Microtubule organization in cultured soybean and black spruce cells: Interphase —mitosis transition and spindle morphology

Summary Microtubule (MT) distribution during the cell cycle, especially spindle organization, has been investigated using immunofluorescence light microscopy in cultured cells of two higher plant species, soybean (angiosperm) and black spruce (gymnosperm). In soybean, the prophase and metaphase spindles were different in morphology and structure. The prophase spindle covering the nucleus was barrel-shaped and MTs extended between poles. The metaphase spindle consisted mainly of short MT bundles on either side of the chromosome mass. During prometaphase, the polarity and shape of the prophase spindle disappeared, suggesting that the metaphase spindle is newly formed in prometaphase and not derived from the prophase spindle. A striking feature of MT organization in black spruce was sharply defined poles during prometaphase and anaphase. They were located close to the cell edge, suggesting that a structure in the cytoplasm or associated with the plasma membrane is responsible for their formation. In black spruce the metaphase spindle was long with pointed poles and MT fir tree structures. In contrast, the metaphase spindle of soybean was short with very broad poles and lacked MT fir trees. These results suggest that MT fir tree structure may not be necessary for a functional spindle.     

Anastral spindle assembly and γ-tubulin in Drosophila oocytes

Background  

Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral Drosophila oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain.     

NPY in rat retina is present in neurons, in endothelial cells and also in microglial and Müller cells

NPY is present in the retina of different species but its role is not elucidated yet. In this work, using different rat retina in vitro models (whole retina, retinal cells in culture, microglial cell cultures, rat Müller cell line and retina endothelial cell line), we demonstrated that NPY staining is present in the retina in different cell types: neurons, macroglial, microglial and endothelial cells. Retinal cells in culture express NPY Y(1), Y(2), Y(4) and Y(5) receptors. Retina endothelial cells express all NPY receptors except NPY Y(5) receptor. Moreover, NPY is released from retinal cells in culture upon depolarization. In this study we showed for the first time that NPY is present in rat retina microglial cells and also in rat Müller cells. These in vitro models may open new perspectives to study the physiology and the potential pathophysiological role of NPY in the retina.     

The Ras pathway and spindle assembly collide?

Although alterations in Ras signalling are found in about 30% of human cancers, the transforming activity of oncogenic Ras is not fully understood. In a recent paper, a putative Ras1 effector in S. pombe, named Scd1, was reported to localize to mitotic spindles. Scd1 physically associates with Moe1, a factor that may contribute to the inherent instability of microtubules (MTs) and appears to be needed for proper spindle function. Altered MT dynamics within the spindle are likely to affect spindle assembly and chromosome capture, processes that need to be delicately controlled if cells are to guard against genome instability and transformation. BioEssays 23:307-310, 2001.     

Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro and in vivo

Background

Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.

Methodology/Principal Findings

Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.

Conclusions/Significance

These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.     

Involvement of PKCζ and GSK3β in the stability of the metaphase spindle

In the somatic cell, the mitotic spindle apparatus is centrosomal, and several isoforms of protein kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is still unclear. Other protein kinases such as, glycogen synthase kinase 3β (GSK3β) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3β are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3β was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3β. Second, the involvement of inactive GSK3β in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCζ acting through GSK3β. Phospho-PKCζ is in close molecular proximity to GSK3β, whereas the other isoforms of PKC such as pPKCβII, pPKCγ, pPKCμ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3β may be a substrate of PKCζ.     

Trolox and ascorbate: are they synergistic in protecting liver cells in vitro and in vivo?

From in vitro studies involving multilamellar liposomes or other artificial systems, several groups of workers have deduced that Trolox (a water-soluble analogue of vitamin E) and ascorbate are synergistic antioxidants. Here, we demonstrate that while Trolox and ascorbate individually protect cultured hepatocytes against oxyradicals generated either with xanthine oxidase plus hypoxanthine or with hydrogen peroxide, the two antioxidants do not appear to be synergistic when used in equimolar combinations. Also, in a rat model of hepatic ischemia-reperfusion, we observed that infusion of Trolox or ascorbate (7.5-10 mumol/kg body weight) into the postischemic liver reduced the reperfusion injury by 76 or 67%, respectively. However, when both compounds were used together (each at the same dose as used separately), the organ salvage amounted to only 79%. Therefore, there is no evidence of synergism between Trolox and ascorbate in our in vitro and especially in vivo systems.     

Protein phosphatase 4 is phosphorylated and inactivated by Cdk in response to spindle toxins and interacts with γ-tubulin

Many pharmaceuticals used to treat cancer target the cell cycle or mitotic spindle dynamics, such as the anti-tumor drug, paclitaxel, which stabilizes microtubules. Here we show that, in cells arrested in mitosis with the spindle toxins, nocodazole, or paclitaxel, the endogenous protein phosphatase 4 (Ppp4) complex Ppp4c-R2-R3A is phosphorylated on its regulatory (R) subunits, and its activity is inhibited. The phosphorylations are blocked by roscovitine, indicating that they may be mediated by Cdk1-cyclin B. Endogenous Ppp4c is enriched at the centrosomes in the absence and presence of paclitaxel, nocodazole, or roscovitine, and the activity of endogenous Ppp4c-R2-R3A is inhibited from G₁/S to the G₂/M phase of the cell cycle. Endogenous γ-tubulin and its associated protein, γ-tubulin complex protein 2, both of which are essential for nucleation of microtubules at centrosomes, interact with the Ppp4 complex. Recombinant γ-tubulin can be phosphorylated by Cdk1-cyclin B or Brsk1 and dephosphorylated by Ppp4c-R2-R3A in vitro. The data indicate that Ppp4c-R2-R3A regulates microtubule organization at centrosomes during cell division in response to stress signals such as spindle toxins, paclitaxel, and nocodazole, and that inhibition of the Ppp4 complex may be advantageous for treatment of some cancers.     

“Reductional anaphase” in replication-defective cells is caused by ubiquitin-conjugating enzyme Cdc34-mediated deregulation of the spindle

Equal partitioning of the duplicated chromosomes into two daughter cells during cell division is a coordinated process and is initiated only after completion of DNA synthesis. However, this strict order of execution breaks down in CDC6-deficient cells. Cdc6, an evolutionarily conserved protein, is required for the assembly of pre-replicative complexes (pre-RCs) and is essential for the initiation of DNA replication. Yeast cells lacking Cdc6 function, though unable to initiate DNA replication, proceed to undergo “reductional anaphase” by partitioning the unreplicated chromosomes and lose viability rapidly. This extreme form of genomic instability in cdc6 cells is thought to be due to inactivation of a pre-RC based, Cdc6-dependent checkpoint mechanism that, during normal cell cycle, inhibits premature onset of mitosis until pre-RC is assembled. Here, we show that chromosome segregation in cdc6 mutant is caused not by precocious initiation of mitosis in the absence of a checkpoint, but by the deregulation of spindle dynamics induced via a regulatory network involving the ubiquitin-conjugating enzyme Cdc34, microtubule-associated proteins (MAPs) and the anaphase-promoting complex (APC) activator Cdh1. This regulatory circuit governs spindle behavior in the early part of the division cycle and precipitates catastrophic chromosome segregation in the absence of DNA replication.     

Transport and distribution of α-tocopherol in lymph,serum and liver cells in rats

Rats were cannulated in the major mesenteric lymph duct and given an intraduodenal bolus of unlabeled and α-[³H]tocopherol, and [¹⁴C]oleic acid in soybean oil. The appearance of α-tocopherol in lymph was negligible during the first 2 h and peaked 4–15 h after feeding, whereas no detectable amount was recovered in the portal vein. Intestinal absorption via the lymphatic pathway was 15.4 ± 8.9% (n = 10) and 45.9 ± 10.8% (n = 4) for α-tocopherol and [¹⁴C]oleic acid, respectively. About 99% of α-tocopherol in lymph was associated with the chylomicron fraction (d < 1.006 g/ml). In non-fasting rats, 51% of serum α-tocopherol was associated with chylomicrons/VLDL (very-low-density lipoprotein, d < 1.006 g/ml) and 47% with HDL (high-density lipoprotein, 1.05 < d < 1.21 g/ml). Our study revealed that the liver, skeletal muscle and adipose tissue contain approx. 92% of the total mass of α-tocopherol measured in ten different organs. Parenchymal and nonparenchymal liver cells contributed to 75% and 25% of the total mass of α-tocopherol in the liver, respectively.     

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.     

Structure–activity relationship study of glaziovianin A against cell cycle progression and spindle formation of HeLa S3 cells

Various derivatives of glaziovianin A, an antitumor isoflavone, were synthesized, and the cytotoxicity of each against HeLa S₃ cells was investigated. Compared to glaziovianin A, the O⁷-allyl derivative was found to be more cytotoxic against HeLa S₃ cells and a more potent M-phase inhibitor.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

DNA polymerase β and PARP activities in base excision repair in living cells

To examine base excision repair (BER) capacity in the context of living cells, we developed and applied a plasmid-based reporter assay. Non-replicating plasmids containing unique DNA base lesions were designed to express luciferase only after lesion repair had occurred, and luciferase expression in transfected cells was measured continuously during a repair period of 14 h. Two types of DNA lesions were examined: uracil opposite T reflecting repair primarily by the single-nucleotide BER sub-pathway, and the abasic site analogue tetrahydrofuran (THF) opposite C reflecting repair by long-patch BER. We found that the repair capacity for uracil-DNA in wild type mouse fibroblasts was very strong, whereas the repair capacity for THF-DNA, although strong, was slightly weaker. Repair capacity in DNA polymerase β (Pol β) null cells for uracil-DNA and THF-DNA was reduced by approximately 15% and 20%, respectively, compared to that in wild type cells. In both cases, the repair deficiency was fully complemented in Pol β null cells expressing recombinant Pol β. The effect of inhibition of poly(ADP-ribose) polymerase (PARP) activity on repair capacity was examined by treatment of cells with the inhibitor 4-amino-1,8-naphthalimide (4-AN). PARP inhibition decreased the repair capacity for both lesions in wild type cells, and this reduction was to the same level as that seen in Pol β null cells. In contrast, 4-AN had no effect on repair in Pol β null cells. The results highlight that Pol β and PARP function in the same repair pathway, but also suggest that there is repair independent of both Pol β and PARP activities. Thus, before the BER capacity of a cell can be predicted or modulated, a better understanding of Pol β and PARP activity-independent BER pathways is required.     

‘3H-testosterone distribution and binding in RAT thymus cells in vivo’

Summary Autoradiography and biochemical investigations showed that [³H]-testosterone where injected intraperitoneally into male white rats was incorporated rapidly into thymus lymphocytes. Thymic cortex contained more silver grains than medulla, and larger lymphocytes were more labelled than medium or small lymphocytes.Cytosol fraction of thymus cells labelledin vivo with [³H]-testosterone, contained the largest quantity of labelled hormone. A 4S cytosol fraction binds [³H]-testosterone. This could be separated by Sephadex chromatography or by linear sucrose gradient centrifugation. Nuclear extract contained also a small quantity of the labelled hormone.