cell survival curve

cell survival curve

1. CELL SURVIVAL CURVE PRESENTER :DR.VIJAY.P.RATURI MODERATOR :- MR.TEERTHRAJ SIR J.R 2 ,KGMU lucknow

2. DEFINITION Cell survival curve describes the relationship between the radiation dose and the proportion of cells that survive. Cell “Death” : loss of reproductive integrity Clonogenic : S urvivor able to proliferate indefinitely to produce a large clone or colony.

3. Mitotic death : Death while attempting to divide(dominant following irradiation) Apoptosis : Programmed cell death In general, a dose of 100 Gy is necessary to destroy cell function in nonproliferating systems. By contrast, the mean lethal dose for loss of proliferative capacity is usually less than 2 Gy .

4. The I n V itro Survival Curve Plating efficiency PE = x 100 Surviving fraction SF =   100 cells are seeded into an unirradiated culture, and 10 colonies are formed , then the PE is 10/100 . If there are 5 colonies after a 450 cGy dose of radiation , the SF is 5/[100 × 10/100] = 1/2. Thus, the SF of 450 cGy is 50%. NO IRRIDATION IRRADIATED

5. The In Vitro Survival Curve

6. The number of cells in cell lines within cell cultures can increase in one of two way: Arithmetically or exponentially (geometrically ). The number of cells increases linearly (by a constant number ) with each generation in an arithmetic. In exponential , the number of cells doubles with each generation , and so exponential growth is faster than arithmetic growth

7. If the SF is calculated for various doses, then it can be presented as a cell–dose plot . Combining the points on the plot leads to a cell survival curve. SIGMOID CURVE SEMILOGARITHMIC CURVE

8. EXPONENTIAL SURVIVAL CURVE S urvival curves resulting from the single target–single hit hypothesis of target theory . They show that cell death dueto irradiation occurs randomly . At certain doses with one unit increase, both same number of cell deaths and same proportion of cell death occur.

9. D0 = dose that decreases the surviving fraction to 37 %. This is the dose required to induce an average damage per cell . A D0 dose always kills 63% of the cells in the region in which it is applied, while 37% of the cells will survive . 1/D0 = the slope of the survival curve . As the value of D0 decreases → 1/D0 increases → slope → radiosensitive cell . As the value of D0 increases → 1/D0 decreases → slope → radioresistant cell.

10. SHOULDERED SURVIVAL CURVES WITH ZERO INITIAL SLOPE These survival curves are based on the multiple target–single hit hypothesis of target theory They are produced by the hypothesis of requiring multiple targets per cell, and only one of these targets needs to be hit to kill the cell .

11. Dq : half-threshold dose → the region of the survival curve where the shoulder Starts (indicates where the cells start to die exponentially) (= quasi-threshold dose ). n: extrapolation number (the number of D0 doses that must be given before all of the cells have been killed ).

12. Dq → the width of the shoulder region . Dq = Do log n 2.7 If n increases → Dq increases → a wide shouldered curve is observed . If n decreases → Dq decreases → a narrow shouldered curve is observed . If Dq is wide and D0 is narrow, the cell is radioresistant . The D0 and Dq values for the tumor should be smaller than those of normal tissue to achieve clinical success.

13. SHOULDERED SURVIVAL CURVE WITH NON ZERO INITIAL SLOPE

14. COMPONENTS OF SHOULDERED SURVIVAL CURVES WITH NONZERO INITIAL SLOPE • Component corresponding to the single target–single hit model (blue in the figure) - This shows lethal damage. - This shows the cells killed by the direct effect of the radiation . - This shows the effect of high-LET radiation . • Component corresponding to the multiple target–single hit model (red in the figure) - This shows the accumulation of SLD. This shows the cells killed by the indirect effect of the radiation . - This shows the effect of low-LET radiation.

15. SHAPE OF THE SURVIVAL CURVE At “low doses” for sparsely ionizing(low LET ) radiations, such as x-rays, the survival curve starts out straight on the log-linear plot with a finite initial slope. The surviving fraction is an exponential function of dose. At higher doses, the curve bends. At very high doses, the survival curve often tends to straighten again. For densely ionizing (high-LET) radiations, such as α-particles or low-energy neutrons, the cell survival curve is a straight line from the origin.

16. THE SHAPE OF THE SURVIVAL CURVE A :The linear quadratic model. B :The multitarget model. A. G ood fit to experimental data for the first few decades of survival.

17. MECHANISMS OF CELL KILLING The principal sensitive sites for radiation-induced cell lethality are located in the nucleus as opposed to the cytoplasm. The evidence implicating the chromosomes, specifically the DNA , as the primary target for radiation-induced lethality may be summarized as follows:

18. Cells are killed by radioactive tritiated thymidine incorporated into the DNA. The radiation dose results from short-range α -particles and is therefore very localized. Certain structural analogues of thymidine, particularly the halogenated pyrimidines , are incorporated selectively into DNA in place of thymidine if substituted in cell culture growth medium. This substitution dramatically increases the radiosensitivity of the mammalian cells.

19. Factors that modify cell lethality, such as variation in the type of radiation, oxygen concentration, and dose rate , also affect the production of chromosome damage in a fashion qualitatively and quantitatively similar. The radiosensitivity of a wide range of plants has been correlated with the mean interphase chromosome volume, which is defined as the ratio of nuclear volume to chromosome number. The larger the mean chromosome volume, the greater the radiosensitivity

20. BYSTANDER EFFECT Defined as the induction of biologic effects in cells that are not directly traversed by a charged particle, but are in proximity to cells that are. Nagasawa and Little, 1992 Low dose of α -particles, a larger proportion than estimated of cells showed an biologic change.

