Esther Leonardo

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase

p85/p110 phosphoinositide 3‐kinase (PI3K) is a heterodimer composed of a p85‐regulatory and a p110‐catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65‐PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85α‐protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

Conserved regulation of proximodistal limb axis development by Meis1/Hth.

Vertebrate limbs grow out from the flanks of embryos, with their main axis extending proximodistally from the trunk. Distinct limb domains, each with specific traits, are generated in a proximal-to-distal sequence during development. Diffusible factors expressed from signalling centres promote the outgrowth of limbs and specify their dorsoventral and anteroposterior axes. However, the molecular mechanism by which limb cells acquire their proximodistal (P–D) identity is unknown. Here we describe the role of the homeobox genes Meis1/2 and Pbx1 in the development of mouse, chicken and Drosophila limbs. We find that Meis1/2 expression is restricted to a proximal domain, coincident with the previously reported domain in which Pbx1 is localized to the nucleus, and resembling the distribution of the Drosophila homologues homothorax (hth) and extradenticle (exd); that Meis1 regulates Pbx1 activity by promoting nuclear import of the Pbx1 protein; and that ectopic expression of Meis1 in chicken and hth in Drosophila disrupts distal limb development and induces distal-to-proximal transformations. We suggest that restriction of Meis1/Hth to proximal regions of the vertebrate and insect limb is essential to specify cell fates and differentiation patterns along the P–D axis of the limb.

Blocked negative selection of developing T cells in mice expressing the baculovirus p35 caspase inhibitor

Clonal deletion in the thymus by apoptosis is involved in purging the immune system of self‐reactive T lymphocytes (negative selection). Cysteine proteases (caspases) belonging to the CPP32 family are activated during this process. We have produced transgenic mice expressing baculovirus p35, a broad‐range caspase inhibitor. Thymocytes from p35 transgenic mice were resistant in vitro to several apoptosis‐inducing agents; this resistance correlated with the inhibition of CPP32‐like activity. Negative selection in vivo of thymocytes triggered by two exogenous antigens, staphylococcal enterotoxin B superantigen and an antigenic peptide in the F5 T‐cell receptor transgenic model, was specifically inhibited in p35 transgenic mice. Our results provide direct evidence for caspase involvement in negative selection during thymocyte development.

Dido gene expression alterations are implicated in the induction of hematological myeloid neoplasms

The myelodysplastic/myeloproliferative diseases (MDS/MPDs) are a heterogeneous group of myeloid neoplasms that share characteristics with chronic myeloproliferative diseases and myelodysplastic syndromes. The broad spectrum of clinical manifestations makes MDS/MPDs extremely difficult to diagnose and treat, with a median survival time of 1-5 years. No single gene defect has been firmly associated with MDS/MPDs, and no animal models have been developed for these diseases. The association of deletions on chromosome 20q with myeloid malignancies suggests the presence of unidentified tumor suppressor genes in this region. Here we show that the recently identified death inducer-obliterator (Dido) gene gives rise to at least 3 polypeptides (Dido1, Dido2, and Dido3) through alternative splicing, and we map the human gene to the long arm of chromosome 20. We found that targeting of murine Dido caused a transplantable disease whose symptoms and signs suggested MDS/MPDs. Furthermore, 100% of human MDS/MPD patients analyzed showed Dido expression abnormalities, which we also found in other myeloid but not lymphoid neoplasms or in healthy donors. Our findings suggest that Dido might be one of the tumor suppressor genes at chromosome 20q and that the Dido-targeted mouse may be a suitable model for studying MDS/MPD diseases and testing new approaches to their diagnosis and treatment.

Ceramide-induced cell death is independent of the Fas/Fas ligand pathway and is prevented by Nur77 overexpression in A20 B cells.

The role of ceramide in triggering apoptosis is still a matter of debate. While in some experimental systems, ceramide was shown to mediate Fas-induced cell death, in other instances it was claimed to induce the expression of Fas ligand (FasL), killing cells in a caspase-dependent fashion. We found that, in mature A20 B cells, ceramide-induced apoptosis is independent of the caspase pathway, since we observed no ICE-like, CPP32-like and Mch2 activities and no PARP proteolysis. Moreover, we were unable to protect these cells from ceramide-induced apoptosis using caspase inhibitors, while they blocked Fas-induced apoptosis and no FasL induction could be detected following ceramide treatment. These results suggest that ceramide does not induce apoptosis through the Fas/FasL pathway. We also found that overexpression of Nur77, a zinc-finger transcription factor described to upregulate FasL, antagonizes ceramide-induced apoptosis, but not Fas-induced apoptosis. This further supports the hypothesis that Fas and ceramide death pathways are independent in A20 cells. Ceramide-induced cell death was associated with increased c-myc, p53, Bax and p27kip1 levels; in contrast, cells transfected with Nur77 (A20Nur77), resistant to ceramide-induced apoptosis, showed a marked downregulation of p53 after ceramide treatment, with neither Bax nor p27kip1 induction. In conclusion, our results suggest that, in the A20 B cell line, Fas and ceramide trigger two distinct pathways and that Nur77 overexpression confers protection against ceramide-mediated apoptosis which correlates with inhibition of p53, Bax and p27kip1 induction.

Quantitative determination of tumor cell intravasation in a real-time polymerase chain reaction-based assay.

Tumor cells acquire the ability to enter blood vessels surrounding the primary tumor, endowing them with the capacity to disseminate and become established in distant sites, originating a metastasis. Determination of the intravasation ability of tumor cells is thus important, as it can be correlated with their potential malignancy. To analyze the intravasation phenotype of human tumor cells in vivo, we performed chick embryo chorioallantoic membrane (CAM) assays. Cells were inoculated on the CAM of 9-day-old chick embryos and the membrane at the opposite side of the egg was recovered after 48 h incubation. To measure intravasation ability, we calculated the amount of human DNA in each CAM sample by real-time PCR of Alu sequences and SYBR Green I fluorescence detection. This analysis showed a detection limit of 1 human cell per 105 total cells, and we were able to distinguish between tumor cells of distinct invasive capacity. This assay has several advantages over current methods to measure intravasation ability, including the elimination of post-PCR analysis, sensitivity and easy scale-up of sample numbers.

Synaptonemal complex assembly and H3K4Me3 demethylation determine DIDO3 localization in meiosis

Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3−/− spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.

Inhibitor of apoptosis protein from Orgyia pseudotsugata nuclear polyhedrosis virus provides a costimulatory signal required for optimal proliferation of developing thymocytes.

The inhibitors of apoptosis proteins (IAPs) constitute a family of endogenous inhibitors that control apoptosis in the cell by inhibiting caspase processing and activity. IAPs are also implicated in cell division, cell cycle regulation, and cancer. To address the role of IAPs in thymus development and homeostasis, we generated transgenic mice expressing IAP generated from the baculovirus Orgyia pseudotsugata nuclear polyhedrosis virus (OpIAP). Developing thymocytes expressing OpIAP show increased nuclear levels of NF-κB and reduced cytoplasmic levels of its inhibitor, IκBα. In mature thymocytes, OpIAP induces optimal activation and proliferation after TCR triggering in the absence of a costimulatory signal. OpIAP expression in immature thymocytes blocks TCR-induced apoptosis. Taken together, our data illustrate the pleiotropism of OpIAP in vivo.

Exacerbated inflammatory responses in transgenic mice expressing an inhibitor of apoptosis protein (OpIAP)

AbstractMembers of the inhibitor of apoptosis protein family are involved not only in suppressing apoptosis, but also in signal transduction, cell division, and are associated with some types of cancers. Here we show that transgenic expression of the inhibitor of apoptosis protein OpIAP in murine T lymphocytes leads to a significant increase in T-cell receptor-induced cell activation, proliferation and cytokine production. Transgenic T lymphocytes expressing OpIAP have a lower proliferation threshold in response to T-cell receptor stimulation. Unstimulated OpIAP transgenic T lymphocytes show elevated nuclear levels of NF-κB transcription factor that increase after in vivo antigen peptide treatment. OpIAP transgenic animals present an exacerbated inflammatory response in an experimental contact hypersensitivity model, suggesting increased T-cell activation in vivo. These data indicate a new role for the inhibitor of apoptosis proteins in T-lymphocyte activation and proliferation.

Analysis and Characterization of the Interleukin 2 Receptor α and β Chain Expression inCD4−CD8− Cells

The differential effects that the binding of interleukin 2(IL‐2)to its β or αβ receptors might induce in two different CD4− CD8− T‐cell lines were analysed. While LDl. T3b, a double‐negative T cell derived from MRL/Ipr mice, constitutively expressed high levels of the IL‐2R β chain, YAC‐1, a Moloney sarcoma virus‐transformed CD4− CD8−T cell, expressed (as an activated T cell) the β and α chains. The presence of IL‐2 in the culture medium was lethal for LDl. T3b cells, while it had no effect on the growth of YAC‐1 cells. IL‐2 increased the exprcssion of the β chain and, to a lesser extent, of the α chain in YAC‐1 cells. In addition, other markers such as CD4 and CD5 were induced by IL‐2 in this celt line.