A STUDY OF TRHEATED THYMIDINE‘ {NCORPORATION IN MOUSE FEMUR BONE - ‘ " MARROW DEOXYRIBONUCLEIC ACID Thesis for ”19 Degreé of ph..D. MICHIGAN STATE UNIVERSITYj Leo Herman Blackwell, Jr‘. 1964 THESIS This is to certify that the thesis entitled A SWDY OF TRITIATED THYMIDINE INCORPORATION IN HOUSE MR BONE MARROW DEOXYRIBONUCLEIC ACID presented by Leo Herman Blackwell, Jr. has been accepted towards fulfillment of the requirements for Eh . D . degree mm? gaff/flag” Major professor 0-169 . \ LIBRARY Michigan State University ABSTRACT A STUDY OF TRITIATED THYMIDINE INCORPORATION IN MOUSE FEMUR BONE MARROW DEOXYRIBONUCLEIC ACID1 by Leo Herman Blackwell, Jr. The incorporation of H3-thymidine in mouse femur bone marrow deoxyribonucleic acid (DNA) and whole mouse femur was studied by: 1) Extracting DNA from femur bone marrow cells and assaying DNA for tritium, utilizing liquid scintillation counting, and 2) Combusting whole dried femurs to tritiated water and carbon dioxide and assaying the tritiated water by liquid scintillation counting. The data permit the following observations and conclusions: 1) Tritiated thymidine is actively incorporated into bone marrow DNA both directly from the plasma at a very rapid rate and indirectly from a precursor pool at a much slower rate . 2) The greatest contribution to total DNA turnover in the normal mouse femur marrow is made by the turnover of erythroid cell DNA. 3) A very large endogenous pool of DNA precursors, transported through the plasma to the marrow, must be postulated. Leo Herman Blackwell, Jr. 4) Additional DNA, as DNA, enters the normal mouse femur from some other source. Tritium label from this source appears in the mouse femur between eighteen and twenty-four hours after injection of tritiated thymidine. 1This work was performed under the auSpices of the United States Atomic Energy Commission. A STUDY OF TRITIATED THYMIDINE INCORPORATION IN“MOUSE FEMUR BONE MARROW DEOXYRIBONUCLEIC ACID By Leo Herman Blackwell, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1961+ (c (u a) CV V‘ To my wife, June, who gave many hours of encouragement and criticism. 11 ACKNOWLEDGEMENTS The author wishes to express sincere thanks to Dr. L. F. Wolterink for encouragement and guidance throughout his graduate studies. Special thanks are due Dr. Harveny. Patt, of the Division of Biological and Medical Research, Argonne National Laboratory, who directed the research. The author wishes also to express his appreciation to Dr. Robert L. Straube for his guidance, assistance and encouragement during the time this work was conducted. The helpful discussions with Dr. Walter E. Kisieleski, Dr. P. K. Lala, Miss Mary A. Maloney, Dr. Harold G. Sutton and Mr. S. A. Tyler, the technical assistance of Mrs. Betty Newell and Mrs. Eugenia Cook and the secretarial help of Miss Donna Daniel are gratefully acknowledged. iii TABLE OF CONTENTS INTRODUCTION AND REVIEW OF LITERATURE . Tritium and Tritiated Thymidine Specificity of Thymidine for Deoxyribonucleic AC1d O O O O O O O O I O O O 0 Cell Renewal Systems . . . . . Studies of Deoxyribonucleic Acid Synthesis with Tritiated Thymidine . . . . .111 Vitro StUdies o o 0 o In Vivo Studies . . . . . General Scheme for Bone Marrow Deoxyribonucleic Acid Turnover Objective of Thesis . . . . . MATERIALS AND METHODS . . . . . . . Animals . . . . . . . . . . . Sacrifice . . . . . . . . . . Blood . . . . . . . . . . . . DNA Extraction . . . . . . . . Diphenylamine Reaction . . . . Radioactivity Counting . . . . Femmr Combustion . . . . . . . Combustion Technique . . . . . Tritiated Thymidine . . . . . Hypertransfusion . . . . . . . Autoradiography . . . . . . . iv 0 Page 11 13 15 15 15 16 16 18 18 19 19 20 22 22 Page RESUIJTS O I O O O O 0 O O O O O O O C O O O O O O O O O O 23 Time for Maximum Uptake of Tritiated Thymidine Activity into Femur Deoxyribonucleic Acid . . . . . 23 Activity Incorporated into Femur Bone Marrow Deoxyribonucleic Acid . . . . . . . . . . . . . . . 27 Activity Incorporated in Whole Pemur . . . . . . . . 31 Bone Marrow Cell DNA Content Per Femur Estimated From the Activity in DNA and Activity in Femur . . . 33 Activity Incorporated into the Whole Pemur - Hypertransfused Mice . . . . . . . . . . . . . . . . 3h Granulocyte and Lymphocyte Appearance in the Peripheral Blood . . . . . . . . . . . . . . . . . . ho Summary of Pertinent Data and Their Statistics . . . ho DISWSSION O O O O O O O O O O O O C O O O O O O O O O 0 us Difference in Femur Bone Marrow Deoxyribonucleic Acid Activity and Femur.Activity . . . . . . . . . . h? Consideration of a "Pre-DNA" Pool Feeding Labeled Substrate into Pemur Bone Marrow Deoxyribonucleic AC1d O O O O O O O O O O O O O O O O O O O O O O O O 52 Evaluation of Change in Activity in Bone Marrow Deoxyribonucleic Acid Between 18 and 2% Hours . . . 55 Estimation of the Relative Contributions of Granuloid, Erythroid and Lymphoid Cells to the Deoxyribonucleic Acid Turnover in the Normal Mouse . S6 SWRY AND CONQIUS IONS O O O O O O O O O O O I O O O O O 59 REFERENCES 0 O O O O O O O C O O O O O O O 0 O O O O O O 62 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . 69 LIST OF TABLES TABLE Page I Distribution of Cells or DNA in the Femur of Normal and Hypertransfused Mice . . . . . . . 36 II Constants of Least Squares Lines and DNA Output - Figures 5, 7 and 9 . . . . . . . . . . h3 III Comparison of Hemocytometer Count to Coulter Electronic Cell Counter Count at Various Dilutions O O O O O O O O O O O O O O O Q g o . 71 IV Standardization of DNA Activity Techniques . . . 7h V Results of Individual Experiments Using DNA Extraction in Normal Animals . . . . . . . . . . 81 VI Results of Individual Experiments Using Pemur Combustion in Normal Animals . . . . . . . . . . 82 VII Results of Individual Experiments Using Femur Combustion in Hypertransfused Animals . . . . . 85 vi Figure 1. 100 11. LIST OF FIGURES Combustion flask arranged for ignition . . . . . . . Clearance of H3-thymidine and non-volatile breakdown products of H3-thymidine from mouse blood plasma . . Femur bone marrow DNA activity as a function of time. H3-thymidine was injected at time 0 and saline or cold thymidine was injected at 15 minutes . . . . . . . . . . . . . . . . . . . . . . Whole femur activity as a function of time. H3-thymidine was injected at time 0 and saline or cold thymidine was injected at 15 minutes . . . . Concentration of tritium activity in femur bone marrow DNA as a function of time following the administration of H3-thymidine . . . . . . . . . . . The concentration of non-volatile tritium activity in normal bone marrow after an injection of H3-thymidine. The results of three experiments are combined with a different symbol for each . Tritium activit incorporated in the whole femur (normal animalsl as a function of time following the administration of H3-thymidine . . . . . . . . . Pemur bone marrow cell DNA activity incorporated per femur as a function of time after a single injection of H3-thymidine . . . . . . . . . . . . . Tritium activity incorporated in the whole femur (hypertransfused animals) as g function of time after a single injection of H -thymidine . . . . . . Granulocyte labeling in the peripheral blood of normal and hypertransfused mice given a single injection of H3-thymidine . . . . . . . . . . . . . Lymphocyte labeling in the peripheral blood of normal and hypertransfused mice given a single injection of H3-thymidine . . . . . . . . . . . . . vii Page 21 2h 26 26 28 3O 32 35 38 hl A2 Figure Page 12. The movement of labeled tritium compounds in the femur bone marrow cells after a single injection of tritiated thymidine . . . . . . . . . . A6 13. A model for the movement of tritium-label in the femur bone marrow following a single injection of H3-thymid1ne s o o s o o e e o e e o o s e a a e o o 5"" 1h. Paired Coulter counts covering the range used in these experiments . . . . . . . . . . . . . . . . . . 7O 15. Standard curve for DNA . . . . . . . . . . . . . . . 76 16. Amount of DNA extracted as a function of the cell umber . O O O O C O C C O O C C C C C C O O C C O C 79 viii LIST OF APPENDICES Appendix Page I Coulter Electronic Cell Counter Standardization for Nucleated Mouse Bone Marrow Cells . . . . . . . . . . . . . . . . 69 II Standardization of DNA Activity Technique . . . . 73 III Standard Curve - DNA . . . . . . . . . . . . . . . 75 IV Standardization of Femur Combustion . . . . . . . 77 V DNA Content Per Cell . . . . . . . . . . . . . . . 78 VI Basic Data . . . . . . . . . . . . . . . . . . . . 80 ix INTRODUCTION AND REVIEW OF LITERATURE Tritium and Tritiated Thymidine In 1919, Lord Rutherford (see Rutherford, Chadwich and Ellis, 1951) found the (d,d) reaction produced tritium in addition to helium-3. It was not until 1939 that Alvarez and Cornog proved that helium-3 was stable and tritium'was the radioactive component. Tritium can now be produced by pile reactions such as 3Li6 + on1 —*> Heh + H3. Because tritium is a very weak beta emitter (E a 2 1 maximum 18 kiloelectronvolts, half life - 12.h years), it has been utilized for autoradiographic analysis of biological specimens. In 1951, Robertson designed a proportional counting system for tritium analysis. Since that time, liquid scintillation counting systems have been developed. These have made possible the analysis of tritium in biological samples which complement work done utilizing autoradiography. Thymidine was tritiated by Verly in 1957. The method used was a catalytic exchange between tritium water and thymidine. Friedkin (1960) showed that the preparation produced by Verly, as well as commercial preparations of tritiated thymidine (RB-thymidine) contained tritium in the methyl group. The tritium thus bound is highly stable, and only metabolic reactions are capable of liberating tritium from labeled thymidine introduced into cells (Verly 9.2 9.1... 