Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10⁻⁵M, 10⁻⁶M or 10⁻⁷M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10⁻⁵ M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G₀/G₁ phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P 0.05), 13.9% at 24 h (P 0.001), and 76.5% at 48 h (P 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P 0.005) and 12 h (P 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G₁/S transition by prolonging G₂/M and through decreasing the DNA synthesis rate     

Age-related changes in the cell kinetics of rat foot epidermis

Abstract. The durations of the cell cycle and its component phases have been determined for the basal layer of the epidermis of the skin from the upper surface of the hind foot of the rat using single pulse [³H]-thymidine labelling and the percent labelled mitosis (PLM) technique. Rats of three age groups were used, namely 7, 14 and 52 weeks. The duration of DNA synthesis (T_s) and the G₂ plus M phase (Tg₂± m) were comparable in 7-week and 52-week-old rats ( P > 0–1). The major difference between 7-week and 52-week-old rats was in the duration of the G₁ phase (Tg₁). In 7-week-old rats Tg₁ was 15.0 ± 0.8 h and in 52-week-old rats Tg₁ was 31.2 ± 3.5 h. A consequence of this variation was that the overall duration of the cell cycle was longer in 52-week-old rats (53.9 ± 5.3 h) than in 7-week-old rats (30.1 ± 1.3 h).
Difficulties were found in fitting a simple curve to the PLM data for 14-week-old rats. This suggests that the proliferative cell population of the epidermis of rats of this age group may be heterogeneous. A satisfactory fit to the data was obtained using a computer model which assumed that the proliferative population of the epidermis of 14-week-old rats was a mixture of cells with cell cycle parameters the same as those of the 7-week and the 52-week-old rats. These two sub-populations of relatively slowly and rapidly proliferating cells were present in the ratio of 2:1.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Abstract. Cellular uptake of [³H]thymidine ([³H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10^-18 mol/cell. The pool activity of G₁ cells was unmeasurable but rose to maximum values at the border of the G₁-S phase. It decreased again during G₂. The [³H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min^-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Elucidation of the DNA Synthetic Cycle of Entamoeba Spp. Using Flow Cytometry and Mathematical Modeling

ABSTRACT. We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G₁, S, and G₂ phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G₁, S, and G₂ proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 × 10^-14 g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 × 10^-14 g DNA/cell.     

Cell Cycle-Dependent Expression of Specific Opiate Binding with Variable Coupling to Adenylate Cyclase in a Neurotumor Hybrid Cell Line NG108–15

Abstract: Monolayer cultures of neuroblastome × glioma hybrid (clonal) cell line NG108-15, synchronized by the isoleucine/glutamine deprivation method, showed maximal expression of opiate binding sities at the same point in the cell cycle at which prostaglandin E₁(PGE₁) had a maximum stimulatory effect of cyclinc AMP synthesis. However, the capacity of enkephalin (D-Ala²D-Leu⁵] to block the stimulation of cyclic AMP synthesis by PGE₁ was not related to the number of opiate receptors expressed. The K₁ for the inhibition of cyclic AMP synthesis by opioid peptides increased substanitilly during the period of the cell cycle at which maximal expression of opiate binding sites occurred, making the effectivel level of inhibition of adenylate cyclase activity by 0.1μM enkephalin [D-Al²D-Leu³] the same throght the cell cycle. Data are presented to suggest the enkephalin receptor coupling to adenylate cyclase, via a GTP-binding protein, is maximal during G₁ phase (which may approximate the state of the differentiated neuron) and minimal during S + G₂ phase, just prior to cell division, when many receptors are uncoupled.     

Cell cycle progression in human cells following re-oxygenation after extreme hypoxia: consequences concerning initiation of DNA synthesis

Abstract. The initiation of DNA synthesis and further cell cycle progression in cells during and following exposure to extremely hypoxic conditions in either G₁ or G₂+M has been studied in human NHIK 3025 cells. Populations of cells, synchronized by mitotic selection, were rendered extremely hypoxic (< 4 p.p.m. O₂) for up to 24n h. Cell cycle progression was studied from flow cytometric DNA recordings. No accumulation of DNA was found to take place during extreme hypoxia. Cells initially in G₁ at the onset of treatment did not enter S during up to 24 h exposure to extreme hypoxia, but started DNA synthesis in a highly synchronous manner within 1.5 to 2.25 h after reoxygenation. The duration of S phase was only slightly affected (increased by ≅10%) by the hypoxic treatment. This suggests that the DNA synthesizing machinery either remains intact during hypoxia or is rapidly restored after reoxygenation. Cells initially in G₂ at the onset of hypoxia were able to complete mitosis, but further cell cycle progression was blocked in the subsequent G^ Following reoxygenation, these cells progressed into S phase, but the initiation of DNA synthesis was delayed for a period corresponding to at least the duration of normal G₁ and did not appear in a synchronous manner. In fact, cell cycle variability was found to be increased rather than decreased as a result of exposure to hypoxia starting in G₂. We interpret these findings as an indication that important steps in the preparation for initiation of DNA synthesis take place before mitosis. Furthermore, the change in cell cycle duration induced by hypoxia commencing in G₁ is of a nature other than that induced by hypoxia commencing in other parts of the cell cycle.     

Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle

Abstract. Multivariate analysis of the expression of cyclin proteins and DNA content has opened new possibilities for the study of the cell cycle. By virtue of their cell cycle phase specificity, the expression of cyclins may serve, in addition to DNA content, as another marker of a cell's position in the cycle, and provide information about the proliferative potential of cell populations. Several applications of the methodology based on bivariate analysis of DNA content v . expression of B, E and D type cyclins are reviewed: 1 expression of cyclins by individual cells during their progression through the cycle can be studied, using exponentially growing cells without the necessity of cell synchronization or other perturbations of the cycle; 2 cells having the same DNA content but residing in different phases of the cycle (e.g. G₂ diploid v. G₁ tetraploid) can be distinguished; 3 cell transition from G₀ to G₁ and progression through G₁ (e.g. mitogen stimulated lymphocytes) can be assayed; 4 the population of proliferating cells can be distinguished from noncycling cells based on dual cell labelling with a G₁ and G₂ cyclin antibody; 5 cyclin restriction points can serve as additional cell cycle landmarks to map the point of action of antitumour drugs; 6 unscheduled expression of cyclins (e.g. the presence of cyclin B1 during G₁ and S) can be detected in several tumour transformed cell lines, possibly indicating disregulation of the machmery of cell cycle progression. The last finding 6 is of special importance, because such disregulation may be of prognostic consequence in human tumours.     

Normal G1/S transition and prolonged S phase within one cell cycle after seeding cells in the presence of an ornithine decarboxylase inhibitor

Abstract. We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G₁/S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine-flow cytometric method to more specifically study the G₁/S transition, the S phase length, and the progression of cells from S phase through G₂+ M and into G₁, after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G₁/S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G₁ phase occurred later than the effect on S phase progression. These results imply that the G₂+ M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO.     

Variability of ecdysteroid-induced cell cycle alterations in Drosophila Kc sublines

Abstract. The cell cycle of two lines isolated from Drosophila Kc cells was followed by flow cytofluorometry and cell counting. The first line is the 8-9K clone which grew in a medium supplemented with 5% serum; the second, named subline Kc0, grew in a serum-free medium. The stationary phase is characterized by a G₂ cell accumulation: 73% in the 8-9K clone and 50% in the Kc0 subline.
When the medium was supplemented with the steroid moulting hormone 20-hydroxyecdysone, more than 90% of 8-9K cells and 65% of Kc0 cells were progressively arrested in G₂. In the continuous presence of 20-hydroxyecdysone, most of the 8-9K cells remain G₂-arrested; no massive G₂ release into M was observed and only a few cells were able to divide. When treated for only 3 or 7 days, a transient release into M and proliferation occurred after hormone-free medium renewal, largely masked by G₂ cell death. These results are discussed in comparison with other reports on cell cycle alteration induced by ecdysteroids.     

