Cell Responses to RT Flashcards
1
Q
How to get plating efficiency?
A
Plate cells and do not radiate them (control).
The colony # that form gives you the plating efficiency!
2
Q
Formula to calculate surviving fraction (SF)?
A
Colonies counter / (cells plated x plating efficiency)
3
Q
What are the axes for L-Q survival curves plots?
A
log of SF against linear dose!
4
Q
For a L-Q cell survival curve, is the shoulder the linear or the quadratic part?
A
It is the linear part! it is directly proportional to D.
This is counter intuitive but it is so because we plot log of the surviving fraction!
5
Q
For a linear-quadratic (LQ) cell survival curve, is the “straight” slope the linear or the quadratic part?
A
It is the quadratic part! It is directly proportional to D2
This is counter intuitive but it is so because we plot log of the surviving fraction!
6
Q
What is the α/β ratio?
A
It is the dose in Gy at which the linear and quadratic components of cell killing are equal.
7
Q
For high LET radiation (neutrons, α particles), which part of the cell survival curve fraction (α or β) is predominant?
A
It is almost 90-99% α!
8
Q
What is the other popular model of cell survival?
A
The target theory model developed by Puck and Markus.
9
Q
What is Do?
A
It comes from the Puck and Markus model of cell survival curve.
It is the dose at which the surviving fraction is reduced to 37%.
Conceptually, it is the dose to hit each cell in the culture one time.
10
Q
Why was 37% chosen as the surviving fraction for D0?
A
Because ln(0.37) = 1.
It makes calculations easier
11
Q
Is Do the same at any portion of the cell survival curve?
A
Yes, as long as you are on the slope (NOT the shoulder) of the curve.
12
Q
What is D1 ?
A
It is the initial slope, due to single event killing, the dose to reduce survival to 37%
13
Q
What is Dq (quasithrehold dose)?
A
You extend the straight portion of the curve back towards the y axis.
Where this line crosses SF 1 on y-axis, you get Dq from the x-axis.
Where this line intersects the y axis, that value gives you the n. n is a surrogate for how big the shoulder of the curve is.
14
Q
How do we define cell death in radiation?
A
Loss of reproductive ability
aka senescence
15
Q
What is the single-hit, single-target model?
A
Observed biological effect results from a single energy deposit. There is no shoulder to this curve!
It only occurs in three scenarios:
- high-LET radiation (alpha, neutrons)
- cells are in the M phase
- cells have a defect in their DNA repair
16
Q
What is the single-hit, multi-target model?
A
A cell has multiple targets that need to be hit for it to be killed. All must be hit before the cell dies!
17
Q
What are the assumptions of the target theory model?
A
- Ionizations near molecules cause structural changes (in DNA)
- There is a mathematical relationship between dose and effect
- Each ionization event is random (Poisson distribution)
18
Q
Between the L-Q and the target theory cell survival models, which one is the most accurate?
A
L-Q!
These curves are usually identical. It’s how we think about them that differs.
The T-T model was developed for a single-cell type. Thus, it works better for single-cell types or cells in culture.
In practice, for tissues with multiple cell types, the L-Q model is much more accurate.
19
Q
For two given Do, which cell line is more radiosensitive?
A
The one with the lower Do
20
Q
For a given set of n values from target theory model, which one is likely to belong to a radiation with high LET?
A
n = 1
21
Q
What kind of damage is represented by α and β?
A
α represents non-repairable damage. A single hit with high LET radiation does all the damage required to kill.
β represents repairable damage. Such damage usually requires two hits from two different radiation tracks separated by time. The damage may be repaired during this time!
22
Q
Why do we fractionate radiation instead of giving it as a single dose?
A
This allows for much more normal tissue sparing than if we were to give it as a single dose.
23
Q
Does apoptosis cause inflammation?
A
No, no cell contents are spilled out. Therefore, there is no inflammation.
24
Q
What is a mitotic catastrophe?
A
This happens when the DNA is damaged to the point that when the cell goes to divide, it cannot and must die off. It usually occurs after one or a few abortive mitotic cycles.
The cell remains active until it goes to divide.
25
What is necroptosis?
It is a programmed (regulated) form of necrosis and inflammatory cell death. It is caspase-independent and often used as a defense mechanism (suicide) against viruses.
26
What proteins (kinases, etc) are involved in necroptosis signaling?
