Proportion Of Cells In Mitosis Research Articles

Bluetongue virus infection induces aberrant mitosis in mammalian cells

BackgroundBluetongue virus (BTV) is an arbovirus that is responsible for ‘bluetongue’, an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division.MethodsWe used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2.ResultsOur data showed that these ‘aberrant mitoses’ can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects.ConclusionsWe hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells.

Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.

Irc15 Is a Microtubule-Associated Protein that Regulates Microtubule Dynamics in Saccharomyces cerevisiae

Microtubules are polymers composed of alpha-beta tubulin heterodimers that assemble into microtubules. Microtubules are dynamic structures that have periods of both growth and shrinkage by addition and removal of subunits from the polymer. Microtubules stochastically switch between periods of growth and shrinkage, termed dynamic instability. Dynamic instability is coupled to the GTPase activity of the beta-tubulin subunit of the tubulin heterodimer. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs) that interact with microtubules to regulate dynamic instability. MAPs in budding yeast have been identified that bind microtubule ends (Bim1), that stabilize microtubule structures (Stu2), that bundle microtubules by forming cross-bridges (Ase1), and that interact with microtubules at the kinetochore (Cin8, Kar3, Kip3). IRC15 was previously identified in four different genetic screens for mutants affecting chromosome transmission or repair [11-14]. Here we present evidence that Irc15 is a microtubule-associated protein, localizing to microtubules in vivo and binding to purified microtubules in vitro. Irc15 regulates microtubule dynamics in vivo and loss of IRC15 function leads to delayed mitotic progression, resulting from failure to establish tension between sister kinetochores.

The influence of 4‐hydroxy‐2‐nonenal on proliferation, differentiation and apoptosis of human osteosarcoma cells

The product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE) is known to cause cell death at high concentrations, while at lower concentrations it can influence cell proliferation and differentiation. In our experiments we used human osteosarcoma cells (HOS), to test the influence of HNE on cell proliferation, differentiation and induction of apoptosis. Apoptosis induction was estimated by TiterTACS TUNEL test. The cells were in parallel counted and the DAPI staining method was used to distinguish between apoptotic and necrotic cells as well as to define the proportion of cells in mitosis. To test the influence of HNE on HOS cell differentiation, cells were treated every second day with HNE. After 10 days, the cells were stained for alkaline phosphatase, a marker for osteoblast differentiation. Cell growth inhibition was caused by supraphysiological concentrations of 10 or 100 microM HNE, while apoptosis was induced with supraphysiological as well as by the physiological amount of the aldehyde (1 microM). Necrosis appeared when cells were treated with 10 or 100 microM, but not with 1 microM HNE. The proportion of cells in mitosis gradually declined with increased HNE concentration. Multiple exposures of HOS cells to 10 microM HNE prevented HOS cell differentiation. These results indicated that HNE inhibits proliferation and differentiation of HOS cells in the same concentration dependent manner as it causes apoptosis. We thus assume that HNE might be one of the important signaling molecules regulating the growth of the human osteosarcoma cells.

Monolayer versus aggregate balance in survival process for EGF-induced apoptosis in A431 carcinoma cells: Implication of ROS-P38 MAPK-integrin alpha2beta1 pathway.

