Carbon Ion Radiation Therapy With Concurrent Gemcitabine for Patients With Locally Advanced Pancreatic Cancer.

PURPOSE: To determine, in the setting of locally advanced pancreatic cancer, the maximum tolerated dose of carbon ion radiation therapy (C-ion RT) and gemcitabine dose delivered concurrently and to estimate local effect and survival. METHODS AND MATERIALS: Eligibility included pathologic confirmation of pancreatic invasive ductal carcinomas and radiographically unresectable disease without metastasis. Concurrent gemcitabine was administered on days 1, 8, and 15, and the dose levels were escalated from 400 to 1000 mg/m(2) under the starting dose level (43.2 GyE) of C-ion RT. The dose levels of C-ion RT were escalated from 43.2 to 55.2 GyE at 12 fractions under the fixed recommended gemcitabine dose determined. RESULTS: Seventy-six patients were enrolled. Among the 72 treated patients, dose-limiting toxicity was observed in 3 patients: grade 3 infection in 1 patient and grade 4 neutropenia in 2 patients. Only 1 patient experienced a late grade 3 gastric ulcer and bleeding 10 months after C-ion RT. The recommended dose of gemcitabine with C-ion RT was found to be 1000 mg/m(2). The dose of C-ion RT with the full dose of gemcitabine (1000 mg/m(2)) was safely increased to 55.2 GyE. The freedom from local progression rate was 83% at 2 years using the Response Evaluation Criteria in Solid Tumors. The 2-year overall survival rates in all patients and in the high-dose group with stage III (≥45.6 GyE) were 35% and 48%, respectively. CONCLUSIONS: Carbon ion RT with concurrent full-dose gemcitabine was well tolerated and effective in patients with unresectable locally advanced pancreatic cancer.

Phase 1 trial of preoperative, short-course carbon-ion radiotherapy for patients with resectable pancreatic cancer.

BACKGROUND: The authors evaluated the tolerance and efficacy of carbon-ion radiotherapy (CIRT) as a short-course, preoperative treatment and determined the recommended dose needed to reduce the risk of postoperative local recurrence without excess injury to normal tissue. METHODS: Patients radiographically defined with potentially resectable pancreatic cancer were eligible. A preoperative, short-course, dose-escalation study was performed with fixed 8 fractions in 2 weeks. The dose of irradiation was increased by 5% increments from 30 grays equivalents (GyE) to 36.8 GyE. Surgery was to be performed 2 to 4 weeks after the completion of CIRT. RESULTS: The study enrolled 26 patients. At the time of restaging after CIRT, disease progression with distant metastasis or refusal ruled out 5 patients from surgery. Twenty-one of 26 patients (81%) patients underwent surgery. The pattern of initial disease progression was distant metastasis in 17 patients (65%) and regional recurrence in 2 patients (8%). No patients experienced local recurrence. The 5-year survival rates for all 26 patients and for those who underwent surgery were 42% and 52%, respectively. CONCLUSIONS: Preoperative, short-course CIRT followed by surgery is feasible and tolerable without unacceptable morbidity.

[Two cases of advanced colorectal cancer which demonstrated the reversibility of oxaliplatin-mediated increase in splenic volume].

Hepatic sinusoidal injury arises occasionally after oxaliplatin-based chemotherapy. As a result, portal hypertension associated with splenomegaly occurs in some cases. We report two cases of advanced colorectal cancer which showed splenomegaly after administration of oxaliplatin-based chemotherapy. In both cases, mFOLFOX6/bevacizumab was administered as a firstline chemotherapy. Splenic volume was determined by loading the CT images onto a commercially available workstation. In case 1, initial splenic volume was 137.82mL. Two months later, it increased to 160.96mL. After six cycles of chemotherapy, we removed oxaliplatin due to peripheral neuropathy. Consequently, the splenic volume decreased to 151.58mL. Subsequent to the reintroduction of oxaliplatin, the splenic volume increased to 177.48mL. Following two cycles of mFOLFOX6/bevacizumab, oxaliplatin was removed again. In a similar way, splenic volume decreased to 158.52mL. In case 2, initial splenic volume was 105.84mL. Ten months later, it increased to 228.54mL. After administration of mFOLFOX6/bevacizumab, we continued chemotherapy with sLV5FU2/bevacizumab and irinotecan. The splenic volume decreased to 197. 06mL. In conclusion, oxaliplatin- based chemotherapy induces an increase in splenic volume, however, it may be reversible after discontinuation of oxaliplatin.

