Synchronization of mouse 3T3 and SV40 3T3 cells by way of centrifugal elutriation

Populations of G1 phase 3T3 and SV40 3T3 mouse fibroblasts have been isolated from exponentially growing cultures by the technique of centrifugal elutriation. Return of the G1 phase cells to growth conditions results in their synchronous passage through the cell cycle, as determined from monitoring of cell number, [³H]thymidine ([³H]TdR) incorporation and fraction of [³H]TdR labeled nuclei. The durations of G1, S and G2 phases are consistent with values obtained by previous investigators using conventional induction techniques for synchronization. The method for isolation of the G1 phase cells is rapid, the yield is high and the process does not appear to alter the temporal aspects of the cell cycle in either cell type.     

Cell cycle parameters of 3T3 cells cultured as aggregates

Summary BALB/c 3T3 cells cultured as aggregates were examined by two independent techniques to determine whether or not cells accumulated at a specific point in the cell cycle, and if so to determine the point at which they accumulate. Replating cells onto dishes followed by pulse labeling with [³H]thymidine and autoradiography indicated that aggregate-cultured cells were in the same phase of the cell cycle as cells cultured as confluent monolayers. Flow microfluorometry confirmed that 75% of the aggregate-maintained cells were arrested in G₀ or G₁, with 25% distributed throughout the rest of the cell cycle. Labeling and mitotic indices of cells in aggregates were also consistent with about 20 to 25% of the cells being in S+G₂=M phases of the cell cycle at any time. This work was supported by PHS Grant CA20323 and NSF Grant PCM 74-15092 to H. G., who is also a Harry H. Pinney Cancer Scholar.     

SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([³H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([³H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [³H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G₁ because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.     

THE EFFECTS OF CYTOSINE ARABINOSIDE ON THE CELL CYCLE OF CULTURED HUMAN LEUCOCYTES: A MICRODENSITOMETRIC AND AUTORADIOGRAPHIC STUDY

PHA-stimulated leucocytes treated with cytosine arabinoside showed a changed uptake of [³H]thymidine, a lowered mitotic index and chromosome damage. Combined autoradiography and Feulgen microdensitometry demonstrated inhibition of DNA synthesis. Cells in the S period of the cell cycle were arrested in their progress, and cells newly entering S accumulated at the beginning of this period. At stronger concentrations these effects on the cell cycle were complicated by the cytotoxic effect of cytosine arabinoside. The inhibition of DNA synthesis is considered in the light of the chromosomal damage and megaloblastic changes caused by cytosine arabinoside, and is compared to the different pattern of DNA arrest found in megaloblastic anaemia.     

S phase synchrony in monolayer CHO cultures

Parameters are described for reproducible S phase synchrony of Chinese hamster ovary cells growing in monolayer, adapting a method described by Tobey & Crissman [1] for CHO cells growing in suspension culture. Cells are collected at the G1/S boundary in hydroxyurea after reversal of an early G1 block induced by isoleucine deprivation. The entire population enters the S period within 60 min after removal of hydroxyurea and proceeds through the S period with minimal decay of synchrony, as evidenced by autoradiographic and rate studies on [³H]TdR uptake. In addition, a method is described for obtaining cells synchronized during two successive S periods. The presence of hydroxyurea during G1 does not measurably affect the rate of uptake of [³H]uridine or [³H]leucine into TCA-insoluble material; however, cultures released from the hydroxyurea block at 10 h incorporate slightly more [³H]uridine (but not [³H]leucine) in the next 6 h than cultures maintained in hydroxyurea over this interval. Delaying entry into S with hydroxyurea for as long as 15 h does not significantly change the initial rate or duration of DNA synthesis upon removal of hydroxyurea, arguing against the build-up of substances responsible for initiation of replicons. Furthermore, if DNA synthesis is delayed with hydroxyurea in one cell cycle, a constant minimal interval of 15 h elapses before the population enters into the next S phase, suggesting that the timing of the S period is coupled to the timing of the previous S.     

Coexpression of two thermosensitive defects in a Chinese hamster cell line : Manifestation of a G1 block associated with a mitosis defect

Asynchronous cultures of ts12, an anchorage-dependent derivative of the thermosensitive Chinese hamster cell line ts111, show a rapid drop in [3H]thymidine incorporation with accumulation of the cells in the G1 and in the G2 phases of the cycle, when shifted from 34.5 to 39.4 degrees C. Shift-up experiments carried out after either isoleucine deprivation or synchronization at 39.4 degrees C, locate the execution point of a ts function in late G1 (2.5-3 h before S). However, stimulation of proliferation of a high density-arrested population allows a fraction of the cells to enter S. In addition to the G1 ts defect, ts12 expresses a slight cytokinesis defect at 39.4 degrees C (8-15% binucleate cells). The results suggest that altered processes are taking place at a post-metaphasic stage during the first hours after the shift-up. When populations are synchronized by a thymidine block and released at 39.4 degrees C, multinucleate cells in addition to binucleate cells are observed. Part of these multinucleate cells result from abnormal karyokinesis without inhibition of cytokinesis. Evidence is presented suggesting that excess thymidine allows the re-expression of the multinucleation phenotype of ts111.     

