Cross talk between the bombesin neuropeptide receptor and Sonic hedgehog pathways in small cell lung carcinoma.

Small cell lung carcinoma (SCLC) often features the upregulation of the Sonic hedgehog (Shh) pathway leading to activation of Gli transcription factors. SCLC cells secrete bombesin (BBS)-like neuropeptides that act as autocrine growth factors. Here, we show that SCLC tumor samples feature co-expression of Shh and BBS-cognate receptor (gastrin-releasing peptide receptor (GRPR)). We also demonstrate that BBS activates Gli in SCLC cells, which is crucial for BBS-mediated SCLC proliferation, because cyclopamine, an inhibitor of the Shh pathway, hampered the BBS-mediated effects. BBS binding to GRPR stimulated Gli through its downstream Gαq and Gα12/13 GTPases, and consistently, other Gαq and Gα13 coupled receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active GαqQL and Gα12/13QL mutants stimulated Gli. By using cells null for Gαq and Gα12/13, we demonstrate that these G proteins are strictly necessary for Gli activation by BBS. Moreover, by using constitutively active Rho small G-protein (Rho QL) as well as its inhibitor, C3 toxin, we show that Rho mediates G-protein-coupled receptor (GPCR)-, Gαq- and Gα12/13-dependent Gli stimulation. At the molecular level, BBS caused a significant increase in Shh gene transcription and protein secretion that was dependent on BBS-induced GPCR/Gαq-12/13/Rho mediated activation of nuclear factor κB (NFκB), which can stimulate a NF-κB response element in the Shh gene promoter. Our data identify a novel molecular network acting in SCLC linking autocrine BBS and Shh circuitries and suggest Shh inhibitors as novel therapeutic strategies against this aggressive cancer type.

EC-SOD gene therapy reduces paracetamol-induced liver damage in mice.

BACKGROUND: Paracetamol overdose causes acute liver damage which leads to severe centrilobular hepatic necrosis. The hepatotoxic effect is caused by reactive metabolites and oxidative stress. Since extracellular superoxide dismutase (EC-SOD) protects tissues against the harmful effects of superoxide anion, the hypothesis that systemic adenovirus-mediated EC-SOD gene transfer could reduce liver damage was tested. METHODS: Mice were given paracetamol (600 mg/kg) enterally 2 days after adenovirus-mediated gene transfer of EC-SOD (2 x 10(9) pfu). Five days after gene transfer, plasma and tissue samples were collected for clinical chemistry analyses and tissue pathology evaluation. RESULTS: EC-SOD was expressed in a dose-dependent manner with the highest enzyme activity occurring 3 days after the gene transfer. Clinical chemistry and tissue pathology analyses showed that adenoviral EC-SOD gene transfer significantly attenuated release of liver enzymes and inhibited necrosis and apoptosis caused by paracetamol overdose. CONCLUSION: The results indicate the involvement of superoxide anion in paracetamol-mediated liver damage and suggest a possible protective role for EC-SOD gene transfer in paracetamol-induced liver damage.

Extracellular superoxide dismutase deficiency and atherosclerosis in mice.

Lipoprotein peroxidation in the arterial wall has been implicated in atherogenesis. The superoxide radical is formed in arteries and can induce such oxidation. Extracellular superoxide dismutase (EC-SOD) occurs in high concentration in the vascular wall interstitium, and in this study, we examined the importance of the enzyme in atherogenesis. On an apolipoprotein E-null background, the limited aortic lesions induced by a 1-month atherogenic diet were larger in EC-SOD wild-type mice than in EC-SOD-null mice, whereas there were no differences between the EC-SOD genotypes in the larger lesions seen after 3 months on the diet or after 8 months on normal chow. Despite smaller or equal lesions in the EC-SOD-null mice, their cholesterol levels were somewhat higher. Also, on a wild-type background, there were no effects produced by the absence or presence of EC-SOD on atherogenic diet-induced aortic root lesions. The urinary excretion of the lipid peroxidation biomarker 8-isoprostaglandin F(2alpha) was related to the rates of atherogenesis in the mice but was not influenced by the EC-SOD genotype. Likewise, the EC-SOD status had no effect on the staining for oxidized low density lipoprotein epitopes in aortic root sections. Our findings suggest that EC-SOD has little influence on atherogenesis in mice.

