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Summary of informal discussion
Experimental
107
Cell Research, Suppl. 9, 107-110 (1963)
SUMMARY
OF INFORMAL
DISCUSSION
Dr T. Hauschka pointed out that they had not made chromosome counts on the Morris 5123 hepatoma or the H 35, 7800, 7316 or any of the other socalled minimal deviation hepatomas. However, karyotypic analysis of precancerous stages in livers of rats fed on azo-dyes indicates that neoplasia is preceded by an increase in nuclear abnormalities, as recently found by Porter and Bruni and by Stich and Maini. Among these altered cells may be some with special genotypic combinations, including protein deletions, as a result of their unusual chromosome constitutions. It would be well to compare the minimum deviation hepatomas with the normal karyotypic pattern. It was mentioned by Dr Potter that Dr H. C. Pitot and Miss Fallon have found a normal diploid number in the Morris hepatoma 5123. There might have been abnormalities in chromosome size or shape but they have not seen any and the count is normal. In the past Dr Hauschka has pointed out that there are tumors that have a normal diploid number and it is assumed, on the basis of other data, that there are probably heteroploid mutations or something similar that leads to polyploidy. In the figure on the human ascites exudates studied by Ishihara and Sandberg, there are listed 11 benign exudates that were diploid and 34 malignant ones that were cytologically abnormal. The 5123 minimal deviation hepatoma is a malignant hepatoma in that it invades, metastasizes and kills the animal in about three months, although it is a slow growing hepatoma. It is proven that tumors can have mutations that lead to biochemical changes that are irrelevant to the possession of their malignant properties, although they may be relevant to the chemotherapeutic problem. We have to divide the biochemistry as well as the cytogenetics of the cancer problem into the problem of chemotherapy and the problem of carcinogenesis. The question as to whether it is any longer possible to be able to do definitive research on mechanisms of carcinogenesis using aneuploid neoplasms is highly relevant to the purposes of this conference. Dr Busch has done some studies on 5123 hepatoma, but much more work needs to be done. Dr Hauschka replied that we should continue working with aneuploid tumors because most tumors unfortunately are aneuploid so we have no other choice. Even when we work with tumors that are superficially diploid, we are no closer to the problem or no further away from it, as biochemists at least, than if we were working with a more altered tumor. Experimental
Cell Research, Suppl. 9
R. E. Stowell The earlier findings through cloning of transplantable tumors have been confirmed very nicely by later studies on primary tumors. All of them are mosaic populations; even the precancerous stages are mosaic cell populations. There are normal human beings among us without obvious disease syndromes who have mosaic chromosome patterns. Conversely, one should not expect that there are no diploid tumors. What do mutations do that lead diploid cells to change their growth patterns? Surely, genes which affect growth patterns are scattered throughout’the genome. So you would expect just what we find: some slightly deviational, and some cytologically very abnormal tumors.. We should study them all, but we cannot press this study any further by mere systematic chromosome counting. What then can we do? We appear to have reached the limits of light-microscopic resolution. Now we must get down to liner ultra-structural and genie levels before we can reach decisions about specific nuclear changes in carcinogenesis. Dr Hsu stated that sometimes when an inexperienced worker is looking at the chromosomes, when there is a constriction or a similar phenomenon, he will call it a chromosome break. But this does not necessarily mean a break of the continuity of the DNA molecule because one can sometimes see a connection between the two broken ends. When a fragment really separates from the chromosome or twists around, there is no question about the broken chromosome macromolecule per se. All breaks that are scored are true breaks because questionable breaks were omitted. One of Dr Hsu’s colleagues did work for some time on the effects of cytoxin on chromosomes. However, they only recorded the number of breaks but not their distribution because they could not effectively localize the breaks with many human chromosomes. The effect of alkylating agents may be like analogs, breaking the G-C region or the A-T region. There has been some preliminary observation that TEM causes breaks in the regions similar to that found in BUDR. Szybalski has proposed that TEM interferes with thymine metabolism. It is difficult to envisage the mechanism of breaking of chromosomes resulting from bromouracil. As to whether spontaneous mutations may be nonrandom to some extent, it was suggested that all spontaneous mutations are induced by an unknown treatment or cause. If it is chemically induced, as by accidental contact or by ingestion of an unrecognized chemical, there may be a nonrandom distribution of mutations or chromosome damage. According to Dr Hauschka, the chemically induced tumors in the mouse Experimental
Cell Research, Suppl. 9
Summary of informal discussion
109
