Bernard Rossi

The multiple endocrine neoplasia type 2B point mutation switches the specificity of the Ret tyrosine kinase towards cellular substrates that are susceptible to interact with Crk and Nck

The RET proto-oncogene encodes a Tyrosine Kinase Receptor (RTK) which plays an important function in the proliferation and/or differentiation of neuroectodermic cells. Germline mutation of a methionine to a threonine within the RET TK domain predisposes to the Multiple Endocrine Neoplasia type 2B (MEN 2B). It has been demonstrated that, unlike c-Ret, the MEN 2B mutated Ret displays constitutive TK activity, tyrosine autophosphorylation and transforms fibroblasts. However, this oncoprotein is more than a fully activated wild-type (WT) Ret TK since it also displays modified substrate specificity. Change in substrate specificity leads to the tyrosine autophosphorylation of MEN 2B Ret on new sites as well as the phosphorylation of several novel downstream targets. But, none of these substrates have been identified and the ability of MEN 2B Ret phosphoprotein to interact with Src Homology 2 (SH2) domain containing molecules has been poorly investigated. In this report, using a constitutively activated Ret TK form, Ret-ptc 2, we demonstrate that the MEN 2B as the activated WT Ret TK binds to several SH2 signalling proteins such as Shc, Grb-2, Phospholipase Cγ, Crk and Nck. However, in contrast to the activated WT form, expression of the MEN 2B mutated Ret-ptc 2 results in the tyrosine phosphorylation of a panel of proteins which interestingly interact with Crk and Nck. We identified Paxillin, a cytoskeletal protein as one of the Crk associated proteins that is dramatically phosphorylated in MEN 2B but not in WT Ret expressing cells. These data suggest that MEN 2B mutated Ret triggers distinct signalling pathways that might be related to its transforming power.

High expression of the antigen recognized by the monoclonal antibody GB24 on human breast carcinomas: A preventive mechanism of malignant tumor cells against complement attack?

SummaryGB24 is a mouse monoclonal antibody raised against a common trophoblast-lymphocyte cross-reactive antigen. GB24 detects the membrane cofactor protein (MCP, CD46), a member of the complement regulatory protein family, which serves as a cofactor for factor 1 mediated cleavage of C3b. This study investigated the reactivity of GB24 on 38 breast carcinomas and 34 normal/benign breast tissues by immunochemistry as well as the reactivity of F2B7-2, an antibody specific to the decay accelerating factor (DAF, CD55) of the complement. GB24 staining was present on both normal tissue and benign lesions, but very strong diffuse reactivity was observed on carcinomas. This reactivity increased with the tumor grade. By contrast, malignant tumor cells lacked DAF expression. F2B7-2 antibody reacted weakly with benign epithelial cells. Results were studied by computer assisted image analysis to accurately define the mean optical densities. The densitometric analysis of MCP positive carcinomas showed a high intensity of the staining. Expression of MCP and DAF on MCF-7 cell lines was analyzed by flow cytometry. MCF-7 cell lines were strongly stained by mAb GB24 only. These data suggest that selectively enhanced expression of the antigen recognized by GB24 is associated with malignant breast disorders. This high expression, which may reflect a protective mechanism by which tumor cells survive complement activation, may prove useful as a marker of malignant transformation.

The sensitivity of activated Cys Ret mutants to glial cell line-derived neurotrophic factor is mandatory to rescue neuroectodermic cells from apoptosis.

ABSTRACT Hirschsprungs disease (HSCR), a frequent developmental defect of the enteric nervous system is due to loss-of-function mutations of RET, a receptor tyrosine kinase essential for the mediation of glial cell-derived neurotrophic factor (GDNF)-induced cell survival. Instead, gain-of-function Cys mutations (e.g., Cys609, Cys620, and Cys634) of the same gene are responsible for thyroid carcinoma (MEN2A/familial medullary thyroid carcinoma) by causing a covalent Ret dimerization, leading to ligand-independent activation of its tyrosine kinase. In this context, the association of Cys609- or Cys620-activating mutations with HSCR is still an unresolved paradox. To address this issue, we have compared these two mutants with the Cys634 Ret variant, which has never been associated with HSCR, for their ability to rescue neuroectodermic cells (SK-N-MC cells) from apoptosis. We show here that despite their constitutively activated kinase, the mere expression of these three mutants does not allow cell rescue. Instead, we demonstrate that like the wild-type Ret, the Cys634 Ret variant can trigger antiapoptotic pathways only in response to GDNF. In contrast, Cys609 or Cys620 mutations, which impair the terminal Ret glycosylation required for its insertion at the plasma membrane, abrogate GDNF-induced cell rescue. Taken together, these data support the idea that sensitivity to GDNF is the mandatory condition, even for constitutively activated Ret mutants, to rescue neuroectodermic cells from apoptosis. These findings may help clarify how a gain-of-function mutation can be associated with a developmental defect.

