Cell Survival Curves and Normal-Tissue Response

1. 1 Cell Survival CurvesandNormal-Tissue Response Chapter 3 MOVE THE CHROMOSOME EVIDENCE STUFF TO LECTURE #2MOVE THE CHROMOSOME EVIDENCE STUFF TO LECTURE #2

2. 2 Lecture Topics Reproductive integrity In-vitro survival curves Survival curve shapes; multi-fraction regimen Mammalian cells in culture Evidence for aberrations leading to cell death Mitotic death and apoptosis Dose-response relationships in vivo Clonogenic and functional endpoints

3. 3 Reproductive Death Cell death can have different meanings: loss of a specific function - differentiated cells (nerve, muscle, secretory cells) loss of the ability to divide indefinitely � proliferating cells such as stem cells in hematopoietic system or intestinal epithelium loss of reproductive integrity - �reproductive death�

4. 4 Reproductive Death Converse - �Survival� - retention of reproductive integrity the capacity for sustained proliferation in cells that normally proliferate

5. 5 Reproductive Death Mitotic Death or Apoptosis End result the same � cell loses ability to proliferate indefinitely

6. 6 Relevant Dose 100 Gy destroys cell function in non-proliferating systems (for example: nerve, muscle cells) 2 Gy mean lethal dose for loss of proliferative capacity

7. 7 Why Cell Cultures In-vitro cultures allow us to quantify the effects of radiation on individual cell colonies

8. 8 In-Vitro Cultures Proof of reproductive integrity - the capability of a single cell to grow into a large colony, visible to the naked eye A surviving cell that has retained its reproductive integrity and is able to proliferate indefinitely is said to be clonogenic

9. 9 Tissue Culture Techniques Specimen taken from organism Chopped into small pieces Single-cell suspension prepared by use of enzyme trypsin dissolves and loosens cell membrane Cells seeded onto culture dish Covered with growth medium Maintained at 37o C Attach to surface, grow and divide

10. 10 Survival Curves Describes relationship between radiation dose and the fraction of cells that �survive� that dose Used to assess biological effectiveness for different radiation types The shape of survival curves are tell-tale

11. 11 Survival Curve Shape These are the general shapes of survival curves for mammalian cells exposed to radiation Semi-log plots are typical More detail later

12. 12 Estimating Survival In order to determine the in vitro surviving fraction, we must know the plating efficiency PE is the percentage of cells (in control batch) that grow into colonies in other words, those cells that survive the plating process Indicates the experimental success of cells being grown in a seed dish

13. 13 Surviving Fraction Equal to the fraction of cells that plate successfully and survive irradiation (without losing their reproductive integrity) to grow into colonies

14. 14 Derivation of Survival Curves cells taken from stock culture and placed in seed dishes then irradiated and allowed to grow into colonies colonies counted for survival data

15. 15

16. 16 Characteristics of Survival Curves Low-LET radiations: low dose region survival curve begins as linear on semi-log plot surviving fraction is an exponential function of dose mid dose region shoulder region appears high dose region survival curve becomes linear again and surviving fraction returns to an exponential function of dose surviving fraction is a dual exponential

17. 17 Characteristics of Survival Curves High-LET radiations: survival curve is linear surviving fraction is a pure exponential function of dose

18. 18 Survival Curves and LET Increasing LET: increases the slope of the survival curve results in a more linear curve shoulder disappears due to increase of killing by single-events

19. 19 Survival Curve Explanation Simple to qualitatively describe curves Difficulty lies in explaining underlying biophysical events Many models have been proposed Biologic data not sufficiently precise to choose among the models

20. 20 Two General Survival Models Linear-quadratic model �dual radiation action� first component - cell killing is proportional to dose second component - cell killing is proportional to dose squared Multi-target model based on probability of hitting the �target� widely used for many years; still has merit

21. 21 Linear Quadratic Model S = e-(aD + bD2) where: S represents the fraction of cells surviving D represents dose a and b are constants that characterize the slopes of the two linear portions of the semi-log survival curve biological endpoint is cell death

22. 22 Linear Quadratic Model Linear and quadratic contributions to cell killing are equal when the dose is equal to the ratio of a to b D = a/b or aD = b D2 a component is representative of damage caused by a single event (hit, double-strand break, �initiation/promotion�, etc.) b component is representative of damage caused by multiple events (hit/hit, 2 strand breaks, initiation then promotion, etc.)

