Hiroyuki Marusawa

MicroRNA-33 encoded by an intron of sterol regulatory element-binding protein 2 (Srebp2) regulates HDL in vivo

Sterol regulatory element-binding protein 2 (SREBP-2) transcription factor has been identified as a key protein in cholesterol metabolism through the transactivation of the LDL receptor and cholesterol biosynthesis genes. Here, we generated mice lacking microRNA (miR)-33, encoded by an intron of the Srebp2, and showed that miR-33 repressed the expression of ATP-binding cassette transporter A1 (ABCA1) protein, a key regulator of HDL synthesis by mediating cholesterol efflux from cells to apolipoprotein A (apoA)-I. In fact, peritoneal macrophages derived from miR-33–deficient mice showed a marked increase in ABCA1 levels and higher apoA-I–dependent cholesterol efflux than those from WT mice. ABCA1 protein levels in liver were also higher in miR-33–deficient mice than in WT mice. Moreover, miR-33–deficient mice had significantly higher serum HDL cholesterol levels than WT mice. These data establish a critical role for miR-33 in the regulation of ABCA1 expression and HDL biogenesis in vivo.

Transmission of hepatitis B virus from hepatitis B core antibody- positive donors in living related liver transplants

BACKGROUND In order to clarify the risk of hepatitis B virus (HBV) transmission from hepatitis B core antibody-positive (HBcAb(+)) donors and to evolve a new strategy to counter such a risk, we undertook a retrospective (1990-1995) and prospective (1995-1996) analysis of our experience with living related liver transplantation involving HBcAb(+) donors. METHODS Between June 15, 1990, and June 30, 1995, HBcAb(+) individuals were not excluded as donor candidates at our institutions. For 171 liver transplants, 16 donors were HBcAb(+). Between July 1, 1995, and June 30, 1996, HBcAb(+) individuals were generally excluded as donor candidates; however, three recipients were given liver grafts from HBcAb(+) donors because other donor candidates presented even higher risks. In the latter period, recipients with transplants from HBcAb(+) donors underwent prophylactic passive immunization with hyperimmune hepatitis B immunoglobulin (HBIG). The serum of 10 HBcAb(+) donors was examined by nested polymerase chain reaction for the presence of HBV-DNA, but it was not detected in any of them. However, the same examination of the liver tissue of five such donors yielded positive results in all cases. RESULTS In the first 5-year period, out of 16 recipients with HBcAb(+) donors, 15 became hepatitis B surface antigen-positive after transplant. The three recipients with HBcAb(+) donors during the second 1-year period, who were treated by prophylactic passive immunization with HBIG, remained hepatitis B surface antigen-negative and negative for serum HBV-DNA after transplant. CONCLUSIONS HBV exists in the liver of healthy HBcAb(+) individuals, but not in the blood. Therefore, HBV is thought to be transmitted to recipients by liver grafts from the HBcAb(+) donors at a significantly high rate. The prevention of viral activation and clinical disease development by means of passive immunization with HBIG seems promising, although the follow-up period in our study may be too short for any definitive conclusions.

STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells

Signal transduction and activator of transcription 3(STAT3) signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. There are few reports, however, on the role of STAT3 signaling in gastric cancer. The aim of the present study was to clarify the role of STAT3 signaling in apoptosis and cellular proliferation in gastric cancer. Here we reported that STAT3 was constitutively activated in various human gastric cancer cells and its inhibition by ectopic dominant-negative STAT3 or Janus kinase inhibitor, tyrphostin AG490, induced apoptosis. Furthermore, STAT3 inhibition markedly decreased survivin expression, and forced expression of survivin rescued AGS cells from apoptosis induced by STAT3 inhibition. Although some reports demonstrated that the PI3K/Akt pathway regulates survivin expression, inhibition of the PI3K/Akt pathway did not affect survivin expression in AGS and MKN1 cells. Finally, activated form of STAT3, Tyr-705 phospho-stat3, was found in the nucleus of cancer cells in 11 of 40 (27.5%) human gastric cancer specimens. These findings suggest that constitutively activated STAT3 signaling supports gastric cancer cell survival in association with survivin expression.

