ASTRO 2023 (4-15) Flashcards
IV. Chromosome and Chromatid Damage
IV-1) Which of the following statements concerning chromosome aberrations produced in cells after whole body X irradiation is TRUE?
A. The formation of terminal deletions follows an exponential dose response
B. Translocations are an unstable type of chromosome aberration
C. The number of dicentric chromosomes detected in peripheral blood lymphocytes remains relatively constant with time
D. SKY (spectral karyotyping) is a useful method for detection of stable aberrations decades following irradiation
E. The minimum dose that can be estimated by scoring dicentric chromosomes is 2 Gy
IV-1) D
Spectral karyotyping (SKY) uses fluorescence staining of chromosomes employing uniquely-colored probes specific for individual chromosomes, thus allowing them to be distinguished from each other on the basis of color.
Stable translocations are revealed using SKY as a single chromosome that appears to be multi-colored. The formation of terminal deletions follows a linear dose response since these are single-hit aberrations.
Translocations can be stable aberrations since they do not necessarily lead to cell death
The number of dicentric chromosomes detected in peripheral blood lymphocytes decreases with time after irradiation since these are unstable aberrations that ultimately cause the death of the lymphocyte progenitors and stem cells.
The minimum dose that can be detected through scoring dicentric chromosomes is roughly 0.25 Gy .
IV-2) Which of the following types of chromosome aberrations are most responsible for the formation of micronuclei observed after irradiation?
A. Sister chromatid exchanges
B. Chromatid gaps
C. Inversions
D. Quadriradials
E. Acentric fragments
IV-2) E
Micronuclei are created due to the presence of acentric fragments, which form in the progeny of irradiated cells that undergo mitosis in the presence of one or more asymmetrical chromosome aberrations.
Sister chromatid exchanges are reciprocal exchanges between chromatids of the same chromosome that are not readily induced by ionizing radiation.
Chromatid gaps appear as loss of genetic material from a single chromatid arm and may be caused by incomplete breaks.
Inversions result when two breaks are produced in a single chromosome and the resulting excised chromosomal fragment reinserts itself back into the chromosome, but with the opposite polarity
A quadriradial is a chromatid-type aberration that may arise from illegitimate interchromosomal recombination, accompanied by crossing over.
IV-3) Which of the following is the most reliable measure for the presence of radiation-induced chromosome aberrations in interphase cells?
A. Reciprocal translocations
B. Ring chromosomes
C. Dicentric chromosomes
D. Micronuclei
E. Chromatid breaks
IV-3) D
Individual chromosome aberrations can, in general, be detected readily only during mitosis. However, some chromosome aberrations lead to the formation of micronuclei, which develop when a pseudo nuclear membrane forms around acentric chromosome fragments or whole chromosomes that did not segregate properly into daughter cells during the previous mitosis. Micronuclei are observed in peripheral lymphocytes and thus can be seen in interphase cells.
IV-4) Which one of the following statements concerning the induction of chromosome aberrations is INCORRECT?
A. Primary radiation-induced breaks can reconstitute without apparent morphological change to the chromosome, rejoin illegitimately with another break site to produce an intra- or inter-chromosomal aberration, or remain “open,” leading to a simple terminal deletion
B. The induction and interaction of DNA double-strand breaks is the principal mechanism for the production of chromosome aberrations
C. Dicentrics, centric rings, and translocations are formed following Xirradiation of cells in the G0/G1 phase of the cell cycle, and their formation follows a linear-quadratic dose response
D. FISH using multi-colored probes has allowed chromosome aberration complexity to be studied in detail
E. Chromatid type aberrations are observed when cells are irradiated during the G1 phase of the cell cycle
IV-4) E
Chromatid type aberrations are produced in cells only when irradiation follows DNA synthesis in S phase. In this case, only one of the two chromatid has the anomaly. Anaphase bridges are caused by chromatid type aberrations. Chromosomal type aberrations are produced when irradiaiton occurs prior to DNA synthesis and the anomaly is duplicated on both chromatids of the chromosome. Examples included dicentric or ring chromosomes.
IV-5) The formation of dicentric chromosome aberrations follows a linear quadratic dose response curve. This has been interpreted to mean that the production of dicentric chromosomes results from:
A. Two chromosome breaks, produced either by one or by two separate radiation tracks
B. Two chromosome breaks produced by two separate radiation tracks
C. Two chromosome breaks produced by a single radiation track
D. One chromosome break produced by two separate radiation tracks
E. One chromosome break produced by a single track of radiation
IV-5) A
The formation of dicentric chromosomes is linear at low radiation doses but follows a quadratic function at higher doses. Two distinct mechanisms are thought to be responsible for these two components of the linear quadratic dose response curve. The linear portion of the dose response relationship is assumed to result from the simultaneous induction of two chromosome breaks by a single track. The quadratic portion is assumed to result from the two chromosome breaks being produced by two separate radiation tracks.
IV-6) Which of the following statements concerning chromosome aberrations is TRUE?
A. A ring chromosome is an example of a chromatid-type aberration
B. A dicentric is a stable chromosome aberration
C. Breakage of a single chromatid in G2 often leads to the formation of an anaphase bridge
D. Terminal deletions are induced as a linear function of dose
E. For low LET radiation, the yield of dicentric chromosomes is inversely proportional to the dose-rate
IV-6) D
Terminal deletions are induced as a linear function of dose since they result from a single chromosomal break. A ring chromosome is an example of a chromosome-type aberration, not a chromatid-type aberration.
A dicentric is an unstable aberration since it results in the formation of an acentric fragment and ultimately causes cell death.
Breaks in two chromatids, followed by illegitimate rejoining, produce an anaphase bridge
The yield of dicentric chromosomes increases with increasing dose-rate for low LET radiation
IV-7) Increased numbers of chromosome aberrations, especially quadriradials, are frequently found even in the absence of radiation in which of the following human syndromes?
A. Xeroderma pigmentosum
B. Fanconi anemia
C. Cockayne’s syndrome
D. Niemann-Pick disease
E. Li-Fraumeni syndrome
IV-7) B
Blood cells from individuals with Fanconi anemia, an AR disorder, are harboring a mutation in one of 22 FANC genes, which are part of the FA/BRCA pathway. Defects in this pathway result in chromosomal instability due to decreased capacity to repair interstrand DNA crosslinks. This results in the development of high numbers of chromosome aberrations, especially quadriradials. These complex aberrations increase dramatically with exposure to DNA cross-linking agents such as mitomycin c.
V. Mechanisms of Cell Death
V-1) Pathways that trigger apoptosis culminate in widespread intracellular proteolysis. Which of the following proteases is a downstream executioner that directly participates in the breakdown of numerous cellular proteins?
A. caspase-8 (CASP8)
B. caspase-9 (CASP9)
C. caspase-3 (CASP3)
D. caspase-10 (CASP10)
E. XIAP (BIRC4)
V-1) C
There are at least 14 human caspases, which fall into two categories: the initiator caspases (caspases-2, -8, -9 and -10), which activate the downstream caspases, and the executioner caspases(caspases-3, -6 and -7), which cleave cellular substrates.
XIAP is a protein that binds to and inhibits the action of caspases.
V-2) Which of the following statements regarding cell death following RT is TRUE?
A. The majority of solid epithelial tumors regress during treatment because of radiation-induced apoptosis
B. The intrinsic apoptotic pathway can be triggered either by radiationinduced DNA damage or by sphingomyelin-mediated damage to the outer plasma membrane
C. A novel drug that abolishes the G1 checkpoint would be expected to reduce the incidence of mitotic catastrophe in irradiated cells.
D. Cells that undergo replicative senescence following radiotherapy are characterized by increased membrane blebbing and DNA fragmentation
E. The presence of gamma-H2AX histone foci in irradiated cells is indicative of sphingomyelin activation
V-2) B
The intrinsic apoptotic pathway can be triggered either by damage to DNA or by damage to the plasma membrane.
Radiation acts directly on the plasma membrane, activating acid sphingomyelinase, which generates ceramide by enzymatic hydrolysis of sphingomyelin. Ceramide then acts as a second messenger in initiating an apoptotic response via the mitochondrial system.
Mitotic catastrophe, and not apoptosis, is the major mechanism of cell death in epithelial tumors.
Inhibition of the G1 checkpoint in irradiated cells may increase the probability of mitotic catastrophe since cells are more likely to enter mitosis with damaged chromosomes.
Radiation-induced senescent cells cease dividing and can remain metabolically active for extended periods before dying, but do not show membrane blebbing and DNA fragmentation, which are characteristic of apoptosis.
H2AX foci noted in the nuclei of irradiated cells are indicative of the presence of DNA double-strand breaks.
V-3) Radiation-induced cellular senescence is often the result of:
A. Cellular nutrient deprivation
B. Oxidative stress secondary to mitochondrial dysfunction
C. p16-mediated cell cycle arrest
D. Telomere shortening
E. Mitotic catastrophe
V-3) C
The term “senescence” refers to the loss of cellular replicative potential leading to a reduced capability to repopulate a tissue after exposure to genotoxic agents, including ionizing radiation. Senescence is most often the result of a permanent arrest in G1, associated with elevated expression of the cell cycle inhibitors p16INK4A (CDKN2A) and p21 (CDKN1A, WAF1/CIP1). Importantly, senescence is not a type of cell death per se because cells remain morphologically intact and metabolically active when senescent. Depending on the level of tumor suppressor proteins and the oncogenic signal, senescence can be reversible in a small subset of cells though in most cells this process is irreversible.
A clinically relevant scenario for radiation-induced senescence is the loss of salivary gland function and xerostomia commonly seen in head and neck cancer patients undergoing radiotherapy. Another one is radiation induced premature senescence in fibroblasts that triggers proinflammatory and profibrotic senescence associated secretory phenotype (SASP) and ultimately drives fibrosis in the lung.
Mitochondrial dysfunction is a hallmark of apoptotic cell death, not senescence.
Telomere shortening occurs in most normal somatic cells as part of each cell cycle (“end replication problem”) and triggers senescence once a critical low threshold is reached, but telomere shortening tends not to be the cause for radiation-induced senescence which is driven by DNA damage and cell cycle arrest.
Nutrient deprivation can lead to autophagy, and ultimately autophagic death cell distinct from apoptosis.
V-4) The extrinsic pathway of apoptotic cell death requires:
A. Signals derived from changes in chromatin conformation
B. Activation of death receptors that translocate from the plasma membrane to the nucleus and degrade DNA
C. Engagement of death receptors located on the plasma membrane that lead to activation of the initiator caspase-8 (CASP8)
D. p53 (TP53) activation
E. The triggering of changes in mitochondrial membrane potential
V-4) C
There are two principal pathways that can lead to apoptotic death. One of these, the extrinsic pathway, involves extracellular signaling through death receptors located on the plasma membrane such as TRAILR-1 (TNFRSF10A), TRAILR-2 (TNFRSF10B) or FAS (CD95/APO-1). These death receptors are activated in response to ligand binding of TRAIL (TNFSF10) or FAS ligand (FASLG/CD95-L) and signal through a series of adapter molecules such as the adapter molecule Fas- associated death domain (FADD) within the death-inducing signalling complex (DISC). Upon recruitment and oligomerization FADD then binds pro caspases-8 and -10, causing their homodimerization and activation.
The activation of procaspase-8 is thought to occur via an induced proximity model leading to its conversion to the active enzyme, caspase- 8. Ionizing radiation can elicit activation of the extrinsic pathway leading to apoptosis.
The other pathway by which ionizing radiation can elicit an apoptotic response is the intrinsic pathway. This can be stimulated by DNA damage leading to signaling to mitochondria, changes in mitochondrial membrane potential, release of cytochrome c, and activation of procaspase-9.
In most cases, activated caspase-8 induces apoptosis through activation of pro-caspase-3 at the DISC independently of mitochondria. However, in some cells, especially when only a low amount of active caspase-8 is generated (and hence not sufficient amounts of pro-caspase-3), caspase-8 cleaves the ‘Bcl-2 homology (BH) 3-only protein’ Bid, generating an active fragment (tBid) that activates the (intrinsic) mitochondrial death pathway. In this manner, the extrinsic death signal may be amplified through formation and activation of the apoptosome which contributes to effector caspase activation. In other words, the extrinsic pathway can feedinto the intrinsic one and additionally change mitochondrial membrane potential.
V-5) One hallmark of the apoptotic process is the display of phosphatidylserine residues on the outer surface of the plasma membrane. This is an important event in terms of the tissue response to ionizing radiation because it:
A. Helps recruit death ligands expressed by neighboring cells to receptors on the cell surface
B. Stimulates an inflammatory response to remove dying cells from the tissue
C. Signals the recruitment of phagocytes that engulf the dying cells without causing an inflammatory response
D. Is required for DNA condensation and fragmentation
E. Leads to increased ceramide levels
V-5) C
Apoptosis helps maintain tissue homeostasis because cells that are undergoing an apoptotic response, recruit phagocytes that clear the dying cells, also known as “apoptotic corpses”, from the tissue without stimulating an inflammatory response. In fact, uptake of apoptotic cells by macrophages can actually lead to the release of anti-inflammatory mediators such as TGF-β and IL-10, and the attenuation of the RIG-I/IRF- 3 pathway and the cGAS/STING pathway through proteolytically inactivating RIP kinase 1 or the degradation of cytoplasmic DNA. As a result, apoptosis can decrease the expression of interferons and other inflammatory factors.
Of note, the concept that apoptosis is entirely non-inflammatory isn’t always strictly true. An example is the induction of apoptosis in hepatocytes following FAS activation that causes a strong inflammatory response probably because they can’t get cleared fast enough by phagocytes.
The exposure of phosphatidylserines (phospholipids) on the exterior of the plasma membrane is the signal that initially recruits phagocytes. Ordinarily, phosphatidylserine is sequestered on the inner leaflet of the phospholipid bilayer and is not displayed on the cell’s surface. The process of necrosis, which involves rupture of the cell membrane and the leakage of cellular contents into the surrounding tissue, does elicit an inflammatory response.
While DNA condensation and fragmentation are important steps in the apoptotic process, they are not coordinated directly through the exposure of phosphatidylserine on the plasma membrane. A number of stimuli lead to increased ceramide levels, including TNF, FasL and ionizing radiation, but not phosphatidylserine.
V-6) Regarding the regulation of apoptosis, which of the following pairs of
mammalian proteins and their apoptosis-related functions is FALSE?
A. p53 (TP53) — upregulation of PUMA
B. DIABLO — caspase activation
C. XIAP (BIRC4) — caspase inhibition
D. BAX — cytochrome c release
E. caspase-3 (CASP3) — initiator caspase
V-6) E
The characteristic changes associated with apoptosis are due to activation of a family of intracellular cysteine proteases, known as caspases. Initiator caspases are the first to be activated, and include caspases-2, -8, -9 and - 10. Initiator caspases cleave and activate the effector/executioner caspases, including caspases-3, -6, and -7, which then cleave, degrade or activate other cellular proteins. Activation of caspases is regulated by members of the BCL2 family and by the inhibitors of apoptotic protein (IAP) family. BCL2 family members can be either pro- or anti-apoptotic.
BAX is one of a series of pro-apoptotic members of the BCL2 family. These pro-apoptotic BCL2 family members regulate the release of cytochrome c from mitochondria and elicit the subsequent activation of caspases. Another important function of p53 is that it causes upregulation of pro-apoptotic PUMA. X-linked IAP (XIAP) inhibits the activity of caspases directly. DIABLO is a pro-apoptotic protein that prevents IAPs from inhibiting caspases. BAX and p53 are required for some forms of DNA damage-induced apoptosis.
V-7) Which ONE of the following is a morphological or biochemical feature
of apoptosis?
