TY - JOUR
T1 - CXCL12/SDF-1 over-expression in human insulinomas and its biological relevance
AU - Ilhan, Aysegul
AU - Nabokikh, Anastasiya
AU - Maj, Magdalena
AU - Vidakovic, Melita
AU - Nielsen, Jens H
AU - Prikoszovich, Thomas
AU - Niederle, Bruno
AU - Base, Wolfgang
AU - Luger, Anton
AU - Wagner, Ludwig
N1 - Keywords: Animals; Cell Proliferation; Cell Survival; Cells, Cultured; Chemokine CXCL12; Cloning, Molecular; Gene Dosage; Gene Expression Regulation, Neoplastic; Humans; Insulinoma; Oxidation-Reduction; Pancreatic Neoplasms; Rats; Reactive Oxygen Species; Superoxide Dismutase; Transfection; Up-Regulation
PY - 2009
Y1 - 2009
N2 - This study was performed on the basis of previously obtained investigative gene array data concerning the over-expression of CXCL12/SDF-1 in human insulinomas versus human pancreatic islet preparations. The presence of CXCL12/SDF-1 was studied by RT-qPCR in human insulinomas (n=8) versus pancreatic islets (n=3), and was found to be significantly up-regulated in the former (p<0.012). The mRNA data were confirmed by immunostaining and confocal microscopy of human normal pancreatic islets, which showed the absence of CXCL12 protein and high expression in insulinoma tissue. Individual human insulinoma cells at cytospins stained positive for CXCL12 in the paranuclear region. These morphological data were extended by consecutive immunoblotting for cell-compartment-specific marker proteins of fractions obtained by sucrose gradient fractionation using Rin-5F insulinoma cells. CXCL12-containing fractions were positive for the membrane marker NSF but negative for SNAP-25 and appeared at a lighter density in the gradient than heavy insulin granules, suggesting packaging in specific granules different from insulin. In order to determine the biological relevance of the protein in insulinomas, we investigated the colony-forming potential of human CXCL12 stable-transfected rat Rin-5F insulinoma cells. These clones secreted human CXCL12 and contained 50-1000-fold higher copy numbers compared to its endogenous rat homologue. In colony-forming assays, these transfectant clones developed greater colony numbers, which were larger than wild-type and sham transfectants. To elucidate the mechanism of action, we identified a CXCL12 transfectant-specific increase in the pro-survival factor Mn-SOD, which is considered important for the inactivation of reactive oxygen species, thereby prolonging cell survival. These data demonstrate the importance of CXCL12 in the tumor biology of insulinoma.
AB - This study was performed on the basis of previously obtained investigative gene array data concerning the over-expression of CXCL12/SDF-1 in human insulinomas versus human pancreatic islet preparations. The presence of CXCL12/SDF-1 was studied by RT-qPCR in human insulinomas (n=8) versus pancreatic islets (n=3), and was found to be significantly up-regulated in the former (p<0.012). The mRNA data were confirmed by immunostaining and confocal microscopy of human normal pancreatic islets, which showed the absence of CXCL12 protein and high expression in insulinoma tissue. Individual human insulinoma cells at cytospins stained positive for CXCL12 in the paranuclear region. These morphological data were extended by consecutive immunoblotting for cell-compartment-specific marker proteins of fractions obtained by sucrose gradient fractionation using Rin-5F insulinoma cells. CXCL12-containing fractions were positive for the membrane marker NSF but negative for SNAP-25 and appeared at a lighter density in the gradient than heavy insulin granules, suggesting packaging in specific granules different from insulin. In order to determine the biological relevance of the protein in insulinomas, we investigated the colony-forming potential of human CXCL12 stable-transfected rat Rin-5F insulinoma cells. These clones secreted human CXCL12 and contained 50-1000-fold higher copy numbers compared to its endogenous rat homologue. In colony-forming assays, these transfectant clones developed greater colony numbers, which were larger than wild-type and sham transfectants. To elucidate the mechanism of action, we identified a CXCL12 transfectant-specific increase in the pro-survival factor Mn-SOD, which is considered important for the inactivation of reactive oxygen species, thereby prolonging cell survival. These data demonstrate the importance of CXCL12 in the tumor biology of insulinoma.
U2 - 10.1016/j.mce.2008.10.015
DO - 10.1016/j.mce.2008.10.015
M3 - Journal article
C2 - 19013212
SN - 0303-7207
VL - 298
SP - 1
EP - 10
JO - Molecular and Cellular Endocrinology
JF - Molecular and Cellular Endocrinology
IS - 1-2
ER -