1958). Specificityjof Thymidine for Deoxyribonucleic Acid Thymidine labeled with Nitrogen-15 is a specific precursor of deoxyribonucleic acid (DNA) in rats (Reichard and Estborn, 1951). Later work by Friedkin, Tilson and Roberts (1956) using thymidine-2- carbon-lh proved that thymidine was incorporated into DNA of bone marrow and other tissues, with only a negligible amount of the radioactivity being incorporated into RNA or components other than DNA. Because of this high degree of specificity for DNA, thymidine has come to be widely used for the study of cell proliferation, since DNA can be tagged only during DNA synthesis (i.e., DNA replication) immediately prior to mitosis. - It has been established that free bases are not utilized in the production of nucleic acids, but that ribosides and ribotides, as well as simple precursors are utilized in nucleic acid formation (Schuhman, 1961). Thymidine is degraded to carbon dioxide and B-mminoisobutyric acid (probably in the liver in mammals) (Rubini 'gg‘gl., 1960). The normal pathway for thymidine incorporation prior to the final assembling of new DNA in a proliferating cell system is thought to be by methylating deoxyuridylic acid to thymidylic acid which is then incorporated directly into new DNA (Rubini, Keller and McCall, 1961;). When 113-thymidine is injected into an animal, it is evidently phosphorylated to thymidylic acid for subsequent incorporation into DNA. 3 Cell Renewal Systems The study of tissue proliferation began, perhaps, with Ham- and Leeuwenhoek's discovery and description of spermatozoa in 1677 (see Needham, 1963). These authors, however, did not realize that the small cells they observed.were the consequence of cell proliferation. Since these early studies, the growth of cell populations in the adult organism has been studied in relation to hormone response, repair after trauma (regeneration in lower animals, repair of radiation damage), and tumor growth. New cells (thus new DNA) can arise only by mitosis, and since some tissues exhibit a greater rate of mitosis than others, DNA replication varies in a similar fashion. Patt (l95h), and other workers in the field of radiation biology, noted that some tissues (gut, blood cells, skin) are constantly renewed, and that there exists a steady state between production and loss, as long as the system is not perturbed. Failure of these cell systems to produce new cells after irradiation is due to the blockage of mitosis, as well as death of proliferative cells. In 1956, Leblond and Walker applied the term "cell renewal system" to these cell populations that are kept in normal balance by continuous and equal rates of production and loss. These renewal systems may be divided into further compartments as follows: Birth —-> Proliferation —-) Differentiation —-> Function -—9 Death and Removal. The birth compartment is represented by an undifferentiated cell (called a stem cell) which can undergo mitosis (thus producing new cells and therefore new DNA) and give rise to a somewhat more differentiated cell capable of further mitosis. The proliferative compartment is represented by the cells differentiated from the stem cells. The function of the cells in the proliferative compartment is to amplify the number of cells which will eventually become the mature functional components of the cell system. Thus, the major characteristic of the proliferative compartment is mitosis, although some differentiation also takes place. The proliferative compartment, and the birth compartment, are the only sourcesof new DNA. Obviously, the chief site of DNA replication is the proliferative compartment which is numerically much larger than the birth compartment. Although there may be several different recognizable cells in the proliferating compartment, they all undergo similar cell cycles. G1 -———-—>'S T l M.<%—-—-——-62 In most mammalian cell renewal systems, the mitotic time (M) is about 1 hour, the time (G2) between DNA synthesis (S) and mitosis is about 1 to 2 hours, and the DNA synthesis time (S) is about A to 10 hours. These times are thought to be fairly constant with G1 being the variable between cell cycle lengths in different cell systems (Patt and Quastler, 1963). Cells in the differentiating and functional compartments are not capable of further DNA synthesis, therefore, to keep the system in balance, new cells (or new DNA) must be furnished from the proliferating compartment, to replace cells lost from the functional compartment. In general, all cells in a particular renewal system contain about the same amount of DNA per nucleus. Consequently, a chemical DNA analysis of a tissue includes both the DNA capable of replication (i.e., the proliferative compartment) and the DNA incapable of replication (i.e., the differentiating and functional compartments). Until radioactive isotopes became readily available, the only way to study the dynamics of a cell renewal system was to evaluate number of cells in mitosis total number of cells in the population the mitotic index ( ) for recognizable cell types. With an assumed mitotic time (usually 1 hour) and the mitotic index, one can calculate the turnover time (the time taken for the replacement of a number of cells equal to the whole population). If the number of cells produced per unit of time is known, the total number of cells in the population can be estimated (Patt, 1957). N - 2&- where: N =- Number of cells in papulation t I Mitotic time P - Cells produced per unit time M - Mitotic index The difficulties one encounters by analyzing a cell renewal system in this fashion are associated with such things as the assumed mitotic time and the growth fraction of the population (i.e., that fraction of a cell population which will undergo DNA replication to the total population). If, for example, certain of the cells that appear to be capable of dividing never participate in cell division, the mitotic index will be underestimated (Mendelsohn, 1960). In effect, two populations of morphologically like cells ‘may exist, those that will divide and those that will not divide. Labeling cells with radioactive markers has enhanced the study of cell renewal systems since by utilizing autoradiography one can actually follow a population of given cell types as it moves from one compartment to another. In addition, one can obtain the labeling total number of cells labeled ) total number of cells in population The dur't1°“ of DNA index ( synthesis can be estimated rather precisely using timed autoradiography (Maloney, Patt and Weber, 1962; Wimber, 1963). Knowing the labeling index and the DNA synthesis time, one can again calculate the turnover time. However, the problem of the growth fraction is still present. These parameters do provide a precise estimation of the proliferation rate of the population. Number of cells labeled DNA synthesis period Proliferation rate - (cells/hour) If the relative number of cells of different types in a mixed population is known, their relative proliferation rates can be determined (Patt and Maloney, 1963). Modern studies of cell renewal systems are usually based on one or both of the above methods of analysis. Some studies have followed the activity of a radioactive precursor in DNA synthesis by standard counting methods, but until tritiated thymidine became available, there were no tracers which were specific for incorporation into DNA alone. Therefore, the interpretation of those results was difficult. Because of the high degree of specificity of thymidine for DNA as discussed above, many workers have used tritiated thymidine to study cell proliferation (Hughes £5 31., 1958; Bond 35 11., 1959; Patt and Maloney, 1959). Generally, they have made the following assumptions (Cronkite gt 51., 1959a): "1) The tritium label on thymidine does not exchange. 2) Thymidine base does not exchange after incorporation into DNA. 3) DNA turnover is solely the result of mitosis and cell death. A) Re-utilization of tritium labeled materials in DNA synthesis is insignificant.* 5) Re-utilization of large chunks of DNA is unlikely in most cell renewal systems. 6) DNA synthesis in normal cells destines a cell to divide once again. 7) Tritium-labeled thymidine is uniformly distributed throughout the body and is either incorporated into DNA or degraded. 8) The effective availability time of H3-thymidine for DNA synthesis is short and a mmall fraction of the time for synthesis of DNA. 9) There is no significant radiation injury of these cells. 10) In 33552 labeling determines the proliferative potential of normal cells. 11) [In yiyg labeling after a single intravenous injection makes possible the study of the kinetics of cell proliferation." The validity of several of these assumptions has been questioned. Some of these (h, 5 and 8) will be considered in this study. * In Cronkite's list of assumptions, this reads "significant." This is evidently a misprint. See some of his other papers (Cronkite 35 21., 1959b; Cronkite gt 51., 1960). Studies of Deoxyribonucleic Acid Synthesis with Tritiated Thymidine In £959. 8 tudies The fate of H3-thymidine in yitgg has been studied utilizing tissue cultures and autoradiography of bone marrow cells of various animals (Rubini ££.El-: 1962). It was found that if cold thymidine was added at the same time as tritiated thymidine almost complete suppression of labeling occurred. If, on the other hand, cold thymidine was added 20 minutes after H3-thymidine, maximum labeling was not affected. The implication is that only 20 minutes are required to incorporate thymidine into DNA. In other words, "flash labeling" is characteristic of such systems. After 1 hour of incubation, the majority of the activity was present still as H3-thymidine (as determined by chromatography), but when fresh cells were added to the medium, no labeling occurred. If fresh H3-thymidine was added, additional labeling did occur. Rubini 35's}. (1962) postulated that in the "used" fraction, (i.e., the "thymidine" was still present but unavailable) there was a build-up of "cold" deoxyuridine and thymidine which diluted the specific activity of the H3-thymidine, so that further labeling did not occur, despite constant incorporation of thymidine into DNA. These authors calculated that there is an endogenous pool of thymidine which contained two times the amount of thymidine necessary for DNA synthesis during the hour of the culture. Cronkite 35 31. (1959b) calculated that ig‘gigg not all cells could have an endogenous pool of thymidine in view of the grain counts one observes over a cell on an autoradiograph. The results of the experiments reported in this work question the validity of this calculation, at least for cells of the bone marrow. 