Inflammation-Associated Cancer Development in Digestive Organs: Mechanisms and Roles for Genetic and Epigenetic Modulation

Chronic inflammation, regardless of infectious agents, plays important roles in the development of various cancers, particularly in digestive organs, including Helicobacter pylori-associated gastric cancer, hepatitis C virus-positive hepatocellular carcinoma, and colitis-associated colon cancers. Cancer development is characterized by stepwise accumulation of genetic and epigenetic alterations of various proto-oncogenes and tumor-suppressor genes. During chronic inflammation, infectious agents such as H pylori and hepatitis C virus as well as intrinsic mediators of inflammatory responses, including proinflammatory cytokines and reactive oxygen and nitrogen species, can induce genetic and epigenetic changes, including point mutations, deletions, duplications, recombinations, and methylation of various tumor-related genes through various mechanisms. Furthermore, inflammation also modulates the expressions of microRNAs that influence the production of several tumor-related messenger RNAs or proteins. These molecular events induced by chronic inflammation work in concert to alter important pathways involved in normal cellular function, and hence accelerate inflammation-associated cancer development. Among these, recent studies highlighted an important role of activation-induced cytidine deaminase, a nucleotide-editing enzyme essential for somatic hypermutation and class-switch recombination of the immunoglobulin gene, as a genomic modulator in inflammation-associated cancer development.

Expression of activation-induced cytidine deaminase in human hepatocytes via NF-κB signaling

Activation-induced cytidine deaminase (AID) is involved in somatic DNA alterations of the immunoglobulin gene for amplification of immune diversity. The fact that constitutive expression of AID in mice causes tumors in various organs, including lymphoid tissues and lungs, suggests the important role of the aberrant editing activity of AID on various tumor-related genes for carcinogenesis. AID expression, however, is restricted to activated B cells under physiological conditions. We demonstrate here that ectopic AID expression is induced in response to tumor necrosis factor-α stimulation in cultured human hepatocytes. The proinflammatory cytokine-mediated expression of AID is achieved by IκB kinase-dependent nuclear factor (NF)-κB signaling pathways. Hepatitis C virus, one of the leading causes of hepatocellular carcinoma (HCC), enhanced AID expression via NF-κB activation through expression of viral core protein. The aberrant expression of AID in hepatoma-derived cells resulted in accumulation of genetic alterations in the c-myc and pim1 genes, suggesting that inappropriate expression of AID acts as a DNA mutator that enhances the genetic susceptibility to mutagenesis in human hepatocytes. Our current findings indicate that the inappropriate expression of AID is induced by proinflammatory cytokine stimulation and may provide the link between hepatic inflammation and the development of HCC.

Antibody to hepatitis B core antigen and risk for hepatitis C-related hepatocellular carcinoma : A prospective study

