUrdgreenDNAP.I redTime
42Cell Cycle M phase 0.5-1 hr G2 phase 1-2 hrs S phase DNA synthesis G0 quiescentS phaseDNA synthesis6-8 hrsG1 phasevariable lengthIf all cells in a population are dividingMitotic Index (M.I.) = lTm / TcLabeling Index (L.I.) = lTs /TcWhere l is a correction for uneven cell numbers due to mitosis (0.69)
43Cell Cycle Synchronisation The best estimates of kinetics come from use of cells synchronized in a specific cell cycle phaseMitotic cells can be shaken off from some cell lines - M phase cellsSerum deprivation - G1 phase cellsHydroxyurea synchronizes cells at the G1/S transition
44Cell Cycle and Radiosensitivity .01.11Variations 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 SEARLY SG1 PHASEG2/M PHASE48121620Dose (Gy)Increasing radioresistanceG S G2 M
45Cell Cycle ArrestCells 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 checkpointsCells tend to arrest atG1 - especially if they have wt p53. This may lead to apoptosisIntra S phase - initiation and elongation stages of DNA replication are affected by p53 independent mechanismsG2 - most cells arrest here - allows chromatid repair prior to segregation in MM phase - block in anaphase until all sister chromatids have aligned properly on the spindle - Monitors spindle integrity for cytokinesis
46Cell Cycle Arrest DNA Damage Dependent Checkpoints Irradiated (7Gy)P.I stain at 9hrwild-type irradiatedDecrease in SIncrease in G2Mi.e. G1 and G2M arrestP53 or ATM deficient irradiatedloss of G1/S checkpointand only G2M arrest
47What Drives Cell Cycle Progression? Growth factors are required for G0 through G1 to S (and cell survival)To activate resting cells to enter G1To allow cells to pass through G1 phaseTo gain competence to progress into S phaseThe growth factors that are required vary with the cell type. For example, for fibroblasts:PDGF (platelet derived GF) activates cellsEGF (epidermal GF) and insulin act as competence factors to progress into S phaseIGF (insulin GF) promotes progression into SCycling is growth factor independent through S, G2, M
48Molecular Mechanism of Cell Cycle Progression Progression through each checkpoint requires:Retinoblastoma (Rb) tumor suppressor gene familyespecially G1-S transitionRegulatory FactorsCyclins 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 phaseRegulators of CDKsInhibitory kinasesActivated phosphatasesNon-kinase inhibitors
49Retinoblastoma Protein pRb Cyclin D/cdk4/6 and cyclin E/cdk2 phosphorylate Rb, which is essential for cell cycle progression into SPhosphorylation 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.
50Cyclins Have no intrinsic enzymatic activity Cyclins A to J have been identified (no I)Synthesized and degraded during each cell cycle phaseBind and activate cdks
51Cyclin Dependent Kinases Cyclins bind and activate Cdks, whichAre serine/threonine kinases with multiple substratese.g. pRb, p53, E2F, etc. that they activate/inactivateHave regulatory domainsE.g. inhibitory and activating phosphatesAre present throughout cell cycleTo move cells from G0 to G1 to SCyclin D activates cdks 4/6 andCyclin E activates cdk2Inhibitory phosphatePCyclinkinase sitePcdkactivating phosphate
53G0 quiescent Cyclin D Cyclin B Cyclin A Cyclin D Cyclin A Cyclin E cdk1 phosphorylates substrates leads toNuclar envelope breakdownChromosome separationSpindle assemblyChromosome condensationCyclin BCDK1Cyclosome (APC)pRb dephosphorylationCyclin ACDK1/2G0 quiescentCyclin DCDK4/6Cyclin ACDK1/2Early - mid G1Cyclin DCDK4/6Responsible for pRbphosphorylationCyclin ECDK2Responsible for pRbphosphorylation
54Cyclin Kinase Inhibitors Inhibitors (CKIs) belong to 2 familiesINK4 and KIP/CIPGenerally compete with cyclins for CDKsPhase Complexes InhibitorsG1 cyclin D-CDK4, 6 p16 (INK 4a),p19ARF (INK 4a)p15 (INK4b)G1/S cyclin E-CDK2, 3 p21CIP1, p27KIP1S cyclin A-CDK2 p21, p57G2/M cyclin B-CDK1 p21p53 is a transcription factor for p21, which is why it is involved in cell cycle arrest after IR
56Cell Cycle in CancerIf 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 cancere.g. cyclin D amplification and/or p16 deletion or silencing and/or p53 mutation in Head and Neck Ca
57Growth Factor/Cytokine CancerGrowth Factor/CytokineReceptorSurvivalSignalsOncogenesPI3KNF-BRasRafMAPKProliferationCell death
59Loss of Proliferative Ability can Occur in Different Ways Quiescence Senescence Terminal DeathDifferentiationIrreversible,non-physiological processIrreversible, physiologicalactive processCell cycle inhibition is a secondary effectApoptosisAutophagyNecrosisProperty of stem cellsReversible, physiological processApoptosis and differentiation is inhibitedHigh free radical scavenger levels
60Tissue Kinetics Cell cycle Growth fraction (G.F.) Cell loss factor Kinetics in tumors or normal tissues depend uponCell cycleGrowth fraction (G.F.)G.F. is the proportion of proliferating cellsG.F. = P / (P + Q) where P = proliferating cells and Q = non-proliferating cells (quiescent/senescent/differentiated cells)Cell loss factorCell Loss Factor is due to death or loss of cellsIf = 0, Td = Tpot where Td is the actual volume doubling time and Tpot is potential volume doubling time= 1 - Tpot / Tdif G.F. = 1 then Tpot = Tc = lTs / 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, < 1.0
61VARIES GREATLY WITH TUMOR Tumor KineticsHuman SCC36 hrs0.256 days60 days0.9Tc Cell cycle timeG.F Growth fractionTpot Pot. doubling timeTd Actual doubling timeCell loss factor(36hr x 4)(1-6/60)Rate of tumor growth, and the rate of tumor regression, are determined largely by the cell loss factor!VARIES GREATLY WITH TUMOR
62Tumor RegressionThe rate of tumor growth and regression is determined byrate of cell loss (G.F.cell cycle kineticsSlow growing tumors may regress rapidlyRapidly growing tumors are expected to regress and regrow rapidlySlow regression is not an indication of treatment failureThe rate of tumor regression after Tx is not, in general, prognostic
63Tumors can regenerate at the same time as they regress! Tumor RegenerationX-raysRelative tumor volumeControlIrradiatedTumors can regenerate at the same time as they regress!Growth delaySurviving clonogensmeasured in vitroTimeRat rhabdomyosarcomaHermans and Barendsen, 1969
64EVIDENCE FOR ACCELERATED REPOPULATION IN TUMORS Time to tumor recurrence after therapy is shorter than than would be expected from the original growth rateSplit-course radiation therapy often gives poor resultsProtraction of treatment time often results in poor resultsAccelerated treatment has been shown to be of benefit in some circumstances.
65Accelerated Tumor Repopulation T2 T3local controlno local controlT2 and T3 SCC head and neck (excluding nasopharynx and vocal cord). TCD50 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/dyWithers et al, 1988
66Accelerated 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.
67Accelerated repopulation in human tumors provided the rationale for accelerated fractionation protocols