Gene transfer of extracellular superoxide dismutase to atherosclerotic mice.

Clinical and epidemiological studies have provided circumstantial evidence that oxidized low-density lipoprotein (LDL) and antioxidants are involved in the pathogenesis of atherosclerosis. Superoxide dismutases (SODs) have been shown in vitro to protect LDL from deleterious effects of superoxide anions. In the present study, we have used adenoviral gene transfer to determine effect of extracellular SOD (EC-SOD) on atherogenesis in LDL receptor -/- mice. Intravenous administration of EC-SOD adenovirus (2 x 10(9) plaque forming units) into tail vein targeted transgene mainly to liver and induced a 3.5- to sevenfold increase in plasma total SOD activity. EC-SOD was secreted into circulation for 2-3 weeks mostly in a truncated B-form, suggesting that endogenous proteolytic mechanisms control the level and distribution of the enzyme. Therapeutic potential was determined by measuring plasma resistance against copper oxidation and analyzing atherosclerotic lesion areas in aortas of LDL receptor -/- mice. Mice were kept on a cholesterol diet for 10 weeks before gene transfer and 3 or 6 weeks after the gene transfer. Results showed a tendency for a reduction in the overall lesion area after EC-SOD gene transfer as compared with LacZ transduced control mice, but the difference did not reach statistical significance. It is concluded that short-term overexpression of EC-SOD in vivo does not affect atherogenesis in LDL receptor -/- mice.

Gene encoding the catalytic subunit p110beta of human phosphatidylinositol 3-kinase: cloning, genomic structure, and screening for variants in patients with type 2 diabetes.

Phosphatidylinositol (PI) 3-kinase is a key signaling molecule in insulin-stimulated glucose transport. Therefore, we investigated the catalytic subunit p110beta, of human PI 3-kinase as a candidate gene for type 2 diabetes. Human p110beta gene was cloned from the placental genomic library. All 22 exons, intronic regions flanking the exons and 1.5 kb of the proximal/5' region of the p110beta gene, were screened for variants by single-strand conformation polymorphism analysis in 79 Finnish patients with type 2 diabetes . Allele frequencies of the variants were also determined in 77 nondiabetic control subjects. No variants were found in exons in diabetic patients. However, we identified two nucleotide polymorphisms in the proximal/5' region of the p110beta gene and a variation in the number of 2-bp repeat sequence (TA)n in intron 4. The allele frequencies did not differ between diabetic and control subjects. Our results may indicate that the catalytic subunit p110beta of PI 3-kinase plays such a fundamental role in the insulin-signaling pathway that structural variants are not likely to exist in that gene. The importance of the polymorphisms in the proximal/5' region of the p110beta gene for insulin signaling remains to be determined.

Rabbit extracellular superoxide dismutase: expression and effect on LDL oxidation.

Extracellular superoxide dismutase (EC-SOD) is a secreted antioxidative enzyme with an abundant mRNA expression in kidney and arterial wall. In order to study expression and antioxidative function of EC-SOD, we cloned the rabbit ec-sod cDNA and produced the recombinant protein in cell culture. In vitro studies did not show a direct relationship between the amounts of synthesized mRNA and secreted protein activity, suggesting post-transcriptional regulation. The antiatherogenic role of EC-SOD was studied by determining the effect of EC-SOD on the oxidation (ox) of low density lipoprotein (LDL), and subsequent degradation of oxLDL in RAW 264 macrophages in vitro. It was found that recombinant EC-SOD reduced both the degradation of LDL in RAW 264 macrophages by 28-36% and its electrophoretic mobility caused by endothelial cell-mediated oxidation. It is therefore suggested that EC-SOD can act as a protective enzyme against the development of atherosclerosis.

