Radiation Targets 2 Cell Proliferation Cell Death and

  • Slides: 67
Download presentation
Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill Mc. Bride Dept. Radiation

Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill Mc. Bride Dept. Radiation Oncology David Geffen School Medicine UCLA, Los Angeles, Ca. [email protected] ucla. edu www. radbiol. ucla. edu

Objectives: • • Know that senescence as well as cell death can lead to

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 www. radbiol. ucla. edu

Intrinsic Radiosensitivity The outcome of radiation exposure depends on • The DNA lesions that

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 www. radbiol. ucla. edu

FRACTION OF CELLS SURVIVING 2 GY IN VITRO LYMPHOMA NEUROBLASTOMA MYELOMA SMALL CELL LUNG

FRACTION OF CELLS SURVIVING 2 GY IN VITRO LYMPHOMA NEUROBLASTOMA MYELOMA SMALL CELL LUNG CANCER MEDULLOBLASTOMA 0. 2 (0. 08 - 0. 37) BREAST CA SCC PANCREATIC CA COLORECTAL CA NON-SMALL CELL CA 0. 43 (0. 14 - 0. 75) MELANOMA OSTEOSARCOMA GLIOBLASTOMA HYPERNEPHROMA 0. 52 (0. 2 - 0. 86) Tumor cells vary dramatically in intrinsic radiosensitivity depending on their tissue of origin. The number of DNA lesions are the same but the outcome is different. www. radbiol. ucla. edu

20 -40 Gy Seminoma, Dysgerminoma, Acute Lymphocytic leukemia, Wilms’ tumor, Neuroblastoma 40 -50 Gy

20 -40 Gy Seminoma, Dysgerminoma, Acute Lymphocytic leukemia, Wilms’ tumor, Neuroblastoma 40 -50 Gy Hodgkin's, Lymphosarcoma, Seminoma, Histiocytic cell sarcoma, Skin ca. (basal and squamous cell) 50 -60 Gy Squamous cell ca. (cervix, head and neck), Breast ca. , Ovarian ca. , Medulloblastoma, Retinoblastoma, Ewing's tumor 60 -65 Gy Larynx (<1 cm), breast cancer lumpectomy 70 -75 Gy Oral cavity (<2 cm, 2 -4 cm), Oro-naso-laryngo-pharyngeal ca. , Bladder ca. , Cervix ca. , Uterine ca. , Ovarian ca. , Lung ca. (<3 cm) >80 Gy 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. www. radbiol. ucla. edu

Robert Hooke (1635 -1703) was the first to use Not! the term ‘cell’ in

Robert Hooke (1635 -1703) was the first to use Not! the term ‘cell’ in the 1665 Micrographia Antony van Leeuwenhoek (1632 -1723) Made powerful lenses, discovered bacteria father of microbiology Rudolph Virchow (1821 -1902) - Recognized leukemia and mechanism of embolism Developed theory that cells come from cells (“omnis cellula a cellula”) Walther Flemming (1843 -1905) - identified chromatin and mitosis (Gk, thread) (“omnis nucleus a nucleo”) 1906 Bergonie and Tribandeau. Action des rayou X sur le testicle Elect. Med. 14, 779 - radiosensitivity is related to cell proliferation www. radbiol. ucla. edu

 • DSB repair, checkpoint arrest, and cell death are all part of the

• 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. www. radbiol. ucla. edu

Loss of Proliferative Ability can Occur in Different Ways Quiescence Senescence Property of stem

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

Radiation-Induced Senescence Is particularly relevant to radiation fibrosis, but also occurs in cells other

Radiation-Induced Senescence Is particularly relevant to radiation fibrosis, but also occurs in cells other than fibroblasts. TGF- Proliferative Progenitor Fibroblast p 21 Post-mitotic Fibroblast Stress-induced (Including radiation) Proliferation-induced Cancer-induced www. radbiol. ucla. edu Collagen production and fibrosis Tumor progression

Early Observations on Cell Death after Irradiation • Radiobiologists like Puck and Marcus (1956)

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 424 hrs after irradiation. www. radbiol. ucla. edu

