Metaphase arrest and delay in cell cycle kinetics of root apical meristems and mouse bone marrow cells treated with leaf aqueous extract of Clerodendrum viscosum Vent

Objectives

To study cell cycle delay and metaphase arresting activity of leaf aqueous extract of Clerodendrum viscosum Vent. (LAECV) in root apical meristems and mouse bone marrow cells.

Materials and methods

Cell cycle delay and metaphase arresting activities of LAECV were analysed, in root apical meristems of onion and wheat, and in mouse bone marrow cells, by scoring mitotic index, metaphase frequency and transition of cells from metaphase to anaphase. Colchicine was used as the standard metaphase arresting drug. Phytochemicals present in LAECV were detected and their phytotoxic activity was evaluated by analysing green‐gram (Vigna radiata) seedling's root growth retardation and branch root swelling phenomenon.

Results

LAECV treatment resulted in dose‐dependent root growth retardation of green‐gram seedling root length (P < 0.01) and half maximal growth inhibitory concentration (IC₅₀) of LAECV was 0.87 mg/ml at 144 h. In onion and wheat root meristem cells the mitotic index decreased, metaphase frequency increased and transition from metaphase to anaphase reduced. Experimentation with mouse bone marrow cells indicated that LAECV induced metaphase arrest (164.3% increase in arrested metaphases per 300 mg/kg body weight, over 2.5 h). Phytochemicals like carbohydrates, glycosides, saponins, terpenoids, triterpenoids, tannins and trace amounts of alkaloids were detected in LAECV.

Conclusion

It may be said that LAECV contains mitostatic and metaphase arresting components that are able to induce significant metaphase arrest in root apical meristems and also in mouse bone marrow cells.

     

Mitotic catastrophe and apoptosis induced by docetaxel in hormone-refractory prostate cancer cells

Studies performed in different experimental and clinical settings have shown that Docetaxel (Doc) is effective in a wide range of tumors and that it exerts its activity through multiple mechanisms of action. However, the sequence of events induced by Doc which leads to cell death is still not fully understood. Moreover, it is not completely clear how Doc induces mitotic catastrophe and whether this process is an end event or followed by apoptosis or necrosis. We investigated the mechanisms by which Doc triggers cell death in hormone-refractory prostate cancer cells by analyzing cell cycle perturbations, apoptosis-related marker expression, and morphologic cell alterations. Doc induced a transient increase in G2/M phase followed by the appearance of G0/1 hypo- and hyperdiploid cells and increased p21 expression. Time- and concentration-dependent apoptosis was induced in up to 70% of cells, in concomitance with Bcl-2 phosphorylation, which was followed by caspase-2 and -3 activation. In conclusion, Doc would seem to trigger apoptosis in hormone-refractory prostate cancer cells via mitotic catastrophe through two forms of mitotic exit, in concomitance with increased p21 expression and caspase-2 activation.     

In vitro effects of 2-methoxyestradiol on cell morphology and Cdc2 kinase activity in SNO oesophageal carcinoma cells

The effects of 1 x 10(-6) M exogenous 2-methoxyestradiol (2 ME) were determined on cell morphology and cell division cycle (Cdc) 2 kinase activity in SNO oesophageal carcinoma cells. Mitotic indices revealed an increase in metaphase cells (11.2%) when compared to the 0.5% vehicle-treated cells after 18 h of exposure to 2 ME. Vehicle-treated control cells did not show any hallmarks of apoptosis after 18 h of exposure to dimethyl sulphoxide. Only 0.5% of 2 ME-treated cells showed characteristics of apoptosis. Conversely, increased morphological hallmarks of apoptosis were observed in SNO-treated cells after 21.5 h of 2 ME exposure. When compared to the 0.5% in vehicle-treated cells, 4.7% of cells were in apoptosis. Furthermore, 34.1% of cells were blocked in metaphase after 21.5 h of 2 ME exposure compared to 0.6% of vehicle-control cells. In addition, Cdc2 kinase activity was statistically significantly increased (1.3-fold) (p<0.005) in 2 ME-treated cells when compared to vehicle-treated controls. The present preliminary study suggests that the accumulation observed in metaphase cells and the increase in Cdc2 kinase activity caused by 2 ME are consistent with morphological hallmarks of mitotic arrest and disrupted mitotic spindle formation, thus leading to induction of apoptosis in SNO cells.     