Context Retrospective studies suggest that exposure to hepatitis B virus (HBV) may contribute to the development of hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)positive patients with cirrhosis. Contribution These investigators prospectively studied patients with chronic HCV infection and evidence of occult HBV infection (negative results for hepatitis B surface antigen and HBV DNA but positive results for antibody to hepatitis B core antigen [anti-HBc] on serologic testing). Patients with HCV-related cirrhosis and positive results for anti-HBc on serologic testing were at high risk for HCC. Anti-HBc positivity was associated with increased risk for HCC, even in patients with a virologic response to interferon therapy. Caution The effect of alcohol cannot be fully assessed because of the small number of study patients who drank moderately. Implication Anti-HBc serologic testing may be a valuable indicator of special risk for HCC in patients with HCV-related cirrhosis. The Editors Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide, and its incidence has been increasing (1, 2). In Japan, an endemic area for hepatitis B virus (HBV) and hepatitis C virus (HCV), it is well known that more than 75% of cases of HCC are attributable to HCV-related chronic liver disease, and nearly 15% are attributable to HBV-related liver disease (3). Several reports have focused on the clinical role of HBV as a unique infection, in which HBV DNA is detectable in the liver despite the absence of serum hepatitis B surface antigen (HBsAg) (46). It is increasingly recognized that after a person is exposed to HBV, infection persists in the liver for a prolonged period (710). This unique persistent infection, known as occult (or latent) HBV infection, is characterized by HBV DNA in the liver but no HBsAg in the serum (1113). In most cases, antibody to hepatitis B core antigen (anti-HBc) is detectable, and thus anti-HBc is believed to be a surrogate marker for latent carriers (14). In fact, we recently showed that HBV infection invariably occurred through grafts from anti-HBcpositive donors in HBV-naive recipients through living-donor liver transplantation (15). In addition to our data, other reports showing frequent HBV transmission from anti-HBcpositive cadaveric donors to recipients indicate that most healthy persons who are positive for anti-HBc, even at low titers, have latent HBV infection in liver tissue (11, 1618). Indeed, we have shown that most anti-HBcpositive healthy persons have a latent episomal form of HBV infection accompanied by ongoing viral replication (19, 20). In contrast, the prevalence of latent HBV infection in anti-HBcpositive patients with HCV-related chronic liver disease, including chronic hepatitis, cirrhosis, and HCC, remains controversial (21). However, several reports have revealed that the HBV genome is frequently detectable in liver tumors in anti-HBcpositive, HBsAg-negative patients with HCV-related liver disease, which suggests that occult HBV infection may contribute to the progression of liver damage and the development of HCC in HCV-positive patients (14, 2227). In a large-scale retrospective study of the prevalence of anti-HBc among 2014 patients with chronic HCV infection, we found that nearly 50% of patients with HCV-related liver disease had anti-HBc (28). Moreover, we found a strong correlation between the prevalence of anti-HBc and the clinical progression of liver disease. The prevalence of anti-HBc was approximately 60% in patients with HCV-related HCC (28). This high prevalence of anti-HBc in HCV-positive patients, particularly those with HCC, strongly suggests that previous exposure to HBV plays an important role in the development of HCC in patients with HCV-related chronic liver disease. Therefore, we performed a prospective study to determine whether previous exposure to HBV affects the clinical course, especially in development of HCC, in patients with chronic HCV infection. Methods Patients Patients with chronic HCV infection who presented to Kyoto University, Kyoto, Japan, and 14 affiliated core hospitals from May 1995 to June 1995 were enrolled. To be eligible, patients had to have serologically confirmed HCV infection without HBsAg and HBV DNA in sera. All patients had been followed with biochemical tests, including -fetoprotein, and ultrasonography or computed tomography (CT) every 3 to 6 months before and after enrollment. We excluded patients who had elevated -fetoprotein levels or those in whom HCC had been diagnosed before enrollment. As a result, 872 patients with chronic HCV infection were enrolled. We discontinued follow-up in patients who moved from the study districts but included their clinical data until they moved. The end of follow-up was defined as the date of diagnosis of HCC, date of death, date of move from the study district, or the closing date of the study (15 May 2005). A total of 384 patients were classified into the hepatitis group or cirrhosis group on the basis of histologic findings on liver biopsy. The differential diagnosis of cirrhosis or hepatitis was made in the remaining 488 patients by using the cirrhosis discriminant score (29, 30). This score is based on 3 laboratory variables: platelet count, alanine aminotransferaseaspartate aminotransferase ratio, and prothrombin time. It has been shown to be highly sensitive in identifying cirrhosis in patients with HCV infection. In accordance with the original definition, patients with a high score (8) were classified into the cirrhosis group. Patients with ascites confirmed by ultrasonography or CT or previous variceal bleeding were given a diagnosis of cirrhosis, regardless of their score, because these findings are strong indicators of portal hypertension and most likely cirrhosis. As a result, 597 (68.5%) patients had a diagnosis of chronic hepatitis and 275 (31.5%) patients had a diagnosis of cirrhosis at the time of enrollment. The patients had regular clinical assessments, biochemical tests, and ultrasonography or CT of the liver every 3 to 6 months during the follow-up period. Patients were stratified into 3 categories according to their smoking habits: nonsmokers, light smokers who smoked fewer than 20 pack-years, and heavy smokers who smoked 20 pack-years or more. Similarly, we stratified patients into 3 categories according to their drinking habits: nondrinkers, moderate drinkers with an average ethanol intake less than 30 g/d, and heavy drinkers with an average ethanol intake greater than 30 g/d. Information on average alcohol intake was based on the patients drinking habits during the 15 years before study entry. All patients provided informed consent to participate in the study, and the study was designed in accordance with the Declaration of Helsinki (31). Serologic Studies At study entry, serum samples from each patient were tested for serologic markers of HCV. Detection of HBsAg, anti-HBc, and antibody to hepatitis B surface antigen (anti-HBs) was performed by using commercial enzyme immunoassay kits (Dainabot, Tokyo, Japan) (28). Results of the anti-HBc assays were expressed as the percentage of inhibition, and the specimen was considered to be anti-HBc positive when the percentage of inhibition was greater than 50% (19). Detection of HBV DNA was done by using DNA probe assay (32). Anti-HCV was assessed by using second-generation assays (Dainabot) (28). Serum HCV RNA levels were determined in 254 patients by using a competitive reverse transcriptionpolymerase chain reaction assay, and positivity of HCV RNA was confirmed in all patients who were examined at study entry (33). History of Interferon Therapy Of the 576 patients with chronic hepatitis, 224 had a history of interferon therapy. One hundred ninety-two patients received 5 to 10 million U of interferon- intramuscularly every day for the first 2 weeks and then 3 times weekly for the following 22 weeks. The remaining 32 patients were treated with 3 to 6 million U of interferon- intravenously every day for 8 weeks. No patient received pegylated interferon or combination therapy with ribavirin. Patients who received interferon were divided into 3 groups based on their virologic response to therapy. Patients with a sustained virologic response were defined as those with no detectable HCV RNA by qualitative assay at least 24 weeks after cessation of therapy. Patients with relapse were defined as those with disappearance of viremia at the end of treatment followed by reappearance of viremia with 24 weeks. Nonresponders included patients whose serum HCV RNA remained positive during therapy. Statistical Analysis The incidence rates for HCC are expressed as the number of HCC cases per 1000 person-years. Incidence rate ratios were calculated by dividing rates, and the exact 95% CIs for the rate ratios were calculated on the basis of a binomial distribution, which is a conditional distribution for 2 independent Poisson distributions. The Cox proportional hazards model was used to calculate the incidence rate ratios for the association between HBc seropositivity and HCC incidence. The multivariate model, with adjustment for potential risk factors, included male sex, age, alcohol intake (none, 0 to 30 g/d, and 30 g/d), smoking (none, 0 to 20 pack-years, and 20 pack-years), and history of interferon therapy (yes or no). The associated 95% Wald CIs were calculated. Analyses were done by using PC-SAS, version 8.2 (SAS Institute, Inc., Cary, North Carolina) and JMP, version 4.0 (SAS Institute, Inc.). Role of the Funding Source The Japan Society for the Promotion of Science provided funding for the study. The funding source had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Results Characteristics of Patients at Enrollment We followed 846 of the 872 enrolled patients. The remaining 26 patients were excluded from the analysis. Twenty-on

