Raymond G. Schipper

Proton MR spectroscopy of prostatic tissue focused on the detection of spermine, a possible biomarker of malignant behavior in prostate cancer

To investigate whether polyamines may be valuable diagnostic and prognostic markers in prostate cancer, the presence of alyamines was studied in various human prostatic tissues using both proton magnetic resonance (MR) spectroscopy and righ-pressure liquid chromatography (HPLC). The HPLC results showed that normal and benign hyperplastic prostatic tissues re characterized by a high content of spermine. Spermine levels were reduced in tumor tissue, especially in prostatic carcinoma h metastases, and in xenografts of human prostatic carcinoma cells. These preliminary results indicate that spermine may be d as a biomarker for malignant behavior. The MR spectroscopy study showed that it is possible to detect spermine resonances prostatic biopsy material by one-dimensional and two-dimensional J-resolved MR spectroscopy at high field (600 MHz). ocalized one-dimensional in vitro MR spectra obtained at the clinical field strength of 1.5 T showed spermine signals in the region between 3.0 and 3.3 ppm. In in vivo MR spectra of the human prostate, however, these signals were obscured by esonances of choline (3.2 ppm) and creatine (3.0 ppm).

Intracellular Localization of Ornithine Decarboxylase and Its Regulatory Protein, Antizyme-1

The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical studies in cultured human cervix carcinoma and human prostatic carcinoma (PC-346C) cells. ODC localization patterns varied from predominantly cytoplasmic to both cytoplasmic and nuclear staining, whereas AZ1 was mostly found in the nucleus. In cells that were synchronized in the mitotic phase, localization of both ODC and AZ1 changed from perinuclear at the beginning of mitosis into nucleoplasmic at close proximity to the chromosomes during meta-, ana- and telophase. Upon completion of mitosis, localization of ODC and AZ1 was reverted back to the cytoplasm, i.e., predominantly perinuclear immediately after cytokinesis. When PC-346C cells were treated with polyamines to induce AZ1-regulated ODC degradation, ODC was predominantly found in the nucleus and colocalized with immunoreactive AZ1. A comparable accumulation of ODC and AZ1 in the nucleus was found in PC-346C cells treated with the polyamine analog SL-11093. The present study suggests that AZ1 is involved in nucleocyto-plasmic shuttling of ODC, which may be a prerequisite for ODC regulation and/or function.

Distribution Patterns of Ornithine Decarboxylase in Cells and Tissues: Facts, Problems, and Postulates

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Increased polyamine levels are required for growth, differentiation, and transformation of cells. In situ detection of ODC in cells and tissues has been performed with biochemical, enzyme cytochemical, immunocytochemical, and in situ hybridization techniques. Different localization patterns at the cellular level have been described, depending on the type of cells or tissues studied. These patterns varied from exclusively cytoplasmic to both cytoplasmic and nuclear. These discrepancies can be partially explained by the (lack of) sensitivity and/or specificity of the methods used, but it is more likely that (sub)cellular localization of ODC is cell type-specific and/or depends on the physiological status (growth, differentiation, malignant transformation, apoptosis) of cells. Intracellular translocation of ODC may be a prerequisite for its regulation and function.

Immunocytochemical detection of ornithine decarboxylase.

Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouins fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats.

Polyamines in Regulation of Prostatic Cell Growth

The prostate and prostate secretions have played an important role in the initial identification of polyamines. One of the polyamines, spermine, was reported in human semen as early as the 17th century by Antoni Van Leeuwenhoek. After the elucidation of the chemical structure of polyamines, about 200 yr later, the biochemical mechanism of action of polyamines in the prostate gland and semen has been studied extensively. Apart from this historical perspective, the prostate has one of the highest polyamine concentrations of any tissue. In rats, the amount of polyamines, especially spermidine, is highest in the ventral prostate followed by the dorsal and lateral prostate, whereas the coagulating glands and seminal vesicles show low values (1). The human prostate gland synthesizes exceptionally high levels of spermine on an average of 130 mg/100 g per day (2).