Takenari Yamanaka

Cellular FLICE/Caspase-8–Inhibitory Protein as a Principal Regulator of Cell Death and Survival in Human Hepatocellular Carcinoma

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8–inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R–mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-κB and cFLIP down-regulation attenuated NF-κB activation induced by TNF-α or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-κB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-α, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-κB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor–mediated apoptosis but also by regulating NF-κB activation in human HCCs.

Expression of survivin during liver regeneration.

Survivin functions to suppress cell death and regulate cell division, and is observed uniquely in tumor cells and developmental cells. However, the expression and regulation of survivin in non-transformed cells are not well elucidated. Therefore, we investigated the expression of survivin in a murine liver regeneration model after partial hepatectomy and intraperitoneal carbon tetrachloride (CCl(4)) injection. We found that the expression of survivin transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases during liver regeneration. In a normal mouse liver cell line, over-expression of survivin resulted in a decrease in the G0/G1 phase and an increase in the S and G2/M phases, resulting in Rb phosphorylation. These findings suggest that survivin is dramatically expressed in a cell cycle-dependent manner during liver regeneration and provide a new insight into the regulation of cell proliferation and differentiation.

p21WAF1/CTP1 expression and hepatitis virus type.

Since p21WAF1/CIP1 (p21) is a universal inhibitor of cyclin-dependent kinases and is regulated transcriptionally by p53, which is activated by DNA stress, its expression reflects DNA stress in chronic hepatitis. Recently an association with both hepatitis B and C virus and the expression of p53 or p21 was reported. We analyzed p21 expression in 18 cases of HBV-associated chronic liver diseases and 32 cases of HCV-associated chronic liver diseases by immunohistochemical analysis, and investigated the possible association between hepatocyte p21 expression and hepatic inflammation, fibrosis, and especially hepatitis virus type. The p21-positive hepatocytes were more numerous in areas of intense inflammation and spotty necrosis and areas close to fibrosis, and they increased according to the degrees of grading and staging. The p21 labeling index (LI) in patients with liver cirrhosis was significantly higher than that in patients with chronic hepatitis of both hepatitis viral types (5.84 ± 0.61 vs 12.0 ± 0.83, P < 0.0001 in hepatitis B, 10.28 ± 0.80 vs 15.6 ± 1.09, P = 0.0004 in hepatitis C), Furthermore, the p21 LI was significantly higher in HCV-associated liver disease than in HBV-associated liver disease in every group (4.02 ± 0.48 vs 7.74 ± 0.96, P = 0.021 in low grade group, 7.35 ± 0.46 vs 12.8 ± 0.57, P < 0.0001 in high grade, 12.0 ± 0.83 vs 15.6 ± 1.09, P = 0.034 in liver cirrhosis). In, conclusion, p21 expression was up-regulated by the stress of inflammation and fibrosis and might be influenced by viral proteins in human chronic liver disease.

Functional Expression of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand in Human Colonic Adenocarcinoma Cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. Therefore, we investigated TRAIL sensitivity, TRAIL-induced nuclear factor-κB (NF-κB) activation, and expression of TRAIL in human colonic adenocarcinoma cell lines (HT-29, LS180, SK-CO-1). All four TRAIL receptors (TRAIL-R1 through TRAIL-R4) are expressed in these cell lines. TRAIL sensitivity was assessed by assay of cell viability. Cancer cell viabilities were 83 ± 3.1% (HT-29), 90 ± 4.3% (LS180), and 88 ± 6.3% (SK-CO-1) at 24 hours after the addition of 100 ng/ml TRAIL, indicating that these cell lines were relatively resistant to TRAIL. Activation of NF-κB was variably influenced by TRAIL administration, with no consistent tendency among the cell lines, indicating that TRAIL-induced NF-κB activation might be cell-type dependent. In contrast, TRAIL was expressed in the human colonic adenocarcinoma cell lines by Western blotting and RT-PCR. Increased expression of TRAIL on tumor cells was observed by flow cytometry after cytokine stimulation (IFN-γ, TNF-α) or the addition of chemotherapeutic agents (camptothecin, doxolubicin hydrochloride). TRAIL on HT-29 cells was functional and able to induce apoptosis in Jurkat cells. Jurkat cell viability was increased by the addition of TRAILR1-R4-Fc. In the presence of various cytokines or chemotherapeutic agents, functional TRAIL is expressed on the surface of tumor cells, and this expressed TRAIL might contribute to tumor immune privilege by inducing apoptosis of activated human lymphocytes.

Peroxisome proliferator-activated receptor gamma augments tumor necrosis factor family-induced apoptosis in hepatocellular carcinoma.