A431 cells escape EGF-induced apoptosis by forming cell aggregates. We show that these clusters migrate and merge with neighboring ones, resulting in larger structures composed of a multilayer central (3D) population surrounded by a cell monolayer (2D). We found that after 48 hr of 10 nM EGF treatment, 3D structure formation correlates with alpha2beta1 integrin upregulation. Blockade of alpha2 integrin impairs 3D structure formation. We studied the involvement of reactive oxygen species (ROS) in this process. We show that A431 cells express the NADPH oxidase catalytic subunits Nox1. EGF-induced dose-dependent ROS production was inhibited by the NADPH oxidase inhibitor, diphenylene iodonium (DPI), in these cells while rotenone was ineffective. Inhibition of ROS level in A431 cells with DPI or ebselen (glutathione peroxydase mimic) as well as P38 MAP kinase inhibition by SB203580 decreases alpha2 integrin subunit expression and induces a shift to 3D versus 2D populations. Cell cycle analysis of 2D cells shows that DPI, ebselen and SB203580 decrease the number of cells in S/G2 phase without affecting the cell number in mitosis phase. On the contrary, for 3D cells, these treatments increased the proportion of cells in mitosis without modification of the cell number in S/G2 phase. For both populations, apoptosis was increased by DPI and ebselen. Resistance of cell aggregates by paclitaxel to cell death is usually described. We show that DPI abolishes paclitaxel resistance of 3D cell aggregates. We observed a greater than additive effect between paclitaxel and DPI resulting in an increased proportion of cells in S/G2 phase for 3D populations. These results suggested that the ROS-P38 MAP kinase-alpha2beta1 integrin pathway was implicated in the A431 survival process by modulating the balance between 2D/3D cells.

Exisulind and CP248 induce growth inhibition and apoptosis in human esophageal adenocarcinoma and squamous carcinoma cells.

We examined the effects of exisulind (sulindac sulfone) and a potent derivative CP248 on the Barrett's esophagus (BE)-related adenocarcinoma cell lines Seg-1 and Bic-1, and on HCE7 esophageal squamous carcinoma cells. Marked growth inhibition and apoptosis occurred in all cell lines with IC50 values of 100-300 microM for exisulind and 100 nM for CP248. Bic-1 and HCE7 cells were more sensitive to the growth inhibitory properties of exisulind. Treatment of all cell lines with CP248 for 24 h increased the proportion of cells in mitosis. Exisulind had no effect on cell-cycle progression. Treatment with either compound induced rapid activation of the c-Jun NH2-terminal kinase 1 (JNK1), suggesting that JNK1 activation plays a role in the induction of apoptosis by these compounds. Only Seg-1 cells expressed a detectable basal level of cyclooxygenase-2 (cox-2), providing further evidence that cox-2 is not the critical target for the growth inhibitory and apoptotic effects of these compounds. Cellular levels of reduced glutathione (GSH) increased approximately five-fold in all cell lines after 24 h of treatment with either compound. These studies provide support for the use of exisulind in BE chemoprevention trials, and of exisulind or CP248 in the therapy of patients with esophageal carcinoma.

Zeolite A increases proliferation, differentiation, and transforming growth factor beta production in normal adult human osteoblast-like cells in vitro.

Silicon in trace amounts enhances bone formation, and the silicon-containing compound zeolite A (ZA) increases eggshell thickness in hens. In the studies reported here, treatment of nearly homogeneous strains of normal human osteoblast-like cells for 48 h with ZA at 0.1-100 micrograms/ml induced a dose-dependent increase (r = 0.35, P < 0.001) in DNA synthesis (n = 31) to 162 +/- 16% (mean +/- SEM) of control and in the proportion of cells in mitosis (n = 4) from 9.1 +/- 1.8 to 27.0 +/- 4.5% (r = 0.69, P < 0.005). ZA treatment also increased alkaline phosphatase activity (P < 0.05) and osteocalcin release (P < 0.05) but did not significantly affect collagen production per individual cell. The mitogenic action of ZA was dependent on cell seeding density over the range of 1250-40,000 cells per cm2, which is consistent with induction of an autocrine factor(s). TGF-beta is a potent mitogen for osteoblasts. ZA treatment increased the steady-state mRNA levels of transforming growth factor beta 1 (TGF-beta 1) and induced the release of the latent form of TGF-beta protein into the conditioned medium within 6 h. We conclude that ZA induces the proliferation and differentiation of cells of the osteoblast lineage.

