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High Yield Summary Reviews in Oncology

Radiation and Biology Gayle E. Woloschak, PhD

Tuesday, June 18, 2019 11:00 a.m. – 1:00 p.m. Eastern Daylight Time

June 25, 2019 Gayle E. Woloschak PhD

Professor of Radiation Oncology, , and Cell & Associate Dean for Graduate Student and Postdoctoral Affairs Northwestern University Feinberg School of

June 25, 2019

Definitions

• Bq-2.7x10-11 Ci—equal to one disintegration per sec • Ci—3.7x1010 disintegrations per sec • Gy—absorbed radiation dose, the quantity which deposits 1 Joule of energy per kg—1Gy=100rad • —dose equivalence—multiply in Gy by the Quality Factor (Q)— 1Sv=100rem • LET—measure of the rate of energy transfer from an to the target material—keV of energy lost/micron track length Unit Conversion Factors: SI and Conventional Units • 1 Bq = 2.7 × 10–11 Ci = 27 pCi • 1 Ci = 3.7 × 1010 Bq = 37 GBq • 1 Sv = 100 rem • 1 rem = 0.01 Sv • 1 Gy = 100 • 1 rad = 0.01 Gy • 1 Sv Bq–1 = 3.7 × 106 rem μCi–1 • 1 rem μCi–1 = 2.7 × 10–7 Sv Bq–1 • 1 Gy Bq–1 = 3,7 × 106 rad μCi–1 • 1 rad μCi–1 = 2,7 × 10–7 Gy Bq–1 Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Units Used in

• —average dose x radiation weighting factor—Sv • Effective dose—sum of equivalent doses to organs and tissues exposed, each multiplied by the appropriate tissue weighting factor—Sv • Committed equivalent dose—equivalent dose integrated over 50 years (relevant to incroporated )—Sv • Committed effective dose—effective dose integrated over 50 years (relevant to incorporated radionuclides)--Sv • Collective effective dose—product of the average effective dose and the number of individuals exposed—person-Sv • Collective effective dose committed—integration of the over 50y (relevant to incorporated radionuclides)—person-Sv uR/hr US Geological Survey, 1993 2-6 Annual Report 2008 NCRP, July 2006 Indirect Action

For Low LET radiation, 67% damage is indirect action

For High LET radiation, most (all?) damage is direct action

Critical distance of indirect action is within 2nm radius from DNA.

Hall and Giaccia, 2018 Stochastic vs Deterministic

• Heritable effects and cancer • Non-cancer somatic effects • Single cell effect • Organ effect • 2 severity states • Different severity states (present/absent) (graded) • No threshold dose • Threshold • Low dose or high dose • Intermediate or high dose • Dose-response: Linear • Dose-response: threshold Quadratic sigmoid • Relevant to radiation • Relevant to normal tissue early protection, diagnostic radiology and late effects in radiotherapy • Example: cancer • Example: , normal tissue damage Summary of Aberrations

• Chromosomal Aberrations • cell is irradiated in G1 (before duplication of chromosomes) • break occurs in single strand of chromatin replicated in S, seen in M phase • translocations dicentrics and rings – both lethal • dicentrics used for dose estimates, 25cGy is sensitivity • Chromatid Aberrations • cell is irradiated after S (after duplication) • radiation causes break in both chromatids • observe it in M phase • anaphase bridge--lethal • Correlation of chromosome aberrations and : • physical abnormalities • mental retardation • cancer • lethality Issues of Radiation Genetics

• No unique mutations • Most mutations are not detected; those that are detected are most often dominant (single gene) • Mutations are rare events • Within the same species, genes vary both in their spontaneous mutation rate and in their vulnerability to radiation. • Number of mutations is proportional to dose. Summary of Radiation Genetics

• Doubling dose for mutations in male mice (low LET): acute 20-30cGy; protracted 90-100cGy • Estimated mutagenic hazard in humans:2-3x10-7 mutations/gene/cGy • Period at risk for expression of a radiation mutation: 10 genetic generations of 30y each=300 y • Genetic maximum permissible dose limits: occupational 50mSv/y; general population 1mSv per year Radiation and Cell Cycle

