[A case of gallbladder carcinoma following cholecystogastric fistula].

A 79-year-old woman with pneumobilia and liver dysfunction was admitted to our hospital. ERCP and gastrointestinal endoscopy revealed choledochal stones and a cholecystogastric fistula at the greater curvature of the gastric antrum. The risk of cholecystectomy and fistulectomy appeared to be extremely high for this patient because of her advanced age and low respiratory function due to interstitial pneumonia. Therefore, only an endoscopic lithotomy was performed, and the cholecystogastric fistula remained. However, after 2 years of follow-up, she developed an advanced gallbladder carcinoma. This finding suggests that cholecystogastric fistula is a risk factor for gallbladder carcinoma. Because of the difficulty of early detection of gallbladder carcinoma associated with cholecystogastric fistula, both fistulectomy and cholecystectomy are necessary when cholecystogastric fistula is diagnosed.

[Double esophageal carcinosarcomas showing rapid growth of a new polypoid tumor after sloughing of another polypoid tumor].

A 55-year-old man presented at our hospital with a large polypoid esophageal tumor. Initial upper gastrointestinal endoscopy revealed that this tumor had sloughed off to be replaced with ulceration in the thoracic esophagus. However, after the tumor at the thoracic esophagus sloughed off, a semi-circular, superficial, flat squamous cell carcinoma was observed adjacent to the ulceration. In addition, a separate carcinosarcoma, 2cm in diameter, was found at the esophagogastric junction. Approximately one month later, endoscopic re-examination revealed a new polypoid tumor approximately 4cm in diameter that was growing rapidly in the center of the superficial thoracic esophageal carcinoma lesion. Standard subtotal esophagectomy was performed. Histopathological examination revealed that both lesions were esophageal carcinosarcomas. This is a rare case of double esophageal carcinosarcoma associated with rapid polypoid tumor growth from a superficial squamous cell carcinoma lesion.

[Eosinophilic cholangitis from almost normal appearance to the bile duct sclerosis similar to primary sclerosing cholangitis].

A 48-year-old man was admitted to our hospital because of eosinophilia and liver dysfunction. Initial abdominal CT and MRI (MRCP) finding showed almost normal liver and bile duct. Liver biopsy demonstrated mild portal infiltration of lymphocytes and eosinophils. Definitive diagnosis was difficult, but we suspected autoimmune disease. Oral steroid administration was started, which led to a rapid improvement of eosinophilia and liver dysfunction. Dose reduction of steroid administration resulted in exacerbation of eosinophilia and liver dysfunction. Follow-up MRCP and ERCP study revealed biliary strictures similar to primary sclerosing cholangitis (PSC). A second liver biopsy revealed dense infiltration composed of lymphocytes and eosinophils in the portal area. Therefore we diagnosed eosinophilic cholangitis. This is the first case of eosinophilic cholangitis, observed after changes of the bile duct from an almost normal appearance to diffuse sclerosing and narrowing similar to PSC by imaging and pathological studies.

[A case of combined syndrome of juvenile polyposis and hereditary hemorrhagic telangiectasia associated with SMAD4 mutation].

A 47-year-old man was found to have abnormal findings on chest radiography. Chest computed tomography (CT) and magnetic resonance imaging (MRI) showed that he had a pulmonary arteriovenous malformation. He had experienced epistaxis when he was a junior high school student, and since then, the symptom had frequently recurred. Further, he had telangiectasia on the lips. Thus, he was given a diagnosis of hereditary hemorrhagic telangiectasia (HHT). Endoscopy revealed gastric telangiectasia, and in addition, his colon had many juvenile polyps. When he was 49 years of age, he underwent genetic analysis for HHT. A diagnosis of juvenile polyposis-HHT combined syndrome (JP-HHT) was made since a heterozygous germline 4-base deletion in exon 9 of SMAD4 was detected. To the best of our knowledge, this is the first case of JP-HHT associated with SMAD4 mutation in Japan.

Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells.

