Ernestina Saulle

Detection of Bovine Mitochondrial DNA in Ruminant Feeds: A Molecular Approach to Test for the Presence of Bovine-Derived Materials

A ban on ruminant-derived proteins in ruminant feeds has been introduced as a preventive measure to avoid the spread of bovine spongiform encephalopathy (BSE), as well as to minimize any potential risk of BSE transmission from bovines to humans. In the absence of commercially available efficient methods for identification of bovine-derived proteins in animal feeds, we developed a rapid and sensitive polymerase chain reaction (PCR)-based assay which allows detection and identification of a bovine-specific mitochondrial DNA sequence from feedstuffs. The amplified product encodes for the whole ATPase subunit 8 and the amino-terminal portion of the ATPase subunit 6 proteins, which are known to exhibit a relatively low degree of conservation among vertebrates. The specific amplification of such a bovine mitochondrial sequence from reference feedstuff samples was demonstrated by means of both direct sequencing and single-strand conformational analysis of the PCR product. Specificity was also confirmed by the absence of detectable homologous PCR product when using reference feedstuff samples lacking bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method allows detection of the presence of bovine mitochondrial DNA in feedstuffs containing less than 0.125% of bovine-derived meat and bonemeals. Furthermore, it does not appear to be considerably affected by prolonged heat treatment. DpnII and SspI restriction endonuclease digestions of the unpurified PCR product may be used routinely to confirm the bovine origin of the amplified sequence. Since this method is specific, rapid, and sensitive, it could be successfully utilized as a routine control assay to evaluate the presence of bovine-derived meat and bonemeals in ruminant feeds.

Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis

In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.

Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex

Background The human hemoglobin switch (HbF→HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor. Design and Methods We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34+ cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production. Results In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80–90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects. Conclusions Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221–222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.

High sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide

In the present study we have explored the sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide (CDDO-Im). For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/ADR and A2780/CISP, OVCAR3, SKOV3 and HEY cancer cell lines and primary ovarian cancer cells, providing evidence that: (i) the majority of these cell lines are highly sensitive to the pro-apoptotic effects induced by CDDO-Im; (ii) TRAIL, added alone exerted only a weak proapoptotic, but clearly potentiated the cytotoxic effect elicited by CDDO-Im; (iii) the apoptotic effect induced by CDDO-Im involves GSH depletion, c-FLIP downmodulation and caspase-8 activation; (iv) CDDO-Im inhibits STAT3 activation and CDDO-Im sensitivity is inversely related to the level of constitutive STAT3 activation. Importantly, studies on primary ovarian cancer cells have shown that these cells are sensitive to the pro-apoptotic effects of CDDO-Im. These observations support the experimental use of synthetic triterpenoids in the treatment of ovarian cancer.

Salinomycin Potentiates the Cytotoxic Effects of TRAIL on Glioblastoma Cell Lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to exhibit therapeutic activity in cancer. However, many tumors remain resistant to treatment with TRAIL. Therefore, small molecules that potentiate the cytotoxic effects of TRAIL could be used for combinatorial therapy. Here we found that the ionophore antibiotic salinomycin acts in synergism with TRAIL, enhancing TRAIL-induced apoptosis in glioma cells. Treatment with low doses of salinomycin in combination with TRAIL augmented the activation of caspase-3 and increased TRAIL-R2 cell surface expression. TRAIL-R2 upmodulation was required for mediating the stimulatory effect of salinomycin on TRAIL-mediated apoptosis, since it was abrogated by siRNA-mediated TRAIL-R2 knockdown. Salinomycin in synergism with TRAIL exerts a marked anti-tumor effect in nude mice xenografted with human glioblastoma cells. Our results suggest that the combination of TRAIL and salinomycin may be a useful tool to overcome TRAIL resistance in glioma cells and may represent a potential drug for treatment of these tumors. Importantly, salinomycin+TRAIL were able to induce cell death of well-defined glioblastoma stem-like lines.

A Small Molecule SMAC Mimic LBW242 Potentiates TRAIL- and Anticancer Drug-Mediated Cell Death of Ovarian Cancer Cells

Background Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays. Principal Findings LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis. Conclusion LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.

Fibroblast growth factor receptor mutational screening in newborns affected by metopic synostosis.

Abstract A number of craniosynostotic disorders have recently been ascribed to mutations in genes coding for the fibroblast growth factor receptors(FGFRs). The common feature of these FGFR-associated conditions is the unilateral or bilateral premature ossification of the coronal suture. One distinct craniosynostotic condition is trigonocephaly, which results from the premature fusion of the metopic suture. Trigonocephaly mostly occurs as isolated cranial defect; however, the premature closure of the metopic suture may represent a feature of more complex craniosynostotic conditions in which a progressive involvement of other cranial sutures with age is observed. The possible involvement of mutated FGFRs in trigonocephaly was investigated in nine newborns affected by isolated premature synostosis of the metopic suture. All except one of these cases carried no mutations in the FGFR1–3 domains indicated as hot spots for craniosynostosis-associated mutations. A T(978)C transition in the FGFR2 exon IIIa was found in a patient who had a phenotype that apparently fitted the trigonocephalic condition at birth, but showed additional facial anomalies, which worsened progressively with age towards a Crouzon-like profile. The present finding points out the importance, from both diagnostic and prognostic points of view, of early FGFR mutational screening in craniosynostotic conditions, even in forms that apparently do not involve closure of the coronal suture at birth.

