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.