BACKGROUND: There are a growing number of reports on the sub-physiological temperature culturing of mammalian cells for increased recombinant protein yields. However, the effect varies and the reasons for the enhancement are not fully elucidated. Expression of cold-inducible RNA-binding protein (cirp, also called cirbp or hnRNP A18) is known to be induced in response to mild, but not severe, hypothermia in mammalian cells. To clarify the molecular mechanism underlying the induction and to exploit this to improve the productivity of recombinant proteins, we tried to identify the regulatory sequence(s) in the 5' flanking region of the mouse cirp gene. RESULTS: By transiently transfecting HEK293 cells with plasmids expressing chloramphenicol acetyltransferase as a reporter, we found that the cirp 5' flanking region octanucleotide 5'-TCCCCGCC-3' is a mild-cold responsive element (MCRE). When 3 copies of MCRE were placed upstream of the CMV promoter and used in transient transfection, reporter gene expression was increased 3- to 7-fold at 32°C relative to 37°C in various cell lines including HEK293, U-2 OS, NIH/3T3, BALB/3T3 and CHO-K1 cells. In stable transfectants, MCRE also enhanced the reporter gene expression at 32°C, although more copy numbers of MCRE were necessary. Sp1 transcription factor bound to MCRE in vitro. Immunohistochemistry and chromatin immunoprecipitation assays demonstrated that more Sp1, but not Sp3, was localized in the nucleus to bind to the cirp regulatory region containing MCRE at 32°C than 37°C. Overexpression of Sp1 protein increased the expression of endogenous Cirp as well as a reporter gene driven by the 5' flanking region of the cirp gene, and down-regulation of Sp1 had the opposite effect. Mutations within the MCRE sequence in the 5' flanking region abolished the effects of Sp1 on the reporter gene expression both at 37°C and 32°C. CONCLUSIONS: Cold-induced, as well as constitutive, expression of cirp is dependent, at least partly, on MCRE and Sp1. The present novel enhancer permits conditional high-level gene expression at moderately low culture temperatures and could be utilized to increase the yield of recombinant proteins in mammalian cells.

Low temperature protects mammalian cells from apoptosis initiated by various stimuli in vitro.

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we examined the effects of low temperature (32 degrees C) on cells exposed to a variety of stress in vitro. We found that 32 degrees C suppressed induction of apoptosis by cytotoxic stimuli such as adriamycin, etoposide, thapsigargin, NaCl, H(2)O(2), and anti-Fas antibody. In adriamycin-treated BALB/3T3 cells, the down-shift in temperature from 37 degrees C to 32 degrees C increased the Bcl-xL protein level and decreased the mRNA level of Puma and mitochondrial translocation of Bax, suppressing caspase-9-mediated apoptosis. Furthermore, the protein level and stability of p53 were decreased, and its nuclear export was increased concomitant with Mdm2 mRNA upregulation. The low temperature effect was not observed in p53(-/-)/Mdm2(-/-) mouse embryonic fibroblasts, suggesting that the effect is mediated by suppression of the p53 pathway. In contrast, while thapsigargin-induced apoptosis was suppressed by the low temperature, no effect on the p53 protein level was observed. Furthermore, the survival rate of p53(-/-)/Mdm2(-/-) cells exposed to thapsigargin was increased when cultured at 32 degrees C compared with 37 degrees C. In conclusion, mild hypothermia protects cells from a variety of stress by p53-dependent and p53-independent mechanisms.

The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53.

Gankyrin is an ankyrin repeat oncoprotein commonly overexpressed in hepatocellular carcinomas. Gankyrin interacts with the S6 proteasomal ATPase and accelerates the degradation of the tumor suppressor Rb. We show here that gankyrin has an antiapoptotic activity in cells exposed to DNA damaging agents. Downregulation of gankyrin induces apoptosis in cells with wild-type p53. In vitro and in vivo experiments revealed that gankyrin binds to Mdm2, facilitating p53-Mdm2 binding, and increases ubiquitylation and degradation of p53. Gankyrin also enhances Mdm2 autoubiquitylation in the absence of p53. Downregulation of gankyrin reduced amounts of Mdm2 and p53 associated with the 26S proteasome. Thus, gankyrin is a cofactor that increases the activities of Mdm2 on p53 and probably targets polyubiquitylated p53 into the 26S proteasome.

Apg-2 has a chaperone-like activity similar to Hsp110 and is overexpressed in hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. We constructed subtracted cDNA libraries enriched with genes overexpressed in HCCs. Among the 17 genes identified were molecular chaperones, Hsp110, Hsp90B, and Hsp70-1. Expression of the Hsp110 family members was further analyzed, and increased transcript levels of Hsp110 and Apg-2, but not Apg-1, were found in 12 and 14, respectively, of 18 HCCs. Immunohistochemical analysis demonstrated the overexpression of the proteins in tumor cells. Apg-2 had chaperone ability similar to Hsp110 in a thermal denaturation assay using luciferase, and showed anti-apoptotic activity. These results suggest that the Hsp110 family members play important roles in hepatocarcinogenesis through their chaperoning activities.