23. 23 a and b Determination

24. 24 Multi-target Model Quantified in terms of: measure of initial slope due to single-event killing, D1 measure of final slope due to multiple-event killing, D0 width of the shoulder, Dq or n D1 and D0 are reciprocals of the initial and final slopes the doses required to reduce the fraction of surviving cells by 37% the dose required to deliver, on average, one inactivating event per cell

25. 25 Why 37%? S = exp[-aD], but how do we define a? if we define a as the slope, where a = x/D0, then x is the fractional reduction of surviving cells if all cells are assumed to take one lethal hit, then x = 1 and a = 1/D0, so that S = exp[-D/D0] = e-1 = 0.37 In radiation therapy, it is useful to calculate the multi-fractioned dose required to kill 90% of the population (10% survival) 0.1 = exp[-D10/D0] D10 = 2.3D0

26. 26 Multi-target Model Shoulder-width measures: the quasi-threshold dose (Dq) the dose at which the extrapolated line from the straight portion of the survival curve (final slope) crosses the dose axis at 100% survival the extrapolation number (n) �broad shoulder� results in larger value of n �narrow shoulder� results in small value of n n = exp[Dq / D0]

27. 27 Multi-Target Model

28. 28 Model Parameters

29. 29 Mechanisms of Cell Killing

30. 30 DNA as the Target Abundant evidence for sensitive sites located in nucleus Early experiments with non mammalian systems Evidence for chromosomal DNA as principal target

31. 31 Evidence for Site of Cell Killing Habrobracon (wasp) eggs Average number of incident a�s needed to reduce hatchability to 37%: cytoplasm: 17.6 x 106 nucleus: 1

32. 32 Evidence Implicating Chromosomes Shown that cells were killed by tritiated thymidine incorporated into DNA (very localized dose) Structural analogues of thymidine substantially increase radiosensitivity of cells when incorporated into the DNA - similar structures that do not incorporate, however, do not effect radiosensitivity In plants, those with a larger �mean chromosome volume� have greater radiosensitivity Transplantation of irradiated nucleus into unirradiated cells is lethal at doses that an unirradiated nucleus can survive

33. 33 Bystander Effect Past teachings in radiation biology have taught that hereditary biologic effects require direct damage to DNA Recent experiments demonstrated a �bystander effect� Defined as induction of biologic effects in cells not directly traversed by a charged particle (but in close proximity)

34. 34 Bystander effect, continued One study (Nagasawa and Little) showed that following low dose of a particles a larger proportion of cells showed damage than were estimated to have been hit by a particles 30% of cells showed increase in sister chromatid exchange even though <1% were calculated to have been hit

35. 35 Bystander effect, continued Additional studies with microbeams confirmed effect and was extended to protons and soft x-rays Using soft x-rays bystander effect demonstrated for chromosomal abberations, cell killing, mutation, oncogenic transformation and alteration of gene expression

36. 36 Aside � Gap Junctions Multicellular organisms have many advantages over single celled organisms, but certainly one of the major advantages is that in a co-operative "family" of cells, each is free to specialize in ways that would be impossible if each cell had to live alone. It is customary for groups of specialized cells to be organized into tissues, which can, in turn, be further organized in to organs and organ systems. This kind of association and co-operativity requires that similar cells be held together in close and direct physical contact with one another. Neighbors must not only work together, they must be joined together.

37. 37 Aside � Gap Junctions There are two major ways in which cells in tissues can be held together; an extracellular matrix of macromolecules can form a lattice-work that can then be used by the associated cells to move, change position and a framework in which cells can interact with one another, and cell junctions can create firm, direct, specialized points of fusion between two cells in direct physical contact.

38. 38 Aside � Gap Junctions Junctions between cells most occur on or very near the cell's plasma membrane, but can also involve the tiny space between cells and sometimes the layer of cytoplasm that lies just below the plasma membrane. Gap junctions are probably the most common type of join between two cells, and are found in almost all animal tissues. Each junction allows small, water soluble molecules to move directly between the cytoplasms of the two cells in contact, which means that both cells share metabolites and even electrical properties.