1.Cells are most sensitive to radiation at or close to M 2.Cells are most resistant to radiation in late S 3.For prolonged G1 → a resistant period is evident early G1 followed be a sensitive period in late G1 4.Cells are usually sensitive to radiation in G2 (almost as sensitive as in M) There are exceptions to these rules in some cell lines. Frequency of chromosomal aberrations is a linear quadratic function of dose. Low Dose: both aberrations caused by the same e- High Dose: caused by different

FIGURE 2.20 The frequency of chromosomal aberrations (dicentrics and rings) is a Iinearquadratic function of dose because the aberrations are the consequence of the interaction of two separate breaks. At low doses, both breaks may be caused by the same ; the probability of an exchange aberration is proportional to dose (D). At higher doses, the two breaks are more likely to be caused by separate electrons. The Q probability of an exchange aberration is proportional to the square of the dose (D2). Hall and Giaccia 2018 Contribution to Killing

• M phase/G2—alpha-large beta-nil • G1 phase—alpha-large beta-small • Early S—alpha and beta are medium • Late S—alpha-small beta-large Cell-survival curves

Survival curve describes the relationship between the proportion of cell survival and radiation dose

linear-quadratic model:

2 S = e−D−D S = fraction of surviving D = dose ,  = constants

D = D2 → D = / (dose at which the linear and quadratic contributions to cell killing are equal)

densely ionizing radiation: /  sparsely ionizing radiation: / 

Hall and Giaccia 2018 Single hit. multitarget Single target

2-component L-Q model

Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Cells are more sensitive to Radiation in the presence of Oxygen than in its absence High dose region of survival curve

Low dose region of survival curve

Hall and Giaccia 2018 Oxygen Concentration and Sensitivity to Radiation

Approx. partial pressure of O2 Cells reach full at which gamma-irradiated cells radiosensitization at exhibit halfway 20-40mmHg, near the between their fully aerobie O2 concentration in and fully hypoxic response venous blood. is near 3mmHg

1mmHg=1Torr

Radiosensitivity increases until about 30mmHg is achieved, and then it remains the Same for all oxygen concentrations. The most dramatic increase is seen at low O2 Concentrations near 0.5% (3mmHg). From Hall and Giaccia, 2018 Sizes of Tumors and presence of necrotic core regions

All tumors with a radius of 200um or greater have necrotic centers. Tumors with a radius of less than 160um usually do not have necrotic centers.

From Hall and Giaccia, 2018 Diffusion of Oxygen through Tumor Tissue

Regions of hypoxia also have low pH, glucose, GFs

Oxygen can generally diffuse 150um at the arterial end of the capillary and less at the venous end. From Hall and Giaccia, 2018 HPH or PHD

From Hall and Giaccia, 2018 Relationship of OER/RBE with LET

The point at which the RBE increases is where the OER drops most dramatically.

Hall and Giaccia 2018 Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Oxygen Effect During Cell Cycle

OER – • Oxygen enhancement ratio • Varies throughout the cell cycle G2 phase OER = 2.3 - 2.4 S phase OER = 2.8 – 2.9 • For any given phase of the cell cycle, OER was the same for all doses • For asynchronous cells, OER does vary with doses: OER is smaller at higher levels of survival OER is higher at lower levels of survival • Little therapeutic significance High vs. Low LET

Effect High LET Low LET DNA damage Mostly direct 2/3 is indirect Ionizations Dense (blobs) Sparse (spurs) survival curve Steeper slope; reduce Smaller slope, larger shoulder shoulder Alpha/beta Alpha is high, beta is Alpha is less important constant Cell cycle effects +/- ++++ SLDR none ++ PLDR none ++ OER O2 has little or no effect on O2 enhances low LET high LET effects Radioprotectors Little effect (?) effective Categories of Cellular 1. Lethal—irreversible, irreparable, leads to 2. Sublethal (SLD)—repaired in hours; if a second dose is given, can interact with more damage to create lethal damage; represents shoulder on cell survival curve Sublethal Damage Repair—increase survival when a dose is fractionated over time 3. Potentially Lethal Damage (PLD)—can be modified by the post-irradiation environment Dose-Rate Effect for HeLa Cells

