Sesquiterpene lactones and the diterpene 5-epi-icetexone affect the intracellular and extracellular stages of Trypanosoma cruzi

Chagas disease is a major health problem in Latin America and is caused by the parasitic protozoan Trypanosoma cruzi. Although many drugs have been used to alleviate the disease, these have been ineffective in the chronic phase and have also presented numerous side effects on patients. In this study we tested the effect of three sesquiterpene lactones (dehydroleucodine, helenalin and mexicanin) and a diterpene (5-epi-icetexone) on parasites (Y-strain) grown in host cells. At 48h of treatment, the number of amastigotes inside the cells was lower than in the controls. This effect was observable at concentrations of 1.5-3.8μM, which are of low cytotoxicity to host cells. In addition, the compounds caused a decrease in the percentage of infected cells. The treatments also reduced the presence of trypomastigotes in the extracellular medium. In all cases, helenalin was the most potent. The number of parasites per cell at 24h indicates the occurrence of multiple infection, which would also be affected by the compounds. However, we should not discard an effect on the proliferation and survival of parasites within the host cells. On the other hand, an additional effect on the differentiation of parasites and/or the survival of extracellular trypomastigotes might be possible. We conclude that these compounds are very effective against T. cruzi possibly by multiple mechanisms.     

Prostaglandins A and J arrest the cell cycle of cultured vascular smooth muscle cells without suppression of c-myc expression.

The effects of prostaglandins (PGs) A and J, which are anti-tumor eicosanoids, on the proliferation of cultured vascular smooth muscle cells were investigated. Serum-stimulated DNA synthesis was potently inhibited by PGA1, PGA2, PGJ2, and delta 12-PGJ2 in similar dose-dependent fashions. The effects of PGA1 and PGA2 were reversible when they were removed from the culture media, whereas recoveries were only partial in the cells treated with PGJ2 and delta 12-PGJ2. PGs were effective even if they were added immediately before entry into S phase. Inhibition of DNA synthesis was sustained when hydroxyurea, which blocks cell cycle at the G1/S border, was added after the removal of PGA2, and vice versa; PGs blocked DNA synthesis when they were added after the removal of hydroxyurea. Levels of c-myc mRNA formed two peaks during the G1 phase, at 1-2 h and at 8-12 h. The PGs did not affect the first elevation, but enhanced the second and sustained it up to 18-24 h, whereas in controls, c-myc mRNA decreased quickly after entry into S phase. The rate of degradation of c-myc mRNA was much smaller in PG-treated cells than in nontreated cells. We conclude, therefore, that PGA and PGJ inhibit a crucial event(s) in the cell cycle occurring at the G1/S border, but that this inhibition is not accompanied by the reduction in c-myc gene expression in contrast with some types of tumor cells treated with PGs.     

Diterpenes from Alomia myriadenia (Asteraceae) with cytotoxic and trypanocidal activity

Further investigation of the aerial parts of Alomia myriadenia revealed an halimane diterpene identified as ent-8S,12S-epoxy-7R,16-dihydroxyhalima-5(10),13-dien-15,16-olide along with the known ent-16-hydroxylabda-7,13-dien-15,16-olide, ent-12R-hydroxylabda-7,13-dien-15,16-olide, 6,7-methylenedioxycoumarin (ayapin), and kaempferol-7-methylether (rhamnocitrin). Evaluated in a panel of human cancer cell lines, the 16-hydroxylabade diterpene was the most active, showing an ED(50) value of 0.3 mug/ml against Lu1 (human lung cancer) cells. Tested in vitro against Trypanosoma cruzi in infected murine blood, this compound caused lysis of 100% of the parasites at 250 mug/ml.     

Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication

Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells.     

Isoprenoids and astroglial cell cycling: diminished mevalonate availability and inhibition of dolichol-linked glycoprotein synthesis arrest cycling through distinct mechanisms.

