Hiromitsu Saisho

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell–like properties†‡

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti‐apoptotic properties compared with those of non‐SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 × 10 3 SP cells were sufficient for tumor formation, whereas an injection of 1 × 10 6 non‐SP cells did not initiate tumors. Re‐analysis of SP cell–derived tumors showed that SP cells generated both SP and non‐SP cells and tumor‐initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non‐SP cells, and several so‐called “stemness genes” were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy. (HEPATOLOGY 2006;44:240–251.)

Natural History of Minute Hepatocellular Carcinoma Smaller Than Three Centimeters Complicating Cirrhosis

Twenty-two patients with cirrhosis and minute hepatocellular carcinoma less than 3 cm in diameter were followed for periods of 6-37 mo without specific treatment. The survival curve drawn by the Kaplan-Meier method showed a 1-yr survival of 90.7%, a 2-yr survival of 55.0%, and a 3-yr survival of 12.8%. The ultrasonic patterns of these masses in the liver were correlated with the size and showed a tendency to change from a low echo pattern to a low periphery and, finally, to a massive pattern. The growth speed calculated from the doubling time for tumor volume varied considerably from case to case with an average of 6.5 +/- 5.7 mo; it also changed in some cases during the observation period. Serum alpha-fetoprotein levels were generally low, rarely assisted in diagnosis, but tended to increase when the mass attained a diameter of greater than 3 cm; sudden acceleration in the rate of increase in alpha-fetoprotein level often coincided with a change of ultrasonic pattern to the massive one.

Diagnosis and Clinical Features of Small Hepatocellular Carcinoma With Emphasis on the Utility of Real-Time Ultrasonography: A Study in 51 Patients

The clinical features and usefulness of various methods for diagnosis of small hepatocellular carcinoma, no greater than 5 cm in diameter, were studied in 51 patients. The diagnosis was verified in 28 patients by hepatic resection and in 4 patients at autopsy. The tumor was less than 3 cm in diameter in 23 patients. In 37 patients (72.5%), the tumor was first discovered by real-time linear scan ultrasonography, generally during a routine periodic follow-up examination of patients with chronic liver disease, mostly cirrhosis. Clinically, 62.7% of the patients were asymptomatic, and symptoms in the remainder were not suggestive of carcinoma. Most patients showed only mildly abnormal liver function tests that did not suggest the diagnosis. Serum alpha-fetoprotein level was normal in 25.5%, and it was elevated above 200 ng/ml in only 33.3%. Among the various imaging modalities, ultrasonography had the highest detection rate (92.2%) for these small hepatocellular carcinomas compared with computed tomography (73.2%), scintigraphy (50.0%), and angiography (86.0%). False-positive lesions, however, were also found frequently by ultrasonography, requiring ultrasonography-guided biopsy for differential diagnosis in some of the patients. Routine examination at regular intervals of patients with chronic liver disease using a combination of real-time ultrasonography and alpha-fetoprotein measurement is currently the most effective approach to detecting small hepatocellular carcinoma.

Abdominal wall fat index, estimated by ultrasonography, for assessment of the ratio of visceral fat to subcutaneous fat in the abdomen

PURPOSE To establish a new index of regional fat distribution using ultrasonography for assessment of the ratio of the visceral fat area (V) to the subcutaneous fat area (S) (V/S ratio). SUBJECTS AND METHODS The subjects examined were 62 patients (23 males and 39 females); 51 patients had hyperlipidemia and 11 patients had glucose intolerance. The mean body mass indices ranged from 20.3 to 42.9. The mean age of the patients was 44 +/- 13 years. The thicknesses of the preperitoneal fat layer (P) and subcutaneous fat layer (S) in the abdomen were measured by ultrasonography and the P/S ratio was calculated. The V/S ratio was obtained with radiographic computed tomography. RESULTS Of the various P/S ratios examined, the ratio of the maximum thickness of preperitoneal fat to the minimum thickness of subcutaneous fat was most closely correlated with the V/S ratio (r = 0.746, p < 0.0001). This ratio was termed the abdominal wall fat index (AFI). AFI was positively correlated with serum triglyceride levels and negatively correlated with high-density lipoprotein cholesterol (r = -0.312, p < 0.05), whereas the V/S ratio was correlated with triglyceride levels. AFI was positively correlated with basal insulin levels in both men and women. CONCLUSION These results suggest that AFI measured by ultrasonography may be a new indicator of visceral fat deposition, and may reflect metabolic disorders such as lipid metabolism and glucose metabolism disorders.

