The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species

Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2‘,7‘-dichlorodihydrofluorescein diacetate, to detect the level fluctuation of reactive oxygen species (ROS) during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry, with TdT mediated dUTP nick end labeling (Tunel) and propidium iodide(PI), indicated that As2O3 (2μM) could induce apoptosis in NB4 cells prevailingly from G2/M phase, and this efficacy was enhanced upon co-administration of 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ) (2.5μM) which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determin the selective induction of G2/M apoptosis and the cells‘ susceptibility to apoptosis by As2O3.     

Loss of pentameric symmetry of C-reactive protein is associated with promotion of neutrophil-endothelial cell adhesion.

The classic acute-phase reactant C-reactive protein (CRP) is a cyclic pentameric protein that diminishes neutrophil accumulation in inflamed tissues. When the pentamer is dissociated, CRP subunits undergo conformational rearrangement that results in expression of a distinctive isomer with unique antigenic and physicochemical characteristics (termed modified CRP (mCRP)). Recently, mCRP was detected in the wall of normal human blood vessels. We studied the impact and mechanisms of action of mCRP on expression of adhesion molecules on human neutrophils and their adhesion to human coronary artery endothelial cells. Both CRP and mCRP (0.1-200 microg/ml) down-regulated neutrophil L-selectin expression in a concentration-dependent fashion. Furthermore, mCRP, but not CRP, up-regulated CD11b/CD18 expression and stimulated neutrophil extracellular signal-regulated kinase activity, which was accompanied by activation of p21(ras) oncoprotein, Raf-1, and mitogen-activated protein kinase kinase. These actions of mCRP were sensitive to the mitogen-activated protein kinase kinase inhibitor PD98059. mCRP markedly enhanced attachment of neutrophils to LPS-activated human coronary artery endothelial when added together with neutrophils. This effect of mCRP was attenuated by an anti-CD18 mAb. Thus, loss of pentameric symmetry in CRP is associated with appearance of novel bioactivities in mCRP that enhance neutrophil localization and activation at inflamed or injured vascular sites.     

A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle

Immunofluorescence microscopy revealed the presence of protein phosphatase 2A (PP2A) on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. Biochemical analysis showed that PP2A could be reversibly bound to microtubules in vitro and that approximately 75% of the PP2A in cytosolic extracts could interact with microtubules. The activity of microtubule-associated PP2A was differentially regulated during the cell cycle. Enzymatic activity was high during S phase and intermediate during G1, while the activity in G2 and M was 20-fold lower than during S phase. The amount of microtubule-bound PP2A remained constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport.     

Loss of cell cycle controls in apoptotic lymphoblasts of the bursa of Fabricius.

Lymphoblasts of the normal embryonic follicles of the chicken bursa of Fabricius undergo rapid apoptosis when exposed to gamma-radiation or when cell-cell contacts are disrupted by mechanical dispersion in short term culture. We have observed previously that overexpression of v-myc sensitizes preneoplastic bursal lymphoblasts to induction of cell death, whereas resistance to induced cell death is acquired during progression to neoplasia. In this study we observed extensive DNA degradation in the large majority of the lymphoblast population within the first hour after dispersion-induced apoptosis. Paradoxically these cells continued to progress into S-phase with the bulk of DNA cleavage and death occurring in S-phase cells (i.e., in cells with more than 2C and less than 4C DNA content). We confirmed the S phase status of apoptotic cells by determining that detection of nuclear cyclin A in individual cells also corresponded with detection of DNA breakage. Levels of cyclin E, cyclin E-dependent H1 histone kinase, and p53 proteins were maintained during dispersion-induced DNA cleavage. gamma-radiation failed either to inhibit cell cycle progression or to raise p53 levels in dispersed bursal lymphoblasts. In intact bursal follicles low doses of gamma-radiation induced p53 whereas higher, apoptosis-inducing doses failed to induce p53 or prevent G1 to S-phase progression. These results suggest that normal DNA damage-induced cell cycle checkpoint controls are lost or overridden when apoptosis is induced in bursal lymphoblasts.     