39. 39 Aside � Gap Junctions These types of junctions are made from proteins that completely cross the plasma membrane of one cell, and then make contact with an identical protein that crosses the plasma membrane of the neighbor cell. A small group of these proteins come together to form a channel or connexon through the membrane. Water soluble materials can move through the membrane using this channel, and then pass directly into a similar channel, or connexon, in the opposite membrane of the adjacent cell

40. 40 Bystander effect, continued Effect most pronounced when bystander cells are in gap-junction communication with irradiated cells. Up to 30% of bystander cells can be killed Effect much smaller when cell monolayers are sparsely seeded and separated by several hundred microns Killing reduced to 5 � 10% of bystanders

41. 41 Bystander effect, continued Presumption is effect due to cytotoxic materials released into the medium Implication is that the target for cell killing is larger than the nucleus, or even the cell Importance appears mainly at low doses Implications are for risk estimation

42. 42 Bystander effect, continued Additional experiments involving the transfer of medium from irradiated cells results in biologic killing when added to unirradiated cells Suggest that irradiated cells secrete a molecule into medium that is capable of killing cells Majority of these experiments involve low LET x- or ?-rays

43. 43 Apoptotic and Mitotic Death Apoptosis described as a set of changes at the microscopic level associated with cell death Also called programmed cell death is common in embryonic development in which some tissues become obsolete; e.g., tadpoles losing tails

44. 44 Apoptotic and Mitotic Death Apoptosis characterized by stereotyped sequence of morphological events Cell ceases to communicate with its neighbors Rounds up and detaches from neighbors Chromatin condenses at nuclear membrane Fragmentation of nucleus takes place Cell shrinks and separates into membrane bound fragments called apoptotic bodies

45. 45 Apoptotic and Mitotic Death Sequence continued DNA double strand breaks occur between nucleosomes Fragments are multiples of 185 base pairs Characteristic �ladder� fragments seen in gels

46. 46 Apoptotic and Mitotic Death Apoptosis occurs in normal tissues as well as in tumors due to radiation damage Highly cell-type dependent Hemopoietic and lymphoid cells are particularly prone to rapid radiation �induced apoptotic death In tumors, mitotic cell death is as important as apoptosis

47. 47 Apoptotic and Mitotic Death Most common form of cell death following radiation exposure is mitotic death Cells die attempting to divide with damaged chromosomes Death may occur in 1st or subsequent divisions Relationship between cell killing and induction of specific chromosomal aberrations have been observed

48. 48 Chromosomal Aberrationsand Cell Death Close relationship observed between cell killing and induction of specific lethal chromosomal aberrations Log of the surviving fraction plotted against the average number of lethal aberrations per cell shows one-to-one ratio (asymmetric exchange such as rings and dicentrics)

49. 49 Chromosome Aberration and Cell Survival

50. 50 Survival Curves for Mammalian Cells in Culture Measured for many established cell lines grown in culture Derived from human or other mammals (e.g., small rodents) Parent tissues sometimes neoplastic othertimes normal All mammalian cells exhibit x-ray survival curves similar to next graph

51. 51 Survival Curves Initial shoulder Portion of straight line Shoulder size variable Some have almost continuous curving Do for most cells is 1-2 Gy (100 � 200 rad)

52. 52 Survival Curves There are exceptions � cells from patients with cancer prone syndromes such as ataxia telangiectasia (AT) Do for x-rays is about 0.5 Gy (50 rad) The in vitro radiosensitivity correlates with hypersensitivity to radiotherapy

53. 53 Intrinsic Cell Radiosensitivity Different cell types often have vastly different radiosensitivities Cells from normal tissue show a narrow range of radiosensitivities. Cells from tumors show a broad range (see below)

54. 54 Aside - Cell Types

55. 55 Aside - Cell Types

56. 56 Aside - Cell Types

57. 57 Aside - Cell Types

58. 58 Cell Classification �Vegetative inter-mitotic� produce cells like themselves, go through mitosis regularly (e.g. intestinal crypt cells) �Differentiating inter-mitotic� divide regularly, some differentiation (e.g., spermatocytes) �Reverting post-mitotic� don�t divide regularly, but can if needed (e.g., liver cells) �Fixed post-mitotic� do not divide, highly differentiated (e.g., skin, red blood cell, muscle, nerve)