A long-term controlled trial of endoscopic pancreatic stenting for treatment of main pancreatic duct stricture in chronic pancreatitis.

BACKGROUND/AIMS: Endoscopic pancreatic stenting (EPS) has been used to treat main pancreatic duct (MPD) stricture in chronic pancreatitis (CP), with favourable reported results. However, most studies were retrospective and uncontrolled. We conducted a longterm prospective controlled study of EPS for treatment of MPD stricture in CP. METHODOLOGY: Consecutive patients with CP were treated to remove pancreatic stones by extracorporeal shock-wave lithotripsy or endoscopic basket extraction. After treatment, 41 patients were enrolled in the study upon meeting the criteria of complete removal of stones, pain relief after the treatment, and dominant stricture of the MPD. Twenty patients chose EPS, while 22 control patients did not. We compared recurrence of pain and pancreatic function between groups for over 3 years of follow-up. RESULTS: The mean follow-up period was 62.5 ± 20.9 months. Pain recurred in 15% of EPS patients (3/20) and in 50.0% of control patients (11/22), a significant difference (p<0.05). Progression of exocrine insufficiency in the EPS group was significantly slower than in the control group (p<0.05), while endocrine function showed no difference between groups. CONCLUSIONS: EPS reduced pain recurrence and slowed down the progression of exocrine insufficiency in CP patients with MPD stricture.

[Two cases of colorectal cancer presenting with periodic, transient elevation of serum iron after chemotherapy including irinotecan].

It is known that the serum iron level shows a transient elevation after chemotherapy in some cases; however, the cause of this phenomenon has not been clearly described. We report two cases of colorectal cancer whose serum iron level demonstrated recurrent elevation after administration of irinotecan as a second-line chemotherapy. The transferrin saturation rate showed marked elevation together with serum iron. This fact indicates that the release of non-transferrin bound iron (NTBI) occurs and then, NTBI binds with transferrin immediately thereafter. Additionally, elevation of indirect bilirubin in case 1, and mild anemia in case 2 were observed after every course of chemotherapy. All these phenomena were synchronized with the fluctuation of the serum iron level. These observations suggest that the transient elevation of the serum iron was related with the release of the NTBI from red blood cells after chemotherapy including irinotecan.

[A case of mucosa-associated lymphoid tissue lymphoma with penicillin allergy successfully treated with levofloxacin, minomycin and rabeprazole].

A 52-year-old Japanese woman was referred to our Institute because of Helicobacter pylori(H. pylori)-positive gastric mucosa-associated lymphoid tissue(MALT)lymphoma. Since she had a penicillin allergy, we could not eradicate H. pylori using the standard triple therapy including amoxicillin. Additionally, H. pylori was resistant to both clarithromycin and metronidazole. So she was treated with minomycin (MINO), levofloxacin (LVFX), and rabeprazole (RPZ) based on a drug sensitivity test. MINO+LVFX+RPZ appear to be a promising, appropriate, and well-tolerated eradication regimen for H. pylori demonstrating resistance to both clarithromycin and metronidazole, and for patients who are allergic to penicillin.

[Two cases of colorectal cancer presenting with periodic, transient elevation of serum iron due to hemolysis after chemotherapy including 5-FU].

The serum iron level reportedly shows transient elevation after chemotherapy in some cases. However, the cause of this phenomenon has not been clearly described. We report two cases of colorectal cancer whose serum iron level demonstrated recurrent elevation after chemotherapy. Both were advanced colorectal cancer cases with liver metastases, so we started chemotherapy with modified FOLFOX6+bevacizumab. After several courses, we changed the regimen to simplified LV5FU2+ bevacizumab in both cases. The serum iron level showed transient, periodical elevation irrespective of the therapeutic regimen. Additionally, indirect bilirubin also showed transient elevation, which was completely synchronized with the fluctuation of the serum iron level. These observations suggest that hemolysis is the main cause of periodic, transient elevation of serum iron level after chemotherapy including 5-FU.

Long-term follow-up evaluation for more than 10 years after endoscopic treatment for postoperative bile duct strictures.

