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Newer Measurements of Cell Proliferation in the Colon
Vol. 51, No. 5, Part 2 Printed in U.S.A.
GASTROENTEROLOGY
Copyright © 1966 by The Williams & Wilkins Co.
NEWER MEASUREMENTS OF CELL PROLIFERATION IN THE COLON MARTIN LIPKIN, M.D.
GastTointestinal ReseaTch Unit, Cornell Medical Division, Bellevue Hospital, and DepaTtment of Medicine, Cornell University Medical College, New Y oTk, New Y ark
Quastler. 2 The cycle has customarily been divided into a number of phases, each of which reflects the occurrence of specific biochemical, morphological, and functional processes taking place within the cell. During one portion of the proliferative cell cycle, DNA replication occurs. This part of the cycle has been referred to as the S phase. Cells synthesize DNA in preparation for mitosis. In proliferating colonic epithelial cells of man, the duration of the S phase is 10 to 15 hr. Following the replication of the new complement of DNA, the cells enter a phase during which DNA is no longer synthesized, although RNA and protein synthesis take place. This phase of the cell cycle has been called the "gap 2" or G2 phase. In proliferating colonic epithelial cells of man, it lasts 1 to 7 hr. The cells then enter into mitosis, the duration of which appears to be in the order of 1 hr, in these cells. Included among the many characteristic features of mitosis are a reduction to the normal content of DNA and a marked reduction of RNA synthesis. Following the completion of mitosis, the cell enters a phase referred to as the "gap 1" or G 1 phase, which is the most variable one in length. During this phase, RNA and protein synthesis continues. Some cells also elaborate specific DNA precursor enzymes in preparation for reentry into the S phase, while other cells enter into a nonproliferative phase, where the cells elaborate other Address requests for reprints to: Dr. Martin biochemical derivatives as they differentiate Lipkin, Gastrointestinal Research Unit, Cornell further into more specialized and maMedical Division, Bellevue Hospital, 28th Street ture cell types. This portion of the proliferand First Avenue, New York, New York 10016. ative cycle has been subdivided by BulThis study was supported by Public Health Service Grant AM-06284, of the National Insti- lough3 into the apophase (the part of the tute of Arthritis and Metabolic Diseases, and cycle immediately after mitosis, when the Research Career Program Award K3-AM-4468. cells recover from the previous mitosis,
Definitions of the functions that describe cell renewal in gastrointestinal mucosa are of both theoretical and practical interest. Because of the direction of our most recent studies, my presentation will be concerned largely with questions related to renewal patterns in normal gastrointestinal tissue. A number of topics taken from our current laboratory work on the colon as well as other mucosal tissues will be considered. In contrast to specialized cells, such as parietal or zymogen cells, the epithelial cells of the gastrointestinal mucosa renew very rapidly. Various experimental approaches have been used to define parameters of cell renewal in different parts of the gastrointestinal tract in man and animals. Injection of tritiated thymidine (H3TdR) into animals and man is one such approach, and provides a marker to study the life cycle of cells. H 3TdR is incorporated into cells during the DNA synthesis phase of the proliferative cycle. Microautoradiographs processed from tissues removed at various periods after injection of IPTdR have facilitated measurements of the durations of the phases of the proliferative cycle, the generation time of the cells, and the location of the areas in mucosa where cells proliferate and mature. In studies of colonic epithelial cells of man , we have been able to measure the durations of the phases of the proliferative cycle. 1 Various subdivisions of the cell cycle have been made, for example, as given by
851
852
LIPKIN
grow in bulk and return to the normal state), the dichophase (when the cells become committed to specialize for tissue function or mitosis again), and early prosphase (when enzymes are elaborated to facilitate DNA synthesis). Recent studies in our laboratory 4 have considered the problem of variations in cell cycle duration, by the analysis of measurements made on the gastrointestinal mucosa of newborn golden Syrian hamsters. These studies have investigated cells tagged with H 3TdR as they pass through the mitotic phase of the proliferative cycle. The durations of the phases of the proliferative cycle have been measured, and estimates made of the range of cycle durations of groups of proliferating cells, as well as the fractional contribution of each group to the total proliferating population. The comparative rates of cell renewal in different areas of the same crypt, as well as rates of removal of labeled cells from the mucosa have been studied. \Ve have recently constructed theoretical models containing known populations of proliferating cells, the populations containing fixed repetitive cycle durations, or random distributions of cycle durations. Certain well known anatomical similarities in the organization of gastrointestinal epithelium serve to unify otherwise noteworthy differences in the mucosa of different regions. In like manner, our studies indicate that a variety of proliferative kinetic parameters both unify and also bring out differences in gastrointestinal epithelium, in both the same and distant anatomical areas. Within the columns of proliferating cells in stomach, small intestine, and colon, there is a distinct anatomical demarcation, denoting a zone where many cells are proliferating. This zone, containing a high density of cells synthesizing DNA, is located deep in the mucosa. In all parts of ~he mucosa, there is also a characteristic area between the deeper area that contains a high density of DNA-synthesizing cells, and the surface of the mucosa. In this latter area, the area of transition, there is a gradual falloff of the density of proliferating cells. Near the surface, the proliferative potential ends, and no cells synthesize
Vol. 51, No. 5, Part 2
DNA. These relationships are not maintained in polyp tissue, and cells that synthesize DNA are found largely at the surface.5 In addition, in tissue near polyps that appears histologically normal, an abnormal organization of cells within the tissue may also be present, with DNA-synthesizing cells found at the surface. 5 In normal tissue, in the area of transition cells leave the proliferative cycle and develop into mature or nonproliferating cells. In all areas of the gastrointestinal tract that we have examined, including the colon, the duration of the proliferative cell cycle does not radically change as cells approach the surface in the transitional zone. Some decay of synchronization of the phases of the cell cycle, however, does occur. In our work, the analysis of repetitive vvaves of labeled mitoses has demonstrated that the synchronization of cell cycle durations is greatest in the center of the proliferating columns. The findings indicate that as proliferative cells approach the surface, they lose their proliferative potential rather suddenly, and over a few cell positions become nonproliferative and then mature cells. These features characterize normal epithelial cells of the colon. Whether the cessation of proliferation as cells mature can be reversed, and how this may relate to the occurrence of gastrointestinal disease is not clear. The potential for cells to slide back into previous phases of the proliferative cycle is there, for it can occur following radiation damage. 6 Following injection of I-PTdR, analysis of the fraction of mitoses labeled over a period of several days or longer allows for a further definition of the cell cycle times. In newborn hamster, a smaller amount of variance is associated with the duration of the DNA synthesis phase of the cycle compared to the postmitotic or G 1 phase. In all areas of the mucosa we have examined in newborn hamsters, some proliferating cells have a short postmitotic ( G1) duration, and the DNA synthesis phase occupies the major part of the cell cycle time. Other proliferating cells have much longer G1 durations, and S phase occupies much less of the cycle time. Our theoretical models that simulate the repet-
November 1966
853
DISCUSSTON
itive waves of labeled mitoses have 2- or 3fold spreads of cell cycle durations. 4 Further analysis of the fraction of mitoses labeled after H 3TdR, over a prolonged period yields repetitive waves of labeled mitoses that differ in their cycle frequency . In the jejunum of newborn hamsters, more than 3 cycles have been completed by 50 hr. In stomach and sigmoid, slower redivision of cells has occurred. Observing the rate of removal of labeled cells in each area similarly demonstrates more rapid renewal of epithelial cells in small intestine compared to stomach and colon. As noted above, measurement of the density of labeled cells supported this finding, and fewer DNA synthesizing cells are found in each part of the proliferating columns of stomach and colon compared to duodenum and jejunum. In each area of the gastrointestinal tract, the repetitive waves of labeled mitoses also lose their periodic character in a characteristic duration, and settle around a value that corresponds to the fraction of cells synthesizing DNA at the time of injection. The loss of the periodic character of repetitive waves of labeled mitoses can be explained by the repetitive divisions of cells whose cycle durations change in random manner. On the other hand, we have been able to simulate the repetitive waves of labeled mitoses seen in the colon, by constructing theoretical models made up of groups of cells whose repetitive cycle durations are fixed and unchanging. In the latter instance, too, the waves of labeled mitoses lose their periodic character and become progressively damped, finally settling around an ordinate fraction that corresponds to the initial fraction of cells in DNA synthesis phase at the time of label-
ing. The question of how rigid and unchanging are repetitive cycle durations under normal and abnormal conditions is providing us with an interesting area of investigation at the present time. Another interesting and important area concerns the sequential divisions of proliferating cells. A cell division can yield two daughter cells both of which again divide, and so on, in exponential manner as tumor cells may do. Or, a cell division can yield one daughter cell destined to divide again, and one destined to differentiate further and become mature. The definition of detailed patterns of sequential divisions of cells in colon, and the influence of intracellular and extracellular factors on cell proliferation are important areas for future investigation. REFERENCES 1. Lipkin, M. 1965. Cell replication in the gastrointestinal tract of man. Gastroenterology 48 : 616-624. 2. Quastler, H. 1963. The analysis of cell population kinetics, p. 18. In L. F. Lamerton and R. J. M. Frye [ed.], Cell proliferation. A Guiness Symposium. F. A. Davis Company, Philadelphia. 3. Bullough, W. S. 1965. Mitotic and functional homeostasis: A speculative review. Cancer R es. 25 : 1683-1727. 4. Lipkin, M ., and E. Deschner. Comparative analysis of cell proliferation in the gastrointestinal tract of newborn hamsters. Submitted for publication. 5. Deschner, E., M. Lipkin, and C. Solomon. 1966. In vitro study of human rectal epithelial cells. II. H 3 thymidine incorporation into polyps and adjacent rectal mucosa. J. Nat. Cancer Inst . 36 :849-857. 6. Patt, H., and H. Quastler. 1963. Radiation effects on cell renewal and related systems. Physiol. Rev. 43 : 357-396.
DISCUSSION OF PAPER PRESENTED BY DR. LIPKIN DR. MARTIN BROTMAN (Rochester, Minn.) I would like to ask some questions regarding the interpretation of the normal values, which have now appeared in the literature for large intestinal cell proliferation rates
in the human, and to inject a note of caution into interpretation at this stage. First, if you compare the colchicine techniques and the tritium-labeled thymidine techniques for single species and different parts
854
DISCUSSJOjV
of the intestinal tract, the widest variation between the rates observed occurs in the large intestine; as much as 10 hr versus 16 days for turnover in the large intestine of a rat. Secondly, the data that are now available regarding in vivo studies of the normal human colon usually are limited to intravenous injection of tritium-labeled thymidine, followed by a biopsy of the transverse colostomy stoma in patients who have malignant disease. I think these "normal" values fail to take into consideration the observation by Knutzen and his coworkers in the rat. Using tritium-labeled thymidine, they observed a marked difference in the proliferation rates in the ascending and descending colon. The descending colon had a 3-fold proliferation rate compared to the ascending colon. V\,T e have accumulated biochemical data, as yet unpublished, that the rates of incorporation of amino acids into protein are similarly different in the ascending and descending colon. This observation suggests different characteristics of cells in the ascending and descending colon. Although Dr. Lipkin has not mentioned his studies of cell turnover rates in colon cultures, I wanted to ask a question on the interpretation of these findings. There has been an observation that tritium-l abeled thymidine will inhibit the growth of bela cell cultures, and I am wondering how this observation will temper our future interpretation of observations of tritium-labeled in vitro studies? DR. LIPKIN: Regarding the comparison of colchicine and thymidine, our data in mice indicate more rapid renewal in small intestine than colon or stomach with both methods. Additional methods should be used to secure a complete view of this problem. One approach would be to continually infuse the tissue with thymidine and note the time of labeling of all the cells. In regard to the question about inhibition with thymidine, the literature has not indicated
Vol. 51, No.5, Part 2
inhibition of cell proliferation in the doses we have used. DR. EDWARD WILSON (St. Louis): Dr. Sprinz has mentioned the effect of reserpine on cell regeneration, and I wondered if you had any information about possible central nervous system regulation of cell proliferation? DR. LIPKIN: The information t hat Dr. Sprinz gave is very interesting. It is a topic we are working on at the present time, but I do not have additional information to add at this moment. DR. MAx CooPER (Minneapolis) : I was confused by your definition of differentiation. Did you mean to say that different iation begins only after the period of DNA synthesis? I , also, wanted to ask in which of these phases do you see nucleoli? Finally, in referring to a closed cycle, did you mean that a cell once differentiated to the stage of specific product synthesis, still can reverse and go back through the cycle? DR. LIPKIN: It is better to use the term transitional cell rather than differentiating cell. Once mature, a cell would not normally reenter the proliferative cycle. Nucleoli can be seen in cells incorporating thymidine. DR. RuvEN LEVITAN (Boston) : Does the age of the subject have something to do with the proliferation of cells, and secondly, is it fair to call this normal data if the person has carcinoma? I s it not possible that such people have nutritional deficiencies that may influence what you are measuring? DR. LIPKIN: It has been shown that younger animals have proliferation rates that are more rapid than older animals. All of our patients have not had carcinoma. We had a patient with brain damage. I would suspect that the spread of values in persons free of disease is not too different from what we have here.
GASTROENTEROLOGY
Copyright © 1966 by The Williams & Wilkins Co.
NEWER MEASUREMENTS OF CELL PROLIFERATION IN THE COLON MARTIN LIPKIN, M.D.
GastTointestinal ReseaTch Unit, Cornell Medical Division, Bellevue Hospital, and DepaTtment of Medicine, Cornell University Medical College, New Y oTk, New Y ark
Quastler. 2 The cycle has customarily been divided into a number of phases, each of which reflects the occurrence of specific biochemical, morphological, and functional processes taking place within the cell. During one portion of the proliferative cell cycle, DNA replication occurs. This part of the cycle has been referred to as the S phase. Cells synthesize DNA in preparation for mitosis. In proliferating colonic epithelial cells of man, the duration of the S phase is 10 to 15 hr. Following the replication of the new complement of DNA, the cells enter a phase during which DNA is no longer synthesized, although RNA and protein synthesis take place. This phase of the cell cycle has been called the "gap 2" or G2 phase. In proliferating colonic epithelial cells of man, it lasts 1 to 7 hr. The cells then enter into mitosis, the duration of which appears to be in the order of 1 hr, in these cells. Included among the many characteristic features of mitosis are a reduction to the normal content of DNA and a marked reduction of RNA synthesis. Following the completion of mitosis, the cell enters a phase referred to as the "gap 1" or G 1 phase, which is the most variable one in length. During this phase, RNA and protein synthesis continues. Some cells also elaborate specific DNA precursor enzymes in preparation for reentry into the S phase, while other cells enter into a nonproliferative phase, where the cells elaborate other Address requests for reprints to: Dr. Martin biochemical derivatives as they differentiate Lipkin, Gastrointestinal Research Unit, Cornell further into more specialized and maMedical Division, Bellevue Hospital, 28th Street ture cell types. This portion of the proliferand First Avenue, New York, New York 10016. ative cycle has been subdivided by BulThis study was supported by Public Health Service Grant AM-06284, of the National Insti- lough3 into the apophase (the part of the tute of Arthritis and Metabolic Diseases, and cycle immediately after mitosis, when the Research Career Program Award K3-AM-4468. cells recover from the previous mitosis,
Definitions of the functions that describe cell renewal in gastrointestinal mucosa are of both theoretical and practical interest. Because of the direction of our most recent studies, my presentation will be concerned largely with questions related to renewal patterns in normal gastrointestinal tissue. A number of topics taken from our current laboratory work on the colon as well as other mucosal tissues will be considered. In contrast to specialized cells, such as parietal or zymogen cells, the epithelial cells of the gastrointestinal mucosa renew very rapidly. Various experimental approaches have been used to define parameters of cell renewal in different parts of the gastrointestinal tract in man and animals. Injection of tritiated thymidine (H3TdR) into animals and man is one such approach, and provides a marker to study the life cycle of cells. H 3TdR is incorporated into cells during the DNA synthesis phase of the proliferative cycle. Microautoradiographs processed from tissues removed at various periods after injection of IPTdR have facilitated measurements of the durations of the phases of the proliferative cycle, the generation time of the cells, and the location of the areas in mucosa where cells proliferate and mature. In studies of colonic epithelial cells of man , we have been able to measure the durations of the phases of the proliferative cycle. 1 Various subdivisions of the cell cycle have been made, for example, as given by
851
852
LIPKIN
grow in bulk and return to the normal state), the dichophase (when the cells become committed to specialize for tissue function or mitosis again), and early prosphase (when enzymes are elaborated to facilitate DNA synthesis). Recent studies in our laboratory 4 have considered the problem of variations in cell cycle duration, by the analysis of measurements made on the gastrointestinal mucosa of newborn golden Syrian hamsters. These studies have investigated cells tagged with H 3TdR as they pass through the mitotic phase of the proliferative cycle. The durations of the phases of the proliferative cycle have been measured, and estimates made of the range of cycle durations of groups of proliferating cells, as well as the fractional contribution of each group to the total proliferating population. The comparative rates of cell renewal in different areas of the same crypt, as well as rates of removal of labeled cells from the mucosa have been studied. \Ve have recently constructed theoretical models containing known populations of proliferating cells, the populations containing fixed repetitive cycle durations, or random distributions of cycle durations. Certain well known anatomical similarities in the organization of gastrointestinal epithelium serve to unify otherwise noteworthy differences in the mucosa of different regions. In like manner, our studies indicate that a variety of proliferative kinetic parameters both unify and also bring out differences in gastrointestinal epithelium, in both the same and distant anatomical areas. Within the columns of proliferating cells in stomach, small intestine, and colon, there is a distinct anatomical demarcation, denoting a zone where many cells are proliferating. This zone, containing a high density of cells synthesizing DNA, is located deep in the mucosa. In all parts of ~he mucosa, there is also a characteristic area between the deeper area that contains a high density of DNA-synthesizing cells, and the surface of the mucosa. In this latter area, the area of transition, there is a gradual falloff of the density of proliferating cells. Near the surface, the proliferative potential ends, and no cells synthesize
Vol. 51, No. 5, Part 2
DNA. These relationships are not maintained in polyp tissue, and cells that synthesize DNA are found largely at the surface.5 In addition, in tissue near polyps that appears histologically normal, an abnormal organization of cells within the tissue may also be present, with DNA-synthesizing cells found at the surface. 5 In normal tissue, in the area of transition cells leave the proliferative cycle and develop into mature or nonproliferating cells. In all areas of the gastrointestinal tract that we have examined, including the colon, the duration of the proliferative cell cycle does not radically change as cells approach the surface in the transitional zone. Some decay of synchronization of the phases of the cell cycle, however, does occur. In our work, the analysis of repetitive vvaves of labeled mitoses has demonstrated that the synchronization of cell cycle durations is greatest in the center of the proliferating columns. The findings indicate that as proliferative cells approach the surface, they lose their proliferative potential rather suddenly, and over a few cell positions become nonproliferative and then mature cells. These features characterize normal epithelial cells of the colon. Whether the cessation of proliferation as cells mature can be reversed, and how this may relate to the occurrence of gastrointestinal disease is not clear. The potential for cells to slide back into previous phases of the proliferative cycle is there, for it can occur following radiation damage. 6 Following injection of I-PTdR, analysis of the fraction of mitoses labeled over a period of several days or longer allows for a further definition of the cell cycle times. In newborn hamster, a smaller amount of variance is associated with the duration of the DNA synthesis phase of the cycle compared to the postmitotic or G 1 phase. In all areas of the mucosa we have examined in newborn hamsters, some proliferating cells have a short postmitotic ( G1) duration, and the DNA synthesis phase occupies the major part of the cell cycle time. Other proliferating cells have much longer G1 durations, and S phase occupies much less of the cycle time. Our theoretical models that simulate the repet-
November 1966
853
DISCUSSTON
itive waves of labeled mitoses have 2- or 3fold spreads of cell cycle durations. 4 Further analysis of the fraction of mitoses labeled after H 3TdR, over a prolonged period yields repetitive waves of labeled mitoses that differ in their cycle frequency . In the jejunum of newborn hamsters, more than 3 cycles have been completed by 50 hr. In stomach and sigmoid, slower redivision of cells has occurred. Observing the rate of removal of labeled cells in each area similarly demonstrates more rapid renewal of epithelial cells in small intestine compared to stomach and colon. As noted above, measurement of the density of labeled cells supported this finding, and fewer DNA synthesizing cells are found in each part of the proliferating columns of stomach and colon compared to duodenum and jejunum. In each area of the gastrointestinal tract, the repetitive waves of labeled mitoses also lose their periodic character in a characteristic duration, and settle around a value that corresponds to the fraction of cells synthesizing DNA at the time of injection. The loss of the periodic character of repetitive waves of labeled mitoses can be explained by the repetitive divisions of cells whose cycle durations change in random manner. On the other hand, we have been able to simulate the repetitive waves of labeled mitoses seen in the colon, by constructing theoretical models made up of groups of cells whose repetitive cycle durations are fixed and unchanging. In the latter instance, too, the waves of labeled mitoses lose their periodic character and become progressively damped, finally settling around an ordinate fraction that corresponds to the initial fraction of cells in DNA synthesis phase at the time of label-
ing. The question of how rigid and unchanging are repetitive cycle durations under normal and abnormal conditions is providing us with an interesting area of investigation at the present time. Another interesting and important area concerns the sequential divisions of proliferating cells. A cell division can yield two daughter cells both of which again divide, and so on, in exponential manner as tumor cells may do. Or, a cell division can yield one daughter cell destined to divide again, and one destined to differentiate further and become mature. The definition of detailed patterns of sequential divisions of cells in colon, and the influence of intracellular and extracellular factors on cell proliferation are important areas for future investigation. REFERENCES 1. Lipkin, M. 1965. Cell replication in the gastrointestinal tract of man. Gastroenterology 48 : 616-624. 2. Quastler, H. 1963. The analysis of cell population kinetics, p. 18. In L. F. Lamerton and R. J. M. Frye [ed.], Cell proliferation. A Guiness Symposium. F. A. Davis Company, Philadelphia. 3. Bullough, W. S. 1965. Mitotic and functional homeostasis: A speculative review. Cancer R es. 25 : 1683-1727. 4. Lipkin, M ., and E. Deschner. Comparative analysis of cell proliferation in the gastrointestinal tract of newborn hamsters. Submitted for publication. 5. Deschner, E., M. Lipkin, and C. Solomon. 1966. In vitro study of human rectal epithelial cells. II. H 3 thymidine incorporation into polyps and adjacent rectal mucosa. J. Nat. Cancer Inst . 36 :849-857. 6. Patt, H., and H. Quastler. 1963. Radiation effects on cell renewal and related systems. Physiol. Rev. 43 : 357-396.