59. 59 Survival Curve Shape and Mechanisms of Cell Death

60. 60 Survival Curve Shape Mammalian cells cultured in vitro vary considerably in their sensitivity (see Fig 3.8) Asynchronous mouse tumor cells are most radioresistant Then glioblastoma cells of human origin Followed by neuroblastoma cell lines Mitotic cells from all these have basically the same radiosensitivity Implication is that if chromosomes condense in mitotsis then radiosensitivity is governed by DNA content However, in interphase, radiosensitivity varies due to different conformations of DNA

61. 61 Mitotic Death and Apoptosis Mitotic death Results from exchange type aberrations Cell survival curve has broad initial shoulder Characterized by substantial dose rate effect Apoptotic death mechanisms not clearly understood Associated cell survival curve is linear on semi-log plot Little or no dose-rate effect

62. 62 Mitotic Death and Apoptosis Most cell lines show contributions of both mitotic and apoptotic death following radiation exposure Dose response characterized as S = e-(aM+aM)D + bmD2) Where S is the fraction surviving D is dose aM+aM describe the contributions to cell killing that are linear functions of dose bm describes the contributions to cell killing that is a function of the dose squared

63. 63 Oncogenes and Radioresistance Has been reported that transfection of activated oncogenes into cells cultured in vitro increases their radioresistance Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. Transfection typically involves opening transient "holes" or gates in cells to allow the entry of extracellular molecules, typically supercoiled plasmid DNA, but also siRNA, among others An oncogene is one that contributes to the production of a cancer. Oncogenes are generally mutated forms of normal cellular genes (proto-oncogenes). A gene capable, when activated, of transforming a cell.

64. 64 Oncogenes and Radioresistance Oncogenes are found in the oncogenically activated state in retroviruses and transformed cells and in their normal non-oncogenically activated state in non-transformed cells in which they are called proto-oncogenes However not clear that oncogene expression is directly involved in induction of radioresistance Even less clear if oncogenes play a role in radioresistance in human tumors

65. 65 Genetic Control of Radiosensitivity Molecular biology of repair processes extensively studied in lower organisms such as bacteria and yeast Some cases a very radiosensitive mutant can result from mutation in a single gene that functions as a repair or checkpoint gene More complicated in mammalian systems � involve large number of genes Many radiosensitive mutants have been isolated Some patients exhibit severe normal tissue reaction to radiation therapy

66. 66 Inherited Human Syndromes Associated with Sensitivity to X-rays Ataxia telangiectasia (AT) Basal cell nevoid syndrome Cockayne�s syndrome Down�s syndrome Fancono�s anemia Gardner�s syndrome Nijmegan breakage syndrome Usher�s syndrome

67. 67 AT Example Fibroblasts from AT patients are 2-3 times as radiosensitive as normal AT patients receiving radiation therapy show considerable tissue damage unless doses are reduced AT patients also have an elevated incidence of spontaneous cancer

68. 68 The Effective Survival Curve If the dose is delivered as equal fractions with sufficient time between for repair of the sub-lethal (non-killing) damage, the shoulder of the survival curve is repeated many times. The effective survival curve becomes a composite of all the shoulder repetitions. Often used in radiation therapy treatment regimes (�multifraction regimes�).22

69. 69 In Vivo Assays forDose-Response Relationships Reconstruction of cell survival curves multi-fraction experiments Obtaining dose-response relationships clonogenic endpoints functional endpoints skin reactions, fibrosis, deformities, others

70. 70 Survival Curve Reconstruction Used for in vivo assays where original number of cells is unknown Determining survival curve descriptors Reconstructing the shape of a survival curve Accomplished by fractionating total dose Determination of values depends on knowledge of surviving fraction

71. 71 Determining D0, Dq and n

72. 72 Determining D0, Dq and n

73. 73 Multi-Fraction Doses Shape of the survival curve, and values of a and b, can be reconstructed/inferred from plots of multiple-fraction effects also see Figs. 18-7, 18-10, and 18-26