BACKGROUND: Endoscopic stent therapy is routinely used to treat postoperative bile duct strictures. However, no studies have detailed long-term follow-up evaluation for more than 10 years. METHODS: This study enrolled 22 consecutive patients with a diagnosis of postoperative bile duct strictures from 1987 to 2006. Cases involving digestive tract reconstruction were excluded. Dilation was performed after passage of a guidewire through the stricture followed by temporary stent placement. The final objective was to achieve stent free status after sufficient dilation. The stent was removed when the cholangiogram showed apparent resolution of the stricture. If stent removal was not possible within 12 months, the authors proposed a surgical treatment option. RESULTS: Initial therapy was performed for 21 patients (21/ 22, 95%). The remaining patient had complete occlusion, which required surgical repair. For 3 of the 21 cases, guidewire passage through the narrow stricture under fluorographic guidance alone was impossible. However, visualization by peroral cholangioscope enabled passage of the guidewire in all three cases. In two cases, the stricture persisted longer than 12 months, rendering stent removal impossible. Therefore, stent removal within 12 months was achieved in 90% of the cases (19/21). Two patients requested prolonged stenting in lieu of the authors' proposal to repeat the surgery. This resulted in sufficient dilation after an additional 6 months. Consequently, a total of 21 patients were enrolled for long-term follow-up evaluation. The posttreatment follow-up period was 121 + or - 64 months (range, 31-254 months; median, 120 months). Three patients died of causes unrelated to hepatobiliary disease. The remaining patients were successfully followed up until this writing. The overall long-term success rate was 95% (20/21). No hepatobiliary malignancies developed within the follow-up period. CONCLUSIONS: Endoscopic stent therapy is available for postoperative bile duct strictures. Long-term prognosis for more than 10 years is excellent. Repeat surgical interventions may be unavoidable in some cases, but endoscopic treatment should be proposed as the first-line treatment.

Transarterial chemotherapy alone versus transarterial chemoembolization for hepatocellular carcinoma: a randomized phase III trial.

BACKGROUND/AIMS: Transcatheter arterial chemoembolization (TACE) is a combination of transarterial infusion chemotherapy (TAI) and embolization, and has been widely used to treat patients with hepatocellular carcinoma (HCC). However, since the impact of adding embolization on the survival of patients treated with TAI had never been evaluated in a phase III study, we conducted a multi-center, open-label trial comparing TACE and TAI to assess the effect of adding embolization on survival. METHODS: Patients with newly diagnosed unresectable HCC were randomly assigned to either a TACE group or a TAI group. Zinostatin stimalamer was injected into the hepatic artery, together with gelatin sponge in the TACE group and without gelatin sponge in the TAI group. Treatment was repeated when follow-up computed tomography showed the appearance of new lesions in the liver or re-growth of previously treated tumors. RESULTS: Seventy-nine patients were assigned to the TACE group, and 82 were assigned to the TAI group. The two groups were comparable with respect to their baseline characteristics. At the time of the analysis, 51 patients in the TACE group and 58 in the TAI group had died. The median overall survival time was 646 days in the TACE group and 679days in the TAI group (p=0.383). CONCLUSIONS: The results of this study suggest that treatment intensification by adding embolization did not increase survival over TAI with zinostatin stimalamer alone in patients with HCC.

Protein profiling in pancreatic juice for detection of intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND/AIMS: Malignant intraductal papillary mucinous neoplasm of the pancreas (IPMN) has a very poor prognosis, and there is no useful biomarker for an early diagnosis at present. A biomarker is expected to allow an early diagnosis of IPMNs and consequently lead to an improvement of the patients' prognosis. Recent advances in proteomic analysis are remarkable; therefore we explored novel biomarkers for IPMN using Surface-Enhanced Laser Desorption and Ionization (SELDI) Mass Spectrometry. METHODOLOGY: We collected pancreatic juice samples from 33 patients with IPMNs, 54 patients with pancreatic ductal carcinoma, and 31 with chronic pancreatitis. We analyzed the pancreatic juice samples using a SELDI ProteinChip system (Ciphergen Biosystems, Fremont, CA). RESULTS: We identified a 6240-Da peak whose expression in pancreatic juice from patients with IPMNs was significantly higher compared with that in other pancreatic diseases (P<0.01). This 6240-Da protein was partially purified and was identified as pancreatic secretory trypsin inhibitor (PSTI) by amino acid sequencing. The pancreatic juice PSTI levels, as measured by radioimmunoassay, were significantly higher in the IPMN group than in the other groups (P<0.001). When the diagnostic cutoff value of PSTI in pancreatic juice was set at 25000 ng/mL, the positive predictive value, negative predictive value, sensitivity, and specificity were respectively 89%, 83%, 48%, and 98%. CONCLUSIONS: PSTI levels of pancreatic juice in patients with IPMN were significantly higher than those in patients with other pancreatic diseases. The PSTI level in pancreatic juice may be useful for the diagnosis of IPMN.