Induction of replicative senescence by 5-azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH-3T3 cells

Abstract. To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96 h exposures to 4 μM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G₁ compartments of the second and third cell cycles of serum stimulated cells. The exit from the G₀/G₁ compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2–3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G₂ phase arrests and delayed G₀/G₁ phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G₁ phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.     

ON THE RESTING STAGES OF THE JB-1 ASCITES TUMOUR

Cytophotometric determination of single-cell DNA after repeated ³H-thymidine labelling of the JB-1 ascites tumour in the plateau phase of growth showed a massive accumulation of unlabelled cells with both G₁ and G₂ content. Autoradiography combined with cytophotometry or colcemid block demonstrated that some of these unlabelled cells were rapidly triggered into the cell cycle when plateau tumours were transferred to new hosts. This indicated that tumour cells may be held up in non-cycling stages corresponding to both the G₁ and the G₂ phase of the cell cycle.     

Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D₃ (VD₃). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G₁/G₀ phase, and a recently described G₂+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well-differentiating HL60-G cells exposed to one of ten analogs of VD₃, and compared the cell cycle effects of each compound with its potency as a differentiation-inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G₂ compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G₂+ M compartment, but preceded the G₁ to S, and the G₂ compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.     

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines

Abstract.   Objectives : This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G₂/M phases and consecutive cell survival. Materials and methods : Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. Results : By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G₂ phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G₂ accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly ( P <  0.01) increased the fraction of radiation-induced G₂ arrested cells 30–56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by γ-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. Conclusions : These results indicate that separase has additional targets involved in regulation of G₂ to M progression after DNA damage. Prolonged G₂ phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.     

cAMP IN THE CELL CYCLE OF THE DINOFLAGELLATE CRYPTHECODINIUM COHNII (DINOPHYTA)

The second messenger cAMP is a key regulator of growth in many cells. Previous studies showed that cAMP could reverse the growth inhibition of indoleamines in the dinoflagellate Crypthecodinium cohnii Biecheler. In the present study, we measured the level of intracellular cAMP during the cell cycle of C. cohnii . cAMP peaked during the G₁ phase and decreased to a minimum during S phase. Similarly, cAMP-dependent protein kinase activities peaked at both G₁ and G₂+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O₂'-dibutyryl (Bt₂)-cAMP directly stimulated the growth of C. cohnii . Flow cytometric analysis of synchronized C. cohnii cells suggested that 1 mM cAMP shortened the cell cycle, probably at the exit from mitosis. The size of Bt₂-cAMP treated cells at G₁ was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates.     

Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

A dual block to cell cycle progression in HL60 cells exposed to analogues of vitamin D,

Abstract. The physiologically active form of vitamin D₃, 1,25-dihydroxy-vitamin D₃, (1,25(OH)₂, D₃), induces differentiation of several types of myeloid leukaemia cells. The acquisition of monocyte-like phenotype is accompanied by slower progression through the cell cycle, and G₁, block has been reported to be the basis of this effect. It is shown here that human promyelocytic leukaemia HL60 cells treated with analogues of vitamin D₃, which are potent inducers of monocytic differentiation, have an additional cell cycle block. Exposure to 10-⁷m 1,25(OH)₂, D₃, or 1,25-(OH)₂,-16-ene-D₃ resulted in monocytic differentiation and the expected G₁, block evident at approximately 48 h in a rapidly differentiating variant of HL60 cells (HL60-G), and at 96 h in the more slowly differentiating HL60-240 cells. In addition, a G₂,+M block was noted at approximately 72 h in HL60-G and HL60-240 cells. Exposure to vitamin D₃, analogues also markedly increased the number of dikaryons, suggesting that cytokinesis was impaired more than karyokinesis. Treatment with a third analogue 25-hydroxy-16,23-diene-D₃, produced little differentiation and had minimal effects on the cell cycle parameters. These findings indicate that vitamin D₃, analogues regulate cell proliferation by control of the transition of G₁, and G₂,+M phases, reminiscent of the cdc2/CDK2 type of cell cycle control.     

