Dr. George O. Gey (Johns Hopkins University): Does this virus always give a necrotizing response?

Dr. Dawe: I presume you mean, Does it always give some necrosis? I would have to say that there has always been some necrosis. There has never been any pure proliferative response. This is a thing that is quite difficult to evaluate in tissue culture, since there is some necrosis in areas of many normal cultures. However, it is my impression, based on the nuclear lesions, that there is always some specifically virus-induced necrosis along with the proliferation.

Dr. Gey: When you get rid of the virus, what have you got? Here we have a disease which is produced in many organs and considered to be a neoplastic disease due to the polyoma virus. What are these cells in the hamster? Are they completely free of virus? If so, are they not new cells with a genome built into them which makes them different from what they were before?

I would like to try to delineate between noncytopathic and cytopathic change. Our experience does include tumor-producing viruses.

Dr. Andervont: If I understand Dr. Gey's question, he wants to know whether a cell that has proceeded to malignancy under the influence of the virus needs the continued presence of the virus to remain malignant. Is that the point?

Dr. Gey: Exactly.

Dr. Irena Koprowska (The Wistar Institute): Dr. Dawe, you have described intracellular inclusions. Can you compare them with the inclusions described by Leuchtenberger in tissue cultures infected with adenoviruses?

Dr. Dawe: I have not done any studies on the histochemical nature of the nuclear lesions. Dr. Love (National Cancer Institute) is presently doing some studies with the toluidine blue-molybdate method and the only thing that can be said is that the amphophilic inclusion, by his interpretations, contains RNA. The clear zone around this inclusion is much more difficult to interpret, and I do not believe Dr. Love is prepared at present to say what all is in there, so I must stop with that. I might add that Dr. Banfield (National Cancer Institute), who has done some electron microscopy on these cultures, has seen particles characteristic of the polyoma virus in areas that seem to correspond to the clear zones around the inclusions

Dr. M. M. Sigel (University of Miami): I should like to suggest that there may be an alternate explanation, for the lack of cytopathogenic effect, to the one offered by the author, which is based on an interrelationship of mesenchyme and epithelium. This explanation may lie in the type of system used: the organotypic tissue culture.

It is well known that HeLa cells grown on glass in monolayers and exposed to polio-virus demonstrate cytopathogenic changes and disintegrate. In our experience, when the same cells arc planted in the brain of an animal they show considerably less destruction or cytopathogenic change while still supporting growth of the poliovirus. Such cells form structural entities in the rat brain. It is therefore quite possible that structures, such as are retained in your cultures and that developed in the brains of our rats, may be a major factor in determining the fate of cells infected by virus.

Dr. Dawe: Yes, the interplay of mesenchyme and epithelium need not be a peculiar one in the sense that such a relationship might also exist for HeLa cells even in a heterologous host. It may be basically a nutritional relationship-a feeder-cell type of culture system. I do not know that your observation particularly contradicts this interpretation; it may parallel it to some extent. The same sort of thing might apply (some of the experts on Shope papilloma might have something to add here) to the Shope papilloma virus which is seen in its complete form in the outer layers of the epidermis rather than in the basal cells, in which the mesenchymal-epithelial relationship remains intact.

Dr. L. L. Coriell (South Jersey Medical Research Foundation): I would like to underscore the observation of the relationship between the epithelial cell and the mesenchyme in another system. HeLa cells in the normal rat grow well for a few days and then die, whereas in the cortisone-X-ray-treated rat, HeLa cells continue to grow until the death of the animal.

There is a striking difference between a normal animal, which discards the transplanted tumor cells, and the animal whose defensive mechanisms have been altered. In the latter there is little connective tissue or leukocytic reaction; the cells continue growing and the host provides a rich blood supply but no collagen formation.

Dr. W. R. Earle (National Cancer Institute): With a culture of very low cell density on glass with a fluid overlay, unless there is a certain specific cell density the cells will not live, or, if they live, they will show all sorts of disintegrative changes. When there is a mixed culture of epithelium and mesenchymal cells, is there a possibility that, instead of specific reaction between the mesenchyme and the epithelium, the mesenchyme destroyed the epithelium the way almost any type of cell would have done in order to survive?

Dr. Dawe: You are referring to the situation in which we isolated the epithelium completely and obtained a gradual death of the cells. This was one of the reasons that we took multiple epithelial rudiments and piled them up together, so that we would have approximately a cell mass about equivalent to what we had when we had an intact rudiment. Even under these conditions, the epithelium does not survive as long as it does when combined with mesenchyme.

Dr. Leon Dmochowski (The M. D. Anderson Hospital): I want to comment on the relationship of this virus to the kidney cells of the epithelial and connective-tissue origin. We have recently carried out a series of morphological investigations by means of the electron miscroscope in collaboration with Dr. B. E. Eddy and Dr. S. E. Stewart. We found that after the infection of mice, rats, or hamsters with polyoma virus, within 2 or 3 weeks the nuclei of epithelial cells (that is, of cells of the proximal and distal convoluted tubules) are filled with virus particles or viruslike particles. The cells are distinct by the appearance of these characteristic structures in their nuclei.

After the appearance of proliferative lesions in the epithelial elements of the kidneys of animals from the 3 species, the number of these characteristic structures decreases, and when tumors appear it is hard to find the viruslike particles in the nuclei of the kidney cells. Just before we came to New York, we found these characteristic structures in the nuclei of the cells of hamster kidney tumors induced by polyoma, but it took Dr. Gey about 3 weeks to find them. It appears therefore that only very few tumor cells contain these characteristic structures, but, during the initial course of infection, numerous cells contain these particles. The important question is, therefore, whether virus is necessary its a continuous stimulus of neoplasia or whether it acts only as an initiating stimulus.

Dr. Dawe: I would like to say something in reply to Dr. Gey's and Dr. Dmochow-ski's questions as to whether or not the virus has to be present in the cells after the tumor is established or whether the tumor can get along without the virus. In the oldest cultures that we have carried, only up to 75 days, we have a population made up entirely of altered cells. There are no unaltered cells left, but many of the altered ones show the inclusion bodies and the nuclear clear areas that are characteristic of the presence of virus. On the other hand, as Dr. Dmochowski has pointed out, in the fully developed tumors one does not see the nuclear lesions except occasionally at the margins of necrosis.

However, if one takes a piece of fully developed tumor, places it in a tube of medium, lets it drift there for anywhere from 1 day to 2 weeks, then removes it and examines it with the light microscope, one sees the appearance of the nuclear inclusions and the other nuclear lesions. Dr. Banfield, with the electron microscope, has found that many cells in such cultures contain typical polyoma-virus particles, whereas it is very difficult to demonstrate similar particles in tumors freshly removed from the animal.