Lecture 4.pdf

Cell Survival Curves 1 REPRODUCTIVE INTEGRITY • A cell survival curve illustrates the relationship between radiation dose and cell survival proportion. • Definition of "survival" varies based on cell type; for proliferating cells, loss of reproductive integrity is significant. • Reproductive int...

Cell Survival Curves 1 REPRODUCTIVE INTEGRITY • A cell survival curve illustrates the relationship between radiation dose and cell survival proportion. • Definition of "survival" varies based on cell type; for proliferating cells, loss of reproductive integrity is significant. • Reproductive integrity refers to the cell's capacity for sustained proliferation and the ability to produce a large number of progeny. • Cell Death Definition: • Differentiated cells: (such as nerve, muscle, or secretory cells) death can be defined as the loss of a specific function • Proliferating cells : (such as stem cells in the hematopoietic system or the intestinal epithelium) death can be defined as the loss of the capacity for sustained proliferation (Reproductive death) • Reproductive death implies the inability to divide indefinitely and generate a large clone of cells. 2 • Clonogenic cells retain reproductive integrity and can proliferate indefinitely. REPRODUCTIVE INTEGRITY • Implications and Dose Considerations • Cell Death Mechanisms: • Mitotic Death: Dominant mechanism in most cells, death while attempting to divide. • Apoptosis: Programmed cell death is significant in specific cell types. • Clinical Significance: • Understanding reproductive integrity aids in tumor eradication strategies. • Radiotherapy focuses on rendering tumor cells unable to divide, inhibiting further growth. • Dose calibration crucial to achieving desired outcomes in cancer treatment. • Radiation Dose Requirements: • Nonproliferating Systems: Approximately 100 Gy needed to destroy cell function. 3 • Loss of Proliferative Capacity: Mean lethal dose usually less than 2 Gy. THE IN VITRO SURVIVAL CURVE • Cell Culture Techniques: • Tissue specimens from tumors or normal tissues can be dissociated into single-cell suspensions using trypsin. • Seeded into a culture dish with appropriate growth medium, maintained at 37° C under aseptic conditions, cells attach, grow, and divide. • Cultures undergo periodic farming to maintain viability, resulting in established cell lines (stock cell lines ) used in cellular radiobiology experiments . 4 THE IN VITRO SURVIVAL CURVE • • Experimental Approach and Observations Experimental Setup: • Cells from a growing stock culture are trypsinized, counted, and seeded into dishes. • Control: Cells form visible colonies indicating reproductive integrity. • Irradiation: Cells exposed to radiation (e.g., 8 Gy of x-rays) undergo varied responses. • Observations: 1. Unirradiated Cells: o Single cells divide and form colonies. o Each colony originates from a single ancestor. o Plating efficiency measures the percentage of cells forming colonies. 2. Irradiated Cells: o Some cells remain single or show apoptotic signs. o Others form abortive colonies after limited divisions. o Some cells grow into colonies, indicating retained reproductive integrity. A. In this unirradiated control dish, 100 cells were seeded and allowed to grow for 7 days before being stained. There are 70 colonies; therefore, the plating efficiency is 70/100 or 70%. B: Two thousand cells were seeded and then exposed to 8 Gy of x-rays. There are 32 colonies on the dish. Thus, Surviving fraction 5 = Colonies counted / [Cells seeded × (PE / 100)] = 32/ (0.7 × 2,000) = 0.023 THE SHAPE OF THE SURVIVAL CURVE • Mammalian cell survival curves are vital in radiation therapy, depicting how cells respond to various doses of radiation. • Survival Curve Characteristics: • Shape: Linear scale for dose and logarithmic scale for surviving fraction. • Sparsely Ionizing Radiations (Low-LET): X-rays show exponential decrease in surviving fraction with dose. • Densely Ionizing Radiations (High-LET): αparticles or low-energy neutrons display a straightline curve, approximating exponential cell killing. A: The linear-quadratic model. The experimental data are fitted to a linear-quadratic function. There are two components of cell killing: One is proportional to dose (αD); the other is proportional to the square of the dose (βD2). The dose at which the linear and quadratic components are equal is the ratio α/β. The