1. RIP-1 → RIP-3 activation
2. RIP3 → MLKL activation (trimer formation)
3. Plasma membrane permeabilization
Mnemonic: RIP MLK
27
What is pyroptosis?
It is an inflammatory form of apoptosis associated with the antimicrobial response. it requires induction of caspase-1 by pyroptosome.
It involves membrane rupture and the release of damage-associated molecular pattern molecules (DAMP).
28
What are some initial changes to a cellular membrane that are observed during apoptosis?
Phospholipid phosphatidylserine translocates to the outer leaflet of the plasma membrane.
29
What is the hallmark feature of apoptosis?
Formation of apoptosis blebs.
30
How is apoptosis observed on Agarose gel electrophoresis?
DNA laddering.
This happens because DNA breakdown happens in a very orderly fashion, generally around nucleosomes.
it fragments the DNA in multiples of 185 bp fragments (May contain 1, 2, 3, or more of these 185 bp units)
31
Does the cell shrink or swell up in apoptosis?
Shrinks
32
Does the cell swell or shrink during necrosis?
Swell
33
What are the most common pro-apoptotic proteins?
Bax, Noxa, Bid, Puma
Bid goodbye
34
What are the most common pro-survival proteins?
Bcl-2, Bcl-XL
35
What is the DNA domain difference between pro-survival and pro-apoptotic proteins?
The BH4 domain
36
What role does p53 protein play in apoptosis?
It turns on Puma, Noxa, and Bax
37
What is the importance of apoptosis in different cancers?
Lymphomas > Carcinomas > Sarcomas (NO apoptosis)
38
What are the steps involved in the extrinsic apoptosis pathway?
1. TNFR, TRAIL, and/or Fas are activated by TNF, TRAIL R1/R2, and Fas ligands respectively.
2. These activate caspase 8 (extrinsic pathway initiator caspase)
3. This rapidly activates killer caspases 1, 3, 4, 6, and 7 (effector caspases)
4. This sets into motion a cascade of protease events that result in cell death.
Mnemonic: **E***X*trinsic → **E**ight, *X* (ten)
39
What are the steps involved in the intrinsic apoptosis pathway?
1. ATM senses DNA damage and activates p53.
2. p53 activates Bax/Bak/Bad and other pro-apoptotic proteins.
3. Proteins cause mitochondria to leak cytochrome c
4. Cytochrome c activates caspase 9 (intrinsic pathway initiator caspase)
5. This rapidly activates killer caspases 1, 3, 4, 6, and 7 (effector caspases)
6. This sets into motion a cascade of protease events that result in cell death.
Mnemonic: **IN***T*rinsic → n**IN**e, *T*wo
40
What type of cell death involves the release of cytochrome-C from mitochondria?
Apoptosis
41
Where do endonucleases cut DNA?
Within the DNA.
42
Where do exonucleases cut DNA?
At the ends of the DNA.
43
What is the bp difference between different rungs of the apoptosis DNA ladder on gel electrophoresis?
185 bp
44
What kind of DNA fragmentation is seen on Agarose gel electrophoresis in necrosis?
No patterns. Just a smear of DNA from one end to the other 2/2 random nature of DNA degradation.
45
What is an annexin assay?
It is an assay for detecting apoptosis.
It detects phosphatidylserine on cell surfaces as they undergo apoptosis.
46
What sort of genetic changes cause intrinsic radioresistance?
- Raf overexpression
- N-, H-, and K-ras point mutations
- Bcl2 overexpression
- Her2/neu overexpression
47
What is the role of Bcl2 overexpression in apoptosis control?
Overexpression leads to radioresistance as it is a pro-survival (NOT pro-apoptotic protein).
48
What is the number of tumor cells and doublings in 1g of tumor?
- 109 cells
- 30 doublings
49
What is the formula for tumor control probability?
P = e-n
50
What is TD50?
Tumor Dilution 50%
Number of tumor cells required to induce a tumor in 50% of recipient mice
51
What is TCD50?
Tumor Control Dose 50%.
The dose required to control 50% of tumors.
52
How many cells/tumor would be left by TCD37?
1 cell per tumor
This translates into a tumor control probability of 0.37 (37%)
53
How is cell survival related to TCD?
Survival = 1 - TCD
54
How many cells/tumor would be left by TCD90?
0.1
Or in 100 treated patients, only 10 tumor cell is left behind.
