Cell Survival Curves (Chap. 3)

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1 Cell Survival Curves (Chap. 3)

2 Example of pedigress of EMT6 mouse cells Clone

3 The in vitro cell survival curve

4 Cell Survival Assay Dye exclusion assay: membrane integrity MTT assay: respiratory activity (reducing power) DNA synthesis: cell proliferation Colony formation assay: reproductive integrity

5 Clonogenic Assay In vitro: In vivo: in vivo/in vitro in vivo/in vivo assay (ex( vivo assay) in situ in vivo assay

$3-23 Surviving fraction = Colonies counted Cells$

6 In vitro clonogenic Assay: Fig Surviving fraction = Colonies counted Cells seeded x (PE/100)

7 Clonogenic Assay The plating efficiency varies for different cell lines and cell densities. The assay is not suited to all cell lines. Many normal cell types do not form colonies unless provided with the correct growth factors.

8 First in vitro cell survival curve: Fig Puck and Marcus, 1956

9 The Shape of the survival Curve Random nature of cell killing Cell Survival models Target theory models Repair theory model

10 Target Theory Models Single target, single hit (linear model) Single hit, multiple (n) targets Two component models Single target, two hits (quadratic model) Linear-quadratic model

11 Repair theory model

12 Typical Cell Survival Curve: Fig L-Q model TC model ln n = D q /D 0

13 Comparison of L-Q L Q and TC models Neither the LQ not the TC model has any established biologic basis. At high doses the LQ model predicts a survival curve that bends continuously, whereas the TC model become linear. At low doses the LQ model describes a curve that bends more than a TC curve.

14 Mechanisms of Cell Killing DNA as the target

15 Experimental design to demonstrate that the nucleus is the target

16 Fig. 3-4: 3 Relationship between cell survival and DNA damages

17 Fig.3-5: DNA strand break follow L-Q L Q model

18 Fig. 3-7: summary of D 0 values of various cell lines

19 Fig. 3-8: 3 Survival curve shape and mechanism of cell death S = e ( a + a ) D β D M A M 2

20 Why don t t all cells die by apoptosis after RTx? Major regulatory control over release of cytochrome c is exerted through members of the Bcl-2 2 family (acronym for B cell lymphoma oncogene). Bcl-2 2 is the prototypical inhibitor of apoptosis. The family contains at least 7 anti-apoptotic apoptotic (eg( Bcl-2, Bcl-x(L x(l)) and 10 pro-apoptotic (eg( Bax, Bak,, Bid, Bad) members. Many members localize in the outer membrane of the mitochondria or endoplasmic reticulum. They appear to control the leakiness of the membranes i.e. cytochrome c release

21 Why don t t all cells die by apoptosis after RTx? The apoptotic rheostat in a cell - the balance of pro-and anti- apoptotic forces (eg( eg.. Bcl-2/Bax or TNFR1 to TNFR2). There are many survival pathways that affect both intrinsic and extrinsic death pathways. For example, IAPs (inhibitors of apoptosis) block caspase activation. This survival pathway is activated through NF-kB in some cells following irradiation. Proliferative signals tend to alter the rheostat in favor of survival These factors determine the intrinsic radiation response of cells

22 Oncogenes and Radioresistance Chiang, CS Mol. Diag

23 Relationship between radiosensitivity and radiation-induced induced G 2 /M block

24 Genetic Control of Radiosensitivity

25 Effective Dose Survival Curve: Fig ed 0 ed 10

26 Survival curve of mammalian cells and microorganisms: Fig Micrococcus radiodurans Potato virus phage Yeast E. Coli B/r E. coli Mammalian cells

27 End

28 Effects of Radiation on the Cells Increase cell cycle time transient cell cycle delay Decrease cell cycle time accelerated proliferation Cells die in different ways: Reproductive cell death (mitotic cell death) Giant cell formation Interphase cell death (apoptosis) Senescence

29 Colony Assay Control: 70 colonies 8 Gy: 32 colonies

30 Puck and Marcus, 1956

Random nature of cell killing Death is all or nothing p SF = 0.37 SF = 0.37 x 0.37 SF = 0.37 3 of x = e m m x!