linear-quadratic curve bends continuously but is a good fit to experimental data for the first few decades of survival. B: The multitarget model. The curve is described by the initial slope (D1), the final slope (D0), and a parameter that represents the width of the shoulder, either n or Dq. 6 THE SHAPE OF THE SURVIVAL CURVE • • • • • • • Model 1: The single-target/single-hit model. The single-target/single-hit model has little practical application Assumes a single target in the cell. Cell dies if this target is hit, no repair opportunity. The single-target single-hit model is inadequate to explain most cell survival data from mammalian cells, because it does not account for the shoulder portion of the curve at low doses. In this model, D is the dose delivered and D0 is a constant, defined as the dose that gives on average one hit per target. A dose D = D0 reduces the surviving number of cells to 37% of the initial population. 7 THE SHAPE OF THE SURVIVAL CURVE • Model 2: The Multitarget Model • cell contains two or more targets that must be hit before the cell is killed. Assumes multiple targets per cell. Cell requires hits on multiple targets in a short time for death. Repair can occur between hits, causing sublethal damage. The simplest equation for the multitarget model is • • • • • • • Multitarget models can fit both high-LET (no shoulder) and lowLET (shoulder) radiations. Low-LET Curve: Bending "shoulder" at low dose. n=6, D q =2.5Gy. High-LET Curve: No shoulder. n=1,D q =0. 8 • THE SHAPE OF THE SURVIVAL CURVE The Linear-Quadratic Model • LQ model provides a better fit for the initial shoulder region of the survival curve. • Combination of linear and quadratic components.. • Accounts for both sublethal and lethal damage. Single-hit survival: SF1=exp(−αD) Two-hit survival: SF2=exp(−βD^2) Overall survival curve: SF=SF1×SF2=exp(−αD−βD^2) • α: Represents the single-hit component, indicating cell killing due to single hits. • β: Represents the multiple-hit component, accounting for two-hit cell killing. α/β Ratio • • • Definition: Dose at which linear and quadratic contributions to the survival curve are equal (α = β). Significance: Indicates the width of the shoulder in the survival curve. α/β Values: Low values (1–5 Gy) indicate radioresistant or late responders, high values (6–12 Gy) indicate radiosensitive or early responders. 9 MECHANISMS OF CELL KILLING DNA as the Target: • 1. 2. 3. 4. Evidence for chromosomal DNA as the principal target for cell killing is circumstantial but overwhelming and may be summarized as follows: Radioactive Tritiated Thymidine Incorporation: Cells are killed by radioactive tritiated thymidine incorporated into DNA due to localized short-range β-particles. Selective Incorporation of Structural Analogues: Halogenated pyrimidines, analogues of thymidine, increase radiosensitivity when incorporated into DNA, indicating DNA as a primary target. Relation Factors to Chromosome Damage: Factors modifying cell lethality, radiation type, oxygen concentration, repair mechanism, and dose rate affect chromosome damage, linking it to cell lethality. Correlation with Chromosome Volume: Radiosensitivity in various organisms correlates with mean interphase chromosome volume, indicating the importance of chromosome damage in lethality. 1 0 MECHANISMS OF CELL KILLING The Bystander Effect Bystander Effect defined as the induction of biological effects in non-irradiated cells near irradiated ones. Factors Influencing Bystander Effect o Cell Communication: Effect is pronounced in cells connected via gap junctions. o Medium Transfer Experiments: Irradiated cells secrete molecules causing cell death in non-irradiated cells. 1 1 MECHANISMS OF CELL KILLING Apoptotic and Mitotic Death Apoptosis: Programmed Cell Death • Morphological Changes: Condensation of chromatin, cell shrinkage, fragmentation. • Role in Radiation: Common in embryonic development, radiation-induced in some normal tissues and tumors. • p53 and Bcl-2: Key regulators; apoptosis often p53-dependent. • *Unnecessary cells undergo apoptosis when their function serves no purpose. Mitotic Death • • Chromosomal Aberrations: Asymmetric exchange-type aberrations lead to cell death. • Relationship with Cell Killing: Quantitative correlation observed between aberrations and cell survival. • Linear-Quadratic Relationship: At low doses, linear relationship due to a single electron; at higher doses, quadratic