Recombinant human arginase inhibits proliferation of human hepatocellular carcinoma by inducing cell cycle arrest.

Human hepatocellular carcinoma (HCC) has an elevated requirement for arginine in vitro, and pegylated recombinant human arginase I (rhArg-PEG), an arginine-depleting enzyme, can inhibit the growth of arginine-dependent tumors. While supplementation of the culture medium with ornithine failed to rescue Hep3B cells from growth inhibition induced by rhArg-PEG, citrulline successfully restored cell growth. The data support the roles previously proposed for ornithine transcarbamylase (OTC) in the arginine auxotrophy and rhArg-PEG sensitivity of HCC cells. Expression profiling of argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and OTC in 40 HCC tumor biopsy specimens predicted that 16 of the patients would be rhArg-sensitive, compared with 5 who would be sensitive to arginine deiminase (ADI), another arginine-depleting enzyme with anti-tumor activity. Furthermore, rhArg-PEG-mediated deprivation of arginine from the culture medium of different HCC cell lines produced cell cycle arrests at the G(2)/M or S phase, possibly mediated by transcriptional modulation of cyclins and/or cyclin dependent kinases (CDKs). Based on these results, together with further validation of the in vivo efficacy of rhArg-PEG against HCC, we propose that the application of rhArg-PEG alone or in combination with existing chemotherapeutic drugs may represent a specific and effective therapeutic strategy against HCC.

Confocal analysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and aortic smooth muscle cells.

Detyrosinated and acetylated alpha-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium-centrosome and centrosome-Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

Albumin can reverse the release of potassium from human erythrocytes treated with the non-ionic detergent, Brij 58.

Exchange of erythrocyte intracellular (i/c) K+ for extracellular (e/c) Na+ in human erythrocytes treated with sub-CMC concentrations of the non-ionic detergent Brij 58 can be stopped by reincubation in serum or albumin containing solutions. The progressive equilibration of the K+ contents of detergent-treated human erythrocytes with the incubation medium was reversed by an albumin-mediated withdrawal of detergent molecules from the cell. Re-establishment of near normal [K+] in terms of K+/kg water proceeds in two ways: (i) a metabolism-dependent net accumulation of K+ ions; and (ii) a metabolism-independent shrinkage of erythrocytes, this being the more significant factor.

Heat-induced damage to HeLa-S3 cells: correlation of viability, permeability, osmosensitivity, phase-contrast light-, scanning electron- and transmission electron-microscopical findings.

The responses of HeLa S-3 to mild hyperthermia for relatively critical times at 43 and 45 degrees C were analysed in detail, including growth and colony-forming ability, permeability, osmotic sensitivity and microscopical appearances. For comparative purposes lower temperatures (e.g. 41 degrees C) and higher temperatures (50 and 55 degrees C) were used in some experiments. The evidence from many different aspects, including scanning and transmission electron microscopy, suggests that critical heat exposures do not per se cause severe membrane damage and loss of cell integrity, but changes quickly become manifest when cells are 'recovered' by returning to 37 degrees C. Attention is drawn to the ability of heat-treated cells to show osmotic-like swelling and restoration towards normal volume in medium of 30 per cent normal strength, which would not be expected on the hypothesis that hyperthermia primarily disrupts membrane structure and functioning. Ultrastructural changes during and after hyperthermia--including nucleolar changes, the appearance of perichromatin granules, the formation of electron-dense cytoplasmic clusters, and the development of intranuclear actin rods--corroborate and extend other findings. However, mitochondrial changes were found to be particularly significant, appearing early and correlating well with the loss of viability and metabolic functioning found after heat treatment. These include the early development of intramitochondrial dense granules, followed by vesicularization of the cristae, swelling of the intracristal spaces, myelin degeneration and the formation of bodies which could otherwise be mistaken for secondary lysosomes. The findings indicate the need for more intensive investigations of mitochondria and mitochondrial functioning in hyperthermia-induced cell damage, and their careful correlation with the 'recovery' of energy-dependent process in cells subsequently returned to 37 degrees C.