193134a0Nature1934811196201131341350028-0836196210.1038/193134a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v193/n4811issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue193134a0Estimation of Deoxyribonucleic acid Synthetic Period for Myelocytes in Dog Bone Marrow
AU  - MALONEY, MARY A.
AU  - PATT, HARVEY M.
AU  - WEBER, CHARLOTTE L.Division of Biological and Medical Research, Argonne National Laboratory, Argonne, IllinoisA SINGLE injection of tritiated thymidine can be thought of as a flash labelling, and the initial labelling index (per cent positive) will be approximately proportional to the percentage of cells in synthesis of deoxyribonucleic acid (DNA). If the cell population in question is homogeneous, the duration of the period of DNA synthesis relative to the total generation cycle will be given by the labelling index. It is not known whether the DNA synthesis time is constant for proliferating cells in the various tissues. If this were so, the labelling index per se, with appropriate correction for cell heterogeneity, would provide a measure of relative turnover or growth. Synthesis times of 5-10 hr. have been reported for the several rapidly growing cell systems that have been studied1. This article is concerned with the time of synthesis of DNA of the myelocyte in the dog.
About 20 per cent of myelocytes are labelled within 30 min. after thymidine injection (0-1 mc./kgm. of a 360 mc./mM solution IV). In previous work2, we estimated a DNA synthesis time of some 5 hr. from the early time-course of labelled mitoses in bone marrow autoradiographs. Since, however, only about half myelocyte mitoses were labelled during the first several hours after injection of thymidine, the DNA synthetic period has been evaluated in another way in recent experiments. When tritiated thymidine is infused at a constant rate for a period longer than the DNA synthesis time, the degree of incorporation should attain a maximum. Cells in synthesis at the beginning of the infusion will incorporate tritiated thymidine for a variable period. However, a cell that has just entered synthesis will label throughout its synthetic period. Such a cell should label maximally and all cells entering synthesis after the infusion has begun should reach a similar maximum. The duration of the synthetic period should correspond to the time when labelling reaches the maximum value. A similar analysis has been used in surviving marrow cultures (Lajtha, L. G., personal communication).
Five beagles were infused intravenously at a constant rate with tritiated thymidine in 5 per cent glucose. One dog was infused with 12 me. (360 mc./mM) over a 12-hr, period. The remaining four dogs were divided into two pairs: one of each pair received tritiated thymidine with a specific activity of 360 mc./iruV/, the other with a specific activity of 36 mc./mM. All four dogs received a total dose of 10 me. of tritiated thymidine over a 6-6J hr. period. Bone marrow samples were aspirated at frequent intervals during the infusion. Autoradio-graphs were prepared3 and grain counts were made on one thousand myelocytes in each marrow sample.
Fig. 1. Distribution of myelocyte grain counts during infusion of tritiated thymidine
Fig. 2, Changes in the grain count of myelocytes during infusion of tritiated thymidine
The degree of labelling from cell to cell as revealed by grain count indicates the relative amount of DNA synthesized, assuming uniform geometry and availability of precursor. Geometry is an important variable because the average range of tritium p-particles is only about IJA, which is less than the differences in cell thickness in smear or section. Owing to the relatively high specific activity of tritiated thymidine, only small amounts of the precursor are ordinarily administered and, as a result, labelling may also be affected by intra- and extracellular thymidine pools. These factors, however, should not influence appreciably the overall trend of changes in grain concentration during infusion of thymidine, since differences due to geometry and availability would be random for any given cell. Such differences would, of course, contribute to the variance. The importance of thymidine availability is perhaps borne out by comparison of the initial (30 min.) grain count distributions with the 36 and 360 mc./mM preparations. The coefficient of variation was 46 per cent for the former and 62 per cent for the latter; this difference is highlv significant (P < 0-001).
The degree of labelling from time to time also indicates the relative amount of DNA synthesized; but, in this case, it is necessary to consider that cells are continually entering synthesis and mitosis during the infusion. Cells entering synthesis will be added to the labelled population, but the added cells will be minimally labelled at the outset. The picture is complicated further by mitosis. Some of the labelled cells will complete division during the course of thymidine infusion2. The progeny of minimally labelled cells will be lost from the population because of dilution of label by mitosis. However, other cells will be added to the population as the more heavily labelled myelocytes complete mitosis. Thus, marrow specimens will include cells that are being labelled during the entire period between successive sampling as well as cells that have initiated or completed synthesis in this interval. Later, marrow samples will also include the progeny of cells labelled earlier; it is conceivable that some of these may also be in synthesis, but relabelling would not seem to be an important factor duiing a 6-12 hr. infusion.
These factors, in the aggregate, would tend to decrease the mean or median grain count. Accordingly, attention has been focused on representative myelocytes the relative position of which in respect to grain count distribution is constant throughout the infusion period. This was done by following the nth most heavily labelled cell as determined by the position of the median cell initially; this analysis compensates for changes in the number of labelled cells as the infusion progresses. A cell that represents this position in the grain count distribution for one of the dogs is designated by an arrow in Fig. 1. The changes in grain concentration of such representative cells in the five dogs is shown in Fig. 2; the curve was determined by calculation of the moving average of the grain counts for 2 adjacent points with each stated interval [plusmn] 15-30 min. Grain counts increase to a maximum which is reached after about 4-5-5 hr. infusion. This estimate of the DNA synthetic period for myelocytes in dog bone marrow confirms our previous approximation based on analysis of labelled mitoses.
This work was performed under the auspices of the U.S. Atomic Energy Commission.Lajtha, , L. G., The Kinetics of Cellular Proliferation, 173 (Grune and Stratton, Inc., New York, 1959).Patt, , H. M., and Maloney, , M. A., The Kinetics of Cellular Proliferation, 201 (Grune and Stratton, Inc., New York, 1959).Maloney, , M. A., and Patt, , H. M., Proc. Soc. Exp. Biol. Med., 98, 801 (1958).PubMedISIChemPort