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9 p.-9 fig.-1 tab., We recently reported that the heparin (Hep) III domain of fibronectin contains the H2 cell adhesion site in repeat III5 which binds activated α4 integrins. We have now further characterized the heparin and cell binding activities of this domain. A recombinant fragment containing repeats III4-III5 (FN-III4–5) induced Jurkat cell adhesion upon integrin activation with Mn2+ or TS2/16 monoclonal antibody (anti-β1). Adhesion of Mn2+-treated cells to FN-III4–5 or FN-III5 fragments was inhibited by chondroitinase ABC and ACII but not by the anti-α4 monoclonal antibody HP2/1. In contrast, HP2/1 completely blocked adhesion of TS2/16-treated cells while chondroitinase had a partial (FN-III4–5) or minor (FN-III5) effect. Thus, the role of each receptor depended on the stimulus used to activate α4β1. The combination of HP2/1 and chondroitinase at dilutions which did not inhibit when used individually abolished adhesion of Mn2+ or TS2/16-treated cells to both fragments, indicating a cooperative effect between α4β1 and chondroitin sulfate proteoglycans (CSPG). Furthermore, we have identified a 20-amino acid sequence in III5 (HBP/III5) which binds heparin and induces cell adhesion via CSPG exclusively. Although soluble HBP/III5 was a poor inhibitor, when combined with H2, it abolished adhesion to FN-III4–5 and FN-III5 fragments. These results establish that adhesion to the Hep III domain involves the cooperation of activated α4β1 and CSPG and show that HBP/III5 is a novel heparin and CSPG-binding site contributing to cell adhesion to this domain., This work was supported by Grants SAF97-0064-C03-02 from the Comisión Interministerial de Ciencia y Tecnología (CICYT), 94/0277 from Fondo de Investigaciones Sanitarias (FIS), and partially by funds from the Associazione Italiana per la Ricerca sul Cancro (to L. Z).

12 páginas, 8 figuras -- PAGS nros. 2344-2354, The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of ∼100 aa, named scavenger receptor cysteine-rich (SRCR). Two types of SRCR domains (A or B) have been reported, which differ in the number of coding exons and intradomain cysteines. Although no unifying function has been reported for SRCR-SF members, recognition of pathogen-associated molecular patterns (PAMPs) was recently shown for some of them. In this article, we report the structural and functional characterization of mouse S5D-SRCRB, a new group B member of the SRCR-SF. The s5d-srcrb gene maps at mouse chromosome 7 and encompasses 14 exons extending over 15 kb. The longest cDNA sequence found is 4286 bp in length and encodes a mature protein of 1371 aa, with a predicted M(r) of 144.6 kDa. Using an episomal mammalian-expression system, a glycosylated soluble recombinant form >200 kDa was obtained and used as immunogen for the generation of specific rat mAbs. Subsequent immunohistochemical and real-time PCR analysis showed significant S5D-SRCRB expression in murine genitourinary and digestive tracts. S5D-SRCRB was shown to bind endogenous extracellular matrix proteins (laminin and galectin-1), as well as PAMPs present on Gram-positive and Gram-negative bacteria and fungi. PAMP binding by S5D-SRCRB induced microbial aggregation and subsequent inhibition of PAMP-induced cytokine release. These abilities suggest that S5D-SRCRB might play a role in the innate defense and homeostasis of certain specialized epithelial surfaces, This work was supported by grants from the Spanish Ministry of Education (SAF 2007-62197 to F.L.), the Generalitat de Catalunya (Grants 2009/SGR/524 to J.Y. and 2009/SGR/252 to F.L.), and the Spanish Research Network on Infectious Diseases (Red Espan˜ola de Investigacio´n en Patologı´a Infecciosa, RD06/0008/1013 to F.L.). C.M.-J. and C.E.-F. are recipients of fellowships from the Spanish Ministry of Education (FPU AP2007-02223 and FPI BES2008-005544, respectively).

10 páginas, 5 figuras -- PAGS nros. 4382-4391, Purpose: Arsenic trioxide (ATO) induces B-cell chronic lymphocytic leukemia (B-CLL) cell apoptosis in vitro. We sought to study the mechanism involved in this effect and whether ATO is suitable for combination therapies with protein kinase inhibitors. Experimental Design: B-CLL cells were isolated from the peripheral blood of 28 patients. Cell viability studies with ATO alone or in combination with kinase inhibitors were done by flow cytometry, Western blotting, and immunofluorescence analyses. Results: After 48 hours, 3 μmol/L ATO induced apoptosis (average 75%) in all B-CLL samples studied and with minimal effect on normal peripheral blood lymphocytes. Apoptosis entailed Akt and NF-κB inactivation, XIAP downregulation, and PTEN upregulation, thus implying inhibition of the phosphoinositide 3-kinase (PI3K) survival pathway. Indeed, the combination of ATO and PI3K inhibitors increased the apoptotic effect of either agent alone. ATO also induced c-jun-NH2 terminal kinase (JNK) activation, and this was crucial and required for subsequent apoptotic events, as inhibiting JNK activity by either gene silencing or specific inhibitors prevented Akt and NF-κB inactivation, caspase activation, and mitochondrial damage. Moreover, JNK activation was the earliest response to ATO, preceding and determining reactive oxygen species production. Conclusions: We identified the mechanism involved in ATO action on B-CLL cells and show that the combination of low doses of ATO and PI3K inhibitors efficiently induces B-CLL cell death. ATO may therefore constitute an efficient treatment for B-CLL, particularly in combined therapies, Grants PI060400, RD06/0020/0011, and SAF2009-07035 (A. García-Pardo), RD06/0020/0041 (A. Pandiella), and PI061637 and RD06/0020/0080 (M.J. Terol) from the Ministerio de Ciencia e Innovación, and grant from the Fundación de Investigación Médica Mutua Madrileña (A. García-Pardo). JRM was supported by a fellowship from the Fundación Ramón Areces, Madrid, Spain