RT Lethal Sectoring in Mitotic Death RECURRENCE! The fear of death is the most

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

Courtesy: Randi Syljuasen Control Cells Control Nuclei Stained www. radbiol. ucla. edu Irradiated Cells

Courtesy: Randi Syljuasen Control Cells Control Nuclei Stained www. radbiol. ucla. edu Irradiated Cells Irradiated Nuclei Stained

Alternative Deathstyle Mechanisms Programmed cell death type 1: Apoptosis Programmed cell death type 2:

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. www. radbiol. ucla. edu

Alternative Deathstyle Mechanisms Physiologic Type 1: Apoptosis Type 2: Autophagy • • Pathologic Type

Alternative Deathstyle Mechanisms Physiologic Type 1: Apoptosis Type 2: Autophagy • • Pathologic Type 1: Apoptosis 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. www. radbiol. ucla. edu

Physiologic Programmed Cell Death PCD is involved in: • Morphogenesis • Tissue sculpting •

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

Pathologic Programmed Cell Death • Self sacrifice by infected/damaged cells • Self sacrifice by

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 www. radbiol. ucla. edu

Programmed Cell Death Type I: Apoptosis Morphology Apoptosis is a tightly regulated “active” cell

Programmed Cell Death Type I: Apoptosis Morphology Apoptosis is a tightly regulated “active” cell death process that is associated with Ø Cell and nuclear shrinkage Ø Nuclear fragmentation with formation of apoptotic bodies Ø Blebbing of cell membrane, but no early loss of membrane integrity Ø Deletion of single cells in isolation Ø 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 www. radbiol. ucla. edu

Programmed Cell Death Type I: Apoptosis Molecular Hallmarks During apoptosis, endonucleases are induced that

Programmed Cell Death Type I: Apoptosis Molecular Hallmarks During apoptosis, endonucleases are induced that cleave between nucleosomes. On agarose gel electrophoresis, the DNA separates into fragments with sizes that are multiples of 180 -200 bp. This is called a “ladder. ” Histones H 2, H 3, H 4 Nucleosome DNA Core DNA Spacer Region (140 bp) (60 -100 bp) 55 A HISTONE H 1 110 A Sites of endonuclease cleavage www. radbiol. ucla. edu +

Detection of Apoptosis - TUNEL Assay • Apoptosis can be visualized in tissue sections

Detection of Apoptosis - TUNEL Assay • Apoptosis can be visualized in tissue sections using terminal deoxynucleotidyl transferase (Td. T) to add fluorescein-labeled (d. UTP) nucleotides onto 3’-OH ends of DNA that result from the action of the apoptotic endonuclease • An Apoptotic Index (AI) can be derived www. radbiol. ucla. edu

Apoptosis in Gut after IR • Radiation-induced apoptosis occurs in normal tissues in specific

Apoptosis in Gut after IR • Radiation-induced apoptosis occurs in normal tissues in specific sites and in cells that have a proapoptotic tendency • In gut this is in the base of the crypts Sites of apoptosis www. radbiol. ucla. edu

Programmed Cell Death Type 2: Autophagy Morphology Autophagy – A tightly regulated process –

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 www. radbiol. ucla. edu

Pathological Cell Death Type 3: Necrosis Morphology Necrosis is a rapid non-physiological process associated

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. www. radbiol. ucla. edu

Triggers for Cell Death • Type 1 - Apoptosis: – Extrinsic triggering of “death”

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 PI 3 K/Akt/m. TOR pathway is activated by growth factors allowing increased expression of transporters for glucose, amino acids, etc. Akt increases glycolysis. m. TOR drives protein translation rates. • Type 3 - Necrosis: – Extrinsic activation of immune cells leads to release of cytotoxins perforins, etc. that cause necrosis www. radbiol. ucla. edu

What Deathstyles are Associated with Radiation-Induced Death? Any of them • Mitotic death after

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 www. radbiol. ucla. edu

How do cells commit suicide? • Apoptosis is Mediated by Caspases - “Roads to

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 www. radbiol. ucla. edu

Executioner Caspases • Executioner caspases cleave >40 substrates (including each other) leading to the

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

Radiation-Induced Apoptosis INITIATORS DNA Damage Sphingomyelin Ceramide JNK ATM P 38 MAPK EFFECTORS FADD

