Pulse Labeling Index Research Articles

A comparison of different methods to determine cell proliferation by flow cytometry

Abstract. The proliferation of human melanoma cells (MeWo) in vitro was studied with a number of different techniques. In particular, we compared the expression of PCNA and the Ki‐67 antigen on the one hand with BrdU pulse and continuous labelling on the other. Two‐dimensional flow cytometry (with DNA content as a second parameter) was employed to discriminate between cycling and non‐cycling cells as well as cells in the G1, S and G2 phases of the cycle. Cell cultures in different stages of growth were analyzed. We found that the percentage of anti‐PCNA and Ki‐67 positive cells agreed very well with the BrdU pulse and continuous labelling index, respectively. Our data further support the assumption that under certain conditions PCNA is a marker of S‐phase cells, whereas Ki‐67 can be used to quantify the growth fraction. Possible pitfalls of the techniques are discussed.

Alveolar macrophage kinetics after inhalation of 239PuO2 by CBA/Ca mice: changes in synthesis of DNA.

For workers in the nuclear industry, the primary route for the entry of radioactive materials into the body is by inhalation, and the rate of clearance of particles from the pulmonary region of the lung is an important factor in determining radiation dose. It is the function of alveolar macrophages (AM) to maintain the sterility of the lung and to remove insoluble particles from the respiratory surfaces and airways. The AM population is not static, and under normal conditions the loss of macrophages from the alveoli via the conducting airways is balanced by renewal. Studies of the effects of external irradiation on the kinetics of AM are numerous, but to date little is known about the effects of inhaled radioactive particles. In this investigation the effects of inhaled 239PuO2 (plutonium dioxide) particles on the synthesis of DNA by AM were studied at times up to 77 days after exposure. We also measured the number of cells recovered by bronchoalveolar lavage and the incidence of AM with nuclear aberrations. The latter provides a sensitive indicator of the effects of radiation. One of the earliest effects observed after exposure to 239PuO2 is a reduction in the number of AM recovered by lavage. This reduction is associated with a 3-fold reduction in the proportion of AM undergoing DNA synthesis at early times after exposure. The overall mean pulse labeling index of AM recovered from sham-exposed mice is 1.68%, and no trend is observed with time.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and partial purification of a potent mitogenic factor for human thyroid follicular cells

Normal adult human thyroid follicular cells have an extremely limited proliferative capacity in vitro. No previously studied mitogen, including thyrotropin (TSH) or epidermal growth factor (EGF), has in our hands resulted in a significant improvement over the 3–4% nuclear [ 3H]thymidine pulse-labelling index (LI) obtainable with 10% fetal calf serum. Here we report the detection in the conditioned medium from a sub-clone of NIH3T3 fibroblasts of a mitogenic activity capable of increasing this response up to 10-fold, to an LI of over 20%, together with an even greater relative stimulation of mitotic activity. Preliminary characterisation has excluded EGF and TGFα, and demonstrated that the activity is bound reversibly by heparin-Sepharose, thus pointing to a member of the heparin-binding fibroblast- or hepatocyte-growth factor families. This material should have wide practical application in facilitating primary culture of follicular cells, and may reveal new mechanisms of stromal-epithelial interaction regulating normal and neoplastic thyroid growth in vivo.

Effects of 17β-estradiol on c-MYC and c-Ha-RAS expression in the liver of ovariectomized female rats

Twenty four hours after i.p. injection of 17β-estradiol (E 2) (500 μg/100 g b.w.) to ovariectomized rats, the hepatocytes [ 3H]-thymidine pulse-labeling index (L.I.) was significantly increased, reaching a value of 4.3 ± 1.6 percent (i.e. much lower than 32.0 ± 2.0 percent 24 h. after partial hepatectomy -PH-). E 2-treatment was followed by an increase in liver content in c-myc transcripts, with a peak at 650 percent basal value at 8 h, very similar to that observed after PH. In contrast, E 2 induced an increase in liver c-Ha-ras expression with a similar time-course evolution but markedly lower amplitude than that seen after PH. These data are consistent with a role of c-myc in proliferative competence i.e. the ability to perform the G0-G1 transition, and with a role of c-ras in further progression of the cells in the cycle.