The Wilms tumor protein is persistently associated with the nuclear matrix throughout the cell cycle

In order to investigate the subnuclear interactions of the WTI gene product, nuclear fractionation analyses were performed with human osteosarcoma HOS and myelogenous leukemia K562 cells. The WT1 protein was tightly associated with the nucleus and was resistant to high-salt or derergent extraction and DNase I digestion. Both the expression level and stability of WT1 and its resistance to high salt and DNase I treatments remained constant during the cell cycle. In addition, human WT1 ectopically expressed in mouse NIH3T3 cells was also resistant to these treatments. These results suggest that WT1 functions in tight association with the nuclear matrix.     

Mcl-1 involvement in mitochondrial dynamics is associated with apoptotic cell death

The B-cell lymphoma-2 (Bcl-2) family proteins are critical regulators of apoptosis and consist of both proapoptotic and antiapoptotic factors. Within this family, the myeloid cell leukemia factor 1 (Mcl-1) protein exists in two forms as the result of alternative splicing. The long variant (Mcl-1L) acts as an antiapoptotic factor, whereas the short isoform (Mcl-1S) displays proapoptotic activity. In this study, using splice-switching antisense oligonucleotides (ASOs), we increased the synthesis of Mcl-1S, which induced a concurrent reduction of Mcl-1L, resulting in increased sensitivity of cancer cells to apoptotic stimuli. The Mcl-1 ASOs also induced mitochondrial hyperpolarization and a consequent increase in mitochondrial calcium (Ca²⁺) accumulation. The high Mcl-1S/L ratio correlated with significant hyperfusion of the entire mitochondrial network, which occurred in a dynamin-related protein (Drp1)–dependent manner. Our data indicate that the balance between the long and short variants of the Mcl-1 gene represents a key aspect of the regulation of mitochondrial physiology. We propose that the Mcl-1L/S balance is a novel regulatory factor controlling the mitochondrial fusion and fission machinery.     

MyoD induced cell cycle arrest is associated with increased nuclear affinity of the Rb protein.

In studying the mechanism through which the myogenic determination protein MyoD prevents entry into the S phase of the cell cycle, we have found a relationship between MyoD and the retinoblastoma (Rb) tumor suppressor protein. By direct needle microinjection of purified recombinant MyoD protein into quiescent fibroblasts, which were then induced to proliferate by serum, we found that MyoD arrested progression of the cell cycle, in agreement with studies utilizing expression constructs for MyoD. By studying temporal changes in cells injected with MyoD protein, it was found that MyoD did not prevent serum induced expression of the protooncogene c-Fos, an event that occurs in the G0 to G1 transition of the cycle. Injection of the MyoD protein as late as 8 h after the addition of serum still caused an inhibition in DNA synthesis, suggesting that MyoD inhibits the G1 to S transition as opposed to the G0 to G1 transition. MyoD injection did not prevent the expression of cyclin A. However MyoD injection did result in a block in the increase in Rb extractibility normally seen in late G1 phase cells. As this phenomenon is associated with the hyperphosphorylation of Rb at this point in the cell cycle and is correlated with progression into S phase, this provides further evidence that MyoD blocks the cycle late in G1.     

Hematopoietic stem cell aging is associated with functional decline and delayed cell cycle progression

The molecular mechanisms underlying hematopoietic stem cell (HSC) aging remain to be elucidated. In this study, we investigated age-related changes in the functional and phenotypic properties of murine HSCs. Consistent with previous studies, we found that the number and frequency of CD34^−/lowc-Kit⁺Sca-1⁺lineage marker⁻ (CD34⁻KSL) cells, a highly enriched HSC population, significantly increased in old mice, though their repopulating ability was reduced. Continuous bromodeoxyuridine labeling revealed a significant delay in the cell cycle progression of CD34⁻KSL cells in old mice. This delay was also observed in young recipients transplanted with whole bone marrow cells from old mice. When cultured in vitro, CD34⁻KSL cells from old mice showed a greater capacity to give rise to primitive CD48⁻KSL cells with reduced HSC activity. Gene expression profiling identified age-related changes in the expression of several cell cycle regulatory genes, including p21/Cdkn1a and p18/Cdkn2c. These results support the notion that HSC aging is largely regulated by an intrinsic genetic program.     

Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5

Endothelial cells (ECs) are quiescent in normal blood vessels, but undergo rapid bursts of proliferation after vascular injury, hypoxia or induced by powerful angiogenic cytokines like fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Deregulated proliferation of ECs facilitates angiogenic processes and promotes tumor growth. In dividing cells, cell cycle-associated protein kinases, which are referred as cyclin-dependent kinases (cdks), regulate proliferation, differentiation, senescence, and apoptosis. Cyclin-dependent kinase-5 (cdk5) is expressed in neuronal cells and plays an important role in neurite outgrowth, of neuronal migration and neurogenesis, its functions in non-neuronal cells are unclear. Here, we show for the first time that the cdk5 is expressed at high levels in proliferating bovine aortic endothelial (BAE) cells, by contrast insignificant low levels of cdk5 expression in quiescent BAE cells. In addition, bFGF up-regulates cdk5 expression in a dose-dependent fashion. Interestingly, temporal expression data suggests that cdk5 expression is very low between 24-48 h, but high level of cdk5 expression was detected during 60-72 h. This later time corresponds to the time of completion of one cell cycle (doubling of cell population) of BAE cell culture. Angiostatin (AS), a powerful inhibitor of angiogenesis inhibits ECs proliferation in dose-dependent manner with concomitant down-regulation of cdk5 expression. The role of cdk5 in ECs, proliferation and apoptosis was confirmed by selective inhibition of cdk5 expression by the purine derivative roscovitine, which inhibits bFGF-stimulated BAE cells proliferation and induces apoptosis in dose-specific manner. By contrast, the roscovitine analog olomoucine, which is a specific inhibitor of cdk4, but not of cdk5 failed to affect ECs proliferation and apoptosis. These data suggest for the first time that neuron specific protein cdk5 may have significant role in the regulation of ECs proliferation, apoptosis, and angiogenesis and extends beyond its role in neurogenesis.     

Loss of rachis cell viability is associated with ripening disorders in grapes

Rachises of grape (Vitis vinifera L.) clusters that appeared healthy or displayed symptoms of the ripening disorders berry shrivel (BS) or bunch-stem necrosis (BSN) were treated with the cellular viability stain fluorescein diacetate and examined by confocal microscopy. Clusters with BS and BSN symptoms experienced a decrease of cell viability throughout the rachis, and their berries contained 70-80% less sugar than healthy berries. The xylem-mobile dye basic fuchsin, infiltrated via the cut base of shoots with one healthy and one BS cluster, moved to all berries on the healthy cluster but generally failed to move into the peduncle of the BS cluster. Peduncle girdling did not interrupt dye movement in the xylem, but stopped solute accumulation in berries and led to berry shrinkage. In contrast, surgically destroying the peduncle xylem at the onset of ripening did not affect berry growth and solute accumulation. These results indicate that cessation of sugar and water accumulation in BS and BSN is associated with phloem death in the rachis. Although xylem flow to the berries may also cease, a functional xylem connection to the shoot may not be required for normal ripening, while water loss from berries by transpiration and xylem efflux may explain the characteristic berry shrinkage that is associated with these ripening disorders. The similarity of internal tissue breakdown in BS and BSN rachises and the correlation observed here between the proportion of shrinking berries on a cluster and the severity of rachis necrosis suggest that there may be a gradual transition between the two ripening disorders. Seeds from healthy and BS clusters showed no differences in colour, morphology, weight, viability, and ability to germinate, which indicates that the disorder may not appear until seeds are mature.     

Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for progression from G1 to S phase in the cell cycle. We present evidence that the active form of the Cdc28 protein kinase is a complex of approximately 160 kd containing an endogenous substrate, p40, and possibly other polypeptides. This complex phosphorylates p40 and exogenous histone H1 in vitro. Cell cycle arrest during G1 results in inactivation of the protein kinase accompanied by the disassembly of the complex. Furthermore, assembly of the complex is regulated during the cell cycle, reaching a maximum during G1. Partial complexes thought to be intermediates in the assembly process phosphorylate histone H1 but not p40. Addition of soluble factors to these partial complexes in vitro restores p40 phosphorylation and causes the complex to increase to the mature size. A model is presented in which p40 phosphorylation is required during G1 for cells to initiate a new cell cycle.     

Pan1 is an intrinsically disordered protein with homotypic interactions

The yeast scaffold protein Pan1 contains two EH domains at its N‐terminus, a predicted coiled‐coil central region, and a C‐terminal proline‐rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp³¹² and Trp⁶⁴² are each in an EH domain, Trp⁹⁵⁷ is in the central region, and Trp¹²⁸⁰ is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern‐Volmer constants due to unique electrostatic environments of the tryptophan residues. Time‐resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis. Proteins 2013; 81:1944–1963. © 2013 Wiley Periodicals, Inc.     

Loss of pentameric symmetry of C-reactive protein is associated with delayed apoptosis of human neutrophils

Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. Here we report that a novel signal for delaying neutrophil apoptosis is the classic acute phase reactant, C-reactive protein (CRP). However, this anti-apoptotic activity is expressed only when the cyclic pentameric structure of CRP is lost, resulting in formation of modified or monomeric CRP (mCRP), which may be formed in inflamed tissues. By contrast, native pentameric CRP and CRP peptides 77-82, 174-185, and 201-206 failed to affect neutrophil apoptosis. The apoptosis delaying action of mCRP was markedly attenuated by an antibody against the low affinity IgG immune complex receptor (CD16) but not by an anti-CD32 antibody. mCRP evoked a transient concurrent activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways, leading to inhibition of caspase-3 and consequently to delaying apoptosis. Consistently, pharmacological inhibition of either ERK or Akt reversed the anti-apoptotic action of mCRP; however, they did not produce additive inhibition. Thus, mCRP, but not pentameric CRP or peptides derived from CRP, promotes neutrophil survival and may therefore contribute to amplification of the inflammatory response.     

Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation

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Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells

Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic cell lineages while retaining their capacity for self renewal. One of the predictions of this model is the existence of a heterogeneous pool of HSCs, some members of which are destined to become lineage restricted progenitor cells while others function to renew the stem cell pool. To test whether HSCs are heterogeneous with respect to cell cycle status, we determined the fraction of phenotypically defined murine HSCs (Thy1.1lo Lin-/lo Sca- 1+) that contain > 2n amount of DNA as measured by propidium iodide staining, Hoechst dye uptake and [3H]thymidine labeling; that fraction is 18-22%. In contrast, in the developing fetal liver, 40% of HSCs are in the S/G2/M phases of the cell cycle. Those HSCs which exhibit a low level of staining with rhodamine 123 are almost exclusively in G0/G1 (97%) whereas only 70% of HSCs which stain brightly for rhodamine 123 are in G0/G1. The injection of 100 G0/G1 HSCs rescued 90% of lethally irradiated mice in contrast to 100 S/G2/M HSCs, which protected only 25% of lethally irradiated recipients. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of 100 G0/G1 HSCs compared to recipients of 100 S/G2/M cells. These data indicate that a significant proportion of HSCs are actively proliferating during steady state hematopoiesis and that this subpopulation of cells exhibits reduced stem cell activity.     

POTE protein, a cancer-testis antigen, is highly expressed in spermatids in human testis and is associated with apoptotic cells

The POTE gene family encodes very closely related proteins that are highly expressed in testis and in many cancers. Recent studies indicate that the POTE proteins have a pro-apoptotic function. To examine if POTE is associated with cells that are undergoing apoptosis in testis, we determined the cellular location of POTE and of Cleaved Caspase-3 in testicular tissues from 26 azoospermic men. We found intense expression of POTE in round spermatids that are undergoing apoptosis, which are positive for Cleaved Caspase-3. This study suggests POTE may have a role in apoptosis in the human testis.