Primary astroglial cultures were used to compare the relationships to cell cycling of dolichol-linked glycoprotein synthesis, and of availability of mevalonate, the precursor of dolichol and other isoprenoid lipids. With shift-up to 10% serum (time 0) after 48 h of serum depletion, the proportion of cells in S phase (bromodeoxyuridine immunofluorescence) remained under 15% for 12 h, then increased by 20 h to 72 +/- 10%; DNA synthetic rates (thymidine incorporation) increased 5-fold. S phase transition was prevented by addition at 10-12 h of tunicamycin, an inhibitor of transfer of saccharide moieties to dolichol. Mevinolin, an inhibitor of mevalonate biosynthesis, also blocked cycle progression when added at this time. However, mevinolin markedly inhibited the isoprenoid pathway, as reflected by over 90% reduction of sterol synthesis, without inhibiting net glycoprotein synthesis. Removal of mevinolin after a 24 h exposure delayed S phase until 48 h, following recovery of sterol synthesis, even though kinetics of glycoprotein synthesis were unaffected. Tunicamycin removal after 24 h spared sterol synthesis, but caused delay of S phase until 72 h, following recovery of glycoprotein synthesis. In mevinolin-treated cultures, S phase transition was restored by 1 h of exposure to mevalonate at 10 h, although cycling was thereby rendered sensitive to inhibition by cycloheximide and by tunicamycin. Cell cycle progression following hydroxyurea exposure and release was unaffected by mevinolin, tunicamycin, or cycloheximide. Thus, in these developing astroglia, mevalonate and its isoprenoid derivatives have at least two cell cycle-specific roles: dolichol-linked glycoprotein synthesis is required at or before the G1/S transition, while a distinct mevalonate requirement is apparent also in late G1.     

Dependence of Cell Division on the Completion of Chromosome Replication in Caulobacter crescentus

The relationship between chromosome replication and cell division in the stalked bacterium Caulobacter crescentus has been investigated. Two compounds, hydroxyurea and mitomycin C, were found to inhibit completely deoxyribonucleic acid (DNA) synthesis while allowing continued cell growth and elongation. When these inhibitors were added to exponentially growing cultures, cell division stopped after 38 min when hydroxyurea was used and after 33 min when mitomycin C was used. The period of continued cell division corresponds closely to the period previously determined for the postsynthetic gap (G2) in the DNA cycle of this organism. These results indicate that cell division is coupled to the completion of chromosome replication in C. crescentus.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

Effect of interferon on growth and division cycle of Friend erythroleukemic murine cells in vitro

The administration of appropriate doses of interferon to cultures of Friend leukemia cells causes a pronounced inhibition of cell growth. Several lines of evidence indicate that this effect is due to interferon itself, rather than to unknown contaminants of interferon preparations. Autoradiograph analysis of growth parameters of Friend leukemia cells during treatment with interferon demonstrates that the rate of entry into the S phase, the percent decline of unlabeled mitoses, and the mitotic indexes are significantly lower in interferon- treated cell cultures than in control untreated cultures when tritiated thymidine was added 12 h after the administration of interferon. These data indicate that fractions of interferon-treated cell population are delayed in both G1 and in G2 phases of the cell cycle. This was confirmed by exact measurements of the length of the various phases of the cycle. The interferon-induced inhibition of growth of Friend leukemia cells is reversible after removal of the compound. Autoradiograph data obtained from control cultures and from cultures previously treated with interferon that had been washed free of interferon and reseeded in interferon-free medium, demonstrate that during the first 12 h after removal of interferon, a large majority of the cells previously treated with interferon had a deranged flow into the S phase, a high number of unlabeled mitoses, and a low mitotic index. These data provide further evidence for the above-mentioned prolongations of G1 and G2 phases of the cell cycle. All growth parameters tested reverted to normal values within 12 h after washing out interferon.     

Hydroxyurea treatment affects the G1 phase in next generation CHO cells

DNA replication kinetics were studied in populations of synchronized CHO cells treated in the previous generation with hydroxyurea. These CHO cells were re-synchronized by selective detachment of mitotic cells after previously synchronized G1 traversing cultures were treated with 0.1 mM and 2 mM hydroxyurea for 9 and 13 h. Our results show that these cells exhibit a shortening of G1 of at least 1 h relative to cells selected in mitosis from untreated exponentially growing cultures. Survival studies indicated that the hydroxyurea treatments did not affect plating efficiencies. Cell viability was reduced when the initially synchronized populations were blocked with 2 mM, but not 0.1 mM hydroxyurea for greater than 13 h. DNA replication measurements after these blocks showed that all cultures treated with 2 mM hydroxyurea for either 9, 13 or 15 h were blocked at the same point near the G1/S boundary, and then progressed through S phase with similar kinetics. The observed shortening of G1 in the next generation of these cells was independent of both the concentration (0.1 or 2.0 mM) and the time (9 or 13 h) of the hydroxyurea block. These results suggest that specific events relating to the next cell generation can be uncoupled from DNA synthesis and can occur when hydroxyurea inhibits normal cell cycle traverse of G1 cells into and through S phase.     