Natural Product Celastrol Destabilizes Tubulin Heterodimer and Facilitates Mitotic Cell Death Triggered by Microtubule-Targeting Anti-Cancer Drugs

Background

Microtubule drugs are effective anti-cancer agents, primarily due to their ability to induce mitotic arrest and subsequent cell death. However, some cancer cells are intrinsically resistant or acquire a resistance. Lack of apoptosis following mitotic arrest is thought to contribute to drug resistance that limits the efficacy of the microtubule-targeting anti-cancer drugs. Genetic or pharmacological agents that selectively facilitate the apoptosis of mitotic arrested cells present opportunities to strengthen the therapeutic efficacy.

Methodology and Principal Findings

We report a natural product Celastrol targets tubulin and facilitates mitotic cell death caused by microtubule drugs. First, in a small molecule screening effort, we identify Celastrol as an inhibitor of neutrophil chemotaxis. Subsequent time-lapse imaging analyses reveal that inhibition of microtubule-mediated cellular processes, including cell migration and mitotic chromosome alignment, is the earliest events affected by Celastrol. Disorganization, not depolymerization, of mitotic spindles appears responsible for mitotic defects. Celastrol directly affects the biochemical properties of tubulin heterodimer in vitro and reduces its protein level in vivo. At the cellular level, Celastrol induces a synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifests an efficacy toward Taxol-resistant cancer cells. Finally, by time-lapse imaging and tracking of microtubule drug-treated cells, we show that Celastrol preferentially induces apoptosis of mitotic arrested cells in a caspase-dependent manner. This selective effect is not due to inhibition of general cell survival pathways or mitotic kinases that have been shown to enhance microtubule drug-induced cell death.

Conclusions and Significance

We provide evidence for new cellular pathways that, when perturbed, selectively induce the apoptosis of mitotic arrested cancer cells, identifying a potential new strategy to enhance the therapeutic efficacy of conventional microtubule-targeting anti-cancer drugs.     

Arrest in metaphase and anatomy of mitotic catastrophe: mild heat shock in two human osteosarcoma cell lines

The exits from metaphase arrest and anatomy of mitotic catastrophe were studied in two human osteosarcoma cell lines, nontumorigenic HOS TE85 and its chemically transformed strain MNNG-HOS, applying mild genotoxic damage by heat shock at 41.8 degrees C for 24 h. Under these conditions, both cell lines doubled or tripled their mitotic index entering arrest in metaphase. On return to 37 degrees C, the arrest was either released or ended in apoptosis. The transformed strain showed a greater capacity to arrest in metaphase as well as a greater probability of developing the third pathway: to restitute this arrest in polyploid interphase. This, in turn, either entered an 'endocycle' or, following a delay, apoptosis. Thus, arrest in metaphase was a cross-point of the mitotic cycle, apoptosis, and endocycle. Mitotic catastrophe can morphologically manifest combinations of elements of these three processes.     

