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F Fazioli

Publications and source records attributed to F Fazioli.

At least 37 records · Page 2Linked to original sources
PubMed1993

Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals.

A method which allows direct cloning of intracellular substrates for receptor tyrosine kinases (RTKs) was developed. By applying this technique to the study of the epidermal growth factor receptor (EGFR) signaling pathway, we have isolated a cDNA, designated eps8, which predicts a approximately 92 kDa protein containing an SH3 domain. Eps8 also contains a putative nuclear targeting sequence. Antibodies specific to the eps8 gene product recognize a protein of M(r) 97 kDa and a minor 68 kDa component, which are closely related, as demonstrated by V8 proteolytic mapping. The product of the eps8 gene is tyrosine-phosphorylated in vivo following EGF stimulation of intact cells and associates with the EGFR, despite the lack of a functional SH2 domain. Several other RTKs are also able to phosphorylate p97eps8. Thus, the eps8 gene product represents a novel substrate for RTKs. Adoptive expression of the eps8 cDNA in fibroblastic or hematopoietic target cells expressing the EGFR resulted in increased mitogenic response to EGF, implicating the eps8 gene product in the control of mitogenic signals.

3T3 Cells↗
PubMed1993

eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.

3T3 Cells↗
PubMed1993

The ezrin-like family of tyrosine kinase substrates: receptor-specific pattern of tyrosine phosphorylation and relationship to malignant transformation.

A method for the isolation of tyrosine kinases substrates was developed. The method takes advantage of immuno-affinity purification of an entire set of proteins phosphorylated by tyrosine kinases, followed by generation of antisera against the purified protein pool and immunological screening of bacterial expression libraries with these antisera. By applying this methodology to the study of the phosphorylation events triggered by activation of the epidermal growth factor receptors, we have isolated several cDNAs encoding novel putative tyrosine kinase substrates. One of these cDNAs encodes radixin, a protein belonging to the band 4.1 family of proteins and highly related to ezrin and moesin. We demonstrated that, despite a high degree of relatedness, these three proteins exhibit a distinct receptor-specific pattern of phosphorylation, raising the possibility that they might mediate receptor-specific cellular changes. In addition the generation of antibodies specific for either radixin, ezrin or moesin allowed us to show that a previously described tumor transplantation antigen is indeed ezrin, thus implicating this protein in the determination of the biological phenotype of certain tumors.

Amino Acid Sequence↗
PubMed1992

Identification and biochemical characterization of novel putative substrates for the epidermal growth factor receptor kinase.

To gain insight into the mechanisms which control the mitogenic response to epidermal growth factor (EGF), we have partially purified and characterized several intracellular proteins which are phosphorylated on tyrosine residues following activation of the epidermal growth factor receptor (EGFR). Partial purification was achieved by immunoaffinity chromatography using immobilized anti-phosphotyrosine antibodies. Antisera generated against the partially purified proteins were used to identify at least five novel EGFR putative substrates, designated, on the basis of their apparent molecular weight, p97, p68, p61, p56, and p23. All of these proteins became specifically phosphorylated on tyrosine after EGF treatment of intact cells, as assessed by phosphoamino acid analysis, and none of them represented an EGFR degradation product. The phosphorylation of these proteins appeared to be relatively specific for the EGFR. In particular, an EGFR-related kinase, erbB-2 was much less efficient than EGFR at phosphorylating p97, p56, and p23 and incapable of phosphorylating p68. The identification of these novel EGFR putative substrates should lead to a better understanding of the mechanisms controlling the specificity of EGFR-mediated mitogenic signaling.

3T3 Cells↗
PubMed1992

Constitutive expression of IL-1 beta, M-CSF and c-fms during the myeloid blastic phase of chronic myelogenous leukaemia.

During the myeloid blast crisis (BC) of chronic myelogenous leukaemia (CML) non-random additional chromosome abnormalities occur in over 80% of patients. However, these cytogenetic changes have been reported to precede the clinical signs of CML-BC by several months to years suggesting that other biological events may participate in the multistep process of acute transformation of CML. The autocrine production of growth factors has been recently shown to occur in several haematological malignancies and particularly in acute myeloblastic leukaemia (AML). In the present report we demonstrate that IL-1 beta gene is expressed in almost all cases of CML in myeloid blast crisis. The secretion of IL-1 from CML blasts in culture supernatants was confirmed in all five of the patients we studied. A high proportion of cases showed constitutive expression of the M-CSF gene and many of the same patients often had a simultaneous co-expression of the proto-oncogene c-fms which encodes for the M-CSF receptor. After exposure of leukaemic cells to phorbol myristate acetate (PMA), release of M-CSF protein was documented in three of five patients studied. No significant interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF), was detected in these patients demonstrating that a different pattern of growth factors secretion exist in AML and CML, where distinct molecular events are likely involved in the control of leukaemic proliferation.

