We comparatively investigated the extent of apoptotic cell loss in human colorectal cancers evaluated by two methods, namely the conventional terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) method and immunohistochemistry for single-stranded (ss) DNA. The apoptotic index (AI) obtained with the TUNEL method was higher than that shown by the immunohistochemistry for ssDNA. However, a significant correlation in AIs evaluated by these methods was found. The AIs obtained by both methods were significantly higher in the advanced cancers than in the early cancers. Cellular proliferation activity was assessed in terms of positivity rate (PR) for expression of proliferating cell nuclear antigen (PCNA). The PR of advanced cancers was significantly higher than that of early cancers. The present results indicate that immunohistochemistry for ssDNA is useful (as is the TUNEL method) for evaluation of apoptotic tumor cells in colorectal carcinomas. In addition, it was confirmed that there is a remarkable increase of not only proliferation activity, but also tumor cell apoptosis in the process of progression of colon cancer from early to advanced stages of the disease.Key words: Apoptosis -TUNEL -Immunohistochemistry -Single-stranded DNA -Colorectal cancer Apoptosis is characterized morphologically by large chromatin fragments, 1) by extensive margination and fragmentation of the chromatin viewed electromicroscopically,2) and by a characteristic "ladder formation" of DNA fragments of about 180-200 base pairs by endonuclease activation in gel electrophoresis.3)The terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick endlabeling (TUNEL) method, which detects apoptosis and programmed cell death has been utilized to identify apoptotic cells at the single-cell level. 4) In contrast, Tomei has speculated that apoptosis involves the modification of chromatin, which results in a break-down of the supercoiling organization and the formation of individual super breaks. He has further proposed that single-stranded (ss) DNA modification in the nucleosomal linker region might constitute a critical early step in apoptosis.5) Naruse et al. showed that the antibody against ssDNA is a good marker of both drug-induced apoptosis and programmed cell death during embryogenesis. A polyclonal antibody against ssDNA has been used to immunohistochemically detect apoptotic cell death in epithelial cells. 6)There is, to our knowledge, no report on the relationship between apoptosis detected by immunohistochemistry for ssDNA and cellular proliferation activity assessed in terms of the positivity rate (PR) of proliferating cell nuclear antigen (PCNA) in colorectal carcinoma. In the present study, we investigated the extent of apoptotic cell loss in human colorectal cancers by the conventional TUNEL method and by immunohistochemistry for ssDNA. In addition, we compared the apoptotic indices (AIs) obtained by these two methods to determine whe...

Cyclophosphamide induced fragmented nuclei in mouse thymic epithelial cells. Agarose gel electrophoresis showed the fragmentation of the DNA extracted from mouse thymus exposed to cyclophosphamide. The cell death induced by cyclophosphamide was considered to be apoptotic. Polyclonal antibody against single-stranded DNA was used immunohistochemically to detect apoptotic cell death in thymic epithelial cells. This antibody also detected programmed cell death in the interdigital necrotic zone of the mouse limb plate on day 14 of gestation, and in the ganglion of the trigeminal nerve on day 13 of gestation. These results show that the antibody specific for single-stranded DNA detected both drug-induced apoptosis and programmed cell death during embryogenesis.

Copper is an essential trace element in prokaryotes and eukaryotes and is strictly regulated by biological mechanisms. Menkes and Wilson diseases are human disorders that arise from disruption of the normal process of copper export from the cytosol to the extracellular environment. Recently a gene for Wilson disease (WD)(also named the ATP7B gene) was cloned. This gene encodes a copper transporter of the P-type ATPase. We prepared monoclonal and polyclonal anti-(WD protein) antibodies and characterized the full-length WD protein as well as a shorter form that is produced by alternative splicing in the human brain. We found that the WD protein is localized mainly in the Golgi apparatus, whereas the shorter form is present in the cytosol. These results suggest that the alternative WD proteins act as key regulators of copper metabolism, perhaps by performing distinct roles in the intracellular transport and export of copper.

Ceruloplasmin, a blue copper oxidase found in plasma, is synthesized in hepatocytes as a single polypeptide chain consisting of a 19 amino acid leader peptide plus 1046 amino acids of mature protein (132 kDa). Holoceruloplasmin is secreted into the plasma with 6-7 atoms of copper bound per molecule. In this study we identified apo- and holoceruloplasmin and examined the mechanism of copper incorporation during ceruloplasmin biosynthesis using the Long-Evans Cinnamon (LEC) rat which does not incorporate copper into newly synthesized ceruloplasmin. We followed the conversion from ceruloplasmin precursor (with little or no carbohydrate) to the larger product (after carbohydrate addition), which occurred in the secretory compartments of hepatocytes, by native gel electrophoresis. We found that copper accumulates in the hepatocellular Golgi apparatus of LEC rats due to a disorder in the process of copper incorporation. The data indicate that copper is incorporated into ceruloplasmin late in the course of its transport through the secretory compartments.

Background/Aims: The aim of this study was to examine the relationship between the degree of urinary copper excretion and stages of diabetic nephropathy. Methods: Copper, ceruloplasmin and albumin concentrations were measured in serum and urine samples from 41 type 2 diabetic outpatients with different stages of nephropathy and from 10 healthy controls. The copper/albumin and copper/ceruloplasmin ratios in serum and urine were determined. Furthermore, we examined whether free copper ions are dissociated from ceruloplasmin under various pH conditions. Results: Urinary copper concentrations significantly increased only in macroalbuminuric patients. The copper/ceruloplasmin and copper/albumin ratios in urine were consistently greater than those in serum which were not different between patients and healthy controls except the copper/albumin ratio in macroalbuminuric patients. The ratios in urine decreased in parallel with the progression of nephropathy. Copper was found to be released from ceruloplasmin under acidic conditions. Conclusion: Urinary copper excretion in healthy controls may be the result of dissociation from the albumin-copper complex of serum during its passage through the kidney. In diabetic patients with advanced nephropathy, urinary copper excretion may be due to dissociations from both copper-albumin and ceruloplasmin-copper complexes filtered through the damaged glomerulus. Overloading of urinary copper to damaged renal tubules may play some roles in the progression of nephropathy in patients with advanced nephropathy.