Partial elimination of G1 and G2 periods in higher plant cells by increasing the S period

Meristematic cells from Allium cepa L roots can attain a steady-state of growth at both 15 and 25 °C in the presence of drugs, hydroxyurea and 5-amino-uracil, which reduce the rate of DNA synthesis. These drugs, at used concentrations, significantly lengthen the S period without altering the cell growth rate, as indicated by the maintenance of the generation time. It has been observed that steady-state populations respond to a gradual increase in S by a reduction of G2 until a minimum value; with larger lengthening of S, both G1 and G2 are reduced. Natural synchronous populations have been used to study cell cycle parameters during transition from the physiological steady-state to the new one created by the presence of the drug. G2 (but not G1) is reduced during transition even in the presence of maximum drug concentrations that do not alter the cell growth rate. Both the S period and the division time are lengthened during transition. These observations support the concept that certain fractions of G1 and G2 are expendable, because they have no role in the DNA-division sequence of cell cycle events. We conclude that cell size regulates the length of these fractions by means of a negative correlation.     

Molecular and cellular events during the yeast to mycelium transition in Sporothrix schenckii

Unbudded singlets from exponentially growing yeast cells of Sporothrix schenckii were harvested, selected by filtration and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 degrees C. These conditions supported only the development of the mycelial form of S. schenckii in a reproducible manner which allowed further analysis of the early cellular events occurring during the yeast-to-mycelium transition. The relationship between macromolecular synthesis (DNA and RNA synthesis) and nuclear division, hyphal growth and septum formation were investigated during germ tube formation. RNA synthesis started 0 to 3 h after the induction of germ tube formation, followed by DNA synthesis and the first nuclear division, which took place between 3 and 6 h. Germ tube formation followed nuclear division and was first evidenced 6 h after the induction of germ tube formation, but was not completed until 12 h after inoculation. Septation was first observed in these germ tubes at the mother cell-germ tube junction 6 h after induction. Addition of hydroxyurea, an inhibitor of DNA synthesis, to the medium, also inhibited nuclear division and germ tube growth, suggesting that these processes in S. schenckii are dependent upon DNA synthesis.     

A method of synchronization of normal and malignant human cells in culture.

A method is described for providing reproducible S phase parasynchrony in both normal mesenchyme and transformed epithelia. Cells were seeded at low density in medium containing 10% serum. 24 h later the serum concentration was reduced to 0.5%. After 110th the cells were collected at the G1/S boundary in fresh medium containing 10% serum plus 2.5mM hydroxyurea over 20h. After removal of hydroxyurea and trypsinization the re-plated cells entered the S phase with a high degree of synchrony, as judged by autoradiography, pulse-labelling with 3H-thymidine, cell growth and time lapse cinematography. By 6h after synchronization 80% of the population had entered the S phase and between 10-13h 70% went through mitosis.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

The "Point of no Return" Concept in Cell Division. The Effects of Some Metabolic Inhibitors on Synchronized Chlorella pyrenoidosa

The coarse of growth and cell division in synchronized cultures of Chlorella pyrenoidosa was studied after the addition of metabolic inhibitors at differing times during the cell cycle (14 h light - 10 h darkness with nitrate as nitrogen source. 12 h light: 12 h darkness with urea as nitrogen source). Dinitrophenol (DNP) added to a final concentration of 0.3 mM at any time in the synchronization cycle, the compound remaining in the suspension from the time of addition to the end of the dark period, inhibited spore formation completely. Growth measured as increase in cell volume was less sensitive to the action of the inhibitor. Chloramphenicol (CAP) added dining the 0–5 h interval to a final concentration of 0.1 mM resulted in 80 per cent inhibition of cell division. Similar treatment started at successive times thereafter resulted in a gradual decrease of the inhibition. Treatment at the 14th hour and during the dark period did not affect the sporulation. Similar experiments with 0.9 mM puromycin added at various times during the illumination period gave almost complete inhibition of cell division, while the growth was reduced by only 25 per cent. para-Fluorophenylalanine (p-FPhe) at 3.3 × 10^?2 mM stopped cell division nearly completely irrespective of addition time in the light period. Addition during the dark period also prevented an increase in the number of tree cells. In this case about half of the cells produced spores which were not released. It is concluded that DNP inhibits all stages of preparation for cell division, as well as the division process itself. With CAP a genuine transition point of preparation for cell division was observed, although its interpretation as related to protein synthesis is somewhat uncertain. With puromycin and p-FPhe no transitions were observed.     

Trypanosoma cruzi: interaction with vertebrate cells. DNA synthesis and growth of intracellular amastigotes and their relationship to host cell DNA synthesis and growth.