False-positive apoptosis signal in mouse kidney and liver detected with TUNEL assay.

Apoptosis is a physiological, programmed process for the elimination of cells from living organisms. Currently, one of the most frequently used methods to detect apoptosis is TUNEL assay. It has provided valuable information about apoptosis in various tissues. However, the sensitivity and the specificity of TUNEL technique have also been criticized. We detected an intense false-positive apoptotic signal in nude and Balb/c mice kidney and liver. In kidney the signal was confined to the proximal, distal and collecting tubular cells, and in liver to hepatocytes. Both tissues appeared normal in light microscopy, and no DNA ladder formation or increase in caspase-3 enzyme activity was detected. BrdU labelling and Ki-67 immunostaining did not reveal increased cell proliferation in these tissues. On the other hand, false-positive signal was not detected in testis, spleen, pancreas or renal cell carcinoma from the same animals. Also, no false-positive signal was seen in human liver or kidney samples. Although factors known to produce false-positive staining related to sample harvesting, preparation and staining protocols were eliminated, the cause of the false- positive apoptotic signal remains unknown. We conclude that caution must be exercised when examining apoptosis in mouse tissues with TUNEL assay.

Local hypomethylation in atherosclerosis found in rabbit ec-sod gene.

Extracellular superoxide dismutase (EC-SOD) protects arteries against deleterious effects of superoxide anions and the development of atherosclerosis. In this study, we cloned and characterized rabbit ec-sod gene. We identified 6 rabbit C-elements and 5 CpG clusters in the cloned sequence. One of the CpG clusters is located on the coding sequence. Because CpG clusters are potential sites for methylation and may explain the occurrence of mutations, methylation status of each of the CpG dimers located in the coding sequence CpG cluster was characterized using direct genomic sequencing. Unexpectedly, a marked reduction in the amount of methylated CpG dinucleotides in ec-sod gene was detected in atherosclerotic aortas as compared with normal aortic intima-media. Although alterations in DNA methylation are well characterized in malignant tumors, the presence of methylation changes in atherosclerosis has not been studied even though both diseases are characterized by excess cellular proliferation and alterations in gene expression. Further analysis of the whole genomic methylation by high-pressure liquid chromatography in normal and atherosclerotic aortas revealed a tendency for a decreased 5-methylcytosine (5-mC) content in atherosclerotic aortas as compared with normal arteries. Hypomethylation in atherosclerotic aortas occurred at the same level as has been reported from malignant tumors. Although a causal relationship between the methylation level and expression of EC-SOD cannot be proven, our results show that ec-sod hypomethylation is associated with the development of atherosclerosis and suggest that it may affect structure and function of ec-sod and other genes possibly involved in the development of atherosclerotic lesions.

Cloning and sequencing of the chicken egg-white avidin-encoding gene and its relationship with the avidin-related genes Avr1-Avr5.

The gene encoding chicken egg-white avidin (Avd) was amplified from chromosomal DNA, cloned and sequenced. The entire coding region of preavidin (pre-Avd) containing four exons was identified by comparing the Avd gene (1119 bp) with the cDNA. It has a high identity percentage (91-95%) with the previously isolated Avd-related genes 1-5 (Avr1-Avr5). Interestingly, comparison of Avd with the Avr genes showed that the introns were better conserved (on average 97%) than the exons (90%). The Avd gene, as well as the cDNA, encodes a Gln residue at position 53 of the mature protein, which is in contrast to the previously determined amino-acid sequence.

Molecular cloning and nucleotide sequence of chicken avidin-related genes 1-5.

Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg-white avidin. Two overlapping genomic clones were found to contain five genes (avidin-related genes 1-5, avr1-avr5), which have been cloned, characterized and sequenced. All of the genes have a four-exon structure with an overall identity with the avidin cDNA of 88-92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68-78% with avidin. The amino acid residues responsible for the biotin-binding activity of avidin and the bacterial biotin-binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone-specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo.