Enrico Cundari

The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction.

Genotoxicity testing aims to detect a large range of genetic damage endpoints and evaluate such results in context of cell survival. The cytokinesis block micronucleus test offers the advantage to provide simultaneously information on both cell cycle progression and chromosome/genome mutations. Indeed, 1. frequencies of cytokinesis-blocked binucleated cells (and polynucleated) are good estimators of the mitotic rate; 2. frequencies of apoptotic figures in mononucleated and binucleated cells provide a measure for cell death before or after cell division; 3. combination of fluorescence in situ hybridization (FISH) for centromere/telomeres and micronucleus scoring allows discrimination between clastogenic and aneugenic events; 4. detection of FISH signals for chromosome specific sequences in both macronuclei and micronuclei, discriminates between aneuploidy due to chromosome non-disjunction or to chromosome loss. The cytokinesis block in vitro micronucleus test is thus a cytogenetic multi-test providing mechanistic information with a simple, rapid, objective, microscopical analysis.

Dissecting the Biological Functions of Drosophila Histone Deacetylases by RNA Interference and Transcriptional Profiling

Zinc-dependent histone deacetylases (HDACs) are a family of hydrolases first identified as components of transcriptional repressor complexes, where they act by deacetylating lysine residues at the N-terminal extensions of core histones, thereby affecting transcription. To get more insight into the biological functions of the individual HDAC family members, we have used RNA interference in combination with microarray analysis in Drosophila S2 cells. Silencing of Drosophila HDAC1 (DHDAC1), but not of the other DHDAC family members, leads to increased histone acetylation. Silencing of either DHDAC1 or DHDAC3 leads to cell growth inhibition and deregulated transcription of both common and distinct groups of genes. Silencing DHDAC2 leads to increased tubulin acetylation levels but was not associated with a deregulation of gene expression. No growth of phenotype and no significant deregulation of gene expression was observed upon silencing of DHDAC4 and DHDACX. Loss of DHDAC1 or exposure of S2 cells to the small molecule HDAC inhibitor trichostatin both lead to a G2 arrest and were associated with significantly overlapping gene expression signatures in which genes involved in nucleobase and lipid metabolism, DNA replication, cell cycle regulation, and signal transduction were over-represented. A large number of these genes were shown to also be deregulated upon loss of the co-repressor SIN3 (Pile, L. A., Spellman, P. T., Katzenberger, R. J., and Wassarman, D. A. (2003) J. Biol. Chem. 278, 37840–37848). We conclude the following. 1) DHDAC1 and -3 have distinct functions in the control of gene expression. 2) Under the tested conditions, DHDAC2, -4, and X have no detectable transcriptional functions in S2 cells. 3) The anti-proliferative and transcriptional effects of trichostatin are largely recapitulated by the loss of DHDAC1. 4) The deacetylase activity of DHDAC1 significantly contributes to the repressor function of SIN3.

Integrin α3β1 Is an Alternative Cellular Receptor for Adenovirus Serotype 5

ABSTRACT Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of α3β1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin α3β1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-α3 and anti-β1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with α3β1, which involved multiple additional contact sites.

ATM is activated by default in mitosis, localizes at centrosomes and monitors mitotic spindle integrity

We previously showed that ATM is responsible for p53 phosphorylation at Ser15 andlocalization at centrosomes during mitosis. When p53 centrosomal localization is preventedby inhibiting polymerization of spindle microtubules, a stabilized form of p53 is transmittedto daughter cells that arrest in the next G1 phase of the cell cycle after exit from mitosis. ATcells are unable to both localize p53 at centrosomes in mitosis and arrest after exposure tomitotic-spindle poisons. Here we show that during mitosis ATM is activated byphosphorylation at Ser1981 and localizes at centosomes. When mitotic spindle is disrupted bynocodazole, ATM is displaced from centrosomes and co-localizes with phospho-Ser15-p53under the form of spots dispersed in the mitotic cytoplasm. After release from nocodazoleblock,as soon as cells exit mitosis, p53 is redirected to the nucleus and its Ser15phosphorylation is substituted by phosphorylation at Ser46. We suggest that ATM is activatedby default at each mitotic onset and phosphorylates p53 at Ser15 so as to keep it inactive atcentrosomes when the spindle is correctly in place or, in case of inactivation of the mitoticspindle, to maintain the memory of a perturbed mitosis.

