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The insulin-like growth factors (IGF-I and -II) are structurally related peptides participating in the regulation of metabolism, growth and cellular differentiation. In the present study, the human hepatoma cell line PLC was studied for the expression of individual components of the IGF axis. Northern blot analysis using IGF-I and -II coding cDNAs failed to detect IGF-I- or -II-specific transcripts in total RNA from PLC cells. Biosynthesis of type I and II IGF receptors was demonstrated by northern blotting and binding studies as well as cross-linking of the respective radiolabeled ligand. Both IGF-I and -II stimulated [3H]-thymidine incorporation dose-dependently. The mitogenic activity of exogenously added IGFs was reduced by the presence of IGF-binding proteins of 24, 30, 34, 41 and 45 kDa in supernatants of PLC cells identified as IGFBP-4, -1, -2 and -3, respectively, by [125I]IGF-I ligand-, immuno- and northern blotting. Biosynthesis of IGFBP-3 was stimulated dose-dependently by IGF-I and -II, while IGFBP-1, -2 and -4 were not affected. The increase of IGFBP-3 in response to IGF-I and -II was due to a stimulation of IGFBP-3 specific mRNA as well as to an inhibition of IGFBP-3 endocytosis. Proteolytic activity for rhIGFBP-3 was detected in media from PLC cells at acidic pH that was inhibited by the aspartyl protease inhibitor pepstatin A as well as after immunodepletion of cathepsin D from media of PLC cells. Thus, a role of cathepsin D for the regulation of IGFBP-3 bioavailability via endocytosis in acidic prelysosomal compartments was suggested. The susceptibility of PLC for IGF-I and -II was restricted by their ability to increase the abundance of inhibitory IGFBPs and to decrease the level of IGF-I receptor expression. The present data point to the IGF axis as a complex regulatory system that self limits the mitogenic activity of exogenous IGFs.

The mitogenic and metabolic activities of insulin-like growth factors (IGF) are modulated by a family of six high affinity IGF binding proteins (IGFBPs). This study describes the expression of the mouse IGFBP-6 which is unique among IGFBPs in its preferential binding of IGF II, in insect cells using the baculovirus system. The purified, O-glycosylated IGFBP-6 was functional as shown by IGF binding and by inhibition of IGF II-stimulated DNA synthesis in human fibroblasts. Specific antibodies generated in chicken against the recombinant IGFBP-6 were used for Western blotting analysis and immunohistochemistry. Strong immunoreactivity was found in ossifying bones of the cranial base, in cell clusters of the pancreas anlage, in the trigeminal ganglion, on myoblasts, on motoneurons of the spinal cord of embryonic mice. In tissues of adult mouse, strong IGFBP-6 immunostaining was present in epidermal and peridermal layers of the skin, in meningeal layers, in long-striated skeletal muscle, and in the Langerhans' islets of the pancreas. No immunopositive staining was observed in lung and liver indicating that sites of synthesis and IGFBP action are different.

Background/Aims: The liver is the main source of circulating insulin-like growth factor binding proteins. In man, the cellular origin of insulin-like growth factor binding proteins has remained obscure. Methods: Human hepatocytes isolated from surgical specimens were purified and cultured using a collagen gel immobilization technique. Gene expression of individual insulin-like growth factor binding proteins and of the acid-labile subunit of the insulin-like growth factor ternary binding protein complex was studied by Northern blotting, secretion of insulin-like growth factor binding proteins by Western ligand blotting and immunoblot analysis. Neutral size chromatography of medium samples was used to detect insulin-like growth factors binding protein complexes. Results: In cultured hepatocytes transcripts for insulin-like growth factor binding protein-1, -2, -3, -4 and for acid-labile subunit could be demonstrated. Ligand blotting revealed the secretion of insulin-like growth factor binding proteins of molecular weights of 24 kD, 30 kD, 34 kD, 43 kD and 46 kD, respectively. Using polyclonal antisera, these proteins were identified as insulin-like growth factor binding protein-1, -2 and the insulin-like growth factor binding protein-3 doublet. Neutral size chromatography of culture supernatants showed the presence of an insulin-like growth factor binding protein complex of approximately 40 kD, but absence of the high molecular weight ternary complex of 150 kD. Conclusions: It is concluded that in man parenchymal liver cells have to be regarded as a source of acid-labile subunit and of circulating insulin-like growth factor binding proteins including insulin-like growth factor binding protein-3.

