Mitotic cell death induction by targeting the mitotic spindle with tubulin-inhibitory indole derivative molecules

Tubulin-targeting molecules are widely used cancer therapeutic agents. They inhibit microtubule-based structures, including the mitotic spindle, ultimately preventing cell division. The final fates of microtubule-inhibited cells are however often heterogeneous and difficult to predict. While recent work has provided insight into the cell response to inhibitors of microtubule dynamics (taxanes), the cell response to tubulin polymerization inhibitors remains less well characterized. Arylthioindoles (ATIs) are recently developed tubulin inhibitors. We previously identified ATI members that effectively inhibit tubulin polymerization in vitro and cancer cell growth in bulk cell viability assays. Here we characterise in depth the response of cancer cell lines to five selected ATIs. We find that all ATIs arrest mitotic progression, yet subsequently yield distinct cell fate profiles in time-lapse recording assays, indicating that molecules endowed with similar tubulin polymerization inhibitory activity in vitro can in fact display differential efficacy in living cells. Individual ATIs induce cytological phenotypes of increasing severity in terms of damage to the mitotic apparatus. That differentially triggers MCL-1 down-regulation and caspase-3 activation, and underlies the terminal fate of treated cells. Collectively, these results contribute to define the cell response to tubulin inhibitors and pinpoint potentially valuable molecules that can increase the molecular diversity of tubulin-targeting agents

The telomeric protein AKTIP interacts with A- and B-type lamins and is involved in regulation of cellular senescence

AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence

Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit.

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent-or independent-on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition

X-ray irradiated cultures of mouse cortical neural stem/progenitor cells recover cell viability and proliferation with dose-dependent kinetics

Exposure of the developing or adult brain to ionizing radiation (IR) can cause cognitive impairment and/ or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical NSPCs are resistant to low/moderate IR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53- related molecular pathways and cell cycle alterations. Irradiated NSPC cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. These results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult NSPC niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to IR

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination

Towards a unifying model for the metaphase/anaphase transition

The term mitosis actually covers a complex sequence of events at the level of the cell membrane, the cytoplasm, the nuclear membrane and the chromosomes; recently attention has been focused more and more on the checkpoints that control their orderly progression. The term 'checkpoint' refers here to the inhibitory pathways that coordinate coupling between the sequence of events, ensuring dependence of the initiation of each upon successful completion of others. This paper will mainly focus upon the possible checkpoint which controls a brief but essential step, dissociation of the sister chromatids into two identical chromosomes. This step will be called the metaphase/anaphase transition. First, the molecular components that are important in metaphase/anaphase transition will be reviewed: accurate segregation of sister chromatids between the daughter cells is dependent on coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibres, topoisomerases, proteolytic processes and motor proteins. Deficiencies in or impairment of any of these structures or in their control systems may lead to a more or less important genomic imbalance. A model combining the ultrastructural components, the molecular components and the controlling molecules will be proposed. The unifying concept emerging from this synthesis indicates that sister chromatids separate independently of the tubulin fibres, as a result of proteolytic processes controlled by the anaphase promoting complex. The spindle fibres are thus necessary to move the separated chromatids to the spindle poles but probably not to initiate separation. A number of remaining questions are also highlighted.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Elimination of micronucleated cells by apoptosis after treatment with inhibitors of microtubules

Two major mechanisms responsible for chromosome segregation errors are non-disjunction and chromosome loss, both leading to aneuploidy. Previous studies in our laboratory showed the existence of thresholds for the induction of chromosome non-disjunction and chromosome loss and the induction of apoptosis by microtubule inhibitors. From a mechanistic point of view one can expect that apoptosis contributes to the elimination of cells with premutagenic/mutagenic lesions. If aneuploid cells were eliminated by the induction of apoptosis below the threshold concentrations for chromosome loss and non-disjunction, the defined thresholds would not be applicable to cells unable to undergo apoptosis. The aim of this study was to investigate whether apoptosis was induced directly or indirectly as a response to aberrant chromosome segregation below the thresholds for the induction of chromosome loss and non-disjunction, as previously defined by us. Therefore, human lymphocytes were exposed in vitro to five concentrations of nocodazole and five concentrations of carbendazim representing the threshold concentrations for chromosome non-disjunction and chromosome loss, two concentrations below the lowest threshold and one concentration between the two threshold values. After 48 h exposure to the aneugens, induction of apoptosis was analysed by the annexin-V test. The frequencies of chromosome non-disjunction and chromosome loss were estimated in cytokinesis-blocked human lymphocytes in combination with FISH; this methodology was applied to whole cell cultures as well as to apoptotic and viable cell fractions obtained using magnetic annexin microbead cell sorting. Our results suggest that elimination of aneuploid cells does occur. However, the efficiency of disappearance of micronucleated cells is higher than for cells presenting chromosome non-disjunction. The correlation found between early apoptotic events and micronucleus formation could account, at least in part, for the specific elimination of aneuploid cells.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Yeast lsm pro-apoptotic mutants show defects in S-phase entry and progression

Expression of the histone genes is tightly coupled to rates of DNA synthesis in yeast and histone mRNAs are modulated both transcriptionally and post-transcriptionally. Trf4 and Trf5, poly(A) polymerases, that mediates polyadenylation and consequent degradation) and Rrp6, an exosome component, play a role in the regulation of histone mRNA levels. In this paper we show that in the mRNA degradation mutant Kllsm4 Delta 1, histone mRNAs are induced early in the S-phase and maintained at high level all along the entire cell cycle due to a delay in the exit from S-phase and/or entry into M-phase. The overexpression of the HIR1 gene (Histone transcriptional repressor), previously isolated as a multicopy suppressor of the apoptotic phenotypes observed in Kllsm4 Delta 1, can also restore the normal cycling of histone genes expression. We also found that low doses of hydroxyurea neutralize the onset of the apoptotic phenotypes in Kllsm4 Delta 1, as well in another mRNA decapping mutants (lsm1) and, in addition, increase the chronological lifespan in both strains suggesting that an entry delay into the S phase can recover some cellular defects in decapping mutants

In Silico Design in Homogeneous Catalysis Using Descriptor Modelling

This review summarises the state-of-the-art methodologies used for designinghomogeneous catalysts and optimising reaction conditions (e.g. choosing the right solvent).We focus on computational techniques that can complement the current advances in high-throughput experimentation, covering the literature in the period 1996-2006. The reviewassesses the use of molecular modelling tools, from descriptor models based onsemiempirical and molecular mechanics calculations, to 2D topological descriptors andgraph theory methods. Different techniques are compared based on their computational andtime cost, output level, problem relevance and viability. We also review the application ofvarious data mining tools, including artificial neural networks, linear regression, andclassification trees. The future of homogeneous catalysis discovery and optimisation isdiscussed in the light of these developments