Survival of aneuploid, micronucleated and/or polyploid cells: Crosstalk between ploidy control and apoptosis

Microtubule inhibitors are known to block the cell cycle at M-phase, by damaging the mitotic spindle. However, under certain circumstances, cells can escape these effects and become aneuploid, polyploid and/or micronucleated. It is well known that aneuploidy can have adverse effects on human health such as pregnancy wastage, birth defects and the development of human tumours. The present paper aims at reviewing the data our laboratory has accumulated during the last years about the relation between aneuploidy/polyploidy/presence of micronuclei and the induction of apoptosis in human cells after in vitro exposure to the microtubule inhibitor nocodazole. Exposure to high doses of nocodazole results in polyploidy due to mitotic slippage in the absence of a functional spindle. Depending on their p53-status polyploid cells may eventually arrest, die or continue cycling. In these experimental conditions, our data showed that polyploidy does not constitute a strong apoptotic signal. In case of exposure to low concentrations of nocodazole, microtubule depolymerization is disturbed resulting in a spindle with damaged microtubules. This can give rise to chromosome loss and non-disjunction. Our data showed that in particular micronucleated cells, originating from chromosome loss can be eliminated by apoptosis. In addition, nocodazole-induced apoptosis involves the apical caspase-8 and -9 and the effector caspase-3. We show evidence that caspase-3, in addition to its function in apoptosis, plays a role in the formation of micronuclei.

E1B55K-Deleted Adenovirus (ONYX-015) Overrides G1/S and G2/M Checkpoints and Causes Mitotic Catastrophe and Endoreduplication in p53-Proficient Normal Cells

In order to take advantage of cell replication machinery, viruses have evolved complex strategies to override cell cycle checkpoints and force host cells into S phase. To do so, virus products must interfere not only with the basal cell cycle regulators, such as pRb or Mad2, but also with the main surveillance pathways such as those controlled by p53 and ATM. Recently, a number of defective viruses has been produced which, lacking the latter ability, are incapable of replicating in normal cells but should be able to grow and finally lyse those cells that, such as the tumor cells, have lost their surveillance mechanisms. A prototype of these oncolytic viruses is the E1B55K-defective Adenovirus ONYX-015, which was predicted to selectively replicate and kill p53-deficient cancer cells. We found that, despite wt p53 and notwithstanding the activation of the checkpoint regulators p53, ATM, and Mad2, ONYX-015 actively replicated in HUVEC cells. Furthermore, ONYX-015 replication induced a specific phenotype, which is distinct from that of the E4-deleted adenovirus dlE4 Ad5, although both viruses express the main regulatory region E1A. This phenotype includes overriding of the G1/S and G2/M checkpoints, over-expression of MAD2 and retardation of mitosis and accumulation of polyploid cells, suggesting the occurrence of alterations at the mitotic-spindle checkpoint and impairment of the post-mitotic checkpoint. Our data suggest that viral E1A and E4 region products can override all host cell-checkpoint response even at the presence of a full activation of the ATM/p53 pathway. Furthermore, the E4 region alone seems to act independently of the E1B55K virus product in impairing the ATM-dependent, p53-independent G2/M checkpoint since dlE4 Ad5-infected cells arrested in G2 while ONYX-015-infected cells did enter mitosis.

