Biomedical subjects

Elisabetta Rovida

Publications and source records attributed to Elisabetta Rovida.

7 recordsLinked to original sources

PubMed2006

The c-Jun-N-terminal-Kinase inhibitor SP600125 enhances the butyrate derivative D1-induced apoptosis via caspase 8 activation in Kasumi 1 t(8;21) acute myeloid leukaemia cells.

We recently showed that the histone deacetylase inhibitor D1 induced apoptosis in the t(8;21) Kasumi 1 acute myeloid leukaemia (AML) cell line and activated caspase 9. The present study characterised the effects of the combined administration of D1 with PD98059, SB203580 or SP600125, specific inhibitors of mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 or Jun N-terminal kinase (JNK), respectively. Among these inhibitors, SP600125 was the only one to markedly induce apoptosis and decrease cell proliferation. These experiments showed that SP600125 activated caspase 8 and confirmed that D1 activated the intrinsic pathway of apoptosis, as caspase 8 was not affected while Bcl-2 was down-regulated following D1 administration. The combination of the two drugs enhanced caspase-8 activation and induced apoptosis in an additive fashion. JNK was constitutively activated in the Kasumi 1, NB4, HL60 and THP-1 human AML cell lines, as well as in primary blasts from a t(8;21) AML patient. In all these cells, the pro-apoptotic effect of the two drugs alone was increased when they were combined. On this basis, the combined administration of D1 with SP600125 seems to be very promising as a potential anti-leukaemic tool in AML.

Anthracenes↗

PubMed2005

Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF.

The macrophage colony-stimulating factor (M-CSF, CSF-1) regulates survival, proliferation and differentiation of mononuclear phagocytes, as well as macrophage motility and morphology. The latter features are usually regulated by ECM-mediated activation of integrins and subsequent tyrosine phosphorylation of cellular proteins, including focal adhesion kinase (FAK). FAK is phosphorylated by downstream receptor tyrosine kinases as well. We addressed the question whether M-CSF regulates FAK tyrosine phosphorylation in macrophages, and found that M-CSF induces FAK phosphorylation at all known tyrosine residues. This phosphorylation was dependent on Src. Extracellularly-regulated kinase (ERK), Jun N-terminal kinase (JNK) and phosphatidylinositol-3-kinase (PI3K) were found to be negatively involved in M-CSF-induced FAK phosphorylation, as their inhibition resulted in FAK hyper-phosphorylation. Following M-CSF treatment, FAK and the active forms of M-CSFR and Src were redistributed to the cytoskeleton, where active ERK, JNK and PI3K were detectable. Immunofluorescence showed the presence of FAK and its active form in focal complexes following M-CSF treatment. Moreover, cell spreading and adhesion were impaired when FAK tyrosine phosphorylation was abrogated by either transfection with FRNK, a dominant negative form of FAK, or treatment with a number of inhibitors of upstream FAK-activating signals. These results point to a relevant role for FAK in the regulation of cell spreading and adhesion in macrophages.

Animals↗

PubMed2003

Butyrates, as a single drug, induce histone acetylation and granulocytic maturation: possible selectivity on core binding factor-acute myeloid leukemia blasts.

Acute myeloid leukemia (AML) is a disease characterized by a block of maturation. Genes coding for core binding factors are rearranged in a considerable subset of AML cases and result in an altered interaction of core binding factor (CBF) subunits with transcriptional coregulators (NCoR/SMRT). Recruitment of histone deacetylase is also altered in AML, and a subsequent transcriptional repression of target genes involved in myeloid maturation is determined. We determined here the effects of two histone deacetylase inhibitors, sodium butyrate and the stable prodrug xylitol butyrate derivative (D1), on a t(8;21)-positive cell line (Kasumi-1) as well as primary AML blasts. Exposure (24-96 h) to butyrates (1 mM) of Kasumi-1 cells induced histone H4 acetylation, whereas H3 acetylation was unchanged. Induction of morphological and immunophenotypic granulocytic maturation (96 h), also confirmed by an increased expression of CAAT/enhancer binding protein alpha, was observed. Inhibition of proliferation and apoptosis via activation of caspase-9 was also observed. In primary AML blasts, butyrates (0.5 mM) increased histone H4 acetylation of 18 of 19 cases tested. Terminal granulocytic maturation was observed in all cases (5 of 5) characterized by chromosomal translocations involving CBF, whereas in non-CBF cases, maturation was incomplete (4 of 8) or absent (4 of 8). Our data indicate the possibility to effectively remove, in CBF AML cases, the maturation block generated by histone deacetylase stable recruitment, contributing to a possible development of molecularly targeted therapies of AML.

