Effects of mevinolin on cell cycle progression and viability of tobacco BY-2 cells

Mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, was used to study the importance of mevalonic acid (MVA) for cell cycle progression of tobacco (Nicotiana tabacum L.) BY-2 cells. After treatment with 5 microM mevinolin, the cell cycle progression was completely blocked and two cell populations accumulated (80% in phase G0/G1 and 20% in G2/M). The arrest could be released by subsequent addition of MVA. Effects were compared to those caused by aphidicolin, an inhibitor of alpha-like DNA polymerases that blocks cell cycle at the entry of the S phase. The 80% proportion of mevinolin-treated TBY-2 cells was clearly arrested before the aphidicolin-inducible block. By the aid of a double-blocking technique, it was shown that the mevinolin-induced cell arrest of highly synchronized cells was due to interaction with a control point located at the mitotic telophase/entry G1 phase. Depending on the developmental stage, mevinolin induced rapid cell death in a considerable percentage of cells. Mevinolin treatment led to a partial synchronization, as shown by the increase in mitotic index. The following decrease was correlated with the above-mentioned induction of cell death.     

Indomethacin-induced G1/S phase arrest of the plant cell cycle

In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.     

Estimating cell death in G(2)M using bivariate BrdUrd/DNA flow cytometry.

BACKGROUND: In an accompanying paper (Asmuth et al.) it was found necessary to include cell death explicitly to estimate parameters of cell proliferation. The use of bivariate flow cytometry to estimate the phase durations and the doubling times of cells labeled with thymidine analogues is well established. However, these methods of analysis do not consider the possibility of cell death. This report demonstrates that estimating cell death in G(2)/M is possible. METHODS: Mathematical models for the experimental quantities, the fraction of labeled undivided cells, the fraction of labeled divided cells, and the relative movement were developed. These models include the possibility that, of the cells with G(2)/M DNA content, only a certain fraction will divide, with the remainder dying after some time T(R). Simulation studies were conducted to test the possibility of using simple methods to estimate phase durations and cell death rates. RESULTS: Cell death alters the estimates of phase transit times in a rather complex manner that depends on the lifetime of the doomed cells. However, it is still possible to obtain estimates of the phase durations of cells in S and G(2)/M and the death rates of cells in G(2)/M. CONCLUSIONS: The methods presented herein provide a new way to characterize cell populations that includes cell death rates and common measurements of cell proliferation.     

Induction of survivin expression by taxol (paclitaxel) is an early event, which is independent of taxol-mediated G2/M arrest

Survivin is a novel anti-apoptotic protein that is highly expressed in cancer but is undetectable in most normal adult tissues. It was reported that taxol-mediated mitotic arrest of cancer cells is associated with survivin induction, which preserves a survival pathway and results in resistance to taxol. In this study, we provide new evidence that induction of survivin by taxol is an early event and is independent of taxol-mediated G(2)/M arrest. Taxol treatment of MCF-7 cells rapidly up-regulated survivin expression (3.5-15-fold) within 4 h without G(2)/M arrest. Lengthening the treatment of cells (48 h) with taxol resulted in decreased survivin expression in comparison with early times following taxol treatment, although G(2)/M cells were significantly increased at later times. Interestingly, 3 nm taxol induces survivin as effectively as 300 nm and more effectively than 3000 nm. As a result, 3 nm taxol is ineffective at inducing cell death. However, inhibition of taxol-mediated survivin induction by small interfering RNA significantly increased taxol-mediated cell death. Taxol rapidly activated the phosphatidylinositol 3-kinase/Akt and MAPK pathways. Inhibition of these pathways diminished survivin induction and sensitized cells to taxol-mediated cell death. A cis-acting DNA element upstream of -1430 in the survivin pLuc-2840 construct is at least partially responsible for taxol-mediated survivin induction. Together, these data show, for the first time, that taxol-mediated induction of survivin is an early event and independent of taxol-mediated G(2)/M arrest. This appears to be a new mechanism for cancer cells to evade taxol-induced apoptosis. Targeting this survival pathway may result in novel approaches for cancer therapeutics.     

