WR-1065, an active metabolite of the cytoprotector amifostine, affects phosphorylation of topoisomerase IIα leading to changes in enzyme activity and cell cycle progression in CHO AA8 cells

The effects of WR-1065 (2-((aminopropyl)amino)ethanethiol) on cell cycle progression, topoisomerase (topo) IIα activity, and topo IIα phosphorylation in Chinese hamster ovary (CHO) cells have been investigated. Exposure of CHO cells to 0.4 μ m of WR-1065 for 30 min did not effect cell cycle progression nor topo IIα activity and phosphorylation status. However, concentrations ranging from 4 μ m to 4 m m were equally effective in significantly altering these three end points. Cell cycle progression was analysed by flow cytometry. Following a 30 min exposure to this range of concentrations, cells redistributed throughout the cell cycle with the most prominent changes being an accumulation of cells in G₂. Topo IIα activity was measured using a kinetoplast DNA (kDNA) decatenation assay. Enzyme activity was reduced by 50% relative to control levels throughout the 4 μ m to 4 m m dose range tested. Likewise, topo IIα phosphorylation levels, analysed using an immunoprecipitation assay and an antibody specific to the 170 kDa band of topo II, decreased between 42% to 48% of control levels. Inhibition of topo IIα activity in cells exposed to WR-1065 is consistent with the associated observation of WR-1065 mediated cell cycle progression delay and build-up of cells in the G₂ phase of the cell cycle.     

CELL CYCLE COMPARTMENT ANALYSIS OF CHINESE HAMSTER CELLS IN STATIONARY PHASE CULTURES

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G₁ and G₂ portions of the cycle; the relative proportion of these cells in G₁ increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G₂ than in G₁. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.     

Method For Probing Cells In Radiation-Induced G2Arrest: Demonstration of Potentially Lethal Damage Repair

Abstract. Chinese hamster ovary cells were arrested in the G₂ phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G₂, for the investigation of G₂ arrest. Comparisons were made of the number of cells retrieved from G₂ arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. the retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. the repair curves (surviving fraction us time) were each described by a single exponential. G₂ cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

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.     

Accumulation of CHO cells in G2 phase following exposure to WR-1065

The radioprotector WR-1065 (2-[(aminopropyl)amino]ethanethiol) is known to protect mammalian cells from the cytotoxic and mutagenic effects of radio- and chemotherapeutic agents, but the exact mechanisms involved in this protection are not fully known. To help determine the effects of WR-1065 alone on cells, we examined its effect on a variety of cellular processes. Incubation of AA8 cells in 4 mM WR-1065 did not significantly affect the rate of DNA synthesis. Autoradiographic analysis of heavily labeled (S-phase population) nuclei of AA8 cells showed no significant difference in the S-phase population of WR-1065-treated versus control cells for up to 3 h. An examination of the effect of WR-1065 on repair synthesis, as measured by unscheduled DNA synthesis (UDS) in cells exposed to 15 Gy, showed no difference between treated and sham-treated cells for up to 2 h exposure. A significant reduction in the amount of UDS was seen in cells treated with the protector for 2.5 and 3 h. Incubation of cells in WR-1065 did alter the cell cycle distributions. An increase in the G2-phase population with a corresponding decrease in the G1-phase population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065. After the removal of WR-1065 at 3 h, a redistribution of the cells throughout the cell cycle occurred as has been observed in cells treated with other synchronization agents. These data suggest that perturbations in cell cycle progression, rather than direct effects on the rate of DNA synthesis, could play a role in the increased survival and reduced mutation frequencies observed in the presence of WR-1065.     

The effect of chemical radiation protectors on cell cycle progression after gamma or neutron irradiation

Abstract. The effects of two chemical radiation protectors, WR-1065 and WR-151326, were characterized in V79 Chinese hamster cells after either cobalt-60 (⁶⁰Co) gamma or fission spectrum neutron irradiation. Each protector was administered at a concentration of 4 mM to exponentially growing cultures for 30 min prior to and during irradiation with either ⁶⁰Co gamma or JANUS fission spectrum neutrons. After irradiation the cells were either plated immediately for survival or returned to the incubator and assayed for cell progression. Aliquots of cells were removed at selected times, counted, fixed and stained with 4'6-diamidino-2-phenylindole (DAPI). Analysis of DNA histograms indicate that the presence of the protector during irradiation reduced the division delay experienced at the G₂-M interface. Implications of these effects are discussed.     