55
How many cells/tumor would be left by TCD95?
0.05
Or 5 in 100 tumor cells
56
How many cells/tumor would be left by TCD99?
0.01
Or in 100 treated patients, only 1 tumor cell is left behind.
57
What are giant cells?
They are huge cells with multiple nuclei.
They represent cells undergoing mitotic catastrophe after the formation of chromosome aberrations.
58
What is the primary mechanism of cell death following irradiation?
Mitotic catastrophe 2/2 missegregation of genetic material 2/2 chromosome aberrations or damage to replication machinery.
59
What is the role of caspases in apoptosis?
They activate DNAses, which cleave DNA between nucleosomes.
60
What is the final step in apoptosis?
Loss of membrane integrity
The membrane blebs off and disappears
61
Does apoptosis occur singly or in clusters?
Singly.
Necrosis, by contrast, occurs in clusters.
62
What is the initial intracellular event that kickstarts the intrinsic apoptotic pathway?
Leakage of cytochrome c from mitochondria.
63
How does Bcl.XL prevent apoptosis?
Inhibits the release of cytochrome c from mitochondria.
64
What is synthetic lethality as it relates to two genes?
Two genes are considered synthetically lethal if the inactivation of one out of the two does not cause cell death. However, inactivation of both does cause cell death.
65
How does caspase 3 paradoxically cause tumor growth?
It is induced in irradiated, dying cells.
Caspase 3 acts via paracrine signaling and stimulates the growth of neighboring cells.
66
What is the role of beclin?
It is an important part of autophagy.
It promotes the development of autophagosomes.
Mnemonic: Bec → beak (phagy?!)
67
What part of the cell cycle are most cells in the body in?
G0 (out of the cell cycle)
68
What part of the cell cycle are most tumor cells in?
G1
69
Which part of the cell cycle or G0 is most radioresistant?
G0
70
What is the R point in the cell cycle?
Restriction point.
It is a point in the G1 phase up till which cells need growth factors to continue in the cell cycle
After this, the cells do not need growth factors to complete the cell cycle.
71
Which part of the cell cycle is the most variable in length between different cell types?
G1
72
Which phases of the cell cycle are included in the interphase?
G1, S, and G2
73
What is 3H-TdR used for?
It can be used to measure DNA synthesis.
Cells incorporate it into synthesizing DNA.
It emits β particles, which can be read.
It is called "auto-radiography."
It takes weeks to months.
74
What is BrdU?
Like 3D-TdR, it is taken up by synthesizing DNA.
An antibody directed against it is then used to label the DNA.
It is also used as a radiosensitizer.
75
How does hydroxyurea affect cell cycle progression?
It kills cells in the S phase and blocks G1-S progression.
All the cells in culture progress to G1 and arrest there.
76
How long does hydroxyurea take to synchronize the cell cycle?
The length of G2, M, and G1 phases.
77
What is a drug-dependent method of synchronizing the cell cycle?
Hydroxyurea.
78
What is the order of sensitivity of different phases of the cell cycle to radioactivity?
M > G2 > G1 > late-S phase
In practice, M and G2 are almost equally radiosensitive.
79
What is the most radioresistant phase of the cell cycle?
Late S -phase
80
What kind of conditions can make M-phase cells more radioresistant?
Hypoxic conditions.
81
How do the survival curves of cells in different phase of the cell cycle compare to one another?
M phase usually has no shoulder (single-hit kinetics).
The more radioresistant a phase is, the more shoulder it has.
82
How does cell sensitivity to radiation vary through the G1 phase?
Cells are more resistant in the early G1 phase, followed by a sensitive period in the late G2 phase.
83
Which cell cycle-specific problem does fractionation avoid?
Cell-cycle synchronization.
Sensitive cells are killed off.
Resistant cells cycle to the more sensitive phases of the cell cycle.
84
What is sublethal damage?
Damage that can be repaired in hours.
If it is combined with a second dose, it can create lethal damage.
Sublethal damage is represented by the shoulder of the cell survival curve
85
What is potentially lethal damage?
This damage is usually lethal, but modulation of the post-radiation environment increases survival.
86
Why do we fractionate with respect to reassortment?
To allow reassortment of tumor cells into the more radiosensitive phases.
To spare normal tissue by allowing time for tissue repair.
87
What effect does fractionation have on repopulation?
