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.
|