New methods for calculating kinetic properties of cells in vitro using pulse labelling with bromodeoxyuridine

Abstract. The transit times of Chinese hamster ovary cells through the phases of their cell cycle were measured using dual parameter flow cytometry to measure DNA content and the presence of monoclonal antibodies to bromodeoxyuridine. Up to four separate populations can be accurately measured: unlabelled cells in G₂+ M; labelled cells that have not yet divided; labelled cells that have already divided; and the unlabelled cells that were originally in G₁ plus the cells that were originally in G₂+ M and have since divided. The fractions of cells in these populations can be easily followed in time and the usual kinetic properties can be estimated from these fractions, or combinations thereof, including the times through G₁, S, G₂+ M and the cycle time. We present equations for analysing this type of data and comment on which equations are most appropriate for measuring specific kinetic properties of the cells.     

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.     

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.     

Cytokinetic studies on the switch from glucose to uridine metabolism, and vice versa, of Ehrlich ascites tumour cells in vitro

Abstract. Glucose is normally required as the energy source and for the proliferation of neoplastic cells. For Ehrlich ascites tumour cells, kept under glucose-free culture conditions, this requirement was alleviated by uridine, indicating that the supply of ribose is obligatory for sustaining growth capacity.
In a 96-hr culture experiment with mouse-derived cells, the increase in cell number from cultures supplemented with 5 mM uridine was 50–70%, whilst lactate production was 5% that of controls. An increase in the number of multinucleate cells was observed from cell-smears; DNA histograms indicated the presence of cells with a DNA content higher than 4c and an increased portion of cells in G₂ phase. For precise determination of changes in cell cycle distribution on transfer of cells from glucose-supplemented to glucose-free conditions, the progression of phase-accumulated cells (by centrifugal elutriation) was monitored by DNA distribution analysis; G₂ cells continued the cycle at a rate comparable to controls but were delayed, in the following cycle, predominantly in S and G₂ phases. This was also observed with G₁ cells from a G₁-accumulated fraction in the first cycle.
The addition of glucose to cells kept for some hours in glucose-free, uridine-supplemented medium resulted in an immediate increase in mitotic index (amplification by the colcemid method).
The results are interpreted and support our concept that the delivery of compounds, necessary for normal growth, i.e. hexoses for glycoproteins and glycolipids, are limited as a consequence of the 'metabolic channelling' of pentose from uridine in Ehrlich ascites tumour cells. Therefore, the constantly lowered growth-rate in uridine-supplemented cells observed with long-term culture experiments could reflect an adaption of growth-cycle to these limitations.     

Association of WR-1065 with CHO AA8 cells, nuclei, and nucleoids

The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.     

Unbudded G2 as well as Gj arrest in the stationary phase of the basidiomycetous yeast Cryptococcus neoformans

Abstract Stationary-phase cells of Cryptococcus neoformans displayed two morphological characteristics: virtually all the cells were unbudded even in the early stationary phase and even when grown in rich media, and average cell size increased from that of exponential-phase cells. DNA contents for small and large stationary-phase cells were determined by quantitative fluorescence microscopy after DNA staining with propidium iodide or DAPI. Small cells contained G, DNA, whereas large unbudded cells had either a G₂ or G₁ DNA content, indicating that Cr. neoformans can enter into the stationary phase from either the G₁ or G₂ period.     

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.     

Chemical protection and cell-cycle effects on radiation-induced mutagenesis

Chinese hamster ovary cells in the exponential phase of growth were harvested and separated by the method of centrifugal elutriation into subpopulations enriched with up to 95% G1 phase, 70% S phase and 65% G2 + M phase cells. Cell cycle distributions were routinely monitored by flow cytometry. Following elutriation, aliquots of cells from each of the enriched cell fractions were incubated in the presence or absence of 4 mM of 2-[(aminopropyl)amino] ethanethiol (WR-1065) for 30 min at 37 degrees C. The cells were then irradiated with 60Co gamma-rays or fission-spectrum neutrons from the JANUS research reactor. Both cell killing and mutagenesis were determined. Regardless of the radiation quality used, cells enriched in G1 phase were the most sensitive to radiation-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus. The relative magnitude of protection exerted by WR-1065 differed for each of the elutriator separated cell populations. The greatest magnitude of protection, however, was observed for G1-enriched populations, regardless of the radiation quality used or the biological end-point tested.     

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.     

High temperature causes arrest of cell cycle in G2 phase in BmN cells derived from the silkworm, Bombyx mori

Abstract.  The influence of temperature on the insect cell line, BmN, derived from the silkworm, Bombyx mori is investigated. These cells proliferate at an accelerated pace as the temperature increases from 22 to 30 °C, but the growth rate slows at 34 °C, and proliferation stops at 38 °C. At high temperatures, abnormal cellular morphology is observed. Cells treated at 38 °C have cytoplasmic bilateral protrusions and they gradually aggregate and float in the medium. BmN cells without proliferation at 38 °C are viable but have reduced DNA synthesis. At high temperatures, the cell cycle of BmN cells halts at the G₂ phase. After heat treatment of the larvae, an accumulation of larval haemocytes with high DNA content is found, which suggests that the cell cycle arrest at G₂ also occurs in the silkworm at high temperatures.     

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.     

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.