-Decrease dose-rate, survival curve is More shallow, shoulder disappears, Survival is an exponential function of dose Shallow shoulder to survival curve -For HeLa Cells: dose-rate effect is most for low dose rate Evident at .01-1 Gy/min. Above or below That there is little effect. -Dose rate effect varies from one cell type To another

similar

Hall and Giaccia 2018 Amifostine •WR2721 → WR1065 (in vivo) •Active transport of drug to normal tissues vs. tumor (?) Give drug minutes before radiation, ↓ normal tissue toxicity •Alkaline phosphatase (liver) converts inactive prodrug amifostine into the active drug in the body. •Tissue variability: good protection: hematopoietic system, gut lining, salivary glands no protection: CNS (doesn’t cross blood-brain barrier) •Hydrophilic nature gives ↑ uptake in normal tissues •Dose-limiting toxicity: •Also call ethyol •Is FDA approved for use as a radioprotector

Law of Bergonie and Tribondeau

The radiosensitivity of a cell varies: • DIRECTLY with its rate of mitosis. • DIRECTLY with its mitotic future. • INDIRECTLY with its degree of morphologic and functional differentiation.

• EXCEPTION: Lymphocytes which are very radiosensitive but are fixed post- mitotic cells. Cassarett’s formulation:

The more differentiated, the more radioresistant the cell type. Cells with increased capability for division are more radiosensitive than non-dividing cells. Exception: Lymphocytes (D0 of 80cGy) Hall and Giaccia 2010 10-49h

Doses given in Gy, but they represent Gy of gamma-rays or x-rays Narrow window of dose over which BM transplants are useful following TBI

LD50 increases with antibiotics and nursing. Hall and Giaccia 2010 Effects of 200R on fetal development given at different times post-fertilization:

Hall and Giaccia 2010 Comparisons of Lab Animal and Human Data on Radiation Effects

Mental retardation: High risk:40%/Sv Low risk: 10%/Sv

FIGURE 12.9 Chart illustrating the similarities and differences between data from small laboratory animals and data from the Japanese survivors of the atomic bomb attacks. The gestation weeks are for the human; the equivalent gestation periods have been matched for animal studies. Both sets of data indicate that irradiation early in gestation may result in the death of the embryo but that malformations do not occur. The animal data show a high incidence of a wide spectrum of malformations during organogenesis. The principal finding in the Japanese is microcephaly, which occurred up to 15 weeks, and mental retardation, which occurred most frequently following irradiation at 8 to 15 weeks of gestation and, to a lesser extent, at 16 to 25 weeks.

-Radiation early in gestation may cause embryonic death but not malformations. -Malformations occur during organogenesis -During fetal period, microcephaly and mental retardation result. Hall and Giaccia 2018 Hall and Giacia 2010 Table 15.5

15-25 w for mental retardation is 10%/Sv

Hall and Giaccia 2010 Cell death vs. time following radiation damage in F- and H-type tissues

Taken from work done By Curt Sigestadt

Extrapolation from rodents to humans Early Responding: Extra dose Conventional needed therapy is never to counter long enough to proliferation in include early proliferation responding of late-reacting tissues begins tissues. within a few weeks and occurs during the time course of Hall and conventional Giaccia 2010 therapy

Prolonging time-range of normal therapy has little sparing effect on late reactions, but large sparing effect on early reactions. Dose-Response for Early vs. Late Responding Tissues:

Alpha/beta Alpha/beta is near small 10, more near 2, more linear than curved curved at low doses

Alpha/Beta > for early than late responding tissues where: alpha is the linear component, beta is the quadratic component alpha/beta is the dose where linear and quadratic components of cell killing are equal or dose at which single and multiple event killing are equal Hall and Giaccia 2010 Late vs. Early Reacting Tissues

• For late reacting tissues: -Fraction size dominates the late effects -Overall treatment time has little influence on late effects • For early-reacting tissues/tumors: -Fraction size is somewhat important -Overall treatment time is important Isoeffect Curves: Early vs. Late Effects

