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Home , Cell synchronization, G1 phase, G2 phase, Radiosensitivity, S phase

Introduction

• Mammalian cells propagate by • The in all dividing mammalian Radiosensitivity and Cell Age in cells is divided into four phases: – M phase can be identified through the light the Mitotic Cycle microscope – After the cells pass through M, there is a period of apparent inactivity, termed G1 (the first "gap" in activity in the cell cycle) Chapter 4 – Next, the cells actively synthesize DNA during the , identified through labeling – Between the S phase and the onset of the next Eric J. Hall., Amato Giaccia, mitosis, there is another gap in activity, G2 • The time between successive divisions for the Radiologist defines the cell cycle time TC

Cell labeling techniques Cell labeling techniques

• Cell visualization during the S phase is based on feeding the cells with radioactive labels or fluorescent dyes, which are incorporated into duplicating • The medium is flushed after a short time period • Cells are fixed and stained for ease of viewing after varying time interval • Tritiated thymidine (3HTdR) incorporated into chromosomes emits b- particles forming a latent image on a layer of photo-emulsion coated over the • Labeled cells are counted sample (takes ~3-4 weeks) • Labeling with dyes (e.g., BrdU) is more convinient; use fluorochrome-tagged to BrdU, observable under a fluorescence microscope

Cell labeling techniques Cell labeling techniques

• BrdU labeling movie: • To ensure free access of the antibody to its http://academics.wellesley.edu/Biology/Concepts/Html/brdu.html antigen, the cells must be fixed and permeabilized • are extremely sensitive to location of a • Sample preparation entails fixing the target cells cellular component or a specific molecule (a tissue to the substrate (microscope slide) antigen) • Perfect fixation immobilizes the antigens, while – Target can be inside or on the surface of a cell retaining authentic cellular and subcellular – Antibody is tagged with a fluorescing dye molecule architecture and permitting access of antibodies to all cells and subcellular compartments

1 Cell labeling techniques • Only a small portion • A biophysical technique that allows counting and of cells in a sample sorting cells in a sample based on their size, uptakes the label structure, and functionality • The technique is very – Functionality is determined through labeling with antigen- labor-intensive, specific fluorescent dyes relying on the manual cell counting • Can discriminate cells at speeds up to 70,000 • New automated cells/second (compare to 200 cells/min manually) approaches • Flow cytometry tutorials: significantly speed up https://www.youtube.com/watch?v=EQXPJ7eeesQ https://www.youtube.com/watch?x-yt-cl=84838260&feature=player_embedded&v=sfWWxFBltpQ&x-yt-ts=1422327029 characterization

The cell cycle times and kinases

• Based on the number of counted labeled • Cyclins and -dependent kinases were discovered by mitosis and varying the time between Hartwell, Hunt, and Nurse, who thus won the 2001 Nobel labeling and counting, can characterize Prize in Physiology or Medicine "for their discoveries of the temporal transitions within a cycle key regulators of the cell cycle" • The difference among mammalian cell • Cyclins are a group of regulatory that control the cycle times, varying from ~10 to 100 progression of cells through the cell cycle by activating hours among different cells in different cyclin-dependent kinase (CDK) enzymes circumstances, is the result of a dramatic • Protein kinases are enzymes that modify the function of variation in the length of the G1 period other proteins by attaching phosphate groups (PO4-) to them (phosphorylate proteins) • The remaining components of the cell – They are key controllers of most biochemical pathways and cycle vary comparatively little important in health and disease Origin of HeLa cells: http://www.youtube.com/watch?v=0gF8bCE4wqA

Progression through the cell cycle Synchronizing cells within cycle

• Each cyclin protein is • Survival curves are collected with the assumption synthesized at a discrete that the population of irradiated cells is phase of the cycle: and E in G , in S asynchronous: cells distributed throughout all 1 phases of the cycle and G2, and in G2 and M. Transitions in the • Study of the variation of radiosensitivity with the cycle occur only if a given kinase activates the proteins position or age of the cell in the cell cycle was made block required for progression possible only by the development of techniques to • Tumor suppressor produce synchronously dividing cell cultures: (, Rb) can block cell populations of cells in which all of the cells occupy division if DNA is damaged the same phase of the cell cycle at a given time Current concept of the cell cycle and its regulation by protein kinases, activated by cyclins

