Radiation Biology Board Review

Front 1

Back 1

Front 2

Back 2

Front 3

Name three lethal chromosomal aberrations

Back 3

1) DIcentric
2) Ring
3) Anaphase Bridge (technically a chromatid aberration)

Front 4

What molecular technique can be used to detect chromosomal translocations?

Back 4

FISH.

Front 5

53BP-1

Back 5

DNA repair protein that partially regulates whether a DSB is handled via homologous recombination or non-homologous end-joining
- Inhibits homologous recombination

Front 6

A DNA DSB occurs. Which repair process is most likely used?
In G1?
In S/G2?

Back 6

Front 7

Acute vs Late Radiation Effects

Back 7

Acute Effects: The target cell is usually apparent and can be repaired fairly rapidly.
- The timing of onset is based on the turnover rate of the mature functional cells.
Late Effects: May be due to both vascular damage and/or parenchymal cell loss.
- May improve but is never completely reversed.
- More sensitive to changes in fractionation.

Front 8

After a dose of 1-2 Gy, how many damaged bases are induced per cell?

Back 8

>1000 per cell

Front 9

After a dose of 1-2 Gy, how many double strand breaks are induced per cell?

Back 9

~40 per cell

Front 10

After a dose of 1-2 Gy, how many of single strand breaks are induced per cell?

Back 10

~1000 per cell

Front 11

Amifostine (WR-2721)

Back 11

- Radioprotectant
- Prodrug and aminothiol
- FDA approved for salivary gland cancers
- Selectivity: Normal cells have a higher pH and higher levels of alkaline phosphatase -> greater activation of amifostine into its active form
- Toxicity: N/V, hypotension
- NB: Does NOT protect the CNS (cannot cross BBB)

Front 12

Apoptosis

Back 12

- Organized cell death
- Characterized by cell shrinkage and "laddering" of DNA on a gel
- Generally dependent on p53
- Steps: 1) Cell rounds up and detaches from surounding tissue
2) Chromatin condensese and nucleus fragments
3) Cytoplasmic condensation leads to cell shrinkage
4) Separation of the cell into apoptotic bodies, which are phagocytized

Front 13

Artemis

Back 13

- DNA endonuclease that plays a role in NHEJ
- Processes any 5' or 3' overhangs, hairpins, etc.
- Binds DNA-PKcs

Front 14

Ataxia Telangectasia

Back 14

- Familial cancer syndrome caused by mutation of ATM
- Leads to cerebellar ataxia and oculocutaneous telangectasias as well as heightened sensitivity to ionizing radiation
- NOT sensitive to UV light
- ATM is involved in the rapid response to DSBs and other radiation damage
- AT cells are notable bc they do NOT arrest in S phase in response to DNA damage (radioresistant DNA synthesis)

Front 15

Autophagy

Back 15

- Process by which cells cannibalize themselves to procure ATP and/or molecular precursors
- Unclear whether this is a drastic attempt at survival or a step to promote death

Front 16

Base Excision Repair

Back 16

Front 17

bcl-2 function

Back 17

Anti-apoptotic gene

Front 18

Bone Marrow Assay

Back 18

- Transplant assay used to determine the dose response of acutely responding tissues
- A donor mouse is given a test dose D --> BM cells are injected into a supralethally-radiated recipient mouse --> 9-10 days later, the donor is sacrificed --> the number of splenic colonies are counted --> the number of colonies are plated vs dose

Front 19

Bone marrow transplants may be useful after exposure to what dose range?

Back 19

8-10 Gy

Front 20

BRCA-1

Back 20

- Located on 17q21
- Codes for a protein that assists with DNA repair. Most associated with HOMOLOGOUS RECOMBINATION. Thus, cells with mutated BRCA show increased genomic instability.
- Mutated in 5-10% of breast cancer cases (BRCA1 and 2 together)

Front 21

Caretaker Genes: What are they? Examples?

