Tomoyuki Shirase

Redox regulation of annexin 2 and its implications for oxidative stress-induced renal carcinogenesis and metastasis

Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal damage leading to a high incidence of renal cell carcinoma (RCC) in rats. Differential display analysis of such RCCs revealed elevated expression of annexin 2 (Anx2), a substrate for kinases and a receptor for tissue-type plasminogen activator and plasminogen. We conducted this study to clarify the significance of Anx2 in Fenton reaction-based carcinogenesis. Messenger RNA and protein levels of Anx2 were increased time-dependently in the rat kidney after Fe-NTA administration as well as in LLC-PK1 cells after exposure to H2O2. The latter was inhibited by pretreatment with N-acetylcysteine, pyrrolidine dithiocarbamate or catalase. Immunohistochemistry revealed negligible staining in the normal renal proximal tubules, but strong staining in regenerating proximal tubules, karyomegalic cells and RCCs. Metastasizing RCCs showed higher Anx2 protein levels. Anx2 was phosphorylated at serine and tyrosine residues in these cells and coimmunoprecipitated with phosphorylated actin. Overexpression of Anx2 induced a higher cell proliferation rate in LLC-PK1 cells. In contrast, a decrease in proliferation leading to apoptosis was observed after Anx2 antisense treatment to cell lines established from Fe-NTA-induced RCCs. These results suggest that Anx2 is regulated by redox status, and that persistent operation of this adaptive mechanism plays a role in the proliferation and metastasis of oxidative stress-induced cancer.

Two distinct mechanisms for loss of thioredoxin-binding protein-2 in oxidative stress-induced renal carcinogenesis

Thioredoxin is a major component of thiol-reducing system. Recently, we identified thioredoxin-binding protein-2 (TBP-2) as a negative regulator of thioredoxin. Here, we report the role of TBP-2 in oxidative renal tubular injury and the subsequent carcinogenesis by ferric nitrilotriacetate. TBP-2 was abundantly expressed in the rat kidney. Immunohistochemical analysis revealed that TBP-2 was present in association with nuclei and mitochondrial intermembrane space in the proximal tubular cells and coimmunoprecipitated with cytochrome c. After acute oxidative tubular damage, TBP-2 protein, but not messenger RNA, markedly decreased, demonstrating shortened half-life of this protein. Most cases of the induced renal cell carcinoma showed undetectable levels of TBP-2 protein, which was associated with the methylation of CpG island in the promoter region. Genome sequence analyses identified the poly-A tract in the 3′ untranslated region as a mutation hot spot in this rather nonselective environment. Collectively, the amounts of TBP-2 protein were inversely associated with proliferation of tubular cells, as evaluated by proliferating cell nuclear antigen. These results suggest that loss of TBP-2 is essential for proliferation of not only neoplastic but also non-neoplastic renal tubular cells, and that TBP-2 is a target gene in oxidative stress-induced renal carcinogenesis by ferric nitrilotriacetate.

Periportal edema and necrosis as diagnostic histological features of early humoral rejection in ABO‐incompatible liver transplantation

Humoral rejection caused by antidonor blood group A/B antibodies is one of the most important obstacles for successful ABO‐incompatible liver transplantation. However, no specific morphologic features of liver biopsies to distinguish humoral rejection from other conditions such as ischemia or sepsis have been satisfactorily documented. To histologically clarify the early changes in humoral rejection, we studied 41 cases of living donor ABO‐incompatible liver transplantation whose allograft biopsies during the first episode of suspected acute rejection were available within the first postoperative month. Postoperative isohemagglutinin IgM titers were ×64 or more in 21 patients (51%; high‐titer group) and less than ×64 in 20 cases (49%; low‐titer group). In the high‐titer group, elevation of postoperative titers ×64 or more occurred within postoperative days 5.7 ± 4.1 (range: 1–17). An increase in the incidence of cholangitis was observed in the high‐titer group (90% vs. 30%, P < .0001), as well as poorer overall graft survival than in the low‐titer group (38% vs. 70%, P < .05). Seven biopsies obtained from the high‐titer group within 3 days after the onset of elevation of the antibody titers and one biopsy obtained at the peak of the antibody titers demonstrated periportal edema and necrosis, neither of which was found in the low‐titer group. All grafts of these patients caused massive hepatocyte necrosis or severe biliary complications. In conclusion, a high morbidity rate of ABO‐incompatible liver transplantation is associated with high postoperative levels of antibody titers. Periportal edema and necrosis observed during elevation of antibody titers can be regarded as histological indications of early changes in severe humoral rejection. (Liver Transpl 2004;10:16–27.)

