Dr. Kaplan: It seems to me that Dr. Colter and Dr. Epstein have made opposing statements on one very interesting point, namely, the question of cytoplasmic versus nuclear locus of action. Dr. Colter, I believe, made the statement in connection with his discussion of the Stent hypothesis that all available evidence concerning replication of viral RNP indicates that it occurs in the nucleus. I was interested in this statement, since in a review which I prepared (Cancer Res. 19: 791, 1959) I searched very hard for any evidence as to the locus of virus nucleoprotein replication, and failed to find critical evidence on this point.

Contrariwise, Dr. Epstein has come to the same conclusion that I was inclined to approach, i.e., that at least with the RNA-containing viruses there is perhaps more reason to believe that their locus of action is cytoplasmic, and that if they are to be regarded as auxiliary genetic determinants of the cell, they do so by acting somewhere in the cytoplasm.

Dr. Duryee (George Washington University): It is important to think about Dr. Kaplan's remarks and his cogent analysis of the critical problem of where a tumor virus operates. In view of Dr. Epstein's concluding statement that tumor viruses operate by causing "a cytoplasmic change in the host cell purely incidental to virus replication," we need to have a better rationale. I wish, therefore, to make some general comments on Dr. Epstein's and Dr. Colter's papers.

In the frog renal adenocarcinoma, which Dr. Lucke studied for many years, we now know the locus for tumor-virus action. In spontaneous "transforming" tubules, and in tumor-filtrate injected kidneys, the first observable change is appearance of "new" DNA around the nucleolus where it attaches to its chromosome. By all criteria this attachment point is a gene locus.

The second event that happens is an increase in the RNA content of the nucleolus. It is a striking phenomenon, one which is being noted by other investigators. In human gynecological tumors it has been beautifully confirmed by Dr. Margaret E. Long and Dr. H. C. Taylor, Jr., when they measured up to 70 times the RNA in nucleoli of Grade III cancers compared to Grade I.

The third sequential event is the extrusion or appearance of RNA in the cytoplasm in the form of inclusion bodies. Thus nuclear RNA transfers to the cytoplasm to accelerate protein synthesis and to stimulate cell growth. We find that while the amount of RNA or RNP varies from cell to cell and tumor to tumor, it always correlates in amount with the degree of malignancy. Perhaps of prime importance is the heretofore unnoticed fact that DNA-aggregates may escape from the nucleus along with RNP. We have identified this DNA in amphibia as tumor virus.

One cannot escape from the observation that parallel changes in experimental frog tumors are found in a wide variety of human neoplasms, always correlating with degree of malignancy. (Duryee, Long, Taylor, McKelway, Ehrmann, 1960; Page, Regan, and McCarty, 1938.)

Finally these changes precede and lead to initiation of mitosis and eventual metaplasia or even neoplasia. In brief, nucleolar stimulation is the trigger, we believe, for all hyperplasia, although to carry out Dr. Beard's thought, it is necessary to do as we have done-prove metastasis and invasion before one can claim to have produced actual malignancy.

Thus there is invariably a spectrum of cell changes which may result from a tumor-virus infection. This question should be pondered by all of us: Do the viruses, whether they are RNA-type or DNA-type, initiate similar sequences in every host cell? In other words, let us be more diligent in examining host cell changes in their mitotic trigger mechanism. It may be worth stating again that viral infections may act simply as another carcinogen, e.g., just as methylcholanthrene or intense X irradiation may act. We cannot solve the problem of cancer by direct study of methylcholanthrene; neither can we solve it by merely studying the nature of viruses. We are going to have to look more often at the point of host cell response at the DNA-gene locus where RNP is first manufactured.

Dr. George Weber (Indiana University): I would like to mention our recent studies with Dr. Levine and Dr. Ashmore on cytoplasmic enzymes of Rous sarcoma and the chorio-allantoic membrane. These studies may be of interest because of the RNA nature of the Rous virus. It was shown that the enzyme, phosphohexose-isomerase, which channels glucose-6-phosphate into glycolysis is markedly increased. A similar elevation was noted in the lactic dehydrogenase activity. These increases amounting to 300 to 400 percent are calculated on per average cell basis. This is an important consideration because the Rous sarcoma contains twice as many cells per wet weight as the chorio-allantoic membrane.

In addition to these increased activities of phosphohexose-isomerase and lactic dehydrogenase, the presence of other glycolytic enzymes was also demonstrated in this tumor (fructose-1, 6-diphosphatase and phosphoglucomutase). The shunt enzymes were also present. The increased glycolytic enzyme activities correlated well with results obtained on incubation of tissue slices with differentially labeled glucose. Both enzymatic and isotope data indicate the operation of an active shunt and increased glycolysis. It may be worthwhile to mention that similar enzymatic and metabolic behavior was found when Rous sarcomas were grown in the wing web of chickens. (Weber, G., Banerjee, G., Levine, A. S., and Ashmore, J.: Metabolic studies on the Rous sarcoma. Proc. Am. Assoc. Cancer Res. 3: 161, 1960.)

Dr. Pollard (University of Texas): The mechanism of cell-virus interaction constitutes a fundamental process which is more significant than any other single facet of virology.

Our group has been studying cell response to psittacosis virus infection by means of acridine orange stain and ultraviolet microscopy. With this procedure, RNA of the cytoplasm and of the nucleolus stains red and the DNA of the nucleus stains green-yellow. Psittacosis virus invades the cytoplasm and within hours after exposure the DNA virus particle is surrounded by a red-staining, RNase-sensitive "matrix." Within the following 48 hours the RNA-staining masses change color from red to orange to yellow-green, and at the latter stage mature virus particles become visible. This exemplifies a procedure whereby cellular response to viral activity can be studied visually. The histochemical observations indicate that the DNA of psittacosis virus initiates a mobilization of RNA by the host cell and that as the virus particles mature this transforms to DNA. Thus an intracellular indicator system becomes available through the use of fluorochrome techniques, which may have application in problems not directly associated with this particular virus.

Dr. Mellors: For some time Dr. Monroe and I have been studying Rous sarcoma in chicken wing web and in the chorio-allantoic membrane of the chick embryo with fluorescent antibodies. After Dr. Stanley's comment about the immunologists being left behind, I am hesitant to mention this procedure, but the findings on Rous sarcoma cells were interesting in this respect: Various forms of cytoplasmic particulates containing Rous viral antigen were readily demonstrated, but it was also clear that perhaps about one in a thousand sarcoma cells contained nuclear viral antigen, i.e., Rous viral antigen was demonstrable in the nucleus of perhaps one in a thousand sarcoma cells as a discrete focus which sometimes lies, or, as one focuses up and down, appears to lie near the nucleolus. This apple-green focus of viral antigen may be seen above or next to a blue-violet autofluorescent nucleolar image, so that 1 believe, as far as the protein moiety of Rous virus is concerned, there certainly is a time when viral protein occurs in the nuclei of sarcoma cells.

Work on the cellular formation of other types of proteins, for example, rheumatoid factor or 19S gama-globulin which is formed in plasma cells, has also shown that on occasion protein antigen can be detected in the nucleus as well as in the cytoplasm of cells that synthesize these proteins.

Dr. Bryan: Dr. Malmgren and Dr. Fink have a paper in press in which they confirm the observation which you have described, that in the rare cell fluorescence can be seen in an area which appears to be the nucleolus.