Adult↗
PubMed1992

erbB-2 autophosphorylation is required for mitogenic action and high-affinity substrate coupling.

Autophosphorylation of gp185erbB-2 in vivo is confined to its carboxy terminus and is required for optimal erbB-2 transforming activity under conditions of receptor overexpression. It remains unresolved, however, to what extent autophosphorylation regulates erbB-2 mitogenic signaling in normal cells, nor is the biochemical basis for such a regulatory function known. To address these issues, we utilized a chimeric molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and intracellular domains of the erbB-2 product. In this EGFR/erbB-2 chimera, erbB-2 kinase activity is regulated by EGF binding. An EGFR/erbB-2 mutant bearing multiple Tyr----Phe substitutions at erbB-2 autophosphorylation sites (EGFR/erbB-2 5P) displayed markedly reduced phosphotyrosine content following EGF stimulation in comparison with the non-mutated chimera. When expressed in NR6 cells, the EGFR/erbB-2 5P mutant was unable to deliver a sizeable mitogenic signal when activated by EGF at physiological levels. In intact cells, the 5P mutant was still able to stimulate phosphorylation of the gamma isozyme of phospholipase C (PLC-gamma), a prototype erbB-2 substrate, although with a delayed time course, indicating that the 5P mutation decreased the affinity of the erbB-2 kinase for this substrate. This conclusion was further supported by the inability of the 5P mutant to associate with PLC-gamma in co-immunoprecipitation experiments. We infer that a major role of autophosphorylation is to increase the affinity of the erbB-2 kinase for its cellular substrates, so that, under physiological conditions, autophosphorylation is absolutely required for erbB-2 mitogenic signaling.

Amino Acid Sequence↗
PubMed1991

The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.

Animals↗
PubMed1991

The juxtamembrane regions of the epidermal growth factor receptor and gp185erbB-2 determine the specificity of signal transduction.

The epidermal growth factor receptor (EGFR) and gp185erbB-2 are closely related tyrosine kinases. Despite extensive sequence and structural homology, these two receptors display quantitative and qualitative differences in their ability to couple with mitogenic signalling pathways. By using chimeric molecules between EGFR and erbB-2, we found that the determinants responsible for the specificity of mitogenic signal transduction are located in the amino-terminal half of the tyrosine kinase domain of either receptor. In the EGFR, mutational analysis within this subdomain revealed that deletion of residues 660 to 667 impaired receptor mitogenic activity without affecting its tyrosine kinase properties. This sequence is therefore likely to contribute to the specificity of substrate recognition by the EGFR kinase.

Amino Acid Sequence↗
PubMed1990

A paracrine circuit in the regulation of the proliferation of macrophages infiltrating murine sarcomas.

Tumor-associated macrophages (TAM) isolated from two murine sarcomas (mFS6 and MN/MCA1) had high levels of proliferative activity (7 to 11% of cells in S phase) compared to peritoneal macrophages (1 to 2% of cells in S phase). In an effort to elucidate the mechanisms responsible for the proliferative activity of TAM, expression of c-fms and macrophage (M)-CSF was investigated in TAM and sarcoma cells. TAM had high levels of mRNA transcripts of the c-fms protooncogene, which encodes a tyrosine kinase probably identical to the M-CSF receptor, but did not express M-CSF transcripts whereas sarcoma cells had high levels of M-CSF mRNA. Sarcoma cell conditioned medium had M-CSF activity on bone marrow cells and induced proliferation of peritoneal exudate and bone marrow-derived macrophages. These activities were blocked by anti-M-CSF antibodies. These findings outline a paracrine circuit in the regulation of TAM proliferation, involving M-CSF, secreted by sarcoma cells and acting on c-fms expressing TAM. Inasmuch as TAM from these murine sarcomas have tumor growth promoting activity, a "ping pong" reciprocal feeding interaction may occur between macrophages and neoplastic cells in these tumors.

Animals↗
PubMed1990

The carboxy-terminal domains of erbB-2 and epidermal growth factor receptor exert different regulatory effects on intrinsic receptor tyrosine kinase function and transforming activity.