Effects of cytosine arabinoside and daunorubicin on survival and cell cycle progression of chinese hamster ovary cells

Abstract. Kinetic and cytotoxic effects of cytosine arabinoside (Ara-C) and daunorubicin (DNR) on exponentially growing Chinese hamster ovary (CHO) cells were measured by flow cytometry and by a colony-forming assay, respectively. With Ara-C alone, increasing drug concentrations between 10^-7 M, for up to 27 hr, were associated with increased inhibition of cell progression through the S phase. Even at the very toxic concentration of 10^-4 M, however, cells were able to enter and progress slowly through S. DNR, which appears to enter these cells relatively slowly, was highly toxic even at 2 times 10^-7 M. It decreased the rate of progression through S phase and caused cells to accumulate in G₂, except at the highest concentration (2 times 10^-5 M), at which progression was inhibited throughout the cycle. Simultaneous exposure of the cells to Ara-C and DNR yielded cell cycle distributions similar to those of the former drug alone. When cells were exposed to a non-lethal dose of Ara-C and to a dose of DNR which was lethal to a fraction of the cell population (or conversely), either simultaneously or separated by a drug-free interval, small, but in some cases significant, drug interactions were observed. These effects were not caused by druginduced redistribution of cells within the cell cycle, but may have been related to the effects of the non-lethal drug on DNA synthesis rate.     

Influence of denervation on the regeneration of Pleurodele limbs

Abstract. A cytophotometric study of Feulgen-stained mesenchymal cell nuclei from regeneration blastemas of both innervated and denervated limbs over the 1st 7 days following the midbud stage showed a diminution of the percentage of cells in the S + G₂ phases and a corresponding augmentation of the percentage of cells in the G₀+ G₁ phases. This change, which was temporally correlated with the redifferentiation of the innervated blastemas, was greater in denervated blastemas, even though they do not redifferentiate. From these results, it is concluded that the denervation of midbud blastemas brings about either an extension of the G₁ phase or an exiting from the cell cycle to G₁ (G_0–1), or both phenomena.     

The effects of interleukin 4 on the cell cycle of a human B-cell lymphoma line

Abstract. Exposure of Farage, a human B-cell lymphoma line, to IL-4 for 3–11 days led to inhibition of tritiated thymidine ([³H]dT) uptake by the cells. Study of the incorporation of 5-bromodeoxyuridine by Farage cells showed that IL-4 reduced significantly the number of cells in the S phase of the cell cycle and increased the proportion of cells in the G₁ phase. Limiting dilution analysis of proliferation demonstrated that IL-4 decreased the frequency of clone-forming cells by 40%. IL-4 did not reduce the viability of Farage cells. On the contrary, IL-4 diminished the spontaneous death of Farage cells in culture, as determined by pulse chase analysis of cells which were labelled with [³H]dT. Moreover, the pre-treatment of Farage cells with IL-4 prevented their death induced by exposure to a high dose of staurosporine. IL-4 abrogated the staurosporine-induced arrest of cells in the G₂+ M phase and replaced it by accumulation of cells in the G₁ phase. IL-4 protected Farage cells from the radioactive suicide caused by the uptake of [³H]dT by dividing cells. The cytokine failed to prevent the damage to Farage cells exerted by mitomycin C, which affected cellular DNA regardless of the phase of the cell cycle. The data obtained showed that IL-4 inhibited the division of B cells by arresting their progression through the early stages of the cell cycle. This inhibition of the cell efflux from G₁ phase plays an important role in the protection against cell death during further stages of the cell cycle.