XXXV Reunião Anual da SBBqResumoID:9181


Overexpression and Cellular Localization of CGI-109 in Leukemia-stimulated Bone Marrow Stromal Cells
Roversi F.M.1,2; Trindade D.M.3; Jotta P.Y. 1, Kobarg J.3;Yunes J.A.1

1 Laboratório de Biologia Molecular, Centro Infantil Boldrini, Campinas, SP; 2 Universidade Federal de São Paulo/ Escola Paulista de Medicina (UNIFESP/EPM), São Paulo, SP; 3 Centro de Biologia Molecular e Estrutural, Laboratório Nacional de Luz Síncrotron, Campinas, SP.

Acute lymphoblastic leukemia (ALL) is associated to a significant increase of angiogenesis (sprouting of capillaries from preexisting vessels) in bone marrow microenvironment, which regulate proliferation, maturation, and trafficking of blood cells in the bone marrow (BM). Among these stromal cells, the bone marrow endothelial cells (BMEC) portray a perfect environment in BM for ALL growth. Microarray was used to analyze about 22.000 genes. In one experiment, leukemia effect in BMEC's gene expression was investigated in vitro, by co-culturing BMEC with primary leukemia cells. In other experiment, BMEC's gene expression was analyzed after ALL patients' plasma addition. Among selected genes which were induced 4 times over controls, was the CGI-109 gene not yet characterized. Its similarity with p24 proteins family suggested that it is involved in cargo molecules transportation from ER to Golgi, selecting and packaging proteins into COPII. The other genes related with COPII had no expression alteration. CGI-109 increased expression could be related with transportation of angiogenic factor, whose expression is stimulated by ALL. In this context, we validated CGI-109 gene expression results by RQ-PCR assays of bone marrow stromal cell stimulated by ALL. The CGI-109 gene was cloned to investigate CGI-109 protein intracellular localization. The CGI-109 coding sequence was amplified through PCR, using primers containing restriction enzyme (BamHI-XbaI) regions, cloned into pGEM-T, and confirmed by sequencing. The presence of C/G-rich regions required use of DMSO for amplification. The BamHI-XbaI fragment was subcloned into pEF6/V5-His TOPO to allow expression of CGI-109 with a C-terminal fusion tag. Efficiency of transfection of primary bone marrow stromal cell was too low. Therefore, we used human PC3 cells, which were efficiently transfected with DOTAP liposomes and used for subsequent experiments. Double-staining immunofluorescence was used to compare the intracellular localization of the transfected CGI-109 with that of the trans-Golgi marker WGA. CGI-109 exhibited paranuclear staining colocalizing with WGA fluorescence, suggesting the presence of CGI-109 in the Golgi, corroborating its putative participation in the secretory pathway. Co-immunoprecipitation experiments will be performed to investigate whether CGI-109 is involved in the transportation of chemokines found to be overexpressed by leukemia-stimulated bone marrow stromal cells.