A. Random cleavage of DNA
B. Cellular swelling
C. Lack of dependence on ATP as an energy source
D. Chromatin condensation
E. Rupture of the plasma membrane
V-7) D
During the apoptotic process, endonucleases cut the DNA at precise sites corresponding to the linker region between nucleosomes. This leads to the formation of fragments that are multiples of 80 bp units. There is no cell swelling, such as occurs in necrosis, but rather cell shrinkage after the apoptotic process begins followed by condensation of chromatin at the periphery of the nucleus. Apoptosis is an energy-dependent process requiring ATP. During the apoptotic process, the plasma membrane initially remains intact but later fragments and surrounds the apoptotic bodies.
V-8) The TUNEL assay used to identify apoptotic cells detects:
A. The action of BAX on the mitochondria
B. Membrane integrity
C. Mitochondrial release of cytochrome c
D. Binding of TNF (TNF) to its receptor
E. DNA fragmentation
V-8) E
The terminal deoxynucleotidyl transferase (TdT) mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) technique has been used to identify apoptotic cells. It is based upon the binding of TdT to the 51exposed 3’-OH terminal ends of DNA fragments generated during apoptosis and catalyzes the addition of modified deoxynucleotides, conjugated with biotin or fluorescein, to the DNA termini.
V-9) Which of the following best describes radiation-induced bystander
effects?
A. Damage to unirradiated normal tissue noted after irradiation of a
tumor
B. Cell killing resulting from irradiation of the cell’s cytoplasm in the
absence of direct irradiation of the nucleus
C. Radiation-induced increase in cell membrane permeability that
causes increased sensitivity to cytotoxic drugs
D. DNA and/or chromosomal damage that occurs in unirradiated cells
that are nearby irradiated cells
E. Intercellular communication that modifies the shoulder region of
the radiation survival curve
V-9) D
While damage to cellular DNA was long considered the major initiator of cellular responses to ionizing radiation, more recent evidence suggests the involvement of non-targeted pathways, including radiation-induced bystander effects. Bystander effects are defined as radiation-like effects observed in cells that are not themselves irradiated, but that are in communication with irradiated cells through their location near these cells or by stimuli transferred from the irradiated cells through the intracellular medium. Various endpoints have been measured as bystander effects, including enhanced cell killing, induction of apoptosis, presence of chromosome aberrations and micronuclei, presence of DNA double strand breaks, increased oxidative stress, genetic effects (including induction of mutations, and neoplastic transformation) and altered gene expression
V-10) Mitotic death in irradiated cells results primarily from:
A. The mis-rejoining of DNA single strand breaks.
B. DNA ladder formation.
C. Stimulation of the extrinsic death pathway.
D. Mis-assortment of genetic material into daughter cells.
E. An alteration in cell membrane permeability.
V-10) D
Mitotic death in most irradiated cells results primarily from mis assortment of genetic material into daughter cells as a result of the formation of asymmetrical chromosome aberrations. This aberrant mitosis triggers mitotic catastrophe, which is characterized by cells exhibiting multiple tubulin spindles and centrosomes as well as the formation of multinucleated giant cells that contain uncondensed chromosomes. Mitotic death can be of any molecular mechanism, including apoptosis or necrosis.
Single strand breaks are repaired rapidly and do not appear to play an important role in cell lethality.
DNA ladder formation is characteristic of apoptosis.
An alteration in cell permeability occurs in cells undergoing necrosis.
V-11) Which of the following concerning autophagy is INCORRECT?
A. Autophagy is a reversible process that can contribute both to tumor cell death and survival
B. The U.S. Food and Drug Administration–approved anti-malarial drugs chloroquine and hydroxychloroquine are inhibitors of autophagy
C. Autophagy contributes to cellular metabolism by degradation of damaged protein aggregates and organelles
D. Mitophagy refers to autophagy in mitotic cells
E. Autophagy is controlled by the Atg family of proteins
V-11) D
Autophagy can be nonselective or selective. Nonselective, bulk degradation of cytoplasm and organelles by autophagy provides material to support metabolism during periods of cellular stress. For example, autophagy provides internal nutrients, when external ones are unavailable.
Whether mechanisms exist to prevent bulk autophagy from consuming essential components, such as a cell’s final mitochondrion, remains unclear, and in some cases such consumption may lead to cell death.
Selective autophagy of proteins and of organelles such as mitochondria (mitophagy), ribosomes (ribophagy), endoplasmic reticulum (reticulophagy), peroxisomes (pexophagy), and lipids (lipophagy) occurs in specific situations.
Autophagy (‘self-eating’) tends to refer to macroautophagy: the sequestration process of cytoplasmic material for degradation. (Microautophagy and chaperone-mediated autophagy are other types.)
After initiation, an isolation membrane encloses a small portion of cytoplasmic material, including damaged organelles and unused proteins, to form a double-membraned structure called an “autophagosome” that subsequently fuses with lysosomes to become an “autolysosome”, in which the cytoplasmic material is degraded by lysosomal enzymes. The whole process is tightly regulated through at least 30 Atg-autophagy related genes that orchestrate initiation, cargo recognition, packaging, vesicle nucleation expansion and fusion and breakdown. The initial steps center around the Atg1 complex (Atg1–Atg13–Atg17– Atg29–Atg31) that translocates to the ER, (thought to be the major membrane source for autophagy). This leads to recruitment of the autophagy-specific form of the class III PI(3)K complex, which includes Vps34, Vps15, Atg6/Beclin- 1 and Atg14, to the ER. To form an autophagosome, elongation and closure of the isolation membrane requires 2 protein conjugation systems, the Atg12–Atg5–Atg16 complex and the Atg8/LC3– phosphatidylethanolamine (PE) complex. Detection of autophagy relies on the redistribution of GFP-LC3 fusion proteins into vesicular structures (which can be autophagosomes or autolysosomes).
‘Autophagic cell death’ is the excessive version of autophagy, that occurs in the absence of chromatin condensation. In contrast to apoptotic cells, there is little or no association of autophagic cells with cells phagocytes. Although the expression ‘autophagic cell death’ is a linguistic invitation to believe that cell death is executed by autophagy, the term simply describes cell death with autophagy
V-12) Which of the following is NOT a feature of apoptosis?
A. Chromatin condensation
B. Cell shrinkage
C. DNA fragmentation
D. Rapid engulfment by neighboring cells
E. Pro-inflammatory response
V-12) E
There is no pro-inflammatory response in pure apoptosis. Apoptosis can in fact be anti-inflammatory through its recruitment of macrophages via “find-and-eat-me” signals; macrophages in turn produce anti inflammatory TGF-β. Pro-inflammatory forms of cell death include necrosis, necroptosis, and pyroptosis.
V-13) Which of the statements is TRUE regarding the activation of one type of apoptotic pathway?
A. Apoptosis is initiated by PARP
B. Fas ligand binding its receptor initiates apoptosis
C. Caspases involved in the execution of apoptosis are also involved in the execution of necrosis
D. Bcl2 is a pro-apoptotic protein.
E. Anti-apoptotic Bax dimerizes and translocates to the mitochondria.
V-13) B
Fas ligand binds its receptor and triggers the external death receptor pathway.
Cleavage of PARP-1 by caspases is considered to be a biochemical hallmark of apoptosis.
Different caspases play different roles in the initiation and execution of apoptosis and are not involved in necrosis. Necrosis is the unregulated digestion of cellular components as a result of external factors
Bcl-2 inhibits apoptosis while Bax stimulates apoptosis
VI. Cell and Tissue Survival Assays
VI-1) Which of the following in vivo assays of radiation response does NOT depend on a functional endpoint?
A. LD50
B. Skin nodule formation
C. Myelopathy
D. Breathing rate
E. Cognitive impairment
VI-1) B
A functional endpoint for radiation response is a measured endpoint that is downstream of clonogenic survival and may involve measurement of tissue/organ function, the incidence of toxicity, or whole animal survival.
Clonogenic endpoints directly measure the replicative capacity of cells (e.g., colony formation). Skin nodule formation is not a functional endpoint; it is a clonogenic assay measuring survival of individual epidermal cells regrowing in situ. All of the other assays cited represent non-clonogenic, functional endpoints for assaying radiation damage.
VI-2) Using the linear-quadratic survival curve model, what would the cell
surviving fraction be following a dose of 2 Gy delivered acutely (use
=0.3 Gy-1 and =0.1 Gy-2)?
A. 0.01
B. 0.10
C. 0.37
D. 0.50
E. 0.90
VI-2) C
Using the equation S = e-(D+D2) , the surviving fraction would be: e-[(0.3)(2)+(0.1)(2)2] = e-[(0.6)+(0.4)] = e-1= 0.37
VI-3) For the same α and β values used in the previous problem, what would be
the approximate surviving fraction if the 2 Gy dose were delivered at a
low dose rate over a 6-hour period instead of acutely (assume no
repopulation takes place during the irradiation)?
A. 0.10
B. 0.20
C. 0.37
D. 0.55
E. 0.90
VI-3) D
If the dose was delivered at a low dose rate, the surviving fraction would increase due to repair of sublethal damage during the course of irradiation. If one assumes that there is full repair of sublethal damage during the 6 hr irradiation (which is probably an oversimplification), sublethal damage would not contribute to cell killing. The β component of the LQ equation would therefore approach zero, leaving the α component to dominate. The surviving fraction can therefore be estimated as e-(0.3)(2) = e-0.6 = 0.55
VI-4) Which clonogenic assay has been used to measure the radiation sensitivity
of bone marrow stem cells in vivo?
A. Dicentric assay
B. BrdU (BrdUrd) assay
C. Endpoint dilution assay
D. In vivo/in vitro excision assay
E. Spleen colony assay
VI-4) E
The spleen colony assay involves the ability of donated bone marrow stem cells, injected intravenously into lethally-irradiated recipient mice, to form discrete splenic colonies. The higher the radiation dose received by the donated marrow, the fewer colonies (relative to the number of cells injected) will form in the recipients’ spleens. This technique allows a cell survival curve to be generated in vivo.
VI-5) The components typically required for the analysis of a standard, adherent
cell clonogenic survival assay require all of the following, EXCEPT:
A. Calculation of a plating efficiency
B. Colony formation rates at a range of cell densities, for several
radiation doses
C. A cell line capable of multiple cell divisions
D. Intact apoptosis pathways
E. Nonirradiated control
VI-5) D
The clonogenic survival assay measures the ability of single cells to divide continuously after a given exposure, and typically measures colony formation 7-14 days after exposure to the agent. It requires a normalization in which the number of colonies formed is divided by the number of cells seeded (in the absence of any DNA damaging agent), which yields the plating efficiency. Surviving fraction is then calculated for each dose of a given agent by dividing the number of colonies formed by the number of cells seeded and normalizing to the “0 Gy” plating efficiency. Multiple doses and cell densities typically are needed for the adequate analysis of cell survival. This is not a short-term growth delay assay, and thus a cell capable of multiple cell divisions is needed. DNA damaging-agents induce cell death via a number of pathways, including apoptosis. However, apoptosis is not the sole cell death pathway
VII-1) If a cell line exhibiting a strictly exponential radiation survival curve is exposed to a dose that produces an average of one lethal “hit” per cell, the surviving fraction after this dose would be approximately:
A. 0.01
B. 0.10
C. 0.37
D. 0.50
E. 0.90
VII-1) C
Assuming that all cells in the cell population are identical and that cell killing is a random, probabilistic process that follows a Poisson distribution. One model that can calculate the radiation dose that produces an average of one lethal hit is the single-target single-hit model. From the equation that describes this model, S = e , the dose, D, at which there would be an average of one hit per cell would be equal to D0, the constant of proportionality. Therefore, S = e-1 ~ 0.37.
VII-2) The / ratio is equal to the:
A. Surviving fraction at which the amount of cell killing caused by the
induction of irreparable damage equals the amount of cell killing
caused by the accumulation of sublethal damage
B. Optimal fraction size to use in a fractionated radiotherapy regimen
C. Dose below which a further decrease in fraction size will not affect
the surviving fraction for a particular total dose
D. Dq
E. Dose at which the D component of cell kill is equal to the D2
contribution to cell killing
VII-2) E
The / ratio represents the dose at which the D component of cell killing, assumed to result from single hit killing, is equal to the lethality produced by the D2 component of cell killing that results from multi hit killing.
VII-3) Cells from individuals diagnosed with which of the following bdiseases/syndromes would not be expected to have increased sensitivity to X-ray irradiation?
A. Nijmegen breakage syndrome
B. LIG4 syndrome
C. ATR-Seckel syndrome
D. Xeroderma pigmentosum
E. Ataxia telangiectasia
VII-3) D
Cells derived from an individual diagnosed with xeroderma pigmentosum are defective in nucleotide excision repair (NER). These cells are sensitive to UV radiation because this form of radiation produces damage such as pyrimidine dimers that are removed through the nucleotide excision repair pathway. Because DNA double-strand breaks are important lesions responsible for lethality in cells exposed to X-rays and because DNA double-strand break repair is generally normal in cells derived from a person diagnosed with xeroderma pigmentosum, their cells are not expected to be especially sensitive to X rays.
People with Nijmegen breakage syndrome, LIG4 syndrome, ATR-Seckel syndrome and ataxia telangiectasia, who possess mutations in either NBS1, LIG4, ATR or ATM, respectively, are all characterized by defects in strand break repair or repair-related signaling. Therefore, their cells would be expected to be sensitive to X rays.
VII-4) Which of the following statements concerning cell survival curve analysis
is TRUE?
A. The parameter generally increases as the radiation dose rate
decreases
B. The inverse of the Dq corresponds to the final slope of the survival
curve
C. The extrapolation number, n, of a survival curve increases with
increasing LET of the radiation
D. D0 is a measure of the incremental increase in cell survival when a
given dose is fractionated
E. If n = 1, then D37 = D0
VII-4) E
Parameters to define a radiation cell survival curve include: the initial slope (D1), a final slope (D0), and some quantity that is a measure of the width of the shoulder. This quantity can be the extrapolation number (n) or the quasi-threshold dose (Dq). If n=1, the survival curve has no shoulder and D37 (dose resulting in a survival fraction of 0.37) equals the D0.
D0 is a measurement of radiosensitivity based on the exponential part of the survival curve. In a target theory, it can be defined as a dose that gives an average of one lethal hit per cell in a population. Thus, an increase of an exposed dose of D0 would result in a 37% further reduction of survival.
A smaller D0 value indicates a higher sensitivity to radiation. For the same radiation dose, radiation delivered at a lower dose rate may produce less cell killing than radiation delivered at a higher dose rate because sublethal damage repair occurs during the protracted exposure.
As the dose rate is reduced, the slope of the survival curve becomes shallower and the shoulder tends to disappear because does not change significantly but trends to zero in the linear quadratic model.
The inverse of the D0, not the Dq, is equal to the final slope of the survival curve.
For densely ionizing radiation (increasing LET), the shoulder of the survival curve tends to disappear. N, therefore, decreases, until it reaches a value of 1.0 for very high LET radiations. The D0 would not necessarily be a good predictor for the effect of fractionation on survival; Dq or n would be better.
VII-5) Reducing the dose rate at which a continuous -irradiation is delivered may affect its cell killing efficacy due to several different biological processes. For a total dose of 6 Gy, which pair of dose rate ranges and biological processes resulting in altered cell killing is INCORRECT?
A. 10 - 1 Gy/min : reoxygenation
B. 1 - 0.1 Gy/min : repair
C. 0.1 - 0.01 Gy/min : redistribution
D. 0.01 - 0.001 Gy/min : repopulation
VII-5) A
Reoxygenation generally occurs over a period of hours to days. Little to no reoxygenation of hypoxic cells is therefore likely during irradiation performed at dose rates in the 1-10 Gy/min range since, for a total dose of 6 Gy, the irradiation times would only vary from 0.6-6 minutes.
If the total treatment time is long enough that significant repair of sublethal damage (half-time on the order of 0.5-1.0 hour) can occur during irradiation, repair does influence cell survival.
The irradiation time would vary from 6-60 minutes for dose-rates in the range of 1-0.1 Gy/min and significant repair would occur.