Suppression of SLC11A2 Expression Is Essential to Maintain Duodenal Integrity During Dietary Iron Overload

Iron is essential for the survival of mammals, but iron overload causes fibrosis and carcinogenesis. Reduced iron absorption and regulated release into circulation in duodenal mucosa constitute two major mechanisms of protection against dietary iron overload; however, their relative contribution remains elusive. To study the significance of the former process, we generated SLC11A2 transgenic mice (TGs) under the control of the chicken beta-actin promoter. TGs were viable and fertile, and displayed no overt abnormalities up to 20 months. No significant difference in iron concentration was observed in major solid organs between TGs and their wild-type littermates, suggesting that increased number of iron transporters does not lead to increased iron absorption. To test the sensitivity to iron overload, TGs and wild-type mice were fed with an iron-rich diet containing 2% ferric citrate. Iron supplementation caused suppression of endogenous duodenal SLC11A2 expression, down-regulation of duodenal ferroportin, and overexpression of hepatic hepcidin, precluding excessive iron uptake both in the TGs and wild-type mice. However, iron-treated TGs revealed increased mortality, resulting from oxidative mucosal damage leading to hemorrhagic erosion throughout the whole intestinal area. These findings suggest that reduced iron release from duodenal cells into circulation plays a role in mitigating excessive iron uptake from the diet and that finely regulated duodenal absorption is essential to protect intestinal mucosa from iron-induced oxidative damage.

Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens

Although the human genome project has been completed, the functions of many genes remain undetermined. In situ hybridization (ISH) is a key method for identifying cells in which a given messenger RNA is transcribed. Paraffin‐embedded specimens remain precious materials for research, but preservation of high‐quality RNA in these specimens is not expected unless ample caution was taken during fixation. Peptide nucleic acid (PNA) is a recently developed hybrid molecule with genetic information that has high stability and high affinity to the complementary DNA or RNA. We applied a PNA probe to mRNA ISH of liver specimens obtained by autopsy and embedded in paraffin 28–48 years ago. An 18‐mer PNA probe for glyceraldehyde 3‐phosphate dehydrogenase was used. Staining was then analyzed in association with morphology by hematoxylin and eosin staining, and with the time between death of the patient and tissue fixation. Notably, specimens fixed with formalin and embedded in paraffin 48 years ago yielded excellent results if the time before fixation was short enough (<8 h). There was a significant inverse correlation between the intensity of ISH staining and the time before fixation. Oligonucleotide PNA probe, albeit at high cost, would increase the value of paraffin‐embedded specimens in storage for use in human medical research.

Immunoglobulin G4-related disease presenting as bilateral ovarian masses and mimicking advanced ovarian cancer

Immunoglobulin G4‐related disease (IgG4‐RD) is characterized by extensive infiltration of IgG4+ plasma cells and fibrosis in various organs. However, the involvement of the ovary in IgG4‐RD has never been reported. A 59‐year‐old woman presented with urinary retention. Magnetic resonance imaging and computed tomography revealed a huge multinodular pelvic mass and common iliac/para‐aortic lymph node swelling. A laparotomy was performed under the suspicion of advanced ovarian cancer, and the pelvic mass was identified as ovary in origin. Histopathology of the excised tumor revealed massive lymphoplasmacytic infiltration (>90% were IgG4+ plasma cells), storiform fibrosis, and obliterative phlebitis; thus leading to a diagnosis of IgG4‐RD. We conclude that IgG4‐RD can present as a bilateral ovarian mass along with lymphadenopathy, therefore mimicking ovarian cancer.