2 Effect of x-rays on synchronously Synchronizing cells within cycle dividing cell cultures

• Two principal techniques of : • The proportion of cells – Mitotic harvest: physical separation of cells preparing for that survive the dose in- mitosis (works only on monolayer cell cultures) creases rapidly with time – Use of a drug, eliminating all cells in S phase, and as the cells move into S imposing a block on cells at the end of G1 (works for cell phase and tissue samples) • When the cells move out of S into and subsequently to a second mitosis, the proportion of surviving cells decreases again

Effect of x-rays on synchronously Variation of radiosensitivity dividing cell cultures with cell age in the mitotic cycle

• Cells are the most sensitive in • Cells are most sensitive at or close to mitosis Calculated for M and G2: survival curves are M phase under steep and have no shoulder • Resistance is usually greatest in the latter part of S phase. The hypoxia • Cells in the latter part of S increased resistance is thought to be caused by homologous phase (LS) exhibit a survival recombination repair between that is curve that is less steep, but more likely to occur after the DNA has replicated has a very broad shoulder • If G1 phase has an appreciable length, a resistant period is • The range of sensitivity evident early in G1, followed by a sensitive period toward the between the most sensitive end of G1 cells (mitotic) and the most resistant cells (late S) is of the • is almost as sensitive as M phase same order of magnitude as • Exceptions to these generalizations have been noted for some Cell survival curves for Chinese hamster cells at various stages of the cell cycle the oxygen effect cell lines

Molecular checkpoint genes Molecular checkpoint genes

• Mammalian cells exposed to radiation tend to • Mutant cells that lose this G2 checkpoint function experience a block in the G2 phase move directly into mitosis • In several strains of yeast, mutants have been with damaged chromosomes isolated that are more sensitive than the wild type – They are at a higher risk of dying - hence their greater to both and UV light by a factor sensitivity to radiation and other between 10 and 100 DNA-damaging agents • The mutant gene has been cloned and sequenced – Cells that survive mitosis are likely to give rise to errors in and found to be a “G2 molecular checkpoint gene" segregation - hence more prone to carcinogenesis

3 The age-response function for a Effect of oxygen tissue in vivo • Effect of oxygen on cell survival curves is • The pattern of response in characterized by oxygen enhancement ratio this organized normal tissue, with a sensitive period D ( hypoxic ) OER  % S between G1 and S and D ( aerated ) % S maximum • The OER was measured at 2.3 to 2.4 for G2 phase late in S, is very similar to cells, compared with 2.8 to 2.9 for S phase, with G1 that characteristic of many cell lines cultured in vitro phase cells showing an intermediate value (label uptake) • This small variation of oxygenation through the • High LET radiation cycle is of little clinical significance in radiation decreases the variation of The survival of jejunal crypt cells exposed to g- radiosensitivity through the therapy rays or neutrons as they pass through the cell cycle after synchronization with hydroxyurea cell cycle

Mechanisms for the age- The implications of the age- response function response function in radiotherapy

• The patterns of radiosensitivity and radio- • Since general population of cells in tissues is resistance correlate with the mechanism of asynchronous, cells in more sensitive phases of the repair of DNA DSBs: cycle are preferentially killed – Radiosensitivity correlates with nonhomologous end joining, which dominates early in the cell cycle and is • Variations in sensitivity through the cell cycle error prone may be important in because – Radioresistance correlates with homologous they lead to "sensitization resulting from recombinational repair, which occurs after replication reassortment" in a fractionated regimen (in S phase) and is more faithful • Not fully understood • Reassortment is one of the “4 R’s of radiobiology”

Introduction

• Radiation damage to mammalian cells can Fractionated Radiation and the operationally be divided into three categories: – (1) lethal damage, which is irreversible and irreparable Dose-Rate Effect and, by definition, leads irrevocably to cell death – (2) potentially lethal damage (PLD), the component of Chapter 5 radiation damage that can be modified by postirradiation environmental conditions – (3) sublethal damage (SLD), which under normal Eric J. Hall., Amato Giaccia, circumstances, can be repaired in hours unless additional SLD is added (e.g., from a second dose of radiation) Radiobiology for the Radiologist