Movement of the surviving cells through the cell cycle (causing redistribution of viable cells from resistant phases into sensitive phases) can influence the radiosensitivity of the cell population when irradiation times are increased to several hours (for the dose-rate range of 0.1-0.01 Gy/min, times of 1-10 hours would be needed to produce 6 Gy.
Repopulation can lead to an increase in the number of cells during irradiation and, hence, to an increase in the total number of surviving cells when a radiation dose is delivered over days (10-100 hours are required to produce a total dose of 6 Gy over a range of 0.01-0.001Gy/min.
VII-6) The survival curve for a cell population irradiated with a form of high LET radiation is characterized by a D10 of 3 Gy. For a starting population of 10^8 cells, approximately how many cells will survive when a single dose of 18 Gy is given?
A. 10^0
B. 10^1
C. 10^2
D. 10^3
E. 10^4
VII-6) C
For high LET radiation it can be assumed that the survival curve is exponential, or near exponential, and cell survival can be modeled using the single-target, single-hit equation (S = e), or the simplified form of the linear quadratic equation in which is zero (S = e-D). Using either of these equations, 3 Gy reduces the surviving fraction to 10-1, and a dose of 18 Gy therefore would reduce survival to 10-6. Therefore, irradiating 108 cells with 18 Gy would result in the survival of: (108 cells) x (10-6 surviving fraction) = 102 cells.
VII-7) A total dose of 12 Gy of X-rays delivered in 3 Gy fractions reduces cell survival to 10-4. Assuming that cell killing can be modeled using an exponential survival curve, what dose would be required to reduce the surviving fraction to 10-6?
A. 9 Gy
B. 18 Gy
C. 24 Gy
D. 36 Gy
E. 72 Gy
VII-7) B
An exponential survival curve can be modeled using the single-target, single-hit equation (S = e), or the simplified form of the linear quadratic equation in which is zero (S = e-D). Since four 3 Gy fractions reduce the surviving fraction to 10-4, and assuming an equal effect per fraction, each 3 Gy fraction reduces the surviving fraction by 10-1. Accordingly, two additional 3 Gy fractions (producing a total dose of 18 Gy) would yield a surviving fraction of 10-6.
VII-8) In an attempt to generate a radiation survival curve for a new cell line,
four cell culture dishes were seeded with 102
, 103
, 104
and 105
cells, and
X-irradiated with 0, 3, 6 and 9 Gy, respectively. At the end of a two-week
incubation period, a total of 40 colonies was counted on each dish. Which
one of the following statements is TRUE?
A. The D0 for this cell line is 3 Gy
B. The survival curve for this cell line is exponential
C. The n and Dq values for this survival curve are large
D. The cell surviving fraction after a dose of 3 Gy is 0.04
E. The alpha-beta ratio for this cell line is small
VII-8) B
The survival curve for this cell line is exponential because each incremental dose of 3 Gy decreased the surviving fraction by an additional factor of 0.1. Thus, this survival curve can be modeled using an exponential equation which can be expressed as either S= e-D (linear quadratic model) or S = e-D/D0 (target theory model). The D0 is equal to the D10/2.3, or 1.3 Gy, not 3 Gy. The n and Dq values for this survival curve are equal to 1 and 0 Gy, respectively, and are therefore small, not large. The surviving fraction after a single dose of 3 Gy can be calculated from the colony forming efficiency of the irradiated cells (40/1000), divided by the plating efficiency (PE) of the unirradiated cells (40/100), which is equal to 0.04/0.4 or 0.1. Since this survival curve can be represented by S = e-D, the term of the linear-quadratic equation must approach zero, so the / would be very high, and in fact will be undefined if the term is actually zero.
VII-9) What is the approximate eD10 (effective D10) for a particular cell line if the
eD0 is 4 Gy?
A. 2 Gy
B. 4 Gy
C. 6 Gy
D. 9 Gy
E. 12 Gy
VII-9) D
eD10 is the dose required to kill 90% of population eD10 = 2.3 x D0
The eD10 is equal to the eD0 multiplied by 2.3, or 4 Gy x 2.3 = 9.2 Gy
VII-10) When irradiating a cell population with a dose that causes an average of
one lethal event per cell, this will likely result in a survival fraction of:
A. 0% of cell survival.
B. 10% of cell survival.
C. 37% of cell survival
D. 63% of cell survival
E. 100% of cell survival
VII-10) C
When the irradiated cell population receives an average of 1 lethal hit, it results in 37% cell survival based on Poisson statistics.
VII-11) A 1 cm-diameter tumor that contains 107
clonogenic cells is irradiated
with daily dose fractions of 1.8 Gy. The effective dose response curve has
been determined and is exponential with a D10 of 8 Gy. What total dose
will correspond to the TCD90 (90% probability of tumor control),
assuming no cell proliferation between dose fractions?
A. 32 Gy
B. 40 Gy
C. 48 Gy
D. 56 Gy
E. 64 Gy
VII-11) E
In order to achieve a 90% tumor control rate, the total dose delivered must reduce the number of surviving clonogenic cells to an average of 0.1. This is based on the equation P = e-(N)(SF), where P is the probability of tumour cure (90% or 0.9 in this case), N is the initial number of tumor clonogens (10^7) and SF is the surviving fraction resulting from the irradiation protocol. Thus, for 10^7 clonogenic cells, a total dose that reduces the surviving fraction to 10^-8 (i.e., an average of 0.1 clonogen surviving in each tumor or 1 cell surviving out of every 10 tumors irradiated) must be used to achieve a 90% control rate. Since the effective survival curve is exponential with a D10 of 8 Gy, it would be necessary to use a dose of 64 Gy.
VII-12) Based on the information presented in the previous question, what would
be the TCD90 if a surgical excision removed 99% of the tumor clonogens
prior to radiotherapy (assume that the surgery did not otherwise affect the
growth fraction of the tumor).
A. 24 Gy
B. 32 Gy
C. 40 Gy
D. 48 Gy
E. 56 Gy
VII-12) D
A surgical excision of 99% of the tumor would reduce the initial number of clonogens to 10^5. Thus to achieve a 90% control rate, a dose of 48 Gy would be required, corresponding to a final clonogen surviving fraction of 10^-6.
VII-13) For a tumor that requires 18 days to double its diameter, what is the
approximate cell cycle time of its constituent cells (assume no cell loss
and that all cells are actively dividing)?
A. 6 days
B. 9 days
C. 12 days
D. 15 days
E. 18 days
VII-13) A
A doubling of a tumor’s diameter reflects about an 8-fold increase in cell number, which would require 3 cell divisions to accomplish. Thus, if 18 days were required to complete 3 cell cycles, the cell cycle time must be 6 days.
VIII. Linear Energy Transfer
VIII-1) Concerning RBE, OER, and LET, which of the following statements is
TRUE?
A. Maximum cell killing per dose delivered occurs at an LET
corresponding to approximately 1000 keV/µm
B. RBE changes the most over the LET range of 0.1 to 10 keV/µm
C. The relationship between OER and LET is bell-shaped
D. RBE decreases with increasing LET above about 100 keV/µm
E. OER increases with LET
VIII-1) D
RBE decreases with increasing LET above approximately 100 keV/µm. This is thought to be due to the “overkill” effect in which many more ionizations (and damage) are produced in a cell traversed by a very high LET particle than are minimally necessary to kill it, thereby “wasting” some of the energy.
Maximum cell killing occurs at an LET of approximately 100 keV/µm, not 1000 keV/µm.
RBE shows the greatest changes for LET values between roughly 20 and 100 keV/µm.
OER decreases slowly with increasing LET for low LET values, but falls rapidly after LET exceeds about 60 keV/µm and, therefore, does not follow a bell-shaped curve.
VIII-2) Which of the following statements is CORRECT? Compared with
damage from low LET radiation, damage from high LET radiation:
A. Is reduced to a greater extent in the presence of sulfhydryl compounds
B. Shows more potentially lethal damage recovery
C. Exhibits a greater OER
D. Is less subject to split-dose recovery
E. Shows greater sparing when the irradiation is given at a low dose rate
VIII-2) D
There is little or no split-dose recovery following high LET radiation exposure because the single dose survival curves for high LET radiations have little or no shoulder. There is also little or no potentially lethal damage recovery, oxygen effect or radioprotection afforded by the presence of sulphydryl compounds. Delivery of a radiation dose at a low dose rate leads to less sparing for a high LET radiation compared with a low LET radiation.
VIII-3) Which of the following statements concerning RBE is TRUE? The RBE:
A. Is lower for neutrons than for protons over the therapeutic energy
range
B. Is greater for high LET particles in hypoxic cells as compared to
oxygenated cells of the same type
C. Is diminished for carbon ions when delivered over several fractions as
compared to a single dose
D. Is greatest for heavy charged particles at the beginning of the particle
track
E. Increases for MeV alpha-particles with increasing dose
VIII-3) B
Relative Biological Effectiveness (RBE) is defined as: Dose of Reference Radiation (250 keV X-Rays)/ Dose of Test Radiation to give the same biological effect
The reference radiation for calculation of RBE is low LET radiation, suchas 250 keV X-rays or Co-60. The dose of the reference radiation that will achieve the same level of cell killing as high LET particles in hypoxic cells will be greater because there is little to no oxygen effect for high LET radiation.
The RBE is greater for neutrons than it is for protons in the therapeutic energy range because the high energy protons used in radiotherapy are of a relatively low LET and therefore possess an RBE of approximately 1.1.
The RBE for carbon ions, or any other type of high LET radiation, is greater for fractionated irradiation compared with an acute exposure due to the substantial sparing exhibited with reference X-rays with fractionation.
The RBE for charged particles is low at the beginning of the particle track and greatest near the end of the track, in the Bragg peak region.
RBE does show a fractionation dependence; it decreases with increasing fraction size. The RBE for 4 MeV alpha particles will decrease with increasing dose because there is more sublethal damage repair with low LET X-rays at lower doses, and therefore more survival compared with high-LET radiation.
VIII-4) Which of the following pairs of radiation type and approximate LET value
is CORRECT?
A. 150 MeV protons – 0.5 keV/m
B. 1 GeV Fe ions – 20 keV/m
C. 60Co -rays – 15 keV/m
D. 2.5 MeV -particles – 5 keV/m
E. 250 kV X-rays – 10 keV/m
VIII-4) A
150 MeV protons have an LET of approximately 0.5 keV/m. 1 GeV Fe ions, 60Co -rays, 2.5 MeV -particles and 250 kV X-rays have LET values of approximately 143, 0.2, 166, and 2 keV/m, respectively.
VIII-5) Which of the following statements concerning LET is INCORRECT?
A. LET is proportional to charge density of a medium
B. LET is proportional to charge (squared) of the particle moving
through a medium
C. LET is inversely proportional to speed (squared) of the particle
D. LET is inversely proportional to mass of the particle moving through
a medium
E. LET is related to density of ionizations along the particle’s track
VIII-5) D
LET is a measure of local energy deposition along a track of medium. It is inversely proportional to the energy of a given charged particle. The local transfer of energy to medium is more probable at lower energies.
VIII-6) Which statement concerning the linear energy transfer (LET) is
CORRECT?
A. LET is equal to the energy transferred by ionizing radiation to soft
tissue per unit mass of soft tissue
B. LET is equal to the number of ion pairs formed per unit track length
C. Once a photon transfers all its energy to an electron, the LET is that
of the electron
D. LET is the quotient of the average energy that a particle lost in causing
ionization to the average distance it travels between two consecutive
ionizations
E. The track average method and the energy average method for
calculating LET give different numerical values for therapy protons
in soft tissue
VIII -6) D
LET is the quotient of the average energy that a particle lost in causing ionization to the average distance it travels between two consecutive ionizations. Photons, such as 250 KV X-rays, in passing through tissue produce no ionizations directly but only by setting in motion atomic electrons of tissue molecules. Electrons set in motion by incident photons have a broad energy distribution which is dissipated in tracts with LET ranging from about 0.4 to 40 keV/μm. Radiation therapy high energy photons can generate neutrons with energy between 0.1 to 2 MeV through photon interactions with nuclei of high atomic number materials that constitute the linac head and collimator systems. These neutrons in passing through tissue also produce no ionization directly but by setting protons in motion by knock on collisions with hydrogen nuclei of the cellular water molecules. Protons set in motion by photoneutrons dissipate energy over a range of LET up to about 70 keV/μm. Answer choice E mainly pertains to neutrons, not protons, where the average method and the energy method for calculating LET give significantly different numbers.
VIII-7) How many ion clusters are formed by 55 keV/μm silicon ion along a 1 μm
segment of the ion trajectory through the cell nucleus? Assume silicon ion
irradiation with the beam parallel to a cellular monolayer and that ion
clusters are uniformly spaced along the silicon ion track
A. 0.5 cluster every 1 μm or 1 cluster every 2 μm
B. 5.5 clusters every 1 μm
C. 500 clusters every 1 μm
D. 5,500 clusters every 1 μm
E. 55,000 clusters every 1 μm
VIII-7) C
On average, the formation of a three-ion cluster requires dissipation of 110 eV. Therefore, or 1 cluster every 20 Ǻ (1 μm = 10,000 Ǻ). This spacing of ion clusters along the silicon ion track corresponds to a 20 Ǻ diameter of the DNA helix.
IX. Modifiers of Cell Survival: Oxygen Effect
IX-1) What is the approximate maximum diffusion distance of oxygen from a
normally-oxygenated capillary through a typical respiring tissue?
A. 5 nm
B. 15 m
C. 200 m
D. 900 m
E. 2.6 mm
IX-1) C
In a typical respiring tissue, the approximate distance that oxygen can diffuse from a normally oxygenated capillary before cellular hypoxia is detectable is approximately 70-200 µm. The oxygen diffusion distance will depend on the partial pressure of oxygen in the capillary and on the rate of oxygen consumption by the tissue, and therefore shows some variability. Thomlinson and Gray measured 150 µm in their landmark experiments in 1955.
Olive et al. (IJROBP 1992) determined that the maximum oxygen diffusion distance using solid tumor cubes incubated with fluorescent probes and found it to range from 107 um to 192 µm, depending on the cell line.
Torres Filho et al. (Proc. Natl. AcadUSA 1994) measured in vivo oxygen concentration in a SCID mouse model and found hypoxia to occur at distances >200 µm.
IX-2) A dose of 10 Gy of X-rays reduces the tumor cell surviving fraction to
0.001 in an animal irradiated while breathing air, and to 0.1 in an animal
irradiated under nitrogen. An estimate of the hypoxic fraction for this
tumor in the air-breathing mice would be:
A. 0.0001
B. 0.01
C. 0.25
D. 10
E. 25
IX-2) B
The fraction of cells in a tumor that are hypoxic can be estimated using the paired survival curve method. This corresponds to the surviving fraction of cells irradiated in normally oxygenated tumors divided by the surviving fraction of cells from a tumor made fully hypoxic by asphyxiating the host with nitrogen immediately prior to irradiation, which is assumed to render all of the tumor cells radiobiologically hypoxic. Thus, the estimate for the fraction of hypoxic cells would be 0.001/0.1 = 0.01.
IX-3) The Km for radiosensitization by oxygen (the oxygen concentration at
which cellular radiosensitivity is halfway between the fully aerobic and
fully hypoxic response) corresponds to an oxygen concentration of
approximately:
A. 0.02%
B. 0.5%
C. 3%
D. 15%
E. 30%
IX-3) B
The Km value occurs at an oxygen concentration of roughly 0.5-1% or 3- 8 mm Hg.
IX-4) The most dramatic change in radiation sensitivity occurs over which of
the following ranges of oxygen tension (in units of mm Hg or Torr)?
A. 0-30
B. 30-60
C. 60-100
D. 100-250
E. 250-760
IX-4) A
The most dramatic change in radiation sensitivity occurs over an oxygen tension range of 0-30 mm Hg (Torr). Cells irradiated under an oxygen partial pressure at the low end of this range are maximally radioresistant, whereas irradiation at 30 mm Hg oxygen results in near maximum radiosensitization.