Radiation-Induced Apoptosis INITIATORS DNA Damage Sphingomyelin Ceramide JNK ATM P 38 MAPK EFFECTORS FADD x Members of TNFR family with Death Domains (TNFR 1, Fas, TRAIL) Activation of Pro-caspase 8 p 53 Bax Mitochondria JNK - jun kinase Cytochrome c Pro-caspase 9 Caspase 9 Apaf-1 Caspase 8 FADD - Fas activated death domain Apoptosome Complex TERMINAL PHASE Caspase 3, 6, 7 www. radbiol. ucla. edu ATM - mutated in ataxia telangiectasia Apaf - apoptosis activating factor

 • The decision to commit apoptosis is determined by an internal “rheostat” within

• The decision to commit apoptosis is determined by an internal “rheostat” within the cell i. e. cells have a pro-apoptotic or antiapoptotic 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 www. radbiol. ucla. edu

Why don’t all cells die by apoptosis after RTx? • Mitochondrial Control: Members of

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 (PI 3 K) – 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 www. radbiol. ucla. edu

Control Over Radiation-Induced Apoptosis INITIATORS DNA Damage Stress Sphingomyelin Ceramide JNK ATM P 38

Control Over Radiation-Induced Apoptosis INITIATORS DNA Damage Stress Sphingomyelin Ceramide JNK ATM P 38 MAPK x Members of TNFR family With Death Domains FADD NF- B EFFECTORS p 53 Bax Bcl-2/Bcl-xl Cytochrome c Mitochondria Caspase 9 IAPs Caspase 8 Apaf-1 Apoptosome Complex TERMINAL PHASE IAP - inhibitors of apoptosis FLIP - FLICE (procaspase Caspase 3, 6, 7 8) inhibitory protein www. radbiol. ucla. edu

“Survival Pathways” Growth Factors, Cytokines, Proliferative Signals TNFR 2 TNFR 1 Sphingomyelin Ceramide PI

“Survival Pathways” Growth Factors, Cytokines, Proliferative Signals TNFR 2 TNFR 1 Sphingomyelin Ceramide PI 3 -kinase Ras Raf Proliferation PDK 1 Metabolic Pathway NF B Inhibitors of Apoptosis (IAPs) AKT m. TOR P 90 RSK Bad Bcl-2/Bcl-XL caspases Survival www. radbiol. ucla. edu ERK Context is everything “Location, location”

Clinical Significance of Cell Death • Intrinsic cellular radiosensitivity is determined in part by

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 www. radbiol. ucla. edu

 • 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 survival • Survival pathways are appropriate targets for tumor radiosensitization • EGFR • Iressa, Tarceva, C 225, Farnesyl Transferase Inhibitors • NF- B • COX-2 inhibitors • Survival pathways form appropriate targets for normal tissue radioprotection • Keratinocyte growth factor (KGF) in bone marrow transplant patients www. radbiol. ucla. edu

 • Volume 354: 567 -578 February 9, 2006 • Radiotherapy plus Cetuximab for

• Volume 354: 567 -578 February 9, 2006 • Radiotherapy plus Cetuximab for Squamous-Cell Carcinoma of the Head and Neck • James A. Bonner, M. D. , Paul M. Harari, M. D. , Jordi Giralt, M. D. , Nozar Azarnia, Ph. D. , Dong M. Shin, M. D. , Roger B. Cohen, M. D. , Christopher U. Jones, M. D. , Ranjan Sur, M. D. , Ph. D. , David Raben, M. D. , Jacek Jassem, M. D. , Ph. D. , Roger Ove, M. D. , Ph. D. , Merrill S. Kies, M. D. , Jose Baselga, M. D. , Hagop Youssoufian, M. D. , Nadia Amellal, M. D. , Eric K. Rowinsky, M. D. , and K. Kian Ang, M. D. , Ph. D. • The median duration of locoregional control was 24. 4 months among patients treated with cetuximab plus radiotherapy and 14. 9 months among those given radiotherapy alone …. . • the median duration of overall survival was 49. 0 months among patients treated with combined therapy and 29. 3 months among those treated with radiotherapy alone …. . • Radiotherapy plus cetuximab significantly prolonged progression-free survival … With the exception of acneiform rash and infusion reactions, the incidence of grade 3 or greater toxic effects, including mucositis, did not differ significantly between the two groups. www. radbiol. ucla. edu

Cell Proliferation and Cell Death: Two Sides of the Same Coin? www. radbiol. ucla.

