Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill McBride Dept. Radiation...

www.rwww.radbioladbiol.ucla.edu.ucla.edu

Radiation Targets 2: Radiation Targets 2: Cell Proliferation, Cell Death and Cell Proliferation, Cell Death and

SurvivalSurvival

Bill McBrideBill McBrideDept. Radiation OncologyDept. Radiation Oncology

David Geffen School MedicineDavid Geffen School MedicineUCLA, Los Angeles, Ca.UCLA, Los Angeles, Ca.

[email protected]@mednet.ucla.edu


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

reproductive colongenic cells and affect the outcome of RTreproductive colongenic cells and affect the outcome of RT• Be able to distinguish between interphase and mitotic (catastrophic) cell Be able to distinguish between interphase and mitotic (catastrophic) cell

death following irradiationdeath following irradiation• Understand the physiologic, morphologic, and mechanistic differences Understand the physiologic, morphologic, and mechanistic differences

between apoptosis, autophagy, and necrosis as deathstyles and how cells between apoptosis, autophagy, and necrosis as deathstyles and how cells die in response to irradiationdie in response to irradiation

• Understand how survival pathways operate to affect cellular Understand how survival pathways operate to affect cellular radiosensitivity and how these can be targeted for radiotherapeutic benefit.radiosensitivity and how these can be targeted for radiotherapeutic benefit.

• Know the molecular basis for cell cycle arrest following IR and its Know the molecular basis for cell cycle arrest following IR and its importance in repair and carcinogenesisimportance in repair and carcinogenesis

• Understand the importance of cell cycle kinetics, cell loss factors in tumor Understand the importance of cell cycle kinetics, cell loss factors in tumor growth and regressiongrowth and regression

• Recognize the importance of changes in these parameters during the Recognize the importance of changes in these parameters during the course of a fractionated RT regimencourse of a fractionated RT regimen


Intrinsic RadiosensitivityIntrinsic Radiosensitivity

The outcome of radiation exposure depends onThe outcome of radiation exposure depends on • The DNA lesions that are caused and their The DNA lesions that are caused and their

persistencepersistence• How cells and tissues How cells and tissues ‘‘sensesense’’ danger and danger and

respond by activating cell survival or death respond by activating cell survival or death pathways pathways


FRACTION OF CELLS SURVIVING 2 GY IN VITROFRACTION OF CELLS SURVIVING 2 GY IN VITRO

LYMPHOMALYMPHOMANEUROBLASTOMANEUROBLASTOMAMYELOMAMYELOMASMALL CELL LUNG CANCERSMALL CELL LUNG CANCERMEDULLOBLASTOMAMEDULLOBLASTOMA

BREAST CABREAST CASCCSCCPANCREATIC CAPANCREATIC CACOLORECTAL CACOLORECTAL CANON-SMALL CELL CANON-SMALL CELL CA

MELANOMAMELANOMAOSTEOSARCOMAOSTEOSARCOMAGLIOBLASTOMAGLIOBLASTOMAHYPERNEPHROMAHYPERNEPHROMA

0.2 (0.08 - 0.37)0.2 (0.08 - 0.37)

0.43 (0.14 - 0.75)0.43 (0.14 - 0.75)

0.52 (0.2 - 0.86)0.52 (0.2 - 0.86)

Tumor cells vary dramatically in intrinsic radiosensitivity Tumor cells vary dramatically in intrinsic radiosensitivity depending on their tissue of origin. The number of DNA depending on their tissue of origin. The number of DNA

lesions are the same but the outcome is different.lesions are the same but the outcome is different.


20-40Gy20-40Gy Seminoma, Dysgerminoma, Acute Lymphocytic leukemia, Seminoma, Dysgerminoma, Acute Lymphocytic leukemia, WilmsWilms’’ tumor, Neuroblastoma tumor, Neuroblastoma

40-50Gy40-50Gy Hodgkin's, Lymphosarcoma, Seminoma, Histiocytic cell Hodgkin's, Lymphosarcoma, Seminoma, Histiocytic cell sarcoma, Skin ca. (basal and squamous cell)sarcoma, Skin ca. (basal and squamous cell)

50-60Gy50-60Gy Squamous cell ca. (cervix, head and neck), Breast ca., Ovarian Squamous cell ca. (cervix, head and neck), Breast ca., Ovarian ca.,Medulloblastoma, Retinoblastoma, Ewing's tumorca.,Medulloblastoma, Retinoblastoma, Ewing's tumor

60-65Gy60-65Gy Larynx (<1 cm), breast cancer lumpectomy Larynx (<1 cm), breast cancer lumpectomy70-75Gy70-75Gy Oral cavity (<2 cm, 2-4 cm), Oro-naso-laryngo-pharyngeal ca., Oral cavity (<2 cm, 2-4 cm), Oro-naso-laryngo-pharyngeal ca.,

Bladder ca., Cervix ca., Uterine ca., Ovarian ca., Lung ca. (<3 Bladder ca., Cervix ca., Uterine ca., Ovarian ca., Lung ca. (<3 cm)cm)

>80Gy>80Gy Head and neck ca. (~4 cm), Breast ca. (~5 cm), Glioblastomas, Head and neck ca. (~4 cm), Breast ca. (~5 cm), Glioblastomas, Osteogenic sarcomas (bone sarcomas), Melanomas, Soft tissue Osteogenic sarcomas (bone sarcomas), Melanomas, Soft tissue sarcomas (~5 cm), Thyroid Ca.sarcomas (~5 cm), Thyroid Ca.

(In Rubin P, et al, eds: Clinical Oncology: A Multidisciplinary Approach, (In Rubin P, et al, eds: Clinical Oncology: A Multidisciplinary Approach, edition 7, p 72. Saunders, 1993)edition 7, p 72. Saunders, 1993)

Clinically, tumors show the same histological correlation Clinically, tumors show the same histological correlation with respect to sensitivity to RT.with respect to sensitivity to RT.


Not!Not!Robert Hooke (1635-1703) was the first to use Robert Hooke (1635-1703) was the first to use the term the term ‘‘cellcell’’ in the 1665 in the 1665 MicrographiaMicrographia

Antony van Leeuwenhoek (1632-1723) - Antony van Leeuwenhoek (1632-1723) - Made powerful lenses, discovered bacteria - Made powerful lenses, discovered bacteria - father of microbiologyfather of microbiology

Rudolph Virchow (1821-1902) - Recognized Rudolph Virchow (1821-1902) - Recognized leukemia and mechanism of embolism - leukemia and mechanism of embolism - Developed theory that cells come from cells Developed theory that cells come from cells

((““omnis cellula a cellulaomnis cellula a cellula””))

Walther Flemming (1843-1905) - identified Walther Flemming (1843-1905) - identified chromatin and mitosis (Gk, thread)chromatin and mitosis (Gk, thread)

((““omnis nucleus a nucleoomnis nucleus a nucleo””))

19061906 Bergonie and Tribandeau. Action des Bergonie and Tribandeau. Action des rayou X sur le testicle rayou X sur le testicle Elect. Med.14, 779- radiosensitivity is related to cell proliferation- 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 The relationship of repair, cell proliferation and cell

death following IR has been the subject of many death following IR has been the subject of many studies, primarily because, clinically, loss of studies, primarily because, clinically, loss of reproductive, clonogenic cellsreproductive, clonogenic cells following RT following RT determines the outcome of cancer treatment.determines the outcome of cancer treatment.


