Peak Of Mitosis Research Articles

APC/C CDH1 ubiquitinates IDH2 contributing to ROS increase in mitosis

NADPH is a cofactor used by reactive oxygen species (ROS) scavenging enzymes to block ROS produced in cells. Recently, it was shown that in cancer cells, ROS progressively increases in tune to cell cycle leading to a peak in mitosis. Loss of IDH2 is known to cause severe oxidative stress in cell and mouse models as ROS increases in mitochondria. Therefore, we hypothesized that IDH2, a major NADPH-producing enzyme in mitochondria is ubiquitinated for ROS to increase in mitosis. To test this hypothesis, in cancer cells we examined IDH2 ubiquitination in mitosis and measured the ROS produced. We found that IDH2 is ubiquitinated in mitosis and on inhibiting anaphase-promoting complex/Cyclosome (APC/C) IDH2 was stabilized. Further, we observed that overexpressing APC/C coactivator CDH1 decreased IDH2, whereas depleting CDH1 decreased IDH2 ubiquitination. To understand the link between IDH2 ubiquitination and ROS produced in mitosis, we show that overexpressing mitochondria-targeted-IDH1 decreased ROS by increasing NADPH in IDH2 ubiquitinated cells. We conclude that APC/C CDH1 ubiquitinates IDH2, a major NADPH-producing enzyme in mitochondria contributing to ROS increase in mitosis. Based on our results, we suggest that mitosis can be a therapeutic window in mutant IDH2-linked pathologies.

ROS and Oxidative Stress Are Elevated in Mitosis during Asynchronous Cell Cycle Progression and Are Exacerbated by Mitotic Arrest.

Although elevated levels of reactive oxygen species (ROS) have been observed in cancer cells and cancer cells aberrantly proliferate, it is not known whether the level of reactive oxygen species and the accumulation of oxidative damage to macromolecules vary across the cell cycle. Here, we measure the prevalence of reactive oxygen species and of biomolecule oxidation across the cell cycle in freely cycling cancer cells. We report that reactive oxygen species vary during the cell cycle and peak in mitosis, resulting in mitotic accumulation of oxidized protein cysteine residues. Prolonged mitotic arrest further increased the levels of ROS and the abundance ofoxidatively damaged biomolecules, including cysteine-sulfenic-acid-containing proteins and 8-oxoguanine. These finding suggest that mitotic arrest agents may enhance the effects of ROS-dependent anticancer therapies.

FRUIT SET AND EARLY BERRY DEVELOPMENT IN TWO GRAPEVINE CULTIVARS

The growth of the ovary/fruitlet of the beiry of two grapevine cultivars (‘Barbera’ and ‘Freisa’) was investigated at the karyological and histochemical level, in order to study the relationship between mitosis and cell-wall development and the synthesis of polysaccharides. The experiment started when flower buds appeared and continued throughout the formation of fruitlets until early July. In general, the peak of mitosis occurred a few days after fruit set, and the nucellar tissues were affected by the mi- totic processes longer than any other ovary tissues. The growth of the berry was mainly due to the mesocarp cell enlargement It seems that the main components of the cell-wall were pectins, which appeared to be increasing during the mitotic phase of tissue growth. Afterwards, stretching of cell-walls proceeded at a faster pace than pectin synthesis. Differences between the varieties were small but definite, indicating that they may be genotypical.

Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells

To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

Development of the model of cell cycle synchronization in early embryos of Danio rerio (Teleostei)

An attempt was undertaken to develop a model system based on artificial cell cycle synchronization by means of reversible mitosis blocking in zebrafish embryos for studying the role of cell cycle synchrony in embryogenesis. Dechorionized and intact embryos at the stages of 512-cell blastula and 75% epiboly were treated with nocodazole and then washed within several times of exposure. When working on dechorionized embryos, we succeeded to obtain complete block mitosis in the presence of low nocodazole concentrations: 0.5-1.0 microg/ml. Block of mitosis was relieved in all experimental series within a certain time after the beginning of washing. This inertia depended on both nocodazole concentration and duration of treatment. The nocodazole elimination was significantly accelerated only after five (or more) changes of washing medium containing DMSO. As a result, the conditions were established for obtaining a parasynchronous cell population in the zebrafish gastrulas with a peak of mitosis up to 17.2%.

The Plk1 Polo Box Domain Mediates a Cell Cycle and DNA Damage Regulated Interaction with Chk2

Polo-like kinase 1 (Plk1) regulates multiple processes during mitosis. Chk2 is a tumorsuppressor that participates in DNA damage checkpoint signaling cascades. Plk1phosphorylates, co-localizes with, and interacts with Chk2, suggesting interconnection ofDNA damage checkpoints and mitotic regulation. However, the function of theirassociation is unknown. Here, we show that the interaction between Chk2 and Plk1 is cellcycle-regulated, with a peak in mitosis. DNA damage in G2 and M phases but not in Sphase induces dissociation of Plk1 and Chk2. In vitro, the Plk1 PBD bindsphosphorylated Chk2, and mediates an interaction independent of other eukaryoticproteins. Additionally, a phosphopeptide encompassing phosphoT68 of Chk2 binds Plk1in a PBD-dependent manner, and stimulates Plk1 activity. These results identify potentialmechanisms for interaction and inter-regulation of these two protein kinases.

High glucose accelerates the life cycle of the in vivo exposed mesothelium

High glucose accelerates the life cycle of the in vivo exposed mesothelium.BackgroundMouse mesothelium exposed in vivo for 30 days to high glucose solutions develop morphological changes that characterize a population of cells near the end of their life span.MethodsThe present study was designed to explore, in mesothelial cell imprints, whether these changes could derive from an early acceleration of the cell population life cycle in mice exposed for periods of up to 30 days to a 4.25% glucose fluid (236 mmol/L/L) prepared in Hank's balanced salt solution (HBSS). Three critical points of the cell's life cycle were evaluated: the G1 checkpoint [proliferating cell nuclear antigen (PCNA) expression], DNA synthesis (3H-thymidine incorporation), and the prevalence of mitosis.ResultsCell populations exposed to a high glucose concentration showed an initial acceleration of their life cycle, as sustained by a peak of mitosis at two hours, an early increase of DNA incorporation sustained during the first 24 hours, as well as a top level of PCNA expression after three to four hours. These significantly higher values, compared with the control animals treated with HBSS, collapsed after 24 hours and were nil after 30 days of exposure.ConclusionsExposure to a high glucose concentration induced an early and short-lived acceleration of the mesothelial cell cycle, and with a longer exposure this was followed by a depletion of the growth capabilities of the exposed monolayer.“The cell, the mysterious protagonist of life, is hidden obstinately in the double invisibility of smallness and homogeneity.”Recollections of my Life, by SANTIAGO RAMON Y CAJAL Translated by E. Horne Craigie in Memoires of the American Philosophical Society (Vol. VIII, part II), Philadelphia, 1937, pp 526–527 High glucose accelerates the life cycle of the in vivo exposed mesothelium. Mouse mesothelium exposed in vivo for 30 days to high glucose solutions develop morphological changes that characterize a population of cells near the end of their life span. The present study was designed to explore, in mesothelial cell imprints, whether these changes could derive from an early acceleration of the cell population life cycle in mice exposed for periods of up to 30 days to a 4.25% glucose fluid (236 mmol/L/L) prepared in Hank's balanced salt solution (HBSS). Three critical points of the cell's life cycle were evaluated: the G1 checkpoint [proliferating cell nuclear antigen (PCNA) expression], DNA synthesis (3H-thymidine incorporation), and the prevalence of mitosis. Cell populations exposed to a high glucose concentration showed an initial acceleration of their life cycle, as sustained by a peak of mitosis at two hours, an early increase of DNA incorporation sustained during the first 24 hours, as well as a top level of PCNA expression after three to four hours. These significantly higher values, compared with the control animals treated with HBSS, collapsed after 24 hours and were nil after 30 days of exposure. Exposure to a high glucose concentration induced an early and short-lived acceleration of the mesothelial cell cycle, and with a longer exposure this was followed by a depletion of the growth capabilities of the exposed monolayer.

High glucose accelerates the life cycle of the in vivo exposed mesothelium

Mouse mesothelium exposed in vivo for 30 days to high glucose solutions develop morphological changes that characterize a population of cells near the end of their life span. The present study was designed to explore, in mesothelial cell imprints, whether these changes could derive from an early acceleration of the cell population life cycle in mice exposed for periods of up to 30 days to a 4.25% glucose fluid (236 mmol/L/L) prepared in Hank's balanced salt solution (HBSS). Three critical points of the cell's life cycle were evaluated: the G1 checkpoint [proliferating cell nuclear antigen (PCNA) expression], DNA synthesis ((3)H-thymidine incorporation), and the prevalence of mitosis. Cell populations exposed to a high glucose concentration showed an initial acceleration of their life cycle, as sustained by a peak of mitosis at two hours, an early increase of DNA incorporation sustained during the first 24 hours, as well as a top level of PCNA expression after three to four hours. These significantly higher values, compared with the control animals treated with HBSS, collapsed after 24 hours and were nil after 30 days of exposure. Exposure to a high glucose concentration induced an early and short-lived acceleration of the mesothelial cell cycle, and with a longer exposure this was followed by a depletion of the growth capabilities of the exposed monolayer.

Direct Binding of CDC20 Protein Family Members Activates the Anaphase-Promoting Complex in Mitosis and G1

Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis. We show that APC is activated during mitosis and G1 by two regulatory factors, hCDC20 and hCDH1. These proteins directly bind to APC and activate its cyclin ubiquitination activity. hCDC20 confers a strict destruction-box (D-box) dependence on APC, while hCDH1 shows a much more relaxed specificity for the D-box. In HeLa cells, the protein levels of hCDC20 as well as its binding to APC peak in mitosis and decrease drastically at early G1. Thus, hCDC20 is the mitotic activator of APC and directs the degradation of substrates containing the D-box. The hCDH1 protein level remains constant during the cell cycle and may target specific substrates lacking the D-box in G1, such as polo-like kinase, for ubiquitination.

Cell division and DNA topisomerase I activity in root meristems of pea seedlings during water stress

ABSTRACT Low water potential, generated by PEG addition to the liquid medium of hydroponically grown pea seedlings, induces a fall in moisture content in the roots, followed by the arrest of elongation. This water stress reduces the mitotic index of root meristems during the treatment and induces the appearance of a peak of mitosis at 12 hours from the beginning of recovery. This peak suggests that during water stress the cell cycle is blocked in G2 or late S phase. In a first attempt to understand the biochemical events leading to cell cycle arrest, we tested the in vitro activity of DNA topoisomerase I extracted from stressed or control root meristems. The activity of this enzyme in extracts from stressed seedlings was lower than in controls, whereas it was higher in extracts from seedlings which had recovered from water stress for a few hours. The highest specific activity was observed with seedlings at 24 hours from the start of recovery. The fact that during stress treatments and recovery there was no variation in the synthesis of a 45 kDa protein, indicated as DNA topoisomerase I, suggested that the activity of this enzyme could be posttranslationally regulated. The hypothesis that variations in the concentration of unknown endogenous regulators of the activity of this enzyme may take place during water loss or uptake in the cytosol of meristematic cells is discussed.

Effect of paclitaxel (Taxol) alone and in combination with radiation on the gastrointestinal mucosa

Purpose : Paclitaxel is a potentially useful drug for augmenting the cytotoxic action of radiotherapy because it has independent cytotoxic activity against certain cancers and blocks cells in the radiosensitive mitotic phase of the cell cycle. However, all rapidly proliferating tissues, both normal and neoplastic, may be affected by this therapeutic strategy. The aim of this study was to define the in vivo response of rapidly dividing cells of the small bowel mucosa to paclitaxel given alone and in combination with radiation. Methods and Materials : Mice were given single IV doses of 10 or 40 mg/kg paclitaxel or four doses of 10 mg/kg paclitaxel at 6, 12, or 24 h intervals. The kinetics of mitotic arrest and apoptosis in jejunal crypts of mice at 1–24 h after treatment were defined histologically. An in vivo stem cell microcolony assay was used to assess the radiosensitizing potential of paclitaxel when radiation was delivered at the peak of mitosis and at 24 h after drug treatment. Results : Paclitaxel blocked jejunal crypt cells in mitosis and induced apoptosis in a dose-dependent manner. Fractionating the paclitaxel dose over 1–4 days did not result in any greater accumulation of mitotically blocked cells than did a single dose. Mitosis peaked 2–4 h after paclitaxel and returned to near normal by 24 h. Apoptosis lagged several hours behind mitosis and peaked about 6 h later than mitosis. Despite these kinetic perturbations, there was little or no enhancement of radiation effect when single doses were delivered 2–4 after paclitaxel administration. The maximum sensitizer enhancement ratio of 1.07 observed after a single paclitaxel dose of 40 mg/kg is consistent with independent crypt cell killing. Conversely, when radiation was given 24 h after paclitaxel, a significant protective effect of the drug (SER 0.89–0.92), most probably due to a regenerative overshoot induced by paclitaxel, was observed. Conclusion : Stem cells of the jejunal mucosa determining radiation response were not radiosentized by paclitaxel with the drug concentrations and dose delivery schedules used, although additive cytotoxicity was observed with the highest drug dose. A radioprotective effect was observed when radiation was given 24 h after paclitaxel administration.

Kinetics of spermatogenesis in the Indian desert gerbil, Meriones hurrianae (Jerdon): Seminiferous epithelial cycle, frequency of stages, spermatogoniaL renewal and germ cell degeneration

The present study provides information on the germ cell associations, the pattern of spermatogonial cell renewal, percentage frequency distribution of different cellular associations and germ cell degeneration in the adult gerbil, M. hurrianae. Based on the formation of acrosomal system and the development of the spermatids as visualized with PAS-hematoxylin, 15 steps of spermiogenesis were identified. The first 12 steps were associated with 12 stages, and the other three steps were between the first 6 stages. The relative frequency was maximal for stage VII (14.24) and minimal for stage VIII (4.38), indicating stage VII to be of the longest and stage VIII of the shortest duration. On the basis of their shape, size and nuclear morphology, 6 types (A0, A1, A2, A3 In and B) of spermatogonia were identified in the gerbil. A-type spermatogonia are oval in shape. From A1 and A3 spermatogonia, progressive heterochromatinization was evident. The A3-spermatogonia divide to give rise to In spermatogonia which are smaller than A-type cells. The B-type spermatogonia are derived from In-type cells and are round in shape. These divide to give rise to first generation primary spermatocytes. Spermatogonia divide at fixed stages during the CSE, and they exhibit 5 peaks of mitosis. A-type spermatogonia divide during stages VI, IX, XII and I. In- and B-Types divide during stages II and V respectively. During stages XII and I, A3-type cells divide to give rise to In- as well as to A1-type cells, thus restoring the A-spermatogonial population. In this way A3-cells serve to renew the stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ki-S1, a novel proliferative marker: flow cytometric assessment of staining in human breast carcinoma cells.

There is considerable interest in immunohistochemical markers of proliferation which are suitable for use on routinely fixed clinical material. The novel proliferation-associated antibody Ki-S1 shows promise in this respect. In this study we have: (i) defined the pattern of Ki-S1 labelling relative to the cell cycle phase; (ii) investigated the labelling pattern with Ki-S1 on a human breast cell line (ZR75) under varying proliferative conditions induced by serum deprivation and refeeding; (iii) examined in a flow cytometric study Ki-S1 staining in archival, clinical breast carcinoma samples. In exponentially growing cells Ki-S1 showed a marked cell cycle phase-specific variation in staining intensity which increased linearly through the S-phase, was high in G2 and reached its peak in mitosis. Ki-S1 staining intensity mirrored the changes in proliferative activity of ZR75 cells during serum deprivation and refeeding. In a small series of human breast carcinoma, Ki-S1 staining intensity correlated with S-phase fraction (SPF) derived from DNA profiles. The antigen labelled by Ki-S1 is extremely robust, resisting degradation by fixation and by an aggressive enzymic tissue disaggregation method. Ki-S1 warrants further investigation as a proliferation-related marker, particularly for routine clinical application.

Mitotic activity of stomach epithelium in the ground squirrel, citellus erythrogenys brandt

1. 1. During hibernation the epithelial cells of the stomach in ground squirrels divide upon spontaneous emergence from hibernation. In torpor periods, mitoses are blocked up. 2. 2. In animals which have come out of hibernation in spring, one peak of mitosis has been recorded in the first few hours after emergence, and another 3 or 4 days later. 3. 3. In summer the mitotic index is low.

