Thymidine Infusion Research Articles

Do myoblasts in vivo withdraw from the cell cycle? A reexamination.

The proliferative fraction of mononucleated cells in differentiating chick embryonic wing muscle (day 11) was measured following continuous infusion of tritiated thymidine into the embryonic circulation. During progressively longer intervals of infusion of the isotopically labeled precursor, the percentage of cells that enter S becomes larger, reaching 92% at the longest time period measured (21 hr). These observations suggest that until they are withdrawn into nonreplicative muscle syncytia, virtually all of the single cells in differentiating embryonic muscle remain in the proliferative pool. Earlier calculations of the size of this pool in developing muscle, based on the percentage of cells in S during a brief pulse, indicated, however, that less than half of the mononucleated cells are still replicating. We therefore compared the size of the proliferative fraction determined by continuous labeling with the calculation of this same parameter using our own pulse-labeling data. We find that the calculation underestimates the size of the proliferative pool and is, in fact, an estimate of only that portion of the cells whose generation times cluster around the average. This underestimate is particularly pronounced in differentiating muscle in which, concomitant with myogenic fusion, the distribution of G1 times (and consequently generation times as well) becomes longer and more highly variable. Our results suggest that the mode of administering the labeled DNA precursor profoundly affects the measurement of cell cycle parameters in vivo when these parameters exhibit considerable variability. The data presented here do not support the notion that any sizeable fraction of the myoblast population is withdrawn from the cell cycle for any significant period of time prior to fusion.

In vivo cell synchrony in the L1210 mouse leukaemia studied with 5-fluorouracil or 5-fluorouracil followed by cold thymidine infusion.

[3H]-TdR and [3]-udR labelling indices and mitotic indices were followed in tumour-bearing mice after application of either 5-fluorouracil (FU) alone or of FU followed by cold TdR infusion. With FU alone, accumulation of cells at the beginning of S was found, but there was no indication of a synchronous passage of the accumulated cells further round the cycle. When FU injection was followed by cold TdR infusion, a synchronous passage of the accumulated cells through the cycle was observed. However, there was a large variation in the response of individual mice to this treatment.

Granulocyte dynamics and the question of ineffective granulopoiesis.

ABSTRACTA simple analysis was developed to compare granulocyte production in marrow with granulocyte turnover in peripheral blood of the dog. the analysis is anchored to the relative number of granulopoietic and erythropoietic cells that are flashlabeled with tritiated thymidine. This provides a convenient measure of relative production rates, since there is little, if any, difference in the duration of the respective DNA synthesis periods. the latter was confirmed in the present work by comparison of myeloid and erythroid cells in respect to (1) labeled mitosis curves and (2) changes in labeling intensity with constant infusion of tritiated thymidine. the ratio of flash‐labeled granulocyte to erythrocyte progenitors was unity (1.02 ± 0.05) in the eight dogs studied, which means that gross production rates were essentially the same. Since net production as revealed by peripheral blood turnover is greater for erythrocytes than for granulocytes, it follows that there must be some ineffective granulopoiesis. This may correspond to over half of the total production. It is suggested that this phenomenon, which apparently occurs in man as well as in dog, could represent an important feature of granulocyte regulation.

Comparison of multiple injections with continuous infusion of tritiated thymidine, and estimation of the cell cycle time.

ABSTRACT Labelled nuclei were counted in stratified squamous epithelium in mice killed after 24 h intraperitoneal administration of tritiated thymidine to label cells synthesizing deoxyribonucleic acid. Multiple injections produced the same result as an infusion of tritiated thymidine given after 24 h infusion of saline, but infusion of tritiated thymidine alone produced a different result. Thus, cell proliferation was depressed during the first 24 h of continuous infusion but was normal during the second 24 h. Comparison of proliferation of the oesophageal epithelium at the level of the thyroid and at the level of the diaphragm showed no difference between the two. Comparison of male with female mice given 72-h infusions of tritiated thymidine showed that cell proliferation occurred at the same rate in both. The cell cycle time was estimated in the epithelium of the oesophagus and tongue by comparison of mice given a single injection with mice given multiple injections of tritiated thymidine.