As we fractionate, we give the tumor more time to repopulate.
88
How does decreasing the dose rate affect survival?
There is a trend towards increased survival at low dose rates.
Testes and embryos are notable exceptions.
89
Between what doses is the dose-rate effect most evident?
0.01 - 1 Gy/min
90
How does dose rate affect testes?
Testes more sensitive to low-dose rates than high-dose rates (an exception to the rule).
91
How does dose-rate affect the fetus?
Low-dose rates are more damaging because you will affect many phases of pregnancy as opposed to just one (receiving dose all at once).
This is also an exception to the usual dose-rate rule!
92
Can stem cells within the jejunum be morphologically distinguished from their progeny?
They cannot be distinguished morphologically. However, they can be distinguished functionally, by their ability to reproduce and repopulate the crypts.
93
What did the Till & McCulloch studies show?
They injected bone marrow cells into lethally-irradiated mice. Some of these cells developed colonies and nodules in the bone marrow of the recipient mice.
The donor mice were also irradiated, and it was shown that increasing doses required an increased number of harvested cells for Spleen colony formation.
94
What feature distinguishes autophagy from other forms of cell death?
Reversability.
95
How does Do relate to the slope of the survival curve?
slope = 1/Do
96
What is the relationship between the extrapolation factor, n, quasi threshold dose, Dq, and D0?
ln(n)= Dq/D0
97
What is the qualitative meaning of Dq?
It is the total amount of sublethal damage that cells must acquire before lethality occurs.
It is the threshold under which the multitarget, single-hit model is not applicable.
98
What is another name for mean D0 or D37?
Mean lethal dose
99
What is the relationship between D10 and D37?
D10 = 2.3 x Do
100
Which cancer cell lines have apoptosis as the primary mechanism of cell death?
Leukemia and lymphoma
101
What is the qualitative meaning of n, the extrapolation number), in the target theory model?
It is the number of hits to one target required to cause cell killing.
102
What is the D0 for most mammalian, well-oxygenated cells?
1-2 Gy range
103
What is the equation for a single hit, single target equation for the target theory model?
S = e-D/D0
104
How is D10 related to D0?
D10 = 2.3 x D0
105
In the expression SFx, what is the dose of radiation being used?
it is x Gy
106
What are the initiator caspases? What's their function?
Caspases 2, 8 , 9, & 10.
They initiate downstream executioner caspases that directly produce cellular effects.
107
Which caspases are the executioners?
Caspases 1, 3, 4, 6, & 7
108
What is the function of XIAP (BIRC4) during apoptosis?
It inhibits caspases.
XIAP = X-linked Inhibitor of Apoptosis
109
Is apoptosis a common mechanism of cell death in epithelial tumors?
No.
Mitotic catastrophe is the main mechanism of cell death in such tumors.
110
What triggers the intrinsic apoptotic pathway?
Damage to DNA or plasma membrane
111
How does radiation damage plasma membranes?
It does so by activating sphingomyelinase, which hydrolyzes sphingomyelin and generates ceramide.
Ceramide then acts as a second messenger for apoptosis via the mitochondrial system.
112
What are the morphological markers of mitotic catastrophe/senescence?
Multinucleation
Giant cells
113
What's the difference b/w senescence and quiescence?
- Senescent → metabolically active but cannot divide
- Quiescent → metabolically active and can re-enter the cell cycle, if needed
114
What are some molecular markers of mitotic catastrophe/senescence?
p16
β-galactosidase
115
How is autophagy induced?
1. Inhibition of mTOR
2. Activation of PI3K
3. Mitochondrial activation of ERK
4. Activation of JNK
116
Which molecules mediate radiation-induced senescence?
it is caused by elevated p16 and p21, and permanent arrest in G1.
117
What activates the extrinsic apoptotic pathway?
Membrane death receptors:
- TNFR
- TRAIL R-1
- TRAIL R-2
- FAS
These are activated by binding to TNF, TRAIL and FAS ligands.
Downstream, they activate caspase 8.
TRAIL = TNF-related apoptosis-inducing ligand
118
How are cells undergoing apoptosis removed from tissue?
They are engulfed by phagocytes, which are recruited by increased expression of phosphatidylserine residues on the outer surface of the plasma membrane.
119
What is the function of DIABLO?
It is the protein that inhibits the inhibitors of apoptotic proteins (IAPs) from inhibiting caspases, thereby, promoting apoptosis.