Antennal Bud Development in Bracon hebetor (Hymenoptera: Braconidae) Examined by Light and Electron Microscopy during the Third and Fourth Instars

Antennal development of Bracon hebetor (Say) was examined at a cellular and ultrastructural level. Imaginal buds from parthenogenetically produced male larvae of known age were examined by dissection, light microscopy, and transmission and scanning electron microscopy. The adult male antenna consists of 18-20 cylindrical flagellar segments with proximal scape and pedicel. In the 56- to 60-h-old larva, the antennal bud is an undifferentiated, ovoid cellular mass. By 92-96 h, the bud has developed into an elongated, tubular, fully segmented appendage. Cell death, uniformly distributed in the bud, occurs at a low level throughout the 40 h but does not appear to have a major morphogenetic role. Cell size remains constant throughout the 40 h. A high level of mitotic activity is responsible for growth of the bud. A peak in mitosis is detected between 72 and 76 h, just before segmentation. Segments appear to be formed synchronously. Segmentation does not appear to involve localized zones of proliferation, or cell death, or statistically significant changes in cell size. Alterations in cellular interactions with resultant shifts in cell shape or position may be involved.

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with 32P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8–10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation—dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.

Liver sinusoidal cell ultrastructure in partially hepatectomized rats

Two-thirds of the liver was removed from male Wistar rats weighing 160–200 g. In response to the operation phasic changes occurred in the structure of the lysosomal apparatus in the Kupffer cells. The number of primary lysosomes in the Kupffer cells was increased 2.5 h after partial hepatectomy, and the size and polymorphism of the lysosomes were increased after 9 h. At the peak of mitotic activity of the hepatocytes (30 h after partial hepatectomy) mainly secondary lysosomes were identified in the Kupffer cells, but 48 h after the operation, on the other hand, their number was reduced, although “young” forms of primary lysosomes appeared. Manifestations of fatty infiltration predominated in the endothelial cells and reached a maximum at the peak of mitosis of the hepatocytes. The results are evidence that ultrastructural changes in the sinusoidal cells depend on the phases of reparative regeneration of the liver.

Evidence of a G 1regulation of circadian proliferation in mouse methylcholanthrene-induced sarcomas

In two methylcholanthrene induced sarcomas of C 57BL male mice presenting a circadian proliferation (peak of mitoses at noon and peak of labelling indices between midnight and 4 a.m.), the authors performed per cent labelled mitosis curves every 6th hr during the 8th diurnal period of growth. The G 1 phase showed the most important variation with a significant increase in the groups receiving 3H -thymidine at 2 a.m. The possibility that this periodic variation of G 1 phase regulates the circadian rhythm of cell proliferation is discussed.

Mitotic factors from mammalian cells induce germinal vesicle breakdown and chromosome condensation in amphibian oocytes.

Cytoplasmic extracts of HeLa cells synchronized in various phases of the cell cycle were injected into fully grown Xenopus laevis oocytes to monitor the presence of factors that can induce meiotic maturation: i.e., germinal vesicle breakdown and chromosome condensation. Extracts from GI and S phase cells had no activity. The maturation-inducing activity, which was found to be low during early and mid G2 phases, increased rapidly during late G2 and reached a peak in mitosis. The results of this study suggest that the factors that regulate the breakdown of nuclear membrane and chromosome condensation during mitosis, meiosis, and premature chromosome condensation appear to be very similar, if not identical, throughout the animal kingdom.

Temporal relationship between glucose 6-phosphate dehydrogenase activity and DNA-synthesis.

Wounds have been inflicted in the skin of guinea-pigs. Measurements were made on the basal cells 39-54 h later, at different times of the day. It has been shown that there can be two peaks of mitosis, one about mid-day and the other about 22 h. Synthesis of DNA, measured by Feulgen microdensitometry, preceded mitotic activity. Marked changes were found in glucose 6-phosphate dehydrogenase activity, measured by quantitative cytochemistry and microdensitometry. The greatest activity preceded DNA-synthesis, indicating that pentose-shunt metabolic activity is involved in biosynthetic processes required for cell proliferation.