Cell Proliferation and Cell Death: Two Sides of the Same Coin? www. radbiol. ucla. edu

Timeframe of Cellular Life The Cell Cycle • Under the microscope, Flemming identified cells

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 32 P for DNA synthesis (S phase) and there is a time gap (G 2) before the beginning of cell division (M) and there is another gap (G 1) 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 G 2), 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 www. radbiol. ucla. edu

Labeling Index Mitotic Index Flash label with 3 H-Td. R or Bd. UR for

Labeling Index Mitotic Index Flash label with 3 H-Td. R or Bd. UR for 20 mins Fix and stain mitosis Mitotic Index (M. I. ) = TM/TC If 3 H-Td. R labeled If Bd. UR labeled AR film *Anti-Bd. UR ØU. V. microscopy Autoradiography … …. . . … … … …… …… …. . . . Labeling Index (L. I. ) = TS/TC www. radbiol. ucla. edu …. . . . … … …. . Where is a correction factor for cell division, about 0. 69

Frequency of Labeled Mitosis Technique (FLM) • By counting the number of mitoses that

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 estimated • But, it is easier to use BUd. R and flow cytometry www. radbiol. ucla. edu

From FLM to FACS PM tubes Label cells with dye and use a laser

From FLM to FACS PM tubes 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 www. radbiol. ucla. edu

Flow Cytometry for DNA Quantity 1. label DNA with propidium iodide (fluorescent dye) 2.

Flow Cytometry for DNA Quantity 1. label DNA with propidium iodide (fluorescent dye) 2. measure light output by flow cytometry 3. analyze DNA histograms 4 n 4 n G 2 G 1 M S 2 n + n www. radbiol. ucla. edu # cells G 1 G 2 M S 2 n 2 n 4 n degree of fluorescence

Cell Cycle Kinetic Analysis by Flow Cytometry P. I. (DNA - red) combined with

Cell Cycle Kinetic Analysis by Flow Cytometry P. I. (DNA - red) combined with Bromodeoxyuridine uptake followed by staining with fluorescently labeled anti-Brd. Urd (green) Brd. Urd green G 1 s G 1 Brd. Urd green s s G 2/M DNA red G 2/M DNA P. I. red Time www. radbiol. ucla. edu DNA P. I red G 2/M

Cell Cycle G 2 phase 1 -2 hrs M phase 0. 5 -1 hr

Cell Cycle G 2 phase 1 -2 hrs M phase 0. 5 -1 hr G 0 quiescent S phase DNA synthesis 6 -8 hrs G 1 phase variable length If all cells in a population are dividing Mitotic Index (M. I. ) = l. Tm / Tc Labeling Index (L. I. ) = l. Ts /Tc Where l is a correction for uneven cell numbers due to mitosis (0. 69) www. radbiol. ucla. edu

Cell Cycle Synchronisation The best estimates of kinetics come from use of cells synchronized

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 - G 1 phase cells • Hydroxyurea synchronizes cells at the G 1/S transition www. radbiol. ucla. edu

Cell Cycle and Radiosensitivity 1 S. F. LATE S . 1 EARLY S G

Cell Cycle and Radiosensitivity 1 S. F. LATE S . 1 EARLY S G 1 PHASE G 2/M PHASE . 01 0 4 8 12 Dose (Gy) 16 20 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. Increasing radioresistance G 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. S www. radbiol. ucla. edu G 2 M

Cell Cycle Arrest • Cells have “checkpoints” where they “proof-read” DNA for damage before

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 • G 1 - especially if they have wt p 53. This may lead to apoptosis • Intra S phase - initiation and elongation stages of DNA replication are affected by p 53 independent mechanisms • G 2 - 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 www. radbiol. ucla. edu