(all with distinct, and common, gene patterns)(all with distinct, and common, gene patterns)

IR is a pathological signal and can cause senescence

Loss of Proliferative Ability can Loss of Proliferative Ability can Occur in Different WaysOccur in Different Ways

Quiescence Senescence Quiescence Senescence Terminal Terminal Death DeathDifferentiationDifferentiation

Property of stem cellsProperty of stem cellsReversible, physiological Reversible, physiological processprocessApoptosis and Apoptosis and differentiation is inhibiteddifferentiation is inhibitedHigh free radical scavenger High free radical scavenger levelslevels

Irreversible, Irreversible, physiologicalphysiologicalactive processactive processCell cycle inhibition is a Cell cycle inhibition is a secondary effectsecondary effect

Irreversible,Irreversible,non-physiological non-physiological processprocess

ApoptosisApoptosisAutophagyAutophagyNecrosisNecrosis


Stress-induced Stress-induced (Including radiation)(Including radiation)

Proliferation-inducedProliferation-inducedCancer-inducedCancer-induced

Proliferative Proliferative ProgenitorProgenitorFibroblastFibroblast

Post-mitoticPost-mitoticFibroblastFibroblast

TGF-TGF-

Radiation-Induced Radiation-Induced SenescenceSenescence

p21p21

Collagen production and fibrosisCollagen production and fibrosisTumor progressionTumor progression

Is particularly relevant to radiation fibrosis, but also Is particularly relevant to radiation fibrosis, but also occurs in cells other than fibroblasts.occurs in cells other than fibroblasts.


Early Observations on Cell Early Observations on Cell Death after IrradiationDeath after Irradiation

• Radiobiologists like Puck and Marcus (1956) showed that Radiobiologists like Puck and Marcus (1956) showed that mostmost reproductive cells die a reproductive cells die a mitotic deathmitotic death, also known as , also known as mitotic mitotic catastrophecatastrophe, after IR., after IR.– It may take several cell divisions, the number depending on the It may take several cell divisions, the number depending on the

radiation dose.radiation dose.– After 2 Gy, it may average 2-3 cell divisions before deathAfter 2 Gy, it may average 2-3 cell divisions before death– This may take several days (as opposed to hours)This may take several days (as opposed to hours)– It is due toIt is due to

• Chromosome loss Chromosome loss • Failure of spindle formation during cytokinesisFailure of spindle formation during cytokinesis

• Early radiobiologists also discovered that a few cells of specific Early radiobiologists also discovered that a few cells of specific types die by types die by interphase deathinterphase death (without dividing) (without dividing)– This is generally more rapid than mitotic death, occurring 4-This is generally more rapid than mitotic death, occurring 4-

24hrs after irradiation.24hrs after irradiation.


RECURRENCE!RECURRENCE!

RTRTLethal Sectoring in Mitotic DeathLethal Sectoring in Mitotic Death

The fear of death is the most unjustified of The fear of death is the most unjustified of all fears, for there's no risk of an accident all fears, for there's no risk of an accident for someone who's deadfor someone who's dead. Albert Einstein. Albert Einstein


ControlCells

Control-

Nuclei Stained

IrradiatedCells

Irradiated-

Nuclei Stained

Courtesy: Randi Syljuasen


Alternative Deathstyle Alternative Deathstyle MechanismsMechanisms

Programmed cell death type 1: ApoptosisProgrammed cell death type 1: ApoptosisProgrammed cell death type 2: AutophagyProgrammed cell death type 2: Autophagy

Pathological Death: Necrosis Pathological Death: Necrosis

• Death is often an Death is often an activeactive process: cells process: cells decidedecide to commit suicide to commit suicide• Death pathways prevent carcinogenesis and mutations in them are Death pathways prevent carcinogenesis and mutations in them are

associated with cancer. They provide potential tumor-specific associated with cancer. They provide potential tumor-specific targets for therapeutic intervention.targets for therapeutic intervention.

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


Alternative Deathstyle Alternative Deathstyle MechanismsMechanisms

Physiologic PathologicPhysiologic Pathologic

Type 1: Apoptosis Type 1: Apoptosis Type 1: ApoptosisType 1: ApoptosisType 2: Autophagy Type 2: Autophagy Type 2: AutophagyType 2: Autophagy

Type 3: Necrosis Type 3: Necrosis

• Type 1 and 2 are ProgrammedType 1 and 2 are Programmed• Death is largely an Death is largely an activeactive process: cells process: cells decidedecide to commit suicide to commit suicide• Death pathways prevent carcinogenesis and mutations in Death pathways prevent carcinogenesis and mutations in

molecules in these pathways are associated with cancer. molecules in these pathways are associated with cancer. They They provide potential tumor-specific targets for therapeutic intervention.provide potential tumor-specific targets for therapeutic intervention.

• The same death pathways and mutations affect intrinsic cellular The same death pathways and mutations affect intrinsic cellular radiosensitivity. radiosensitivity. They provide potential tumor-specific targets for They provide potential tumor-specific targets for radiosensitization.radiosensitization.


Physiologic Programmed Cell DeathPhysiologic Programmed Cell Death

Sex differentiationSex differentiation

PCD is involved in:PCD is involved in:• MorphogenesisMorphogenesis• Tissue sculptingTissue sculpting• Homeostatic control of Homeostatic control of

cell numberscell numbers• Preventing Preventing

autoimmunityautoimmunity• PCD is PCD is

immunologically immunologically ““silentsilent””

““It is a myth to think death is just for It is a myth to think death is just for the old. Death is there from the very the old. Death is there from the very beginningbeginning”” Herman Feifel Herman Feifel

Self-reactiveSelf-reactivelymphocyteslymphocytes

IrradiationIrradiation

FingersFingers GutGut

Tadpole TailsTadpole Tails

proliferating cells

This may be why This may be why proliferation often proliferation often correlates with correlates with apoptotic indexapoptotic index

CELL 88:350, 1997CELL 88:350, 1997


Pathologic Programmed Cell Death Pathologic Programmed Cell Death

• Self sacrifice by infected/damaged cellsSelf sacrifice by infected/damaged cells• Self sacrifice by immune cells and other normal Self sacrifice by immune cells and other normal

cells in the battle zonecells in the battle zone• Causes inflammationCauses inflammation

– wound healingwound healing– immunityimmunity


The word comes from The word comes from - from and - from and - falling. - falling.