Association of gallbladder carcinoma and anomalous pancreaticobiliary ductal union

A total of 96 patients with gallbladder carcinoma in whom direct cholangiography clearly opacified the pancreaticobiliary ductal union and the common channel, and 65 patients with an anomalous union of these two duct systems at a distance greater than 15 mm from the papilla of Vater (normally less than 4.6 +/- 2.2 mm, mean +/- SD) were studied. It was found that this anomalous ductal union occurred in 16.7% of the patients with gallbladder carcinoma in comparison with an incidence of 2.8% among 641 consecutive patients with various hepatobiliary and pancreatic diseases studied by endoscopic retrograde cholangiopancreatography who did not have gallbladder carcinoma. It was also found that gallbladder carcinoma occurred in 24.6% of the 65 cases of anomalous ductal union in comparison with a 1.9% incidence of this cancer among 635 consecutive patients similarly studied and found to have normal ductal union (p less than 0.001). Thus, a close etiologic association was suggested between this anomaly in the terminal segment of the biliary tract and gallbladder carcinoma. Of the 65 patients with anomalous ductal union, 50 had the so-called congenital cystic dilatation of the common bile duct and 15 did not. Five of the 50 (10%) and 11 of the 15 (73.3%) had gallbladder carcinoma (p less than 0.01), and this carcinoma seems to be related to anomalous ductal union rather than to cystic dilatation of the common bile duct. As a tumorigenic factor in this anomaly, regurgitation of pancreatic juice has been stressed.

Quantitative Analysis of Bowel Gas Using Plain Abdominal Radiograph in Patients With Irritable Bowel Syndrome

OBJECTIVE:Ideally, the diagnosis of irritable bowel syndrome (IBS) would be achieved using a minimal number of procedures. It is presumed that bowel gas is related to IBS, and it is easily visualized by plain abdominal radiograph. In the present study, to clarify the relationship between IBS and the quantity of bowel gas, the measured bowel gas volume using plain abdominal radiographs was compared with the pathology of IBS.METHODS:Plain abdominal radiographs were digitized and transmitted to a computer (computed radiography) in 30 IBS patients and 30 normal controls. The quantity of bowel gas, determined as the pixel value on images and standardized by physique, was defined as the gas volume score (GVS). Using the mean ± 2SD of GVS in the control group as the normal score, IBS patients were divided into three groups: high, normal, and low. To examine the sequential reproducibility of a similar quantity of bowel gas, a second plain abdominal radiography was performed about 2 months later, and the GVS were compared. The colonic transit time was determined using radiopaque markers.RESULTS:There was a strong correlation between the quantities of bowel gas measured by two independent gastroenterologists. The mean GVS of IBS patients was significantly higher than that in the control group (p < 0.001). The sequential reproducibility was recognized in all 10 IBS patients. There was no significance between colonic transit time and GVS, nor between symptoms and GVS.CONCLUSIONS:Abdominal gas was analyzed objectively by using GVS, and GVS was considered to represent a useful tool for the diagnosis of IBS.