9 ‘13 Viva Studies 3-thymidine Studies in humans on the blood clearance of H (and the clearance of non-volatile breakdown products of H3-thymidine) have utilized liquid scintillation counting (Rubini £5 31., 1960). The subsequent appearance of tritium-labeled DNA in bone marrow was observed autoradiographically. They showed that the blood clearance of non-volatile tritium activity was very rapid, with 90 percent being lost in the first minute. They could not account for 50 percent of the material injected, and assumed that it was incorporated into DNA. Rubini 2£.El- (1960) describe half-times for loss of activity from the plasma for as long as 30 minutes. The assumption is that some semi-logarithmic function with time should characterize plasma disappearance kinetics. However, it appears that their data are better represented by a power function, as will be described later. The possible existence of an endogenous thymidine pool or substrate pool which could be labeled by the initial injection of R3-thymidine and thus feed label into replicating DNA over an extended period has been ignored by most investigators. There is some evidence in the literature which indicates that some DNA precursor pool may be labeled with a single injection of M3-thymidine and then feed into DNA for an extended period. The data from erythroid element proliferation in bone marrow is a case in point. Bypertransfused mice have been shown to have red cell production completely suppressed (Filmanowicz and Gurney, 1961), and the bone ‘marrow is almost entirely free of any erythroid cells (Schooley and Garcia, 1962). When erythropoietin is given to such animals, a wave 10 of erythropoiesis follows, as seen both from the appearance of nucleated erythroid cells in the marrow and from the uptake of radioactive iron in hemoglobin. When Schooley and Garcia (1962) and Gurney (1962) injected H3-thymidine into hypertransfused mice, and 6 hours later gave erythropoietin, they found labeled erythroid precursor cells in the first wave of erythrOpoiesis initiated. Since the stem cell would not have been stimulated to divide until 6 hours after the injection of H3-thymidine, either the stem cell must have been in active proliferation, or there was a labeled substrate pool capable of feeding labeled precursor into DNA. Cronkite 55 31. (1959b) and Quastler (see Lajtha, 1963) present flow diagrams that include the possibility of such a pre-DNA pool. Figure 12 (in the Discussion section) depicts a possible flow of H3-thymidine in the bone marrow. Presumably, this pattern would be similar for all cell renewal systems (e.g., gut, skin, gonads). A thorough evaluation of the time during which H3-thymidine and its breakdown products are available for incorporation during DNA synthesis cannot be found in the literature. This thesis will specifically evaluate these aspects for the case of mouse femur bone marrow cells. In the bone marrow, there are at least two cell renewal systems (myeloid and erythroid). Each is in a steady state, normally. Consequently, total DNA will remain constant 11 in the cases under discussion. Tritium label, however, will change with time, thus providing a true tracer analysis of the over-all DNA kinetics. General Scheme for Bone Marrow Cell Deoxyribonucleic Acid Turnovgg Any H3-thymidine activity incorporated into erythroid DNA should be lost when the maturing red blood cells lose their nuclei. The time for the loss of the red cell nuclei could be as short as 10 hours or as long as 20 hours after injection of labeled thymidine (Bond st 51., 1962). However, labeled orthochromatic normoblasts are seen as early as 3 hours (Bond 33 51., 1959). Therefore, if the nucleus is lost over the "life" of the orthochromatic normoblast population rather than abruptly (just before this labeled portion of the population becomes reticulocytes), the loss of labeled DNA could begin 3 hours after injection. Results of chemical determination of DNA activity after a single injection of H3-thymidine show that the activity incorporated into rat bone marrow DNA (i.e., DNA specific activity) does not change until granulocytes are lost to the blood at h8 hours (Bond g£,gl., 1962). Work by Steel (1962) confirms this observation. This implies either reutilization or some other feed-in of label to the bone marrow DNA since no loss in DNA specific activity is observed when red cell nuclei would have been lost. The fact that the activity does not drop in bone marrow DNA in rats is attributed to reutilization of label by Feinendegen £5 51. (196A). This is based on the assumption that 50 percent of the thymidine is retained in the animal as tritiated DNA. 12 In addition to the loss of bone marrow activity that can be predicted from red cell production, Patt and Maloney (1963), in a model for granulocyte production, show that there could be a substantial loss due to "ineffective" production. Autoradiographic analysis of bone marrow from dogs given tracer amounts of H3-thymidine indicates that about twice as many cells are produced by the proliferating granulocytes as appear in the later non-mitotic stages. Therefore, loss in DNA activity should occur due to loss of granulocyte precursor DNA as well as erythroid precursor DNA before DNA is lost to the blood when mature granulocytes leave the marrow. A simplified model of bone marrow cell production in terms of loss of DNA label after injection of a3-tbynidine would be as follows: 100 percent of DNA lost (Reduction of label due to loss of nuclei) Stem Proliferative Erythroid: Cell ‘-——-+> Cell DNA -—--+>>Nun-Proliferative DNA Stem Proliferative Non-Proliferati Entrance of Myeloid: Cell ———-> Cell DNA —-> ”e mm to blood Cell DNA -—-> DNA (Reduction of label) Approximately 50 percent loss in DNA (Reduction of label due to cell loss) 13 Objective of Thesis From this review of the literature, it is evident that workers using H3 -thymidine in giyg have considered it to be a "flash label" (i.e., a labeling period of about 1 hour) for tagging DNA in the study of proliferating cell systems. However, in ZEEEE studies using bone marrow cells show that there is an endogenous pool large enough to supply two times the thymidine necessary to replicate the DNA in these cultures (Rubini £5 31., 1962). The degree of erythroid labeling obtained in hypertransfused animals 6 hours after H3-thymidine injection (Schooley and Garcia, 1962; Gurney, 1962) can better be explained by a continuous feed-in of labeled precursor. In this work a comparison is made between the tritium activity incorporated into femur bone marrow cell DNA versus the tritium activity incorporated into the total femur following a single injection of H3 -thymidine. The analysis of the whole femur should yield information regarding tritium activity in excess of that incorporated into femur bone marrow cell DNA, since total non-volatile H3-thymidine activity can be analyzed. The far greater part of DNA of the femur is in the bone marrow; therefore, an analysis of femur activity should represent the activity in the femur bone marrow DNA. If these activities are not the same, then perhaps they can be resolved in the light of the above models and a labeled substrate pool feeding into DNA. In addition, studies have been 1h carried out in hypertransfused animals to obtain some information regarding the difference between erythrocyte and granulocyte production in mouse bone marrow, to assess the DNA turnover of these components. MATERIALS AND METHODS Animals Female Carworth Farms CF-l mice, or Argonne stock (ANL/CF-l) mice derived from Carworth Farms CF-l were used throughout. The animals were housed in air-conditioned quarters at 73 t 2 degrees Fahrenheit, h5 i 5 percent humidity and kept on a 12-hour light -- l2-hour dark schedule (6 AML- 6 PM). A standard commercial diet and water were available ad libitum. ‘Mice from outside the Laboratory were isolated for 10 days to be sure they were free from disease. Animals from the Argonne stock were checked routinely from the time of birth for any pathogens. Animals selected for weight and age (21 i 1.5 grams throughout the experiment; 6-8 weeks old) were caged 3 to a cage and left for several days before a particular experiment was begun. Sacrifice Animals were anesthetized with 0.25 milliliter of Nembutal (6 milligrams/milliliter) intraperitoneally and pinned to a cork board. An incision was made on the left side, beginning at the costal margin and continuing to the axilla. The brachial artery was then severed and the blood allowed to flow into the pocket formed by the reflected skin. The blood was collected in a heparinized tube for further analysis or discarded. After bleeding stopped, both femurs were removed and processed by one of the procedures indicated below. 15 16 2122.4 A sample of blood was taken for a hematocrit, then smears were made on all blood samples. These slides were fixed for five minutes in 100 percent methyl alcohol, then stained with Wright's stain for differential counts or saved for preparing autoradiographs. A sample of blood was taken and a leukocyte count done on an electronic cell counter. A Mgdgl_A_Coulter Electronic Cell Counter was used for both leukocyte counts and nucleated marrow cell counts. Usually a 1:500 dilution was used, although if there was an extremely low or high count, appropriate dilutions were made. A 0.5 percent solution of saponin was used to lyse erythrocytes. The procedure and accuracy in the use of the Coulter counter are discussed in several reviews (Coulter, 1956; Brecher, Schneiderman and Williams, 1956;'Mattern, Brackett and Olsen, 1957; Richar and Breakell, 1959). In an early experiment, blood was taken at various times 3 after an injection of H -thymidine and centrifuged to separate the plasma from the cells. A 0.1 milliliter sample of plasma was removed and the radioactivity determined as outlined below in the discussion of radioactivity counting. DNA Extraction The ends were snipped from the femur and the marrow expelled by positive pressure applied by forcing 0.5 milliliter of physiological saline through a 25 gauge needle. The marrow plug was flushed back and forth through the syringe several times to 17 break it up as much as possible. The cells were then washed twice with physiological saline and counted on the Coulter counter. The calibration and reliability of the Coulter counter for the bone marrow cells as counted in these experiments are given in Appendix I. After the aliquot of cells was removed from the femur for the total nucleated cell count, the rest of the cells were taken and the DNA extracted (see also Appendix II), using a modification of Schneider's method as follows (Schneider, l9h5): 1) Add 1 milliliter cold 10 percent trichloroacetic acid to cells, then centrifuge for 15 minutes at 2000 revolutions per minute (rpm) in a refrigerated centrifuge. 2) Decant supernatant. 3) Add 2 milliliters 5 percent trichloroacetic acid to precipitate (shake well). A) Heat in sand bath at 90° Centigrade for 30 minutes. 5) Cool and centrifuge at 2000 rpm for 10 minutes. 6) Transfer supernatant (DNA) to a 5 milliliter volumetric flask. 7) Rinse extraction tube and precipitate with 2 milliliters 5 percent trichloroacetic acid and add to volumetric flask. 8) ‘Make flask up to volume with 5 percent trichloroacetic acid. 9) Take 0.5 milliliter for diphenylamine reaction. 10) Take A milliliters for counting radioactivity. l8 Diphenylamine Reaction The diphenylamine reaction was done following Burton's‘Method (Burton, 1956) (see also Appendix III). All samples were run with a DNA standard to check the consistency of the reaction from day to day. 1) 3) To 0.5 milliliter sample add 1 milliliter diphenylamine reagent (1.5 grams diphenylamine, 100 milliliters acetic acid, 1.5 milliliters sulfuric acid) (at the time of use add 0.010 milliliter aqueous acetaldehyde - 16 milligrams/ milliliter - to each 20 milliliters of reagent). Mix well and incubate at 30° Centigrade for 20 hours. Read on Coleman Junior Spectrophotometer at 600 millimicrons. Radioactivity,Counting A Packard TriCarb Liquid Scintillation Counter was used to count the radioactivity in each sample (see also Appendix II). 1) 2) 3) The h milliliter aliquot was dried overnight in a vacuum dessicator. One milliliter of hymmine-IOX (1 molar solution of p-diisobutyl-cresoxyethoxyethyl dimethylbenzylammonium hydroxide in methanol) was added. The reaction was allowed to proceed for six hours. Fifteen milliliters of scintillator were added and mixed well with the sample. The scintillator consisted of h grams of 2,5 Diphenyloxazole (PPO) and 50 milligrams of l,h-bis 2 (hdmethyl-S-phenyloxazolyl) benzene (POPOP) in 790 milliliters of toluene and 250 milliliters of ethanol. 19 h) The samples were cooled overnight in the TriCarb counter, then counted. 5) An internal standard (0.025 milliliter of a standard tritiated toluene solution containing 2.3h x 106 disintegrations/minute/milliliter) was used to correct for quenching. 6) The activity in each sample was computed by comparison to a known standard (0.01 milliliter of the standard tritiated toluene solution used above in 15 milliliters of scintillator). Femur Combustion Femurs that were removed for combustion were cleaned of as much soft tissue as possible. The bones were then placed in 50 milliliters of physiological saline and left for several hours. The saline was then changed and the bones left in the fresh saline overnight. This was done to remove any non-volatile activity that may have been in the body fluids. The bones were then washed with distilled water and placed in an oven and dried for 2 days at 90° Centigrade to remove all volatile tritium. Combustion Technique A technique for the combustion of samples for tritium analysis has been developed from the Sch6niger analysis (Schaniger, 1955) (see also Appendix IV). The technique is as follows: 1) 2) 3) h) 5) 6) 7) 8) 9) 20 The dried bones are wrapped in black paper and placed in a platinum basket (see Figure l). A heavy walled two liter Erlenmeyer flask is flushed with pure, dry oxygen. The basket containing the sample is hung on the end of the funnel and placed in the flask. A lamp is then used to ignite the black paper. The sample is thus combusted to water and carbon dioxide. The flask is removed to dry ice and left for 1 hour. The flask is then removed to wet ice for 30 minutes. Thirty-six milliliters of scintillator are added through the funnel and mixed well with the sample (tritiated water). Two 15 milliliter aliquots are then taken for counting in the TriCarb counter. Tritiated Thymidine Tritiated thymidine was obtained from Schwartz BioResearch, Inc. with a specific activity of 6 curies/millimole, and activity of 1 millicurie/milliliter. Further dilutions were made in sterile saline, so that the final activity was 50 microcuries/milliliter. The mice were injected with 0.5 microcurie per gram of body weight via the tail vein. 21 FIGURE 1. caiwsnoN PLAsx ARRANGED FOR IGNITION. THE INSERT (UPPER LEFT) snows THE PLATINUM BASKET, sLAcx PAPER AND SAMPLE BEFORE WRAPPING AND PLACING AT THE END or THE FUNNEL. 22 Hypertransfusion The mice were made polycythemic by the method described by Jacobson, Goldwasser and Gurney (1960). Old CF-l or ANL/CF-l male or female mice were sacrificed as described for the experimental animals. The blood collected from these was centrifuged at 3000 rpm for 15 minutes. The plasma and buffy coat were removed. The red cells remaining were washed three times with physiological saline. After the last wash, the cells were adjusted to a hematocrit of 90 percent. The recipient mice were injected intraperitoneally with 0.5 milliliter fresh cells twice a day for two days. On the fourth day after the initial injection, the animals received two more 0.5 milliliter injections. On the sixth day after the initial injection, all the animals had hematocrits of 70 to 80 percent and the experiment was begun. Autoradiggraphy Autoradiographs of blood smears were prepared by placing Kodak AR-lO stripping film on the slides and exposing for three 'weeks. The film‘was deve10ped with Kodak-D-19 developer and fixed ‘with Kodak Acid Fixer (Pelc, 1956). After washing and drying the cells were stained with a modified Giemsa Stain (Gude, 1955). RESULTS Time for Maximum Uptake of Tritiated Thymidine Activity into Femur Deoxyribonucleic Acid Two experiments were done to test the rapidity of thymidine uptake into femur DNA. The first experiment was similar to an experiment reported by Rubini £5 51. (1960). Rubini and co-workers measured the plasma clearance of non-volatile tritium activity in humans. When they analyzed their data as a semi-logarithmic function, they obtained a three component curve. The first two components had half-times of 0.2 and 1 minute respectively. The third component had a half-time of 30 minutes. These workers pointed out that the rapid disappearance of plasma activity could not be correlated with any anatomical or biochemical compartment such as degradation in the liver or uptake by proliferating cell systems and that at 1 minute after injection the plasma concentration corresponded to a dilution as large as the total body water. The plasma clearance data obtained in the present work, as well as Rubini's, are better represented by a power function than by an exponential function. Figure 2 presents a log-log plot of the results of the present experiments. In these experiments, the plasma activity decreases from about 0.15 microcuries/milliliter at 15 minutes to about 0.010 microcuries/milliliter at h hours, indicating that loss occurs very rapidly from the plasma, presumably to cells in DNA synthesis and other sinks for thymidine accumulation or breakdown. 23 tic/ml PLASMA 1.0 0.50' 0.1+O 0.30 0.25 0.20 0.05 -- 0.01.4 0.03“- 0.025" 0.020 0.015 0.010 21+" . 0.1 0.25 0.5 1 . h TIME - HOURS FIGURE 2. CLEARANCE OF H3-THYMIDINE AND NON-VOLATILE BREAKDOWN PRODUCTS OF H3-THYMIDINE Pam HOUSE BLOOD PLASMA. 25 The second experiment to determine the rapidity of uptake of H3 -thymidine in femur bone marrow DNA and whole femur consisted of an injection of either saline or cold thymidine 15 minutes following the H3-thymidine injection. The protocol for this experiment was the same as outlined in the Materials and.Methods, except an injection of 0.2 milliliter of physiological saline or 50 micrograms of cold thymidine per gram of body weight was given 15 minutes after the 3-thymidine. Figures 3 and A show the results of injection of H these experiments. There is only a slight increase in activity in the femur and in femur bone marrow DNA between 30 minutes and 1 hour, and in addition, the activity in femur bone marrow DNA only decreased to about 80 percent of the normal after the injection of 1000 micrograms of cold thymidIne at 15 minutes. Therefore, most of the labeling must have occurred before 15 minutes. After maximum labeling is achieved, the activity in femur bone marrow DNA of cold thymidine injected animals does not drop. This indicates that the constant activity in femur bone marrow DNA in the normal mouse is not due to a feed-in of H3-thymidine from the plasma, since plasma tritium is rapidly falling. The total uptake on the basis of the combusted femur is about 0.0h microcurie/femur. At A hours, it would take approximately 3 milliliters of plasma (0.012 microcurie/milliliter) to be equivalent to the activity concentrated in the femur (0.036 microcurie/femur). The loss in plasma activity does not parallel the loss of activity from the femur; the plasma clearance of tritium being much more rapid. 26 80- . : A f .5, . 0 "“*--------"“-x I . 1 W \D I ' 0 .4 K as. “°"' a s “of ' . D . ’0’ SALINE :i j . a . - ’ ‘ .«X’ COLD THYMIDINE I —'1——'l'——"l. . - . 0 1 2 ‘3' , . h . - TIME ~HOURS FIGURE 3. FEMUR BONE MARROW DNA ACTIVITY AS A FUNCTION OF TIME. R3- THYMIDINE WAS INJECTED AT TIME 0 AND SALINE OR COLD TNTMIDINE WAS INJECTED AT 15 MINUTES. . 0.060 T 0.01.0 -' E In \ U :1 0.020 - ,o’ SALINE , ,x/ COLDTRINIDINE ’ , . 0 l . 2 3 l. ' TIME-HOURS FIGURE h. WHOLE FEMUR ACTIVITY AS A FUNCTION OF TIME. H3-THYMIDINE WAS INJECTED AT TIME 0 AND SALINE OR COLD THYMIDINE WAS INJECTED AT 15 MINUTES. 7 ‘1'“: -_- .. . c. .- ., .. __ . .. .-..-.—”—.g~mn*m—o.