relationship with two separate electrons. Apoptosis and mitotic cell death are not mutually exclusive. Some cells may undergo apoptosis after mitotic cell death 1 2 MECHANISMS OF CELL KILLING Autophagic Cell Death • Autophagy Process: defined as a self-digestive process that uses lysosomal degradation of long-lived proteins and organelles to restore or maintain cellular homeostasis. • Autophagy can be induced by radiation and other stresses. • Autophagic cell death is a type II of programmed cell death. • Autophagy can be a protective mechanism for cells to survive and generate nutrients and energy, but it can also promote cell death. • The role of autophagy in irradiated cells is still under investigation for the enhancement of cancer cell killing. 1 3 MECHANISMS OF CELL KILLING Senescence: Cellular Stress Response • Cellular senescence is a programmed cellular stress response that occurs in response to a variety of stimuli, including telomere shortening, oncogene activation, and DNA damage. • Senescence leads to an irreversible cell cycle arrest, which prevents cells from dividing further. • Senescence is characterized by the activation of the tumor suppressor proteins p53 and Rb that result in the silencing of genes necessary to promote transition from the G1 to the S phase of the cell cycle. • Senescence is thought to be a tumor suppressor mechanism, as it 1 prevents excessive cellular proliferation. 4 SURVIVAL CURVE SHAPE AND MECHANISMS OF CELL DEATH Radiosensitivity and Apoptosis in Mammalian Cells • Mammalian cells cultured in vitro vary considerably in their sensitivity to killing by radiation. • This is due to a number of factors, including 1- The presence of DNA damage repair mechanisms, 2- The cell cycle stage 3- The type of cell death that occurs. • One type of cell death, apoptosis, is closely correlated with radiosensitivity. • Apoptosis is a programmed cell death process that results in the fragmentation of DNA into discrete lengths. • Cell lines that are more radiosensitive show more evidence of apoptosis. 1 5 GENETIC CONTROL OF RADIOSENSITIVITY • Radiosensitive Mutants in Mammalian Cells • Complexity in Mammals: Mammalian cells involve numerous genes in determining radiosensitivity. • Examples: Ku 80, Ku 70, and XRCC7 genes; defects impair DNA doublestrand break (DSB) repair. • These genes are involved in various aspects of DNA repair, and mutations in these genes can lead to a greatly reduced ability to repair DNA double-strand breaks (DSBs). • Inherited Human Syndromes Associated with X-rays 1. AT (Ataxia-telangiectasia): Profound radiosensitivity, elevated cancer risk, ATM gene mutation. 2. Seckel Syndrome, 3. Nijmegen Breakage Syndrome: Radiosensitive conditions due to genetic factors. 1 4. Fanconi Anemia 6 5. Homologues of RecQ-Bloom Syndrome: Linked to X-ray sensitivity; implications in radiation therapy. EFFECTIVE SURVIVAL CURVE FOR A MULTIFRACTION REGIMEN • Multifraction radiotherapy is the most common type of radiotherapy, in which the total dose is delivered in a series of smaller fractions over time. • The effective dose-survival curve is a simplified model of multifraction radiotherapy that takes into account the repair of sublethal damage between fractions. • The effective dose-survival curve is an exponential function of dose, meaning that the fraction of surviving cells decreases exponentially with increasing dose. • The D0 of the effective dose-survival curve is the dose required to reduce the fraction of surviving cells to 37% and has a value close to 3 Gy for cells of human origin cells. • The D10 is the dose required to kill 90% of the population. It is related to the D0 by the expression D10 = 2.3 × D0. 1 7 CALCULATIONS OF TUMOR CELL KILL Problem 1 A tumor consists of 10^8 clonogenic cells. The effective dose–response curve given in daily dose fractions of 2 Gy has no shoulder and a D0 of 3 Gy. What total dose is required to give a 90% chance of tumor cure? Answer To give a 90% probability of tumor control in a tumor containing 10^8 cells requires a cellular depopulation of 10^−9. This means that we need to kill 99.99999% of the tumor cells. The dose resulting in one decade of cell killing (D10) is given by D10 = 2.3 × D0 = 2.3 × 3 = 6.9 Gy The total dose for nine decades of cell killing, therefore, is 9 × 6.9 = 62.1 Gy. 1 8 CALCULATIONS OF TUMOR CELL KILL Problem 2 Suppose that in the previous example, the