31 Random nature of cell killing Death is all or nothing p SF = 0.37 SF = 0.37 x 0.37 SF = of x = e m m x! x m: mean number of hits x: number of hit so p of no hits (x=0) = e - 1 = SF Define D 0 : the mean lethal dose, the dose for which the SF = e - 1 = 37%

32 Single target, single hit (linear model) Dose SF Effect = αd = D/D o SF = e -αd = e -D/D 0

33 Single hit, multiple targets (n) SF Dose Survive of each target = e -D/D 0 Death of each target =1 - e -D/D 0 Death of n targets = (1-e -D/D 0) n SF = 1-1 (1-e -D/D 0) n

34 n Two component model SF [1- (1 SF = e -D/D 0 x D/nD 0 ) n ] (1-e -D/nD

35 Single target, two hits (quadratic model) Dose Effect = βd 2 SF SF = e βd 2

36 Linear-quadratic model Dose Effect = αd+ D+βD 2 SF SF = e ( ad+βd 2 )

37 Types of cell death observed following treatment of cells with DNAD NA-damaging agents NATURE REVIEWS /CANCER/ 2005,5: 231

38 Necrosis vs Apoptosis necrosis apoptosis

Features of Necrosis Necrosis is a non- physiological process associated with Loss of plasma membrane integrity and deregulated ion homeostasis.

39 Features of Necrosis Necrosis is a 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. Necrosis generates an inflammation

Morphological Features of Apoptosis Apoptosis is a tightly regulated active process that is associated with Cell and nuclear shrinkage Nuclear fragmentation with formation of apoptotic

40 Morphological Features of Apoptosis Apoptosis is a tightly regulated active process that is associated with Cell and nuclear shrinkage Nuclear fragmentation with formation of apoptotic bodies Blebbing of cell membrane, but no early loss of membrane integrity Deletion of single cells in isolation Lack of an inflammatory response, phagocytosis of debris by adjacent cells

41 Physiologic Functions of Apoptosis Fingers Gut Tadpole Tails Sex differentiation This is why proliferation often correlates with apoptotic index proliferating cells Self-reactive lymphocytes Irradiation CELL 88 p

Apoptosis in Gut after Irradiation Distribution of Apoptotic cell

Radiation-induced induced apoptosis occurs in normal tissues in

0 2 4 6 8 10 12 14 Position from the Paneth cell It is a myth to

42 Apoptosis in Gut after Irradiation Distribution of Apoptotic cell % of Apoptotic cell Gy_CON 10 Gy_CS Radiation-induced induced apoptosis occurs in normal tissues in specific sites and in cells that have a pro-apoptotic tendency Position from the Paneth cell It is a myth to think death is just for the old. Death is there from the very beginning Herman Feifel

43 Detection of Apoptosis - laddering of DNA in agarose gel electrophoresis During apoptosis, endonucleases are induced that cleave between nucleosomes. On agarose gel electrophoresis, the DNA separates into fragments with sizes that are multiples of bp. This is called a ladder. Histones H2,H3,H4 - DNA Spacer Region ( bp) HISTONE H1 Nucleosome DNA Core (140 bp) 55 A 110 A + Sites of endonuclease cleavage

44 Detection of Apoptosis - TUNEL Assay The breaks in DNA that form can be visualized using terminal deoxynucleotidyl transferase (TdT)) to catalyze the polymerization of fluorescein-labeled labeled nucleotides (dutp)) onto 3 -OH 3 ends. TUNEL detects DNA breaks caused by apoptotic endonuclease It is particularly useful for detecting apoptosis in tissue sections. An Apoptotic Index (AI) can be derived

45 Detection of Apoptosis - Caspase Assay The morphological and biochemical hall-marks of apoptosis result from activation of members of a family of pro-enzyme proteases called Caspases in a cascadic reaction. Caspases can be divided into 2 broad groups Initiator caspases are activated by death signals to cleave and activate Effector caspases that act on multiple substrates. Irrespective of the death signal, all pathways converge to activate a terminal Caspase3-dependent death pathway Caspase activation can be measured by cleavage of artificial fluorescent substrates specific for each caspase