IX-5) Which of the following statements concerning the oxygen effect is
TRUE?
A. OER values obtained for high energy protons used in radiotherapy are
similar to those measured for X-rays
B. During irradiation, an oxygen partial pressure of about 30% is
required to produce full radiosensitization.
C. The OER is defined as the ratio of the surviving fraction of cells
irradiated with a particular X-ray dose under hypoxic conditions
divided by the surviving fraction of cells irradiated with the same dose
under aerated conditions
D. Tumors are thought to contain regions of both acute and chronic
hypoxia; however, only chronically hypoxic cells can reoxygenate
E. The oxygen effect is principally a manifestation of the reaction of O2
with sulfhydryl compounds to form SOO
IX-5) A
Since the high energy protons used in radiotherapy have an LET similar to that of X-rays, their OER values are also similar. However it is important to know that as LET increases, OER values decrease. An oxygen partial pressure greater than about 2-3% during irradiation will result in essentially full radiosensitization.
The OER is defined as the ratio of the radiation dose needed to cause a certain biological effect in hypoxic cells divided by the dose needed to produce the same effect in aerated cells.
Both acutely and chronically hypoxia cells can reoxygenate
The increased cell killing resulting from irradiation in the presence of oxygen is thought to be the result of increased radical damage and damage fixation by oxygen. The initial number of ionizations produced by radiation in the aerated and hypoxic cells would be the same.
IX-6) For single, large radiation doses delivered at a high dose rate, the ratio of
the OER for X-rays divided by the OER for 15-MeV neutrons is
approximately:
A. 0.3
B. 1
C. 2
D. 4
E. 10
IX-6) C
Since the X-ray OER is typically about 3 and the OER for 15 MeV neutrons is about 1.6, the ratio of the OERs is about 2.
IX-7) Which of the following statements concerning the effect of oxygen is
TRUE?
A. Oxygen acts as a radiosensitizer because it inhibits chemical repair
of DNA
B. The OER and RBE both increase with increasing LET
C. Based on pO2 microelectrode measurements, few human tumors
contain regions of hypoxia
D. An oxygen partial pressure of about 30 mM Hg results in a
radiosensitivity halfway between hypoxic conditions and fully
oxygenated conditions.
E. Exposure of cells to hypoxia may stimulate gene transcription
IX-7) E
Exposure of cells to hypoxia, as in other stress situations, leads to changes in expression of a number of stress genes, many of which are responsive to the transcription factor, hypoxia-inducible factor-1 (HIF-1) (HIF1A).
Under normoxic conditions, HIF-1 is hydroxylated on proline residues by oxygen-dependent prolyl hydroxylases. The hydroxylated prolines bind to the von Hippel-Lindau (VHL) protein, which is a component of the E3 ubiquitin-protein ligase complex that ubiquitinates HIF-1 and targets it for degradation.
Oxygen acts as a radiosensitizer principally through its ability to “fix” radiation-induced DNA damage but does not inhibit DNA repair
The OER decreases with increasing LET, whereas the RBE increases with LET until reaching a maximum at approximately 100 keV/µm, and then decreases
.
Measurements with pO2 microelectrodes and bioreductive probes have demonstrated that hypoxic cells are often present in human tumors
The Km of radiosensitivity for cells (i.e., the concentration at which there is 50% radiosensitivity compared to oxic conditions) is about 3mm Hg (about 0.5%), not 30mm Hg.
IX-8) Which of the following statements is FALSE when describing tumor
hypoxia?
A. In rodent tumors, the hypoxic cell fraction is generally within the
range of 5-50%
B. Hypoxia is rarely observed in common human solid tumors
C. Oxygen diffusion and delivery is limited in some parts of tumors
D. Hypoxia can enhance tumor progression by means of hypoxia-related
changes in gene expression
E. Hypoxia induces gene amplification and mutation
IX-8) B
Hypoxia in tumors has been detected using both imaging and direct electrode measurements.
IX-9) Which chemical or compound CANNOT be used to mitgate hypoxiarelated radioresistance?
A. Nicotinamide and carbogen
B. Perfluorocarbon
C. Amifostine
D. Misonidazole
E. Nimorazole
IX-9) C
Amifostine is a drug whose active metabolite contains a sulfhydrl moiety and acts as a free radical scavenger. It has been studied as a radioprotectant in several clinical and preclinical settings. As a radioprotectant, it does not sensitize hypoxic cells.
Carbogen is a mixture of 95% oxygen and 5% carbon dioxide and has been used to mitigate chronic hypoxia. Nicotinamide, used concurrently with carbogen, is intended to mitigate acute intermittent hypoxia seen in tumor vessels by preventing intermittent vessel closure
Perfluorocarbons such as perflubron have been shown to improve tumour oxygenation in preclinical cancer models but have not yet shown clinical utility
Both misonidazole and nimorazole are nitroimidazoles that have radiosensitizing properties in hypoxia cells.
IX-10) Oxygen enhancement ratio (OER) changes depends on the type of
radiation. Which of the following combinations is FALSE?
A. OER 3.0 for x-rays
B. OER 1.6 for neutrons
C. OER 3.0 for protons
D. OER 0.5 for carbon ions
E. OER 1.0 for alpha-particles
IX-10) D
OER for energized ions should be 1.0. By definition, OER cannot be smaller than 1.0.
IX-11) Tirapazamine and other hypoxic cytotoxins have been in preclinical and
clinical development. Which of the following statements is FALSE when
describing the mechanisms and effects of tirapazamine?
A. If it gains one electron in hypoxic conditions, it becomes cytotoxic
B. When two electrons are extracted in aerobic conditions, it becomes
less toxic
C. In normoxic conditions, it can also sensitize cells to radiation
D. Its uptake is greater for cells in hypoxic conditions than cells in
aerobic conditions
E. The potency of some chemotherapy agents can be enhanced by the
presence of this cytotoxin
IX-11) D
There is no difference in the uptake of the chemical by aerobic and hypoxic cells, but there is an obvious difference in the action of cell kill due to the amount of oxygen available in the cells.
Tirapazamine (TPZ) is a benzotriazine di-N-oxide which is reduced (i.e. gaining an electron) preferentially under hypoxia with the help of an intracellular reductase forming the reactive, TPZ radical as well as the very potent hydroxyl radical downstream.
In contrast, under aerobic conditions, the initial TPZ radical is rapidly back-oxidized (i.e. losing electrons) to the parent compound losing its reactive potency, ultimately driving the selectivity for hypoxic cells. A byproduct of this reversal is superoxide which may be responsible for the muscle cramps seen in patients.
IX-12) Which of the following statements about tirapazamine is FALSE?
A. A trial examining the utility of the drug in the definitive treatment of
locally advanced cervical cancer was conducted but failed to fully
accrue due to lack of drug availability.
B. Addition of tirapazamine failed to improve 2-year overall survival in
head and neck patients treated with cisplatin-based chemoradiation
compared to chemoradiation alone without tirapazamine.
C. The use of tirapazamine was not associated with higher rates of
esophagitis in limited stage small cell lung cancer patients treated with
definitive chemoradiation compared to historical controls.
D. In GOG 219, tirapazamine increased gastrointestinal toxicity while
having no effect on progression free survival.
E. In GOG 219, tirapazamine failed to show benefits in progression free
survival but led to significant increase in leukopenia and hepatic/renal
dysfunction.
IX-12) C
In the SWOG 0222 trial, tirapazamine was associated with higher rates of esophagitis compared to historical estimates. Tirapazamine was developed as a hypoxic cytotoxin to potentially enhance tumor responses and showed promise in single-arm phase II trials in several disease sites. However, the phase III trials failed to show efficacy above the control arms. These include GOG 219, which examined the efficacy of the drug when added to standard chemoradiation for locally advanced cervical cancer. The trial failed to accrue due to lack of drug availability. Of a planned accrual of 750 patients, only 379 were eligible and evaluable; no difference in overall or progression-free survival was seen, but there was an increase in grade 3+ leukopenia, GI toxicity, and hepatic/renal dysfunction. The TROG examined tirapazamine’s utility and safety in head and neck cancers in the HeadSTART TROG 02.02 Trial. There was no overall survival or failure-free survival benefit of tirapazamine when added to cisplatin-based chemoradiation in this trial. The authors reported more frequent muscle cramps, diarrhea, and skin rash in the experimental arm.
IX-13) Which of the following is TRUE regarding the use of the hypoxic
radiosensitizer nimorazole in treating head and neck cancers with
radiotherapy?
A. The DAHANCA 5-85 trial showed improved locoregional control but
not overall survival with the addition of nimorazole.
B. Low plasma concentrations of osteopontin were associated with worse
outcomes compared to higher concentrations in the DAHANCA 5 trial
but was also associated with a higher benefit from the addition of
nimorazole.
C. The addition of nimorazole improves locoregional control in p16-
positive tumors but not p16-negative tumors.
D. The addition of nimorazole to radiation is associated with increased
acute mucositis and late fibrosis compared to placebo.
E. The DAHANCA 5-85 trial showed improved locoregional control but
not disease-specific survival with the addition of nimorazole.
IX-13) A
The DAHANCA 5-85 Trial examined the addition of nimorazole to conventionally fractionated radiotherapy for pharyngeal and supraglottic larynx cancers. The trial demonstrated that nimorazole improved locoregional control and disease-specific survival compared to placebo but did not significantly improve overall survival.
Nimorazole was reasonably well-tolerated in the DAHANCA 5-85 Trial, although only 60% of patients completed the treatment, and it was associated with a higher rate of nausea/vomiting, flushing, dizziness, and skin rash; patients also had trouble swallowing the large capsules of the drug. There was no increase in mucositis or late complications with the addition of nimorazole.
Additional subset analyses of the trial showed that high osteopontin concentration was associated with worse disease-specific mortality, but also improved response to nimorazole in terms of locoregional control and disease-specific mortality.
The benefit of nimorazole also appears to be isolated to p16-negative tumors, whereas p16-positive tumors did not appear to benefit.
X. Modifiers of Cell Survival: Repair
X-1) An exponentially-growing, asynchronous population of cells is
maintained under normal physiological conditions. Which of the
following experimental manipulations would potentiate cell killing
following radiotherapy as measured by a clonogenic assay?
A. Cell synchronization in S-phase at the time of irradiation
B. Irradiation under hypoxic conditions
C. Irradiation with the dose split into two fractions with a 24-hour
interval between fractions rather than given as an acute exposure to
the same total dose
D. Incorporation of bromodeoxyuridine into the DNA prior to irradiation
E. Addition of cysteine to the cellular growth medium prior to
irradiation
X-1) D
Bromodeoxyuridine, a synthetic nucleoside analogue, when incorporated into cellular DNA in place of thymidine acts as a radiation sensitizer, so cell killing would be enhanced. S-phase is the most radioresistant phase of the cell cycle, so cell killing would be decreased relative to that for an asynchronous population.
Oxygen is a radiation sensitizer, so cell killing would decrease in cells made hypoxic before irradiation.
Splitting the dose into two fractions separated by 24 hours would allow sublethal damage recovery and possibly enable cellular proliferation to take place between fractions. Cell killing would therefore be less than if the total dose had been delivered acutely.
Cysteine is a sulfhydryl-containing compound that scavenges radiation induced free radicals; it therefore acts as a radioprotector and reduces cell killing.
X-2) For irradiation with X-rays, the increased cell survival observed when a
given total dose is delivered at a low dose-rate (~1 Gy/hr) versus high
dose-rate (~1 Gy/min) is due primarily to:
A. Repair of DNA double-strand breaks
B. Decreased production of DNA double-strand breaks
C. Induction of free radical scavengers
D. Activation of cell cycle checkpoints
E. Down-regulation of apoptosis
X-2) A
Therapeutic radiation at a low dose-rate of ~1 Gy/hr is associated with increased cell survival compared to higher dose-rates primarily due to DNA repair - especially the repair of DNA double-strand breaks produced as a result of radiation.
X-3) Compared to the surviving fraction of cells maintained in a non-cycling
state for several hours after irradiation, decreased cell survival observed
in cells forced to re-enter the cell cycle immediately following irradiation
is evidence for:
A. Rejoining of chromosome breaks
B. Sublethal damage recovery
C. Cell cycle reassortment
D. Translesion of DNA synthesis
E. Expression of potentially lethal damage
X-3) E
Potentially lethal damage recovery is operationally defined as an increase in cell survival under environmental conditions not conducive to progression of cells through the cell cycle for several hours after delivery of a large, single radiation dose. If non-cycling cells are forced to re-enter the cell cycle immediately after irradiation, rather than remaining quiescent, potentially lethal damage will be “expressed” and therefore the surviving fraction will be lower.
Sublethal damage recovery is operationally defined as an increase in cell survival noted when a total radiation dose is delivered as two fractions with a time interval between the irradiations, i.e. splitting the dose, as opposed to a single exposure.
Repair of DNA damage and rejoining of chromosome breaks presumably underlie both the sublethal and potentially lethal damage recovery.
Cell cycle reassortment has a sensitizing effect on a population of cells receiving multi-fraction or protracted irradiation regimens. This is because surviving cells that were in a resistant phase of the cell cycle during the initial irradiation may progress through the cell cycle between fractions and reassert into a more sensitive phase of the cell cycle by the time of delivery of the next fraction. This process is irrelevant under the conditions described here, in which only a single radiation dose was administered.
Translesion DNA synthesis is an error-prone process during which certain DNA polymerases synthesize DNA using a damaged DNA strand as a template, resulting in error-prone DNA synthesis.
X-4) 5 Gy of X-rays is delivered at a high dose rate (1 Gy/min) rather than a low dose
rate (1 Gy/hr). Which of the following statements about the effects of this
change on cell survival is TRUE?
A. The surviving fraction would change the least for a cell line with a
radiation survival curve characterized by a low / ratio
B. Treatment of cells during irradiation with an agent that inhibits DNA
repair would have a greater impact on the surviving fraction of cells
irradiated at the high dose rate
C. More cell killing would occur following treatment at the high dose
rate
D. The difference in the surviving fractions between the two protocols
results primarily from repopulation
E. The total number of ionizations produced is decreased with treatment
at the high dose rate
X-4) C
When a dose of 5 Gy is delivered at a dose rate of 1 Gy/min, irradiation requires 5 minutes. When 5 Gy is delivered at 1 Gy/hr, irradiation requires 5 hours. Extensive repair of sublethal damage will occur during the low dose rate, but will not be able to occur during high dose rate irradiation. Essentially, the chance for separate lesions to interact with one another forming more complex, lethal lesions increases at the higher dose rate. As a result, the component of cell killing will increase. More cell killing would therefore occur when a dose of 5 Gy is delivered at a high dose rate rather than a low dose rate. The surviving fraction would change the least for a cell line with a radiation survival curve characterized by a high, not low, / ratio.
Treatment with an agent that inhibits DNA repair would have little impact during the 5-minute period of irradiation that would occur at the high dose rate. In contrast, such a treatment would markedly reduce cell survival for the 5-hour irradiation required at the low dose rate since, in the absence of the agent, substantial repair would take place during the course of the irradiation. The increase in the surviving fraction for this low dose rate irradiation is primarily a consequence of sublethal damage recovery and not repopulation, as the repopulation would only occur for overall treatment times on the order of days.
The total number of ionizations produced is a reflection of the total dose delivered and does not vary with the dose rate.
X-5) Exponentially growing cells were maintained at 37o
C in 95% air/5% CO2
and irradiated with either a single dose of 8 Gy of X-rays or two 4 Gy
fractions separated by either 2 hours or 8 hours. The surviving fractions
for the three treatments were 0.02, 0.15, and 0.08, respectively. The two
processes that best account for these differences in survival are:
A. Reassortment and repopulation
B. Repair and reassortment
C. Reoxygenation and repair
D. Repopulation and reassortment
E. Repair and reoxygenation
X-5) B
Compared to the cell surviving fraction after the single 8 Gy dose, the increase in cell survival noted for the two 4 Gy doses delivered with a 2- hour interfraction interval was due to sublethal damage repair (SLDR).