Cell Cycle Arrest DNA Damage Dependent Checkpoints • Irradiated (7 Gy) • P. I

Cell Cycle Arrest DNA Damage Dependent Checkpoints • Irradiated (7 Gy) • P. I stain at 9 hr wild-type irradiated Decrease in S Increase in G 2 M i. e. G 1 and G 2 M arrest P 53 or ATM deficient irradiated loss of G 1/S checkpoint and only G 2 M arrest www. radbiol. ucla. edu

What Drives Cell Cycle Progression? Growth factors are required for G 0 through G

What Drives Cell Cycle Progression? Growth factors are required for G 0 through G 1 to S (and cell survival) • • • To activate resting cells to enter G 1 To allow cells to pass through G 1 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, G 2, M www. radbiol. ucla. edu

Molecular Mechanism of Cell Cycle Progression through each checkpoint requires: • Retinoblastoma (Rb) tumor

Molecular Mechanism of Cell Cycle Progression through each checkpoint requires: • Retinoblastoma (Rb) tumor suppressor gene family • especially G 1 -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 G 1. 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 www. radbiol. ucla. edu

Retinoblastoma Protein p. Rb • Cyclin D/cdk 4/6 and cyclin E/cdk 2 phosphorylate Rb,

Retinoblastoma Protein p. Rb • Cyclin D/cdk 4/6 and cyclin E/cdk 2 phosphorylate Rb, which is essential for cell cycle progression into S • Phosphorylation of Rb releases E 2 F, which it normally is bound to. E 2 F is a transcription factor for 20 -30 genes that are required for S phase gene expression. • p. RB mutation often leads to cancer. www. radbiol. ucla. edu

Cyclins • Have no intrinsic enzymatic activity • Cyclins A to J have been

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

Cyclin Dependent Kinases • Cyclins bind activate Cdks, which – Are serine/threonine kinases with

Cyclin Dependent Kinases • Cyclins bind activate Cdks, which – Are serine/threonine kinases with multiple substrates • e. g. p. Rb, p 53, E 2 F, etc. that they activate/inactivate – Have regulatory domains Inhibitory phosphate • E. g. inhibitory and activating phosphates – Are present throughout cell cycle – To move cells from G 0 to G 1 to S • Cyclin D activates cdks 4/6 and • Cyclin E activates cdk 2 P Cyclin kinase site P cdk activating phosphate www. radbiol. ucla. edu

Activating Phosphatases CDC 25 Removes Phosphate from Tyr-15 – CDC 25 A = cyclin

Activating Phosphatases CDC 25 Removes Phosphate from Tyr-15 – CDC 25 A = cyclin E/CDK 2 = G 1/S specific – CDC 25 B = cyclin A/CDK 2 = S-phase exit – CDC 25 C = cyclin B/CDK 1 = G 2/M specific www. radbiol. ucla. edu

cdk 1 phosphorylates substrates leads to • Nuclar envelope breakdown • Chromosome separation Cyclin

cdk 1 phosphorylates substrates leads to • Nuclar envelope breakdown • Chromosome separation Cyclin B • Spindle assembly • Chromosome condensation CDK 1 Cyclin A CDK 1/2 Cyclosome (APC) p. Rb dephosphorylation G 0 quiescent Cyclin D CDK 4/6 Cyclin A CDK 1/2 Early - mid G 1 Cyclin E CDK 2 Responsible for p. Rb phosphorylation www. radbiol. ucla. edu Cyclin D CDK 4/6 Responsible for p. Rb phosphorylation

Cyclin Kinase Inhibitors (CKIs) belong to 2 families • INK 4 and KIP/CIP Generally

Cyclin Kinase Inhibitors (CKIs) belong to 2 families • INK 4 and KIP/CIP Generally compete with cyclins for CDKs Phase G 1 Complexes cyclin D-CDK 4, 6 G 1/S S G 2/M cyclin E-CDK 2, 3 cyclin A-CDK 2 cyclin B-CDK 1 Inhibitors p 16 (INK 4 a), p 19 ARF (INK 4 a) p 15 (INK 4 b) p 21 CIP 1, p 27 KIP 1 p 21, p 57 p 21 p 53 is a transcription factor for p 21, which is why it is involved in cell cycle arrest after IR www. radbiol. ucla. edu