120
Does apoptosis require energy?
Yes, it uses up ATP!
121
What does the TUNEL assay identify?
NEL stands for nick-end labeling.
This assay labels DNA fragments generated during apoptosis.
122
What is the chromosomal reason for mitotic cell death?
Misassortment of genetic material into daughter cells 2/2 asymmetrical chromosomal aberrations.
123
What is an autophagosome?
An organelle that sequesters the cellular component to be removed.
It then fuses with a lysosome and digests the contents.
124
Which family of proteins controls autophagy?
- At least 30 proteins from the Atg family.
- Atg8 and Atg6 care called LC3 and Beclin-1 respectively.
- AuTophaGy = ATG
125
What is Anoikis?
Programmed cell death that occurs when cells are placed in an unfamiliar organ or tissue.
Cancer cells must overcome anoikis to metastasize.
126
Which protein is a/w anoikis?
BIM-EL
BIM-EL → Bak, Bax activation → cytochrome c release → cell death
127
What is a jejunal crypt stem cell assay?
Mouse intestines are irradiated with enough dose (≥11 Gy) to destroy the villi.
After 3.5 days, some of the jejunal crypts will start to regenerate.
1 regenerating crypt = 1 surviving clonogenic cell.
Endpoint = crypts per circumference.
128
What is a bone marrow stem cell assay?
Donor mice irradiated with a test dose
Recipient mice irradiated with a supralethal dose ≥9 Gy (all bone marrow cells are killed).
Donor bone marrow injected into recipient mice.
Donor stem cells form colonies in the spleen which can be easily counted (this takes ~10 days).
1 colony = 1 surviving clonogenic cell. Endpoint = colonies per 10n donor stem cells.
129
What is a skin clone assay?
High-dose radiation is used to create a “moat” of dead skin, with an “island” of intact skin in the middle.
The skin “island” is then irradiated with a test dose.
This area of skin regrows after 12–24 days as a series of nodules.
1 nodule = 1 surviving clonogenic cell.
Endpoint = skin nodules per cm2.
130
What is a kidney tubule assay?
For each mouse, one kidney is irradiated and one is spared.
Wait 60 weeks, and then compare the number of intact kidney tubules on the irradiated side versus the unirradiated side.
Endpoint = % of tubules intact.
131
What are spheroid systems?
Model tumor system
Uses a tumor cell line that grows as spheroids in cell culture (clumps of many cells, not single cells)
Spheroids are more “in vivo like” than a monolayer of cells, but less complex than what is found in an animal tumor model
Spheroids are irradiated while intact, and separated into single cells for plating and colony formation
132
What are the morphological markers of mitotic catastrophe/senescence?
Multinucleation
Giant cell
133
What is an unstable aberration?
Prevents chromosomes from properly segregating during mitosis
They decline in number over time, because it is highly likely to cause cell death.
134
What is a stable aberration?
Do not affect chromosome segregation during mitosis.
Can persist for years, because it is unlikely to cause cell death.
135
How is cell survival related to TCD?
Cell Survival = 1 - TCD
136
What's the mnemonic for cyclins and CDKs?
- DEAB, 6421 (↓ order)
-- Cyclin D + CDK 4/6 → G1-S transition
-- Cyclin E + CDK 2 → G1-S transition
-- Cyclin A + CDK 1→ G2-M transition
-- Cyclin B + CDK 1 → G2-M transition
137
Are microbial cells (bacterial, yeast, etc.) more or less sensitive to radiation than human cells?
Less because of their ↓ DNA content
138
How does the slope of the LQ curve change w/ increasing dose?
- It continues to bend w/ increasing dose, which is not what is observed experimentally
- However, it models the shoulder and the initial portion of the survival curve really well
139
How does the slope of the LQ curve change w/ increasing dose?
- It continues to bend w/ increasing dose, which is not what is observed experimentally
- However, it models the shoulder and the initial portion of the survival curve really well
140
Which phase of the cell cycle is cyclin A synthesized in?
S & G2
141
Which phase of the cell cycle is cyclin B synthesized in?
G2 & M
142
Which phase of the cell cycle is cyclin D/E synthesized in?
G1
143
What's the STING pathway?
- Stimulator of Interferon Genes (STING) pathway is activated in response to radiation
- Promotes inflammation