5 páginas, 3 figuras -- PAGS nros. 708-712, We recently showed that α4β1 integrin induces B-cell chronic lymphocytic leukemia (B-CLL) cell resistance to fludarabine-induced apoptosis via upregulation of Bcl-xL. We have now studied whether p53 was involved in this response. Cells from five B-CLL patients with wild-type p53 determined by DNA sequencing, or from the EHEB cell line, cultured on the α4β1 ligand H/89 during fludarabine treatment, showed significantly higher viability (P⩽0.001) than cells cultured on poly-d-lysine (pLys). Parallel to this different viability, fludarabine increased p53 expression on pLys-cultured cells and this increase was significantly reduced (P⩽0.05) on cells cultured on H/89. Thus, p53 appears to be part of the survival pathway induced by α4β1 ligation and may contribute to the progressive drug resistance in B-CLL, This work was supported by Grants 08.1/012/97 from the Comunidad Autónoma de Madrid (CAM, to A.S. and A.G.P.), SAF2000-0124 (to A.G.P.), and SAF2000-0118 (to A.S.) from the Comisión Interministerial de Ciencia y Tecnologı́a (CICYT). M.T. de la Fuente and B. Casanova were supported by fellowships from CAM; E. Cantero was supported by a fellowship from Fondo de Investigación Sanitaria, Madrid

8 páginas, 7 figuras, 2 tablas -- PAGS nros. 455-462, Apoptosis is a regulated event crucial to the development and proliferation of normal and malignant B cells. We have studied the role of signals delivered via α4 integrin on apoptosis triggered by three different pathways on these cells. For apoptosis induced by serum deprivation, culturing B cells on the recombinant fibronectin fragment H89, a known ligand for α4β1 integrin, resulted in statistically significant (P < 0·005) higher viability values (68%, 65% and 67%) for Ramos, Nalm-6 and EHEB cells, respectively, than culturing cells on poly lysine (42%, 42% and 48%). An antiα4 MoAb reverted the protecting effect, thus confirming that it was due specifically to α4 engagement. Similarly, cells cultured on FN-III4-5, a recently identified fibronectin region which binds activated α4 integrin, also showed statistically significant higher viability than poly lysine cultures. α4 engagement however, did not prevent apoptosis induced on Ramos cells via surface IgM. Adhesion of IM-9 cells, a myeloma cell line carrying functional Fas receptors, to the H89 fragment neither increased cell viability upon triggering apoptosis via Fas when compared to poly lysine. These results indicate that α4 signalling may overcome B cell apoptosis induced by the lack of growth factors but does not seem to affect the IgM or Fas apoptotic pathways, thus suggesting different intracellular mechanisms for these processes, This work was supported by grants SAF97-0064-CO3-02 and SAF2000-0124 from the Comisión Interministerial de Ciencia y Tecnología (CICYT), and 08·1/012/97 from the Comunidad Autónoma de Madrid. M. Garcia-Gila and E.M. López-Martin were supported by a fellowship from CICYT

6 páginas, 2 figuras, 3 tablas -- PAGS nros. 1521-1526, The molecular pathogenesis of B cell chronic lymphocytic leukemia (B-CLL), the most common form of leukemia, remains unknown. We have used the mRNA differential display technique to analyze genes that may be involved in the development/progression of B-CLL. We have identified the tumor suppressor retinoic acid receptor responder 3 (RARRES3) as a B-CLL-related gene. RARRES3 maps to chromosome band 11q23, a region frequently deleted in lymphoproliferative disorders. To assess the potential involvement of RARRES3 in leukemogenesis, we examined 24 cases of B-CLL, 10 of acute lymphocytic leukemia (ALL) and five related cell lines by RT-PCR and sequence analyses. We report a correlation between RARRES3 down-regulation and B-CLL progression. We also found decreased RARRES3 gene levels in ALL cases and in the five cell lines studied. We did not find mutations in any of the leukemia samples assayed, including those with 11q23 deletion. These results indicate that RARRES3 may play a role in B-CLL progression. Leukemia (2001) 15, 1521-1526., This work was supported by grants SAF97-0064-CO3-02 (to AGP) and SAF97-0064-CO3-03 (to AS) from the Comisión Interministerial de Ciencia y Tecnología (CICYT); 08.1/0028/99 (to AGP) and 08.1/012/97 (to AS and AGP) from the Comunidad Autónoma de Madrid (CAM). B Casanova, MT de la Fuente and L Sanz were supported by fellowships from CAM; M García-Gila was supported by a fellowship from CICYT