21. The use of sophisticated single-particle microbeams , which make it possible to deliver a known number of particles through the nucleus of specific cells. The bystander effect has also been shown for protons and soft x-rays. The effect is most pronounced when the bystander cells are in gap-junction communication with the irradiated cells. For example, up to 30% of bystander cells can be killed in this situation.

22. The effect being due, presumably, to cytotoxic molecules released into the medium. The existence of the bystander effect indicates that the target for radiation damage is larger than the nucleus and, indeed, larger than the cell itself. Its importance is primarily at low doses , where not all cells are “hit”.

23. APOPTOTIC DEATH Apoptosis in Greek word : “falling off” Programmed cell death Occurs in normal tissues, also can be induced in some normal tissues and in some tumors by radiation. Double-strand breaks(DSB s) occur in the linker regions between nucleosomes, producing DNA fragments that are multiples of approximately 185 base pairs.  Laddering in gels.

24. Apoptosis is highly cell-type dependent . Hemopoietic and lymphoid cells are particularly prone to rapid radiation-induced cell death by the apoptotic pathway. Apoptosis after radiation seems commonly to be a p53-dependent process ; Bcl-2 is a suppressor or apoptosis.

25. MITOTIC DEATH The most common form of cell death from radiation is mitotic death . Cells die attempting to divide because of damaged chromosomes. The log of the surviving fraction The average number of putative “lethal” aberrations per cell(asymmetric exchange-type aberrations such as rings and dicentrics ) Data such as these provide strong circumstantial evidence to support the notion that asymmetric exchange-type aberrations represent the principle mechanism for radiation-induced mitotic death in mammalian cells.

26. RELATION BETWEEN CHROMOSOMAL ABERRATION & SURVIVAL CURVE

27. SURVIVAL CURVES FOR VARIOUS MAMMALIAN CELLS IN CULTURE First in vitro survival curve for mammlian cells irradiated with x-rays. All mammalian cells studied to date, normal or malignant, regardless of their species of origin, exhibit x-ray survival curves similar to those in figure. Initial shoulder

28. The D of the x-ray survival curves for most cells cultured in vitro falls in the range of 1 to 2 Gy . The exceptions are cells from patients with cancer-prone syndromes such as A taxia-telangiectasia(AT) ; these cells are much more sensitive to ionizing radiations, with a D for x-rays of about 0.5 Gy .

29. In more recent years, extensive studies have been made of the radiosensitivity of cells of human origin, both normal and malignant, grown and irradiated in culture. In general, cells from a given normal tissue show a narrow range of radiosensitivity if many hundreds of people are studied. By contrast, cells from human tumors show a very broad range of D values.

30. SURVIVAL CURVES FOR VARIOUS MAMMALIAN CELLS IN CULTURE

31. SURVIVAL CURVE SHAPE AND MECHANISMS OF CELL DEATH Radioresistant Large dose-rate effect Radiosensitive No dose-rate effect Laddering (after 10 Gy )

32. Characteristic laddering is indicative of programmed cell death or apoptosis during which the DNA breaks up into discrete lengths as previously described. Comparing Fig.A and B, it is evident that there is a close and impressive correlation between radiosensitivity and the importance of apoptosis . Increased “laddering” = Increased radiosensitivity

33. Mitotic death results (principally) from exchange-type chromosomal aberrations ; the associated cell survival curve, therefore, is curved in a log-linear plot, with a broad initial shoulder.

34. GENETIC CONTROL OF RADIOSENSITIVITY Inherited Human Syndromes associated with sensitivity to X-rays Ataxia-telangiectasia(AT) Basal cell nevoid syndrome Cockayne syndrome Down syndrome Fanconi’s anaemia Usher syndrome Nijmegen breakage syndrome

35. EFFECTIVE SURVIVAL CURVE FOR A MULTIFRACTION REGIMEN The effective survival curve is an exponential function of dose whether the single-dose survival curve has a constant terminal slope or is continuously bending. The D of the effective survival curve: the dose required to reduce the fraction of cells surviving to 37%(close to 3 Gy for cells of human origin). D 10 (dose required to kill 90% of the population) D 10 = 2.3 D  

36. EFFECTIVE SURVIVAL CURVE FOR A MULTIFRACTION REGIMEN

37. THE RADIOSENSITIVITY OF MAMMALIAN CELLS COMPARED WITH MICROORGANISMS It is evident that mammalian cells are exquisitely radiosensitive compared with microorganisms. The most resistant is Micrococcus radiodurans , which shows no significant cell killing even after a dose of 1,000 Gy .

38. A , mammalian cells; B, E. coli; C, E. coli B/r; D, yeast; E, phage staph E; F, B. megatherium ; G, potato virus; H, Micrococcus radiodurans .

39. THE RADIOSENSITIVITY OF MAMMALIAN CELLS COMPARED WITH MICROORGANISMS The dominant factor that accounts for this huge range of radiosensitivities is the DNA content . Mammalian cells are sensitive because they have a large DNA content, which represent a large target for radiation damage. E. coli and E. coli B/r have the same DNA content but differ in radiosensitivity because B/r has a mutant and more efficient DNA repair system .

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