Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds.

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

Oxidative/nitrosative stress and peroxiredoxin 2 are associated with grade and prognosis of human renal carcinoma.

Peroxiredoxins (Prxs) 1-6 were assessed in 138 renal cell carcinomas (RCC) using immunohistochemistry and selected samples by Western blotting analysis. Oxidative/nitrosative damage was evaluated using nitrotyrosine immunoreactivity. The expressions of Prxs were correlated with tumor grade and survival and nitrotyrosine reactivity. Non-malignant kidney tubular cells showed positivity with variable intensity for all six Prxs. In RCCs, most cases were positive for Prxs 1 and 2, while only 15-20% of tumors showed expression for Prxs 3 and 4. Prx 2 was associated with tumors of a lower grade (p=0.009) and with a lower frequency of distant metastases (p=0.046). Patients with tumors expressing Prx2 had better prognosis (p=0.027). Instead, nitrotyrosine was significantly associated with high grade tumors (p=0.001). Compared with the non-malignant kidney tubular cells, low Prx expression in the tumor cells can make them more susceptible to oxidative damage. Prx 2 was more abundantly expressed in low grade tumors, suggesting that this protein could play a role in preventing the development of oxidative damage, which in turn can lead to the activation of pathways leading to aggressive tumors.

Antioxidant enzymes in renal cell carcinoma.

The aim of the study was to estimate the significance of oxidative/nitrosative damage and expression of antioxidant enzymes in renal cell carcinomas (RCC). For this we investigated immunohistochemically six antioxidant enzymes (AOEs) including MnSOD, ECSOD, thioredoxin, thioredoxin reductase, and gammaglutamyl cysteine synthetase heavy and light chain in 138 RCCs. As an indicator of oxidative/nitrosative damage, sections were stained with an antibody to nitrotyrosine. The extent of apoptosis was evaluated by TUNEL method and proliferation by immunohistochemistry to Ki67. Variable expression of all AOEs could be seen in RCC with expression of MnSOD being strongest. Nitrotyrosine was significantly associated with high grade tumors. MnSOD was associated with tumors of a lower stage. Cases showing ECSOD reactivity had higher and cases expressing thioredoxin lower apoptotic index than other tumors. No association with patient prognosis was observed. According to the results renal cell carcinomas show oxidative/nitrosative damage which, according to nitrotyrosine staining, was higher in high grade tumors. Of AOEs, MnSOD was more abundantly expressed in low stage tumors suggesting that its antioxidant function could play a main role to prevent development of oxidative damage leading to more aggressive tumors.

Chromosomal gains and losses detected by comparative genomic hybridization and proliferation activity in renal cell carcinoma.

OBJECTIVE: The number of DNA losses found using comparative genomic hybridization (CGH) and the proliferation index MIB-1 have been shown to be prognostic factors in renal cell carcinoma (RCC). We evaluated the associations of these two factors with each other and with histopathology and clinical outcome. MATERIAL AND METHODS: In this prospective study, specimens from 20 primary RCCs were investigated using CGH and MIB-1 assay. The associations of the commonest chromosomal aberrations with histopathology, stage and the clinical outcome of the disease were evaluated. RESULTS: CGH detected genetic aberrations in all tumours. Losses of genetic material (85%) were more common than gains (65%). Most common was loss in the short arm of chromosome 3, which was found in 70% of the tumours. Other frequent changes (20%) were losses of 4q, 13q, 18 and Xp, as well as gains of 5q, 7p, 7q (25%) and chromosome 12. The number of deleted chromosomal areas varied from none to six. The MIB-1 index varied from 0 to 39 (median 4.0). The total number of chromosomal aberrations or deletions showed no association with MIB-1 index or nuclear grade. Most grade 1 and 2 tumours showed a low MIB-1 index. All nuclear grade 4 tumours progressed and were associated with short survival. CONCLUSION: CGH gives an overview of DNA changes in RCC and helps to locate targets for more precise genetic evaluation. CGH findings are also helpful for classifying tumours. In this study, genetic aberrations in primary RCCs were not associated with histopathology, proliferation or clinical outcome, which suggests that CGH does not necessarily give any additional information on the prognosis of the disease. MIB-1 index and TNM stage were associated with survival.

Membranous location of EGFR immunostaining is associated with good prognosis in renal cell carcinoma.

Epidermal growth factor receptor (EGFR) is a key factor in tumorigenesis. The association between EGFR expression and prognosis in renal cell carcinoma (RCC) is not clear. In our study of 134 RCCs, the cellular location of immunostaining was evaluated and patients with EGFR-positive tumours with prominent membranous staining had a good prognosis. Their overall survival was significantly longer (P=0.004) than that of patients with either EGFR-negative tumours or with mainly cytoplasmic staining. However, further studies on the different EGFR expression patterns in RCC are needed to clarify their role in the progression of the disease.

Soluble immunological parameters and early prognosis of renal cell cancer patients.

In local or metastatic cancer, a prognostic tumour marker could be a valuable tool in the selection of different treatments. In renal cell cancer (RCC) no such markers have been available. We therefore evaluated the association between several pretreatment serum markers, tumour classification and short term survival in RCC patients. Serum samples were collected before surgery and three months thereafter from 24 RCC patients. Interleukin-6 (IL-6), IL- 12, soluble IL-2 receptor (sIL-2R) and intercellular adhesion molecule-1 (sICAM-1) were measured in serum samples using specific commercial enzyme immunoassay kits. Serum IL-6, sIL-2R and sICAM-1 levels before nephrectomy were significantly higher in non-local tumours than in local ones (mean IL-6 53 pg/ml versus 6.3 pg/ml, and sICAM-1 443 ng/ml versus 290 ng/ml, sIL-2R 3779 pg/ml versus 1796 pg/ml). In contrast, IL-12 levels were higher in local tumours (148 versus 102 pg/ml) and the levels increased significantly (P < 0.005) after removal of the primary tumour in patients with local disease. All patients with local tumours had normal IL-6 values, while only one with a non-local tumour had IL-6 levels below 10 pg/ml. In addition, IL-6 and sICAM-1 levels before operation were significantly higher in patients with short (less than one year) survival (p=0.007 to IL-6 and p=0.006 to sICAM-1). In contrast, patients with shorter survival had significantly lower IL-12 (p=0.03) levels. Our findings suggest that RCC induces changes in several immunological parameters. These soluble immunological factors, IL-6, IL-12, sIL-2R and sICAM-1, might have a role as prognostic factors in RCC.