Protein kinase C β inhibition by enzastaurin leads to mitotic missegregation and preferential cytotoxicity toward colorectal cancer cells with chromosomal instability (CIN)

Enzastaurin is a selective inhibitor of protein kinase C β and a potent inhibitor of tumor angiogenesis. In addition, enzastaurin shows direct cytotoxic activity toward a subset of tumor cells including colorectal cancer cells (CRC). In spite of promising results in animal models, the clinical activity of enzastaurin in CRC patients has been disappointing although a subset of patients seems to derive benefit. In the present study we investigated the biological and cytotoxic activities of enzastaurin toward a panel of well-characterized CRC cell lines in order to clarify the mechanistic basis for the cytotoxic activity. Our results show that enzastaurin is significantly more cytotoxic toward CRC cells with chromosome instability (CIN) compared to cells with microsatellite instability (MSI). Since CIN is usually attributed to mitotic dysfunction, the influence of enzastaurin on cell cycle progression and mitotic transit was characterized for representative CIN and MSI cell lines. Enzastaurin exposure was accompanied by prolonged metaphase arrest in CIN cells followed by the appearance of tetraploid and micronuclei-containing cells as well as by increased apoptosis, whereas no detectable mitotic dysfunctions were observed in MSI cells exposed to isotoxic doses of enzastaurin. Our study identifies enzastaurin as a new, context dependent member of a heterogeneous group of anticancer compounds that induce “mitotic catastrophe," that is mitotic dysfunction accompanied by cell death. These data provide novel insight into the mechanism of action of enzastaurin and may allow the identification of biomarkers useful to identify CRC patients particularly likely, or not, to benefit from treatment with enzastaurin.     

Mitotic death: a mechanism of survival? A review

Mitotic death is a delayed response of p53 mutant tumours that are resistant to genotoxic damage. Questions surround why this response is so delayed and how its mechanisms serve a survival function. After uncoupling apoptosis from G1 and S phase arrests and adapting these checkpoints, p53 mutated tumour cells arrive at the G2 compartment where decisions regarding survival and death are made. Missed or insufficient DNA repair in G1 and S phases after severe genotoxic damage results in cells arriving in G2 with an accumulation of point mutations and chromosome breaks. Double strand breaks can be repaired by homologous recombination during G2 arrest. However, cells with excessive chromosome lesions either directly bypass the G2/M checkpoint, starting endocycles from G2 arrest, or are subsequently detected by the spindle checkpoint and present with the features of mitotic death. These complex features include apoptosis from metaphase and mitosis restitution, the latter of which can also facilitate transient endocycles, producing endopolyploid cells. The ability of cells to initiate endocycles during G2 arrest and mitosis restitution most likely reflects their similar molecular environments, with down-regulated mitosis promoting factor activity. Resulting endocycling cells have the ability to repair damaged DNA, and although mostly reproductively dead, in some cases give rise to mitotic progeny. We conclude that the features of mitotic death do not simply represent aberrations of dying cells but are indicative of a switch to amitotic modes of cell survival that may provide additional mechanisms of genotoxic resistance.     

Mitotic catastrophe: a special case of apoptosis

Mitotic catastrophe is a poorly defined type of cell death linked to the abnormal activation of cyclin B/Cdk1. Here we propose that a conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe, provided that cell cycle checkpoints are inhibited, in particular the DNA structure checkpoints and the spindle assembly checkpoint. Two subtypes of mitotic catastrophe can be distinguished. First, mitotic catastrophe can kill the cell during or close to the metaphase, in a p53-independent fashion, as this occurs in Chk2-inhibited heterokarya generated by fusion. Second, mitotic catastrophe can occur after failed mitosis, during the activation of the polyploidy checkpoint, in a partially p53-dependent fashion. In these conditions, cells die as a result of caspase activation and mitochondrial membrane permeabilization that constitute hallmarks of apoptosis. Prevention of caspase activation and/or mitochondrial damage avoids mitotic catastrophe, indicating that this form of cell death indeed constitutes a special case of apoptosis. Importantly, the suppression of mitotic catastrophe can favor asymmetric division and the generation of aneuploid cells. This delineates a molecular pathway through which failure to arrest the cell cycle and inhibition of apoptosis can favor the occurrence of cytogenetic abnormalities which are likely to participate in oncogenesis.     