Acetylation↗

PubMed2003

Tryptophan availability selectively limits NO-synthase induction in macrophages.

We studied the effects of tryptophan (TRP) availability on the synthesis and release of nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) in interferon-gamma (IFN-gamma)-activated murine macrophages of the BAC1.2F5 cell line. IFN-gamma (100 U/ml) not only increased the synthesis and release of NO and TNF-alpha from these cells but also induced indoleamine-2,3-dioxygenase, the rate-limiting enzyme of TRP catabolism. This led to an increased metabolic flow through the kynurenine pathway and significantly decreased TRP levels in macrophage incubation media. Low TRP concentrations in the media, however, modified IFN-gamma effects. In TRP-"starved" cultures, in fact, the IFN-gamma-mediated NO synthase induction was significantly reduced, and the increased TNF-alpha synthesis and release were not affected. Our results suggest that a reduced local TRP availability may modify macrophage function and possibly the outcome of immune responses.

Animals↗

PubMed2002

Low Mr phosphotyrosine protein phosphatase associates and dephosphorylates p125 focal adhesion kinase, interfering with cell motility and spreading.

Low M(r) phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M(r) phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, low M(r) phosphotyrosine protein phosphatase both in vitro and in vivo. This event is consistent with an altered association of pp60(src) with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M(r) phosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M(r) phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.

3T3 Cells↗

PubMed2002

Opposite effects of different doses of MCSF on ERK phosphorylation and cell proliferation in macrophages.

We had previously shown that murine macrophages expressing v-Fes, the oncogenically activated counterpart of the c-Fes cytoplasmic tyrosine kinase, proliferate independently of Macrophage Colony-Stimulating Factor (MCSF) and that the Extracellular signal-Regulated Kinase (ERK) pathway mediates the mitogenic effect of v-Fes. In this study, the response of c-fes- and v-fes-overexpressing cells to MCSF was investigated. A critical modulation of the activation of Mitogen-activated ERK Kinase (MEK) and ERK based on the MCSF dose was characterized. ERK activation was increased by MCSF doses capable to elicit a mitogenic response (2-5 U/ml). On the contrary, MCSF doses as low as 0.05 U/ml markedly reduced ERK phosphorylation and nuclear content and moderately but significantly reduced cell proliferation. The reduction of MEK and ERK phosphorylation was very rapid, suggesting the involvement of cytosolic phosphatases. Among these, phospho-tyrosine protein phosphatases and phosphoserine/threonine protein phosphatase-2A were found involved. These findings represent the first observation that different doses of the same growth factor, MCSF in particular, can exert opposite effects on cell proliferation by switching on or off ERK signaling.

Animals↗

PubMed2002

Transmodulation of cell surface regulatory molecules via ectodomain shedding.

Cell responses to exogenous stimuli often result in a rapid decrease of cell surface density of a wide range of diverse regulatory proteins, receptor and adhesion molecules in particular. This decrease may occur in a ligand-dependent fashion (down-regulation), following endocytosis and degradation by lysosomal proteases, or by down-modulation, where molecules are targeted by endoproteases directly on cell surface. These proteases are recruited by trans-modulating agents, different from ligand, which act via their own receptors and the related intracellularly-generated signals. Endoproteolytic activity determines the release of large portions (shedding) of substrate proteins, called ectodomains, which are usually not ligand-bound, and therefore represent biologically-active molecules. Ectodomain shedding is involved in a number of pathophysiological processes, such as inflammation, cell degeneration and apoptosis, and oncogenesis. Common features of the process, such as the involvement of protein kinase C and of transmembrane metalloproteases, have been identified. In this review, we summarize basic concepts on down-modulation and ectodomain shedding, and provide an update of the issue with respect to: (i) new entries to the list of molecules found involved in the process; (ii) current views about the upstream control of shedding, i.e. the pathways linking the signals triggered by the trans-modulating agents to the activation of endoproteolytic activity on the cell surface.

Animals↗