—O’—--o--—. -auo' ._-. v--- 27 The rapid plasma clearance of non-volatile tritium activity and the subsequent sustained level of tritium activity in the femur and in femur bone marrow DNA indicate that thymidine is incorporated very rapidly. Activity Incorporateg_into Femuerone‘Harrow Deoxyribonucleic Acid The activity incorporated into mouse bone marrow DNA was investigated as a function of time. Several individual experiments were run on normal animals. Figure 5 shows the activity in the bone marrow DNA in one femur with time up to 168 hours after H3-thymidine injection. The activity incorporated in femur bone marrow DNA remains essentially constant for about 18 hours (intercept 69.h x 10.6 microcurie/microgram_ DNA -- slope does not differ significantly from zero -- Slope - 0.00508 i 0.00559). (Table II--Page h3--gives:a summary of all pertinent values and their statistics.) Between 18 and 2h hours, additional radioactive DNA is added to the DNA pool in the femoral marrow. The added radioactive DNA must have a much higher activity than*was there previously. This is evident when the points after 2% hours are considered. The extrapolated zero time intercept based on these points is 193.7 x 10"6 microcurie/mdcrogram DNA and the slope is -0.01976 i 0.00190. The twenty-four hour activity is 120.6 x 10-6 mdcrocurie/microgram DNA which is 51.2 x 10"6 microcurie/microgram DNA greater than the previous plateau. This represents an increase in activity of at least 73 percent under steady state conditions 28 oszoAAOh NEH ho 20522 < m< 5.5 30522 mzom ”5:“: 2H Egg”; «SH—.53. mo ZOHHEZMUZOU .mzHQHZVFH m: we onH9H>HHU< <20 duo 3053.2 M28 mafia . w 55on On hoot moo. .3. . Ema/5“ no. :0. mo. we. we. GO. A. 36 > xwoaomm< mom I Haoo\¢zn m: o IOH x m.m magma .x. . . . . . o>auouomwaoum ooH x nm.o : m m m ooH x mm m m mm A SH -coz.cum . . . . . . o>auouomaaoum 00H x mm H H HH A m 00H x oH o m an H mH woe ooH x co.» m.Ho o.om ooH x on.@ o.me m.nm musooeaeau ooH R em.H m.nH H.m ooH R m>.H o.mH m.m aHom 00H x m:.o m.>m H.mH ooH x mo.n o.>m o.m comm on x mm.» H.0w >.Hm 00H x Hm.n m.:n ©.HH ouhoofioaeouoz 00H x co.» m.Ho 0.0m 00H x ~:.n m.on n.oH mumoonAz 00H x om.H H.HH e.n ooH x so.o >.m m.H mumuoHoaaoum ooH x mm.o m.» o.m ooH x ::.o m.n n.H ueeHnonxz HeoH x :.n HeoH R :.n u Houoav Ammoon u aauoav AHouos fly u Houoav Awnoon u HouOHv AHmuOH fiv «HHuo mHHoo ease HHmo wqxumnauz azn m1 HuHuaOAOMMHa «asumeasz azn m1 HaHuamuOmmHn consumauuuuumfiw, HQSHOZ moH2_Qmm=mmzIV Kazan onzz mmh zH OMB xOmmoozH >HH>HHU< ZDHHHMH mane: i MZHH oeH ooH omH . oaH omH omH oHH . ooH om om ow om cm 0: cm cm H . _ b . _ _ P; _ P! P. p P _ . _ _ _ _ 1 ease? $88.? .0 eexum\o1 a wmdo.o I r ll 0 u I amazed-.. 8.8 o a O / .m mmson Os 8. no. mo. Fo.. sauna/afl' 39 (0.058% microcurie/femur -- Figure 7), and the femur activity in the normal animal estimated from DNA activity (0.0581 microcurie/ femur -- Figure 8). It is reasonable for the initial and final activities to be the same only if the DNA pool size is constant, and the numbers of proliferative cells are the same. From Table I, one can see that the overall percentages of cells in the proliferative compartment (red proliferative, myeloblast, promyelocyte, and myelocyte) are not significantly changed between nommal and hypertransfused animals (31.6 percent vs. 30.8 percent). When the points after 2h hours are considered, there is a significant difference in slopes of Figure 7 (normal mice) and Figure 9 (hypertransfused mice). Because of this, the calculated output of femur bone marrow DNA in hypertransfused animals is about 2/3 that of the normal animals (Table II). Since the granulocyte compartment in the hypertransfused animal is twice that of the normal animal (Table I), the DNA output of the hypertransfused animals represents 2 times the normal granulocyte DNA output plus the lymphocyte DNA; whereas the DNA output in the normal animal represents the DNA output of granulocytes, lymphocytes and red cells. Therefore, the slower output in the hypertransfused animal means that the DNA output in the normal animal must be much greater than the DNA output from the granulocytes in the normal animal, since the pool sizes appear to be equal. This is true only if the cell cycle time, the number of cell divisions, the extent of cell loss occurring in the granulocyte compartment, and the 1.0 turnover of other DNA components (e.g., lymphocytes) remain constant. This point will be considered in more detail in the Discussion. Granulocyte and Lymphocyte Appearance in the Peripheral Blood Autoradiographs were prepared on slides of peripheral blood from both control and hypertransfused animals. Figure 10 shows the mean grain count per cell for all granulocytes (labeled and unlabeled) and Figure 11 shows the mean grain count per cell for all lymphocytes (labeled and unlabeled). These results are presented as mean grain count per cell rather than percent labeled to emphasize the difference in activity per cell between lymphocytes and granulocytes. The early loss in femur activity (Figures 7 and 9), is not due to the loss of granulocytes to the blood in either normal or hypertransfused mice since labeled granulocytes do not appear in the peripheral blood until 2h hours. This work confirms the work of Bryant and. Kelly (1958) who used carbon-1h labeled adenine to study the appearance of lymphocytes and granulocytes in the peripheral blood. Summary of Pertinent Data and Their Statistics Table II presents a summary of the intercepts and their errors, the slopes and their errors, the rate constants, the half-times, and the output in micrograms of DNA per hour from the activity in femur bone marrow DNA and femur activity in each experiment at the times indicated in the Results. hl .MZHQHEEIMS .wo ZOHHUMhZH ”“50sz < zm>HU mom: ommammzéfimmmym Qz< .2502 be 98.5 3mmmHmmm WEB 2H OZHAmm<=H x. aumEmzéammEE \X\ em \ "d , .. £6 86 mm.o 1133/ 1.11000 KING WEN 1&2 . mzHaHEnmm mo, 2030”."th NAOZHm < zm>HG HUHZ ommbmmghmmg: 92¢ A5202 no 90015 ngummm HE 2H OzHAmmmmuueemnmw 80 0H e - e 8 g 01 o e 38 0 2.08 m a 090 0 :32 venom-ssuuuumhu Auooa\¢zn mav Aaueuooav unseen use Anusonv wouwm ouaocwum uouum pudendum H usauno «2: ends mam: H omega umoououan an umouuuusH Aeeeeeeeouv HH money “5 DISCUSSION The principal events occurring between an intravenous injection of H3-thymidine and the Observation of labeled bone marrow DNA in the femur are shown in Figure 12. During the period of availability, all cells in DNA synthesis are capable of incorporating labeled nucleoside. In addition, it may be possible for the labeled precursor to be shunted into a storage pool to be used later in DNA formation. Since the DNA pool size is constant, DNA incorporated activity can be altered only by DNA turnover (cell turnOver), feed-in from a labeled substrate pool, or reutilization of labeled breakdown products of DNA. 3 In these experiments an examination has been made of H -thymidine incorporation into a tissue which is known to be undergoing rapid DNA production. From the description of cell renewal systems, and of bone marrow cell proliferation in particular, outlined in the Introduction, it is possible to make certain generalizations about tritium exchange in the bone marrow DNA pool of the femur after initial labeling of DNA is complete: Input - 1) Tritium labeled DNA from the stem cell (if the stem cell is outside femur). The stem cell input is considered to be low (Patt and Maloney, 1963). 2) Tritium labeled DNA from lymphocyte turnover. 3) Tritium labeled DNA from unknown sources (reutilization). h) Tritium labeled substrate from storage pool. 1+6 ADMINISTRATION OF PRECURSOR COMPETITIVE SINKS . COMPETITIVE SOURCES ENOOCENOUS PRECURSORS ELIMINATION P* IN BLOOD . STREAM BREAKDOWN IN LIVER' CATAEOLISN P* 1" CELLS 3* IN CELL ’I 1” I ,n S IN ’ .4‘ STORAGE , . — - 7 w ‘ e L IN NEW REUTILIZATION DNA . - ‘ ! iv 4r Loss TO PERIPHERAL BLOOD ATTRITION NUCLEI LOSS OBSERVATION «r - a» A P - LABELED I‘HYHIDINE S - LABELED SUBSTRATE L* {LABELED DNA FIGURE 12. THE MOVEMENT OP LABELED TRITIUM C(MPOUNDS IN THE FEMUR BONE MARROW CELLS . AFTER A SINGLE INJECTION 0E TRITIATED THYHIDINE. TRIS DIAGRM COULD APPLY EQUALLY WELL TO OTHER CELL RENEWAL SYSTEMS. - 1+7 Output = 1) Tritium labeled DNA from possible granulocyte attrition. 2) Tritium labeled DNA from erythrocyte nuclei loss. 3) Tritium labeled DNA from lymphocyte turnover. h) Tritium labeled DNA from granulocyte loss to the blood. Analysis of femur DNA will also reflect DNA activity in such cells as megakaryocytes and osteoblasts. The change in DNA activity from these sources is assumed to be low in comparison to the changes in DNA activity occurring in the erythroid and myeloid compartments of the femur . Difference in Femur Bone‘Marrgw Deoxyribonucleic Acid Activity and Femur Activity The data obtained from femur bone marrow DNA activity (Figure 8) and femur activity (Figure 7) are different during the first 18 hours in that the femur bone marrow DNA activity remains constant (0.0208 microcurie/femur) while the femur activity drops by about 30 percent of its initial value (0.0h58 microcurie/femur to 0.0317 microcurie/femur). Since the bone marrow cell pOpulation represents a cell renewal system, the DNA incorporated activity should decrease with time, unless there is input of activity. It is not possible to explain the constant activity in femur bone marrow DNA over the first 18 hours on the basis of a long initial period of availability of H3-thymidine in the blood. h8 Reutilization of breakdown products of femur bone marrow DNA might possibly account for the constant activity in femur bone marrow DNA during the first 18 hours. However, on the basis of the uptake in the femur (0.0h58 microcurie /femur) the specific activity of the material injected (6 curies/millimole) is diluted about 500 times. [There are 63 micrograms of bone marrow thymidine per femur (300 micrograms DNA/femur x 0.21 microgram thymidine/microgram DNA), of which about 1/3 are in proliferative cells (Table I). When DNA synthesis occurs, 1/2 of the new DNA contains new thymidine. The total thymidine incorporated into the femur along with the 0.0h58 microcurie by this calculation would be 10.5 micrograms. The Specific activity of the incorporated material would then be 0.00h6 microcurie/microgram of thymidine, as compared to the 6 curie/2h3 milligram of thymidine injected;] This is only an estimate, but there is evidently a great dilution of the injected material. Since labeled lymphocytes are seen in the peripheral blood at early times, and lymphocytes represent about 25 percent of the total femur marrow (Table I) it is possible that labeled lymphocytes