The response of malignant B lymphocytes to ionizing radiation: cell cycle arrest, apoptosis and protection against the cytotoxic effects of the mitotic inhibitor nocodazole

Ionizing radiation and mitotic inhibitors are used for the treatment of lymphoma. We have studied cell cycle arrest and apoptosis of three human B-lymphocyte cell lines after X irradiation and/or nocodazole treatment. Radiation (4 and 6 Gy) caused arrest in the G(2) phase of the cell cycle as well as in G(1) in Reh cells with an intact TP53 response. Reh cells, but not U698 and Daudi cells with defects in the TP53 pathway, died by apoptosis after exposure to 4 or 6 Gy radiation (>15% apoptotic Reh cells and <5% apoptotic U698/Daudi cells 24 h postirradiation). Lower doses of radiation (0.5 and 1 Gy) caused a transient delay in the G(2) phase of the cell cycle for the three cell lines but did not induce apoptosis (<5% apoptotic cells at 24 h postirradiation). Cells of all three cell lines died by apoptosis after exposure to 1 microg/ml nocodazole, a mitotic blocker that acts by inhibiting the polymerization of tubulin (>25% apoptotic cells after 24 h). When X irradiation with 4 or 6 Gy was performed at the time of addition of nocodazole to U698 and Daudi cells, X rays protected against the apoptosis-inducing effects of the microtubule inhibitor (<5% and 15% apoptotic cells, respectively, 24 h incubation). U698 and Daudi cells apparently have some error(s) in the signaling pathway inducing apoptosis after irradiation, and our results suggest that the arrest in G(2) prevents the cells from entering mitosis and from apoptosis in the presence of microtubule inhibitors. This arrest was overcome by caffeine, which caused U698 cells to enter mitosis (after irradiation) and become apoptotic in the presence of nocodazole (26% apoptotic cells, 24 h incubation). These results may have implications for the design of clinical multimodality protocols involving ionizing radiation for the treatment of cancer.     

Epothilone D affects cell cycle and microtubular pattern in plant cells

Epothilones, macrocyclic lactones from culture filtrates of the myxobacterium Sorangium cellulosum, are known as taxol-like microtubular drugs in human medicine. To date, nothing is known about the effect of epothilones on microtubules (MTs) in plant cells and/or on the plant cell cycle. As shown in this report, the treatment of tomato cell suspension cultures with epothilone D produced a continuous increase in the mitotic index. Dose-response curves revealed that epothilone D alters the mitotic index at concentrations as low as 1.5 microM. Mitotic arrest was already visible after only 2 h of treatment, and 55% of the cells were arrested after 24 h. As shown by immunocytological methods, abnormal spindles are formed during metaphase, which leads to a random distribution of chromosomes in the whole cell and prevents the formation of a metaphase plate. The process of chromosome decondensation does not seem to be affected, because micronuclei form at the same place with the distributed chromosomes. This suggests that epothilone D influences the stability of plant MTs mainly during metaphase of the mitotic cycle. In metaphase, the effects of epothilone D seem to be irreversible, because cells with an abnormal spindle could not be recovered after removal of the drug.     

Cell kinetic studies in the murine ventral tongue epithelium: thymidine metabolism studies and circadian rhythm determination

Abstract.  The oral mucosa is a rapidly replacing body tissue that has received relatively little attention in terms of defining its cell kinetics and cellular organization. The tissue is sensitive to the effects of cytotoxic agents, the consequence of which can be stem cell death with the subsequent development of ulcers and the symptoms of oral mucositis. There is considerable interest in designing strategies to protect oral stem cells and, hence, reduce the mucositis side-effects in cancer therapy patients. Here we present details of a new histometric approach designed to investigate the changing patterns in cellularity in the ventral tongue mucosa. This initial paper in a series of four papers presents observations on the changing patterns in the labelling index following tritiated thymidine administration, which suggest a delayed uptake of tritiated thymidine from a long-term intracellular thymidine pool, a phenomenon that will complicate cell kinetic interpretations in a variety of experimental situations. We also provide data on the changing pattern of mitotic activity through a 24-h period (circadian rhythms). Using vincristine-induced stathmokinesis, the data indicate that 54% of the basal cells divide each day and that there is a high degree of synchrony in mitotic activity with a mitotic peak occurring around 13.00 h. The mitotic circadian peak occurs 9-12 h after the circadian peak in DNA synthesis. The data presented here and in the subsequent papers could be interpreted to indicate that basal cells of BDF1 mice have an average turnover time of about 26-44 h with some cells cycling once a day and others with a 2- or 3-day cell cycle time.     

