This section is from the book "Symposium Phenomena Of The Tumor Viruses", by U.S. Dept. of Health. Also available from Amazon: Tumor Suppressing Viruses, Genes, and Drugs: Innovative Cancer Therapy Approaches.
The principal biologic criterion of the virus-cell relationship thus far employed has been that of the rate of virus liberation. Although there is little evidence yet of the precise nature of the factors which govern the rate of liberation, the accumulated data provide the basis for limited conclusions. In the present work, it was seen that, under the prescribed conditions, the rate of virus liberation per cell per hour was constant in a given experiment and was thus precisely in parallel with those cellular processes resulting in exponential growth of the myeloblasts. This occurred in cells freshly drawn from the host and those recovered from a culture series maintained for almost 2 years in vitro. It is evident, table 1, that the rates of cell growth (generation time) were not the same with cells from different sources. Like variations occurred in the rates of virus liberation, but the respective rates of cell growth and virus liberation did not vary in parallel. This principle is well illustrated by the results of experiment 11157 (text-fig. 3) in which the cells were in a state of exponential growth for 204 days. During this period, however, the rate of virus liberation fluctuated through an approximately eightfold excursion from 40 to 5 and back to 30 particles per cell per hour. The events in the earlier stages of the experiment in which the cells were in a mixture of 20 percent chicken serum in Gey's salt solution can only be surmised, since cell counts were not made. To judge from the cell volume observed with each medium change, the population of cells during this period was essentially constant. Calculations made on this assumption showed, as was seen in numerous other experiments (1), that the cells fresh from the bird liberated virus at a high rate in the early period of culture and that the rate declined soon to a level of less than 5 particles per cell per hour. Despite the low level of proliferative activity of the cells under these conditions, virus was still liberated at a rate comparable with that observed with derived cells in a phase of rapid multiplication.
From what has been seen thus far, it may be judged that the processes concerned with virus synthesis are independent of those involved in cell multiplication, at least in the sense that they can proceed at different rates and are by no means wholly regulated by the same metabolic activities. This is but a particularly nice and quantitive corroboration of relations which have been suspected or indicated for other virus tumors in the host. It is well known, for example, that the virus yields from rapidly growing cottontail rabbit warts vary greatly (20), and the same is seen with the Rous sarcoma, in which quantitative studies (21) of thin sections have revealed a relationship between intracellular and extractable virus but none between tumor virus content and rate of tumor growth.
The absolute status of the myeloblast culture system with respect to the ultimate capacity of the cells to multiply and to synthesize virus cannot be fairly assessed at the present. The highest rates of cell proliferation have been a doubling time of approximately 3.5 days. The highest rates of virus liberation have been about 40 particles per cell per hour in cultures in which cell growth was exponential. Under the best conditions the rate of virus synthesis has been constant in the presence of exponential growth of the cells. It is already known that the rate of virus release is subject to control by the environment of the cell. It remains to be seen whether the processes of virus synthesis can be terminated in the intact cell.
The expressions employed for analysis of the myeloblastosis data have been applied to results obtained by Prince (17) relative to virus growth in the chick chorio-allantoic membrane inoculated with Rous sarcoma virus. The results observed in the latter stages of short-term studies with this system were analogous in principle to those with the myeloblast system with respect to a constant rate of formation of Rous sarcoma virus in relation to increase in the mass of the virus-inoculated membranes.
To summarize: Earlier studies have shown that, under some conditions, the myeloblasts of avian myeloblastosis may proliferate at exponential rates in tissue culture, in one instance approximately 500 days, and consistently liberate myeloblastosis virus into the culture medium. In the present work, analyses have been made to clarify some aspects of the kinetics of virus liberation and thus, also, of virus synthesis in association with the cell. The analytical treatment has shown that in some cultures proliferating exponentially for long periods, the myeloblastosis virus was liberated at constant rates in terms of absolute number of virus particles per cell per hour. This activity of the cell can be described by a simple mathematical expression which yields constants applicable to the interpretation and comparison of data from experiments of various types. Application of these analyses has revealed the identity of the principles of behavior of myeloblast cultures of various derivation, such as cells freshly obtained from the circulating blood of birds with myeloblastosis, myeloblasts carried for periods of nearly 2 years under various conditions of culture, and myeloblasts derived by treatment of normal chicken bone marrow with the virus in vitro. In the studies under consideration, the generation times varied from 3.5 to 7.7 days, and the rates of virus liberation were within the range from about 5 to about 40 virus particles per cell per hour. The variations in the rates of virus liberation did not parallel those of the growth rates of the cells in different phases of study. From these findings the interpretation may be made that the respective metabolic processes of virus synthesis and cell growth are not inflexibly coupled and thus may vary independently. The results have served to emphasize the stabilizing influence of balanced culture media consisting of 50 percent chicken serum in medium 199 supplemented by B vitamins and folic acid in high concentration.
Dr. Rubin (University of California): Experiments have been published on the growth rate of Rous sarcoma virus (RSV) in cells removed from chicken sarcomas and maintained in tissue culture. (Rubin, Virology 1: 445, 1955; Rubin, Ann. New York Acad. Sc. 68: 459, 1957). It was reported that RSV is produced at a constant rate which is proportional to the number of cells and the cell mass. Equations were developed to describe the quantitative relationship between virus and cell under conditions of a static cell population and exponential cell growth. The latter equations are similar to those introduced by Beaudreau et al., but they take into account thermal inactivation of RSV since the assay relies on infectivity. In the present case when the assay is based on particle counts this would appear to be unnecessary. However, if we make the likely assumption that the particles can readsorb to the cells, then we must compensate with a term similar to that for heat inactivation. This is particularly true in view of the high cell concentration used in Beaudreau's experiments, which would almost assure rapid loss of particles through readsorption.