Regulation of RasGRP via a phorbol ester-responsive C1 domain.

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.

The chimeric protein tyrosine kinase ETV6-NTRK3 requires both Ras-Erk1/2 and PI3-kinase-Akt signaling for fibroblast transformation.

There is increasing interest in the potential role of the NTRK family of neurotrophin receptors in human neoplasia. These receptor protein tyrosine kinases (PTKs) are well-known mediators of neuronal cell survival and differentiation, but altered NTRK signaling has also been implicated in mesenchymal, hematopoietic, and epithelial malignancies. We recently identified a novel gene fusion involving one of the neurotrophin receptor genes, NTRK3, in the pediatric solid tumor, congenital fibrosarcoma. In these tumors (and subsequently demonstrated in several other human malignancies), a t(12;15)(p13;q25) rearrangement fuses the 3' portion of the ETV6 gene with exons encoding the PTK domain of NTRK3. The resulting ETV6-NTRK3 fusion protein functions as a chimeric PTK with potent transforming activity. However, previous studies failed to detect interactions between ETV6-NTRK3 and molecules known to link wild-type NTRK3 to its two major effector pathways, namely the Ras-Raf1-Mek1-Erk1/2 mitogenic pathway or the phosphatidylinositol 3'-kinase pathway leading to activation of the AKT survival factor. Therefore, it remains unknown whether ETV6-NTRK3 transformation involves altered NTRK3 signaling. We now report that ETV6-NTRK3 expression in NIH3T3 cells leads to constitutive activation of Mek1 and Akt, as well as to constitutively high expression of cyclin D1. ETV6-NTRK3-induced soft agar colony formation was almost completely abolished by inhibition of either the Ras-Raf1-Mek1-Erk1/2 or the phosphatidylinositol 3'-kinase-Akt pathway. Moreover, this inhibition dramatically reduced expression of cyclin D1. Our results indicate that ETV6-NTRK3 transformation involves a link between known NTRK3 signaling pathways and aberrant cell cycle progression and that Mek1 and Akt activation act synergistically to mediate these effects.

Characterization of Ca(2+)-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni.

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.

Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.

Preoperative evaluation in infants and children: recommendations of the Italian Society of Pediatric and Neonatal Anesthesia and Intensive Care (SARNePI).

BACKGROUND: The preoperative assessment involves the process of evaluating the patient's clinical condition, which is intended to define the physical status classification, eligibility for anesthesia and the risks associated with it, thus providing elements to select the most appropriate and individualized anesthetic plan. The aim of this recommendation was provide a framework reference for the preoperative evaluation assessment of pediatric patients undergoing elective surgery or diagnostic/therapeutic procedures. METHODS: We obtained evidence concerning pediatric preoperative evaluation from a systematic search of the electronic databases MEDLINE and Embase between January 1998 and February 2012. We used the format developed by the Italian Center for Evaluation of the Effectiveness of Health Care's scoring system for assessing the level of evidence and strength of recommendations. RESULTS: We produce a set of consensus guidelines on the preoperative assessment and on the request for preoperative tests. A review of the existing literature supporting these recommendations is provided. In reaching consensus, emphasis was placed on the level of evidence, clinical relevance and the risk/benefit ratio. CONCLUSION: Preoperative evaluation is mandatory before any diagnostic or therapeutic procedure that requires the use of anesthesia or sedation. The systematic prescription of complementary tests in children should be abandoned, and replaced by a selective and rational prescription, based on the patient history and clinical examination performed during the preoperative evaluation.

A tripartite complex composed of ETV6-NTRK3, IRS1 and IGF1R is required for ETV6-NTRK3-mediated membrane localization and transformation.

ETV6-NTRK3 (EN), a chimeric tyrosine kinase generated by t(12;15) translocations, is a dominantly acting oncoprotein in diverse tumor types. We previously showed that insulin-like growth factor 1 receptor (IGF1R) is essential for EN-mediated oncogenesis and that insulin receptor substrate 1 (IRS1) is constitutively tyrosine phosphorylated and bound by EN in transformed cells. Given that IRS1 is also an adapter for IGF1R, we hypothesized that IRS1 might localize EN to IGF1R at the membrane to activate phosphatidylinositol 3-kinase (PI3K)-Akt, which is critical for EN oncogenesis. In this study, we examined EN/IRS1/IGF1R complexes in detail. We find that both IRS1 and kinase active IGF1R are required for EN transformation, that tyrosine phosphorylated IRS1 is present in high molecular weight complexes with EN and IGF1R, and that EN colocalizes with IGF1R at the plasma membrane. Both IGF1R kinase activity and an intact cytoplasmic Y950 residue, the IRS1-docking site of IGF1R, are required, confirming the importance of the IGF1R/IRS1 interaction for EN oncogenesis. The dual specificity IGF1R and insulin receptor (INSR) inhibitor, BMS-536924, blocks EN transformation activity, cell survival and its interaction with IRS proteins, and induces a striking shift of EN proteins to smaller sized molecular complexes. We conclude that a tripartite complex of EN, IRS1 and IGF1R localizes EN to the membrane and that this is essential for EN-mediated transformation. These findings provide an explanation for the observed IGF1R dependency of EN transformation. Blocking IGF1R kinase activity may, therefore, provide a tractable therapeutic strategy for the many tumor types driven by the EN oncoprotein.

Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C.

In order to identify cDNAs that can induce oncogenic transformation, a retroviral vector was used to transfer a library of cDNAs from the murine 32D hemopoietic cell line into NIH 3T3 fibroblasts. We have identified and recovered a provirus containing a 1.8-kilobase pair cDNA whose expression causes morphological transformation in NIH 3T3 cells. The transforming cDNA contains a complete open reading frame that encodes a protein (designated Lfc) with a region of sequence similarity to the product of the lbc oncogene. This region includes a domain that is characteristic of the CDC24 family of guanine nucleotide exchange factors in tandem with a pleckstrin homology (PH) domain. The Lfc protein is distinguished from Lbc by a 150-amino acid NH2-terminal extension that contains a cysteine- and histidine-rich domain similar to the diacylglycerol-binding site (zinc butterfly) found in protein kinase C. NH2- and COOH-terminal deletion analysis revealed that both the PH and putative guanine nucleotide exchange factor domains are required, but the zinc butterfly is dispensable, for transformation. Although the removal of the PH domain of the Lfc protein completely eliminated its ability to transform NIH 3T3 cells, replacement of this domain with an isoprenylation site restored all of its transforming activity. This suggests that a PH domain-dependent recruitment of the Lfc protein to the cellular membrane is a necessary step for cellular transformation. The lfc gene is expressed in a broad range of tissues as well as in a variety of hemopoietic and non-hemopoietic cell lines. Lfc appears to be a new member of a growing family of proteins that are likely to act as activators of Ras-like proteins in a developmental or cell-lineage specific manner.

Secretory carcinoma of the breast: a genetically defined carcinoma entity.

Secretory carcinomas (SBC) are characterized by their characteristic histomorphology and more favorable prognosis compared to invasive ductal carcinoma of usual type (IDC). On this basis, 13 SBCs are evaluated by molecular and immunohistochemical (IH) methods. 13 SBCs and 4 IDCs were analyzed for ETV6-NTRK3 gene fusion by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Fluorescence in situ Hybridization (FISH). 8 of 13 microdissected SBCs with evaluable DNA were evaluated for genetic alterations (GA) by comparative genomic hybridization (CGH). IH included estrogen-receptor (ER), progesterone-receptor (PR), Her-2/neu and Ki-67 (MIB-1) in all 13 cases. Molecular and immunohistochemical results in SBCs were compared with previous data regarding immunohistochemical and molecular characteristics of IDCs. 12 of 13 (92 %) SBC cases, but not IDCs expressed the ETV6-NTRK3 fusion gene which encodes a chimeric tyrosine kinase. Retroviral transfer of ETV6-NTRK3 (EN) into murine mammary epithelial cells resulted in transformed cells that readily formed epithelial tumors in nude mice. CGH revealed an average of 2.0 GAs (range 0-6), including recurrent gains of chromosome 8q and 1q and losses of 22q. Four SBCs were positive for ER and 2 were positive for PR. The mean MIB-1-labeling index was 11.4% (range: <1-34%). Her-2/ neu protein overexpression was detected in 1 case (score 3+). Compared to previous findings in IDCs, SBCs are characterized by the recurrent expression of ETV6-NTRK3 fusion gene, a relatively low number of GAs, low proliferative rate, infrequent Her-2/ neu protein overexpression and a lower rate of steroid hormone receptor expression. These results support the hypothesis that SBCs have immunohistochemical and genetic features that specifically distinguish them from IDCs.

Protection against Schistosoma mansoni infection with a recombinant baculovirus-expressed subunit of calpain.

Infections by human schistosomes, in particular Schistosoma mansoni, account for significant morbidity and mortality every year in tropical and sub-tropical areas. The eggs of the parasite induce pathological changes in the infected host; in chronic and heavy infections, these changes may lead to death. A well-designed anti-schistosomal vaccine, alone or in concert with existing control measures such as chemotherapy, may prove to be a safe, inexpensive, and effective means of reducing the occurrence of severe disease and death in S. mansoni infection. Previous studies have demonstrated the importance of the syncytial layer containing the apical plasma membrane (APM) of S. mansoni in both the survival of the parasite in the mammalian host and as a potential source of immunogens which may be utilized as vaccine candidates. In this paper, we present evidence for the protective capacity of several schistosomal antigen preparations, including a calcium binding protein of the APM, S. mansoni calpain (GenBank accession no. M74233). We have constructed and characterized expression of a recombinant baculovirus expressing the large subunit of S. mansoni calpain, Sm-p80. This recombinant Sm-p80 is recognized by IgA, IgM, IgG1, and IgG3 isotype antibodies found in S. mansoni-infected human sera and partially-purified recombinant Sm-p80 provided a 29-39% reduction in worm burden in immunized mice challenged with S. mansoni. Our data indicate that Sm-p80 may be a useful vaccine antigen for the reduction of the morbidity associated with S. mansoni infections of mammalian hosts.