““Like leaves on trees the race of man is found, now green in youth, now withering Like leaves on trees the race of man is found, now green in youth, now withering on the groundon the ground”” The Iliad of Homer. Book vi. Line 181The Iliad of Homer. Book vi. Line 181

Programmed Cell Death Type I: Apoptosis Programmed Cell Death Type I: Apoptosis MorphologyMorphology

ApoptosisApoptosis is a tightly regulated is a tightly regulated ““activeactive”” cell cell death process that is associated withdeath process that is associated with Cell and nuclear shrinkageCell and nuclear shrinkage Nuclear fragmentation with formation of Nuclear fragmentation with formation of apoptotic bodiesapoptotic bodies Blebbing of cell membrane, but no early loss of Blebbing of cell membrane, but no early loss of membrane integritymembrane integrity Deletion of single cells in isolation Deletion of single cells in isolation Lack of an inflammatory response and Lack of an inflammatory response and phagocytosis by local cells (a silent death!)phagocytosis by local cells (a silent death!)


Programmed Cell Death Type I: Apoptosis Programmed Cell Death Type I: Apoptosis Molecular HallmarksMolecular Hallmarks

Histones H2,H3,H4Histones H2,H3,H4

DNA Spacer RegionDNA Spacer Region(60-100 bp)(60-100 bp)

Nucleosome DNA CoreNucleosome DNA Core(140 bp)(140 bp)

110 A110 A

55 A55 A

Sites of endonuclease cleavageSites of endonuclease cleavage

HISTONE H1HISTONE H1

--

++

During apoptosis, During apoptosis, endonucleasesendonucleases are induced that cleave are induced that cleave between between nucleosomesnucleosomes. .

On agarose gel electrophoresis, the DNA separates into On agarose gel electrophoresis, the DNA separates into fragments with sizes that are multiples of fragments with sizes that are multiples of 180-200 bp180-200 bp. This is . This is called a called a ““ladderladder..””


Detection of Apoptosis Detection of Apoptosis - - TUNELTUNEL Assay Assay

• Apoptosis can be visualized in tissue sections Apoptosis can be visualized in tissue sections using terminal deoxynucleotidyl transferase using terminal deoxynucleotidyl transferase (TdT) to add fluorescein-labeled (dUTP) (TdT) to add fluorescein-labeled (dUTP) nucleotides onto 3nucleotides onto 3’’-OH ends of DNA that result -OH ends of DNA that result from the action of the apoptotic endonuclease from the action of the apoptotic endonuclease

• An An Apoptotic IndexApoptotic Index (AI) can be derived (AI) can be derived


Apoptosis in Gut after IRApoptosis in Gut after IR

Sites of Sites of apoptosisapoptosis

• Radiation-induced Radiation-induced apoptosis occurs apoptosis occurs in normal tissues in normal tissues in specific sites in specific sites and in cells that and in cells that have a pro-have a pro-apoptotic apoptotic tendencytendency

• In gut this is in the In gut this is in the base of the cryptsbase of the crypts


Programmed Cell Death Type 2: Autophagy Programmed Cell Death Type 2: Autophagy MorphologyMorphology

AutophagyAutophagy– A tightly regulated processA tightly regulated process– A response to nutrient and growth factor A response to nutrient and growth factor

deprivation, but is also seen in physiologic deprivation, but is also seen in physiologic processes, eg morphogenesis.processes, eg morphogenesis.

– Organelles and other cell components are Organelles and other cell components are sequestered in autophagosomes that fuse sequestered in autophagosomes that fuse with lysosomes (self-digestion)with lysosomes (self-digestion)

– Increased endocytosis, vacuolation, Increased endocytosis, vacuolation, membrane blebbing, nuclear condensationmembrane blebbing, nuclear condensation

– In essence it is a defensive reaction that In essence it is a defensive reaction that eventually can lead to cell deatheventually can lead to cell death


Pathological Cell Death Type 3: NecrosisPathological Cell Death Type 3: NecrosisMorphologyMorphology

NecrosisNecrosis is a rapid non-physiological is a rapid non-physiological process associated withprocess associated with

• Loss of plasma membrane integrity and Loss of plasma membrane integrity and deregulated ion homeostasis.deregulated ion homeostasis.

• Swelling and bursting of cells as water entersSwelling and bursting of cells as water enters• Groups of cells, rather than single cells, are Groups of cells, rather than single cells, are affected.affected.

• DNA forms a random DNA forms a random ““smearsmear”” on agarose gel. on agarose gel. There is no pattern to its fragmentation.There is no pattern to its fragmentation.

• Associated with inflammation.Associated with inflammation.


Triggers for Cell DeathTriggers for Cell Death• Type 1 - Apoptosis:Type 1 - Apoptosis:

– Extrinsic triggering of Extrinsic triggering of ““deathdeath”” receptors (some TNFR family receptors (some TNFR family members)members)

– Intrinsic DNA damage response pathwayIntrinsic DNA damage response pathway– Alterations in mitochondria membrane permeabilityAlterations in mitochondria membrane permeability

• Type 2 - Autophagy:Type 2 - Autophagy:• Removal of growth/survival factor signaling. Removal of growth/survival factor signaling. Often called Often called ““death by death by

neglectneglect..”” Cells have to receive the appropriate stimuli from their Cells have to receive the appropriate stimuli from their environment to survive, if not they die often by autophagy. environment to survive, if not they die often by autophagy. Death is the Death is the default pathway of life!default pathway of life! Cells in the wrong microenvironment die of Cells in the wrong microenvironment die of ““homelessnesshomelessness”” (anoikis), a form of death by neglect. (anoikis), a form of death by neglect.• The PI3K/Akt/mTOR pathway is activated by growth factors allowing The PI3K/Akt/mTOR pathway is activated by growth factors allowing

increased expression of transporters for glucose, amino acids, etc. Akt increased expression of transporters for glucose, amino acids, etc. Akt increases glycolysis. mTOR drives protein translation rates.increases glycolysis. mTOR drives protein translation rates.

• Type 3 - Necrosis:Type 3 - Necrosis:– Extrinsic activation of immune cells leads to release of cytotoxins Extrinsic activation of immune cells leads to release of cytotoxins

- perforins, etc. that cause necrosis- perforins, etc. that cause necrosis


What Deathstyles are Associated What Deathstyles are Associated with Radiation-Induced Death?with Radiation-Induced Death?

Any of themAny of them• Mitotic death Mitotic death after irradiation can be by any after irradiation can be by any molecularmolecular

mechanismmechanism• Interphase deathInterphase death after irradiation is by rapid after irradiation is by rapid apoptosisapoptosis

– Prominent in lymphocytes, spermatogonia, oligodendrocytes, Prominent in lymphocytes, spermatogonia, oligodendrocytes, salivary glandsalivary gland

– Occurs in many tumors and tissues, normally in specific sitesOccurs in many tumors and tissues, normally in specific sites• Cells that are most sensitive to radiation considered to Cells that are most sensitive to radiation considered to

have a have a pro-apoptotic phenotypepro-apoptotic phenotype


How do cells commit suicide?How do cells commit suicide?