Taxol suppresses dynamics of individual microtubules in living human tumor cells

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30-100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.     

Integration of glycosphingolipid metabolism and cell-fate decisions in cancer and stem cells: Review and Hypothesis

The metabolism of glycosphingolipids is strictly regulated during the mitotic cell cycle. Before the G1-to-S transition, the ceramide and glucosylceramide concentration is elevated. Ceramide induces apoptosis synergistically with the pro-apoptotic protein prostate apoptosis response 4 (PAR-4) that may be asymmetrically inherited during cell division. Only one daughter cell dies shortly after mitosis, a mechanism we suggested to regulate the number of neural stem cells during embryonic development. The progeny cells, however, may protect themselves by converting ceramide to glucosylceramide and other glycosphingolipids. In particular, complex gangliosides have been found to sustain cell survival and differentiation. The cell cycle may thus be a turning point for (glyco)sphingolipid metabolism and explain rapid changes of the sphingolipid composition in cells that undergo mitotic cell-fate decisions. In the proposed model termed "Shiva cycle", progression through the cell cycle, differentiation, or apoptosis may rely on a delicate balance of (glyco)sphingolipid second messengers that modulate the retinoblastoma-dependent G1-to-S transition or caspase-dependent G1-to-apoptosis program. Ceramide-induced cell cycle delay at G0/G1 is either followed by ceramide-induced apoptosis or by conversion of ceramide to glucosylceramide, a proposed key regulatory rheostat that rescues cells from re-entry into a life/death decision at G1-to-S. We propose a mechanistic model for sphingolpid-induced protein scaffolds ("slip") that regulate cell-fate decisions and will discuss the biological consequences and pharmacological potential of manipulating the (glyco)sphingolipid-dependent cell fate program in cancer and stem cells.     

Killing cells by targeting mitosis

Cell cycle deregulation is a common feature of human cancer. Tumor cells accumulate mutations that result in unscheduled proliferation, genomic instability and chromosomal instability. Several therapeutic strategies have been proposed for targeting the cell division cycle in cancer. Whereas inhibiting the initial phases of the cell cycle is likely to generate viable quiescent cells, targeting mitosis offers several possibilities for killing cancer cells. Microtubule poisons have proved efficacy in the clinic against a broad range of malignancies, and novel targeted strategies are now evaluating the inhibition of critical activities, such as cyclin-dependent kinase 1, Aurora or Polo kinases or spindle kinesins. Abrogation of the mitotic checkpoint or targeting the energetic or proteotoxic stress of aneuploid or chromosomally instable cells may also provide further benefits by inducing lethal levels of instability. Although cancer cells may display different responses to these treatments, recent data suggest that targeting mitotic exit by inhibiting the anaphase-promoting complex generates metaphase cells that invariably die in mitosis. As the efficacy of cell-cycle targeting approaches has been limited so far, further understanding of the molecular pathways modulating mitotic cell death will be required to move forward these new proposals to the clinic.     

Tumor Treating Fields Perturb the Localization of Septins and Cause Aberrant Mitotic Exit

The anti-tumor effects of chemotherapy and radiation are thought to be mediated by triggering G₁/S or G₂/M cell cycle checkpoints, while spindle poisons, such as paclitaxel, block metaphase exit by initiating the spindle assembly checkpoint. In contrast, we have found that 150 kilohertz (kHz) alternating electric fields, also known as Tumor Treating Fields (TTFields), perturbed cells at the transition from metaphase to anaphase. Cells exposed to the TTFields during mitosis showed normal progression to this point, but exhibited uncontrolled membrane blebbing that coincided with metaphase exit. The ability of such alternating electric fields to affect cellular physiology is likely to be dependent on their interactions with proteins possessing high dipole moments. The mitotic Septin complex consisting of Septin 2, 6 and 7, possesses a high calculated dipole moment of 2711 Debyes (D) and plays a central role in positioning the cytokinetic cleavage furrow, and governing its contraction during ingression. We showed that during anaphase, TTFields inhibited Septin localization to the anaphase spindle midline and cytokinetic furrow, as well as its association with microtubules during cell attachment and spreading on fibronectin. After aberrant metaphase exit as a consequence of TTFields exposure, cells exhibited aberrant nuclear architecture and signs of cellular stress including an overall decrease in cellular proliferation, followed by apoptosis that was strongly influenced by the p53 mutational status. Thus, TTFields are able to diminish cell proliferation by specifically perturbing key proteins involved in cell division, leading to mitotic catastrophe and subsequent cell death.     

The Mitotic Checkpoint in Cancer Therapy

The mitotic checkpoint is a key cell cycle control mechanism that ensures an accurate segregation of chromosomes during mitosis by delaying the onset of anaphase until all chromosomes are properly attached to a bipolar mitotic spindle. While complete loss of this checkpoint is lethal in vertebrates, a weakened mitotic checkpoint is frequently seen in cancer cells and it may contribute to tumorigenesis. Many antitumor drugs, including spindle assembly inhibitors and DNA damaging agents, can activate the mitotic checkpoint. However, since these drugs influence interphase events besides activating the mitotic checkpoint, the role of the mitotic checkpoint in drug-induced cell death remained unclear. Using a KSP antagonist that specifically acts on mitotic cells, we have recently shown that activation of the mitotic checkpoint followed by mitotic slippage or adaptation, activates Bax and initiates apoptosis. Notably, cells with a weakened mitotic checkpoint incur much less apoptotic death than their checkpoint-proficient counterparts, indicating the requirement of a competent mitotic checkpoint in the induction of apoptosis. In light of these findings and other recent reports, the potential influence of the mitotic checkpoint in response to chemotherapies, and the strategy to target the mitotic checkpoint for cancer therapeutics are discussed.     

Studies on cell division in mammalian cells. VII. A temperature- sensitive cell line abnormal in centriole separation and chromosome movement

A temperature-sensitive Syrian hamster mutant cell line, ts-745, exhibiting novel mitotic events has been isolated. The cells show normal growth and mitosis at 33 degrees C, the permissive temperature. At the nonpermissive temperature of 39 degrees C, mitotic progression becomes aberrant. Metaphase cells and those cells still able to form a metaphase configuration continue through and complete normal cell division. However, cells exposed to 39 degrees C for longer than 15 min can not form a normal metaphase spindle. Instead, the chromosomes are distributed in a spherical shell, with microtubules (MT) radiating to the chromosomes from four closely associated centrioles near the center of the cell. The cells progress from the spherical monopolar state to other monopolar orientations conical in appearance with four centrioles in the apex region. Organized chromosome movement is present, from the spherical shell state to the asymmetrical orientations. Chromosomes remain in the metaphase configuration without chromatid separation. Prometaphase chromosome congression appears normal, as the chromosomes and MT form a stable monopolar spindle, but bipolar spindle formation is apparently blocked in a premetaphase state. When returned from 39 degrees to 33 degrees C, the defective phenotype is readily reversible. At 39 degrees C, the mitotic abnormality lasts 3-5 h, followed by reformation of a single nucleus and cell flattening in an interphase- like state. Subsequent cell cycle events appear to occur, as the cells duplicate chromosomes and initiate a second round of abnormal mitosis. Cell cycle traversion continues for at least 5 d in some cells despite abnormal mitosis resulting in cells accumulating several hundred chromosomes.