DSB SSB/Base damage Replication stress, UV, MMC, hypoxia MRN complex HEJ sensors N mediators

DSB SSB/Base damage Replication stress, UV, MMC, hypoxia MRN complex HEJ sensors N mediators Stalled Replication Fork ATM H 2 AX 53 BP 1 MDC 1 MRN BRCA 1 slow HR 53 BP 1 MDC 1 MRN BRCA 1 rapid p 53 CHK 2 ATR ATM ATR MDM 2 transducers DSB Resection CHK 1 53 BP 1 MDC 1 MRN BRCA 1 CHK 2 CHK 1 transactivation effectors p 21 CDC 25 A phosphorylation CDC 25 C phosphorylation and nuclear export CDC 25 A degradation P-thr 14/tyr 15 p 21 CDK 2 CYCLIN E P-thr 14/tyr 15 P CDK 2 CDK! CYCLIN E CYCLIN A/E CYCLIN B G 1/S Arrest Sensescence/transient www. radbiol. ucla. edu S Phase Arrest G 2/M Arrest

Cell Cycle in Cancer • If p 53 or any other molecule governing cell

Cell Cycle in Cancer • If p 53 or any other molecule governing cell cycle arrest is mutated, genetic instability results as well as more rapid cell cycle progression. • Cyclins, 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 p 16 deletion or silencing and/or p 53 mutation in Head and Neck Ca www. radbiol. ucla. edu

Cancer Growth Factor/Cytokine Survival Receptor Oncogenes Raf MAPK Proliferation www. radbiol. ucla. edu Signals

Cancer Growth Factor/Cytokine Survival Receptor Oncogenes Raf MAPK Proliferation www. radbiol. ucla. edu Signals PI 3 K NF- B Cell death

DNA repair Initial damage ROS Cell cycle arrest Cell death /survival DNA damage response

DNA repair Initial damage ROS Cell cycle arrest Cell death /survival DNA damage response ATM, ATR, MRN P 53, Chk 1, Chk 2 P 21, Bax, caspase 8, etc. Immediate early gene response JNK P 38 MAPK NF-k. B AU-rich control: TNF- , IL 1 , IL-2, IL-3, GMCSF, IL-6, IL-8, IL-12, IFN / , VEGF, PDGFB, Inflammatory Cytokines and Growth Factors Cell proliferation NGF, IGFR, DR 5, COX-2 Proteasome inhibition Mitochondrial damage Activation of EGFR, TGF- , etc Tissue recovery /lesion formation www. radbiol. ucla. edu Cell proliferation Cell death /survival

Loss of Proliferative Ability can Occur in Different Ways Quiescence Senescence Property of stem

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

Tissue Kinetics in tumors or normal tissues depend upon • Cell cycle • Growth

Tissue 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 = nonproliferating 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 = l. 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. 0 www. radbiol. ucla. edu

Tumor Kinetics Human SCC Tc Cell cycle time 36 hrs G. F. Growth fraction

Tumor Kinetics Human SCC Tc Cell cycle time 36 hrs G. F. Growth fraction 0. 25 Tpot Pot. doubling time 6 days Actual doubling time 60 days Td Cell loss factor 0. 9 (36 hr 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 www. radbiol. ucla. edu

Tumor Regression • The rate of tumor growth and regression is determined by •

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 www. radbiol. ucla. edu

Tumor Regeneration Relative tumor volume X-rays Control Irradiated Growth delay Surviving clonogens measured in

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

EVIDENCE FOR ACCELERATED REPOPULATION IN TUMORS • Time to tumor recurrence after therapy is

EVIDENCE FOR ACCELERATED REPOPULATION IN TUMORS • Time to tumor recurrence after therapy is shorter 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. www. radbiol. ucla. edu

Accelerated Tumor Repopulation local control T 2 T 3 no local control T 2

Accelerated Tumor Repopulation local control T 2 T 3 no local control T 2 and T 3 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 www. radbiol. ucla. edu

Accelerated Tumor Repopulation Onset may be about day 21. Repopulation may not be constant

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. www. radbiol. ucla. edu

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

Accelerated repopulation in human tumors provided the rationale for accelerated fractionation protocols www. radbiol. ucla. edu