Although SLDR also occurred when the interfraction interval was 8 hours, cells surviving the first dose start progressing through the cell cycle and reassort from the radioresistant phases they were in at the time of the initial irradiation (e.g. late S) into more radiosensitive phases (e.g. G2 and M), thereby resulting in an overall surviving fraction for the 8-hour interval that was lower than that for the split dose protocol with a 2-hour interval between fractions. It is unlikely that much repopulation would take place during the total time of 8 hours needed to complete the irradiations. Reoxygenation would not be an issue for cells maintained in a well-aerated 95% air environment.
X-6) Which of the following pairs of radiobiological process and
corresponding assay method is CORRECT?
A. Reoxygenation – HIF-1 (HIF1A) phosphorylation by ATM
B. Potentially lethal damage recovery – tritiated thymidine uptake
C. Cell cycle “age response” – paired survival curve method
D. Sublethal damage recovery – split dose experiment
E. Repopulation – mitotic shakeoff procedure
X-6) D
Sublethal damage recovery is operationally defined and demonstrated using a split dose protocol.
Potentially lethal damage repair is detected by changing the post irradiation environment and observing the effect on survival. Incorporation of tritiated thymidine into DNA would not specifically measure PLDR, but would reflect DNA synthesis and other forms of DNA repair.
Reoxygenation would best be assayed by performing repeat measurements during the course of radiotherapy by using an oxygen electrode or by treating with a hypoxia maker, such as pimonidazole, that is metabolized and incorporated exclusively into hypoxic cells.
Cell cycle age response is best demonstrated by performing cell synchronization followed by irradiation of cohorts of cells in particular cell cycle phases and then performing the clonogenic survival assay as a readout.
Repopulation can be assayed in vitro by counting the number of cells present as a function of time after irradiation. The mitotic shake off technique is used to collect synchronous populations of cells for use in experiments examining age response functions
X-7) Which of the following is a phosphoinositol 3-kinase like kinase (PIKK)
that serves as the central orchestrator of the signal transduction response
to DSBs?
A. Ku70/80
B. ATM
C. Rad50
D. MSH2
E. p53 (TP53)
X-7) B
Ataxia Telangiectasis Mutated (ATM) serves as the central orchestrator of the signal transduction response to DSBs. Cells deficient in ATM activity display cell cycle checkpoint defects and sensitivity to ionizing radiation.
X-8) Which of the following is TRUE for potentially lethal radiation damage
(PLD)?
A. It is irreversible and irreparable.
B. It is the damage that can be repaired efficiently if cells are allowed to
progress through the cell cycle immediately following IR.
C. It is thought to be primarily complex or “dirty” double strand breaks.
D. It can be observed in a “split dose” experiment.
E. It cannot be detected in tumors in vivo.
X-8) C
PLD is believed to be complex double strand breaks (DSBs) that are repaired slowly as compared to simple DSBs. Therefore, cells that are left in stationary phase after irradiation display enhanced survival as they have time to repair complex DSBs before resuming progression through the cell cycle.
X-9) Which of the following is FALSE for the split dose experiment and
sublethal damage (SLD)?
A. The survivors of the first dose are mainly S-phase.
B. The fraction of cells surviving a split dose decreases as the time
interval between the two doses increases
C. When cells are cycling during the split dose experiments, there is a
dip (decrease) in cell survival caused by reassortment.
D. SLD can be repaired before they can interact to form lethal
chromosomal damage.
E. SLD is demonstrated by low-LET radiation
X-9) B
Sublethal damage (SLD) is the term used to describe the increase in cell survival that is observed if a given radiation dose is split into two fractions separated by a time interval, i.e. allowing time for repair. However, this benefit in survival can be offset when the time between doses extends so much that reassortment, reoxygenation, and repopulation kick in. Ultimately, the outcome of split-fraction experiments depends on the interplay between Repair of DNA damage, Reassortment through the cell cycle and Repopulation (requires a split time period greater than the cell cycle length).
S-phase, especially the latter part of S-phase is the most radioresistant cell cycle phase therefore most survivors of the first fraction are generally in S phase. SLD is seen to a far higher-degree in low-LET irradiated cells than in high-LET irradiation.
The fraction of cells surviving a split dose increases with increasing time between the two doses because of the repair of SLD, hence B is false.
X-10) The survival of mammalian cycling cells at 37o
C increases dramatically
if a X-ray dose is split into two fractions, given within 1 hour of one
another and reaches its maximum if the time between each fraction is
about 2h. What is the mechanism driving this phenomenon?
A. Enhanced survival in split-dose experiments such as this indicate
recovery from potentially lethal damage.
B. The main mechanism driving this is the repair of sublethal damage in
the G1 cell cycle phase.
C. The mechanism that enhances survival is sublethal damage repair that
in cycling mammalian cells increases to a maximum between about 1-
2h after the first exposure.
D. Potentially lethal damage repair tends to be complete by 1h and
survival does not increase further by increasing time allowed beyond
1h.
E. The initial, dramatic benefit in survival is due to sublethal damage
repair while potentially lethal damage repair drives the survival
benefits at intervals of about 2h.
X-10) C
The effect of a given dose of radiation is less if it is split into two fractions, delivered a few hours apart which is due to the repair of sublethal damage. Most drastically this is observed within the first hour when most damage is repaired, starting to plateau after 2h for most proliferating mammalian cells.
The majority of surviving cells will come from S-phase as this is the most radioresistant cell cycle phase.
Potentially lethal damage repair is not assessed in split dose experiments but is more about post-exposure conditions and the time available for repair in arrested populations before cell cycle resumes. (single-dose experiment)
XI-1) Which of the following assays would NOT be useful for the purpose of
quantifying the response of a tumor to irradiation?
A. Lung colony assay
B. Number of tumors per animal
C. Time to reach a certain size
D. Growth delay
E. Colony forming ability of cells explanted from the tumor
XI. Solid Tumor Assay Systems Answers & Explanations
XI-1) B
An increase in the number of tumors per animal would be a reflection of metastatic spread of the tumor, and would not necessarily reflect the radiation response of the primary tumor per se. All of the other assays can be used to quantify the response of tumors to irradiation.
The lung colony assay is a technique to measure the radiosensitivity of tumor cells that are irradiated in situ. Following irradiation, the tumour is removed, made into a preparation of single cells, and then injected into the mouse tail vein. Cells that retain clonogenic capacity form colonies in the lung that are countable. This allows comparison of clonogenic survival in various in situ experimental scenarios
Similarly, colony forming ability of cells explanted from a tumour are radiated in situ; however, radiation response is tested by counting the surviving colonies grown in vitro, also known as the in vivo/in vitro assay.
Lastly, tumor growth assays, quantifies radiation response through the measurement of tumor diameter at various time points following radiation compared to untreated tumors.
XI-2) The TCD50 assay:
A. Measures radiation-induced tumor growth delay
B. Can be conducted using mouse tumors but not human tumor
xenografts
C. Gives a measure of the number of cells required to produce a tumor in
a mouse
D. Yields results independent of the immune competence of the host
animal
E. Measures tumor cure, making it a relevant endpoint for extrapolation
to the clinic
XI-2) E
The TCD50 assay quantifies the dose required to cure 50% of a group of matched tumors and is therefore a highly relevant endpoint for extrapolation to the clinic. The assay can be conducted using mouse tumors or human tumor xenografts, although suppression of the host immune system when using xenografts is crucial in order to minimize misleading results due to rejection of implanted cells. The TD50 assay can be used to measure the number of cells required to cause a tumor in mice and has historically been used to determine tumor cell survival curves, to assess the number of clonogens in a tumor, and to study host factors that influence tumor development
The number of cells required to produce tumor formation in mice is
indicated in the graph below for a glioblastoma cell line (bulk) and two
sub-lines derived from it (CD133+
and CD133-
). Which of the following
statements would best explain the experimental findings?
A. CD133-
cells comprise only a small fraction of the total tumor
B. CD133 is a putative marker for cancer stem cells
C. Unsorted bulk cells contain a large fraction of cancer stem cells
D. CD133-
cells are more radiosensitive than CD133+ cells
E. CD133+
cells are more radiosensitive than CD133-
cells
XI-3 B
CD133 has been described as a putative marker for cancer stem cells in glioblastoma. Injection of 100 CD133+ cells is sufficient to initiate tumour formation in >30% of nude mice, supporting CD133 as a potential cancer stem cell marker. The unsorted bulk cells contain cancer stem cells; given, however, that 100-1,000 more cells are required in order to form the same number of tumors as that seen when purified CD133+ cells are injected, it appears that <1% of the cells in the tumor are stem cells. CD133- cells were derived from a glioblastoma and are therefore not normal, although they possess a very limited ability to form tumors de novo. In this experiment, no data are provided regarding the sensitivity of the cell lines (or sublines) to radiation.
XI-4) A local tumor recurrence after radiotherapy can be caused by:
A. Any surviving cancer cell
B. Any proliferating cancer cell
C. Only cancer cells with the ability to form colonies in vitro
D. Only cancer cells with unlimited proliferative potential
E. Only cancer cells that were well-oxygenated during irradiation
XI-4) D
Results from tumor transplantation experiments indicate that only a small proportion of all cancer cells have an unlimited proliferative capacity and demonstrate the capacity of self-renewal. In analogy to in vitro assays, tumor cells that demonstrate the ability to achieve a local recurrence following radiotherapy have been termed “clonogenic cells” and correspond to putative “cancer stem cells,” or “tumor initiating cells.” The existence of cancer stem cells, defined by the ability for self-renewal and generation of the heterogeneous lineage of cells within a tumor, has been hypothesized.
XI-5) Which assay or endpoint would provide the best estimate of the radiation
response of putative cancer stem cells?
A. Time to first evidence of tumor shrinkage following irradiation
B. Tumor regrowth delay
C. Determining the fraction of proliferating tumor cells 2 weeks after
irradiation
D. 50% tumor control dose
E. Quantifying the number of apoptotic tumor cells 6 hours after
irradiation
XI-5) D
It has been suggested that a small proportion (< 1%) of all cells in a tumour are cancer stem cells. If correct, this hypothesis suggests that all cancer stem cells must be killed or lose replicative potential in order to achieve permanent local tumor control.
Thus, the rate of permanent local tumor control is a direct measure of radiation response of cancer stem cells. In contrast, tumor shrinkage and growth delay are dominated by the response of the bulk of cancer cells and not specific for the radiation response of cancer stem cells. Cancer cells with a limited proliferative capacity, as well as doomed cancer stem cells, might undergo a number of cell divisions before they permanently stop proliferating and ultimately die. Determination of proliferating cells will therefore not provide information regarding the radiation response of cancer stem cells. Cancer cells can die following exposure to radiation in different ways, including interphase death (i.e. apoptosis) and mitotic catastrophe (apoptosis, autophagy, or necrosis). None of these modes of cell death is likely to be specific for cancer stem cells. Given that many solid tumors exhibit resistance to undergoing apoptosis and the controversial data from studies comparing the rate of apoptosis with radiation response of tumors, it is unlikely that the rate of apoptosis after irradiation will be a proper parameter to determine the response of irradiated cancer stem cells.
XI-6) In some experiments, tumors treated with radiation and concurrent
molecularly-targeted drugs against EGFR and VEGFR displayed longer
regrowth delays, but not higher tumor control probabilities, compared to
tumors that were treated with radiation only. Which of the following
statements provides the most likely explanation for this?
A. The treatment is effective for the bulk of tumor cells, but not for cancer
stem cells.
B. The drug did not reach most of the cells due to poor vascular perfusion
in the tumor.
C. Experimental error accounts for this, because growth delay and tumor
control assays usually yield similar results.
D. Tumor cells generally do not express receptors that are targeted by
these drugs.
E. The radiosensitivity of tumor cells does not depend on vascular supply
or physiology.
XI-6) A
There are indeed some examples in the literature showing a discrepancy between growth delay and tumor control probability. In these experiments, various molecular targeting approaches in combination with radiation were investigated. Though difficult to prove, the assumption of a differential effect on cancer stem cells and non-cancer stem cells is the most likely explanation for these results. It is likely that the drug reached the tumor since there was an effect on tumor growth. Cancer cells generally express EGFR and cell survival following irradiation is affected by vascular supply. The observed discrepancy between growth delay and local tumor control in some experimental settings suggests that the latter assay is the preferable endpoint to evaluate new therapeutic approaches with curative intent.
XII. Tumor Microenvironment
XII-1) Which of the following statements concerning tumor hypoxia is TRUE?
A. Hypoxic regions in tumors may be detected using labeled bortezomib
B. As a tumor increases in size, the hypoxic fraction of cells decreases
C. Regions of chronic hypoxia may develop in tumors due to the
intermittent opening and closing of blood vessels
D. In the absence of reoxygenation it is unlikely that all hypoxic cells
would be eliminated from a tumor following a typical course of
radiotherapy
E. Acutely hypoxic tumor cells usually exhibit slow reoxygenation while
chronically hypoxic tumor cells reoxygenate rapidly
XII. Tumor Microenvironment Answers & Explanations
XII-1) D
In the absence of reoxygenation it is unlikely that all hypoxic cells would be eliminated following a typical course of radiotherapy from a tumour possessing even a small percentage of hypoxic cells because hypoxic cells demonstrate approximately 3-fold greater radioresistance compared with aerated cells.
Hypoxic regions in tumors can be detected using a labeled nitroimidazole compound. Bortezomib (Velcade) is a proteasome inhibitor and would not label hypoxic regions within tumors.
Although not without exceptions, as tumors increase in size the hypoxic fraction generally also increases. This is because the typically abnormal tumor vasculature is insufficient to maintain oxygen demand.
Regions of acute or transient hypoxia may develop in tumors due to intermittent blood flow via the intermittent closing down of blood vessels. Chronic hypoxia, on the other hand, is defined as diffusion-limited hypoxia due to the inability of oxygen to diffuse farther than 100 m from a blood vessel.
Acutely hypoxic cells tend to exhibit rapid reoxygenation, whereas chronically hypoxic cells generally reoxygenate more slowly.
XII-2) Bevacizumab (Avastin) is a monoclonal antibody that targets:
A. Basic fibroblast growth factor (bFGF; FGF2)
B. Hypoxia-inducible factor-1 (HIF-1; HIF1A)
C. Von Hippel-Lindau (VHL) protein
D. Ras
E. Vascular endothelial growth factor (VEGF; VEGFA)
XII-2) E
Bevacizumab (Avastin) binds to and neutralizes vascular endothelial growth factor (VEGF)-A ligand, thereby preventing its interaction with cell surface receptors, including the VEGF receptor (VEGFR).
The fibroblast growth factors (FGFs) are a family of pluripotent growth factors that stimulate proliferation of mesodermal or neuroectodermal cells and can play a role in angiogenesis. FGFs have yet to be successfully targeted pharmaceutically.
Hypoxia-inducible factor (HIF)-1 is a transcription factor that detects hypoxia and enhances angiogenesis.
The Von Hippel-Lindau (VHL) protein belongs to a complex that is involved in the ubiquitination and degradation of HIF
The Ras proteins are a family of small GTPases involved in the activation of signaling cascades following activation of receptors. Ras is frequently mutated in human cancers but is difficult to target pharmacologically
XII-3) Which of the following responses is LEAST likely to be observed?
A. Exposure to hypoxia increases the expression of angiogenesispromoting genes
B. Anti-angiogenic therapy improves tumor oxygenation
C. A chronically hypoxic environment increases the metastatic potential
of tumor cells
D. Hypoxia inhibits apoptosis in tumor cells
E. Exposure to hypoxia inhibits cell proliferation
XII-3) D
An increased apoptotic index is often observed in hypoxic regions of tumors.