Risk factors for recurrent bile duct stones after endoscopic papillotomy

Background: The long term outcome of endoscopic papillotomy (EPT) is not well known. The aims of this study were to clarify the clinical course of post-EPT patients and to detect predictors for bile duct stone recurrence. Methods: A total of 1042 consecutive patients who underwent EPT for bile duct stones from December 1975 to September 1998 were prospectively followed up. Patients were divided into four groups according to gall bladder (GB) status: “acalculous GB” group, “calculous GB” group, “cholecystectomy” group, and “prior cholecystectomy” group. Reliable follow up information was obtained for 983 (94.3%) of the 1042 patients. The following factors were considered in the evaluation of predisposing risk factors for recurrence of bile duct stones: age, sex, gall bladder status, periampullary diverticulum, number of bile duct stones, diameter of bile duct stones, diameter of bile duct, lithotripsy, precutting, pneumobilia, and early complications. Results: Recurrence occurred in 111 patients. The “acalculous GB” group was less prone to recurrence than the “prior cholecystectomy” group and the “calculous GB” group. The relative risks (RR) for the latter two compared with the former group were 2.26 (95% confidence interval (CI) 1.24–4.14; p=0.0078) and 2.16 (95% CI 1.21–3.87; p=0.0093), respectively. Other prognostic factors were lithotripsy (RR 2.37; 95% CI 1.47–3.81; p=0.0004) and pneumobilia (RR 1.57; 95% CI 1.01–2.43; p=0.044). Conclusions: Gall bladder status, lithotripsy, and pneumobilia were significantly related to bile duct stone recurrence after EPT.

Influence of steroid therapy on the course of diabetes mellitus in patients with autoimmune pancreatitis: findings from a nationwide survey in Japan.

Objective: The aim of this study was to estimate the number of autoimmune pancreatitis (AIP) patients in Japan and evaluate the influence of steroid therapy on the course of diabetes mellitus (DM) in AIP. Methods: A total of 2972 departments were randomly selected with hospital stratification throughout Japan. We sent a questionnaire asking the selected departments to report the number of patients who had visited their hospital in 2002 and who fulfilled the diagnostic criteria for AIP set by the Japan Pancreas Society. We then sent a second questionnaire asking for the details of the clinical course of DM, if any, and the treatments used for AIP. Results: The annual number of AIP in Japan was estimated as approximately 900 (95% confidence interval 670-1100) or 0.71 per 100,000 individuals in the Japanese population. In the second survey, 66.5% of the 167 patients of AIP were reported as being complicated with DM. Among the AIP patients with DM (n = 93), 52% of the patients started to show DM simultaneously with the onset of AIP, and 33% of the patients had DM before the onset of AIP. Following steroid therapy, 55% and 36% of these patient groups showed improvement of DM control, respectively. On the other hand, less than 20% of patients showed newly developed DM or showed exacerbation of DM control after steroid therapy. The older the patients were, the higher were the rates of new development or exacerbation of DM. Conclusions: These findings indicated that steroid therapy has a beneficial effect on the clinical courses of DM in approximately half of AIP patients. However, it also has negative effect on glucose tolerance in some patients, particularly older patients, and thus, careful observation for involvement of DM should be required in AIP patients treated with steroids.

Immune Complex and Fc Receptor-Mediated Augmentation of Antigen Presentation for in Vivo Th Cell Responses

It has recently been established that FcRs are involved in the triggering of type II and III inflammatory responses. Although FcR is not believed to be involved in the regulation of T cell function, the in vivo contribution of FcRs to T cell function still remains unclear. We analyzed in vivo responses of delayed-type hypersensitivity and proliferation of CD4+ T cells to Ags in FcRγ−/− mice lacking the expression and function of FcγRI, FcγRIII, and FcεRI. We found that the delayed-type hypersensitivity response in FcRγ−/− mice is significantly decreased compared with that in wild-type mice. Moreover, the secondary responses of proliferation and cytokine production as well as the Ab formation by CD4+ T cells from FcRγ−/− mice to Ag and normal APCs were also reduced. In contrast, in vitro primary T cell proliferative responses upon stimulation with anti-TCR Ab or MLR as well as in vivo primary response against staphylococcus enterotoxin B administration were not different between T cells from FcRγ−/− and wild-type mice. In addition, the Ag presentation function of APCs from unimmunized FcRγ−/− mice was normal. On the other hand, Ab-deficient mice also revealed impaired T cell responses. These results demonstrate that the defective T cell responses in FcRγ−/− mice were due to impaired Ag presentation during in vivo priming not to a defect in T cells. Therefore, they suggest that the FcRs on APCs mediate efficient priming of Th cell responses in vivo in an immune complex-dependent manner.

Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells.

Objective: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. Methods: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody. Results: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, α2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, α7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes. Conclusions: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.