Back 21

- Monitor the stability of the genome
- Dysfunction indirectly influences oncogenesis by leading to genomic instability and an increased mutation rate
- No direct influence on tumor growth
- Examples: ATM, XP, BRCA1, BRCA2, MSH6, NBS

Front 22

cdc2

Back 22

- Codes for a kinase
- Same as Cdk1
- Pair with Cyclin A and B

Front 23

Cyclin A

Back 23

- Associates with cdk2 and then cdk1
- Accumulates at G1/S and persists through S phase
- Its activity is needed for entry into S phase, progression through S and entry into M phase
- Can inactivate E2F

Front 24

Cyclin B

Back 24

- Associated with cdk1
- Must be degraded for cells to exit M phase

Front 25

Cyclin D1, D2 and D3

Back 25

- Associate with cdk4 and cdk6
- Targets Rb for phosphorylation

Front 26

Cyclin E

Back 26

- Associates with cdk2
- Required for the G1/S checkpoint
- Maintain Rb in a phosphorylated state, leading to accumulation of active E2F

Front 27

Define: D10

Back 27

- The dose required to kill 90% of a cell population
- Equal to 2.3 x D0

Front 28

Describe the death receptor apoptotic pathway.

Back 28

1) Fas ligand binds Fas (other death-inducing ligand combos exist)
2) The intracellular death domain interacts with pro-caspase 8 to create DISC (death-inducing signalling complex)
3) DISC activates caspase 8
4) Initiator caspase 8 activates the effector caspases --> apoptosis

Front 29

Describe the general process of homologous recombination.

Back 29

1) Recognition --> phosphorylation of ATM
2) MRN complex is recruited to create 3' single-stranded ends
3) The newly created ends are wrapped in RPA
4) RAD51, RAD52 and BRCA 1/2 are recruited and help the homologous strand invade
5) Formation of a Holliday junction
6) Resolution of the Holliday junction and ligation

Front 30

Describe the general steps of mismatch repair.

Back 30

1) Recognize damage
2) Recruit MMR factors
3) Identify which strand is new --> excision of incorrect nucleotides
4) Re-synthesis and ligation

Front 31

Describe the mitochondrial death pathway.

Back 31

1) Disruption of the mitochondrial membrane
2) Release of cytochrome-c
3) Cytochrome-c and APAF-1 interact to activate caspase 9
4) Initiator caspase 9 activates the effector caspases --> apoptosis

Front 32

Describe the regulation of the HIF pathway

Back 32

A: Under normoxic conditions, a group of enzymes called prolyl hydroxylases (HPHs) add hydroxyl groups (OH) to two proline residues of HIF-1α. The hydroxylation of HIF-1α by HPH allows the VHL tumor-suppressor gene to bind and promotes the addition of ubiquitin groups. The addition of ubiquitin groups targets HIF-1α for degradation in the proteasome. B: Under hypoxic conditions, the HPHs cannot hydroxylate HIF-1α on proline residues owing to their requirement for molecular oxygen, and HIF-1α becomes stabilized. It then binds with the HIF-1β subunit in the nucleus and promotes transcription of at least 50 target genes that regulate angiogenesis, erythropoiesis, tissue remodeling, and glycolysis. Most of the studies to date have shown that HIF is essential for tumor growth.

Front 33

Describe the role HIF plays in tumorogenesis

Back 33

When oxygen levels decrease, HIF-1A and HIF-1B combine to promote transcription of 50+ hypoxia-related gene. Among other things, these genes regulate angiogenesis, erythropoesis, tissue remodeling and glycolysis.

Front 34

Describe the steps of non-homologous end joining.

Back 34

1) Recognition of damage --> KU binding
2) Recruitment of DNA-PKcs (holds DNA together?)
3) End processing via Artemis
4) Fill-in Synthesis (if needed)
5) Ligation via PNK/XRCC4/DNA ligase IV/XLF

Front 35

Dose modification factor

Back 35

Used to describe the effect of a radiation protector.