C-fms expression correlates with monocytic differentiation in PML-RAR alpha+ acute promyelocytic leukemia

We have investigated the expression of the M-CSF receptor (c-fms) in 16 freshly isolated acute promyelocytic leukemias (APL) expressing the PML/RARα fusion protein. In parallel, we evaluated the capacity of these cells to differentiate along the granulocytic and monocytic pathways. c-fms was constitutively and constantly expressed in all cases sensitive in vivo to all-trans retinoic acid (ATRA) and its expression was further potentiated following in vitro induction with ATRA. Furthermore, gel-shift analysis of APL cells showed elevated levels of PU.1 binding activity to the M-CSF receptor promoter, particularly after ATRA stimulation. Interestingly, the rise of PU.1 binding activity as well as of PU.1 levels after ATRA treatment was significantly higher in APL patients exhibiting monocytic maturation, as compared to those that did not undergo monocytic differentiation. A variable proportion of ATRA-induced APL cells exhibited monocyte-like morphology and immunophenotype: the proportion of monocytic cells was consistently increased by combined treatment with ATRA and diverse hematopoietic growth factors cocktails, which always comprised M-CSF. Monocytic cells originating from in vitro ATRA-induced maturation of APL cells derive from the leukemic clone as suggested by two lines of evidence: (1) monocytic cells harbor the 15;17 translocation; (2) monocytic cells possess Auer bodies. The c-fmsbright leukemic blasts preferentially showed the capacity for monocytic differentiation as compared to the c-fmsdim/− subset: indeed, enforced expression of c-fms into NB4, a PML/RARα+ cell line, favored the onset of monocytic maturation. Finally, low c-fms expression was observed in an APL relapsing patient resistant to ATRA, as well as in an APL case with t(11;17), PLZF/RARα+. These observations indicate that PML/RARα+ APL blasts are bipotent for differentiation through both neutrophilic and monocytic lineages, whereby monocytic differentiation is linked to c-fms expression and stimulation.

Colocalization of the VEGF-R2 and the common IL-3/GM-CSF receptor beta chain to lipid rafts leads to enhanced p38 activation

Previous studies suggested an important role for vascular endothelial growth factor (VEGF) and its receptors in postnatal haemopoiesis. However, it is unclear how VEGF receptor (VEGFR) signalling could interact with that issued from the activation of haematopoietic growth factor receptors. To elucidate this point we explored VEGF‐R2 and granulocyte‐macrophage colony‐stimulating factor receptor (GM‐CSFR) membrane localization and cell signalling in TF1‐KDR cells (TF1 leukaemic cells that overexpress VEGF‐R2/KDR). Activation of either GM‐CSFR or VEGF‐R2 was shown to determine the migration of both receptor elements (VEGF‐R2 and the common β‐chain of the GM‐CSFR) to lipid rafts. The study of receptor phosphorylation showed that GM‐CSF induced the phosphorylation of its own receptor and the transphosphorylation of VEGF‐R2; on the other hand, VEGF triggered the phosphorylation of its receptor and transphosphorylated the β‐chain of the GM‐CSFR. Co‐stimulation of TF1‐KDR cells with both GM‐CSF and VEGF‐A resulted in massive migration of both the common GM‐CSFR β‐chain and VEGF‐R2 to lipid rafts and sustained p38 mitogen‐activated protein kinase activation. Disruption of lipid rafts inhibited the capacity of both GM‐CSF and VEGF‐A to activate p38. Experiments with specific p38 inhibitors showed that p38 activation was required to sustain the VEGF‐ and GM‐CSF‐dependent proliferation of TF1‐KDR and the survival of primary acute myeloid leukaemia blasts.

Autocrine role of angiopoietins during megakaryocytic differentiation.

The tyrosine kinase Tie-2 and its ligands Angiopoietins (Angs) transduce critical signals for angiogenesis in endothelial cells. This receptor and Ang-1 are coexpressed in hematopoietic stem cells and in a subset of megakaryocytes, though a possible role of angiopoietins in megakaryocytic differentiation/proliferation remains to be demonstrated. To investigate a possible effect of Ang-1/Ang-2 on megakaryocytic proliferation/differentiation we have used both normal CD34+ cells induced to megakaryocytic differentiation and the UT7 cells engineered to express the thrombopoietin receptor (TPOR, also known as c-mpl, UT7/mpl). Our results indicate that Ang-1/Ang-2 may have a role in megakaryopoiesis. Particularly, Ang-2 is predominantly produced and released by immature normal megakaryocytic cells and by undifferentiated UT7/mpl cells and slightly stimulated TPO-induced cell proliferation. Ang-1 production is markedly induced during megakaryocytic differentiation/maturation and potentiated TPO-driven megakaryocytic differentiation. Blocking endogenously released angiopoietins partially inhibited megakaryocytic differentiation, particularly for that concerns the process of polyploidization. According to these data it is suggested that an autocrine angiopoietin/Tie-2 loop controls megakaryocytic proliferation and differentiation.