• Apoptosis is Mediated by Caspases - Apoptosis is Mediated by Caspases - ““Roads to RuinRoads to Ruin”” • The morphological and biochemical hall-marks of The morphological and biochemical hall-marks of

apoptosis are the result of cascadic activation of members apoptosis are the result of cascadic activation of members of a family of of a family of pro-enzymepro-enzyme proteases proteases called called Caspases Caspases byby– ExtrinsicExtrinsic pathway through Tumor Necrosis Factor Receptor pathway through Tumor Necrosis Factor Receptor

(TNFR) family members, which activates (TNFR) family members, which activates caspase 8caspase 8– IntrinsicIntrinsic pathway through cytochrome c leaking from pathway through cytochrome c leaking from

mitochondria, which activates mitochondria, which activates caspasecaspase 9. 9.• Irrespective of the apoptotic death signal, all caspases Irrespective of the apoptotic death signal, all caspases

converge to activate a terminal converge to activate a terminal Caspase 3Caspase 3-dependent -dependent pathwaypathway


Executioner CaspasesExecutioner Caspases• ExecutionerExecutioner caspases cleave >40 substrates (including each other) caspases cleave >40 substrates (including each other)

leading to the morphological features of apoptosisleading to the morphological features of apoptosis• Blocking these caspases does not generally prevent radiation-induced Blocking these caspases does not generally prevent radiation-induced

cell death - by then it is too late!cell death - by then it is too late!

ICAD (inhibitor of caspase activated DNase) ICAD (inhibitor of caspase activated DNase) DNA-PK (DNA protein kinase)DNA-PK (DNA protein kinase)PARP (poly-ADP-ribose polymerase)PARP (poly-ADP-ribose polymerase)

Caspase 3Caspase 3

Caspase 7Caspase 7Caspase 6Caspase 6

Lamin ALamin A ActinActin

CellCellShrinkageShrinkage

iCAD - CADiCAD - CAD DNA-PKcs PARPDNA-PKcs PARP

DNADNARepairRepair

CADCADDNADNA

FragmentationFragmentation


SphingomyelinSphingomyelin

CeramideCeramide

Members of Members of TNFR familyTNFR familywith Death with Death DomainsDomains

(TNFR1, Fas, (TNFR1, Fas, TRAIL)TRAIL)

p53p53

ATMATM

BaxBax MitochondriaMitochondria

Cytochrome cCytochrome c

Caspase 9Caspase 9

Apoptosome ComplexApoptosome Complex

Apaf-1Apaf-1

xx

Caspase 8Caspase 8

INITIATORSINITIATORS

FADDFADD

EFFECTORSEFFECTORS

Caspase 3, 6, 7Caspase 3, 6, 7TERMINAL PHASETERMINAL PHASE

DNA DamageDNA Damage

JNKJNKP38 MAPKP38 MAPK

Pro-caspase 9Pro-caspase 9

JNK - jun kinaseJNK - jun kinase

ATM - mutated in ATM - mutated in ataxia ataxia telangiectasiatelangiectasia

FADD - Fas FADD - Fas activated death activated death domain domain

Apaf - apoptosis Apaf - apoptosis activating factoractivating factor

Activation of Activation of Pro-caspase 8Pro-caspase 8

Radiation-Induced ApoptosisRadiation-Induced Apoptosis


• The decision to commit apoptosis is determined by an internal The decision to commit apoptosis is determined by an internal ““rheostatrheostat”” within the cell i.e. cells have a within the cell i.e. cells have a pro-apoptotic or anti-pro-apoptotic or anti-apoptotic phenotypeapoptotic phenotype

• Radiation increases the AI, but does not change a cell from an Radiation increases the AI, but does not change a cell from an anti-apoptotic to pro-apoptotic phenotypeanti-apoptotic to pro-apoptotic phenotype

• Apoptotic cells reappear between radiation fractionsApoptotic cells reappear between radiation fractions

““There is only one serious philosophical problem. It is suicide. To judge There is only one serious philosophical problem. It is suicide. To judge whether life is, or is not, worth livingwhether life is, or is not, worth living”” Albert Camus Albert Camus


Why donWhy don’’t all cells die by t all cells die by apoptosis after RTx?apoptosis after RTx?

• Mitochondrial Control:Mitochondrial Control: Members of the Members of the Bcl-2 familyBcl-2 family (B cell lymphoma (B cell lymphoma oncogene) localize in the outer membrane of the mitochondria oncogene) localize in the outer membrane of the mitochondria – Bcl-2 is the prototypical inhibitor of apoptosisBcl-2 is the prototypical inhibitor of apoptosis– Bax is from the same family and activates apoptosisBax is from the same family and activates apoptosis– The balance of pro-apototic (bax) to anti-apoptotic (Bcl-2) factors The balance of pro-apototic (bax) to anti-apoptotic (Bcl-2) factors

control the control the ““leakinessleakiness”” of the membranes. of the membranes. • Survival pathways:Survival pathways: These affect intrinsic and extrinsic apoptotic and These affect intrinsic and extrinsic apoptotic and

autophagic pathways and alter the rheostat away from cell death and autophagic pathways and alter the rheostat away from cell death and towards radioresistancy - acting often through the Bcl-2 family. Major towards radioresistancy - acting often through the Bcl-2 family. Major survival pathways aresurvival pathways are– phosphoinositol kinase 3 (PI3K)phosphoinositol kinase 3 (PI3K)– nuclear factor kappa B (NF-nuclear factor kappa B (NF-B)B)

• Cancer is associated with mutations in cell death/survival pathways, as Cancer is associated with mutations in cell death/survival pathways, as is radioresistance, and these are targets for theraputic interventionis radioresistance, and these are targets for theraputic intervention



CeramideCeramide

Members of Members of TNFR familyTNFR familyWith Death With Death DomainsDomains

p53p53

ATMATM

BaxBaxBcl-2/Bcl-xlBcl-2/Bcl-xl

MitochondriaMitochondria

Cytochrome cCytochrome c

Caspase 9Caspase 9

Apoptosome ComplexApoptosome Complex

Apaf-1Apaf-1

xx

Caspase 8Caspase 8

INITIATORSINITIATORS

FADDFADD

EFFECTORSEFFECTORS

Caspase 3, 6, 7Caspase 3, 6, 7

TERMINAL PHASETERMINAL PHASE

DNA DamageDNA DamageStressStress

Control Over Radiation-Induced ApoptosisControl Over Radiation-Induced Apoptosis

NF-NF-BB

IAPsIAPs

IAP - inhibitors of apoptosisIAP - inhibitors of apoptosis

FLIP - FLICE (procaspase FLIP - FLICE (procaspase 8) inhibitory protein8) inhibitory protein



RasRasRafRaf

ERKERK

P90 RSKP90 RSK

SurvivalSurvival

PI 3-kinasePI 3-kinase

PDK1PDK1

AKTAKT

BadBad

TNFR1TNFR1

NFNFBB

TNFR2TNFR2

““Survival PathwaysSurvival Pathways””

Bcl-2/Bcl-XLBcl-2/Bcl-XL

Growth Factors, Cytokines, Proliferative SignalsGrowth Factors, Cytokines, Proliferative Signals