studied by Hellstrom, Stich and others are, on the whole, cytologically more erratic than the virus tumor group. Some recent references indicate that even pattern. For instance, herpes viruses may, on occasion, affect chromosomal simplex infection in hamster tissue culture cells causes chromosome breaks within the first division cycle after the herpes simplex has been added to a cell population in which it normally does not multiply. So the virus tumors will have to be analyzed in greater detail and the precancerous changes in hepatomas will have to be reinvestigated in greater detail. Drs Stich and Maini have done some exacting work along that line and they find during the precancerous stages that there are already some interesting altered cells nonresponsive to mitotic stimuli as well as to mitotic inhibitors. Comments were made by Dr Hotchkiss relative to the usage of the word some of the thoughts expressed by Dr Hsu with respect to che“random”, mical mechanisms of chromosome breakage, and the use of the word “macromolecule” in referring to a chromosome. Nowadays this impingement of biochemistry on morphology and the use of cytochemical techniques is a most fertile movement, but one should be cautious. The ability to use homogeneous samples has made chemistry what it is. In a beaker of hydrochloric acid, every part of the solution has a similar number of molecules. If you take a solution of acetone, you have some of the molecules in enol form and some in keto form. Physicists studying acetone find it complicated enough, but nowadays they have jumped all the way to chromosomes and we are encouraged these days to think of such systems at a molecular and physical level. Dr Hsu and Dr Taylor have been doing elegant work revealing very specific facts about chromosomes. However, Dr Hotchkiss feels that Dr Hsu is discovering the BU-sensitive regionswhich may be much more revealing of special sites of chemical activity rather than merely of AT occurrence. If we remember that there may be 3000 adenines in one single molecule of DNA and a few thousand molecules at least of this size in the chromosome, we cannot really argue that the AT pairs are so grossly concentrated in one place or it should be manifest in other ways. They may be more active or replaceable at this site, a far more informative conclusion. It is true we can talk about how many men of age thirty-five will die of meningitis due to measles, and treat it as random per 100,000 of population. This is “random” if you know nothing about the man and how he got infected. Or the same is true of the number of people who die of falling in the bathtub. Experimental
Cell Research, Suppl. 9
R. E. Stowell But the men who die in the bathtub die because they slip or step on a cake of soap or a rug. We call it random through our ignorance when we don’t know more specific facts. It was suggested that Drs Hsu and Taylor were detecting specific chromosomal events and should be encouraged to formulate them as such rather than as expression of random events in a physico-chemical continuum. Dr Hsu explained that we only know the chromosomes are broken; however, he would like to think that DNA molecule extends unbroken from one end of the chromosome to the other. He agreed with Dr Hotchkiss that we are really very ignorant about how chromosomes are constituted. Therefore, the A-T active zone is only a working hypothesis and not really definitive. As to the DNA molecule, Dr Somers presented some hypothetical ideas about the architecture of chromosomes from her collaboration with Dr Cole. If the Cole model proves to be correct, then the chromosome is made of a continuous DNA molecule; if it is not, then we would have to propose other models to fit the facts. Robert E. Bowel1
Experimental
Cell Research, Suppl. $
107
Cell Research, Suppl. 9, 107-110 (1963)
SUMMARY
OF INFORMAL
DISCUSSION
Dr T. Hauschka pointed out that they had not made chromosome counts on the Morris 5123 hepatoma or the H 35, 7800, 7316 or any of the other socalled minimal deviation hepatomas. However, karyotypic analysis of precancerous stages in livers of rats fed on azo-dyes indicates that neoplasia is preceded by an increase in nuclear abnormalities, as recently found by Porter and Bruni and by Stich and Maini. Among these altered cells may be some with special genotypic combinations, including protein deletions, as a result of their unusual chromosome constitutions. It would be well to compare the minimum deviation hepatomas with the normal karyotypic pattern. It was mentioned by Dr Potter that Dr H. C. Pitot and Miss Fallon have found a normal diploid number in the Morris hepatoma 5123. There might have been abnormalities in chromosome size or shape but they have not seen any and the count is normal. In the past Dr Hauschka has pointed out that there are tumors that have a normal diploid number and it is assumed, on the basis of other data, that there are probably heteroploid mutations or something similar that leads to polyploidy. In the figure on the human ascites exudates studied by Ishihara and Sandberg, there are listed 11 benign exudates that were diploid and 34 malignant ones that were cytologically abnormal. The 5123 minimal deviation hepatoma is a malignant hepatoma in that it invades, metastasizes and kills the animal in about three months, although it is a slow growing hepatoma. It is proven that tumors can have mutations that lead to biochemical changes that are irrelevant to the possession of their malignant properties, although they may be relevant to the chemotherapeutic problem. We have to divide the biochemistry as well as the cytogenetics of the cancer problem into the problem of chemotherapy and the problem of carcinogenesis. The question as to whether it is any longer possible to be able to do definitive research on mechanisms of carcinogenesis using aneuploid neoplasms is highly relevant to the purposes of this conference. Dr Busch has done some studies on 5123 hepatoma, but much more work needs to be done. Dr Hauschka replied that we should continue working with aneuploid tumors because most tumors unfortunately are aneuploid so we have no other choice. Even when we work with tumors that are superficially diploid, we are no closer to the problem or no further away from it, as biochemists at least, than if we were working with a more altered tumor. Experimental
Cell Research, Suppl. 9