Vpr cytopathicity independent of G2/M cell cycle arrest in human immunodeficiency virus type 1-infected CD4+ T cells

The mechanism of CD4(+) T-cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected individuals remains unknown, although mounting evidence suggests that direct viral cytopathicity contributes to this loss. The HIV-1 Vpr accessory protein causes cell death and arrests cells in the G(2)/M phase; however, the molecular mechanism underlying these properties is not clear. Mutation of hydrophobic residues on the surface of its third alpha-helix disrupted Vpr toxicity, G(2)/M arrest induction, nuclear localization, and self-association, implicating this region in multiple Vpr functions. Cytopathicity by virion-delivered mutant Vpr protein correlated with G(2)/M arrest induction but not nuclear localization or self-association. However, infection with whole virus encoding these Vpr mutants did not abrogate HIV-1-induced cell killing. Rather, mutant Vpr proteins that are impaired for G(2)/M block still prevented infected cell proliferation, and this property correlated with the death of infected cells. Chemical agents that inhibit infected cells from entering G(2)/M also did not reduce HIV-1 cytopathicity. Combined, these data implicate Vpr in HIV-1 killing through a mechanism involving inhibiting cell division but not necessarily in G(2)/M. Thus, the hydrophobic region of the third alpha-helix of Vpr is crucial for mediating G(2)/M arrest, nuclear localization, and self-association but dispensable for HIV-1 cytopathicity due to residual cell proliferation blockade mediated by a separate region of the protein.     

Oxidative stress and mitochondrial dysfunctions are early events in narciclasine-induced programmed cell death in tobacco Bright Yellow-2 cells

Narciclasine (NCS) is a plant growth inhibitor isolated from the secreted mucilage of Narcissus tazetta bulbs. It is a commonly used anticancer agent in animal systems. In this study, we provide evidence to show that NCS also acts as an agent in inducing programmed cell death (PCD) in tobacco Bright Yellow-2 (TBY-2) cell cultures. NCS treatment induces typical PCD-associated morphological and biochemical changes, namely cell shrinkage, chromatin condensation and nuclear DNA degradation. To investigate possible signaling events, we analyzed the production of reactive oxygen species (ROS) and the function of mitochondria during PCD induced by NCS. A biphasic behavior burst of hydrogen peroxide (H(2)O(2)) was detected in TBY-2 cells treated with NCS, and mitochondrial transmembrane potential (MTP) loss occurred after a slight increase. Pre-incubation with antioxidant catalase (CAT) and N-acetyl-L-cysteine (NAC) not only significantly decreased the H(2)O(2) production but also effectively retarded the decrease of MTP and reduced the percentage of cells undergoing PCD after NCS treatment. In conclusion, our results suggest that NCS induces PCD in plant cells; the oxidative stress (accumulation of H(2)O(2)) and the MTP loss play important roles during NCS-induced PCD.     

Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.     

Additive effects of radiation and docetaxel on murine SCCVII tumors in vivo: special reference to changes in the cell cycle

The purpose of the present study was to investigate the effects of a combination of docetaxel and irradiation in vivo with special reference to docetaxel-arrested G(2)/M-phase cells. At 24 and 48 h after intraperitoneal administration of docetaxel (90 mg/kg), tumor-bearing mice were irradiated with (60)Co gamma rays. Cell cycle distribution was analyzed by a DNA-Ki-67 double staining method using flow cytometry. An accumulation of cells in the G(2)/M phase of up to approximately 40% was observed 24 h after administration of docetaxel. Between 24 and 72 h, the percentage of cells arrested in G(2)/M phase that expressed Ki-67 decreased from 37.2% to 13.8%, in accordance with the increase in the Ki-67-negative G(2)/M-phase fraction. More than half of the cells arrested in G(2)/M phase lost their expression of Ki-67 protein between 24 and 72 h. The G(1)-phase fraction decreased from 28.4% to 8.6% at 24 h after docetaxel treatment; this remained unchanged at 72 h. These flow cytometry data suggested that docetaxel-arrested G(2)/M-phase cells did not enter the next cell cycle and were killed by docetaxel alone. Our data showed that arrest of cells in G(2)/M phase does not contribute to the synergism that has been reported for combinations of docetaxel and radiation in in vivo tumor models.     

The effect of cell cycle on GFPuv gene expression in the baculovirus expression system.