The gene for vascular endothelial growth factor (VEGF/VEGFA) is one of the major genes under the control of the hypoxia responsive promoter, HRE, which binds the transcription factor, hypoxia-inducible-factor (HIF)-1
Studies in animal models have indicated that treatment with anti angiogenics can cause “normalization” of tumor blood vessels and result in a transient improvement in tumor oxygenation before vessels start to deteriorate.
Pre-clinical studies with animal models as well as clinical studies have linked increased hypoxia in tumors to increased tumor aggressiveness and metastatic potential
Exposure to severe hypoxia halts progression of cells through the cell cycle and therefore inhibits proliferation.
XII-4) Which of the following statements concerning chronically hypoxic cells
in tumors is TRUE? Chronically hypoxic cells:
A. Can be selectively targeted for killing with certain bioreductive drugs
B. Are resistent to hyperthermia
C. Are located within 10 m of capillaries
D. Exist in a high pH microenvironment
E. Are a consequence of intermittent blood flow
XII-4) A
Chronically hypoxic regions in a tumor can be targeted for elimination by administering certain bioreductive drugs that preferentially kill hypoxic, but not aerobic, cells. Chronically hypoxic cells tend to be sensitive to hyperthermia. This is because they exist in an acidic (low pH) microenvironment
Acutely hypoxic cells are a consequence of intermittent blood flow in tumors
It has been shown via model calculations of oxygen consumption rates in respiring tissues and through the use of hypoxia markers that chronically hypoxic cells rarely appear closer than about 70 m from capillaries
XII-5) Which of the following statements concerning tumor angiogenesis is
TRUE?
A. Even without angiogenesis, tumors can grow up to 2 cm in diameter
B. For most tumor types a high microvessel density has been negatively
correlated with metastatic spread
C. Vascular endothelial growth factor (VEGF) is induced under hypoxic
conditions
D. Angiostatin and endostatin are stimulators of angiogenesis
E. Basic fibroblast growth factor (bFGF) is a negative regulator of
angiogenesis
XII-5) C
Expression of vascular endothelial growth factor (VEGF) and downstream angiogenesis is induced under hypoxic conditions via hypoxia-inducible transcription factors that bind to the VEGF promoter to stimulate its transcription. In the absence of angiogenesis, tumors would only be expected to reach a diameter of about 2 mm, not 2 cm
Microvessel density, a measure of angiogenesis, has been correlated positively with metastatic spread for most tumor types Angiostatin and endostatin are inhibitors of angiogenesis while basic fibroblast growth factor (bFGF) is a positive regulator of angiogenesis
XII-6) The regulation of hypoxia-inducible factor-1 (HIF-1; HIF1A) by
oxygen concentration is best described by which of the following
statements?
A. Under hypoxic conditions, HIF-1 transcription and translation are
upregulated as well as translocation of HIF-1 from the cytosol to the
nucleus
B. Under aerobic conditions, the HIF-1 heme moiety becomes
oxygenated. This drives a conformational change in the protein that
limits DNA binding and prevents upregulation of target genes
C. Under hypoxic conditions, HIF-1 is activated by bioreduction,
thereby promoting the up-regulation of target genes
D. Under hypoxic conditions, the HIF-1 heme moiety becomes
deoxygenated. This induces a conformational change in the protein
that leads to enhancing DNA binding and subsequent upregulation of
target genes
E. Under aerobic conditions, HIF-1 is hydroxylated by HIF prolyl
hydroxylases that target the protein for ubiquitination and subsequent
proteosomal degradation, thereby preventing the up-regulation of
target genes
XII-6) E
Hypoxia-inducible factor-1 (HIF-1) is a heterodimer that acts as a key regulator of several oxygen-responsive proteins, including erythropoietin and vascular endothelial growth factor (VEGF). HIF-1 was first identified as a DNA-binding protein that mediated the up-regulation of the erythropoietin gene under hypoxic stress. Subsequent studies have implicated HIF-1 in the regulation of a broad range of oxygen responsive genes including VEGF, VEGF receptors, angiopoietins, nitric oxide synthase, fibroblast growth factors and platelet-derived growth factor (PDGF).
Under aerobic conditions, HIF-1 is hydroxylated by HIF prolyl hydroxylases. Hydroxylation at two prolyl residues targets HIF-1 to the von Hippel-Lindau (VHL) E3 ubiquitin ligase and results in HIF-1 ubiquitination and subsequent proteosomal degradation, thereby limiting upregulation of target genes. Because the hydroxylation catalyzed by prolyl hydroxylases requires molecular oxygen, HIF-1 escapes inactivation under hypoxic conditions.
XII-7) Which of the following statements best describes the “normalization
hypothesis” proposed to explain the survival benefit associated with
combining anti-angiogenics with traditional chemotherapy agents?
A. Anti-angiogenic therapy stimulates the formation of leaky blood
vessels thereby enhancing access of chemotherapy agents to the tumor
parenchyma
B. Anti-angiogenic therapy transiently reduces pericyte coverage of
tumor blood vessels, which would otherwise form a significant
mechanical and biochemical barrier to the delivery of chemotherapy
to the tumor
C. Tumor cell-derived pro-angiogenic factors render endothelial cells
resistant to chemotherapy-induced apoptosis. Anti-angiogenic therapy
eliminates this protection and restores endothelial cell sensitivity to
chemotherapeutic agents
D. Anti-angiogenic therapy reduces the secretion of anti-apoptotic
factors by vascular endothelial cells that would otherwise render
nearby cancer cells relatively resistant to chemotherapeutic agents
E. Anti-angiogenic therapy transiently restores the normal balance of
pro- and anti-angiogenic factors in tumor tissue thereby reducing
tumor vessel leakiness, dilation, and tortuosity as well as increasing
pericyte coverage
XII-7) E
When administered as a single agent, several anti-angiogenic drugs have not yielded a long-term survival benefit. In contrast, delivery of anti angiogenic agents with chemotherapy has produced a significant survival benefit in colon cancer and previously untreated lung and breast cancers.
If the anti-angiogenic agent were destroying tumor vasculature in combination regimens, one would expect decreased tumor blood flow and compromised delivery of chemotherapy to the tumor. The survival benefits produced by the addition of an anti-angiogenic drug to traditional chemotherapeutic regimens therefore appears paradoxical. One possible explanation for this has been termed the “normalization hypothesis.”
Under the pressure of pro-angiogenic factors, tumor vasculature is structurally and functionally abnormal. Anti-angiogenic therapy (transiently) restores the balance of pro- and anti-angiogenic factors. Consequently, immature and leaky blood vessels are pruned, pericyte coverage increases, and the basement membrane becomes more homogenous and normalized. As a result, the resultant vascular bed achieves greater organization by being less leaky, dilated, and tortuous. These morphological changes also result in functional changes, including decreased interstitial fluid pressure, increased tumor oxygenation, and improved penetration of drugs into the tumor parenchyma. Due to improved drug delivery, chemotherapy is more efficacious. Sustained or high-dose anti-angiogenic therapy, however, may drive an imbalance favoring anti-angiogenic factors leading to inadequate tumor blood supply and compromise of the efficacy of systemic therapies.
XII-8) At a distance of 150 µm from the nearest tumor blood vessel, one might
expect all of the following microenvironmental conditions, EXCEPT:
A. Increased hypoxia
B. Decreased pH
C. Decreased interstitial fluid pressure
D. Decreased glucose
E. Increased metabolic acids
XII-8) C
Solid tumors develop regions of increased hypoxia (decreased pO2), decreased pH, decreased glucose, and increased (not decreased) interstitial fluid pressure. Low oxygen levels shift glucose metabolism from oxidative phosphorylation to glycolysis, which increases the production of metabolic acids. Oxygen can diffuse about 70 µm from the arterial end of a capillary.
XII –9) Paclitaxel appears to be effective in radiosensitizing tumors in vivo for all
the following mechanisms, EXCEPT:
A. Induction of apoptosis
B. Upregulation of HIF-1
C. Oxygenation of radioresistant hypoxic cells
D. Arrest of cells in the radiosensitive G2/M phase
E. Decrease of interstitial fluid pressure
XII-9) B
Paclitaxel stabilizes microtubule polymers and protects them from disassembly. As a result, mitosis is consequently blocked and apoptosis is activated.
Paclitaxel has been shown to increase the radiation sensitivity of tumors by inducing apoptosis, increasing oxygenation of hypoxic cells in tumors, arresting cells in the radiosensitive G2/M phase of the cell cycle, and decreasing interstitial fluid pressure.
Some of these studies have been conducted in animal models while others have been performed in clinical trials of human breast cancer patients. No studies have demonstrated upregulation of HIF-1 following treatment with paclitaxel; indeed, one might expect HIF-1 to be degraded more rapidly following reoxygenation.
XII-10) Which of the following statements regarding the tumor microenvironment
is FALSE?
A. Blood vessel supply is heterogeneous and irregular
B. Blood flow through micro-vessels may be sluggish
C. There tends to be an increase in vessel density compared to normal
tissue
D. There are a greater number of hypoxic regions within the
microenvironment of a tumor compared to that seen in normal tissue
E. Nutritional support to the tumor microenvironment is adequate and
homogeneous
XII-10) E
Tumor masses exhibit abnormal blood vessel networks that fail to provide adequate and homogeneous nutritional support.
XII-11) Which of the following statements regarding angiogenesis is FALSE?
A. For a multi-cellular organism to grow, it must have the capacity to
recruit new blood vessels via angiogenesis
B. Angiogenesis is normally regulated by pro-angiogenic, but not antiangiogenic, molecules
C. Angiogenesis is dysregulated in a neoplastic environment
D. Without a nearby blood vessel or effective angiogenesis, a tumor
cannot grow beyond a critical size or metastasize to other organs
E. Tumor cell viability decreases beyond 70m of blood vessels due to
the diffusion limits of oxygen
XII-11) B
Since oxygen is unable to diffuse more than approximately 70 µm from the arterial end of a capillary, tumors require the ability to develop new blood vessels in order to grow. This process is normally regulated by a balance of both pro-(including VEGF, FGF, PDGF) and anti-(thrombospondin, angiostatin, endostatin) angiogenic molecules.
XII-12) Which of the following statements regarding anti-angiogenic therapy
strategies is FALSE?
A. Anti-angiogenic therapies interfere with activators of angiogenesis
B. Anti-angiogenic therapies target receptor tyrosine kinases and related
signal transductions
C. Anti-angiogenic therapies seek to amplify endogenous suppressors of
angiogenesis.
D. Anti-angiogenic therapies use colchicine as an anti-angiogenic agent.
E. Anti-angiogenic therapies ultimately target VEGFR-1 in order to
achieve inhibition of angiogenesis
XII-12) D
Colchicine is an anti-inflammatory agent that binds tubulin. Colchicine itself induces vascular damage but only at doses that are limited by toxicity and therefore not used in the clinical setting for this purpose.
XII-13) Which of the following statements concerning vasculogenesis is TRUE?
A. The process of vasculogenesis is specific to the developing embryo
B. Vasculogenesis refers to a subset of angiogenesis, in that it describes
the formation of only venous vessels as tumors grow beyond 1-2 mm3
C. Tumors use vasculogenesis or angiogenesis in a mutually exclusive
fashion
D. Vasculogenesis is critical for a tumor to achieve local tumor
recurrence following radiotherapy
E. Vasculogenesis utilizes pre-existing blood vessels during the early
stages of tumor development to facilitate tumor growth
XII-13) D
Whereas angiogenesis is the sprouting of endothelial cells from nearby blood vessels, vasculogenesis is the formation of blood vessels from circulating endothelial progenitor cells (i.e. the bone marrow).
Vasculogenesis is of particular importance following treatment with anti angiogenic agents as well as following irradiation. During early tumour development, both vasculogenesis and angiogenesis are likely utilized.
Because tumor irradiation abrogates local angiogenesis, the tumor must rely on the vasculogenesis pathway for re-growth following irradiation.
Tumor irradiation produces a marked influx of CD11b+ myeloid cells into tumors and are critical in order for a tumor to achieve formation of blood vessels after irradiation as well as for tumor recurrence.
XIII. Cell and Tissue Kinetics
XIII-1) Which of the following CDK or cyclin is paired with the correct phase
transition?
A. CDK1 (CDC2) – G2 into M
B. CDK4 – S into G2
C. cyclin A – G2 into M
D. cyclin B – S into G2
E. cyclin D – M into G1
XIII. Cell and Tissue Kinetics Answers & Explanations
XIII-1) A
CDK1 (and cyclin A/B) is associated with the G2 to M cell cycle phase transition.
The other CDKs and Cyclins are appropriately paired as follows:
o G1 phase S phase: CDK4 and Cyclin D1; CDK2 and Cyclin E
o S phase G2 phase: CDK2 and Cyclin A
o G2 phase M phase: CDK1 and Cyclin B/A
XIII-2) Irradiation of an exponentially-growing population of cells in culture with
a dose that kills 90% of cells tends to select surviving cells that are initially
in which phase of the cell cycle?
A. G0
B. G1
C. S
D. G2
E. M
XIII-2) C
A dose that kills 90% of the cells in the population would leave a surviving cell population heavily enriched in the radioresistant cells in late S phase. Radiosensitivity across the cell cycle is ranked as follows from least to most sensitive: Late S, Early S, G1, G2≈M.
XIII-3) The typical cell cycle time (TC) for proliferating cells in human tumors is
in the range of:
A. <1 day
B. 1-5 days
C. 6-25 days
D. 26-100 days
E. >100 days
XIII-3) B
The typical TC for tumor cells in vivo is generally in the range of 1-5 days.
XIII-4) Which of the following statements concerning the cell cycle kinetics of
tumors is TRUE?
A. Often, the cell loss factor (Φ) decreases several weeks after the start
of radiotherapy
B. The growth fraction (GF) is the ratio of the number of viable cells to
the sum of viable and non-viable cells
C. If the volume doubling time (TD) is 60 days and the potential doubling
time (Tpot) is 3 days, then the cell loss factor is 5%
D. Tpot has proven useful in predicting tumor response to accelerated
radiotherapy
E. Typically, the cell loss factor is not of major importance in
determining a tumor’s volume doubling time
XIII-4) A
The cell loss factor (Φ) is the percent of newly produced cells that die or fail to continue dividing. Most human tumors have a ϕ of around 77 % and explains why tumors grow in-vivo far more slowly than would be expected based upon cell doubling time.
CLF=1‐Tpot/Td
Where:
Tpot= potential doubling time = the time necessary to double the number of proliferating tumor cells in the absence of spontaneous cell loss.
Td= volume doubling time = the actual doubling time observed.
Cell loss factor (CLF, ϕ) often appears to decrease several weeks after the start of radiotherapy, which has the net effect of slowing tumor regression. The growth fraction is the ratio of the number of proliferating cells to the sum of proliferating and quiescent cells
.
If the observed tumor volume doubling time (TD) is 60 days and the potential doubling time (Tpot) calculated from the cell cycle time and the growth fraction is 3 days, then the cell loss factor is 95% (Answer Choice C).
Although Tpot (as measured from a tumor biopsy derived from patients previously given bromodeoxyurdine) has not proven to be a robust predictor of long-term outcome after accelerated radiotherapy, it might still be useful for the pre-selection of patients most likely to benefit from accelerated treatment.
For carcinomas, the cell loss factor is usually the major determinant of the discrepancy between a tumor’s potential doubling time and its overall volume doubling time.
XIII-5) Exponentially growing cells are pulse-labeled with tritiated thymidine and
sampled as a function of time thereafter. The time required for the percent
of labeled mitoses to reach 50% of its maximum value corresponds
approximately to:
A. TS
B. TC
C. TG2
D. TG1 + TS/2
E. TG2 + TM/2
XIII-5) E
The question refers to the ‘percent-labeled mitoses technique’ using tritiated thymidine. The radiolabeled thymidine is added to the growth medium of cells for about 20 minutes (flash labeling). Only those cells in S-phase take up the label. The length of time required for the first radioactively-labeled S-phase cells to first enter mitosis, as measured using the percent-labeled mitosis technique, would correspond to the duration of the G2 phase (TG2). The additional time required for the cells to completely fill the mitotic compartment (i.e., 100% labeled mitoses) would be equal to the length of M (TM). The time to reach 50% of the maximum point, therefore, corresponds to TG2 plus TM/2.
