Characterization of heat shock protein 27 phosphorylation sites in renal cell carcinoma.

The phosphorylation of heat shock protein 27 (HSP27) occurs differently in human renal cell carcinoma (RCC) compared to homologous normal kidney tissue. Two-dimensional electrophoresis was used to separate and visualize HSP27, via immunostaining with anti-HSP27 antibody, in tumor and normal renal samples, obtained after surgery resection from patients with RCC. The mean number of protein species was 21 in RCC and 15 in normal tissues. Selected spots were in-gel digested with trypsin, extracted and analyzed by microcapillary liquid chromatography (LC) electrospray ionization tandem mass spectrometry to confirm HSP27 protein identity and reveal phosphorylation sites. Loss of phosphopeptides due to extensive plumbing and/or metal components in automated LC-systems was limited by manual loading of samples directly onto the LC system using a homemade pressure vessel. Mass spectrometry (MS) analysis revealed that in three of the HSP27 protein species phosphorylation occurred at Serine 15 and in five at Serine 82 in a different pattern. The phosphorylation of Serine 15 and 82 was also investigated by immunohistochemistry on tissue sections. The data obtained using anti-HSP27Serine82phos-antibody are consistent with MS results, while the variance between results achieved by anti-HSP27Serine15phos-antibody and by MS is probably due to the low specificity of the antibody. Knowledge of the diversity and modulation of HSP27 phosphorylation protein species might represent useful markers involved in the differentiation of RCC.

Expression of heat shock protein 27 in human renal cell carcinoma.

Heat shock protein 27 (HSP27, Swiss-Prot accession number P04792) is a component of the large and heterogeneous group of chaperone proteins, and its main functions are inhibition of apoptosis and prevention of aggregation of actin intermediate filament. Modified expression of HSP27 has been described in several cancers including testis, breast, and ovaric cancer. In the present work, 18 renal cell carcinoma (RCC) tissues and homologous normal kidney tissues have been investigated for HSP27 expression by combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separation and Western blotting immunodetection. The results showed significant differences either in expression and in HSP27 isoform numbers in RCC compared to normal kidney. The average number of isoforms was 21 in RCC and 15 in normal tissues with 4.5-5.9 pI range and 18-29 kDa M(r) range. The overexpression was also observed by immunohistochemistry on tissue sections. Only two of RCC samples showed less isoforms than homologous normal samples. Two isoforms were not detected using anti-Ser82 phosphorylated HSP27 antibody, neither in normal nor in RCC samples. Five of all the immunodetected isoforms were confirmed by mass spectrometry as HSP27, but no evidence of post-translational modifications was pointed out. The numerous isoforms observed in RCC are not consistent with data reported in the literature so far, and they might be due to different post-translational modifications such as phosphorylation and S-thiolation. Since activation of HSP27 seems to be involved in tumor proliferation and drug resistance, it would be crucial to correlate the severity of disease with the different isoforms from RCC samples to generate diagnostic and prognostic markers.

Increased susceptibility to plasma lipid peroxidation in Alzheimer disease patients.

Oxidative stress, linked to Abeta-lipid interactions, plays a pathogenetic role in Alzheimer's disease. We investigated modifications of lipid peroxidation products in plasma of 52 AD patients, 42 healthy controls and 16 patients with amyotrophic lateral sclerosis, a neurodegenerative disease where oxidative stress also plays a pathogenetic role. Final lipid peroxidation products were measured in plasma by thiobarbituric acid reactive substances (TBARS) assay before and after ex vivo oxidative stress catalysed by copper. There were no significant changes at basal conditions, but after copper-induced oxidation TBARS levels were higher in AD patients (19.0 microM +/- 2.2) versus both controls (5.2 microM +/- 0.8, p<0.001) and ALS patients (7.6 microM +/- 2.1, p<0.01). Stimulated TBARS levels were significantly higher in mild and moderate AD (p<0.0001) with respect to controls, but not in severe AD patients, with a significant inverse correlation between disease severity and lipid peroxidation (p<0.005, r2=0.21). Treatment of a subgroup (13) of mild and moderate AD patients with vitamin C and E for three months decreased plasma lipoperoxidation susceptibility by 60%. Thus, oxidative stress, expressed as ex vivo susceptibility to lipid peroxidation, appears to be an early phenomenon, probably related to AD pathogenetic mechanisms.