Pancreatic intraepithelial neoplasms in the normal appearing pancreas: on their precise relationship with age.

BACKGROUND/AIMS: Pancreatic intraepithelial neoplasms (PanINs) have been considered as age-related lesions as well as precancerous lesions. However, we do not know their true relationship with age because the former studies did not deal with all age groups. Moreover, the numbers of the patients in each age group were not the same. This study was done to examine the "true relationship" between PanINs and age. METHODOLOGY: Ninety autopsied normal appearing pancreases from all age groups (from under 10s to 80s) were examined. Each group had 10 patients. The relationship between PanINs and age was studied. The sexual difference was also studied. RESULTS: Both the incidence of PanIN positive patients and the number of PanINs increased with age from under 10s to 40s, but they did not correlate with patient age after 40s. The number of PanIN positive patients in the under 10s, 40s and 80s were 2, 10 and 9, respectively. The number of PanINs in the under 10s, 40s and 80s were 11, 127 and 130, respectively. A sexual difference was not seen. CONCLUSIONS: The relationship between PanINs and age showed a biphasic correlation pattern before and after 40s. The presence of PanIN positive patients in their first year of age was also a new finding.

Clinical utility of intraductal US to decrease early recurrence rate of common bile duct stones after endoscopic papillotomy.

BACKGROUND AND AIM: The aim of the present study was to determine whether additional intraductal ultrasound (IDUS) to confirm complete stone clearance decreases the recurrence rate of common bile duct stones for a 3-year period after endoscopic papillotomy (EPT). METHODS: IDUS was carried out with a thin-caliber ultrasonic probe (diameter 2.0 mm, frequency 20 MHz) via transpapillary route after stone extraction. If IDUS showed evidence of residual stones and/or sludge, endoscopic management was performed until IDUS examination was negative. A prospective study was conducted on 59 consecutive patients undergoing additional IDUS after stone extraction between January 1996 and May 2003 (IDUS group). The recurrence rate of common bile duct stones was compared with a historical control group (August 1988 to December 1995) consisting of cases that did not undergo IDUS (non-IDUS group). Potential risk factors for recurrence of common bile duct stones were assessed by univariate and multivariate analysis on logistic regression. RESULTS: In 14 of 59 patients (23.7%), IDUS detected small residual stones not seen on cholangiography. The recurrence rate was 13.2% (17 of 129 patients) in the non-IDUS group and 3.4% (two of 59 patients) in the IDUS group (P < 0.05). Multivariate analysis subsequently identified non-IDUS status as an independent risk factor for recurrence (odds ratio 5.12, 95% CI 1.11-23.52, P = 0.036). CONCLUSIONS: Additional IDUS to confirm complete stone clearance after EPT decreases the early recurrence rate of common bile duct stones.

Hepatoblast-like cells enriched from mouse embryonic stem cells in medium without glucose, pyruvate, arginine, and tyrosine.

In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBPbeta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells.

Prevalence of diabetes mellitus and insulin resistance in patients with chronic hepatitis C: comparison with hepatitis B virus-infected and hepatitis C virus-cleared patients.

BACKGROUND/AIMS: Our aim was to evaluate the relationship between hepatitis C virus (HCV) infection and development of diabetes mellitus (DM) or insulin resistance (IR) in comparison with hepatitis B virus (HBV) infection and eradication of HCV infection by interferon treatment. METHODS: This study consisted of 952 outpatients, including 544 HCV-infected (HCV+chronic), 286 HBV-infected (HBV+chronic) and 122 patients whose HCV was cleared by interferon treatment (HCV+cleared) (diabetes study). Among 849 without overt DM, IR was assessed in 423 patients, including 232 HCV-infected (HCV+chronic), 135 HBV-infected (HBV+chronic) and 56 HCV-eradicated patients (HCV+cleared) (IR substudy). RESULTS: The prevalence of DM in the HBV+chronic, HCV+chronic and HCV+cleared groups was 6.3, 13.6 and 9.0%, respectively (HBV+chronic vs HCV+chronic, P<0.005), in the diabetes study, and the prevalence of IR in the HCV+chronic group (54.3%) was also higher than that in the HBV+chronic (36.3%) (P<0.005) and HCV+cleared groups (35.7%) (P<0.05) in the IR substudy. However, HCV infection was not shown to be independently associated with DM development [odds ratio (OR) 1.669; P=0.0936] and with IR (OR 1.531; P=0.2154) by multivariate analysis in comparison with HBV infection as control. CONCLUSIONS: HCV-infected patients showed a higher prevalence of DM and IR than those with HBV infection. However, in Japan, other confounding factors appeared to be more important risk factors for the development of disturbance in glucose metabolism.