could be preferentially taken up and thus keep the activity in femur bone marrow DNA constant. A recent report by Osmond and Everett (196A) indicates that this is unlikely. They show in mice, that 3 after a single injection of H -thymidine,the bone marrow lymphocyte labeling index increases to about no percent in 3 days. If, however, they occlude circulation to one hind limb and administer H3-thymidine, then restore circulation 20 minutes later, no labeled lymphocytes ’49 accumulate in the occluded limb. Labeling, however, proceeds in the normal fashion in the control limb. They maintain that no pathological changes took place in the limb which had its circulation occluded since the distribution of lymphocytes between the normal and control limbsremainsthe same throughout the experimental period. These authors conclude that lymphocytes are not entering the bone marrow from the peripheral blood, and raise the question of intramedullary lymphocytopoiesis. If there is intramedullary lymphocytopoiesis, then the possibility of there being a labeled substrate pool at the tissue level arises since the labeling index continues to increase for 3 days after the injection of H3-thymidine. A long availability time of labeled precursor in the blood, reutilization of labeled breakdown production from femur bone marrow DNA and other sites of cell renewal, or a labeled lymphocyte feed-in to the marrow do not seem to explain the constant level in femur bone marrow DNA activity while the femur activity drops. If any one of these alternatives was accepted to explain the constant femur bone marrow DNA activity, it would imply that the observed drop in femur activity is due to non-volatile tritium clearance. It was pointed out in the Results that the drop in femur activity was not due to the plasma clearance. Since the activity in femur bone marrow DNA should drOp with time, then it is necessary to examine the femur drop for the possibility of this being, in fact, a drop in femur bone marrow DNA activity. 50 If the initial uptake of label into DNA was distributed prOportionally between erythrocyte and granulocyte proliferative cells, then the red cell precursors would have 57 percent of the label or 0.0119 microcurie/femur and the granulocyte precursors would have h3 percent of the label or 0.0089 microcurie/femur (see Table I). The rate of loss of label due to the loss of the red cell nucleus can be evaluated in the following way: Assumptions: Number of circulating red blood cells - 8 x 109 cells/milliliter Blood volume of a mouse - 7 percent of body weight Life span of a mouse red blood cell - h2 days Single femur marrow volume - 5 percent of total marrow volume (LoBue, 1963) Therefore, the total number of red blood cells produced by the bone marrow of one femur in a 20 gram mouse is: _‘(8 x 102) (0.07)3(2Q) (h27’(2fi) (20) a 0.55 x IO6 cells/hour/femur. There are 12 x 106 nucleated erythrocyte precursors per femur (see Table 1). Therefore, the turnover constant for the red cell precursors would be: 0.55 x 195 12 x 10D - 0.0h6/hour. (Half-Time - 15 hours). This should also be the rate at which the label is being lost. Therefore, in 18 hours, 0.0067 microcurie/femur will be lost. (0.0119 - 0.0119e('°‘°h6) (18)). 51 The loss in femur activity in the hypertransfused animal in 18 hours was 25.6 percent (Figure 9). This loss should be due to granulocyte attrition, since mature granulocytes were not lost to the blood until 2h hours (Figure 10), and red cell production was entirely suppressed. If the uptake in hypertransfused mouse femur bone marrow DNA is the same as in the normal animal (0.0208 microcurie/ femur), then the total loss from bone marrow DNA would be 0.0089 microcurie in 18 hours: (0.0h58 x 0.256 - 0.0119 microcurie/femur and 0.0208 - 0.0119 - 0.0089 microcurie/femur). As pointed out in the Results, the loss due to granulocyte attrition in the hypertransfused animal is about twice that of the normal animal. Therefore, the total loss due to granulocyte attrition in the normal animal would be 0.00hh microcurie/femur (0.0089/2). The total loss in 18 hours in the normal animal would be: Loss of red cell nucleus - 0.0067 microcurie/femur Loss of granulocytes - 0.00hh microcurie/femur Total activity lost - 0.0111 microcurie/femur. This is a total loss in femur activity in 18 hours of 2% percent. The observed loss was 0.01h1 microcurie/femur or 31 percent (Figure 7). It is important to estimate the time of expected loss in femur bone marrow DNA activity. Labeled mitoses have been observed as early as 1/2 hour after an injection of H3-thymidine (Patt, 1959), thus in the case of attrition, the loss of labeled cells could presumably occur at any time after the completion of cell division. 52 The time to enuclestion of non-proliferative erythrocytic precursors is about 10 hours (estimated from red cell production of 0.55 x 106 cells/hour/femur and the number of non-proliferative red cell 6 cells/femur - Table 1). The loss of precursors of 5.86 x 10 nuclear material from erythrocyte precursors may be a gradual process extending over the life of the orthochromatic normoblast, rather than an abrupt loss of the nucleus. This would mean that the loss of activity from DNA would be a gradual process that might begin after the last cell division. A calculation of this type should be considered only a first approximation, since many assumptions are made which may be in error. It does serve to indicate that an early loss in femur bone marrow DNA activity is not an unexpected phenomenon. In view of the preceding discussion, and the extended labeling period of the bone marrow lymphocyte reported by Osmond and Everett, and the labeling of nucleated erythroid precursors in erythropoietin stimulated hypertransfused mice reported by Schooley and Giger (1962) and Gurney (1962) it seems that consideration should be given to examining the possibility of an extended labeling period of DNA 3 after a single injection of H ~thymidine. Consideration of a "Pre-DNA" Pool Feeding;Labeled Substrate into Femur Bone'Narrow Deoxyribonucleic Acid It is possible to reconcile the differences between femur bone ‘marrow DNA activity and femur activity during the first 18 hours if a pool feeding labeled substrate into DNA is postulated. A pool of this sort is represented by the difference between the activity 53 in femur bone marrow DNA from 1-18 hours and the total activity in the femur between 1-18 hours. The DNA activity before 18 hours is 0.0208 microcurie/femur (Figure 8) and the femur activity is 0.0h58 microcurie/femur (Figure 7). This difference is represented by: Non-DNA activity - Total activity - DNA activity -0.02107t _ 0.0208 e0.00508: - 0.0h58 e At time 0 an extrapolated value for the total non-DNA activity assumed to represent "Pre-DNA activity" would be 0.0250 microcurie] * femur. This would be equivalent to 129|Ig of "pre-DNA units" (0.0250 microcurie/femur ) l93:h x 10'5 microcurie [microgram DNA ‘ A se“1'108 plot Of the difference between total femur activity and femur bone marrow DNA activity with time is essentially linear for 18 hours. The half-time of the "pre-DNA pool" is 15 hours and its rate constant 0.0h62/hour. The output of "pre-DNA units" then is 5.9 micrograms/hour and the output in femur bone marrow DNA is 5.9 micrograms DNA/hour (see Table 11). Although these outputs are the same, these values should be considered to have at least a 10 percent error (5.h to 6.5 micrograma/ hour on the basis of the femur bone marrow DNA slope after 2h hours). If these outputs are equal, the activity in femur bone marrow DNA will remain constant as long as this pool (pre-DNA pool) supplies labeled precursor to DNA. It should be pointed out that the output of the pre-DNA pool has been determined independently of the DNA output, (i.e., the figures for the determination of the pre-DNA pool output were not used in the determination of the DNA output). A model of total femur activity, and-the pre-DNA pool activity, along with the inflows and outflows of other contributions to the activity are shown in Figure 13. * Units giving rise to 129 micrograms of DNA. .5“ 0000200 0.. «a msaoum E85250 <20 000003 2000.000. 00 E0055- 0 . <20 000.53%. 200.200.5000 02300.3 .203? . x. . z . , x I k \ I \\ - \ I; \ I \ I \ I Ilkf .58 0. an . 20 <20: m m 220 we 82 500.0000 m: 9...: <20 30%: 0000 - .0020 420-53.. _ . . . . 0 mean 500: .man—ngmm mo ZOHHUNHZH magnum < 9330498 30% N28 ME mun. zH nag Esau—mu. ho H.250: ”E. mom AMA—OS < .mm Eva 55 Evaluation of Change in Activity_in Bone Marrow Deoxyribonucleic AciggBetween l8 and 2h Hours The increase in activity in the marrow between 18 and 2% hours is of considerable interest. A similar riSe in DNA activity has been reported for gut and spleen DNA (deLesdain and Pacletti, 1962). Experiments on reutilization of tritium activity in demand situations (i.e., tumor growth and liver regeneration) performed by Bryant (1962) and Rieke (1962) suggest the lymphocyte as a carrier of DNA for the synthesis of new precursors for DNA. However, Osmond and Everett's data (l96h) show that only a few labeled cells (1/100) appear in marrow of a femur that has had its blood supply occluded during the availability period of H3-thymidine. Bond E£.El° (l96h) describe similar results in an experiment using rats in which labeled cells are collected from a donor and transfused into an unlabeled recipient. Evidently, there is no accumulation of labeled cells in the marrow, and the few labeled cells found represent the normal flux of leukocytes into the tissue from the circulating blood. Therefore, even though the mean grain count per cell is higher in lymphocytes than in the granulocytes in the peripheral blood (Figures 10 and 11) it is unlikely that this difference is responsible for the rise in activity at 18 to 2h hours. However, the implication is that there is preformed DNA activity entering the femur bone marrow DNA activity from outside the femur since the femur DNA pool size does not change. In addition, this activity is entering only the DNA pool and not the pre-DNA pool. This is so because the activity in DNA and femur are not significantly different at 2% hours. 