XIII-6) If the mitotic index of a cell line is 5%, the growth fraction is 100%, the
cell cycle time is 14 hours, and the correction factor, , is 0.7, what is the
approximate length of mitosis (TM)?
A. 0.2 hours
B. 1 hour
C. 2 hours
D. 4 hours
E. 8 hours
XIII-6) B
Using the equation MI = TM/TC, where MI is the mitotic index, TM is the length of mitosis and TC is the total cell cycle time, then TM = (MI)(TC/) = (0.05)(14 hours)/0.7 = 1 hour. Of note, the TM for most mammalian cells is typically ~1 hour.
XIII-7) Which of the following is the main reason why the volume doubling time
of a tumor rarely equals its potential doubling time?
A. High cell loss factor
B. High metastatic propensity
C. Long cell cycle time
D. Low hypoxic fraction
E. Low growth fraction
XIII-7) A
A tumor’s volume doubling time rarely equals its potential doubling time because most tumors have a high cell loss factor. Formation of metastases represents only one of many reasons for cell loss, and usually is only a minor contributor.
Human tumor cells typically have cell cycle times of a few days whereas tumor volume doubling times are generally on the order of months.
The presence of a high hypoxic fraction would probably contribute to a low growth fraction, which would affect both Tpot and volume doubling time. If hypoxia were a significant cause of cell death, it would affect the cell loss factor and therefore affect the volume doubling time. The presence of non-proliferating cells affects both the tumor volume doubling time and the potential doubling time and does not cause a difference between them. In addition, non-viable cells (whether hypoxic or aerobic) have similar effects on the tumor volume doubling time and the potential doubling time.
XIII-8) Which of the following statements concerning tumor kinetics is TRUE?
A. Cell-cycle times (Tc) are longer than potential doubling times (Tpot)
because of the presence of non-proliferating cells
B. The Tpot is usually shorter than the volume doubling time because the
growth fraction (GF) is usually less than 100%
C. Tpot can be determined if the mitotic index (MI) and the duration of S
phase (Ts) are known
D. Tumors with long values for Tpot are good candidates for accelerated
radiotherapy
E. In the absence of cell loss, Tpot would equal the volume doubling time
(TD) of the tumor
XIII-8) E
The cell loss factor (Φ) is equal to 1-(Tpot/TD). Therefore, if the cell loss factor were zero, then the Tpot would equal the TD. The mean TC is shorter than the Tpot becauseTpot also considers the presence of quiescent cells, and the growth fraction in tumors is generally less than 100%
For solid tumors the Tpot is generally much shorter than the TD because the cell loss factor is typically quite high. The GF is taken into account in the determination of Tpot, so it does not affect the relationship between the Tpot and the TD.
Tpot can be calculated from the labeling index (LI) and the duration of S phase (TS) using the equation Tpot = TS/LI. (where λ is a constant ranging from about 0.6 to 1.0).
It has been suggested that tumors with short pretreatment values for Tpot, (suggesting the presence of rapidly proliferating cells and a high growth faction), would be most likely to benefit from accelerated radiotherapy, but this has not been confirmed in clinical trials performed to date
XIII-9) Which of the following substrates and target sites of the ATM kinase are
implicated in the control of the G2-checkpoint in irradiated cells?
A. CHK2 (CHEK2) and MDM2
B. NBS1 (NBN) and CHK2
C. CHK2 and CDC25C
D. CHK2 and p53 (TP53)
E. PUMA and p53 (TP53)
XIII-9) C
Regulation of the G2 checkpoint by ATM is thought to occur via the activation of CHK2, which phosphorylates CDC25C phosphatase thereby preventing it from dephosphorylating CDK1 (CDC2), a step necessary for the progression from G2 into M phase. The remaining proteins listed are all targets for phosphorylation by the ATM kinase, and, consequently, are implicated in various cell cycle control pathways although not the G2 checkpoint. CHK2 and MDM2 are involved in control of the G1–S phase transition.
ATM also phosphorylates MDM2, which reduces the ability of MDM2 to negatively regulate p53. NBS1 and CHK2 are implicated in S phase progression. Upon phosphorylation by CHK2, p53 is stabilized and causes cell cycle arrest in G1. PUMA (“p53-upregulated modulator of apoptosis”) is a pro-apoptotic gene that can induce cell death via a p53- dependent pathway.
XIII-10) Which of the following pairs of chemicals could be used with flow
cytometry to determine the S phase fraction of a cell population and
estimate of relative DNA content?
A. Bromodeoxyuridine (BrdU) and propidium iodide
B. Tritiated thymidine and hydroxyurea
C. Dichlorohydrofluorescein and cytochrome c
D. H2AX and ethidium bromide
E. Sphingomyelin and ceramide
XIII-10) A
Bromodeoxyuridine (BrdU) is incorporated into DNA in place of thymidine, so it can be used to label cells in S-phase. The incorporated bromodeoxyuridine is assayed using a fluorescently-labeled anti-BrdUrd antibody. Propidium iodide fluoresces when incorporated into DNA. The amount of fluorescence is directly proportional to the DNA content, which, in turn, is a reflection of the cell cycle phase in which the cell is located.
XIII-11) If a tumor is comprised of cells characterized by a high growth fraction
and a short cell cycle time, which of the following would most likely
describe its behavior prior to and after treatment with a curative dose of
radiation?
A. Slow growth, slow regression
B. Slow growth, rapid regression
C. Rapid growth, rapid regression
D. Rapid growth, slow regression
XIII-11) C
Tumor types with a high growth fraction and short cell cycle time would be expected to grow more rapidly. Such a tumor would also be expected to regress rapidly after irradiation since irradiated cells generally die as they attempt to progress through mitosis.
XIII-12) What is the most probable range of cell cycle time (Tc) and tumor
doubling time (Td) for human tumors?
A. Tc, 1 to 5 days and Td, 20 to 30 days
B. Tc, 1 to 5 days and Td, 40 to 100 days
C. Tc, 0.5 to 1 days and Td, 20 to 30 days
D. Tc, 0.5 to 1 days and Td, 40 to 100 days
E. Tc, 1 to 2 days and Td, 120 to 300 days
XIII-12) B
The volume doubling time (TD) of human tumors is characteristically 40 to 100 days, while the cell cycle time is relatively short, generally between 1 to 5 days. This has important implications, which often are overlooked, in the use of cell cycle-specific chemotherapeutic agents or radiosensitizing drugs for which it is the cell cycle time that is relevant.
XIII-13) What is the main reason for the great disparity between the cell cycle time
of individual dividing cells and the overall doubling time of the tumor?
A. Intratumor oxygen partial pressure (pO2)
B. Growth fraction
C. Cell loss factor
D. Body temperature where the tumor grows
E. Extra- and intra-cellular acidity (pH)
XIII-13) C
The growth of a tumor depends on the fraction of cycling or proliferating cells (growth fraction), the cell cycle time, and cell loss (the number of newly produced cells that die or fail to continue dividing). Physical stresses such as low intra-tumoral oxygen concentration, low pH and high temperature both lengthen the cell cycle and slow down tumor growth. The high rate of cell loss in human tumors largely accounts for the great disparity between Tc and the volume doubling time (TD). Values for the cell-loss factor vary from 0% to more than 90% for tumors in laboratory animals.
XIII-14) The cell loss factor represents the ratio of the rate of cell loss to the rate
of new cell production. Which of the following is not a dominant cause of
cell loss in tumors?
A. Death from inadequate nutrition
B. Apoptosis (programmed cell death)
C. Death from immunologic attack
D. Metastasis
E. Cell migration
XIII-14) E
Cell migration within a tumor has recently been described from the study of microbeam radiation therapy (MRT). Since cell migration occurs within 200 m (the interspace between microbeams), the cells still stay in the tumor mass without affecting cell loss from the tumor.
XIII-15) In an untreated tumor with a potential doubling time of 3 days and a cell
loss factor of 80%, the volume doubling time is:
A. 2.4 days
B. 3.5 days
C. 3.75 days
D. 15 days
E. 20 days
XIII-15) D
The cell loss factor (Φ) is equal to 1-(Tpot/TD). Rearranging this, TD = Tpot/(1-Φ). TD = 3 days/(1-0.8) = 15 days.
XIII-16) For a standard course of radiotherapy, which of the following properties
of a tumor would NOT be expected to adversely affect tumor control?
A. Low SF2
B. Short Tpot
C. Slow reoxygenation
D. Large number of tumor clonogens
E. Early onset of repopulation
XIII-16) A
A low value for SF2 indicates that the surviving fraction of tumor cells following irradiation with 2 Gy is low. This should be advantageous for radiotherapy, as it suggests that the tumor cells are relatively radiosensitive. That being said, a consistent, positive correlation between low SF2 and high tumor control probability has yet to be established. The potential doubling time (Tpot) is the time required for a tumor to double in size, taking into account the number of cells in the cell cycle and the speed of progression through the cell cycle. A short Tpot would be deleterious to tumor control because it suggests a high potential for vigorous repopulation during the course of treatment.
Slow reoxygenation may also limit the effectiveness of treatment as hypoxic cells would remain hypoxic and radioresistant for a longer portion of the overall treatment time than if they had reoxygenated rapidly and efficiently. A large number of clonogenic cells would require a higher total dose for eradication; this might increase the probability of adverse normal tissue effects.
Early onset of repopulation would also be deleterious as the tumor cells would be proliferating for a longer time during the course of radiotherapy and the cell population that must be killed to cure the tumor would therefore be larger.
XIII-17) Which of the following represents a possible mechanism by which a novel
compound could INCREASE tumor response to fractionated radiotherapy
if applied prior to each dose fraction?
A. Prevention of cell cycle redistribution
B. Induction of G2 phase arrest
C. Inhibition of reoxygenation
D. Radioprotection of normal tissues
E. Stimulation of DNA repair
XIII-17) B
An agent that arrested cells in the radiosensitive G2 phase of the cell cycle could increase tumor response to a fractionated treatment protocol if provided prior to each dose of radiation.
Prevention of cell cycle redistribution would diminish response to fractionated radiotherapy because surviving cells would remain in a radioresistant portion of the cell cycle rather than being permitted to traverse into a more radiosensitive phase of the cell cycle.
Inhibition of reoxygenation would reduce tumor response to fractionated radiotherapy due to the prevention of the conversion of surviving radioresistant hypoxic cells to more sensitive aerated cells.
Radioprotection of normal tissues would have no bearing on tumour response per se, although it could improve the therapeutic ratio overall, assuming the tumor was not similarly protected
Stimulation of DNA repair would reduce tumor response to fractionated radiotherapy because a greater proportion of tumor cells may survive if treated with an agent with enhanced capacity for DNA repair.
XIII-18) They are great variations among the mammalian cell cycle times,
varying for about 10 hours for hamster cells grown in vitro to hundreds
of hours for stem cells. This is a result of dramatic variation in which of
the cell cycle phases?
A. M phase
B. G1
C. S phase
D. G2
E. Equally split between all phases
XIII-18) B
Variations in the duration of G1 phase are responsible
XIII-19) There are three principal checkpoints in the cell cycle: a) G1/S b) S-
phase and c) G2/M. What is the purpose of these checkpoints?
A. Maintain cell membrane integrity.
B. Decrease tumor cell antigenicity
C. Enable cells to survive low-pH environment.
D. Maintain genomic integrity.
E. Decrease REDOX stress
XIII-19) D
The purpose of the cell cycle checkpoints is to slow down the cell cycle following DNA damage, hence facilitating DNA repair to maintain genomic integrity
XIV. Molecular Signaling
XIV-1) Following exposure of cells to 3 Gy from a 6 MV X-ray beam, the ATM
protein is activated and phosphorylates multiple intracellular targets.
Which of the following is NOT a target for ATM phosphorylation?
A. Histone H2AX
B. p53 (TP53)
C. VEGF (VEGFA)
D. BRCA1
E. Artemis
XIV. Molecular Signaling Answers & Explanations
XIV-1) C
ATM is a kinase that is activated in response to the presence of DNAdouble-strand breaks, such as following exposure to ionizing radiation.
Activated ATM phosphorylates multiple distinct target proteins, including histone H2AX, p53, BRCA1, and Artemis. Phosphorylation of H2AX (to -H2AX) results in chromatin modification that facilitates the recruitment of factors needed for DNA repair.
The tumor suppressors, p53 and BRCA1, activate cell cycle checkpoint and/or DNA repair processes in response to genotoxic stress.
VEGF is a secreted factor that promotes angiogenesis and is not a direct target of ATM phosphorylation.
XIV-2) Which of the following pairs of molecular events and their functional
consequences is INCORRECT?
A. VHL inactivation —- angiogenesis
B. cyclin D1 repression — inhibition of proliferation
C. cytochrome c release — apoptosis
D. ATM phosphorylation —- epistasis
E. miRNAs mis-expression — carcinogenesis
XIV-2) D
Epistasis is a form of gene interaction in which an allele for one trait (at one locus) influences the expression of an allele, at a different locus, for a separate and independent trait; this process is unrelated to ATM phosphorylation.
Inactivation of the VHL (von Hippel-Lindau tumor suppressor) gene results in overexpression of many environmental stress-inducible mRNAs, including those involved in energy metabolism, apoptosis, and angiogenesis via the activation of vascular endothelial growth factor (VEGF).
Cyclin D1 repression is associated with anti-proliferation effects. Its overexpression has been observed in human cancers, including pancreatic, lung, and esophageal.
Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into the cytoplasm is a primary mitochondrial apoptogenic activity.
MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative regulators of gene expression under normal physiological conditions. Mis-expression of, or mutations in, miRNAs are associated with the development of a variety of human cancers, including B-cell chronic lymphocytic leukemia, colorectal cancer, and breast cancer.
XIV-3) Which of the following pairs of transcription factors and genes they
directly regulate is INCORRECT?
A. HIF-1 and VEGF (VEGFA)
B. p53 (TP53) and p21(CDKN1A)
C. FOS and BRCA2
D. E2F and CDC25A
E. p53 and PUMA
XIV-3) C
FOS is a transcription factor that has been shown to modulate a variety of genes involved in stress responses but has not been shown to modulate BRCA2.
HIF-1 is a hypoxia inducible factor known to regulate the expression of the VEGF gene and thus the regulation of angiogenesis.
p53 is a transcription factor that induces expression of p21.
E2F is known to regulate a large number of proteins involved in cell cycle progression, including CDC25A.
PUMA is the major mediator of p53-dependent apoptosis following ionizing radiation in most cell types. PUMA, a pro-apoptotic BH3-only member of BCL2 family protein promotes BAX/BAK and mitochondria dependent apoptosis in various cell types.
XIV-4) Which of the following statements concerning cytokines is TRUE?
A. NF-B is the critical cytokine responsible for the development of lung
fibrosis following irradiation
B. A paracrine response is the result of a cytokine targeting the same cell
that produced the cytokine
C. Most cytokines are tyrosine kinases
D. Cytokines are proteins released by irradiated cells that stimulate
tissues to produce a biological response
E. An autocrine response is the result of a cytokine targeting cells
adjacent to the cell that produced the cytokine
XIV-4) D
Cytokines are proteins released by cells, including irradiated cells, that stimulate tissues to mount a biological response. NF-B is a transcription factor (not a cytokine). TGF-1 (TGFB1) is an important example of one of the cytokines that has been associated with the development of lung fibrosis following irradiation.
A paracrine response is the result of a cytokine that acts upon a cell, other than itself, within a tissue or organ. In contrast, an autocrine response is the result of a cytokine targeting the cell from which it was produced.