DNA synthesis of intracellular Trypanosoma cruzi amastigotes, following the infection of bovine embryo skeletal muscle (BESM) cells, was studied by autoradiography. After penetration, there was a prereplicative lag period (similar to or approximately 12 h) followed by a synchronous round of DNA synthesis which was found to be independent of parasite number/BESM cell cand the host cell DNA synthesis cycle. Parasite reproduction occurred, for the first time, at approximately 21 h postinfection. It was concluded that T. cruzi trypomastigotes are in the G1/G0 phase of their cell division cycle and that after penetration parasite reproduction occurs independent of events controlling host cell DNA synthesis and growth. The early synchronous growth of intracellular amastigotes should facilitate further studies on the biochemical events controlling trypomastigote-to-amastigote transformation and amastigote reproduction. A further application is envisaged for studies on the mode of action of drugs with trypanocidal activity.     

Changes in the nuclear distribution of cyclin (PCNA) but not its synthesis depend on DNA replication.

Synthesis of cyclin in serum-stimulated quiescent 3T3 cells increases shortly before DNA synthesis after 10 h of stimulation, reaching a maximum after 16 h. Inhibition of DNA synthesis by hydroxyurea does not affect the increase of cyclin following stimulation, as determined by quantitative two-dimensional gel electrophoresis. The levels of cyclin decrease dramatically at the end of the S-phase. Cells kept in the presence of hydroxyurea (G1/S boundary) do not show this decrease in cyclin, indicating that its amounts are regulated by events occurring during the S-phase. Immunofluorescence studies of serum-stimulated quiescent cells in the presence of hydroxyurea, using proliferating cell nuclear antigen (PCNA) autoantibodies, confirm the results obtained by protein analysis. They also reveal that there are dramatic changes in the nuclear distribution of cyclin and that these depend on DNA synthesis or events occurring during the S-phase. Cyclin (PCNA) is no longer detectable at the end of the S-phase. However, pulse-chase experiments indicate that this protein is very stable, suggesting that it possibly interacts with other macromolecules rendering it inaccessible to the antibody. These results strengthen the notion that cyclin is an important component of the events leading to DNA replication and cell division.     

Flow cytometric evaluation of the cytotoxicity of novel antiviral compounds

Two acyclic analogs of bromotubercidin were tested for cytotoxic effects on uninfected cells by monitoring cell growth and measuring cell cycle perturbations using flow cytometry. As reported elsewhere, 5-bromotubercidin analogs in which ribose was replaced by 2-hydroxyethoxymethyl (compound 102) or by 1,3-dihydroxypropoxymethyl (compound 183) were potent inhibitors of human cytomegalovirus (HCMV) replication in vitro (Pudlo et al.: Journal of Medicinal Chemistry 31:2086-2092, 1988). Because these compounds also inhibited the growth of uninfected cells, we performed kinetic studies with an established neoplastic line of human cells (KB) using flow cytometry. Growth of KB cells treated with either compound 102 or 183 were inhibited in a dose-dependent manner. Growth inhibition by compound 183, however, was not fully expressed for at least 24 h. DNA analysis by flow cytometry showed that a 4-h incubation with 10 microM compound 102 caused a decrease of cells in G2/M phase. Cells began to accumulate in early S phase by 12 h of incubation, leading to mid S phase accumulation at 21 h. Compound 183 at 10 microM slightly decreased the number of cells in G2/M phase after a 4-h incubation, and led to accumulation of DNA in S phase after a 12-h incubation. By 24 and 30 h, DNA histograms appeared similar to those of control cells but with a slight accumulation of the population in early S phase. In separate experiments, drugs were removed following a 24-h incubation. After removal of compound 102, KB cell growth resumed with a normal population doubling time. In contrast, the effects of compound 183 were not reversible, suggesting the two compounds acted by different biochemical mechanisms.     

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.     

Comparison of synchronized Chinese hamster ovary cells obtained by mitotic shake-off, hydroxyurea, aphidicolin, or methotrexate

Synchronized cell populations are necessary to study many aspects of cell biology. We have developed a method to obtain highly synchronized Chinese hamster ovary cell populations in S phase or G2 phase by utilizing mitotic selection followed by incubation with either hydroxyurea, aphidicolin, or methotrexate for 12 h. Flow cytometry analysis shows that the coefficient of variation in the spread of the cell population in S phase is as low as 6%. Drug toxicity studies compare the effects of the various drugs on G1 and S phase cells. The use of aphidicolin or hydroxyurea results in the most highly synchronized cell populations, but methotrexate yields inadequate synchronization. These results demonstrate that both aphidicolin and hydroxyurea are useful drugs for obtaining highly synchronized cell populations after an initial synchrony in mitosis. Aphidicolin is perhaps the best choice because of less toxicity to S phase cells when used in low concentrations.