Synchronization of replicons in Ehrlich ascites cells

Ehrlich ascites cells, in which replication units at the beginning of the S phase started and grew synchronously, were obtained by the following protocol: (1) selection of G 1 cells by zonal centrifugation, (2) hypoxia for 12 h, (3) reaeration, (4) addition of cycloheximide (30 μ M) within the first minute after reoxygenation. Studies on the effectiveness of the different steps revealed: (i) G 1 cells reoxygenated after 12 h of hypoxia traverse two succeeding cell cycles highly synchronously. This was shown by monitoring the thymidine incorporation rate, the thymidine pulse-labeling index, and the mitotic index. (ii) Cycloheximide, like hypoxia, suppresses replicon initiation in Ehrlich ascites cells without interfering with DNA chain growth and DNA maturation. The reversibility of the suppression is less complete than in the case of hypoxia. This was shown by DNA fiber autoradiography and by analyzing the length distribution of pulse- or pulse/pulse-chase-labeled daughter DNA in alkaline sucrose gradients. The alkaline sedimentation patterns of daughter-strand DNA, pulse labeled immediately after the cycloheximide addition at the end of the elaborated protocol and 1 and 2 h later, indicated synchronous initiation and growth of a homogeneous population of DNA molecules to replicon-sized lengths.

Ultrastructural and autoradiographic investigations of cell cultures derived from tendons or ligamentous material from patients with fibromatous disorders.

Cell cultures were derived from tendons or ligamentous material from patients with carpal tunnel syndrome (CTS), Dupuytren's contracture (DP), tendopathia nodosa (TN) and hallux valgus (HV). The ultrastructure of the operation specimens as well as of the cell monolayers was investigated, using a floating sheet method in order to preserve both cell-to-cell contacts and the orientation of the monolayers. The histologic features of the tissues obtained in the operations were correlated with the ultrastructure of the cells in culture derived from these specimens. In DP, above all in the nodules, an activation of the capillary endothelium in the vicinity of myofibroblasts and mast cells was observed. In CTS the collagen fibrils varied extremely in diameter. In DP and TN biopsies a splicing process of helicoidly arranged fibrils could be seen. A disintegration of elastic fibers in the fibrillar and amorphous components was found in DP nodules, HV and TN tissues. Transitional forms between fibroblasts and myofibroblasts were observed not only in DP but also-though in a smaller percentage--in the cultures derived from the other patients. The cells showed organelles for active protein synthesis and transport. Autophagocytosis and the formation of multilamellated bodies took place in TN and HV cultures. In CTS, DP and TN cultures cells were connected via gap junctions. In some cultures, above all in those derived from CTS, monocilia were found. In CTS cultures the formation of intracellular collagen occurred. Growth parameters were rather low in HV cultures. PLmax (maximal pulse labelling index) values were higher in TN cultures than in DP and HV cultures. Plating efficiency (PE) values were higher in cultures derived from cell-rich and capillarized tissues than in biopsies with few cells.

Cell kinetics of thyroid epithelial cells during hyperplastic goitre involution.

Thyroid epithelial cell kinetics were investigated in rats when a normal iodine diet was re-established after a long period of iodine deficiency supplemented with propylthiouracil (0.15%) for the last 2 months. In the first (prelabelling) experiment, all rats were labelled with a single injection of [3H]thymidine 2 days before iodine refeeding in order to follow the fate of the prelabelled cells. In the second experiment, the pulse-labelling index at the time of killing was measured; for this purpose the rats received [3H]thymidine 1 h before death. In these two experiments autoradiography was performed on histological sections. Thyroids were excised on day 0 and then at various intervals up to day 73 of iodine refeeding. Plasma tri-iodothyronine (T3) and thyroxine (T4) were very low until day 4 and then increased to reach control values on day 30. Thyroid concentration of iodide rose to 20 times the value on day 0, remained at this high level until day 2, and then diminished on day 4 to reach the control value on day 16. Plasma TSH concentrations were very high in iodine-deficient rats and did not vary significantly until day 8, when they fell rapidly to reach the control value on day 30. Thyroid weight, raised on day 0, decreased relatively quickly until day 4, then more slowly until day 30. Total thyroid epithelial cell number, high on day 0 (30.7 x 10(6) cells) was constant until day 16, then decreased until day 30 at which time a plateau was reached.(ABSTRACT TRUNCATED AT 250 WORDS)

An analysis of proliferative activity in innervated and denervated forelimb regenerates of the newt, Notophthalmus viridescens

ABSTRACT Pulse and continuous labelling with [3H]thymidine combined with mitotic index determinations provided data on the kinetics of cell cycling in innervated and denervated early and mid-bud forelimb blastemas of the adult newt, Notophthalmus viridescens. Most or all blastema cells cycle during regeneration and are thus part of the proliferative fraction. At any given moment, however, only 26 % of the blastema cells are actively progressing through the cell cycle, with the remainder being in a state of transient quiescence (TQ). The small size of the actively cycling (AC) population may in part explain the relatively slow rate of regeneration exhibited by the adult newt. The pulse-labelling index and mitotic index of denervated blastemas paralleled control values for 48 h following nerve withdrawal, but both parameters were significantly reduced by 72 h. By 5 days postdenervation, cell cycle activity was essentially zero. The combined pulse and continuous labelling data suggest that nerves may be primarily involved in the entry of TQ cells into the AC population, with subsequent progression through the cell cycle being less dependent on innervation. Relative to controls, no early postdenervation increases in TCA-precipitable [3H]thymidine incorporation, pulse-labelling index or mitotic index were observed.

Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication.

Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.

Towards a further understanding of the growth-inhibiting action of oxygen deficiency: Evaluation of the effect of antimycin on proliferating Ehrlich ascites tumour cells

The effect of 1 μM antimycin on the proliferative properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in the second in vitro passage was studied. Continuous drug exposure of asynchronous cells caused rapid cessation of cell growth, characterized by the cell number and DNA, RNA and protein content of cultures. Cells cease to consume oxygen and enhance their glycolytic activity. Uptake of labelled thymidine into acid-insoluble material was far below that of the controls, whereas incorporation of labelled uridine exceeded that of controls, as was also observed with other inhibitors of the respiratory chain (sodium cyanide, 2-thenoyltrifluoroacetone, or anaerobiosis). The influence of antimycin on cells at different stages of the cell cycle was tested using cells enriched in either G1, S or G2 phase by centrifugal elutriation. DNA histograms (flow cytometry) and pulse-labelling index curves gave detailed insight into cell-cycle progression of antimycin-treated cells: G1 and early S cells remained stationary; G2 cells still passed from G2 into mitosis to remain subsequently in a non-growing state in G1; S cells were either slowed or halted. Supplementation of antimycin-containing cultures with exogenous pyrimidine nucleosides stimulated reprogression of G1 cells without changing their ATP content. The results of the current experiments are interpreted as supporting the concept that growth cessation of G1 cells under respiratory insufficiency is not predominantly caused by impairment of respiratory phosphorylation but may be the consequence of a lack of precursors for DNA and RNA synthesis.

Experimental studies on the pathogenesis of ochronotic arthropathy

Lapine adult and fetal articular chondrocytes in monolayer culture were employed as an experimental model for studying the effects of homogentisic acid on chondrocyte morphology, proliferation and synthesis of proteoglycans. Concentrations of homogentisic acid in the range from 0.1 to 100 micrograms/ml were investigated and a cytotoxic effect was detected with concentrations of 5 micrograms/ml and above. Thus, with adult articular chondrocytes this effect was seen between 36 and 48 h of subculture with 5 micrograms/ml or more of homogentisic acid present from the beginning of subculture. In fetal articular chondrocyte cultures a concentration of 1 microgram/ml elicited a statistically significant reduction (13%) in cell proliferation without altering sulphated macromolecular synthesis. Experiments with 3H-thymidine autoradiography to measure the pulse labeling index (LI) in fetal chondrocytes in vitro showed that the 5 micrograms/ml concentration of homogentisic acid present for 21 h from the beginning of subculture gave a LI of 2%, compared with 25% for controls. Thus, homogentisic acid can reduce chondrocyte proliferative capacity before morphological alterations can be detected by light microscopy. No significant alterations in the synthesis of proteoglycans were detected at concentrations below the cytotoxic level. The homogentisic acid-induced cytotoxicity and reduction of proliferation in chondrocytes represents a possible pathway by which cartilage is damaged in ochronotic arthropathy.

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells

Oxygen was excluded from cultured Ehrlich ascites cells for 5–7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [ 3H]thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5–7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5–1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.

Adriamycin Effects and the Interactions of Adriamycin with X Irradiation on Murine Mammary Tumors