RADIOTOXICITY OF INCORPORATED [3H]THYMIDINE AS STUDIED BY AUTORADIOGRAPHY AND FLOW CYTOMETRY CONSEQUENCES FOR THE INTERPRETATION OF FLM DATA

The radiotoxic effects of incorporated [³H]thymidine on proliferation kinetics of flash-labelled (30 min, 0.3 μCi/ml, 40 Ci/mM) L-929 cells in vitro were studied by means of autoradiography and flow cytometry. the flow cytometric results obtained by applying the BUdR-33258 Hoechst technique, using new evaluation procedures, showed that the labelled cells are delayed in their progression through the S and G₂+ M phases, leading to mitotic delay. From autoradiographs, the fraction of labelled mitoses was determined and, in addition, the ratio of labelled and of unlabelled mitotic cells to all cells. the radiotoxic effects are not evident from the FLM curve, even if the ratio of labelled mitotic cells to all cells shows a highly distorted shape. A mathematical model has been developed that describes the perturbed cell kinetics due to radiotoxic effects of the incorporated [³H]thymidine. These findings have considerable consequences for the interpretation of autoradiograhic data, especially of labelled mitoses curves.     

Induction of differentiation and arrest of proliferation of mouse myeloid leukemia M1 cells

The relationship between differentiation and the cell cycle of mouse myeloid leukemia M1 cells was studied. The cells were induced to differentiate into macrophage-like cells by treatment with conditioned medium (CM) of hamster embryo cells. CM-treated cells traversed the S phase of the cell cycle at least once, then a fraction of the cells lost the ability to enter the S phase and accumulated in the G1 phase. Incorporation of [³H]thymidine in phagocytosis-induced cells decreased after 12–18 h of CM treatment. The morphology of the differentiated cells changed and the nucleus-cell ratio (NCR) of the individual cells decreased significantly between 12 h and 24 h of CM treatment. The decrease in NCR was well associated with arrest of proliferation in the G1 phase of the cells. The results suggest that G1 arrest of CM-treated M1 cells is an expression of cellular characteristics encoded in the differentiation program.     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

Effect of Methotrexate On Cells In A Keratinized Epithelium With an Active Thymidine Salvage Pathway Assessed By Autoradiography*

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m², injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [³H]TdR and [³H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [³H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)     

Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.     

The effect of microwave radiation on the cell genome

Cultured V79 Chinese hamster cells were exposed to continuous radiation, frequency 7.7 GHz, power density 30 mW/cm2 for 15, 30, and 60 min. The parameters investigated were the incorporation of [3H]thymidine and the frequency of chromosome aberrations. Data obtained by 2 methods (the incorporation of [3H]thymidine into DNA and autoradiography) showed that the inhibition of [3H]thymidine incorporation took place by complete prevention of DNA from entering into the S phase. The normal rate of incorporation of [3H]thymidine was recovered within 1 generation cycle of V79 cells. Mutagenic tests performed concurrently showed that even DNA macromolecules were involved in the process. In comparison with the control samples there was a higher frequency of specific chromosome lesions in cells that had been irradiated. Results discussed in this study suggest that microwave radiation causes changes in the synthesis as well as in the structure of DNA molecules.     

Mitochondrial protein synthesis in chinese hamster cells (line CHO) synchronized by isoleucine deprivation

Mitochondrial protein synthesis was measured in line CHO cells after phases of the cell cycle were synchronized by isoleucine deprivation or mitotic selection. Maximum incorporation of [³H] leucine into mitochondrial polypeptides occurred within 2 hours after isoleucine was added to initiate G₁ traverse. In cells synchronized in G₁ by mitotic selection, the rate of mitochondrial protein synthesis was fairly constant throughout the cell cycle. SDS-polyacrylamide gel electrophoretic profiles of labeled mitochondrial polypeptides were similar in cells synchronized by either isoleucine deprivation or mitotic selection. Obvious changes in the distribution of polypeptides were not detected during various phases of the cell cycle. The increased rate of incorporation of [³H] leucine into mitochondrial polypeptides after reversal of G₁-arrest may indicate that mitochondrial protein synthesis and possibly mitochondrial biogenesis are synchronized in CHO cells deprived of isoleucine.     