Hall and Giaccia 2008

FIGURE 23.7 Isoeffect curves in which the total dose necessary for a certain effect in various tissues in laboratory animals is plotted as a function ofdose per fraction. Late effects are plotted with solid lines, acute effects with dashed lines. The data were selected to exclude an influence on the total dose of regeneration during the multifraction experiments. The main point of the data is that the isodoses for late effects increase more rapidly with a decrease in dose per fraction than is the case for acute effects. (Adapted from Withers HR. Biologic basis for altered fractionation schemes. Cancer. 1985;55:2086- 2095, with permission.)

Isoeffect Curves are steeper for late effects than for early effects. Major Human Population Exposures 1. Japanese A-bomb - very important group for risk estimates - 120,000 people followed long term - registry of exposures no + γ-rays → precise gave rise to ↑ risk estimates of cancer 2. Ankylosing Spondylitis - x-radiotherapy to spine--↑ leukemia - salt injections—bone tumors 3. Thymus enlargement radiotherapy – radiotherapy lead to ↑ thyroid cancer 4. Radiotherapy for adenoids, tonsils—thyroid cancer 5. Tinea capitis (immigrants from N. Africa to Israel) - x-ray treatment of scalp for fungus → thyroid cancer → skin cancer Also had meningiomas, other brain tumors, leukemia, salivary gland tumors American experience had lower incidence of tumors: thyroid, skin 6. Fluoroscopy of TB patients - ↑ 7. Thorotrast patients--Liver tumors Thorotrast (contrast material for x-rays) – radioactive Thorium α-particles 8. Peptic ulcer—patients irradiated for peptic ulcer, higher risk for stomach cancer in those getting <10Gy Cancer and Human Exposures: Occupational Exposures Cancer is the most common late somatic effect in irradiated populations.

1. Skin cancer - early discoveries of radiation, people exposed by putting hand in the beam - Dentists putting their hand in the beam

2. Lung cancer - Uranium miners in Colorado - Radium miners in Britain α-particles / Rn exposure Recent concerns about Rn in the home

3. Bone tumors - Radium dial painters

4. Leukemia - Radiologists before 1920’s - People administering radiotherapy with radionuclides before 1920’s Radiation-induced tumors

• Latent Period – time interval between exposure to radiation and appearance of malignancy • Leukemia: begins at 2y (earlier?), peaks at 7-12 y., gone by 20 y. • Thyroid tumors: 4+y, peak at 7-10y. • Other solid tumors: 20+ y (possibly 12y). • Latent period is affected by age at exposure and by age of tumor expression • Radiation-induced tumors tend to be expressed later in life at the time of spontaneous tumors of the same type. ex/ breast tumors may be hormone dependent Hypothetical Dose-Response Curve for HDR vs. LDR Exposures

Exposures to HDR are more transforming than LDR Excess cancer incidence LNT model predicts is a L-Q function of dose no safe dose of Human data may be radiation adequately fitted by a straight line, a linear function of dose

Hall and Giaccia 2010 Hall and Giaccia 2010 E. J. Hall and A. J. Giaccia, “Radiobiology for the Radiologist,” 8th Edition, Lippincott Williams & Wilkins, Philadelphia, 2018. pg. 245. Table 16.5 Figure 17.4 This chart compiled by Dr. Noelle Metting, Office of Science of the Department of Energy, puts into perspective the different dose ranges relevant to , diagnostic radiology, and . Hall and Giaccia 2010 Chromosomal alterations and oncogene activation events associated with specific tumors

Ret oncogene is associated with thyroid cancer. Hall and Giaccia ErbB is associated with head and neck cancer. 2010 Tumor suppressors: Rb, WT, NF1, FAP, p53, DCC Facts about p53