CeramideCeramide

Inhibitors of Apoptosis (IAPs)Inhibitors of Apoptosis (IAPs)

caspasescaspasesContext is everything - Context is everything -

““Location, location, locationLocation, location, location””

ProliferationProliferation

mTORmTOR

Metabolic Metabolic PathwayPathway


Clinical Significance of Cell DeathClinical Significance of Cell Death

• Intrinsic cellular radiosensitivity is determined in part by the balance of Intrinsic cellular radiosensitivity is determined in part by the balance of the signals transducing cell death or survival pathways the signals transducing cell death or survival pathways

• Clinical RT response is superior in tumors with pathways primed for an 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-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, 2) and local tumor control or patient survival after RT are controversial, perhaps because excessive cell death often correlates with high cell perhaps because excessive cell death often correlates with high cell proliferation or because multiple pathways to cell death are possibleproliferation or because multiple pathways to cell death are possible

• Apoptosis may affect the clinical response of normal tissues to RT e.g. Apoptosis may affect the clinical response of normal tissues to RT e.g. serous cells - serous cells - ““dry mouthdry mouth””

• In general, RT increases the A.I. only in cells with a pro-apoptotic In general, RT increases the A.I. only in cells with a pro-apoptotic phenotype and apoptotic cells reappear between fractions of RT phenotype and apoptotic cells reappear between fractions of RT

• Enhancing PCD in a proportion of cells does not Enhancing PCD in a proportion of cells does not necessarilynecessarily affect the affect the shape of the clonogenic survival curves following radiation - this shape of the clonogenic survival curves following radiation - this depends on the response of the surviving cellsdepends on the response of the surviving cells


• The pathways that govern cell death/survival also govern The pathways that govern cell death/survival also govern radioresistance and radiosensitivity!!!!!radioresistance and radiosensitivity!!!!!

• Manipulation of apoptotic pathways genetically, or with Manipulation of apoptotic pathways genetically, or with drugs, can affect clonogenic cell survival drugs, can affect clonogenic cell survival

• Survival pathways are appropriate targets for tumor Survival pathways are appropriate targets for tumor radiosensitizationradiosensitization• EGFREGFR

• Iressa, Tarceva, C225, Farnesyl Transferase InhibitorsIressa, Tarceva, C225, Farnesyl Transferase Inhibitors

• NF-NF-B B • COX-2 inhibitorsCOX-2 inhibitors

• Survival pathways form appropriate targets for normal Survival pathways form appropriate targets for normal tissue radioprotectiontissue radioprotection• Keratinocyte growth factor (KGF) in bone marrow Keratinocyte growth factor (KGF) in bone marrow

transplant patientstransplant patients


• Volume 354:567-578 Volume 354:567-578 February 9, 2006February 9, 2006• Radiotherapy plus Cetuximab for Squamous-Cell Carcinoma of the Head and NeckRadiotherapy plus Cetuximab for Squamous-Cell Carcinoma of the Head and Neck

• James A. Bonner, M.D., Paul M. Harari, M.D., Jordi Giralt, M.D., Nozar Azarnia, Ph.D., Dong M. 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 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 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. Baselga, M.D., Hagop Youssoufian, M.D., Nadia Amellal, M.D., Eric K. Rowinsky, M.D., and K.

Kian Ang, M.D., Ph.D.Kian Ang, M.D., Ph.D.

• The median duration of locoregional control was 24.4 months among patients The median duration of locoregional control was 24.4 months among patients treated with cetuximab plus radiotherapy and 14.9 months among those given treated with cetuximab plus radiotherapy and 14.9 months among those given radiotherapy alone ….. radiotherapy alone …..

• the median duration of overall survival was 49.0 months among patients treated 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 with combined therapy and 29.3 months among those treated with radiotherapy alone ….. alone …..

• Radiotherapy plus cetuximab significantly prolonged progression-free survival Radiotherapy plus cetuximab significantly prolonged progression-free survival … With the exception of acneiform rash and infusion reactions, the incidence of … With the exception of acneiform rash and infusion reactions, the incidence of grade 3 or greater toxic effects, including mucositis, did not differ significantly grade 3 or greater toxic effects, including mucositis, did not differ significantly between the two groups.between the two groups.


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


Timeframe of Cellular LifeTimeframe of Cellular Life The Cell CycleThe Cell Cycle

• Under the microscope, Flemming identified cells in mitosis (M) and in Under the microscope, Flemming identified cells in mitosis (M) and in interphase - interphase - i.e 2 cell cycle phasesi.e 2 cell cycle phases

• Howard & Pelc, 1951 & 1953, - bean root cells in interphase Howard & Pelc, 1951 & 1953, - bean root cells in interphase incorporate incorporate 3232P for DNA synthesis (S phase) and there is a time gap P for DNA synthesis (S phase) and there is a time gap (G2) before the beginning of cell division (M) and there is another gap (G2) before the beginning of cell division (M) and there is another gap (G1) between M and S to complete the cell cycle - (G1) between M and S to complete the cell cycle - i.e. 4i.e. 4 cell cycle cell cycle phasesphases

• Taylor et al., 1957 looked at tritiated thymidine uptake (in S) and 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), measured the time it takes for labeled cells to enter M (= time in G2), and the other and the other cell cycle kineticcell cycle kinetic parameters parameters

• More recently, bromodeoxyuridine detected by fluorescent antibody is More recently, bromodeoxyuridine detected by fluorescent antibody is used to label cells (in S) and measure cell cycle kinetics by flow used to label cells (in S) and measure cell cycle kinetics by flow cytometry or U.V. microscopycytometry or U.V. microscopy


Fix and stainFlash label with

3H-TdR or BdUR for 20 mins

If BdUR labeled

If 3H-TdR labeled

Mitotic Index (M.I.)= TM/TC

Labeling Index (L.I.) = TS/TC

mitosis*Anti-BdUR

AR film

…

..…..

…..

…..…

..

…..

….. …..…..

…..…..

…..…..

…..…..…..…

..…..…..

…..…..…..

U.V. microscopy

Mitotic IndexMitotic Index Labeling IndexLabeling Index

Autoradiography

Where Where is a correction factor is a correction factor

for cell division, about 0.69for cell division, about 0.69


Frequency of Labeled Mitosis Frequency of Labeled Mitosis Technique (FLM)Technique (FLM)

• By counting the number of mitoses that are By counting the number of mitoses that are labeled at various times after labeled at various times after 33H-thymidine H-thymidine incorporation, the time taken for a cell to incorporation, the time taken for a cell to traverse a specific cell cycle phase, and the traverse a specific cell cycle phase, and the cell cycle time, can be estimatedcell cycle time, can be estimated

• But, it is easier to use BUdR and flow But, it is easier to use BUdR and flow cytometrycytometry


From FLM to FACSFrom FLM to FACSLabel cells with Label cells with dye and use a dye and use a laser to excite it. laser to excite it. Collect output by Collect output by photomultiplier photomultiplier tubes.tubes.