Proliferator-activated receptor &ggr; (PPAR &ggr;) is a nuclear receptor, which mainly associates with adipogenesis, but also appears to facilitate cell differentiation or apoptosis in certain malignant cells. This apoptosis induction by PPAR &ggr; is increased by co-stimulation with tumor necrosis factor (TNF)-&agr;-related apoptosis-inducing ligand (TRAIL), a member of the TNF family. In this study, we investigated the effect of PPAR &ggr; on Fas-mediated apoptosis in hepatocellular carcinoma (HCC) cell lines. PPAR &ggr; was expressed on all seven HCC cell lines and located in their nuclei. 15-Deoxy-&Dgr;-12,14-prostaglandin J2 (15d- PGJ2), a PPAR &ggr; ligand, inhibited cellular proliferation in HepG2, SK-Hep1 or HLE cells, unlike pioglitazone, another PPAR &ggr; ligand, which did not have a significant influence on proliferation of these cells. However, 15d-PGJ2 facilitated Fas-mediated HCC apoptosis that could not be induced by Fas alone. These results suggest that PPAR &ggr; can augment TNF-family-induced apoptosis.

Acute Pancreatitis in Patients with Ulcerative Colitis

Twenty-two patients (13 men and 9 women; median age, 34 years; range, 15–64 years) with ulcerative colitis (UC) were evaluated to determine the incidence of acute pancreatitis with UC at the First Department of Internal Medicine, Mie University School of Medicine, during 1989–2001. Among these, three patients (14%) were diagnosed as having had episodes of acute pancreatitis during the mean follow-up period of 6 years. One patient presented with acute pancreatitis and UC simultaneously. Two patients had drug-induced pancreatitis (one due to azathioprine and the other due to 5-ASA). In conclusion, acute pancreatitis is not a frequent, but an occasional extraintestinal manifestation of UC.

Primary Localized Amyloidosis of the Small Intestine Presenting as an Intestinal Pseudo-obstruction: Report of a Case

Abstract A 47-year-old man with primary amyloidosis confined to the small intestine is reported. Thickening of the folds and multiple polypoid protrusions were found in the duodenum by upper gastrointestinal endoscopy. Because the patient presented with a persistent intestinal pseudo-obstruction, partial jejunectomy was performed. Histological examination of the resected tissue revealed massive deposits of amyloid throughout the jejunal wall. Neither a predisposing condition nor any other sites of deposition were found, and primary amyloidosis of the small intestine was diagnosed. This rare form of amyloid deposition should be recognized so that an early diagnosis can be made.

CASE REPORT: Hepatocellular Carcinoma Associated with Adult-Type Citrullinemia

Hypercitrullinemia is a rare hereditary metabolic disorder caused by the deficiency in the activity of argininosuccinate synthetase. McMurrey et al first reported this disease in infants (1) and Saheki et al classified three types on the basis of qualitative and quantitative analysis of argininosuccinate synthetase (2). The classic neonatal and infantile forms were assigned to type I (abnormal kinetics of the enzyme) and III (undetectable or extremely low levels of the enzyme). Biochemically, the defect of the enzyme in the classical types is found in all tissues and/or cells where argininosuccinate synthetase is expressed (3). Analysis of the amplified cDNA from 14 neonatal/infantile type III citrullinemia patients identified mutations in the mRNA that are heterogeneous (4). Type II citrullinemia is an adult-onset type and is clinically characterized by a sudden onset of consciousness disturbance, a high serum citrulline concentration, and hyperammonemia. Most of this type of citrullinemia occurs in Japan unlike in the United States and Europe. Type II citrullinemia is characterized by a quantitative decrease of argininosuccinate synthetase only in the liver, while argininosuccinate synthetase levels in other tissues, such as kidney, brain, and fibroblasts, are normal. The hepatic content of the enzyme is about 10% of control value, but the translatable mRNA level for the enzyme is similar to the control value and there is no mutation in the argininosuccinate synthetase mRNA. Thus, it is confirmed that the liver contained the normal amount of mRNA coding for argininosuccinate synthetase, but there was increased degradation of the enzyme or inhibited translation (5–8). Although there are reports of the patients of type II citrullinemia complicated by hepatocellular carcinoma, the details were not well elucidated (7, 9). Here, we report on a patient with adult type citrullinemia who developed hepatocellular carcinoma after 29 years of follow-up, and we analyzed argininosuccinate synthetase in hepatocellular carcinoma tissues.

CASE REPORT: Metastatic Gas Gangrene of the Leg Due to Acute Emphysematous Cholecystitis

Subcutaneous emphysema of the lower extremities is mostly associated with gas gangrene following trauma (1). Intraabdominal disease is a rare cause of subcutaneous emphysema of the lower extremities. This may be the result of an enteric fistula following gastrointestinal tract perforation that extends along the anatomic planes of the pelvis to the lower extremities, or there may be extension of a retroperitoneal or subperitoneal abscess with gas-forming organisms along the fascial planes to the lower extremities (2). There are, however, few reports of gas-forming organisms spreading to the leg hematogeneously. This so-called nontraumatic metastatic gas gangrene is a rare entity and is sometimes associated with occult gastrointestinal malignancy. Herein, we report a case of metastatic gas gangrene due to emphysematous cholecystitis without gastrointestinal malignancy.