56 Estimation of the Relative Contributions of Granuloid, Erythroid and Lymphoid Cells to the Deoxyribonucleic Acid Turnover in the Normal House It is of interest to consider the differences in normal and hypertransfused mice in more detail. It was pointed out that the DNA output in the hypertransfused mouse was 2/3 of the output in the normal mouse (Figures 7 and 9). Since the relative granulocyte distribution count in the hypertransfused mouse has been increased by a factor of two, the granulocyte output in the hypertransfused mouse must be about twice the output of the normal animal (Table I). From these data, it is possible to write two simultaneous equations in three unknowns. For the Normal Animal: R-+ C + L - 5.9 micrograms/hour/femur (Pool size x rate constant - output or 300 micrograms DNA/femur x 0.019/hour - 5.9 micrograms/hour/femur) For the Hypertransfused Animal: 20 + L - 3.06 micrograms DNA/hour] femur (300 micrograms DNA/femur x 0.0102l/hour 8 3.06 micrograms DNA/hour/femur) Where R - Red Cell DNA Output G - Granulocyte DNA Output L - Lymphocyte DNA Output. These equations can be put in terms of the DNA output for red cells and the DNA output for granulocytes. R - h.35 - 1/2 L G = 1.5 - 1/2 L 57 If one takes Osmond and Everett's figure (196h) for the average time spent by the lymphocyte in the bone marrow of 72 hours, then the rate constant for lymphocyte DNA output would be 0.01h/hour. Since the total DNA pool in the marrow due to the lymphocyte is about 75 micrograms/femur, the turnover of lymphocyte DNA can be calculated to be 1 microgram/hour. If this value is substituted into the above equation, the red cell DNA output becomes 3.85 micrograms/hour/femur, and the granulocyte DNA output 1.05 micrograms/ hour/femur. The figure presented earlier in the Discussion for red cell production (0.55 x 106 cells/hour/femur) can be put in terms of DNA output using 8.9 x 10-6 microgram DNA/cell -- See Appendix V. This value then becomes h.9 micrograms DNA/hour/femur. It is evident that the erythroid population is the largest contributor to the total DNA turnover on the basis of these data. One can make this estimate for the rat using Steels' data (rate constant 0.02/hour) and Donohue's figure for the total nucleated cell count in the rat (17 x 109 cells/kilogram) (Donahue £5 31., 1958). If there are 8.9 x 10.6 microgram. DNA/cell, the DNA output due to the total bone marrow is 3000 micrograms DNA/hour/kilogram. The red cell production rate is 290 x106 celISIhour/kilogram (Patt, 1957), therefore, DNA output due to red cells would be 2500 micrograms DNA/hour/kilogram. I This arithmetic gives only a rough approximation of DNA turnover of the individual components that make up the bone marrow. The turnover of each component in relation to the other in this instance 58 has been made on the basis of its cellular distribution. Therefore, specific information on cell loss or recycling cannot be calculated from these data. If one knew the activity incorporated into each cell type an evaluation of its turnover could be made. It would be possible to do this if one had autoradiographs of the bone marrow. A quantitative evaluation of the relative amount of thymidine incorporated into each component could then be made. SUMMARY AND CONCLUSIONS A comparison between the uptake of H3-thymidine into mouse femur bone marrow DNA and into whole mouse femur has been made. In addition, certain other physiological effects were observed with the following observations and conclusions. 1. H3-thymidine and its breakdown products are distributed throughout a volume as large as the total body water in not more than three minutes. H3-thymidine and its breakdown products are cleared from the plasma very rapidly and with a time course described by a power function rather than by a simple exponential function. The maximum initial uptake from the plasma is attained in 1 hour in both the whole femur and in femur bone marrow DNA. By 1 hour, the plasma activity is much lower than either the whole femur or the femur bone marrow DNA activity. After the maximum uptake is reached, the activity in femur bone marrow DNA remains constant through 18 hours, while that in the whole femur begins to drop. The rate of loss from the whole femur is much slower than the rate Of loss from the plasma. 2. An examination of hypertransfused mouse whole femur activity shows a slower rate of loss of activity after 2h hours than is observed in the normal femur. A first approximation shows that by far the greatest contribution to DNA turnover in the normal mouse femur bone marrow is made by erythroid DNA turnover. 59 60 3. The amount of DNA per femur was determined to be 300 7 micrograms per femur (3.h x 10 cells per femur). h. A comparison between this work and other published data shows that with the same total dose (0.5 microcuries per gram body weight), the percentage uptake of activity by the femur is independent of the specific activity injected, within the range of 1.9 to 6.0 curies/ millimole thymidine. A large endogenous pool must be present, which dilutes the activity so that a tracer experiment is obtained over a wide range of specific activities. 5. The activity in femur bone marrow DNA remains constant for 18 hours, then between 18 and 2h hours increases to at least twice its pre-l8-hour value. After 2h hours, a decrease in femur bone marrow DNA activity is observed, whose rate is not significantly different from the loss of whole femur activity during the first 18 hours. 6. The whole femur loses activity for the first 18 hours, then between 18 and 2h hours a rise of about 1.3 times the 18-hour value is observed. The amount of activity in the whole femur decreases at about the same rate as the pre-18-hour value. These rates are not significantly different from the rate of loss in activity observed in the femur bone marrow DNA after 2% hours. 7. The differences between the activity incorporated into DNA and its change over the first 18 hours, and the whole femur and its change in activity over the first 18 hours are resolved by postulating a labeled substrate pool for DNA. This pre-DNA pool 61 is labeled at the time of the initial injection. The same amount of activity enters the DNA substrate pool as entered the cells which are in DNA synthesis. The labeled substrate pool is of sufficient size such that it feeds label into DNA for about 18 hours. 8. The activity which enters both femur bone marrow DNA and the whole femur between 18 and 2h hours is in DNA.pg£ gs, and not in the labeled substrate or precursor pool mentioned above. 62 REFERENCES Alvarez, L. W., and R. Cornog. 1939. Cyclotron Bombardment of Deuterium with Deuterons. Phys. Rev. 26: 613-61h. Brecher, G., M. A. Schneiderman, and G. 2. Williams. 1956. Evaluation of Electronic Red Blood Cell Counter. Amer. J. Clin. Path. 26: lh39-lhh9. Bond, V. P., L. E. Feinendegen, and E. P. Cronkite. 1962. Stability of RNA and DNA in Bone Marrow Cells Demonstrated with Tritiated Cytidine and Thymidine. In: Tritium in_the_Physical and Biological Sciences. Vol. II. International Atomic Energy Agency, Vienna. pp. 277-289. Bond, V. P., L. E. Feinendegen, E. Heinze, and H. Cottier. l96h. Distribution of Transfused Tritiated Cytidine--Labeled Leukocytes and Red Cells in the Bone Marrow of Normal and Irradiated Rats. Ann. N. Y. Acad. Sci. ll}: 1009-1019. Bond, V. P., T.?M. Fliedner, E. P. Cronkite, J. R. Rubini, and J. S. Robertson. 1959. Cell Turnover in Blood and Blood-Forming Tissues Studied with Tritiated Thymidine. In: The Kinetics of Cellular Proliferation. Editor: F. Stohlman. New York: Grune and Stratton. pp. 188-200. Bryant, B. J. 1962. Reutilization of Leukocyte DNA by Cells of Regenerating Liver. Exp. Cell. Res. _21: 70-79. Bryant, B. J., and L. 8. Kelly. 1958. Autoradiographic Studies of Leukocyte Formation. Proc. Soc. Exp. Biol. Med. 99: 681-68h. 63 Burton, K. 1956. A Study of the Conditions and Mechanism of the Diphenylamine Reaction for the Colorimetric Estimation of Deoxyribonucleic Acid. Biochem. J. 62: 315-323. Coulter, W. H. 1956. High Speed Automatic Blood Cell Counter and Cell Size Analyzer. Proc. Nat. Elec. Conf. 1g: 103h-10h2. Cronkite, E. P., V. P. Bond, T.‘M4 Fliedner, and J. R. Rubini. 1959a. The Use of Tritiated Thymidine in the Study of DNA Synthesis and Cell Turnover in Hemopoietic Tissues. Lab. Invest. 8: 263-275. Cronkite, E. P., V. P. Bond, T. M. Fliedner, and S. A. Killmann. 1960. The Use of Tritiated Thymidine in the Study Of Haemopoietic Cell Proliferation. In: Haemopoiesis. Editor: G. E. W. Wolstenholme and N. O. Connor. London:Churchill. pp. 70-92. Cronkite, E. P., T.‘M. Fliedner, V. P. Bond, J. R. Rubini, G. Brecher, and H. Quastler. 1959b. Dynamics Of Hemopoietic Proliferation in Man and Mice Studied by H3-Thymidine Incorporation into DNA. Ann. N. Y. Acad. Sci. 11: 803-820. Davidson, J. N., I. Leslie, and J. C. White. 1951. Quantitative Studies on the Content of Nucleic Acids in Normal and Leukemic Cells, from Blood and Bone Marrow. J. Path. and Bact. 63: h71-h83. Donohue, D. N., B.W. Gabrio, and C. A. Finch. 1958. Quantitative Measurement of Hematopoietic Cells of the‘Harrow. J. Clin. Invest. 31: 156h-1570. 63 Burton, K. 1956. A Study of the Conditions and Mechanism of the Diphenylamine Reaction for the Colorimetric Estimation of Deoxyribonucleic Acid. Biochem. J. 63: 315-323. Coulter, W. H. 1956. High Speed Automatic Blood Cell Counter and Cell Size Analyzer. Proc. Nat. Elec. Conf. _1g: 10314-th2. Cronkite, E. P., V. P. Bond, T. H. Fliedner, and J. R. Rubini. 1959a. The Use of Tritiated Thymidine in the Study of DNA Synthesis and Cell Turnover in Hemopoietic Tissues. Lab. Invest. _8_: 263-275. Cronkite, E. P., V. P. Bond, T. H. Fliedner, and S. A. Rillmann. 1960. The Use of Tritiated Thymidine in the Study of Haemopoietic Cell Proliferation. In: H_aemgpoiesis. Editor: G. E. W. Wolstenholme and N. O. Connor. London:Churchill. pp. 70-92. Cronkite, E. P., T. H. Fliedner, V. P. Bond, J. R. Rubini, G. Brecher, and H. Quastler. 1959b. Dynamics of Hemopoietic Proliferation in Man and Nice Studied by H3-Thymidine Incorporation into DNA. Ann. N. Y. Acad. Sci. 11: 803-820. Davidson, J. N., 1. Leslie, and J. C. White. 1951. Quantitative Studies on the Content of Nucleic Acids in Normal and Leukemic Cells, from Blood and Bone Marrow. J. Path. and Bact. 63: h71-h83. Donohue, D. N., B. W. Gabrio, and C. A. Finch. 