The effects of a single intraperitoneal injection of adriamycin (10 mg/kg) on a slow-growing C3H mouse mammary tumor (Slow) were analyzed volumetrically, biochemically, autoradiographically, and flow cytometrically. Adriamycin, at this dose, did not induce regression in tumor volume but did inhibit the growth rate for several days. The (/sup 3/H)TdR pulse-labeling index was initially high (23% at 7 hr vs 16% for control) but then dropped to 8% at 96 hr postinjection. Qualitatively, the flow cytometric data supported these trends with the percentage of cells in S phase being about 95, 85, and 85%, respectively, at 7, 24, and 96 hr postinjection. In contrast, the (/sup 3/H)TdR incorporation even though quite valuable, was initially in agreement but at 96 hr postinjection, it was about 145% of control. The fraction of cells in G/sub 2/M was more than 350% of control at 72 hr and was still over 180% at 120 hr postinjection; however, the mitotic index per se was basically unchanged during this period. Thus the extended effect on the volumetric growth rate appears to be due primarily to the extensive G/sub 2/ arrest. Adriamycin also affects the subsequent X-irradiation response. The dose for local tumor control from a singlemore » irradiation was markedly elevated at 24 hr in the Slow line (slow growing) and at 96 hr after adriamycin injection in the S102F line (fast growing). These X-ray plus drug results are contrary to the results anticipated from the effects of adriamycin, as published in general, and specifically from the cytokinetic effects of adriamycin on the tumors reported here as well as those published previously. These results indicate that the interaction of drugs with X irradiation in solid tumors in vivo is much more complicated than expected from the numerous published in vitro studies.« less

Control of cellular proliferation in HeLa-S3 suspension cultures. Characterization of cultures utilizing acridine orange staining procedures.

Growth control is investigated in detail in fed and unfed HeLa-S3 suspension cultures. Two-step acridine orange staining and flow cytometric analysis indicated declines in cellular red fluorescence (proportional to RNA content) of 40-50% between exponential and plateau phase in both culture types. Cellular green fluorescence (DNA content) assessed simultaneously indicates an increment of cells with Gi-DNA content in plateau phase in the unfed cultures, while fed cultures show a brief increment in G1-phase cells in the transition phase followed by a recovery in plateau phase to a value similar to that of exponential cultures. Temporal declines in the 3H-thymidine pulse-labeling index are observed in both culture systems. These data along with the flow cytometry data indicate a distinct G1-arrest in the unfed plateau cultures and suggest a random arrest of cells about the cell cycle in fed plateau cultures. Acidic acridine orange staining and flow cytometric analysis furthermore indicate the occurrence of a quiescent population comprising approximately 345 of the total cells and consisting of both dead and viable cells in plateau phase unfed cultures. In contrast, fed plateau cultures show approximately 14% quiescent, mostly dead cells. Also, both culture systems show temporal declines in the clonogenic index and a longer cell-cycle transit time in plateau phase relative to exponential phase. These findings confirm earlier work which indicates that the environment has a profound influence on the mode of growth control for mammalian cells in vitro.

Detailed kinetic analysis of Shay chloroleukaemia cell population propagated in permanent suspension culture in vitro.

Detailed kinetic analysis of a growing cell population is difficult, even when assay conditions are nearly ideal. Therefore, it is usually essential to perform several types of experiments and analyse all the results in terms of a mathematical model, the use of which is not limited a priori by a specified application. In the present study we investigated cell population kinetics using rat chloroleukaemia cells propagated in suspension culture in vitro. The parameters were measured: doubling time of the population, fraction of labelled mitoses, changes in labelling index with time after pulse labelling, continuous labelling and stathmokinetic index. Analysis of the results was based on a computer program CECAM, which is a stochastic model capable of simulating essentially all types of kinetic experiments based on presently known assay techniques. The results showed that precise and reliable information on cell population kinetics could not be obtained from the analysis of any single type of experimental data. In particular, the technique of labelled mitoses underestimated the duration of the G1 phase, owing to subtle label-induced changes in population behaviour. These changes could not have been detected with any certainty without rigorous quantitative comparisons with other types of experimental data. As a whole, however, results obtained by the different techniques were in agreement and the kinetic behaviour of chloroleukaemia cells in vitro could be established in detail. In certain circumstances even minute changes in the kinetic parameters of the cells can modify population behaviour drastically. To study these cases adequately the experiments must be designed with utmost care, preferably with the aid of preceding simulations. This is because demonstration of small primary changes in population kinetics may be beyond the limit of detection of any single assay method.

