1. 生物醫學暨環境生物系 助理教授 張學偉 http://genomed.dlearn.kmu.edu.tw

2. Survival curve = radiation dose & cell survival fraction

3. Reproductive Integrity Cell survival  cell death For differentiated cells that do not proliferate e.g., nerve, muscle  lose of specific function (death) For proliferating cells e.g., hematopoietic stem cells, culture cells  lose for sustained proliferation (death)  lose of reproductive integrity (reproductive death)

4. Definition of survival for radiobiology Proliferate indefinitely to produce colony (a large clone = colony = clonogenic) Retain reproductive integrity

5. The in vitro survival curve Cell culture Established cell lines Tissue  trypsin  cell culture in vitro Dose-survival curve

6. PE = plating efficiency

7. Serial dilution

8. The shape of the survival curve log Linear –quadratic function Width of shoulder = Dq or n =semi-threshold dose

10. Multi-target model Initial slope  single-event killing final slope  multiple-event killing = D 1 = D 0

11. D 1 & D 0 = the dose required to reduce the fraction of surviving cells to 37% of it previous value. D 0 (straight at high dose) = the average dose required to deliver one inactivating event (one hit) per cell. = the dose required to reduce survival from 0.1 to 0.037 or 0.01 to 0.0037. = D 0 (37% survival) dose required to reduce survival to e-1 (0.37)

12. Dq (quasithreshold dose) = Width of shoulder =semi-threshold dose (almost the threshold dose) Threshold dose = the dose below which there is no effect. Log e n = D q /D 0 n = extrapolation number D0/0.37(e-1) = Dq/n D0/e-1 = Dq/n n= e-1xDq/D0 ln n = -Dq/D0

13. Linear –quadratic model Two components to cell killing by radiation One that is proportional to dose One that is proportional to square of dose (dual-radiation action; two separate breaks) S = e -  D-  D 2

14. S = the fraction of surviving a dose D ,  = constant When  D =  D 2  D =  /  Linear & quandratic contribution equally to cell killing at same dose

15. Target : DNA (nucleus), nuclear membrane The mechanism of cell killing Chromosome, specifically the DNA, as the primary target for radiation-induced lethality.

16. Apoptotic and mitotic death: AD (falling off) http://www-micro.msb.le.ac.uk/3035/kalmakoff/baculo/baculohostinteract.html

17. http://www.niaaa.nih.gov/publications/arh25-3/image01.gif

18. http://www.copewithcytokines.de/cope.cgi?000638

19. http://www.ucihs.uci.edu/anatomy/histo/corenotes/celldeath2004.pdf

20. Apoptosis: Dsb in linker DNA between nucleosome  185bp [“ladder” in Gel] nercosis  [“smear” in Gel] Fig.3-9 The detection of this DNA ladder is still currently used to distinguish at the molecular level apoptosis from necrosis.

21. Radiation-induced apoptosis is highly cell type dependent. Hemopoietic cells & lymphoid cells  prone to rapid radiation-induced apoptosis. Most tumors  mitotic cell death & apoptosis. or mitotic cell death only.

22. The most common form of cell death from radiation is mitotic death (MD).  Cell die in attempting to divide because of damaged chromosomes. Apoptotic and mitotic death: MD http://www.nature.com/cgi-taf/DynaPage.taf?file=/onc/journal/v23/n16/abs/1207528a.html&dynoptions=doi1097246946 Review Cell death by mitotic catastrophe: a molecular definition

23. Asymmetric exchange-type chromosome aberrations (i.e., dicentrics and rings )  Loss reproductive integrity  Unable to proliferate  death Asymmetric exchange-type chromosome aberrations represent the principle mechanism for radiation-induced mitotic death in mammalian cells.

24. Cell surviving & cell without visible aberration  correlation No apoptosis

25. The probability of an interaction between the two breaks is related to D (low dose) or D 2 (high dose). Exchange type aberrations require two chromosome breaks.

26. Chromosome aberrations in human lymphocytes Ch 2

27. Survival curves for various mammalian cells in culture All mammalian cells, normal & malignant, exhibit similar x-ray survival curve (initial shoulder but size vary) First in vitro survival curve The D o of X-ray survival curves for most culture cells range from 1 to 2 Gy (100-200 rad or cGy). (page 41)

28. radiosensitivity

29. Survival-curve shape and mechanism of cell death mitotic death Most cells fall between apopototic & mitotic death Note! Shoulder

30. Mitotic death results (principally) from exchange-type chromosomal aberration.  log-linear plot with broad shoulder  Characterized by subsequently dose-rate effect (page 74). Apoptotic death result unknow mechanism.  straight line on log-linear plot.  Characterized by expotential function of dose.  little or no dose-rate effect.

32. S = e -(  M  A  D-  M D 2  A = cell killing from apoptotic death (vary linear)  M = cell killing from mitotic death (vary linear)  M = cell killing from mitotic death (vary square) S = e -  D-  D 2 Linear –quadratic model apoptotic death mitotic death

33. Oncogenes and radioresistance Transfection of activated oncogenes to culture cells  Increase radioresistance

34. Genetic control of radiosensitivity ATM (AT-mutated) gene

35. Intrinsic Radiosensitivity and predictive assay Semisolid agar gel with growth factor Nonclonogenic assay Cell growth in multi-wells plate, e.g. MTT assay or chapter 15 Courtenary assay

36. Surviving cell number is then determined indirectly by MTT dye reduction. (Fig23.4) The amount of MTT-formazan produced can be determined spectrophotometrically once and solublilized it in a suitable solvent. 補充

37. The effective survival curve for a multifraction regimen multifraction regimen are most often used in clinical radiotherapy What is multifraction regimen? sublethal damage & time for repair  Shoulder Dilute dose to fraction at time intervals The effective survival curve

38. For human, effective D 0 (37% survival) = 300cGy = 3Gy D 10 (the dose required to kill 90% = 10 % survival) = one decade of cell killing = 2.3 X D 0 Ps: equal Slope Log e n = D q /D 0 (F3.3 & page 37) Natural log 10 = 2.3 equal Slope

39. The D o of effecitive survival curve (slope) has a value close to 300cGy for cells of human origin. This is an avarage value and can differ significantly for different tumors.

40. Calculations of tumor cell kill 10 9

44. The radiosensitivity of mammalian cells compared with microorganisms