The erbB-2 gene product, gp185erbB-2, displays a potent transforming effect when overexpressed in NIH 3T3 cells. In addition, it possesses constitutively high levels of tyrosine kinase activity in the absence of exogenously added ligand. In this study, we demonstrate that its carboxy-terminal domain exerts an enhancing effect on erbB-2 kinase and transforming activities. A premature termination mutant of the erbB-2 protein, lacking the entire carboxy-terminal domain (erbB-2 delta 1050), showed a 40-fold reduction in transforming ability and a lowered in vivo kinase activity for intracellular substrates. When the carboxy-terminal domain of erbB-2 was substituted for its analogous region in the epidermal growth factor receptor (EGFR) (EGFR/erbB-2COOH chimera), it conferred erbB-2-like properties to the EGFR, including transforming ability in the absence of epidermal growth factor, elevated constitutive autokinase activity in vivo and in vitro, and constitutive ability to phosphorylate phospholipase C-gamma. Conversely, a chimeric erbB-2 molecule bearing an EGFR carboxy-terminal domain (erbB-2/EGFRCOOH chimera) showed reduced transforming and kinase activity with respect to the wild-type erbB-2 and was only slightly more efficient than the erbB-2 delta 1050 mutant. Thus, we conclude that the carboxy-terminal domains of erbB-2 and EGFR exert different regulatory effects on receptor kinase function and biological activity. The up regulation of gp185erbB-2 enzymatic activity exerted by its carboxy-terminal domain can explain, at least in part, its constitutive level of kinase activity.

Animals↗
PubMed1990

[An anatomicoclinical analysis of the synovial plica syndrome].

The authors describe embryology and anatomy of synovial plica of the knee joint. They propose a clinic and pathogenic classification and after examining the diagnostic problem of plica syndrome, analyze their long-term results after surgical treatment.

Humans↗
PubMed1989

A morphometric approach to the grading of chondroid tumours on fine-needle smears.

A morphometric study was performed on 600 nuclei from 6 benign chondroid tumours (BCL) (3 enchondromas and 3 exostoses), 600 nuclei from 6 well-differentiated chondrosarcomas (WDC) and 200 nuclei from moderately-differentiated chondrosarcomas (MDC) obtained by fine needle aspiration biopsy. The parameters considered were nuclear area, perimeter and maximum diameter. Moving across the three groups of lesions the nuclei appeared progressively larger. A statistical analysis was performed on the three groups. Significant differences between the means of all the parameters considered were observed. The authors suggest that morphometric analysis may be a possible step in the cytologic diagnosis and grading of chondroid tumours.

Adult↗
PubMed1988

Cell-mediated immunity to chemically xenogenized tumors. I. Inhibition by specific antisera and H-2 association of the novel antigens.

T cell-mediated proliferative and cytotoxic responses occur in vitro to syngeneic tumor cells antigenically altered by mutagen treatment. One such xenogenized variant of the murine L5178Y lymphoma elicits IgG antibodies reactive with determinants on variant cells that are not expressed at detectable levels on parental or normal cells of the same H-2d haplotype and are also unrelated to public specificities of H-2b or H-2k histocompatibility antigens. In the present study we investigated the effect of those antibodies on development of cell-mediated responses in vitro to the xenogenized cells used for induction of the humoral response. The proliferative reaction, generation of cytolytic activity and target cell lysis were all inhibited by the anti-xenogenized tumor immune serum, whereas the corresponding reactions to the parental cells by syngeneic or allogeneic effector lymphocytes were not. In order to investigate the possible H-2 association of T cell-mediated responses to xenogenized cells, we also examined the effect on those reactions of antibodies specific for Class I or Class II products of the H-2d complex. The results obtained suggested a role for I-Ad molecules in the T cell proliferative response to the xenogenized cells, and also indicated a preferential association of the cytotoxic response with H-2Kd determinants.

Animals↗
PubMedDate not supplied

Neoadjuvant chemotherapy for intermediate/high-grade soft tissue sarcomas: five-year results with epirubicin and ifosfamide.

BACKGROUND: Neoadjuvant chemotherapy for intermediate/high-grade soft tissue sarcomas (STS) may provide some advantages for facilitating the surgical resection of the tumor and for disease control. However its role as induction therapy before surgery should still be proved. PATIENTS AND METHODS: Twenty-one patients with intermediate/high-grade STS and tumor size > or = 5 cm were consecutively treated from 1997 to 2001 with neoadjuvant chemotherapy based on epirubicin 60 mg/m2/day on days 1 and 2 and ifosfamide 1.8 gr/m2/day on days 1 through 5 every three weeks. Evaluation of objective tumor response and toxicity were carried out according to WHO criteria. RESULTS: Nine partial responses were documented; stable disease in 11 patients, progressive disease in one patient. Apart from nine cases of grade 4 neutropenia, the treatment was generally well-tolerated. Twelve patients underwent conservative and limb salvage surgery. CONCLUSION: This therapeutic approach seems to be effective in facilitating surgery. Neutropenia was the most significant toxicity but it was preventable or medically treatable with G-CSF support.

Adult↗
PubMedDate not supplied

[A modified Putti-Platt surgical method in the treatment of habitual luxation of the anterior shoulder: our experience].

Fifty cases of recurrent dislocation of the shoulder operated according to the Putti-Platt method modified by lateralization of the insertion of the coracoid processes are reported. Excellent results were obtained in 86% of the cases. There was neither incidence of recurrence, nor degenerative joint alteration. Mild limitation in extrarotation (less than 10 degrees) was observed in 7 cases.

Adolescent↗