EFFECT OF PREDNISOLONE ON THE TUMOR CELLS

Groups of female Donryu rats were inoculated intraperitoneally with 1×107 Yoshida ascites sarcoma cells. Animals were then subjected to the daily treatment with subcutaneous administration of water soluble prednisolone (P). Injection of P was initiated 2 days after inoculation in Group I, while Group II started to receive treatment immediately following inoculation. Each Group was further devided into three subgroups of A, B, and C, to which 1 mg, 0.2 mg, and 0.05 mg of P per 100 g of body weight were administerd, respectively. The result were summerized as follows : 1) Growth rate of the tumor cells was significantly suppressed in A and B rats of Group I and in all subgroups of Group II. This initial suppression, however, was transient and was followed by a rebound increase in the 5th or 6th day after inoculation.2) On the 4th day, when the cell growth was still suppressed, both mitotic index and 3H-TdR pulse labeling index of the tumor cells were significantly decreased in all animals treated with P. The duration of mitosis remained unaffected, being 1 hour. Although no difference was observed in DNA synthetic time among the rats of Group I, it was significantly prolonged in Group II. G2 duration was 2 hours in both P-treated and control rats. Thus, treatment with P appeared to prolong the duration of G1 phase proportionally to the dosis administered. Compartment size of non-proliferative cells was found to be enlarged significantly, when the growth rate was retarded by the treatment.3) In Group II, in vivo 3H-uridine labeling index was significantly depressed on the 4th day, while the mean grain counts were nearly equal to the control. However, the amount of 3H-uridine incorporated into RNA fraction of tumor cells was significantly less in P-treated rats than in the control. Thereafter, the incorporation increased in the P-treated rats, and decreased in the control. On the 5th day after inoculation, no significant difference became appearant between P-treated and control rats.4) It was suggested that P mainly affects the pre-DNA synthetic phase of Yoshida ascites tumor cells and inhibits the initiation of DNA synthesis. These effects were weak, though definitive, and seemed to disappear within several days despite the continued administration.

The kinetics of cultured human glioma cells: autoradiographic studies.

The kinetics of monolayer cell cultures, derived from brain tumors which had been labeled with 3H-thymidine just prior to surgical removal, have been investigated. Analysis of autoradiographs indicates that: 1. the distribution of cell types in primary cultures is similar to that of the viable areas of the parent tumor in vivo; 2. the first week of culture constitutes a lag phase; and 3. vigorous proliferation commences during the second week of culture. Double labeling of the tritiated cultures with 14C-thymidine indicates that the pulse labeling index (PLI) of primary cultures during the second week is lower than that of the parent tumor and that the PLI of the culture increases during the second week. In contrast, we found that long-term established cultures in our laboratory have a high PLI during the first few days after passage, quickly reach a maximum, and then drop precipitously 1 week after passage. These latter variations in PLI correspond to the following growth pattern: 1. exponential growth; 2. a stationary phase due to medium exhaustion; or 3. a a plateau due to overcrowding. There are indications that some cell types, which have the capacity to divide in the parent tumor, lose this ability after transfer to tissue culture.

Identification of a novel cell type in peripheral lymphoid organs of mice. 3. Functional properties in vivo.

Several properties of lymphoid dendritic cells in situ have been determined, and contrasted to information previously established for lymphocytes and mononuclear phagocytes. Dendritic cells are not found in newborn mice, and their concentration in both spleen and mesenteric lymph node does not reach adult levels until 3-4 wk of age. Dendritic cells largely disappear from adherent populations following administration of steroids (2.5 mg hydrocortisone acetate s.c.) and ionizing radiation (D(o) of 100 rads for Co(60)). Splenic dendritic cells can originate from precursors located in both bone marrow and spleen itself, probably the red pulp. The mature splenic population does not actively divide (pulse labeling index with [(3)H]thymidine of 1.5-2.5%), but does turnover at substantial rate, 10+% of the total pool per day. The influx of new cells appears to be derived from a proliferating precursor compartment, but the mechanism for efflux or turnover is not known. Dendritic cells in spleen and node undergo little or moderate increase in numbers during development of a primary immune response. These in vivo characteristics, taken together, further distinguish dendritic cells as a novel cell type, distinct from mononuclear phagocytes and lymphocytes.

Autoradiographic detection of DNA polymerase containing nuclei in sarcoma 180 ascites cells.

ABSTRACTA technique has been developed allowing the autoradiographic detection of incorporation of 3H‐thymidine‐5′‐triphosphate (3H‐TTP) into nuclear DNA of smears of Sarcoma‐180 (S‐180) mouse ascites tumors under the direction of the cell's own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg++, and Ficoll, with the cell's own DNA acting as primer. The incorporation of 3H‐TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg++ and produces a product which is DNase sensitive and RNase resistant.DNA polymerase activity, as studied in a cell free assay, decreases with tumor age. This correlates well with a decreasing 3H‐TTP labeling index in autoradiographs of aging tumors. The 3H‐TTP labeling index has also been shown to exceed but parallel the in vivo3H‐thymidine (3H‐TdR) pulse labeling index for all tumor ages examined.In at least some cell systems DNA polymerase seems characteristic of cells in cycle. The autoradiographic detection of nuclei containing the enzyme offers a new tool for the study of tumor cytokinetics.