clonogenic cells underwent three cell doublings during treatment. About what total dose would be required to achieve the same probability of tumor control? Answer Three cell doublings would increase the cell number by 2 × 2 × 2= 8 Consequently, about one extra decade of cell killing would be required, corresponding to an additional dose of 6.9 Gy. The total dose is 62.1 + 6.9 = 69 Gy. 1 9 CALCULATIONS OF TUMOR CELL KILL • Problem 3 During the course of radiotherapy, a tumor containing 10^9 cells receives 40 Gy. If the D0 is 2.2 Gy, how many tumor cells will be left? • Answer If the D0 is 2.2 Gy, the D10 is given by D10 = 2.3 × D0 = 2.3 × 2.2 = 5 Gy Because the total dose is 40 Gy, the number of decades of cell killing is 40 / 5 = 8. The number of cells remaining is 10^9 × 10^−8 = 10. 2 0 CALCULATIONS OF TUMOR CELL KILL Problem 4 If 10^7 cells were irradiated according to single-hit kinetics so that the average number of hits per cell is one, how many cells would survive? Answer A dose that gives an average of one hit per cell is the D0, that is, the dose that on the exponential region of the survival curve reduces the number of survivors to 37%. The number of surviving cells is therefore 2 1 Radiosensitivity and Cell Age in the Mitotic Cycle 22 • • • THE CELL CYCLE The cell cycle is a fundamental process in all living organisms. It describes the series of events that a cell undergoes as it grows and divides into two daughter cells. Phases of the Cell Cycle: Interphase: The period between mitoses when cells prepare for the next division; not observable through conventional microscopy. 1. G1 Phase (Gap 1): Cell grows and performs its normal functions. 2. S Phase (Synthesis): DNA replication occurs, ensuring genetic material duplication. 3. G2 Phase (Gap 2): Cell continues to grow and prepares for division. Mitosis: The phase during which a cell divides into two progeny cells, identifiable by condensed chromosomes and rounding up of cells using conventional light microscope. 1. Prophase: Chromosomes condense, and the mitotic spindle forms. 2. Metaphase: Chromosomes align at the cell's equator. 3. Anaphase: Chromatids separate and move to opposite poles. 4. Telophase: New nuclei form around separated chromatids. • • o Mitotic Cycle Time (TC): The time between successive cell divisions. • • • Checkpoint Proteins: Monitor the cycle's progress, ensuring accuracy. Cyclins and Cyclin-Dependent Kinases (CDKs): Regulatory proteins controlling cell cycle transitions. Tumor Suppressor Genes (e.g., p53): Prevent cells with damaged DNA from progressing in the cycle. • • • Essential for growth, development, and tissue repair. Maintains genetic stability by ensuring accurate replication and segregation of DNA. Dysregulation can lead to diseases, including cancer. Regulation of the Cell Cycle: Significance of the Cell Cycle: 2 3 SYNCHRONOUSLY DIVIDING CELL CULTURES • • Importance: Synchronously Dividing Cell help in studying radiosensitivity of different cell cycle phases aids in understanding radiation effects. Techniques for Producing Synchronously Dividing Cell Cultures • • • Mitotic Harvest Technique: • Applicable to monolayer cultures attached to growth vessel surfaces. • Mitotic cells detach and float in medium upon gentle shaking. • Plating mitotic cells yields a synchronized population. • Radiation doses given at various phases of the cell cycle. Drug-Induced Synchronization: • Hydroxyurea: Widely used drug. • Kills S-phase cells and blocks G1 phase. • Cells in G2, M, and early G1 accumulate at the block. • Removal of the drug allows synchronized cells to progress through the cycle. Applications of Synchronized Cell Cultures • In Culture: • Widely used in laboratory experiments. • Aid in studying radiation effects on specific cell cycle phases. 2 4 THE EFFECT OF X-RAYS ON SYNCHRONOUSLY DIVIDING CELL CULTURES Radiosensitivity in the Cell Cycle. • • • • Experiment Overview • • Mammalian cells ( Chines hamster cell ) harvested at mitosis. Irradiated with a single dose of 6.6 Gy at different cell cycle phases. • • • 1 hour after mitosis (G1 phase): 6.6 Gy resulted in 13% survival. Survival increases rapidly in S phase, reaching 42%. Falls as cells move to G2 and subsequent mitosis. • • Common pattern in Chinese hamster cells. Surviving fraction peaks late in S phase. Observations and experiment results - Figure 4.7 Characteristic Response Detailed Survival Curves - Figure 4.8 o Mitotic Cells • Steep survival curve, no shoulder. o G1 and G2 Cells o Late S Phase o Oxygen Effect Comparison • Intermediate sensitivity. • Shallower curve with a broad initial shoulder. • Mitotic cells under hypoxia: 2.5 times shallower slope. 2 5 THE EFFECT OF X-RAYS ON SYNCHRONOUSLY DIVIDING CELL CULTURES Radiosensitivity in the Cell Cycle.(cont.) • • Cell Cycle Length Variation - Figure 4.10 Comparison • • • Short G1 Phase (Hamster Cells) vs. Long G1 Phase (HeLa Cells). Overall similar cyclic variation in sensitivity. G2 Sensitivity and Checkpoint • • • Difficult to determine due to synchrony decay and short transit times (about 1 to 2 hours). Retroactive synchronization reveals sharp sensitivity transition around x-ray transition point. (now often called a “checkpoint”) for G2 cell cycle delay. Summary of Radiosensitivity Variations 1. Most sensitive at or near (M phase )mitosis. 2. Greatest resistance in late S phase (homologous recombination repair likely). 3. G1 phase shows early resistance and late sensitivity (if G1 is long). 4. G2 phase usually sensitive, potentially as much as M phase. 2 6 MOLECULAR CHECKPOINT GENES • • • Molecular Checkpoint Genes: Control cell cycle progression. Radiation-induced cell cycle block in G2 phase. Inverse dose-rate effect observed in human cells. • • cells become more sensitive to radiation-induced cell killing as the dose rate is reduced, resulting in their accumulation in G2, which is a radiosensitive Function of Checkpoint Genes • Checkpoint genes halt cells in G2 phase. • Purpose: Inventory of chromosome damage, initiate and complete repairs before mitosis. • Loss of G2 checkpoint gene function leads to direct progression into mitosis with damaged chromosomes. • Loss of G2 checkpoint gene Increased risk of cell death, sensitivity to radiation and DNA-damaging agents. • Mechanism of G2 Checkpoint Genes • • • Involvement of Cdk1 (p34 protein kinase) in checkpoint gene action. Cdk1 levels control progression through mitosis. Checkpoint genes monitor spindle function during mitosis. 2 7 THE AGE-RESPONSE FUNCTION FOR A TISSUE IN VIVO • Radiosensitivity Variation During Mitotic Cycle in Mammalian Cells • Most research conducted on mammalian cells in vitro due to ease of synchronous division. • Synchronization Techniques • • Mitotic Harvest Technique: Applicable to monolayer cultures only. Hydroxyurea Method: Drug-induced synchronization in some tissues (Kills S cells, accumulates cells at G1/S boundary for at least 4 hours). • Hydroxyurea given in 5 intraperitoneal injections, synchronizing cells at G1/S boundary for at least 4 hours. • Case Study: Mouse Jejunum • • • Self-Renewal Tissue: Mouse jejunum's epithelial lining is a self-renewal tissue. Experimental Procedure: Synchronized crypt cells using hydroxyurea injections. Radiation Dose: Single dose of 11 Gy of γ-rays administered at various times post-hydroxyurea injections. • Results • • Radiosensitivity Variation: Ranging from 2 survivors/circumference (2 hours post-injection) to 200 survivors/circumference (6 hours post-injection). DNA Synthesis Monitoring: Tritiated thymidine uptake assay. • Peak coincides with maximum x-ray resistance (about 5 hours post-injection). 2 8 SUMMARY OF PERTINENT CONCLUSIONS • The Cell Cycle in Mammalian Cells • • • Cell Cycle Duration: • • • OER slightly lower for cells in G1 compared to cells in S. Age-Response Function: • • Checkpoint genes halt cell cycling post-DNA damage, allowing chromosome integrity check before mitosis. Oxygen Effect Ratio (OER): • • M and G2 phases: Most radiosensitive. Late S phase: Most resistant. Second peak of resistance in early G1 for cells with long G1 phases. Molecular Checkpoints: • • Fastest cycling mammalian cells and crypt cells: 9 to 10 hours cycle time. Resting mouse skin stem cells: >200 hours cycle time, primarily due to variable G1 phase length. Radiosensitivity in Cell Cycle: • • • • The cell cycle for mammalian cells consists of four phases: Mitosis (M), G1, DNA synthesis phase (S), and G2, followed by mitosis again. Phases regulated by periodic activation of Cdk family members. Crypt cells in mouse jejunum: Similar age-response function as cultured cells. Radiosensitivity Mechanisms: • • Radiosensitivity: Nonhomologous end-joining (early cell cycle (G1 phase), error-prone). Radioresistance: Homologous recombinational repair (after replication in S phase, more faithful). 2 9