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

Radiation 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.

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

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

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 ( ) 0.43 ( ) 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.

Clinically, tumors show the same histological correlation
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.

(“omnis cellula a cellula”)
Robert Hooke ( ) was the first to use the term ‘cell’ in the 1665 Micrographia Not! 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”) 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

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.

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

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

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.

RT 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

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

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.

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.

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 Self-reactive lymphocytes Irradiation Fingers Gut Tadpole Tails proliferating cells Sex differentiation This may be why proliferation often correlates with apoptotic index CELL 88:350, 1997

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

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!) 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 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,H4 Nucleosome DNA Core (140 bp) DNA Spacer Region ( bp) 55 A 110 A HISTONE H1 Sites of endonuclease cleavage

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

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

Programmed Cell Death Type 2: Autophagy Morphology
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

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. 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.

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

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

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

Executioner Caspases Caspase 3 Caspase 7 Caspase 6
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 6 Caspase 7 Lamin A Actin iCAD - CAD DNA-PKcs PARP CAD Cell Shrinkage DNA Fragmentation DNA Repair ICAD (inhibitor of caspase activated DNase) DNA-PK (DNA protein kinase) PARP (poly-ADP-ribose polymerase)

Radiation-Induced Apoptosis
DNA Damage Members of TNFR family with Death Domains (TNFR1, Fas, TRAIL) INITIATORS Sphingomyelin Ceramide FADD JNK P38 MAPK ATM x Activation of Pro-caspase 8 EFFECTORS p53 Bax Mitochondria 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

Apoptotic 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 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

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

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

“Survival Pathways” Survival
Growth Factors, Cytokines, Proliferative Signals TNFR2 TNFR1 Sphingomyelin Ceramide 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

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

Survival 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 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

Volume 354: 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.

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

Timeframe of Cellular Life The 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. 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 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

Labeling Index (L.I.) = l TS/TC
Mitotic Index Labeling 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 U.V. microscopy Autoradiography … .. .. … .. … … .. .. … … .. … .. .. … … .. … .. .. … .. … .. … .. … .. … .. … Where  is a correction factor for cell division, about 0.69 Labeling Index (L.I.) = l TS/TC

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

From 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 tubes LASER Cells in fine stream

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 S G2 M 2n 2n +  n 4n G1 # cells G2M S 2n 4n degree of fluorescence

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

Cell Cycle M phase 0.5-1 hr G2 phase 1-2 hrs S phase DNA synthesis
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)

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

Cell Cycle and Radiosensitivity
.01 .1 1 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 4 8 12 16 20 Dose (Gy) Increasing radioresistance G S G2 M

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

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

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

Molecular Mechanism of Cell Cycle Progression
Progression through each checkpoint requires: Retinoblastoma (Rb) tumor suppressor gene family especially G1-S transition Regulatory 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. 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

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 genes that are required for S phase gene expression. pRB mutation often leads to cancer.

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

Cyclin Dependent Kinases
Cyclins bind and activate Cdks, which Are serine/threonine kinases with multiple substrates e.g. pRb, p53, E2F, etc. that they activate/inactivate Have regulatory domains E.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 and Cyclin E activates cdk2 Inhibitory phosphate P Cyclin kinase site P cdk activating phosphate

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

G0 quiescent Cyclin D Cyclin B Cyclin A Cyclin D Cyclin A Cyclin E
cdk1 phosphorylates substrates leads to Nuclar envelope breakdown Chromosome separation Spindle assembly Chromosome condensation Cyclin B CDK1 Cyclosome (APC) pRb dephosphorylation Cyclin A CDK1/2 G0 quiescent Cyclin D CDK4/6 Cyclin A CDK1/2 Early - mid G1 Cyclin D CDK4/6 Responsible for pRb phosphorylation Cyclin E CDK2 Responsible for pRb phosphorylation

Cyclin Kinase Inhibitors
Inhibitors (CKIs) belong to 2 families INK4 and KIP/CIP Generally compete with cyclins for CDKs Phase Complexes Inhibitors G1 cyclin D-CDK4, 6 p16 (INK 4a), p19ARF (INK 4a) p15 (INK4b) G1/S cyclin E-CDK2, 3 p21CIP1, p27KIP1 S cyclin A-CDK2 p21, 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

DSB SSB/Base damage DSB Resection S Phase Arrest G2/M Arrest
Replication stress, UV, MMC, hypoxia Stalled Replication Fork DSB Resection MRN complex ATR sensors NHEJ ATM ATM HR ATR H2AX 53BP1 MDC1 MRN BRCA1 53BP1 MDC1 MRN BRCA1 53BP1 MDC1 MRN BRCA1 mediators rapid slow MDM2 CHK2 CHK1 transducers p53 CHK2 CHK1 transactivation effectors p21 CDC25A phosphorylation CDC25A phosphorylation CDC25C phosphorylation and nuclear export CDC25A degradation CYCLIN E CDK2 P P-thr14/tyr15 P-thr14/tyr15 CDK! CDK2 p21 CDK2 CYCLIN B CYCLIN A/E CYCLIN E S Phase Arrest G2/M Arrest G1/S Arrest Sensescence/transient

Cell Cycle in Cancer 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 e.g. cyclin D amplification and/or p16 deletion or silencing and/or p53 mutation in Head and Neck Ca

Growth Factor/Cytokine
Cancer Growth Factor/Cytokine Receptor Survival Signals Oncogenes PI3K NF-B Ras Raf MAPK Proliferation Cell death

Inflammatory Cytokines and Growth Factors
Initial damage DNA repair Cell cycle arrest Cell death /survival ROS DNA damage response ATM, ATR, MRN P53, Chk1, Chk2 P21, Bax, caspase 8,etc. Immediate early gene response Inflammatory Cytokines and Growth Factors 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 JNK P38 MAPK NF-kB Cell proliferation Proteasome inhibition Mitochondrial damage Activation of EGFR, TGF-, etc Tissue recovery /lesion formation Cell death /survival Cell proliferation

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

Tissue Kinetics Cell cycle Growth fraction (G.F.) Cell loss factor
Kinetics in tumors or normal tissues depend upon Cell cycle Growth fraction (G.F.) G.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) Cell loss factor Cell 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 time = 1 - Tpot / Td if 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

VARIES GREATLY WITH TUMOR
Tumor Kinetics Human SCC 36 hrs 0.25 6 days 60 days 0.9 Tc Cell cycle time G.F Growth fraction Tpot Pot. doubling time Td Actual doubling time Cell 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

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

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

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

Accelerated Tumor Repopulation
T2 T3 local control no local control T2 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/dy Withers et al, 1988

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.

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

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

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

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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:

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