Long G1 or G0 state: A method of resolving the dilemma for the cell cycle of an in vivo population

A technique is described using standard autoradiographic procedures whereby it is possible to study an in vivo cell population and to decide whether the cells in the population have a long G1 or whether they pass into an out-of-cycle G0 state. The method has been applied to the epithelium of the hamster cheek pouch and involves continuous infusion (or repeated injections) of tritiated thymidine into the animals for a time comparable to the turnover time of the cell population. The population is sampled after various period of infusion and the proportion of cells labelled is compared with the proportion that would be predicted by the G1 and G0 models. Using this technique it is concluded that the proliferating cells of the hamster cheek pouch have a mean cell cycle time of approx. 140 h. More than 90 per cent of this period is occupied by a finite pro-synthetic G1 phase, and few, if any, of the cells are in an out-of-cycle G0 state.

Effects of a glucocorticoid (dexamethasone) on the eosinophils of the rat.

SUMMARY The effects of single and continuous administrations of a synthetic gluco-corticoid (dexamethasone) on eosinophil numbers and distribution have been studied. Absolute blood eosinophil counts were made, and the turnover of the eosinophils was examined by continuous infusion of tritiated thymidine to label the deoxyribonucleic acid of all newly formed cells. Dexamethasone equivalent to less than 200 μg. cortisone/100 g. rat/day produced disappearance of eosinophils from the blood and the normal output of glucocorticoids is therefore probably less than this. Single administrations of dexamethasone produced a blood eosinopenia within 2 hr. due to removal and destruction of eosinophils by the reticulo-endothelial system. Continuous dexamethasone administration reduced the number of proliferating eosinophil cells in the marrow, with a consequent reduction in the number of eosinophils in the tissues.

Leukocyte Labeling in Rats during and after Continuous Infusion of Tritiated Thymidine: Implications for Lymphocyte Longevity and DNA Reutilization

Abstract Leukocyte labeling was studied in rats during and after continuous intravenous infusion of H3-thymidine. The radioisotope was administered for varying periods up to 271 days. The results permit the following conclusions: 1. The median survival of small lymphocytes is about 1 month. Five to 8 per cent of small lymphocytes have a life span of more than 9 months. 2. Following the administration of H3-thymidine, reutilization of the tracer markedly delays the fall-off of labeled cells in the peripheral blood. Reutilization probably involves H3-thymidine released from labeled DNA during cell death, since suppression occurs with massive infusion of non-labeled thymidine. 3. Unlike granulocytes and large lymphocytes, small lymphocytes label nonuniformly, and appear to be comprised of at least two populations with different intensities of labeling and different turnover rates. The more heavily labeled cells have the faster turnover. 4. The complexity of the labeling process indicated by the present observations must be considered in the interpretation of H3-thymidine data. However, the survival of unlabeled cells during continuous H3-thymidine infusion remains a valid means of measuring the life spans of circulating blood cells.

DELAYED INCORPORATION OF TRITIATED THYMIDINE INTO DNA.

Delayed utilization of tritiated thymidine by regenerating mouse liver can be almost completely suppressed by a continuous infusion of nonradioactive thymidine. In addition, as shown earlier for lymphocytes, labeled granulocytes are a potential source of the tritium marker. These observations suggest that the delayed incorporation of label into DNA must be due to the transfer of labeled nucleoside, which may be derived either from the degradation of DNA or from a long-lived intracellular pool. In either case, the transferred material probably originates from all tissues that have a high rate of cell turnover.

Eosinophil turnover in the rat.

THE life-span of the eosinophil is still not known with any certainty: recent estimates vary from a few hours to several days1. In this communication the problem has been investigated by continuous infusion of tritiated thymidine, a method which has previously proved successful in defining lymphocyte turnover2.

Determination of Lymphocyte Turnover by Continuous Infusion of H3 Thymidine

Abstract The use of an osmotic pump for continuous infusion of H3 thymidine in rats is described. Estimates of turnover times of cells in the peripheral blood are 4 days for granulocytes, 60 days for large lymphocytes and in excess of 100 days for small lymphocytes. These turnover times include initial delay in release or maturation after the last mitosis in the marrow or nodes, and any time spent outside the circulation prior to final disappearance of a given cell from the peripheral blood. The data exclude reutilization as an explanation for the observed longevity of lymphocytes.

Chronic infusion of tritiated thymidine into mice with tumors.

A technique for the chronic infusion of mice is presented. Infusion of tritiated thymidine for 3 to 7 days labels most, but not all, of the cells of autochthonous breast tumors. Many of the unlabeled tumor cells are probably nonproliferating, but some may represent cells with exceptionally long intermitotic times.