Pentoxifylline inhibits liver expression of tumor necrosis factor alpha mRNA following normothermic ischemia-reperfusion

BACKGROUNDnPentoxifylline (PTX) has been shown to reduce hepatic injury after normothermic ischemia and reperfusion (I-R) in rat liver.nnnAIMnThe aim of this study was to evaluate the effects of PTX on liver expression of tumor necrosis factor alpha (TNFalpha) mRNA following normothermic liver I-R.nnnMATERIALS AND METHODSnA segmental normothermic ischemia of the liver was induced in male Lewis rats by occluding the blood vessels including the bile duct to the median and left lateral lobes for 90 min. At the end of ischemia the nonischemic liver lobes were resected. Rats were divided into three groups: group 1, control Ringer lactate administration; group 2, PTX treatment; group 3, sham-operated control rats. PTX (50 mg/kg) was injected intravenously 30 min before and 60 min after induction of ischemia. Survival rates were compared and the serum activities of TNFalpha, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured. Histology of the liver was assessed 6 h after reperfusion. Liver TNFalpha mRNA was assessed by PCR amplification at 0, 60, 120, and 210 min after reperfusion.nnnRESULTSnPTX treatment significantly increased 7 day survival (93.3%) compared with nontreated control rats (46.6%, p<0.007). The extent of liver necrosis and the release of liver enzymes were significantly decreased after PTX treatment. Serum activities of TNFalpha were significantly decreased and liver expression of TNFalpha mRNA was inhibited after PTX treatment.nnnCONCLUSIONnPTX protects the liver from ischemic injury and inhibits liver expression of TNFalpha mRNA.

Hypoxia‐reoxygenation differentially stimulates stress‐activated protein kinases in primary‐cultured rat hepatocytes

Abstract Organ injury after ischemia and reperfusion (I/R) remains one of the most important limiting factors in liver surgery and transplantation. Oxygen‐free radical (OFR) generation is considered a major cause of this damage. JNK1/SAPK1, a member of MAPK family, regulates cell adaptation to stressful conditions. The aim of this study was to determine if hypoxia‐reoxygenation (H/R) can activate JNK1/SAPK1 and if OFR are involved in this activation. Primary cultured rat hepatocytes isolated from other liver cells and blood flow were submitted to warm and cold H/R phases mimicking surgical and transplant conditions. JNK1/SAPK1 was activated by both warm and cold H/R. Deferoxamine (1 mM), di‐phenyle‐neiodonium (50 μM) and N‐acetylcysteine (10 mM) significantly inhibited this kinase activation.

Ultrastructure of the polymorphonuclear leucocytes in human immunodeficiency virus infection

Summary The ultrastructure of polymorphonuclear leucocytes (PMNL) was studied in 16 patients infected with the human immunodeficiency virus (HIV). PMNL were isolated from HIV-infected patients with CD4 + lymphocytes counts greater than 200/mm 3 (without signs of active infection)( n = 12)(group 1), or less than 200/mm 3 ( n = 4)(group 2), and from 16 healthy volunteers (group 3). Immunoelectron microscopy staining using an anti-beta2 integrin antibody (anti-CD18) was performed on PMNL from three individuals of group 2 and of three individuals of group 3, before and after incubation with N -formyl-methionyl-leucyl-phenylalanine (f-MLP). The radical oxygen intermediates (ROI) production of PMNL was investigated by luminol-mediated chemiluminescence. A number of ultrastructural abnormalities in PMNL were found in a higher proportion in HIV-infected patients. These were: (a) an increase in the size of the Golgi apparatus and in the number of mitochondria, and in the quantity of endoplasmic reticulum; (b) some dysplastic features including large cytoplamic vacuoles, whorl of myelin, and nuclear pockets; (c) an increase prevalence of multivesicular bodies compared with control PMNL; (d) some cylindrical confronting cisternae and tubuloreticular structures. After anti-CD18 staining, gold particles were seen on the plasma membrane and more rarely inside the cytoplasm of PMNL from each group but no decrease in this staining was noted in HIV PMNL. Incubation with f-MLP similarly increased the immunostaining of the PMNL in each group. In vitro ROI production was significantly depressed for HIV PMNL compared with control PMNL. Some ultrastructural abnormalities observed in this study could support the possibility that one of the mechanisms underlying the qualitative functional defects of PMNL from HIV-infected patients may be related to some cytopathic effect.