The proportion of erythroid mitoses in normal human bone marrow in short-term culture systems

The proportion of erythroid cells in mitosis was determined in direct preparations and in synchronized and unsynchronized short-term cultures from normal human bone marrow. The stimulating effect on the proportion of erythroid mitoses of bovine serum albumin (BSA), erythropoietin (EP) and human leucocyte conditioned medium (HLC) derived from stimulated lymphocytes, was also analyzed in short-term marrow cultures. In synchronized and unsynchronized cultures, the proportion of erythroid mitoses was markedly lower than in direct preparations, with a mean of 75% in the direct compared to 11% in the unsynchronized cultures and 7% in synchronized. Statistical analysis showed that EP had a substantial influence on the erythroid cells while HCL had a minor effect.

Interaction of human adenovirus serotype 2 with human lymphoid cells

Human adenoviruses (e.g., Ad2, Ad5) establish chronic infections in human lymphoid-derived cell lines, including Raji and Jijoye ( R. E. Wallace, 1969, Proc. Soc. Exp. Bich Med. 130, 702–710; N. Faucon, G. Ogier, and Y. Chardonnet, 1982, J. Nad. Cancer Inst. 69, 1215–1220); however, the mechanisms by which chronic infections are established and maintained are not understood. When Raji or MOLT-3 cell cultures were infected with Ad2 at high multiplicity, these cell lines continued to grow exponentially and produced only small amounts of infectious virus. Virus-specific antigens, including the DNA-binding protein and hexon, were expressed in only 5% of the Ad2-inoculated cultures. All Raji and MOLT-3 cells were found to have adenovirus receptors, but the Ad2 virions that adsorbed to most Raji cells were sequestered in caps, suggesting that most cells fail to internalize adsorbed Ad2. Cell synchronization experiments showed a correlation between the proportion of cells that became productively infected and the proportion of cells in mitosis at the time of infection. In contrast, primary blood lymphocytes had few, if any, Ad2 receptors and were not productively infected by Ad2.

Circadian variation in mitotic index of primary spermatogonia in the adult frog(Rana esculenta)

Abstract Circadian rhythmicity in the average proliferation rate of the primary spermatogonial cell crop in the frog testis was studied by the stathmokinetic method. Colchicine was used to specifically block cells after they enter metaphase. The in vivo mitotic activity of primary spermatogonia was studied in three different periods of the year and was analyzed at 8 time points of the 24‐hour day. Circadian variations were suggested in all three periods of the year. Mitotic activity of the primary spermatogonia was also analyzed in vitro at 16 time points through a 48‐hour experimental period. Daily rhythmic fluctuations in the division rate of primary spermatogonia were observed. Thus, the proportion of cells in mitosis within the primary spermatogonial cell crop of the frog testis exhibits not only significant seasonal variations (as reported elsewhere), but also a daily rhythmic variation. The percent difference between low and high values of mitotic circadian rhythm varies with season.

In vitro response of leaf tissues from Lolium multiflorum ? a comparison with leaf segment position, leaf age and in vivo mitotic activity

Immature gramineous leaves provide a convenient system for comparing the response of cells in culture with their state of differentiation. Callusing frequency is compared with leaf segment position, leaf age and in vivo mitotic activity in Lolium multiflorum. (1) In a succession of one millimeter sections from the immature leaf base, callus was formed from the first and second sections but not the third or subsequent sections. The frequency of those explants callusing decreased with distance from the base of the leaf and with leaf age (or leaf extension growth). (2) In vivo, the proportion of cells in mitosis declined from around 10-14% at the base of young leaves to zero at 5 mm from the base and beyond. Mitotic activity also declined in leaves as they aged, and dividing cells were not observed in leaves 30 days from initiation or older. (3) A high frequency of callus formation was associated with a high mitotic index in the explant. But for corresponding mitotic indices, cells further away from the leaf base were less responsive in culture. (4) It is proposed that cells are becoming differentiated even in highly meristematically active regions of the leaf and concomitantly losing their ability to respond in culture.

Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells.

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.

Cell flux studies during chemotherapy with multiple doses of arabinosyl cytosine in chronic myeloid leukemia

Until now, kinetic perturbations induced by cytotoxic drugs during the therapy of leukemia have mainly been investigated by measuring changes in the proportion of cells in mitosis (mitotic index=IM) and/or in DNA-synthesis phase (tritiated thymidine labeling index=IL). This study was designed to determine simultaneously the effect of arabinosyl cytosine (ARA-C) on the IM, the IL, and also the flux of the cells through mitosis. For that purpose, the rate of accumulation of the cells in mitosis following vincristine was measured in vivo before and after a 3-day course of ARA-C in 2 patients with Ph 1 positive chronic myeloid leukemia. It was found that ARA-C increased the IL, as expected, but also the IM of granulocytic precursors, which could not have been predicted. The increased IM was due to prolonged mitosis, whereas the cell flux through mitosis (=cell birth rate) was actually reduced. The data show that cell flux studies are needed for appropriate estimation of cell kinetic effects of cytotoxic drugs and that reliance on cell cycle ‘indices’ alone in some instances may be misleading.

The in vivo induction of mouse lymphocyte transformation by phytohemagglutinin

Summary The effects of the intravenous administration of phytohemagglutinin-M (PHA-M) and phytohemagglutinin-P (PHA-P) on the mesenteric lymph node, spleen, bone marrow, thymus and peripheral blood of the mouse have been studied. Qualitative changes similar to that observed when PHA is used in vitro with lymphocytes were found. There was a marked shift in the differential of the mesenteric node and peripheral blood from small lymphocytes to medium and large lymphocytes, many of which resemble the blast-like cells seen in tissue culture. Both the shift in differential and the increase in proportion of large and medium cells in deoxyribonucleic acid synthesis in the mesenteric node were maximal at 3 days after the administration of PHA. An increase in the weight of the spleen and the proportion of cells in mitosis and in DNA synthesis was also maximum at 3 days, whereas in the bone marrow the maximum effect occurred only 1 day after PHA. No increase in weight of the thymus, shift in the differential or increase in the proportion of cells in DNA synthesis or mitosis was found with PHA-M, but PHA-P did have a slight effect on all three of these parameters. As a result of its erythro- and leukoagglutinating properties, PHA-M caused a decreased hematocrit, red blood cell count and peripheral platelet count with a compensatory increase in the absolute number of megakaryocytes of the bone marrow 3 days after its administration.

The in Vivo Induction of Mouse Lymphocyte Transformation by Phytohemagglutinin

Summary The effects of the intravenous administration of phytohemagglutinin-M (PHA-M) and phytohemagglutinin-P (PHA-P) on the mesenteric lymph node, spleen, bone marrow, thymus and peripheral blood of the mouse have been studied. Qualitative changes similar to that observed when PHA is used in vitro with lymphocytes were found. There was a marked shift in the differential of the mesenteric node and peripheral blood from small lymphocytes to medium and large lymphocytes, many of which resemble the blast-like cells seen in tissue culture. Both the shift in differential and the increase in proportion of large and medium cells in deoxyribonucleic acid synthesis in the mesenteric node were maximal at 3 days after the administration of PHA. An increase in the weight of the spleen and the proportion of cells in mitosis and in DNA synthesis was also maximum at 3 days, whereas in the bone marrow the maximum effect occurred only 1 day after PHA. No increase in weight of the thymus, shift in the differential or increase in the proportion of cells in DNA synthesis or mitosis was found with PHA-M, but PHA-P did have a slight effect on all three of these parameters. As a result of its erythro- and leukoagglutinating properties, PHA-M caused a decreased hematocrit, red blood cell count and peripheral platelet count with a compensatory increase in the absolute number of megakaryocytes of the bone marrow 3 days after its administration.