Quantitative radiation biology was revolutionized in 1956 when Puck
and Marcus published the first cell survival curve, relating radiation
dose to the fraction of cells surviving. The term "survival"
generated a great deal of discussion at that time and led to the definition
of such terms as "reproductive death," "reproductive integrity,"
and "clonogenicity" (among others), all designed to explain that
the end point of cell culture experiments is the loss of the cell's ability
to divide indefinitely and produce a sizable visible clone.
This technique does not distinguish between modes or times of cell death,
however. The end point is bottom line-oriented! It also should be noted
that the Puck and Marcus paper involved HeLa cells, for which apoptosis
is an important form of cell death. It is true that soon thereafter, various
hamster cell lines were developed (with which many of the principles of
radiobiology were demonstrated), which do not undergo apoptosis at all,
but the earliest work was with HeLa cells, which do.
Meanwhile, the development of autoradiography had made possible the
study of cell and tissue kinetics, leading to the concepts of growth fraction
and cell loss factor in tumor biology. From a study of a multitude of animal
tumors, Denekamp proposed that, in general, carcinomas respond rapidly
to radiation because they have a large cell loss factor, while sarcomas
respond slowly because they have a small cell loss factor. (Response,
of course, does not necessarily relate to a final "cure.")
It is within this historical framework that the study of apoptosis has
progressed, with many of the Old Guard being slow to be convinced that
it is important in radiation therapy. The review article by Dr. Meyn is
informative and timely, for he has worked in this area for many years and
is respected for his thoughtful judgment.
The Central Unaddressed Question
Dr. Meyn distinguishes between apoptosis and reproductive cell death.
We disagree somewhat with this point. By all means, distinguish between
apoptosis and mitotic death. The latter is defined as death in attempting
mitosis due to illegitimate chromosomal rejoining. But both apoptosis and
mitotic death are forms of reproductive cell death; no distinction is made
between them in the conventional colony-forming assay. Nor is a distinction
made between primary and secondary apoptosis.
Dr. Meyn does a singular service in pointing out that the distinction
between primary and secondary apoptosis is not trivial. If apoptosis is
simply a means of removing the debris after the cell is already dead from
chromosomal abnormalities, it is not of much interest. To the extent that
apoptosis represents a different, radiosensitive mode of cell death, however,
it is of vital interest. This leads to the central, but unaddressed question:
Why do cells from the same individual, which harbor shared genetic material,
die a rapid apoptotic death if they arise in the hematopoietic system and
a mitotic death if they are of epithelial or fibroblastic origin?
Clearly, further work is required to investigate the molecular basis
of the apoptotic response, particularly following exposure to ionizing
radiation. The tumor-suppressor gene p53 is one gene known to mediate apoptosis;
however, its effect on cellular radiosensitivity remains controversial.
Slichenmyer et al have shown no effect on radiosensitivity when a dominant-negative
mutant of p53 was transfected into a human colorectal carcinoma cell line.
Lee and Bernstein demonstrated increased resistance to radiation in hematopoietic
cells derived from transgenic mice expressing mutant alleles of p53; however,
this effect was not due to loss of programmed cell death.
For the apoptotic index to serve as a useful biologic marker, it must
be able to identify the subset of patients with radiocurable disease.
Further studies are required to investigate the association between spontaneous
apoptosis and radiation-induced apoptosis, as well as the independent prognostic
significance of the spontaneous apoptotic index. If spontaneous apoptosis
and radiation-induced apoptosis are unrelated, the presence of spontaneous
apoptosis could lead to misclassification in assays assessing radiation-induced
apoptosis, subsequently reducing the likelihood that the assay would have
any predictive power.
Study of Apoptosis Still Evolving
It seems to us that in the study of apoptosis, we are long on data and
short on understanding. Put more kindly, perhaps, the study of apoptosis
is in a descriptive phase, with many correlations being found, but with
no discovery that is of much real consequence. We have replaced "sensitive
vs resistant cells" with "cells that die an apoptotic death vs
those that do not," and "tumors that respond rapidly to radiation"
with "tumors showing a propensity toward radiation-induced apoptotic
death." The terminology and nomenclature have changed, but the outcome
is not much altered.
As in so many areas of cancer research in general, and radiation biology
in particular, recent discoveries at the molecular level give a tantalizing
glimpse of the mechanisms underlying the phenomena that we have known about
for so long. But we have not gone beyond that yet. Manipulating the biology
to achieve a therapeutic gain remains an elusive possibility reserved for
some future date.
1. Puck TT, Markus PI: Action of x-rays onmammalian cells. J Exp Med
2. Denekamp J: The relationship between the "cell loss factor"
and the immediate response to radiation in animal tumours. Eur J Cancer
3. Lee JM, Bernstein A: p53 mutations increase resistance to ionizing
radiation. Proc Natl Acad Sci 90:5742-5746, 1993.
4. Slichenmyer WJ, Nelson WG, Slebos RJ, et al: Loss of a p53-associated
G1 checkpoint does not decrease cell survival following DNA damage. Cancer
Res 53:4164-4168, 1993.