Comparative heterokaryon study of cellular senescence and the serum-deprived state

Autoradiographic patterns of DNA replication in serum-deprived human diploid fibroblast-like cells (HDFC) and “senescent” HDFC have been compared in two types of heterokaryons. Each was fused to low passage, proliferating HDFC and, in separate experiments, to HeLa cells. Sequential 1 h pulses with [³H]thymidine were initiated at short intervals following fusion. In all hybridizations serum-deprived and senescent cells behaved identically. Upon fusion to HeLa cells, DNA synthesis in the quiescent nuclei occurred in a wave between 3 and 30 h after fusion. When either serum-deprived or senescent HDFC were fused to young proliferating HDFC, the nuclei of the latter were blocked from entering the S phase if fusion occurred at least 3 h before the G/S boundary. These findings are consistent with the interpretation that one or more crucial steps in G0 occurs 3 h before the G1/S interface. That young serum-deprived (G0) HDFC behave identically to senescent cells in these hybridization studies suggests that the mechanism of arrest in each state might share a final common pathway, and a model based on these observations is proposed.     

Autoradiographic Studies of [methyl-3H]Thymidine Incorporation in a Cyanobacterium (Microcystis wesenbergii)-Bacterium Association and in Selected Algae and Bacteria

The present investigation showed by means of autoradiography that the cyanobacterium Microcystis wesenbergii did not incorporate [³H]thymidine at nanomolar concentrations, whereas its associated heterotrophic bacteria appearing in the gelatinous cover of the cyanobacterium became labeled. Several other tested cyaobacteria and algae did not incorporate [³H]thymidine.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

A Method For Measuring the Generation Time and Length of Dna Synthesizing Phase of Clonogenic Cells In A Heterogenous Population

Information on the cell cycle of progenitor cells in haemopoietic tissue is useful for understanding population control under physiological and abnormal conditions. Unfortunately, methods that have been developed for measuring cell cycle parameters are applicable only to cells of homogenous populations and not to morphologically non-recognizable progenitor cells such as colony forming units (CFU) that are present at low frequency in a heterogenous population. to circumvent this difficulty, a method was developed to measure CFU cell cycle parameters based on specific killing of cells in S phase by [³H]thymidine ([³H]TdR). This was done by estimating the number of CFU killed following exposure of the cell suspension to [³H]TdR for various time periods. Since cycling CFU are continuously entering S phase, a linear curve relating the percentage CFU-kill to the length of exposure of the cells to [³H]TdR in culture can be obtained. the slope of the curve (percentage kill/hr) indicates the rate that CFU enter the S phase and travel through the cell cycle. the inverse of this value will then represent a time period for CFU to move through a complete cell cycle (generation time). the length of S phase can then be obtained by multiplying generation time by the fraction of cells in S phase at time zero. This method has been used to measure generation time and length of S phase of three kinds of haemopoietic progenitor cells: mouse granulocyte-macrophage CFU, human T lymphocyte CFU and CFU from regenerating mouse spleens. This method should be applicable to any normal or neoplastic clonogenic cell populations and the latter could be either of haematological or of solid tumour origin.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Commitment during stenotele differentiation in Hydra is localized near the S/G2 boundary in the terminal cell cycle

The timing of commitment during stenotele differentiation in Hydra was determined. Regeneration of isolated distal regions of the body column induces stenotele differentiation. The kinetics of appearance of committed stenotele precursors was determined in such regenerating pieces. Using [³H]thymidine labeling and hydroxyurea sensitivity, the G1/S and the S/G2 boundaries of the precursor population was also determined. Comparison of these results indicates that stenotele commitment is localized near the S/G2 boundary in the terminal cell cycle of nests of precursor cells.     

Comparative induction of unscheduled DNA synthesis by physical and chemical agents in non-proliferating primary cultures of rat hepatocytes

The incorporation of [³H]thymidine into DNA due to unscheduled DNA synthesis (UDS) induced by N-OH-2-acetylaminofluorene (N-OH-AAF), aflatoxin B₁ (AFB₁), ethyl methanesulfonate (EMS) and ultra-violet light was quantitated by autoradiography and by scintillation spectrometry on acid precipitable macromolecules or DNA insolated by isopycnic banding in cesium chloride (CsCl). Dose-dependent increases in UDS due to N-OH-AAF and AFB₁ treatment were found. Only 2-fold increases at the highest dose levels were found, however, when incorporated [³H]thymidine was quantitated by scintillation spectrometry. Seven, 11, and 25-fold increases in UDS induced by AFB₁, N-OH-AAF and ultra-violet light, respectively, were found when incorporated [³H]thymidine was quantitated by autoradiography, indicating a high sensitivity for detecting ‘long patch’ repair by this technique. Scintillation spectrometry was completely ineffective in detecting EMS-induced UDS, whereas autoradiography demonstrated a small, but significant induction in [³H]thymidine incorporation at high dose levels. The non-proliferative nature of the primary hepatocyte prohibits the uniform radioactive prelabeling of DNA, necessary in other techniques, for the detection of ‘short patch’ repair induced by compounds such as EMS. Therefore, the sensitivity of the primary cultured rat hepatocyte in conjunction with UDS for detecting DNA damage caused by mutagens and carcinogens which induce ‘short patch’ repair may be limited to the autoradiographic analysis of the unscheduled incorporation of [³H]thymidine.