The standard treatment of patients with locally advanced rectal cancers comprises preoperative 5-fluorouracil-based chemoradiotherapy followed by standardized surgery. However, the tumor response to multimodal treatment has varied greatly and ranging from complete resistance to complete pathologic regression. The prediction and treatment of these observed heterogeneous response to therapy in patients is, therefore, an important clinical need. A strategy for studying the molecular basis of this heterogeneous tumor response is to establish the clinical parameters as an in vitro model. For this purpose we used 12 colorectal cancer cell lines and exposed them to 3 µM of 5-fluorouracil and 2 Gy of radiation, which reflect the parameters used in clinical patient treatment. The sensitivity differences of the cell lines to chemoradiotherapy, measured as surviving fractions by a standard colony formation assay, were then correlated with the pretherapeutic gene expression profiles of these cell lines. The microarray measurements were independently validated for a subset of these genes using real-time polymerase chain reactions. In the 12 colorectal cancer cell lines we observed a heterogeneous response, with surviving fractions ranging from 0.28 to 0.81, closely recapitulating clinical reality. Using a linear model analysis, we identified 4,796 features whose expression levels correlated significantly with the sensitivity to chemoradiotherapy (Q < 0.05), including many genes involved in the mitogen-activated protein kinase signaling pathway or cell cycle genes. These data have suggested a potential relevance of the insulin and Wnt signaling pathways for the treatment response, and we identified STAT3, RASSF1, DOK3, and ERBB2 as potential therapeutic targets. We anticipate that this analysis of a gene expression signature for the in vitro chemoradiosensitivity of colorectal cancer cells will unveil molecular biomarkers predictive of the response of rectal cancers to chemoradiotherapy and enable the identification of genes that could serve as targets to sensitize a priori resistant primary tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2508. doi:10.1158/1538-7445.AM2011-2508

Expression, ribosomal frameshifting, and proteolytic processing of HIV-1 GAG and POL proteins were investigated in heterologous mammalian cells in order to elucidate the influence of the cellular background on these events. DNA fragments encoded by the gag and pol region were expressed in two rodent cell lines, LTK- and BHK. Both stably transfected cell lines continuously produce recombinant proteins which react with HIV-specific antisera. The GAG precursor and a 39-kDa proteolytic fragment thereof were the major recombinant proteins detected. Expression of the gag-pol region leads to the production of the GAG-POL precursor. Ribosomal frameshifting at the HIV-1 shifty sequence to a typical extent could be positively demonstrated by an enzyme assay. Despite the presence of the viral protease within the GAG-POL precursors, proteolytic processing of the HIV-derived polyproteins was extremely inefficient. The efficiency could not be enhanced by overexpression of the HIV-1 protease encoding region.

Adenocarcinoma of the small intestine arising from heterotopic gastric mucosa is extremely rare. In this report, we present the case of a 68-year-old woman who complained of abdominal pain, weight loss and subileus. Gross examination of resected small bowel revealed multiple flat polypous lesions with cysts in the ileal submucosa, one of which containing an ulcerated, stenosing tumour. On microscopic examination, an adenocarcinoma of the ileum arising from multifocal gastric heterotopia with secondary gastritis cystica profunda was diagnosed. Comparative genomic hybridization of the adenocarcinoma revealed chromosomal gains at 1q, 3q, 5p, 8q, 11p, 12p, 13q and losses at Xp, 4q, 8p, 10p, 14q, 17p, 20p, compatible with a high degree of genomic instability. peerReviewed