R. E. Stowell The earlier findings through cloning of transplantable tumors have been confirmed very nicely by later studies on primary tumors. All of them are mosaic populations; even the precancerous stages are mosaic cell populations. There are normal human beings among us without obvious disease syndromes who have mosaic chromosome patterns. Conversely, one should not expect that there are no diploid tumors. What do mutations do that lead diploid cells to change their growth patterns? Surely, genes which affect growth patterns are scattered throughout’the genome. So you would expect just what we find: some slightly deviational, and some cytologically very abnormal tumors.. We should study them all, but we cannot press this study any further by mere systematic chromosome counting. What then can we do? We appear to have reached the limits of light-microscopic resolution. Now we must get down to liner ultra-structural and genie levels before we can reach decisions about specific nuclear changes in carcinogenesis. Dr Hsu stated that sometimes when an inexperienced worker is looking at the chromosomes, when there is a constriction or a similar phenomenon, he will call it a chromosome break. But this does not necessarily mean a break of the continuity of the DNA molecule because one can sometimes see a connection between the two broken ends. When a fragment really separates from the chromosome or twists around, there is no question about the broken chromosome macromolecule per se. All breaks that are scored are true breaks because questionable breaks were omitted. One of Dr Hsu’s colleagues did work for some time on the effects of cytoxin on chromosomes. However, they only recorded the number of breaks but not their distribution because they could not effectively localize the breaks with many human chromosomes. The effect of alkylating agents may be like analogs, breaking the G-C region or the A-T region. There has been some preliminary observation that TEM causes breaks in the regions similar to that found in BUDR. Szybalski has proposed that TEM interferes with thymine metabolism. It is difficult to envisage the mechanism of breaking of chromosomes resulting from bromouracil. As to whether spontaneous mutations may be nonrandom to some extent, it was suggested that all spontaneous mutations are induced by an unknown treatment or cause. If it is chemically induced, as by accidental contact or by ingestion of an unrecognized chemical, there may be a nonrandom distribution of mutations or chromosome damage. According to Dr Hauschka, the chemically induced tumors in the mouse Experimental
Cell Research, Suppl. 9
Summary of informal discussion
109
studied by Hellstrom, Stich and others are, on the whole, cytologically more erratic than the virus tumor group. Some recent references indicate that even pattern. For instance, herpes viruses may, on occasion, affect chromosomal simplex infection in hamster tissue culture cells causes chromosome breaks within the first division cycle after the herpes simplex has been added to a cell population in which it normally does not multiply. So the virus tumors will have to be analyzed in greater detail and the precancerous changes in hepatomas will have to be reinvestigated in greater detail. Drs Stich and Maini have done some exacting work along that line and they find during the precancerous stages that there are already some interesting altered cells nonresponsive to mitotic stimuli as well as to mitotic inhibitors. Comments were made by Dr Hotchkiss relative to the usage of the word some of the thoughts expressed by Dr Hsu with respect to che“random”, mical mechanisms of chromosome breakage, and the use of the word “macromolecule” in referring to a chromosome. Nowadays this impingement of biochemistry on morphology and the use of cytochemical techniques is a most fertile movement, but one should be cautious. The ability to use homogeneous samples has made chemistry what it is. In a beaker of hydrochloric acid, every part of the solution has a similar number of molecules. If you take a solution of acetone, you have some of the molecules in enol form and some in keto form. Physicists studying acetone find it complicated enough, but nowadays they have jumped all the way to chromosomes and we are encouraged these days to think of such systems at a molecular and physical level. Dr Hsu and Dr Taylor have been doing elegant work revealing very specific facts about chromosomes. However, Dr Hotchkiss feels that Dr Hsu is discovering the BU-sensitive regionswhich may be much more revealing of special sites of chemical activity rather than merely of AT occurrence. If we remember that there may be 3000 adenines in one single molecule of DNA and a few thousand molecules at least of this size in the chromosome, we cannot really argue that the AT pairs are so grossly concentrated in one place or it should be manifest in other ways. They may be more active or replaceable at this site, a far more informative conclusion. It is true we can talk about how many men of age thirty-five will die of meningitis due to measles, and treat it as random per 100,000 of population. This is “random” if you know nothing about the man and how he got infected. Or the same is true of the number of people who die of falling in the bathtub. Experimental
Cell Research, Suppl. 9
R. E. Stowell But the men who die in the bathtub die because they slip or step on a cake of soap or a rug. We call it random through our ignorance when we don’t know more specific facts. It was suggested that Drs Hsu and Taylor were detecting specific chromosomal events and should be encouraged to formulate them as such rather than as expression of random events in a physico-chemical continuum. Dr Hsu explained that we only know the chromosomes are broken; however, he would like to think that DNA molecule extends unbroken from one end of the chromosome to the other. He agreed with Dr Hotchkiss that we are really very ignorant about how chromosomes are constituted. Therefore, the A-T active zone is only a working hypothesis and not really definitive. As to the DNA molecule, Dr Somers presented some hypothetical ideas about the architecture of chromosomes from her collaboration with Dr Cole. If the Cole model proves to be correct, then the chromosome is made of a continuous DNA molecule; if it is not, then we would have to propose other models to fit the facts. Robert E. Bowel1
Experimental
Cell Research, Suppl. $