46 Initiator Caspases There are 2 discrete pathways to apoptosis, distinguished by their use of different initiator caspases. Extrinsic pathway from TNFR family members that activate caspase 3 through caspase 8. Intrinsic pathway from alterations in mitochondria that activate caspase 3 through caspase 9. The Intrinsic pathway may be activated by positive stimuli or by lack of a required survival pathway - often called death by neglect. One form of death by neglect is when cells do not receive the appropriate stimuli from their environment and die of homelessness (anoikis). Review - Straaser et al. Ann. Rev. Biochem.. 69:217, 2000

47 Effector Caspases Active effector caspases 3, 6, and 7 have >40 substrates whose cleavage defines the features of apoptosis. Blocking these caspases does NOT prevent radiation-induced induced cell death. Caspase 6 Caspase 3 Caspase 7 Lamin A Actin icad - CAD DNA-PKcs PARP Cell Shrinkage ICAD (inhibitor of caspase activated DNase), DNA-PK (DNA protein kinase) PARP (poly-adp ADP-ribose polymerase) CAD DNA Fragmentation DNA Repair

48 The Extrinsic/Death Receptor Pathway Death ligands (eg eg.. TNF, FasL,, etc.) TNFR family members that have death domains (DD) in their cytoplasmic tails 7 of 22 family members have DDs, the best known being fas, TNFR1, and TRAIL. This causes adaptor proteins to bind to the receptors. One adaptor protein, FADD (fas( associated DD), recruits procaspase 8 to a death-inducing signaling complex (DISC), with aggregation and autoactivation of caspase 8.

Intrinsic/Mitochondrial Death Pathway The intrinsic/endogenous pathway is mediated by diverse apoptotic stimuli that converge at the mitochondria resulting in Loss of mitochondrial membrane potential

49 Intrinsic/Mitochondrial Death Pathway The intrinsic/endogenous pathway is mediated by diverse apoptotic stimuli that converge at the mitochondria resulting in Loss of mitochondrial membrane potential Opening of permeability transition pore Release, amongst other molecules, of cytochrome c, which Binds to apaf-1 1 (apoptosis protease activation factor 1), and procaspase 9 to form an apoptosome, activating caspase 9.

50 How Does Radiation Induce Apotosis? Radiation can cause apoptosis through The Intrinsic pathway: through DNA damage, activating p53, which produces bax, which acts on mitochondria to cause leakage through the generation of the lipid ceramide through direct action on mitochondria Or The Extrinsic pathway: by up-regulating expression of TNF and TNFR family members - often through transcription factors p53 or NF-κB.

51 Radiation-Induced Apoptosis INITIATORS DNA Damage JNK p38 mapk ATM Sphingomyelin Ceramide FADD x Members of TNFR family With Death Domains (TNFR1, Fas, TRAIL) EFFECTORS Apaf-1 p53 TERMINAL PHASE Bax Cytochrome c Pro-caspase 9 Apoptosome Complex Mitochondria Caspase 9 Caspase 8 Caspase 3, 6, 7 Autoactivation of procaspase 8 JNK - jun kinase ATM - mutated in ataxia telangiectasia FADD - Fas activated death domain Apaf - apoptosis activating factor

52 Radiation-induced induced Apoptosis The decision to commit apoptosis is determined by an internal apoptotic rheostat within the cell (and influenced by the molecular pathways that are activated). i.e. Cells have a pro-apoptotic or anti- apoptotic phenotype Radiation increases the AI, but does not change a cell from being anti-apoptotic apoptotic into pro-apoptotic There is only one serious philosophical problem. It is suicide. To judge whether life is or is not worth living Albert Camus

53 Radiation-induced induced Cell Death Apoptotic cell death (interphase( cell death) Mitotic cell death (reproductive cell death) Mitotic death can be triggered by failure of the mitotic apparatus improper segregation of chromosomes. Death can be by delayed (as opposed to rapid ) apoptosis or by necrosis.

54 MTT vs Cell survival (a) (b) 1.8 relative MTT OD ratio (%) surviving fraction (%) Surival curve OD ratio of MTT relative mitochondrial activity ( UV / control ) time after UV irradiation (hour) time after UV irradiation (hour)

55 Radiation-induced induced Giant cells In vivo In vitro

Apoptosis: Death Comes for the Cell

Apoptosis: Death Comes for the Cell Joe W. Ramos joeramos@hawaii.edu From Ingmar Bergman s The Seventh Seal 1 2 Mutations in proteins that regulate cell proliferation, survival and death can contribute