Cytokines generally do not have tyrosine kinase activity.
XIV-5) Which of the following statements concerning the response of NF-B to
ionizing radiation exposure is FALSE?
A. NF-B is a transcription factor
B. The Inhibitor of Nuclear factor (NF)-B, IB, is phosphorylated by
ATM and subsequently degraded, allowing NF-B to move from the
cytoplasm into the nucleus
C. NF-B generally acts to stimulate apoptosis and enhance the
radiosensitivity of cells
D. Both DNA double-strand breaks and reactive oxygen species
generated by radiation exposure can activate NF-B
E. NF-B is sequestered as an inactive form in the cytoplasm by
interaction with an inhibitory subunit of the IB
XIV-5) C
Nuclear factor (NF)-B generally exerts a pro-survival influence through interference with apoptotic signals. It accomplishes this via the TNF receptor signaling pathway which, upon activation by an apoptotic signal, is coupled via the FADD adaptor to a caspase cascade involving the initiator caspases-8 or -10. In some cell types, however, this may not occur, since it may be opposed through the parallel triggering by TNF of a signaling pathway that activates NF-B via the TRADD and TRAF adaptors. Active NF-B induces transcription of a set of genes that encode the anti-apoptotic IAPs (“inhibitors of apoptosis”). NF-B can also exert an anti-apoptotic effect by inducing transcription of anti-apoptotic proteins, such as Bcl-xL (BCL2L1), which act to prevent cytochrome c release and the subsequent caspase-9 activation. IB binds to NF-B to prevent its translocation to the nucleus. Following formation of DNA double-strand breaks and reactive oxygen species in irradiated cells, kinases (including ATM) phosphorylate IB, targeting it for ubiquitination and degradation, which allows NF-B to translocate to the nucleus from the cytoplasm where it can act as a transcription factor. NF- B can exist as hetero- or homodimers of five different subunits. Different heterodimers activate different sets of genes while p50 and p52 homodimers, lacking transactivation domains, can selectively repress expression of their target genes. Post-transcriptional modifications and cofactor binding also help shape the specificity of the NF-B response. Competition between p53 (TP53) and NF-B for CBP/p300 may play an important role in determining the balance between apoptosis and cell cycle arrest following irradiation.
XIV-6) Based on transcriptomic studies using microarray profiling, which one of
the following statements best describes the transcriptional response of
irradiated cells and tissues?
A. Many genes are up-regulated by radiation exposure, but downregulation of genes is rarely observed
B. The transcriptional response to radiation is complex, but for a given
cell line similar responses will be seen between 2- and 24-hours postirradiation
C. The transcriptional response is dynamic and varies with time after
irradiation, but overall is similar for most cell lines examined to date
D. Transcriptional responses depend on the time elapsed after irradiation
and on the cell’s tissue of origin but do not vary significantly between
cell types derived from the same tissue or between different
individuals
E. Variability observed in transcriptional profiles between individuals
may provide a basis for prediction of individual therapeutic responses
in the future as a basis for individualized medicine
XIV-6) E
Many genes are both up- and down-regulated following irradiation in both a time and tissue-dependent manner. In addition, variation is also seen between cells derived from the same tissue and between tissue samples taken from different individuals. This inter-individual variation is seen both in the response to stressors such as ionizing radiation and in the normal basal gene expression patterns. One of the major driving factors in the science of microarray profiling is the hope that a better understanding of this variability in gene expression may lead to a more “personalized” diagnosis of disease, prognosis and prediction of the best therapeutic approach for cancer and other diseases.
XIV-7) Concerning the p21 (CDKN1A) protein, which of the following
statements is TRUE?
A. Its transcription is transactivated by p53 (TP53) in response to
ionizing radiation exposure.
B. It is required for entry into S phase of the cell cycle.
C. It is up-regulated only in cells exposed to radiation doses greater than
1 Gy.
D. Overexpression of p21 causes arrest in the G2 phase of the cell cycle.
E. It binds to Bcl-xL (BCL2L1) to promote apoptosis.
XIV-7)
A p21 is one of the most strongly p53-transactivated genes, and codes for the p21 protein. It responds robustly at both the mRNA and protein levels to ionizing and UV radiation as well as to most other stress-inducing agents. p21 is a CDK inhibitor and also binds to PCNA to prevent entry of cells into S phase. The predominant role of p21 appears to be in mediating G1 phase arrest, although it also plays roles in differentiation, senescence, and regulation of apoptosis.
XIV-8) The two most frequently activated signaling pathways in prostate cancer
are driven by androgen receptor (AR) and PI(3)K-Akt. Inhibitors of the
PI(3)K pathway are in early clinical trials, while androgen-deprivation
therapy (ADT) via inhibition of the AR is able to confer a clinical
response in most patients. Which of the following statements most
CORRECTLY describes the relationship between these two pathways and
explains mechanistically why single inhibition of AR or the PI(3)K-Akt
pathways rarely induces tumor regression in preclinical models?
A. ADT represses an AR gene program governing DNA repair and
inhibits repair of ionizing radiation–induced DNA damage
B. AR and PI(3)K pathways regulate each other by reciprocal negative
feedback, such that inhibition of one activates the other
C. ADT represses the PI(3)K/Akt/target of rapamycin (TOR) pathway
D. ADT activates the unfolded protein response
E. All of the above
XIV-8) B
Prostate cancer is characterized by its dependence on androgen receptor (AR) signaling as well as frequent activation of PI(3)K signaling. AR transcriptional output is decreased in human and murine tumors with PTEN deletion. In addition, PI(3)K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinases. Similarly, AR inhibition activates Akt signaling by reducing levels of the Akt phosphatase, PHLPP. These two oncogenic pathways therefore cross regulate each other by reciprocal feedback. Inhibition of one pathway leads to activation of the other thereby maintaining tumor cell survival. Combined pharmacologic inhibition of PI(3)K and AR signaling causes near complete prostate cancer regression in a PTEN-deficient murine prostate cancer model and in human prostate cancer xenografts, indicating that both pathways coordinately support survival.
XIV-9) The phenomenon of “oncogene addiction” is most clearly represented in
which of the following clinical scenarios.
A. A Chronic Myeloid Luekemia (CML) patient treated with imatinib
B. An EGFR-mutant lung adenocarcinoma patient treated with
bevacizumab
C. A BRAF-mutant melanoma patient treated with ipilimumab
D. An EML4-ALK positive lung adenocarcinoma patient treated with
olaparib
E. A FLT3 mutated Acute Myeloid Leukemia (AML) patient treated
with interferon.
XIV-9)
A “Oncogene addiction” was first coined by Bernard Weinstein and refers to the dependence of some tumors on a single dominant oncogene for continued growth and survival and that inhibition of this specific oncogene product is sufficient to halt the neoplastic phenotype.
Answer Choice A is correct because imatinib is correctly paired with its target, BCR-ABL. The other answers are examples of oncogene-addicted cancers that are incorrectly paired with agents that do not target the dominant oncogene product.
EGFR-mutant lung adenocarcinoma patient can be treated with tyrosine kinase inhibitors such as gefitinib or erlotinib. A BRAF-mutant melanoma patient can be treated with a BRAF inhibitor such as venvurafinib or dabrafinib. An EML4-ALK positive lung adenocarcinoma patient treated with an ALK inhibitor such as crizotinib or alectinib. Lastly, a FLT3-mutated AML patient can be treated with FLT inhibitor, midostaurin, along with induction chemotherapy.
XIV-10) Which of the following pairs of genes or portions of genes and
corresponding descriptors is CORRECT?
A. Tumor suppressor genes – activated in many human tumors
B. Exon – the non-coding region of a gene
C. Promoter – involved in regulating gene transcription
D. DNA repair gene – EGFR
E. Oncogene – activated through loss of heterozygosity
XIV-10) C
The promoter region is the regulatory portion of a gene that plays a critical role in directing whether a gene is transcribed or not. Tumor suppressor genes are generally inactivated in many cancers, typically resulting in a loss of control over cell proliferation. Exons are the expressed, or coding, regions of genes, whereas introns are the non-coding sequences. The protein encoded by the EGFR (epidermal growth factor receptor) gene is a cell surface tyrosine kinase receptor that is activated by epidermal growth factor (EGF) ligand, among others, and is important for cell proliferation. Loss of heterozygosity is a common mechanism by which tumor suppressor genes are inactivated. Oncogenes are generally activated by mechanisms including deletion/point mutation, chromosome rearrangement, retroviral integration, or gene amplification.
XV. Cancer
XV-1) Which of the following statements is TRUE concerning the
retinoblastoma protein (RB1)? RB1:
A. Is an important downstream effector controlling the G2 checkpoint
B. Once phosphorylated, releases E2F
C. Is encoded by an oncogene
D. Is phosphorylated by ATM
E. Activity is altered in approximately 10% of cancers
XV. Cancer Answers & Explanations
XV-1) B
RB1 is the product of the RB1 tumor suppressor gene (not an oncogene). Once phosphorylated by CDK4/6, RB1 releases E2F, which then activates genes associated with the G1 checkpoint. RB1 is functionally inactivated in virtually all human cancers, either directly or indirectly, via p53 (TP53).
p53-dependent induction of p21 (CDKN1A) regulates cyclin E/CDK2 and cyclin A/CDK2 complexes, both of which phosphorylate RB1. The RB1 and p53 signaling pathways are dysregulated in the majority of human cancers.
XV-2) Which of the following pairs of cancer type and corresponding genetic
alterations in that cancer is FALSE?
A. Pancreatic — K-RAS
B. Lung adenocarcinoma — ALK
C. Colon — PTCH
D. Thyroid — RET
E. Melanoma - BRAF
XV-2) C
Carcinogenesis is a multistep process with multiple genetic alterations occurring at particular stages of cancer progression. Alterations in PTCH are associated primarily with basal cell skin carcinoma and medulloblastoma. EGFR and VEGF are frequently overexpressed in colon cancer, but their lack of a relationship with progression and survival has led to their prognostic value being questioned. For example, over 90% of human pancreatic cancers harbor an activating point mutation in the K RAS gene at codon 12. Hereditary medullary thyroid carcinoma (MTC) is caused by autosomal dominant gain-of-function mutations in the RET proto-oncogene. BRAF mutation is present in 50% of cutaneous melanomas and provide the therapeutic target for vemurafenib. Alk mutation is present in ~5% of lung adenocarcinomas and provides a target for crizotinib.
XV-3) Which of the following pairs of tumor suppressor proteins and their
corresponding functions is INCORRECT?
A. APC — signal transduction
B. RB1 — cell cycle regulation
C. p53 (TP53) — cell cycle and apoptosis regulation
D. WT1 — post-translational regulation
E. BRCA1 — DNA damage repair
XV-3) D
WT1 is a transcription factor which, when mutated or absent, is associated with the development of Wilms tumor. Loss of APC plays a role in gastrointestinal carcinogenesis due to its normal involvement in cell signal transduction. RB1 and p53 are both tumor suppressors that regulate cell cycle progression; p53 also regulates apoptosis. BRCA1 protein is part of the DNA repair complex, but likely has several other functions as well, including regulation of the cell cycle and maintenance of genomic stability.
XV-4) Which of the following statements is TRUE concerning p53 (TP53)? p53:
A. Is encoded by an oncogene that is activated in the majority of human
cancers
B. Is activated by acute cellular hypoxia
C. Inhibits expression of the GADD45A, p21 (CDKN1A) and PCNA
genes
D. Can be inactivated by Epstein-Barr virus (EBV)
E. Is modified by phosphorylation in response to DNA damage
XV-4) E
p53 is modified post-translationally by phosphorylation or by acetylation in response to DNA damage. p53 is encoded by a tumor suppressor gene (not an oncogene) that is inactivated in more than half of all human cancers. The DNA repair pathways that regulate p53 include not only NHEJ and HRR, but also MMR, BER, and NER so that p53 plays a universal role in DNA damage surveillance and repair. DNA damage causes p53 to become stabilized and active, not inactive. p53 increases expression of GADD45A, p21, and PCNA. Viruses that contain proteins that inactivate p53 include HPV, SV40 and adenovirus, but not EBV.
XV-5) Oncogenes were first discovered from the study of:
A. Chicken Retroviruses
B. Bacteria
C. Yeast
D. Mice
E. Human cells in culture
XV-5) A
Retroviruses, viruses with genomes composed of RNA instead of DNA, can cause cancers in animals (example: Rous sarcoma virus [RSV] in chickens). Usually, this occurs because the retroviruses contain modified (often mutated) proto-oncogenes captured from the genomes of their vertebrate hosts.
XV-6) Which one of the following is NOT a tumor suppressor gene?
A. PTEN
B. BRCA2
C. WT1
D. NF1
E. ABL
XV-6) E
ABL is an oncogene whereas PTEN, BRCA2, WT1, and NF1 are all tumour suppressor genes.
XV-7) Which of the following statements is TRUE concerning p53 (TP53)?
A. MDM2 binding to p53 inhibits its degradation
B. Irradiation of cells stimulates ATM to act as a phosphatase and
remove phosphate groups from p53
C. Following irradiation, p53 activates Cdc25C to stimulate the G2 to M
phase transition
D. p53 stimulates the activity of BAX and BID in irradiated cells,
resulting in apoptosis
XV-7) D
p53 stimulates the activity of BAX and BID in irradiated cells, resulting in apoptosis. MDM2 binding to p53 stimulates degradation of p53. Irradiation of cells activates ATM to add phosphate groups to p53. Following irradiation, p53 inhibits CDC25C which inhibits the G2 to M phase transition. Lymphocytes and thymocytes with a mutant p53 tend to be more radioresistant than their normal counterparts.
XV-8) Which of the following statements is TRUE concerning the products of
the INK4A/ARF locus?
A. p16INK4A (CDKN2A) stimulates the hyper-phosphorylation of the RB
(RB1) protein resulting in release of the E2F transcription factor
B. p14ARF is induced by the RAS/MEK/MAPK pathway and stimulates
cell growth
C. p16INK4A is encoded by a proto-oncogene
D. p16INK4A is activated by the PI(3)K/AKT pathway and increases
synthesis of cyclin D
E. p14ARF inhibits the MDM2-mediated degradation of p53
XV-8) E
p14 ARF inhibits the MDM2-mediated degradation of p53. p16INK4A is a cell cycle inhibitor that prevents phosphorylation of RB by CDK4. p14ARF is an MDM2 inhibitor thereby causing p53 levels to increase, resulting in greater cell cycle inhibition. p16 INK4A is encoded by a tumor suppressor gene.
XV-9) Which of the following represents a potential/actual therapeutic target in
the oncogene-addicted tumor?
A. Mutated KIT and/or PDGFR in gastrointestinal stromal tumors
(GIST).
B. Translocated ABL1 (previous symbol ABL) in T-cell acute
lymphoblastic leukemia.
C. Amplified EGFR in non-small cell lung carcinoma.
D. Translocated ALK in small cell lung carcinoma.
E. Mutated Notch1 in chronic myeloid leukemia.
XV-9) A
The oncogene addiction model postulates that some tumors rely on the continued activity of single dominant oncogene for growth and survival. Thus, according to the oncogene addition model, inactivation of this key single oncogene will halt malignant proliferation by inducing cell-cycle arrest, differentiation, senescence, or other forms of cell death, depending on tissue context. Each of the listed oncogene products in this question is an addictive oncoproteins in human cancer, however Choices B-E are incorrectly paired to the listed cancer.
The receptor kinases KIT and/or PDGFR display activating mutations in more than 90% of gastrointestinal stromal tumors (GIST). This observation supported the use of the multi-target small-molecule tyrosine kinase inhibitor imatinib mesylate (Gleevac) in GISTs. The correct matches in other choices are:
translocated ABL1 in chronic myeloid leukemia; mutated, not amplified, EGFR in non-small-cell lung carcinoma; translocated ALK in non-small cell lung carcinoma; mutated Notch1 in T-cell acute lymphoblastic leukemia