• P53 is a tumor suppressor that is lost or inactivated in over half of human . • Wtp53 is activated by damage to DNA. • Damage to DNA during G1 can lead to blocking cell cycle progression (G1 arrest). Damage to DNA during G2 can lead to . • P53 binds to promoters that contain a 20bp recognition sequence that usually activates transcription. • P53 activates p21, mdm2, gadd45, PCNA, BAX, NOXA, PUMA, others • P53 also binds to ss regions that are generated in damaged DNA including those at telomeres. • Most human cancer cells either have mutations that inactivate p53 directly or have mutations in other loci that lead to loss of p53. • Some viruses lead to p53 inactivation by binding to p53….these include E6 of HPV, Adenovirus E1B, SV40 T Ag • P53 is destabilized by mdm2, which targets it for degradation and also directly inhibits its transactivation activity. • The INK4a-ARF locus codes for p16(INK4a) which controls RB and for p19(ARF) which controls p53 via inactivating Mdm2. • Loss of INK4a-ARF is a common cause of human cancer • P53 is modified predominantly by phosphorylation or by acetylation in response to treatments that damage DNA. Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Basic Clinical Radiobiology, Fourth Edition Edited by Michael Joiner & Albert van der Kogel Table 2.2 , pg. 18 x Hall and Giaccia 2018 Hall and Giaccia 2010 Hall and Giaccia 2010 Cannonical NF-kB pathway

Hellweg CE, Spitta LF, Henschenmacher B, Diegeler S, Baumstark-Khan C. Transcription Factors in the Cellular Response to Charged Particle Exposure. Front Oncol. 2016 Mar 21;6:61. Roles of Specific Cytokines • bFGF—increases growth of endothelial cells, inhibits apoptosis, protects against microvascular damage; induced by radiation, heat, chemo; reduces late effects • PDGF—increases damage to vasculature • TGF-beta—increases inflammation (pneumonitis), decreases growth of connective tissue and epithelial cells; causes fibrosis and vascular changes, requires two different types of receptors for signal transduction • TNF—inflammatory response, cytotoxic for tumor cells, regulated by PKC pathway, associated with radiation-induced complications, induces apoptosis, systemic administration can cause septic - like symptoms • IL1—, inflammatory responses, produced by fibroblasts and inflammatory cells • Interferon—inhibits cell proliferation in general, but can cause immune cell activation • IL6—potent pro-inflammatory cytokine NOTE: Radiation is a potent anti-angiogenic agent shown to induced an increase in VEGF expression leading to neovascularization Radiation Protective Cytokines

Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Relationship between tumor weight, number of cells, and detectability

Generally assume 109 cells per gram Joiner, 2010 Tumor Cure Probability

• Based on Poisson statistics • P=e-n P is probability of cure, n is average number of surviving clonogenic tumor cells • TCD37 would leave about 1 cell/tumor • TCD90 would leave about 10-1 cells/tumor • TCD95 would leave about .05 cells/tumor • TCD99 would leave about 10-2 cells/tumor Tumors

• Small tumors: good correlation between Tpot and volume doubling time • Large tumors: Tvol>>Tpot • Tumor volume increases 3x faster than diameter • Relationship of volume to diameter is volume=(4/3)pr3 • Growth fraction for most tumors is 40-60% • Cell loss factor is the balance between proliferation and cell loss • Sarcomas usually have a lower cell loss factor than carcinomas. Cell loss factor is the dominant growth rate determining factor in carcinomas. • Implications: Carcinomas arise from continuously renewing epithelial cell types. Carcinomas appear to be more radioresponsive since radiation-killed cells are removed by cell loss and tumor shrinkage. Sarcomas appear to be less radioresponsive because dead cells do not disappear. • Tumors with short Tpot may have rapid repopulation time interval between treatments that must be kept short. Latency periods

• Cataracts: 2Gy threshold for single exposure; latency is dose-dependent (months-years); radiation-induced cataracts progress from subcapsular posterior region • Leukemia: begins at 2y, peaks at 7-12 years, disappears by 20 y • Thyroid cancer: 4+y, peaks around 7y • Breast cancer, other solid tumors: 20+y (12y?) Chemotherapy