4 Potentially lethal damage repair Potentially lethal damage repair

• PLD is potentially lethal since • Observed both in under ordinary circumstances vivo and in vitro it causes cell death • Hypothesis: radio- • The cell survival can be resistant tumors increased if postirradiation have efficient conditions are suboptimal for mechanisms to growth repair PLD, while – cells do not attempt to divide radiosensitive – have time to repair the damage tumors do not X-ray survival curves for density-inhibited stationary-phase cells, subcultured either immediately or 6 or 12 hours after irradiation

Sublethal damage repair Sublethal damage repair

• SLD repair is the • If cells are allowed to progress operational term for the in- through the cell cycle, than only crease in cell survival that is those in S phase at the time of irradiation repair the damage Single dose of 15.58 Gy observed if a given dose is and survive split into two fractions • They become radiation sensitive To prevent mitosis separated by a time interval again as they move to M phase • Probability of damage repair (6 hours dose split time) increases if cells do not • The cell population doubles if attempt to divide time > TC

Sublethal damage repair Sublethal damage repair

• Repair of sublethal radiation damage has been • Broad shoulder of a demonstrated both in vivo and in vitro cell survival curve is an indication of • Split dose experiments illustrate three of the SLD repair “four Rs" of radiobiology: • For high LET – Repair radiation there is – Reassortment very little of SLD – Repopulation repair • DSB from two • The effect of the fourth "R" – reoxygenation has separate events: also been demonstrated damage is repaired before the 2nd hit

5 The dose-rate effect The dose-rate effect • For x- or g-rays, dose rate is one one the principal factors that determine the biologic consequences of a given dose • As the dose rate is lowered the biologic effect of a given dose generally is reduced • LDR irradiation may be Chinese considered as an infinite number HeLa cells hamster cells of infinitely small fractions – the survival curve has no shoulder – shallower than for single acute • Different cell lines demonstrate different degree of the dose rate effect Multiple small fractions approximate to a exposures depending on the efficiency of SLD repair continuous exposure to a low dose rate • The effect is most pronounced between 0.01 and 1 Gy/min dose rates

The dose-rate effect The dose-rate effect

(due to block in G2)

• HDR and LDR survival curves for a large number of cell lines of human origin in vitro

• LDR curves fan out due to the difference in SLD repair Red arrow shows “inverse dose rate effect”

Brachytherapy Brachytherapy

• Name comes from the Greek word for “short range” • Utilizes a wide range of dose rates, from HDR (>12 Gy/hr* or 20cGy/min**) to LDR (0.2 – 2 Gy/h) (Repair) Normal tissue: lung • Two distinct types: intracavitary and interstitial • Were developed to an advanced stage in the th The range of dose rates over which The dose-rate effect for pneumonitis in beginning of the 20 century due to availability of different R’s modify radiosensitivity mice: correlates with incomplete repairs the sources • Lowering dose rate allows for more tissue sparing • Lower dose rate better discriminates between tissues with *ICRU report 38; there is also a medium dose rate, MDR high and low radiosensitivity ** EBRT dose rate ~1-5 Gy/min (could be much higher for FFF beams) Kogel et al., Basic Clinical Radiobiology, 4th Ed, 2009

6 Brachytherapy Brachytherapy

• If brachytherapy is used as a sole treatment, a commonly used dose is 35 to 70 Gy in 5 to 9 days • Total dose should he adjusted for dose rate (low DR – higher total dose) In HDR afterloader • Clinical studies show that both tumor control and late effects vary with dose rate for a given total dose • Brachytherapy is often used as a boost to external beam therapy, and only half the treatment is given with brachytherapy

Brachytherapy Summary

• HDR afterloaders use Ir-192; typically, • Labeling techniques are used to measure durations several fractions are delivered of cell cycle phases; they vary dramatically between cell lines, mostly due to differences in G1 length – Examples: partial breast irradiation, cervical treatments • Cell radiosensitivity is a strong function of the position in the cell cycle • Permanent implants (LDR): I-125, Pd-103 • Capability of a cell to repair sublethal damage – Example: prostate cancer treatment strongly correlates with radiosensitivity – Radiobiological advantage of LDR: • Dose rate effect is governed by efficiency of considerable sparing of normal tissues sublethal damage repair; other R’s on dif time scales

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