E.g. DNA can be E.g. DNA can be labeled by propidium labeled by propidium iodide (P.I.)iodide (P.I.)

LASER

Cells in fine stream

PM tubes


Flow Cytometry for DNA QuantityFlow Cytometry for DNA Quantity

1. label DNA with propidium iodide1. label DNA with propidium iodide (fluorescent dye)(fluorescent dye)

2. measure light output by flow cytometry2. measure light output by flow cytometry

3. analyze DNA histograms3. analyze DNA histograms

G1G1SS

G2G2 MM

2n2n

2n + 2n + n n

4n4n 4n4n

2n2n 4n4n

# cells# cells

degree of fluorescencedegree of fluorescence

G1G1

SS

G2MG2M


Cell Cycle Kinetic Analysis by Flow CytometryCell Cycle Kinetic Analysis by Flow Cytometry

G1 s

G2/M

BrdUrdgreengreen

DNAred

G1

s

G2/M

BrdUrdgreengreen

DNAP.I red

G1

s

G2/M

BrdUrdgreengreen

DNAP.I. red

Time

P.I. (DNA - red) combined with Bromodeoxyuridine uptake followed by P.I. (DNA - red) combined with Bromodeoxyuridine uptake followed by staining with fluorescently labeled anti-BrdUrd (green)staining with fluorescently labeled anti-BrdUrd (green)


Cell CycleCell Cycle

G1 phaseG1 phasevariable lengthvariable length

M phaseM phase0.5-1 hr0.5-1 hr

G2 phaseG2 phase1-2 hrs1-2 hrs

S phaseS phaseDNA synthesisDNA synthesis

6-8 hrs6-8 hrs

Where Where is a correction for uneven cell numbers due to mitosis (0.69) is a correction for uneven cell numbers due to mitosis (0.69)

If all cells in a population are dividingIf all cells in a population are dividing

Mitotic Index (M.I.) = Mitotic Index (M.I.) = Tm / TcTm / TcLabeling Index (L.I.) = Labeling Index (L.I.) = Ts /TcTs /Tc

G0 quiescentG0 quiescent


Cell Cycle SynchronisationCell Cycle Synchronisation

The best estimates of kinetics come from use of The best estimates of kinetics come from use of cells cells synchronizedsynchronized in a specific cell cycle phase in a specific cell cycle phase

• Mitotic cells can be shaken off from some cell lines - Mitotic cells can be shaken off from some cell lines - M phase cellsM phase cells

• Serum deprivation - G1 phase cellsSerum deprivation - G1 phase cells• Hydroxyurea synchronizes cells at the G1/S transitionHydroxyurea synchronizes cells at the G1/S transition


Cell Cycle and RadiosensitivityCell Cycle and Radiosensitivity

S.F.S.F.

20201616121288440000.01.01

.1.1

11

Dose (Gy)Dose (Gy)

LATE SLATE S

EARLY SEARLY S

G1 PHASEG1 PHASEG2/M PHASEG2/M PHASE

Variations in sensitivity and in Variations in sensitivity and in cell cycle arrest after irradiation cell cycle arrest after irradiation could be important in radiation could be important in radiation therapy, because fractionated therapy, because fractionated irradiation can lead to irradiation can lead to sensitization by reassortment.sensitization by reassortment.

The oxygen enhancement ratio (OER) The oxygen enhancement ratio (OER) does not vary much with the phase of the does not vary much with the phase of the cell cycle.cell cycle.

High LET responses are less affected by High LET responses are less affected by cell cycle phase than low LET radiation cell cycle phase than low LET radiation responses.responses.

G1 S G2 M

Increasing radioresistance


Cell Cycle ArrestCell Cycle Arrest• Cells have Cells have ““checkpointscheckpoints”” where they where they ““proof-readproof-read”” DNA for damage DNA for damage

before continuing to cycle. This ensures faithful chromosome replication before continuing to cycle. This ensures faithful chromosome replication and maintains genomic integrity.and maintains genomic integrity.

• Irradiation causes cells to arrest at these checkpoints Irradiation causes cells to arrest at these checkpoints • Cells tend to arrest atCells tend to arrest at

• G1 - especially if they have wt p53. This may lead to apoptosisG1 - especially if they have wt p53. This may lead to apoptosis• Intra S phase - initiation and elongation stages of DNA Intra S phase - initiation and elongation stages of DNA

replication are affected by p53 independent mechanismsreplication are affected by p53 independent mechanisms• G2 - most cells arrest here - allows G2 - most cells arrest here - allows chromatid repair prior to chromatid repair prior to

segregation in Msegregation in M• M phase - bM phase - block in anaphase until all sister chromatids lock in anaphase until all sister chromatids

have aligned properly on the spindle - have aligned properly on the spindle - Monitors spindle Monitors spindle integrity for cytokinesisintegrity for cytokinesis


• Irradiated (7Gy)Irradiated (7Gy)• P.I stain at 9hrP.I stain at 9hr wild-type irradiatedwild-type irradiated

Decrease in SDecrease in SIncrease in G2MIncrease in G2Mi.e. G1 and G2M arresti.e. G1 and G2M arrest

P53 or ATM deficient irradiatedP53 or ATM deficient irradiatedloss of G1/S checkpointloss of G1/S checkpointand only G2M arrestand only G2M arrest

Cell Cycle ArrestCell Cycle Arrest DNA Damage Dependent DNA Damage Dependent

CheckpointsCheckpoints


What Drives Cell Cycle Progression?What Drives Cell Cycle Progression?

Growth factors are required for G0 through G1 to S (and cell survival)Growth factors are required for G0 through G1 to S (and cell survival)• To activate resting cells to enter G1To activate resting cells to enter G1• To allow cells to pass through G1 phase To allow cells to pass through G1 phase • To gain competence to progress into S phaseTo gain competence to progress into S phase

The growth factors that are required vary with the cell type. For example, The growth factors that are required vary with the cell type. For example, for fibroblasts:for fibroblasts:

• PDGF (platelet derived GF) activates cells PDGF (platelet derived GF) activates cells • EGF (epidermal GF) and insulin act as competence factors to progress into S EGF (epidermal GF) and insulin act as competence factors to progress into S

phase phase • IGF (insulin GF) promotes progression into SIGF (insulin GF) promotes progression into S

Cycling is growth factor independent through S, G2, MCycling is growth factor independent through S, G2, M


Molecular Mechanism of Cell Cycle ProgressionMolecular Mechanism of Cell Cycle Progression

Progression through each checkpoint requires:Progression through each checkpoint requires:• Retinoblastoma (Rb) tumor suppressor gene familyRetinoblastoma (Rb) tumor suppressor gene family

• especially G1-S transitionespecially G1-S transition• Regulatory FactorsRegulatory Factors

• CyclinsCyclins that are synthesized at the appropriate time for each phase and then that are synthesized at the appropriate time for each phase and then degraded to coordinate cell cycle progression. Growth factors induce cyclin degraded to coordinate cell cycle progression. Growth factors induce cyclin expression in G1.expression in G1.