DMF = [Dose of RT required to produce a given effect in the PRESENCE of a drug]/[Dose of RT required to produce that same effect in the ABSENCE of the drug]

Front 36

E2F

Back 36

- Transcription factor
- Normally bound to Rb protein in G0 and G1 (inactive)
- When Rb is phosphorylated, E2F is released, initiates transcription and leads to progression into S phase

Front 37

Equation for a decade of cell kill

Back 37

D10=2.3*D0

Front 38

Equation for Survival Fraction in-vitro

Back 38

Front 39

Equation: Finding the skin gap for matching fields at depth

Back 39

Front 40

Equation: Survival fraction using the linear-quadratic model

Back 40

Front 41

Explain the oxygen fixation hypothesis

Back 41

After damage by radiation-induced free radicals, many ionized target molecules can subsequently repair themselves and function normally. In the presence of oxygen, however, an organic peroxide is formed, which cannot be easily reversed. The radiation damage is thus said to "fix," or make permanent, the damage.

(pg. 88)

Front 42

For in utero radiation exposure, when is a fetus most susceptible to the development of mental retardation?

Back 42

Greatest susceptibility: 8-15 weeks
Less susceptibility: 16-25 weeks (4x less risk)
Very little risk: <8 weeks or >25 wks

NB: Based on Abomb data

Front 43

Gatekeeper genes: What are they? Examples?

Back 43

- Directly regulate tumor growth by inhibiting cell division or promoting apoptosis
- Both copies of a gatekeeper gene must be inactivated to disable its function
- Examples: APC (colon), VHL (renal cell)

Front 44

Give an example of oncogene activation via translocation.

Back 44

1) t(9;22) bcr-abl in CML
2) t(8;14) c-myc in Burkitt's Lymphoma
3) t(14;18) bcl-2 in DLBCL

Front 45

HNPCC

Back 45

- Familial cancer syndrome
- Caused by defective mismatch repair

Front 46

Homologous recombination: Error-prone or error-free?

Back 46

Mostly error free due to the involvement of a template strand.

Front 47

How does dose rate affect biological effectiveness?

Back 47

In general, biological effectiveness goes down as dose rate is decreased (and exposure time is consequently lengthened)

Front 48

How does Her-2/neu act as an oncogene?

Back 48

Encodes a growth factor receptor that, when mutated, no longer requires its ligand to become active

In other words, the kinase becomes constitutively active.

Front 49

If 10^7 cells were irradiated according to single hit kinetics so that the average number of hits per cell is one, how many cells would survive?

Back 49

Front 50

Inverse Dose-Rate Effect

Back 50

Phenomenon where decreasing the dose rate results in increased cell killing. Postulated to occur because a low dose rate allows the cell to continue to cycle until it arrests in G2, a radiation sensitive period. Higher dose rates, in contrast, make the cell arrest wherever it is at the start of irradiation, which is likely less radiosensitive than G2.

Front 51

Jejunal Assay

Back 51

- Used to determine the dose response of acutely responding tissues
- Radiation mice using 11-16 Gy --> wait 3-5 days --> sacrifice and count the number of regenerating crypts per circumference --> plot number vs dose
- Limitations: Have to use >10 Gy to see an effect, only know the NUMBER of surviving crypts and not the survival FRACTION

Front 52

Kidney Tubule Assay

Back 52

- Used to determine the dose response of LATE responding tissues
- A single kidney per mouse is irradiated --> wait 60 weeks --> sacrifice and count number of regenerated tubules --> plot versus dose

Front 53

KU70 and KU80

Back 53

- Genes involved in repair of DNA DSBs by binding to the free ends.
- First step in non-homologous end joining
- If defective, the cell's ability to repair DSBs is reduced --> increased radiosensitivity

Front 54

Law of Bergonie and Tribondeau

Back 54

Greater radiosensitivity tends to be associated with those cells that:
1) are less differentiated
2) have greater proliferative potential
3) divide more rapidly

Front 55

LET

Back 55

The energy transferred per unit track length.