Mammalian cell transduction and internalization properties of λ phages displaying the full-length adenoviral penton base or its central domain

In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage λ is a promising backbone for the development of new vectors: its genome can host large inserts, DNA is protected from degradation by the capsid and the ligand-exposed D and V proteins can be extensively modified. Current phage-based vectors are inefficient and/or receptor-independent transducers. To produce new, receptor-selective and transduction-efficient vectors for mammalian cells we engineered λ by inserting into its genome a GFP expression cassette, and by displaying the penton base (Pb) of adenovirus or its central region (amino acids 286–393). The Pb mediates attachment, entry and endosomal escape of adenovirus in mammalian cells, and its central region (amino acids 286–393) includes the principal receptor-binding motif (340RGD342). Both the phage chimerae λ Pb and λ Pb (286–393) were able to transduce cell lines and primary cultures of human fibroblasts. Competition experiments showed that the transduction pathway was receptor-dependent. We also describe the different trafficking properties of λ Pb and λ Pb (286–393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of λ Pb (286–393), but not that of λ Pb. The proteasome inhibitor MG-132 improved the efficiency of λ Pb (286–393)-mediated transduction, but not that of λ Pb. In summary, this work shows the feasibility of using λ phage as an efficient vector for gene transfer into mammalian cells. We show that λ Pb and λ Pb (286–393) can both mediate receptor-dependent transduction; while only λ Pb is able to promote endosomal escape and proteasome resistance of phage particles.

Engagement of CD4 Before TCR Triggering Regulates Both Bax- and Fas (CD95)-Mediated Apoptosis

In the present study, we have aimed at clarifying the CD4-dependent molecular mechanisms that regulate human memory T cell susceptibility to both Fas (CD95)-dependent and Bcl-2-dependent apoptotic pathways following antigenic challenge. To address this issue, we used an experimental system of viral and alloantigen-specific T cell lines and clones and two ligands of CD4 molecules, Leu-3a mAb and HIV gp120. We demonstrate that CD4 engagement before TCR triggering suppresses the TCR-mediated neosynthesis of the Flice-like inhibitory protein and transforms memory T cells from a CD95-resistant to a CD95-susceptible phenotype. Moreover, evidence that the apoptotic programs were executed while Fas ligand mRNA expression was inhibited led us to analyze Bcl-2-dependent pathways. The data show that the engagement of CD4 separately from TCR influences the expression of the proapoptotic protein Bax independently of the anti-apoptotic protein Bcl-2, whereas Ag activation coordinately modulates both Bax and Bcl-2. The increased expression of Bax and the consequent dissipation of the mitochondrial transmembrane potential (ΔΨm) suggest a novel immunoregulatory function of CD4 and demonstrate that both passive cell death and activation-induced cell death are operative in CD4+ memory T cells. Furthermore, analysis of the mechanisms by which IL-2 and IL-4 cytokines exert their protective function on CD4+ T cells in the presence of soluble CD4 ligands shows that they were able to revert susceptibility to Bax-mediated but not to CD95-dependent apoptotic pathways.

Mitotic checkpoints and the maintenance of the chromosome karyotype

The goal of the mitotic cell division is the faithful transmission of chromosomes to the daughter cells. To fulfil a correct separation of sister chromatids, kinetochores of all chromosomes should be correctly attached to spindle microtubules of opposite poles and should all be under tension. These events are monitored by the spindle checkpoint, which delays mitotic progression allowing time for corrections when errors occur in the dynamic interactions between chromosomes and microtubules. The G(1) post-mitotic checkpoint constitutes an additional checkpoint preventing further proliferation of cells that have undergone massive spindle damage. This review concentrates on the key structural and protein components which are pivotal for an accurate segregation of chromosomes during anaphase: the chromosome scaffold, sister chromatid cohesion and segregation and the kinetochores in higher eukaryotes. Furthermore, recent advances in understanding spindle and G(1) post-mitotic checkpoint and how they prevent aneuploidization and polyploidization are presented. In a last part the impact of aneuploidy and polyploidy on human health and in particular on cancer development is highlighted.

HIPK2 Controls Cytokinesis and Prevents Tetraploidization by Phosphorylating Histone H2B at the Midbody

Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization.