Search

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.

Aiming to identify signalling pathways relevant for ss-cell growth we performed an explorative micro-array analysis comparing the gene expression profiles of three human insulinomas and one normal pancreatic islet preparation. This revealed an insulinoma-associated down-regulation of the transforming growth factor beta 1 (TGF-beta1) and its target genes. Comparative quantitative real-time PCR (qRT-PCR) including an expanded sample number of both insulinomas (n=9) and pancreatic islet preparations (n=4) confirmed the decreased TGF-beta1 expression and its target molecules (TGFBI, NNMT, RPN2) in insulinomas. Similarly, TGF-beta1 immunofluorescence analysis revealed reduced expression in insulinomas when compared to pancreatic islets. In contrast, TGFBR2 (transforming growth factor beta receptor II) was found up-regulated. However, the consistent down-regulation of the TGF-beta1 targets TGFBI (transforming growth factor, beta-induced), NNMT (nicotinamide N-methyltransferase), RPN2 (ribophorin II) indicates that the parallel up-regulation of TGFBR2 does not compensate for the only marginal TGF-beta1 expression levels in insulinomas. TGFBR2 expression was confirmed at the protein level in insulinomas. SMAD2/3 protein expression was found at higher levels in human pancreatic islets when compared with insulinomas by dual colour confocal microscopy. TGF-beta1 signalling is known to be involved in cell replication and is abrogated in ductal pancreatic tumours. The down-regulation of TGF-beta1 expression and its target molecules in insulinomas is a new aspect of this cytokine. Our data underline parallels in endocrine and exocrine pancreatic tumour development, which may implicate common progenitor cells.

Objective: Long-term in vitro maintenance of human insulin-secreting insulinoma cells., Methods: (1) Cell culture of ex vivo-derived insulinoma cell suspensions from 8 individual human donors, using various cell culture medium supplementations; (2) determination of insulin synthesis and secretion using immunocytochemistry and insulin and pro-insulin radioimmunoassays; (3) nestin-immunostaining of long-term in vitro grown insulinoma cell suspensions, and (4) phase-contrast light microscopy for analyzing the in vitro growth characteristics of the insulinoma cells., Results: (1) Parallel persistence of in vitro insulinoma cell proliferation as well as insulin-synthesizing and -secreting capacity depended on both the co-culture of insulinoma cells with human fibroblasts and the supplementation of the cell culture medium with tissue culture supernatant derived from the rodent pituitary adenoma cell line GH-3; (2) immunostaining for insulin and secretagogin confirmed the neuroendocrine origin of the insulinoma cells grown in vitro; (3) insulin secretion capability persisted up to an observation period of 25 weeks; (4) insulin secretion rates after 6 weeks of in vitro growth ranged from 3.5 to 83.3 muU/ml/h/60,000 cells plated, and (5) after long-term in vitro growth of insulinoma-derived cell suspensions with persistent insulin-secreting capacity, nestin staining was observed predominantly in co-cultured fibroblasts., Conclusion: Our data describe for the first time the long-term in vitro culture of insulin-secreting human insulinomas and highlight the importance of beta-cell trophic factors for insulinoma cell growth.