Evaluation of normal bile duct and cholangitis by intraductal ultrasonography.

BACKGROUND: The intraductal ultrasonographic findings of normal bile duct and cholangitis were examined. METHODS: We studied 9 autopsy cases and evaluated the clinical records of 76 patients who had been subjected to papillotomy for bile duct stones removal retrospectively. RESULT: In vitro study: Under a low pressure, the bile duct wall was thick, and the inner surface and outer contour were irregular. The wall became thinner, and the inner surface and outer contour became smooth as the pressure was increased. The thickness was 0.68 +/- 0.12 mm (mean +/- SD) all along the duct at all pressure, and 0.55 +/- 0.12 mm at a pressure above 15 cm H(2)O. The internal echo was homogenous regardless of the internal pressure used. In vivo study: We could evaluate in 70 patients (92.1%). The wall was 1.30 +/- 0.77 mm thick. There was no relationship between the severity of cholangitis and the wall thickness. The irregular inner surface, heterogeneous internal echo, and irregular outer contour correlated with the severity of cholangitis. CONCLUSION: The normal bile duct wall was between 0.31 and 0.79 mm thick, the inner and outer surfaces were smooth, and internal echo was homogenous. An irregular inner surface, heterogeneous internal echo and an irregular outer contour were important findings of severe cholangitis.

Body mass index in Japanese patients with autoimmune liver disease: overweight patients with primary biliary cirrhosis tend to be asymptomatic.

BACKGROUND/AIMS: The aim of this study was to investigate the effects of being overweight on autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) patients. METHODOLOGY/RESULTS: 44 AIH and 95 PBC patients were enrolled in this study. Body weight and body mass index (BMI) of AIH (57.6 +/- 10.4 kg and 23.8 +/- 2.9 kgm(-2), respectively) were higher than those of PBC (51.6 +/- 7.0 kg and 22.0 +/- 2.6 kgm(-2), respectively) (P < 0.001). The prevalence of overweight patients in AIH was also higher than those in PBC (P < 0.005). Being overweight and having 25 < or = BMI < 30 did not affect the progression of hepatic fibrosis in AIH and PBC. In comparison with the non-overweight with PBC, overweight patients with PBC tended not to be symptomatic, such as having itching or fatigue (P = 0.027). CONCLUSIONS: Clinicians should be aware that not only non-alcoholic fatty liver disease but also PBC patients might be included among the overweight hepatic disease patients with unknown etiology.

A multi-center retrospective analysis of survival benefits of chemotherapy for unresectable biliary tract cancer.

BACKGROUND: This study examined the effect of five systemic chemotherapy regimens on survival in patients with unresectable biliary tract cancer (BTC) as compared with the best supportive care (BSC). METHODS: This study retrospectively reviewed data from 413 consecutive patients with BTC who were seen at any of nine central hospitals in Japan between April 2000 and March 2003. Patients were eligible if they had intra- or extrahepatic cholangiocarcinoma or gallbladder cancer with no prior chemotherapy. Hazard ratios of treatment regimens were estimated using the Cox proportional hazard model and the propensity score method. RESULTS: Three-hundred and four patients were enrolled: 125 (41.1%) received BSC and 179 (58.9%) took chemotherapy. Of those who received chemotherapy, 58 (19.1%) took gemcitabine (GEM), 45 (14.5%) took a cisplatin (CDDP)-based regimen, 30 (9.9%) took a 5-fluorouracil (5-FU)-based regimen, 27 (8.9%) took 5-FU + doxorubicin + mitomycin (FAM) and 20 (6.6%) took S-1. The response rate was 8.4% (n = 15). The CDDP-based regimen was associated with a high frequency of toxicity symptoms. The adjusted hazard ratio for GEM in the Cox regression was 0.53 (95% CI 0.34-0.82) and the hazard ratio for the CDDP-based regimen was 0.49 (95% CI 0.36-0.99). CONCLUSION: Chemotherapy with GEM may benefit patients with BTC.

Enhanced self-renewal capability in hepatic stem/progenitor cells drives cancer initiation.

BACKGROUND & AIMS: Transformed hematopoietic stem/progenitor cells with an enhanced or acquired self-renewal capability function as leukemic stem cells. In a variety of solid cancers, stem/progenitor cells could be also targets of carcinogenesis. However, it remains unclear whether disruption of stem cell function directly contributes to cancer initiation. We sought to elucidate the mechanisms of self-renewal in hepatic stem/progenitor cells and the relation between stem cell function and hepatocarcinogenesis. METHODS: Functional analyses of polycomb-group protein Bmi1 and Wnt/beta-catenin, the molecules that are responsible for the self-renewal capability of many types of stem cells, were conducted in c-Kit(-)CD29(+)CD49f(+/low)CD45(-)Ter-119(-) hepatic stem/progenitor cells using retrovirus- or lentivirus-mediated gene transfer. The tumorigenicity of these cells transduced with the indicated retroviruses was also assessed by transplantation into nonobese diabetic/severe combined immunodeficient mice. RESULTS: Forced expression of Bmi1 and constitutively active beta-catenin mutant similarly promoted the self-renewal of hepatic stem/progenitor cells. The transplantation of Bmi1- or beta-catenin-transduced cells clonally expanded from single hepatic stem/progenitor cells produced tumors, which exhibited the histologic features of combined hepatocellular and cholangiocarcinoma. CONCLUSIONS: These observations imply that the dysregulated self-renewal of hepatic stem/progenitor cells serves as an early event in hepatocarcinogenesis, and they highlight the important roles of Bmi1 and the Wnt/beta-catenin pathway in regulating the self-renewal of normal or cancer stem cells in liver.

Inhibition of subgenomic hepatitis C virus RNA replication in HeLa cells.

BACKGROUND/AIMS: Antiviral therapy such as combination interferon and ribavirin can eradicate hepatitis C virus (HCV) RNA by up to 40-50%. However, many patients still remain non-responders to this treatment for various reasons. The aim of this study was to evaluate the effect of interferon or ribavirin treatment on subgenomic HCV RNA replication in 'non-hepatic' HeLa cells. METHODOLOGY: Huh-7 or HeLa cells harboring HCV replicon were constructed by using cellular RNA of Huh-7 harboring HCV replicon RNAs, named as C13-3 cells. We also tested whether interferon or ribavirin can suppress HCV RNA in HeLa cells. RESULTS: Huh-7 or HeLa cells harboring HCV replicon RNAs were constructed by using cellular RNA of C13-3 cells than using in vitro-transcribed RNA. Ribavirin at 1 microg/mL or 10 microg/mL did not suppress colony formation in HeLa cells, but at 100 microg/mL suppression was observed. Interferon-alpha 2b suppressed HCV replication even at 1 U/mL. CONCLUSIONS: HeLa cells harboring HCV replicon RNAs also might be useful for the development of antiviral drugs.

Insulin-like growth factor (IGF)-II regulates CCAAT/enhancer binding protein alpha expression via phosphatidyl-inositol 3 kinase in human hepatoblastoma cell lines.

To reveal growth factor and its signal pathway to CCAAT/enhancer binding protein alpha (C/EBPalpha) in hepatocyte differentiation, we used Huh-6 and HepG2, human hepatoblastoma (HBL) cell lines that maintain the expression of genes in hepatoblasts and remain at that stage of differentiation. Insulin-like growth factor (IGF)-II, hepatocyte growth factor (HGF), and dexamethasone (Dex) stimulated HBL cells for Northern blot analysis. Bromodeoxyuridine (BrdU) up-take assay and Western blot analysis on albumin was performed to unveil proliferation and differentiation activity of IGF-II. C/EBPalpha and phosphorylation of Akt were analyzed by Western blot analysis. LY294002 and wortmannin, specific inhibitors of PI3 kinase, and PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase, were used to examine the signaling pathway of C/EBPalpha upregulated by IGF-II. Luciferase assay was performed to study the promoter activity of C/EBPalpha. Actinomycin D was used to analyze half-life of C/EBPalpha mRNA. IGF-II up-regualted C/EBPalpha by Northern blot and Western blot while HGF and Dex did not by Northern blot. IGF-II promoted proliferation and differentiation by BrdU up-take assay and Western blot analysis on albumin. Akt phosphorylated by IGF-II, suggested that phosphatidyl-inositol (PI) 3 kinase mediated the signaling pathway of IGF-II. LY294002 and wortmannin suppressed expression of C/EBPalpha. IGF-II activated the promoter activity and prolonged half-life of mRNA, suggesting that IGF-II activated promoter and stabilized mRNA. LY294002 and wortmannin suppressed the promoter activity of C/EBPalpha while PD98059 did not, suggesting that activation of the promoter was mediated by PI3 kinase.