Cell and tissue survival assays Flashcards
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
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.
Using the linear-quadratic survival curve model, what would the cell surviving fraction be following a dose of 2 Gy delivered acutely (use alpha=0.3 Gy-1 and Beta=0.1 Gy-2)?
A. 0.01
B. 0.10
C. 0.37
D. 0.50
E. 0.90
C
Using the equation S = e^(-aD+BD2), the surviving fraction would be:
e^-[(0.3)(2)+(0.1)(2)2] = e^-[(0.6)+(0.4)] = e^-1= 0.37
For the same α (.3) and β (.1) 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
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
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
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.
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
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.
