In situ substrate specificity and ultrastructural localization of polyamine oxidase activity in unfixed rat tissues.

Data concerning the substrate specificity and the exact intracellular localization of the polyamine-catabolizing enzyme polyamine oxidase are conflicting. Biochemical studies have shown that N1-acetylation of spermine and spermidine dramatically increases the specificity of these compounds for peroxisomal polyamine oxidase to produce spermidine and putrescine, respectively. On the other hand, polyamine oxidase activity was demonstrated histochemically both in peroxisomes and in cytoplasm of several tissues, using spermidine and/or spermine as substrate. To elucidate the in situ substrate specificity of polyamine oxidase and the localization of its activity, enzyme activity was detected in rat liver, kidney, and duodenum at the light and electron microscopic levels. For this purpose, unfixed cryostat sections were applied to avoid changes in enzyme activity owing to chemical fixation. Spermine, spermidine, their N1-acetylated forms, and putrescine were used as substrates, and cerium ions as capturing agent for H2O2. Control reactions were performed in the absence of substrate or in the presence of substrate and specific oxidase inhibitors. At the light microscopic level, final reaction product specifically generated by polyamine oxidase activity was found exclusively in a granular form in hepatocytes, epithelial cells of proximal tubules of the kidney, and epithelial cells of duodenal villi with N1-acetylspermidine or N1-acetylspermine as substrates. Final reaction product was not observed in any of the tissues after incubation in the presence of putrescine, spermidine, or spermine. Formation of specific final reaction product was prevented by incubation in the presence of a specific polyamine oxidase inhibitor, but it was not affected by a diamine oxidase inhibitor. Ultrastructural studies revealed that polyamine oxidase activity is localized exclusively to the matrix of peroxisomes of kidney and liver and to microperoxisomes of the duodenum. The localization patterns obtained with unfixed tissues are in agreement with biochemical data. Strong intraperoxisomal, interperoxisomal, and intercellular heterogeneity in polyamine oxidase activity was found in all tissues investigated.

Increased expression of high mobility group protein I(Y) in high grade prostatic cancer determined by in situ hybridization.

In a previous study using the Dunning rat prostate cancer model, we found high mobility group protein I-(Y) [HMG-I(Y)] to be overexpressed in metastatic tumor lines when compared to nonmetastatic lines. Hence, overexpression of this 12-kDa non-histone chromosomal protein may be associated with tumor progression. Firstly, by Northern analysis we showed that HMG-I(Y) expression increases in high grade prostate tumors. These studies, however, required fresh material, and clinical follow-up was limited. To overcome this problem paraffin-embedded material must be made amenable for determination of HMG-I(Y) expression in retrospective studies. RNA in situ hybridization enables the evaluation of mRNA levels in such material. We studied tumors from 71 patients with prostate cancer. The microscopic analysis of each sample included: (a) hybridization on sections with sense HMG-I(Y) and (b) 28S rRNA probes (nonspecific signal); (c) hybridization with antisense 28S rRNA (RNA preservation); (d) hybridization with an antisense HMG-I(Y) probe [quantification of HMG-I(Y) mRNA in the expressing areas]. Data were quantified using an image analysis system. High expression of HMG-I(Y) was observed in regions with high Gleason grade (4 and 5); whereas in lesions of Gleason grade 3, both weak and no expression was observed. In areas of grade 1 and 2, as well as in normal glands, low or no expression was found. We conclude that HMG-I(Y) expression assessed by RNA in situ hybridization is related to tumor differentiation in prostate cancer. These findings indicate that HMG-I(Y) expression may be a marker in prostate cancer diagnosis, and the possible clinical implication of expression of this gene in malignancy is discussed in this report.