• Alkylating agents: inhibit DNA/RNA synthesis; cell cycle non-specific; cisPt, BCNU, procarbazine • Natural products (antibiotics, plant alkaloids): interfere with DNA, inhibit Topo enzymes, mitotic inhibitors, inhibit protein synthesis; cell cycle non-specific; Bleomycin, Taxol, Doxorubicin, vincristine • Antimetabolites: interfere with DNA/RNA, inhibit enzymes for synthesis, cell cycle dependent; 5FU, HU, MTX • Specific inhibitors: Erbitux, Iressa, others • Others: endocrine therapies; tamoxifen, estrogens, androgens Hyperthermia

• Killing is temp/exposure dependent • Thermotolerance is due to the induction of hsps: 39-42C thermotolerance is induced in 2-3h; over 43C thermotolerance takes longer to develop • Hypoxic core of tumor is sensitive to heat • Late S is most sensitive • Heat is more easily carried away from normal tissues than from tumors. • SLDR is inhibited if heat is administered during interfraction interval. • PLDR is inhibited only if heat is given following radiation. Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 dsb Comet sensitivity: 5cGy for alkaline

Sens 25cGy

Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 4Rs

Tissue type Repair Redistributio Repopulatio Reoxygenati n n on

Early + + fast N/A reacting normal Late + little slow N/A reacting normal tumor + + fast +

Time course 1-4h 4-8h Early: 2-4w 1-4d Late: 6w Molecular DNA Repair

1. Base Excision Repair (BER) 2. Excision of nucleotide damage (NER)-- UV 3. Repair of double-strand breaks • Homologous recombination • NHEJ • Single Strand Annealing (yeast)

Nature Reviews Cancer Basic Clinical Radiobiology, Fourth Edition Edited by Michael Joiner & Albert van der Kogel Figure 2.9, pg. 25 FIGURE 2.7 Nucleotide excision repair pathways. The two subpathways of NER, GG-NER/GGR (global genome repair) and TC-NER/TCR (transcription-coupled repair), differ at the initial damage recognition step. GGR uses the XPC-XPE protein complexes, whereas in TCR, the NER proteins are recruited by the stalled RNA polymerase in cooperation with CSB and CSA. Following recognition, the lesion is demarked by binding of the transcription factor IIH (TFIIH) complex, XPA and RPA. The TFIIH complex helicase function unwinds the DNA and generates an open stretch around the lesion, at RPA XPA RPA which point the XPG and XPF-ERCCl endonucleases make incisions at the 3' and 5' ends, respectively, releasing a 24-32 oligomer. The resulting gap is filled by the polymerases ‘8/E aided by RFC and PCNA and the strand is finally ligated. (XPC, xeroderma pigmentosum, complementation group C; XPE xeroderma pigmentosum, complementation group E; CSB, cockayne Syndrome B gene; CSA, cockayne Syndrome A gene; XPG, xeroderma pigmentosum, complementation group G; XPF, xeroderma pigmentosum, complementation group F; ERCC1, excision repair cross-complementation group 1 gene; RFC, replication factor C; PCNA, proliferating cell nuclear antigen.)

Hall and Giaccia 2015 Hall and Giaccia 2010 FIGURE 2.8 Illustration showing that nonhomologous recombination occurs in the G1 phase of the cell cycle, at which stage, there is no sister chromatid to use as a template for repair. In contrast, homologous recombination occurs in the S and G2 phases of the cell cycle, when there is a sister chromatid to use as a template in repair. FIGURE 2.9 Nonhomologous endjoining DNA strand breaks are recognized by the ATM and the MRN (Mrell-Rad50-Nbs1) complex, resulting in resection of the DNA ends. Homologous recombination is inhibited by the activity of 53BP1. The initial step of the core NHEJ pathway starts with the binding of the ends at the DSB by the Ku70/Ku80 heterodimer. This complex then recruits and activates the catalytic subunit of DNA-PK (DNA-PKcs), whose role is the juxtaposition of the two DNA ends. The DNA-PK complex then recruits the ligase complex (XRCC4/XLF-LiGIV/PNK) that promotes the final ligation step.

Hall and Giaccia 2015 FIGURE 2.10 Homologous recombinational repair. The initial step in HR is the recognition of the lesion and processing of the double- strand DNA ends into 3' DNA single strand tails by the MRN (Mrell-RadSO-Nbsl) complex, which are then coated by RPA forming a nucleoprotein filament. Then, specific HR proteins are recruited to the nucleoprotein filaments, such as RADS1, RADS2, and BRCA1/2. RADS1 is a key protein in homologous recombination as it mediates the invasion ofthe homologous strand of the sister chromatid, leading to formation of Holliday junctions. The Holliday junctions are finally resolved into two DNA duplexes. See text for details.

Hall and Giaccia 2015 FIGUR E 2 .1 2 Mismatch repair. The initial step in the mismatch repair pathway is the recognition of mismatched bases through either Msh2Msh6 or Msh2-Msh3 complexes.These recognition complexes recruit MLH1PMS2, MLH1-PMS1 , and MLH1-MLH3, alongside the exonuclease EXOl that catalyzes the excision step that follows. A gap-filling step by polymerases OlE, RCF, and PCNA is followed by a final ligation step.

Hall and Giaccia 2015 E. J. Hall and A. J. Giaccia, “Radiobiology for the Radiologist,” 8th Edition, Lippincott Williams & Wilkins, Philadelphia, 2018. pg. 277. Fig. 17.16 Genome engineering using the CRISPR–Cas9 system Sanchez-Rivera and Jacks, Nature Reviews Cancer 4 June 2015: 387 Methods Summary Animal Models • Allograft or Syngeneic transplant— transplant tumor from same genetic background of mice (mouse to mouse) • Xenograft—transplant human tumors into mice—must be immunodeficient animals (can be nude, NOD/SCID, etc.) (human to mouse, must be immunodeficient; can be humanized with human BM) • Transgenic mouse—made to have tumor with genetic changes, arises in the same animal Science; 03 October 2014; Vol. 346; No. 6205; pg. 26 Promoter Assays

• EMSA: Electrophoretic Mobility Shift Assay • ChIP: Chromatin Immunoprecipitation • Reporter gene assays: promoter on GFP, RFP or similar reporter gene Reporter Gene Assays:

Hall and Giaccia 2015 Promoter Bashing:

E. J. Hall and A. J. Giaccia, “Radiobiology for the Radiologist,” 8th Edition, Lippincott Williams & Wilkins, Philadelphia, 2018. pg. 281. Fig. 17.19 Figure 4.7 (A) Reaction sequence for one cycle of PCR. Each line represents one strand of DNA; the small rectangles are primers and the circles are nucleotides. (B) The first three cycles of PCR are shown schematically. ( C ) Ethidium-bromide-stained gel after 20 cycles of PCR. (See Sec. 4.3.5 for further explanation.)

Tannock IF, Hill RP, Bristow RG and Harrington L: The Basic Science of Oncology Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Basic Clinical Radiobiology, Fourth Edition Edited by Michael Joiner & Albert van der Kogel Table 7.7, pg. 87 Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Biomolecular Action of Ionizing Radiation; Editor:Shirley Lehnert, 2007 Assays for Mutations: Rearrangements vs small mutations

1. Ab to mutant (not wt) protein—small mutations/some rearrangements 2. Change in restriction enzyme fragment length (as opposed to RFLP)—rearrangements, some small mutations 3. Southern blot—rearrangements, amplifications 4. Single-stranded Conformational Polymorphism (SSCP)—small or large mutations 5.Denaturing Gradient Gel Electrophoresis—not used much; small or large mutations 6.Sequencing—small or large mutations others . . . Blots and Assays

• Proteins: 1DE, 2DE, proteomics, Western blots, immunoprecipitation (gene expression) • RNA: microarrays, Northern blots, RT- PCR (gene expression) • DNA: Southern blot, sequencing, PCR (changes in gene sequence) Areas that are emphasized:

• Oxygen effects and hypoxia • P53, ATM, DNA repair • Association of oncogenes and tumors • Oncogenes vs. tumor suppressor genes • Association of irradiated populations with cancer that developed • Cyclins and the cell cycle • Isoeffect curves High Yield Summary Reviews in Radiation Oncology

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