Activity of the Akt-dependent anabolic and catabolic pathways in muscle and liver samples in cancer-related cachexia.

In animal models of cachexia, alterations in the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway have been demonstrated in atrophying skeletal muscles. Therefore, we assessed the activity of proteins in this pathway in muscle and liver biopsies from 16 patients undergoing pancreatectomy for suspect of carcinoma. Patients were divided in a non-cachectic or cachectic group according to their weight loss before operation. Extracts of skeletal muscle and liver tissue from eight cachectic patients with pancreas carcinoma and eight non-cachectic patients were analysed by Western blotting using pan- and phospho-specific antibodies directed against eight important signal transduction proteins of the PI3-K/Akt pathway. Muscle samples from cachectic patients revealed significantly decreased levels of myosin heavy chain (-45%) and actin (-18%) in comparison to non-cachectic samples. Akt protein level was decreased by -55%. The abundance and/or phosphorylation of the transcription factors Foxo1 and Foxo3a were reduced by up to fourfold in muscle biopsies from cachectic patients. Various decreases of the phosphorylated forms of the protein kinases mTOR (-82%) and p70S6K (-39%) were found. In contrast to skeletal muscle, cachexia is associated with a significant increase in phosphorylated Akt level in the liver samples with a general activation of the PI3-K/Akt cascade. Our study demonstrates a cachexia-associated loss of Akt-dependent signalling in human skeletal muscle with decreased activity of regulators of protein synthesis and a disinhibition of protein degradation.

Iodination of proteins in TPO transfected thyroid cancer cells is independent of NIS.

This study shows that organification of radioiodide into proteins of thyroid cancer cells exogenously co-expressing the thyroid peroxidase (TPO) and the sodium/iodide symporter (NIS) is independent of NIS function. When administering (125) I to cells constitutively expressing either NIS, or TPO or NIS/TPO, next to iodide accumulation due to NIS activity, organification was exclusively observed in TPO expressing/co-expressing cells. The use of specific inhibitors for TPO and NIS showed that organification is strictly dependent of TPO and not of NIS. An identical pattern of iodoproteins migrating between approximately 75 and 200 kDa in all cell lines tested was observed. Among the five major iodoproteins, two polypeptides appear to be related and three are most probably unrelated, according to their peptide pattern. Our results significantly indicate that co-expression of TPO in NIS transfected cells mediates iodination on the one hand but on the other hand does not contribute to augmentation of a putative NIS-based radioiodide concentrator gene therapy.

Characterization of a thyroid-specific and cyclic adenosine monophosphate-responsive enhancer far upstream from the human sodium iodide symporter gene.

We describe the cloning and characterization of a human sodium iodide (NIS) upstream enhancer (NUE). This putative enhancer was cloned based on its sequence homology (69% identity) to the rat NUE. A 296 base pair (bp) genomic DNA fragment, which is located 9000 bp upstream from the human hNIS gene, was amplified by polymerase chain reaction (PCR) and inserted into a luciferase reporter gene in front of both the homologous NIS promoter and the heterologous SV40 promoter. No enhancer activity could be found after transfection into HeLa cells, but in FRTL-5 cells representing the thyroid model, a threefold stimulation of the NIS promoter was found. This enhancer activity was present in both directions and was stimulated threefold by thyrotropin (TSH) and 14-fold by the cyclic adenosine monophosphate (cAMP) agonist forskolin. A small element (TGACGCA) in this enhancer was found to be of central importance, because its site-directed mutagenesis abolished the enhancer activity. This element bound specifically to proteins in nuclear extracts from FRTL-5 cells and to a lesser extent also from HeLa cells. In summary, we describe a thyroid-specific and cAMP-responsive enhancer far upstream from the human NIS gene, which is located in the intronic region of another gene coding for a ribosomal protein.

Hydrogen peroxide inhibits activity of the IGF-1 receptor kinase.

IGF-1 receptor (IGF1R) is a transmembrane tyrosine kinase, which is indispensable for cellular growth and differentiation. Using a recombinant GST-tagged cytosolic fragment of IGF1R (GST-IGFK), we now show that oxidation by low doses (50 muM) of hydrogen peroxide markedly inhibits maximum phosphate incorporation in autophosphorylation and substrate phosphorylation assays. A similar inhibition was observed on the activity of intact IGF1R after treatment of T-47D cells. These results are in sharp contrast to the positive influence of hydrogen peroxide on the highly homologous insulin receptor kinase, which was assayed for comparison. This reciprocal influence of physiologically relevant doses of hydrogen peroxide may have important implications on signal transduction of the closely related receptors for insulin and IGF-1.

Interdependent regulation of insulin receptor kinase activity by ADP and hydrogen peroxide.

Insulin signaling requires autophosphorylation of the insulin receptor kinase (IRK) domain. Using purified recombinant IRK fragments and the isolated intact insulin receptor, we show here that autophosphorylation is inhibited by ADP and that this effect is essentially reversed by hydrogen peroxide. Autophosphorylation was inhibited by hydrogen peroxide (60 microM) in the absence of ADP but enhanced in the presence of inhibitory concentrations of ADP (67 microM). Enhancement by hydrogen peroxide required direct interaction of hydrogen peroxide with the kinase domain and was not seen in insulin receptor mutants C1245A and C1308A. A similar enhancement was obtained in intact cells in the absence of insulin upon treatment with 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea, indicating that IRK activity can be alternatively enhanced by a shift in the thiol/disulfide redox status. Molecular modeling of the IRK domain indicated that the ATP-binding site becomes distorted after releasing the nucleotide unless the IRK domain is oxidatively derivatized at Cys1245. Recent clinical studies suggest that these effects may play a role in obesity due to the fact that cytoplasmic creatine kinase in combination with phosphocreatine normally ensures rapid removal of ADP in muscle cells but not in fat cells.