• Cyclin Dependent Kinases (CDK)Cyclin Dependent Kinases (CDK) are activated by cyclins and are activated by cyclins and phosphorylate targets required for the next cell cycle phasephosphorylate targets required for the next cell cycle phase

• Regulators of CDKsRegulators of CDKs • Inhibitory kinasesInhibitory kinases• Activated phosphatasesActivated phosphatases• Non-kinase inhibitorsNon-kinase inhibitors


Retinoblastoma Protein Retinoblastoma Protein pRbpRb

• Cyclin D/cdk4/6 and cyclin E/cdk2 phosphorylate Cyclin D/cdk4/6 and cyclin E/cdk2 phosphorylate Rb, which is essential for cell cycle progression into Rb, which is essential for cell cycle progression into S S

• Phosphorylation of Rb releases E2F, which it Phosphorylation of Rb releases E2F, which it normally is bound to. E2F is a transcription factor normally is bound to. E2F is a transcription factor for 20-30 genes that are required for S phase gene for 20-30 genes that are required for S phase gene expression.expression.

• pRB mutation often leads to cancer. pRB mutation often leads to cancer.


CyclinsCyclins

• Have no intrinsic enzymatic activityHave no intrinsic enzymatic activity• Cyclins A to J have been identified Cyclins A to J have been identified (no I)(no I)

• Synthesized and degraded during each cell Synthesized and degraded during each cell cycle phasecycle phase

• Bind and activate cdksBind and activate cdks


Cyclin Dependent KinasesCyclin Dependent Kinases

• Cyclins bind and activate Cdks, whichCyclins bind and activate Cdks, which– Are serine/threonine kinases with multiple Are serine/threonine kinases with multiple

substratessubstrates• e.g. pRb, p53, E2F, etc. that they activate/inactivatee.g. pRb, p53, E2F, etc. that they activate/inactivate

– Have regulatory domainsHave regulatory domains• E.g. inhibitory and activating phosphatesE.g. inhibitory and activating phosphates

– Are present throughout cell cycleAre present throughout cell cycle– To move cells from G0 to G1 to STo move cells from G0 to G1 to S

• Cyclin D activates cdks 4/6 and Cyclin D activates cdks 4/6 and • Cyclin E activates cdk2Cyclin E activates cdk2

P

P

cdk

Inhibitory phosphateInhibitory phosphate

activating phosphateactivating phosphate

Cyclin

kinase site


Activating Phosphatases Activating Phosphatases CDC25 Removes Phosphate from Tyr-15CDC25 Removes Phosphate from Tyr-15

– CDC25A = cyclin E/CDK2 = G1/S specificCDC25A = cyclin E/CDK2 = G1/S specific– CDC25B = cyclin A/CDK2 = S-phase exitCDC25B = cyclin A/CDK2 = S-phase exit– CDC25C = cyclin B/CDK1 = G2/M specificCDC25C = cyclin B/CDK1 = G2/M specific


Cyclin DCyclin DCDKCDK4/64/6

Cyclin ECyclin ECDKCDK22

Responsible for pRb Responsible for pRb phosphorylationphosphorylation

Cyclin ACyclin ACDKCDK1/21/2

Cyclin BCyclin BCDKCDK11

Cyclin ACyclin ACDKCDK1/21/2

Early - mid G1Early - mid G1

Cyclin D Cyclin D CDKCDK4/64/6Responsible for pRb Responsible for pRb phosphorylationphosphorylation

cdk1 phosphorylates substrates leads tocdk1 phosphorylates substrates leads to• Nuclar envelope breakdownNuclar envelope breakdown• Chromosome separationChromosome separation• Spindle assemblySpindle assembly• Chromosome condensationChromosome condensation

Cyclosome (APC)Cyclosome (APC)pRb dephosphorylationpRb dephosphorylation

G0 quiescentG0 quiescent


Cyclin Kinase InhibitorsCyclin Kinase Inhibitors

PhasePhase ComplexesComplexes InhibitorsInhibitorsG1G1 cyclin D-CDK4, 6cyclin D-CDK4, 6 p16 (INK 4a), p16 (INK 4a),

p19p19ARFARF (INK 4a) (INK 4a)p15 (INK4b)p15 (INK4b)

G1/SG1/S cyclin E-CDK2, 3cyclin E-CDK2, 3 p21p21CIP1CIP1, p27, p27KIP1KIP1

SS cyclin A-CDK2cyclin A-CDK2 p21, p57p21, p57G2/MG2/M cyclin B-CDK1 cyclin B-CDK1 p21p21

p53 is a transcription factor for p21, which is why it is p53 is a transcription factor for p21, which is why it is involved in cell cycle arrest after IRinvolved in cell cycle arrest after IR

Inhibitors (Inhibitors (CKIsCKIs) belong to 2 families) belong to 2 families

• INK4 and KIP/CIPINK4 and KIP/CIP

Generally compete with cyclins for Generally compete with cyclins for CDKCDKss


ATM

MRN complex

NHEJ

53BP1MDC1MRNBRCA1

H2AX

mediatorsmediators

p53 CHK2

CDC25A phosphorylation CDC25A phosphorylation p21

CYCLIN E

CDK2

CYCLIN E

CDK2

P-thr14/tyr15P-thr14/tyr15

p21

G1/S ArrestG1/S ArrestSensescence/transientSensescence/transient

CDC25A degradationCDC25A degradation

rapidslow

transactivation

ATR

53BP1MDC1MRNBRCA1

CHK1

CDC25A phosphorylation CDC25A phosphorylation

CYCLIN A/E

CDK2

S PhaseS PhaseArrestArrest

DSBDSB SSB/Base damageSSB/Base damageReplication stress, UV, MMC, hypoxia

Stalled Replication Fork

HRATM

ATR

53BP1MDC1MRNBRCA1

CHK2 CHK1

CDC25C phosphorylation CDC25C phosphorylation and nuclear exportand nuclear export

CYCLIN B

CDK!

P-thr14/tyr15P-thr14/tyr15 PP

G2/M ArrestG2/M Arrest

DSB ResectionDSB Resection

MDM2

sensorssensors

transducerstransducers

effectorseffectors


• If p53 or any other molecule governing cell cycle arrest is If p53 or any other molecule governing cell cycle arrest is mutated, genetic instability results as well as more rapid cell mutated, genetic instability results as well as more rapid cell cycle progression.cycle progression.