Unit: keV/micrometer

Front 56

Li-Fraumeni Syndrome

Back 56

- Autosomal dominant
- Germ line missense mutation in p53
- Associated with increased incidence of sarcomas, leukemia, brain tumors, lung and breast cancer

Front 57

Mismatch repair

Back 57

- Repair process whereby the cell detects irregularities between the two strands of DNA
- Usually uses methylation to identify the "old" strand
- Defective in HNPCC and Lynch Syndrome
- Defective MMR genes can be detected using a microsatellite instability assay

Front 58

Mitotic Death

Back 58

- Primary mode of radiation-induced cell death
- Causes inflammation of surrounding tissue, cell swelling and a DNA smear in gel
- Often preempted by an asymmetric exchange-type chromosomal aberration (ring or dicentric)

Front 59

Mitotic shake-off

Back 59

- Cells are synchronized in M phase

Front 60

Mouse Skin Cell Assay

Back 60

Allows you to plot the number of surviving skin cells (NOT survival fraction) vs dose

Front 61

MSH, MLH and PSM proteins

Back 61

- Mismatch repair proteins
- Mutations in these proteins can lead to microsatellite instability and carcinogenesis
- Associated with HNPCC

Front 62

myc

Back 62

- Oncogene that can lead to either proliferation or apoptosis depending on the tumor microenvironment

Front 63

Name four mechanisms for oncogene activation.

Back 63

1) Retrovirus integration (viral promoter)
2) Mutation
3) Gene amplification
4) Translocation

Front 64

Name the effector caspases

Back 64

3, 6, and 7

Front 65

Name the initiator caspases

Back 65

2, 8, 9 and 10

Front 66

Name two DNA-associated proteins that can be tagged in order to assay DNA damage.

Back 66

γH2AX and 53BP-1

Front 67

Name two methods for assaying DNA DSBs.

Back 67

1) Pulsed field gel electrophoresis (PGFE)
2) Single cell electrophoresis (comet assay)

Front 68

NBS

Back 68

- Direct phosphorylation target of ATM
- Necessary for DSB repair in response to radiation
- Creates a complex with MRE11 and Rad50

Front 69

NHEJ primarily occurs in what stage of the cell cycle?

Back 69

G1 (no sister chromatid that can be used for repair)

Front 70

Non-homologous end-joining: Faithful or error-prone?

Back 70

Very error prone, as it doesn't use a template strand.

Front 71

Northern Blot

Back 71

Technique for:
- Detecting the presence of a specific RNA sequence (RNA-ssDNA)
- Determining the relative amount of mRNA present
- Detecting relative RNA size

Front 72

Nucleotide Excision Repair

Back 72

- Repairs bulky adducts, such as those caused by UV light and alkylating agents
- Mutations in this system do NOT lead to radiosensitivity; however, they do increase sensitivity to UV light, such as in XERODERMA PIGMENTOSUM
- Two systems: global genome repair and transcription-coupled
- Steps: 1) Recognize damage 2) Excise damage 3) Repair damaged strand via polymerase 4) Ligate

Front 73

Obstetrical x-rays have been conclusively linked with an increased risk of childhood cancer. True or false?

Back 73

True. Initial studies were done by Stewart in the UK in the 1950s. Currently studies suggest that an obstetrical x-ray increases the risk of childhood cancer by 40% over baseline (6% per Gy absolute excess risk).

Front 74

OER for neutrons

Back 74

~1.6

Front 75

OER for sparsely ionizing radiation ie x-rays

Back 75

2.5-3.5
Tends to be larger for larger doses

Front 76

p53

Back 76

- Proapoptotic tumor suppressor gene
- Mutated in Li-Fraumeni syndrome
- Levels are usually kept low due to binding of MDM2 and subsequent proteolysis

Front 77

p53: Tumor suppressor or oncogene?