74. 74 Clonogenic Endpoints Crypt cells of the mouse jejunum Lining of jejunum - self renewing system cells in crypts divide rapidly to provide cells which move up villi, differentiate and become functioning cells cells at top of villi continuously sloughed off continuously replaced by crypt cells

75. 75 Jejunum Villi

76. 76 Experiment: Irradiation of Mice Jejunum Cells Mice given total body dose of 11 - 16 Gy Large fraction of dividing cells sterilized yet no effect to the differentiated cells in the villi Initially, crypt cell populations decrease while epithelial covering of villi shows little change After time, differentiated cells not replaced by the de-populated crypts; the villi shorten, shrink, lost Thus, crypt cells are more radiosensitive than the differentiated epithelial cells

77. 77 Clonogenic EndpointsIn-Situ Skin (Mouse) Colonies

78. 78 Photograph of Skin Nodule

79. 79 Skin Epithelium Survival Curve

80. 80 Functional Endpoints Bone marrow - colonies irradiated bone marrow cells transplanted to sterilized spleen of irradiated animal irradiated mammary & thyroid epithelial cells transplanted to fat pad of another animal Skin reactions (erythema, desquamation) Pneumonitis or fibrosis in mouse lung tissue (based on breathing rate)

81. 81 Pertinent Conclusions Cells from tumors and many normal regenerative tissues grow and form colonies in vitro Fresh tissue explants may grow for weeks in culture, but then die; others become immortal Cells that have retained reproductive integrity are capable of sustained proliferation and are clonogenic Percentage of untreated seeded cells that grow into a colony is called the plating efficiency May be close to 100% or as low as 1%

82. 82 Pertinent Conclusions Fraction of cells surviving a given dose is determined by counting macroscopic colonies and allowing for plating efficiency Cell survival curve is key relationship between fraction of cells retaining reproductive integrity and absorbed dose Shape of survival curve is important

83. 83 Pertinent Conclusions Cell survival curves for high LET radiations (alphas and low energy neutrons) are straight on semi-log plot (survival is an exponential function of dose) Curves for low LET radiations have slope, shoulder and subsequent straight region Many models and theories used to fit data; not possible to discriminate among models

84. 84 Pertinent Conclusions For first 1-2 decades of survival data (1%) and up to doses used in single fraction radiotherapy, survival data can be characterized by the linear-quadratic relationship: S = e-(aD + bD2) Initial slope is given by a (alpha) Quadratic component by � (beta) Ratio of a/� is dose at which linear and quadratic components are equal

85. 85 Pertinent Conclusions Good evidence that DNA is principal target for radiation induced lethality Membrane damage may also be a factor Following exposure cells may Mitotic death Apoptotic death

86. 86 Pertinent Conclusions Cells that die in mitosis have a one-to-one correlation between cell survival and the average number of �lethal� chromosome aberrations Cells that die apoptotic deaths follow choregraphed sequence of events culminating in breakup of DNA into base pairs of ~ 185 For some cells apoptotic death dominates (lymphoid cells) � survival is an exponential function of dose � straight and shoulderless � there is also no dose-rate effect

87. 87 Pertinent Conclusions Some cells have mitotic death as dominant feature Survival is then linear-quadratic Usually a large dose rate effect Many cells populations have both mitotic and apoptotic deaths Correlation between apoptosis and radiosensitivity If apoptosis dominates, cells are radiosensitive If absent, cells are radioresistant

88. 88 Pertinent Conclusions Cells cultured from human tumors show broad range of radiosensitivities which braket the range of normal human cells Genes that influence the radiosensitivity of mammalian cells have been identified If these genes are defective, repair of double strand breaks is problematic Several human syndromes have been associated with radiosensitivity � AT is best example Link between sensitivity to killing by radiation and predisposition to cancer

89. 89 Pertinent Conclusions Effective survival curves for multifraction regimes is an exponential function of dose - a straight line from the origin through a point on the single-dose survival curve Calculated killings of tumor cells can be peformed for fractionated clinical radiotherapy using the �effective survival curve concept� Mammalian cells are much more sensitive that bacteria and yeast � principally because of their larger DNA content