The effects of cell cycle on recombinant protein production and infection yield in the baculovirus-insect cell expression system (BES) were investigated. When, at any cell cycle phase, the host cell was infected by baculovirus, the cell cycle was finally arrested at the S or G(2)/M phase with 4n DNA. In the case of G(1) or S phase-infection, cell cycle of virus-infected cells began to be arrested at S phase from 8 h post-infection or at G(2)/M phase from 4 h post-infection, respectively; while, in the case of M phase-infection, cell cycle was arrested at S phase after 12 h post-infection. When the host cell was infected at the G(1) phase, average intracellular GFPuv fluorescence intensity was 1.3-fold higher than that at G(2)/M phase at 24 h post-infection. The GFPuv expression corresponded to the profile of the G(1) cell cycle in the BES. Infection yield was measured by detection of intracellular DNA binding protein using immunohistochemical method within 7 h post-infection. The infection yield at G(1) or S phase-infection was 1.5-1.8-fold higher than that at G(2)/M phase-infection.     

Sensitization to death receptor cytotoxicity by inhibition of fas-associated death domain protein (FADD)/caspase signaling. Requirement of cell cycle progression

Upon binding of their ligands, death receptors belonging to the tumor necrosis factor (TNF) receptor family initiate a signaling pathway leading to the activation of caspases and ultimately apoptosis. TNF, however, in parallel elicits survival signals, protecting many cell types from cell death that can only be induced by combined treatment with TNF and inhibitors of protein synthesis. Here, we report that in NIH3T3 cells, apoptosis in response TNF and cycloheximide is not inhibited by the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD. fmk). Moreover, treatment with zVAD.fmk sensitizes the cells to the cytotoxic action of TNF. Sensitization was also achieved by overexpression of a dominant-negative mutant of Fas-associated death domain protein and, to a lesser extent, by specific inhibition of caspase-8. A similar, but weaker sensitization of zVAD.fmk to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) or anti-CD95 antibody was demonstrated. The unexpected cell death in response to TNF and caspase inhibition occurs despite the activation of nuclear factor kappaB and c-Jun N-terminal kinases. The mode of cell death shows several signs of apoptosis including DNA fragmentation, although activation of caspase-3 was excluded. TNF/zVAD.fmk-induced cell death is preceded by an accumulation of cells in the G(2)/M phase of the cell cycle, indicating an important role of cell cycle progression. This hypothesis is further strengthened by the observation that arresting the cells in the G(1) phase of the cell cycle inhibited TNF/zVAD.fmk-induced cell death, whereas blocking them in the G(2)/M phase augmented it.     

Glutathione is recruited into the nucleus in early phases of cell proliferation

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S+G(2)/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G(0)/G(1)) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 +/- 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G(0)/G(1) phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.     

Nuclear phospholipase C-beta1b activation during G2/M and late G1 phase in nocodazole-synchronized HL-60 cells

In this study, the activity of nuclear phosphatidylinositol-specific phosholipase C (PI-PLC) was investigated in HL-60 cells blocked at G(2)/M phase by the addition of nocodazole, and released into medium as synchronously progressing cells. Two peaks of an increase in the nuclear PI-PLC activities were detected; an early peak reached a maximum at 1 h after release from the nocodazole block, and a second increase was detected at 8.5 h after the release. Immunoprecipitation studies indicated that the increase in the activity was due to the activation of the nuclear PI-PLC-beta(1). Western blot analysis demonstrated no changes in the level of both a and b splicing variants of PI-PLC-beta(1) in the nuclei of cells isolated at either 1 h or 8.5 h after the block. However, an increase in the serine-phosphorylation of PI-PLC-beta(1b) was detected in the nuclei of HL-60 cells isolated at 1 and 8.5 h after the block, and the presence of MEK-inhibitor PD98059 completely inhibited both the serine phosphorylation and the increase in the PI-PLC activities in vitro. The presence of PI-PLC inhibitor prevented the progression of HL-60 cells through the G(1) into S phase of the cell cycle. These results demonstrate that two peaks of nuclear PI-PLC activities, which are due to a PD98059-sensitive phosphorylation of nuclear PLC-beta(1b) on serine, occur at the G(2)/M and late G(1) phase and are necessary for the progression of the cells through the cell cycle.     

Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells

We investigated the role of the cdk inhibitor protein p21(Cip-1/WAF1/MDA6) (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0-10 min) and secondary (90-240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G(1) that were p21 and MAPK dependent, whereas the elevation of cells present in G(2)/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G(2)/M phase 24-48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G(2)/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G(2)/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G(2)/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G(1)/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G(2)/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.     

The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior to the onset of apoptosis

In contrast to extracellular signals, the mechanisms utilized to transduce nuclear apoptotic signals are not well understood. Characterizing these mechanisms is important for predicting how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death domain-containing protein p84N5 can induce apoptosis that is inhibited by association with Rb. The pattern of caspase and NF-kappaB activation during p84N5-induced apoptosis is similar to p53-independent cellular responses to DNA damage. One hallmark of this response is the activation of a G(2)/M cell cycle checkpoint. In this report, we characterize the effects of p84N5 on the cell cycle. Expression of p84N5 induces changes in cell cycle distribution and kinetics that are consistent with the activation of a G(2)/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine blocks p84N5-induced G(2)/M arrest but not subsequent apoptotic cell death. The p84N5-induced checkpoint is functional in ataxia telangiectasia-mutated kinase-deficient cells. We conclude that p84N5 induces an ataxia telangiectasia-mutated kinase (ATM)-independent, caffeine-sensitive G(2)/M cell cycle arrest prior to the onset of apoptosis. This conclusion is consistent with the hypotheses that p84N5 functions in an Rb-regulated cellular response that is similar to that triggered by DNA damage.     

PLA2 activity is required for nuclear shrinkage in caspase-independent cell death

Apoptosis is defined on the basis of morphological changes like nuclear fragmentation and chromatin condensation, which are dependent on caspases. Many forms of caspase-independent cell death have been reported, but the mechanisms are still poorly understood. We found that hypoxic cell death was independent of caspases and was associated with significant nuclear shrinkage. Neither Bcl-2 nor Apaf-1 deficiency prevented hypoxic nuclear shrinkage. To understand the molecular mechanism of the nuclear shrinkage, we developed an in vitro system using permeabilized cells, which allowed us to purify a novel member of the phospholipase A2 (PLA2) family that induced nuclear shrinkage. Purified PLA2 induced nuclear shrinkage in our permeabilized cell system. PLA2 inhibitors prevented hypoxic nuclear shrinkage in cells and cell death. Hypoxia caused elevation of PLA2 activity and translocation of intracellular PLA2s to the nucleus. Knockdown of the Ca2+-independent PLA2 delayed nuclear shrinkage and cell death. These results indicate that Ca2+-independent PLA2 is crucial for a caspase-independent cell death signaling pathway leading to nuclear shrinkage.     

Application of a continuous bioreactor cascade to study the effect of linoleic acid on hybridoma cell physiology

The aim of the present study is to demonstrate the use of controlled bioreactors for toxicological studies. As a model system the effect of linoleic acid on hybridoma cells is studied in two well-controlled continuously operated bioreactors placed in series. In the first reactor the effect on rapid proliferating cells can be studied, while in the second reactor a special steady state is created, which allows studying the effect on apoptotic cells. Experiments are done at 0, 25, and 50 microM linoleic acid. At the end of the experiment with 50 microM linoleic acid, the concentration of linoleic acid is increased stepwise to determine the cytotoxic level. For rapid proliferating cells exposed to 25 and 50 microM stimulation of growth was observed. At 50 microM there was at the same time an increase in cell death through apoptosis. For stressed apoptotic cells linoleic acid caused partial growth inhibition at 25 and 50 microM and arrest of cell proliferation in the G(2)/M phase at 50 microM. For both, rapid proliferating cells and stressed apoptotic cells, complete growth inhibition occurred at 85 microM, with cells being arrested in the G(2)/M phase and dying mainly through necrosis. Cells in the bioreactor system appeared to be more sensitive towards linoleic acid than cells grown in multi-well plates. (IC(50) = 300 microM; IC(100) = 400 microM). Altogether the results of the present study reveal that the biostat experiments allow detailed analysis of the effect of a bioactive ingredient on cell physiology and behavior.     

Synergistic effects of arsenic trioxide and radiation in osteosarcoma cells through the induction of both autophagy and apoptosis

Osteosarcoma is the most common primary malignant bone tumor, occurring mainly in children and adolescents, and survival largely depends on their response to chemotherapy. However, the risk of relapse and adverse outcomes is still high. We investigated the synergistic anti-cancer effects of ionizing radiation combined with arsenic trioxide (ATO) and the mechanisms underlying apoptosis or autophagy induced by combined radiation and ATO treatment in human osteosarcoma cells. We found that exposure to radiation increased the population of HOS cells in the G(2)/M phase within 12 h in a time-dependent manner. Radiation combined with ATO induced a significantly prolonged G(2)/M arrest, consequently enhancing cell death. Furthermore, combined treatment resulted in enhanced ROS generation compared to treatment with ATO or radiation alone. The enhanced cytotoxic effect of combined treatment occurred from the increased induction of autophagy and apoptosis through inhibition of the PI3K/Akt signaling pathway in HOS cells. The combined treatment of HOS cells pretreated with Z-VAD, 3-MA or PEG-catalase resulted in a significant reduction of cytotoxicity. In addition, G(2)/M arrest and ROS generation could be involved in the underlying mechanisms. The data suggest that a combination of radiation and ATO could be a new potential therapeutic strategy for the treatment of osteosarcoma.     

Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity

It has been shown previously that apoptosis of tobacco cells induced by cadmium ions shows a relatively long lag period between exposure and cell death. This lag phase lasts for 3 d in TBY-2 cell cultures and is characterized by the maintenance of full cell viability despite extensive fragmentation of DNA into pieces of chromatin loop size. Experiments reported here demonstrate that cell death can be prevented if 50 micro M CdSO(4) is removed from the growth medium during the lag phase, suggesting that an irreversible apoptotic trigger is delivered within 24 h, between the third and fourth days of cadmium treatment. The post-cadmium recovery phase was characterized by DNA repair at the level of 50-200 kb and increased telomerase activity. Analysis of high-molecular-weight DNA by pulsed-field-gel electrophoresis revealed that the majority of DNA strand breaks was repaired within 48 h after cadmium withdrawal. Telomerase activity increased 2.5-fold in the recovery phase, but elevated levels were also found in cell extracts from apoptotic cells suggesting that telomerase might be associated with DNA repair, but it is not capable of inhibiting ongoing apoptosis. Limited exposure of TBY-2 cells to cadmium elicits non-random DNA damage of relatively high magnitude that can be repaired. It is proposed that plants might have developed a highly efficient DNA repair system to cope with transient genotoxic stress.     

An effect of zinc on M-phase and G1 of the plant cell cycle in the synchronous TBY-2 tobacco cell suspension

An analysis was undertaken of the effects of a toxic metal,zinc, on plant cell suspension cultures of the TBY–2 cellline of tobacco (Nicotiana tabacum cv. Bright Yellow 2) in orderto determine whether Zn acts in a cell cycle-specific manner.In the control treatment (0 Zn), following a 24 h synchronizationwith aphidicolin and 7 h after the release from the inhibitor,the mitotic index peaked at 45%. The inclusion of Zn in the24 h aphidicolin treatment (100, 200 or 300 µM Zn) resultedin a concentration–dependent decrease in the mitotic peakto 30%, 22% and 10%, respectively, but did not affect the timingof the peak. Hence, despite high concentrations of Zn, cellstraversed from S–phase to mitosis, albeit in smaller proportions,at the same rate as the controls. Cells treated with 0, 100or 200 µM Zn during synchronization and then releasedinto Zn–free media showed successive peaks in mitoticindex at 7 h and 21 h following release, i.e. Zn-treated cellsprogressed through a complete cell cycle at the same rate asthe controls. Synchronization and subsequent release into Zn–containingmedium (100 µM) examined the effect of the metal on predominantlylate G1 cells. In this treatment, the mitotic index peaked at7 h and 19 h, indicating a slightly faster cell cycle (12 h)compared with the control (14 h). Continuous exposure to 100µM Zn through both synchronization and release resultedin a cell cycle of 11 h and a differential effect on the componentphases: M–phase lengthened (1.5 h to 3.5 h) and G1 shortened(6 h to 1 h) compared with the control treatment. Vital staining (Evans Blue) revealed that cell mortality increasedfrom 2.7% (0 Zn) to 6.1% and 6.5% at 100 and 200 µM Zn,respectively. The Zn content of cells increased 40–lfoldfrom 0 to 100 µM Zn. The data are consistent with theeffects of Zn reducing the cycling cell population primarilythrough cell arrest rather than cell death, but also revealthat a substantial population of TBY–2 cells progressedthrough the cell cycle despite accumulating Zn. In particular,the duration of G2 and S-phase was remarkably invariant, clearlyindicating that once plant cells meet the requirements of lateG1 check-points, they are committed to divide, even in the presenceof toxic concentrations of Zn. The synchronous TBY–2 cell suspension, which lacks theheterogeneity and developmental constraints of plant meristems,is an excellent system to study the effects of known toxic metals,and indeed other environmental factors, on the plant cell cycle. Key words: Cell cycle, plant cell suspensions, Nicotiana tabacum, zinc, toxicity