Back 77

Tumor suppressor
- Loss of function lead to anti-apoptotic pathways

Front 78

Parallel Organs at Risk

Back 78

- Functional damage will not occur until a critical number of FSUs have been inactivated
- Consequently, parallel organs exhibit a theshold volume, above which function begins to be compromised
- Once the threshold volume has been exceeded, function declines in a graded way (not all-or-none)
- Examples: Lung, liver

Front 79

Pig Skin Assay

Back 79

- Functional assay used to determine skin effects of radiation
- Acute assay: A rectangle of pig skin is radiated --> the reaction is watched and graded --> avg reaction over a given period of time is plotted vs dose
- Late assay: A rectangle is tattooed on pig skin --> radiated --> the size of the rectangle is charted over time vs dose
- Because pigs are large and expensive, rodent skin can also be used

Front 80

PNK/XRCC4/DNA ligase 4/XLF

Back 80

- Ligase complex used in non-homologous end joining

Front 81

Potentially Lethal Damage

Back 81

The component of radiation damage that can be modified by post-irradiation environmental conditions.
i.e. This damage generally causes cell death under ordinary circumstances, but death can be averted by changing the post-RT environment.

Front 82

RAD51

Back 82

Protein involved in the repair of DNA damage via homologous recombination

Front 83

Rb:

Back 83

- Tumor suppressor gene that regulates the cell cycle
- Requires inactivation of both copies for loss of function
- Active only in G1 cells
- Phosphorylation of the protein releases E2F (transcription factor) -> cell cycle progression into S phase
- Mutations are commonly found in retinoblastoma, osteosarcoma and small cell

Front 84

RBE

Back 84

Relative Biological Effectiveness

RBE= Dose of 250 keV xrays required to produce a given effect/Dose of test radiation required to produce the same effect

Front 85

Refer to the problem below. Now imagine that the cells undergo three rounds of cell division during treatment. Approximately what dose would be needed to achieve the same level of tumor control (90%)?

Back 85

Front 86

Serial Organs at Risk

Back 86

- Exhibit a binary response i.e. above a threshold dose, there is loss of organ function
- As the radiation field size increases to include more FSUs, the probability of tissue complications becomes steeper and moves left (to lower doses)
- Example: Spinal cord

Front 87

Spinal Myelopathy Assay

Back 87

- Functional assay to determine the dose response of a serial OAR
- Irradiate a section of rat spinal cord --> wait 4-12 months --> score muscle atrophy and/or paralysis vs dose
- Late spinal effects in animals peak at about 1-2 years with a large vascular component
- Dose per fraction is more important than the timing of RT
- For small volumes, length of spinal cord irradiated is crucial; however, the reaction becomes semi-independent of dose when >2 cm (see figure)

Front 88

Structurally Defined FSU

Back 88

- Small, self-contained subunit that is independent of its neighbours
- Cells from one FSU cannot migrate to another so a clonogen must remain alive within the FSU to repopulate it
- Examples: Kidney, lung, liver

Front 89

Structurally Undefined FSU

Back 89

- FSUs that are not readily defined so that cells may readily migrate between them
- Cells from one FSU may migrate to repopulate another
- Examples: Skin

Front 90

Sublethal Damage

Back 90

- "Minor" damage that can usually be repaired within 24 hours.
- Can "add up" to lethal damage if not repaired
- Causes the increase in survival seen with fractionation

NB: Little repair of SLD is seen with high LET radiation

Front 91

Sulfhydryls

Back 91

Radioprotectors
Use a thiol group (-SH) to scavenge free radicals
Must be present within 1 second of radiation in order to have an effect
Highly oxygen dependent (bc radioprotectants mitigate the indirect damage of RT)
- Toxicity can be reduced by covering the thiol group with a phosphate

Ex: Cysteine (causes N/V at therapeutic doses)

Front 92

Survival Fraction (in a dish)

Back 92

SF = # of colonies counted/[#of colonies plated * plating efficiency]

Front 93

Telomerase

Back 93

-Reverse transcriptase that rebuikds chromosome ends to counteract the shortening that occurs with cell division

Front 94

Testicular Assay

Back 94

- Used to determine the dose response of acutely responding tissues
- Irradiate mice (8-16 Gy) --> wait ~6 weeks --> sacrifice and count the proportion of tubules with spermatogenic epithelium --> plot versus dose