DISCUSSION OF PAPER PRESENTED BY DR. LIPKIN DR. MARTIN BROTMAN (Rochester, Minn.) I would like to ask some questions regarding the interpretation of the normal values, which have now appeared in the literature for large intestinal cell proliferation rates
in the human, and to inject a note of caution into interpretation at this stage. First, if you compare the colchicine techniques and the tritium-labeled thymidine techniques for single species and different parts
854
DISCUSSJOjV
of the intestinal tract, the widest variation between the rates observed occurs in the large intestine; as much as 10 hr versus 16 days for turnover in the large intestine of a rat. Secondly, the data that are now available regarding in vivo studies of the normal human colon usually are limited to intravenous injection of tritium-labeled thymidine, followed by a biopsy of the transverse colostomy stoma in patients who have malignant disease. I think these "normal" values fail to take into consideration the observation by Knutzen and his coworkers in the rat. Using tritium-labeled thymidine, they observed a marked difference in the proliferation rates in the ascending and descending colon. The descending colon had a 3-fold proliferation rate compared to the ascending colon. V\,T e have accumulated biochemical data, as yet unpublished, that the rates of incorporation of amino acids into protein are similarly different in the ascending and descending colon. This observation suggests different characteristics of cells in the ascending and descending colon. Although Dr. Lipkin has not mentioned his studies of cell turnover rates in colon cultures, I wanted to ask a question on the interpretation of these findings. There has been an observation that tritium-l abeled thymidine will inhibit the growth of bela cell cultures, and I am wondering how this observation will temper our future interpretation of observations of tritium-labeled in vitro studies? DR. LIPKIN: Regarding the comparison of colchicine and thymidine, our data in mice indicate more rapid renewal in small intestine than colon or stomach with both methods. Additional methods should be used to secure a complete view of this problem. One approach would be to continually infuse the tissue with thymidine and note the time of labeling of all the cells. In regard to the question about inhibition with thymidine, the literature has not indicated
Vol. 51, No.5, Part 2
inhibition of cell proliferation in the doses we have used. DR. EDWARD WILSON (St. Louis): Dr. Sprinz has mentioned the effect of reserpine on cell regeneration, and I wondered if you had any information about possible central nervous system regulation of cell proliferation? DR. LIPKIN: The information t hat Dr. Sprinz gave is very interesting. It is a topic we are working on at the present time, but I do not have additional information to add at this moment. DR. MAx CooPER (Minneapolis) : I was confused by your definition of differentiation. Did you mean to say that different iation begins only after the period of DNA synthesis? I , also, wanted to ask in which of these phases do you see nucleoli? Finally, in referring to a closed cycle, did you mean that a cell once differentiated to the stage of specific product synthesis, still can reverse and go back through the cycle? DR. LIPKIN: It is better to use the term transitional cell rather than differentiating cell. Once mature, a cell would not normally reenter the proliferative cycle. Nucleoli can be seen in cells incorporating thymidine. DR. RuvEN LEVITAN (Boston) : Does the age of the subject have something to do with the proliferation of cells, and secondly, is it fair to call this normal data if the person has carcinoma? I s it not possible that such people have nutritional deficiencies that may influence what you are measuring? DR. LIPKIN: It has been shown that younger animals have proliferation rates that are more rapid than older animals. All of our patients have not had carcinoma. We had a patient with brain damage. I would suspect that the spread of values in persons free of disease is not too different from what we have here.