Question

Sketch 2 curves on the same graph for mammalian cells: after X-rays and after alpha-particles irradiation. Explain quantitatively the dependence on radiation dose of the survival for X-ray curve at low doses (up to 0.5Gy), at very high doses (higher than 7Gy). Discuss the mechanisms involved in cell death.

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Answer 1

A cell survival curve is a plot of the number of cells that survive to form colonies as a function of radiation dose. Thus, cell survival curves measure reproductive cell death. Some damaged cells may continue to function for a time, but if they do not reproduce, they are not counted as survivors.

In a cell survival experiment, cells are seeded onto petri dishes and exposed to various doses of radiation. The number of cell colonies that subsequently grow is determined. Each colony is assumed to be derived from a single surviving cell. A cell survival curve is a plot of the fraction of cells that survive (normalized by the fraction of cells that survive with no radiation exposure) versus the radiation dose; each point on a cell survival curve corresponds to a single dose of radiation. Usually, the log of the surviving fraction is plotted on the vertical axis versus dose on the horizontal axis. Mammalian cell survival curves generally show an exponential response to high doses of radiation with a “shoulder” of varying widths in the low-dose range. The effect of radiation on cells can be observed by noting how the cell survival curve changes under different conditions. These changes in condition include using different types of radiation, changing the environment, adding or subtracting oxygen, or delivering the radiation at different times or during different parts of the cell cycle. The study of survival curves revealed many aspects of radiation damage long before the structure of DNA was elucidated.

The In Vitro Survival Curve

“In vitro” means outside a living organism—for example, in a test tube or petri dish. Cell survival can be defined in terms of the reproductive ability of the cells; that is, cells that cannot reproduce are effectively dead. This definition makes sense in the context of common laboratory experiments used to study the effects of radiation on cells prior to the 1980s, when DNA probes began to be available. Cells that continue to reproduce (in petri dishes in the lab) form colonies that are visible to the naked eye, and it is easy to count the surviving colonies after irradiation. This experimental design made it possible to establish many basic facts regarding radiation effects on cells in the early days of radiobiology. By varying the conditions during and after exposure, the factors influencing cell response to radiation can be isolated and studied. The shape of the survival curve contains information regarding the overall sensitivity of the cells, as well as the ability of the cells to repair and recover from radiation damage. Theoretical models have been developed to explain the shapes of the resulting survival curves in terms of cell survival characteristics.

Although reproductive capacity is a good way of defining “cell survival” in the context of in vitro radiobiology experiments, it is not applicable to all cell types. Some cells such as nerve, muscle, and secretory gland cells are nonproliferating. For these cell types, what matters to an organism exposed to radiation is whether the nonproliferating cells maintain function. For proliferating cells (e.g., intestinal lining, stem cells, cells in a cancerous tumor), the reproductive capacity is important, in both the organism and in the laboratory. Reproductive capacity is also referred to as “clonogenic potential.”

The loss of clonogenic potential is a very narrow definition of cell death. A cell that is exposed to radiation may be apparently intact, it may synthesize protein/DNA, and it may even carry out several cycles of mitosis; but if it cannot carry out sustained replication, then it has not survived, according to the definition here.

Cell Survival Curves: Description of Experimental Method

Cell cultures are generated by extracting live cells from various species of mammals such as mice, rats, or humans. The cell population is first grown in a tissue culture flask. The cells are then released from the flask by adding trypsin, the cells are counted, and then a known number of cells is plated onto growth medium in a petri dish and maintained under appropriate conditions (correct temperature, atmosphere, nutrient levels) to survive and grow.

In order to study radiation effects on the cells, many petri dishes are plated under the same conditions using genetically identical cells. Some dishes are not irradiated and are maintained as controls, and the rest of the dishes are exposed to various doses of radiation. The irradiated cells and control cells are then incubated for some time (e.g., a few days up to a couple of weeks) to allow colonies to arise from the surviving cells. A colony is assumed to form by the sustained proliferation of a single cell. Here, populations of identical cells are irradiated with increasing doses of radiation.

Cell Survival Curves: Quantification

In order to quantitatively interpret the results of cell survival assays, it is important to define two quantities: the plating efficiency and the surviving fraction.

Plating Efficiency:

The plating efficiency is simply the percentage of seeded cells that survive to form colonies under control conditions (i.e., no radiation or other modifying factors).

PE= 100× (no.of cells counted/no.of cells seeded)

Knowing the plating efficiency allows one to normalize out effects that lead to cell death but are not attributable to radiation. For example, assume the plating efficiency is found to be 50%. Then, in a later experiment using the same cells and the same incubation conditions, half of the cells irradiated with a 1 Gy dose of X-rays survive.

Surviving Fraction:

Once the plating efficiency is determined, then a meaningful determination of the surviving fraction can be made, that is, the fraction of cells that survive or die due to the radiation that one is testing

SF= no.of cell counted/(no.of cells seeded×[PE/100])

We can plot a curve of different doses of radiation (low or high) against the survival fraction of cells.

Reasons for cell death:-

- loss of specific functions in differentiated cells like nerve, muscle, secretory cells

- loss of the ability to divide in proliferating cells such as stem cells in hematopoietic system or intestinal epithelium i.e. reproductive death.