Front 95

TGF-B

Back 95

- Pro-inflammatory factor
- Promotes growth of connective tissue and fibrosis

Front 96

TGF-B

Back 96

- Normally acts as an antiproliferative factor
- Normally inhibits the phosphorylation of Rb (via synthesis of p21 and p15), thus preventing the release of E2F and preventing progression from G1 to S
- In transformed cells, also acts as a pro-angiogenic and immunosuppressive factor

Front 97

Tissue Rescue Unit

Back 97

Minimum number of FSUs needed to maintain tissue function

Front 98

TNF-Alpha

Back 98

- Strongly proinflammatory and cytotoxic
- Induces the proliferation of fibroblasts, inflammatory cells and endothelial cells
- When used clinically, leads to fatigue, anorexia, and weight loss

Front 99

TSP-1

Back 99

- Normally ANTI-angiogenic and induced by p53
- When down-regulated, cells may undergo oncogenesis and angiogenesis

Front 100

VHL

Back 100

- Mutated in Von Hippel Lindau Syndrome
- Normally inhibits HIF by targeting it for degradation
- When mutated in Von Hippel Lindau, HIF is not targeted --> constitutively elevated levels of HIF --> elevated levels of VEGF --> increased angiogenesis

Front 101

What is the average Do for human cells?

Back 101

3 Gy

Front 102

What is the difference between acute and chronic hypoxia?

Back 102

Acute hypoxia: Results from the temporary closure of tumor blood vessels secondary to malformed vasculature
Chronic hypoxia: Results from the limited diffusion distance of oxygen through respiring tissue.

Front 103

What is the most variable stage of the cell cycle?

Back 103

G1

Highly variable due to differences in nutrient supply, growth factors, genomic stability and microenvironment

Front 104

What is the risk of a fatal cancer based on a given exposure? Genetic disorder? Non-fatal cancer?

Back 104

Fatal cancer: 4%/Sv i.e. 4x10^-2 per Sievert
Genetic disorder: 0.8%/Sv i.e. 0.8 x 10^-2 per Sievert
Non-fatal cancer: Same as genetic

Front 105

What is the survival fraction of irradiated cells?

Back 105

32/(2000*.7)=0.023

Front 106

What proteins are associated with non-homologous end-joining?

Back 106

1) KU70/KU80
2) DNA-PKcs
3) Artemis: Endonuclease that processes the DNA ends
4) DNA polymerase μ or λ: Performs any necessary fill-in synthesis
5) PNK/XRCC4/DNA ligase 4/XLF: Performs the final ligation

Front 107

What proteins are involved in homologous recombination?

Back 107

ATM: Initial response to DNA damage
MRN complex (MRE11, RAD50, NBS1): Endonuclease complex that creates 3' single stranded DNA tails
RPA: Wraps the 3' tails to create a nucleoprotein filament
RAD51, RAD52, BRCA 1/2: Protein complexes that mediates invasion of the sister chromatid adjacent to the undamaged strand --> Holliday junction

Front 108

Which members of the Bcl-2 family are anti-apoptotic?

Back 108

Bcl-2, Bcl-xl, Mcl-1

Prevents cell from dying

Front 109

Which members of the Bcl-2 family are proapoptotic?

Back 109

Bax, Bak, Bid and Bim

Encourage cell to die

Front 110

You would like to assess DNA damage using gel electrophoresis. What conditions will be needed to assess SSBs? DSBs?

Back 110

DSBs: Neutral solution
SSBs: Alkaline solution (needed to separate the strands so that the damaged strands can run out in the gel)

Front 111

Western Blot

Back 111

Technique to detect specific proteins (Protein-Antibody)

NB:
- Native proteins are separated by 3D structure
- Denatured proteins (via SDS) separate by size
- Secondary and tertiary antibodies may be used to amplify the signal

Front 112

Southern Blot

Back 112

Technique used to detect the presence of a specific sequence in a DNA sample (DNA-DNA)

Front 113

Sanger Method (DNA Sequencing)

Back 113

Method for sequencing DNA using dideoxynucleotides (have a -H at the 3' end instead of an -OH, terminating chain elongation)

NB: Reading from the bottom up gives the sequence of the complementary strand