1958. Quantitative Measurement of Hematopoietic Cells of the Harrow. J. Clin. Invest . 31: 156h-157O . 6h Feinendegen, L. W., V. P. Bond, E. P. Cronkite, and W. L. Hughes. l96h. RNA Turnover in Normal Rat Bone Marrow. Ann. N. Y. Acad. Sci. 113: 727-7hl. Filmanowicz, E., and C. W. Gurney. 1961. Studies on Erythropoiesis. XVI. The Response to a Single Dose of Erythropoietin in the Polycythemic Mouse. J. Lab. Clin. Med. 51: 65-72. Friedkin,'M. 1960. The Localization of Tritium in the‘Methyl Group of Thymine. Fed. Proc. 12: 312. Friedkin, N., D. Tilson, and D. Roberts. 1956. Studies of Deoxyribonucleic Acid Biosynthesis in Embryonic Tissues with Thymidine-C1“. J. Biol. Chem. 229: 627-637. Gude, W. D., A. C. Upton, and T. T. Odell. 1955. Giemsa Staining of Autoradiograms Prepared with Stripping Film. Stain Tech. 39: 161-162. Gurney, C. W. 1962. Discussion on Kinetics of Erythropoiesis. In: Erythropoiesis. Editor: L. O. Jacobson and'M. Doyle. New York:Grune and Strattone p. 192. Hughes, W. L., V. P. Bond, G. Brecher, E. P. Cronkite, R. B. Painter, H. Quastler, and F. G. Sherman. 1958. Cellular Proliferation in the'Mouse as Revealed by Autoradiography with Tritiated Thymidine. Proc. Nat. Acad. Sci. 55: h76-h83. Jacobson, L. 0., E. Goldwasser, and C. W. Gurney. 1960. Transfusion- Induced Polycythaemia as a Model for Studying Factors Influencing Erythropoiesis. In: Haemopoiesis. Editor: G. E. W. Wolstenholme and M. O. Connor. London:Churchill. pp. h23-hh5. 65 Lajtha, L. G. 1963. The Use of Radiation in Studies of Cell Proliferation. In: Cell Proliferation. Editor: L. F. Lamerton and RH J. M. Fry. Oxford:Blackwell. pp. 80-91. Leblond, C. P., and B. E. Walker. 1956. Renewal of Cell Populations. Physiol. Rev. 36: 255-279. deLesdain, Nicole, and C. Paoletti. 1962. Evolution de la Radioactivité de l'Acide DéSONyribonucléique chez 1a Soutis Aprés Administration de Thymidine Tritiée. Comptes Rendus QEE‘ 1701-1703. LoBue, J., B. S. Dornfest, A. S. Gordon, J. Hurst, and H. Quastler. 1963. ‘Marrow Distribution in Rat Femurs Determined by Cell Enumeration and Fe-59 Labeling. Proc. Soc. Exp. Biol. Med. 112: 1058-1062. Maloney,‘MH A., H. M. Patt, and C. L. Weber. 1962. Estimation of Deoxyribonucleic Acid Synthetic Period for Myelocytes in Dog Bone Marrow. Nature 123: l3h-135. Mattern, E. F. T., F. S. Brackett, and B. Olson. 1957. Determination of Number and Size of Particles by Electrical Gating: Blood Cells. J. Appl. Physiol. 19: 56-70. Mendelsohn, M. 1960. The Growth Fraction: A New Concept Applied to Tumors. Science 132: 1&96. Needham, J. 1963. Chemical Embryglogz, Vol. I. New York:Hafner. pp. l73-l7h. 66 Osmond, D. G., and N. B. Everett. 196k. Radioautographic Studies of Bone Marrow Lymphocytes 16 1136 and in Diffusion Chamber Cultures. Blood 23: 1-17. Patt, H. M. l95h. Radiation Effects on'Mammalian Systems. Ann. Rev. Physiol. 16: 51-80. Patt, H. M. 1957. A Consideration of Myeloid-Erythroid Balance in Man. Blood 12: 777-787. Patt, H. M., and M. A. Maloney. 1959. Kinetics of Neutrophil Balance. In: The Kinetics of Cellu1gg Proliferation. Editor: F. Stohlman. New York:Grune and Stratton. pp. 201-207. Patt, H. M., and M. A. Maloney. 1963. An Evaluation of Granulocytopoiesis. In: Cell Proliferation. Editor: L. F. Lamerton and R. J. M. Fry. OxfordzBlackwell. pp. 157-171. Patt, H.‘M., and H. Quastler. 1963. Radiation Effects on Cell Systems. Physiol. Rev. 53: 357-396. Pelc, S. R. 1956. The Stripping-Film Technique of Autoradiography. Int. J. Appl. Radiation and Isotopes 1: 172-177. Reichard, P., and B. Estborn. 1951. Utilization of Desoxyribosides in the Synthesis of Polynucleotides. J. Biol. Chem. 168: 839-8h6. Richer, W. J., and E. S. Breakell. 1959. Evaluation of an Electronic Particle Counter for the Counting of White Blood Cells. Amer. J. Clin. Path. 31: 38h-393. Rieke, W. O. 1962. The I! ZEZE Reutilization of Lymphocytic and Sarcoma DNA by Cells Growing in the Peritoneal Cavity. J. Cell. Biol. _1_3: 205-216. 67 Roberson, C. V. 1951. A Methane, Proportional Counting Method for the Assay of Tritium. Rev. Scientific Inst. gg: 353-355. Rubini, J. R., E. P. Cronkite, V. P. Bond, and T. M. Fliedner. 1960. The Metabolism and Fate of Tritiated Thymidine in Man. J. Clin. Invest. 32: 909-918. Rubini, J. R., 8. Keller, A. Eisentraut, and E. P. Cronkite. 1962. 16 Vitro Metabolism of H3-Thymidine. In: Tritium in the Physical and Biolgg1gal Sciences. Vol. 11. International Atomic Energy Agency, Vienna. pp. 2h7-265. Rubini, J. R., 8. Keller, and M. S. McCall. 196M. Inhibition of Tritiated Thymidine Incorporation into DNA by Alkaline Phosphatase Preparations. Cancer Research g3: 655-661. Rutherford, E., J. Chadwick, and C. D. Ellis. 1951. Radiations from Radioactive Substancgg. Cambridge Press. pp. 281-286. Schneider, W. C. 19h5. Phosphorus Compounds in Animal Tissues: I. Extraction and Estimation of Deoxypentose Nucleic Acid and of Pentose Nucleic Acid. J. Biol. Chem. 161: 293-303. Scthiger, W. 1955. Eine‘Mikroanalytische Schnellbestimmung von Halogen in Organischen Substanzen. TMickrochim. Acta. pp. 123-129. Schooley, J. C. and J. F. Garcia. 1962. Immunologic Studies on the Mechanism of Action of ErythrOpoietin. Proc. Soc. Exp. Biol. Med. 119: 636-6h1. Schooley, J. C., and K. Giger. 1962. Autoradiographic Evidence for a Pluropotential Stem Cell in the Bone Marrow. Lawrence Radiation Laboratory Semigpnual Report - Biology and Medicine, UCRL - 10683. pp. 176-179. 68 Schulman, M. P. 1961. Purines and Pyrimidines. In: Metabolic Pathways. Vol. II. New York:Academic Press. pp. 81h-827. Steel, G. G. 1962. in Studies with Tritiated Thymidine. In: The Use of Direct Tritium Assay Techniques Tritium in the Physical and Biological Scienceg. International Atomic Energy Agency, Vienna. Vol. II. pp. 3&9-358. Thomson, R. Y., Heagy, F. C., W. C. Hutchinson, and J. N. Davidson. 1953. The Deoxyribonucleic Acid Content of the Rat Cell Nucleus and its Use in Expressing the Results of Tissue Analysis, with Particular Reference to the Composition of Liver Tissue. Biochem. J. 53: h60-h7h. Verley, W. G., H. Firket, and G. Hunebelle. 1958. Thymidine-H3 in Studies of DNA Synthesis in Tissue Cultures.) Proc. Secogd Geneva Conference on the Peaceful Uses of Atomic Energy. Geneva:United Nations. Vol. 75, pp. 181-185. Verley, W. G., and G. Hunebelle. 1957. Preparation de Thymidine Marquée avec du Tritium. Bull. Soc. Chim. Belg. 66: 6h0-6h9. Wimber, D. E. 1963. ‘Methods for Studying Cell Proliferation with Emphasis on DNA Labels. In: Cell Proliferation. Editor: L. F. Lamerton and R. J. M. Fry. Oxfordznlackwell. pp. 1-17. 69 Appendix I Coulter Electronic Cell Counter Standardization for Nucleated Mouse Bone Marrow Cells Table III presents data from four experiments comparing electronic cell counts at various dilutions with a hemocytometer count. Bone marrow cells were collected from a mouse femur. Aliquots of these cells were taken and various dilutions made for the electronic cell count. A 0.5 percent Saponin Solution was used to lyse red blood cells. A hemocytometer count was made using a 1:20 dilution in h percent acetic acid. These results show that the Coulter counter is reliable over a fairly wide range of dilutions and that the results obtained compare well with the observed cell counts on a hemocytometer, at the usual dilution of 1:500. Richar and Breakell (1959) show that the standard error involved in a hemocytometer count is 20.55 percent as compared to 7.56 percent for the Coulter counter and Coulter (1956) and Mattern, Brackett and Olson (1957) show that the‘most accurate counting range :is from 20,000 to 80,000 counts/0.5'milliliter. Since a 1:500 dilution of the samples gave 20,000 counts/0.5 milliliter, this dilution was chosen even though the least deviation for the hemocytometer could appear to be 1:1000, or slightly greater. The data shown in Figure 1h are paired counts from the Coulter counter. The counts obtained are reproducible over the range studied. 7O .mHszHmmmxm mmmzh 2H QmmD mozoo mezzoo mmbgsou QmmHu». . . . o 4 . a J +~ . . . .00... .O.. . . 8W 0 O .00 e as e s s a s sees as .s . O a O. ”C O . m W ” . . o N. a. - 0 e e on. .0 Os 0 . . W s es s e e . . ... .. ... . s s . ....... .o. a use 00 e 0 00m .. . . . s so a . - : s . . . .’ . a as. N 0. es. a 0mm 0 so a s s as e O. 00:, 80 Appendix VI Basic Data The three tables (v, VI and VII) that make up this appendix are the basic data in the thesis. 81 TABLE V RESULTS OF INDIVIDUAL EXPERIMENTS USING DNA EXTRACTION) IN NORMAL ANIMALS Time Experiment Number-All Values pc/ug DNA X 10-6 (Hours) 1 2 3 & &' 5 11 15 0.5 68.6 1 70.0 61.6 77.8 60.2 2 7&.o 81.0 75.2 6&.2 & 90.2 68.8 78.8 7&.6 70.6 50.0 6 101.2 85.8 65.0 65.2 65.0 55.0 8 59.2 86.8 80.8 10 76.2 811.6 12 75.8 95.& 53.0 66.& 5&.8 16 102.0 18 60.6 90.2 66.& 82.0 2& 75.6 121.6 15&.2 50 77-6 36 108.8 &2 95.h 1+8 95.6 60 85.6 39.1: 72 71.2 3&.8 8& 39.11 96 5o.& 108 19.8 120 15.8 152 8.2 1&& 8.& 168 10.8 82 mmmod cm 98.0 88.0 28.0 820.0 28.0 as 38.0 080.0 B $8.0 $8.0 58.0 .3 «080 $8.0 $8.0 008.0 mm 008.0 : $8.0 2:00 .680 on $8.0 $8.0 .300 88.0 0 920.0 2. 98.0 230.0 030.0 080.0 0 008.0 820.0 090.0 «9.0.0 5.0.0 :80 840.0 a 320.0 38.0 820.0 0.20.0 2.20.0 0 2.40.0 1.80 28.0 030.0 A 38.0 0.30.0 00 R86 and mm 2 S uncommon 830m :fLoMQz set-meanes— n e n Ate-”Me mud—«H24 g2 2H wag—Hung «Drum 95%: ENZHGHMNN 435E595 ho $8.5m: H> Manda. 83 2000.0 0200.0 «02 2000.0 002 0000.0 0020.0 002 «000.0 00 0020.0 0020.0 20 0800 me. 800.0 00 n2mo.o m2 008.0 «2 0n 290.0 mm 230.0 on 008.0 mm 0220.0 00 2000.0 mnn0.0 0200.0 20 250.0 mm «2 22 02 m 0 e 0 Anuaeuv Batten .323 2242:2025: use-.222...» 2.2.2. 22.302330 2> 2.2.2.2. 8h mmooé nmooé mm.— nmooé 22222 N2 22 02 m m p n halve—xv: 2522; 2241122152 uses—Maegan n APHMMV 20o=e2eeoov H> maneu 85 TABLE VII RESULT OF INDIVIDUAL EXPEREMENTS USING FEMUR COMBUSTION IN HYPERIRANSFUSED ANIMALS Time Experiment Number--All Values pc/Pemur (Hours) 9 10 12 1 0.01163 2 0.0h65 h 0.0h11 0.0500 6 0.0103 8 0.0571 0.05h5 10 0.0595 12 0.0570 In 0.0551 0.05h1 16 0.0500 18 0.0h06 0.0591 20 0.0275 22 0.0219 2% 0.0501 0.0551 25 0.0h60 28 0.0551 50 0.0“65 52 0 .01116 #8 0.0500 31. 0.0219 108 0.0179 152 0.011h 168 0.0092 as HUI-.0 I84 1.. .0 . , ’2’. ., 7 fl . no were. 1,...“ .9. A. a. 2. EN- . 2!... C.:\ s, .a I. fi...u...e 1.11. (s . v 1‘s _. . :1 «1...... BR "'11177171777177!77177117711117!!!"NW“ 1