• Cyclins, cdks, cdkis and other molecules involved in cell cycle Cyclins, cdks, cdkis and other molecules involved in cell cycle progression are frequently mutated or have altered expression progression are frequently mutated or have altered expression in cancerin cancer • e.g. cyclin D amplification and/or p16 deletion or silencing e.g. cyclin D amplification and/or p16 deletion or silencing

and/or p53 mutation in Head and Neck Caand/or p53 mutation in Head and Neck Ca

Cell Cycle in CancerCell Cycle in Cancer


Growth Factor/Cytokine Growth Factor/Cytokine ReceptorReceptor

ProliferationProliferation Cell deathCell death

OncogenesOncogenes

RasRasRafRaf

MAPKMAPK

PI3KPI3KNF-NF-BB

SurvivalSurvival

SignalsSignals

CancerCancer


DNA damage responseDNA damage responseATM, ATR, MRNATM, ATR, MRNP53, Chk1, Chk2P53, Chk1, Chk2

Initial damageInitial damage

ROSROS

Cell cycle arrestCell cycle arrest Cell deathCell death/survival/survival

DNA repairDNA repair


NF-kBNF-kB

Tissue recoveryTissue recovery/lesion formation/lesion formation

Cell deathCell death/survival/survival

Cell proliferationCell proliferation

Cell proliferationCell proliferation

Immediate early Immediate early gene responsegene response

AU-rich control:AU-rich control:TNF-, IL1, IL-2, IL-3, GM-CSF, IL-6, IL-8, IL-12, IFN/, VEGF, PDGFB,

NGF, IGFR, DR5, COX-2 Proteasome inhibitionProteasome inhibitionMitochondrial damageMitochondrial damageActivation of EGFR, Activation of EGFR, TGF-TGF-, etc, etc

InflammatoryInflammatoryCytokines andCytokines and

Growth FactorsGrowth Factors

P21, Bax, caspase 8,etcP21, Bax, caspase 8,etc..


Loss of Proliferative Ability can Loss of Proliferative Ability can Occur in Different WaysOccur in Different Ways

Quiescence Senescence Quiescence Senescence Terminal Terminal Death DeathDifferentiationDifferentiation

Property of stem cellsProperty of stem cellsReversible, physiological Reversible, physiological processprocessApoptosis and Apoptosis and differentiation is inhibiteddifferentiation is inhibitedHigh free radical scavenger High free radical scavenger levelslevels

Irreversible, Irreversible, physiologicalphysiologicalactive processactive processCell cycle inhibition is a Cell cycle inhibition is a secondary effectsecondary effect

Irreversible,Irreversible,non-physiological non-physiological processprocess

ApoptosisApoptosisAutophagyAutophagyNecrosisNecrosis


Tissue KineticsTissue KineticsKinetics Kinetics in tumors or normal tissues depend upon in tumors or normal tissues depend upon • Cell cycleCell cycle• Growth fraction (G.F.)Growth fraction (G.F.)

• G.F. is the proportion of proliferating cellsG.F. is the proportion of proliferating cells• G.F. = P / (P + Q) where P = proliferating cells and Q = non-G.F. = P / (P + Q) where P = proliferating cells and Q = non-

proliferating cells (quiescent/senescent/differentiated cells)proliferating cells (quiescent/senescent/differentiated cells)• Cell loss factorCell loss factor

• Cell Loss Factor Cell Loss Factor is due to death or loss of cellsis due to death or loss of cells• If If = 0, Td = Tpot= 0, Td = Tpot where Td is the actual volume doubling time where Td is the actual volume doubling time

and Tpot is potential volume doubling time and Tpot is potential volume doubling time • = 1 - Tpot / Td= 1 - Tpot / Td• if G.F. = 1 then Tpot = Tc = if G.F. = 1 then Tpot = Tc = Ts / L.I.Ts / L.I. • Under steady state conditions, a constant cell number is Under steady state conditions, a constant cell number is

maintained by the balance between cell proliferation and cell loss maintained by the balance between cell proliferation and cell loss i.e. i.e. = 1.0. In tumors and embryos, = 1.0. In tumors and embryos, < 1.0 < 1.0


Tumor KineticsTumor Kinetics

Tc Cell cycle timeTc Cell cycle time

G.F. Growth fraction G.F. Growth fraction

Tpot Pot. doubling timeTpot Pot. doubling time

TdTd Actual doubling time Actual doubling time

Cell loss factorCell loss factor

Human SCCHuman SCC

36 hrs36 hrs

0.250.25

6 days6 days

60 days60 days

0.90.9

Rate of tumor growth, and the rate of tumor regression, are determined Rate of tumor growth, and the rate of tumor regression, are determined largely by the cell loss factor! largely by the cell loss factor!

VARIES GREATLY WITH TUMORVARIES GREATLY WITH TUMOR

(36hr x 4)(36hr x 4)

(1-6/60)(1-6/60)


Tumor RegressionTumor Regression

• The rate of tumor growth and regression is The rate of tumor growth and regression is determined bydetermined by• rate of cell loss rate of cell loss ((• G.F.G.F.• cell cycle kinetics cell cycle kinetics

• Slow growing tumors may regress rapidlySlow growing tumors may regress rapidly• Rapidly growing tumors are expected to regress Rapidly growing tumors are expected to regress

and regrow rapidlyand regrow rapidly• Slow regression is not an indication of treatment Slow regression is not an indication of treatment

failurefailure• The rate of tumor regression after Tx is not, in The rate of tumor regression after Tx is not, in

general, prognosticgeneral, prognostic


Tumor RegenerationTumor Regeneration

Rat rhabdomyosarcomaRat rhabdomyosarcomaHermans and Barendsen, 1969Hermans and Barendsen, 1969

Tumors can Tumors can regenerate at the regenerate at the

same time as same time as they regress!they regress!

ControlControlIrradiatedIrradiated

Surviving clonogensSurviving clonogensmeasured in vitromeasured in vitro

Growth delay Growth delay

TimeTime

Relative tumor Relative tumor volumevolume

X-raysX-rays


EVIDENCE FOR ACCELERATED EVIDENCE FOR ACCELERATED REPOPULATION IN TUMORSREPOPULATION IN TUMORS

• Time to tumor recurrence after therapy is shorter Time to tumor recurrence after therapy is shorter than than would be expected from the original than than would be expected from the original growth rate growth rate

• Split-course radiation therapy often gives poor Split-course radiation therapy often gives poor resultsresults

• Protraction of treatment time often results in poor Protraction of treatment time often results in poor resultsresults

• Accelerated treatment has been shown to be of Accelerated treatment has been shown to be of benefit in some circumstances.benefit in some circumstances.


Accelerated Tumor RepopulationAccelerated Tumor Repopulation

T2 and T3 SCC head and neckT2 and T3 SCC head and neck ( (excluding nasopharynx and vocal excluding nasopharynx and vocal

cord).cord). TCD TCD5050 values are consistent with values are consistent with onset of repopulation at 4 onset of repopulation at 4

weeksweeks followed by accelerated repopulation with a 3-4 day followed by accelerated repopulation with a 3-4 day doubling time, implying a loss in dose of about 0.6 Gy/dydoubling time, implying a loss in dose of about 0.6 Gy/dy

Withers et al, 1988Withers et al, 1988

T2 T3T2 T3local controllocal control

no local controlno local control


Accelerated Tumor RepopulationAccelerated Tumor Repopulation

Onset may be about day 21. Repopulation may not be constant and 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 / 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.day around week 6-7 and thereafter.


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

fractionation protocols fractionation protocols

Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill McBride Dept. Radiation...

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Transcript of Radiation Targets 2: Cell Proliferation, Cell Death and Survival Bill McBride Dept. Radiation...