Parkin as a tumor suppressor gene for hepatocellular carcinoma

The parkin was first identified as a gene implicated in autosomal recessive juvenile Parkinsonism. Deregulation of the parkin gene, however, has been observed in various human cancers, suggesting that the parkin gene may be important in tumorigenesis. To gain insight into the physiologic role of parkin, we generated parkin−/− mice lacking exon 3 of the parkin gene. We demonstrated here that parkin−/− mice had enhanced hepatocyte proliferation and developed macroscopic hepatic tumors with the characteristics of hepatocellular carcinoma. Microarray analyses revealed that parkin deficiency caused the alteration of gene expression profiles in the liver. Among them, endogenous follistatin is commonly upregulated in both nontumorous and tumorous liver tissues of parkin-deficient mice. Parkin deficiency resulted in suppression of caspase activation and rendered hepatocytes resistant to apoptosis in a follistatin-dependent manner. These results suggested that parkin deficiency caused enhanced hepatocyte proliferation and resistance to apoptosis, resulting in hepatic tumor development, partially through the upregulation of endogenous follistatin. The finding that parkin-deficient mice are susceptible to hepatocarcinogenesis provided the first evidence showing that parkin is indeed a tumor suppressor gene.

Expression of activation-induced cytidine deaminase in human hepatocytes during hepatocarcinogenesis

Activation‐induced cytidine deaminase (AID) plays a role as a genome mutator in activated B cells, and inappropriate expression of AID has been implicated in the immunopathological phenotype of human B‐cell malignancies. Notably, we found that the transgenic mice overexpressing AID developed lung adenocarcinoma and hepatocellular carcinoma (HCC), suggesting that ectopic expression of AID can lead to tumorigenesis in epithelial tissues as well. To examine the involvement of AID in the development of human HCC, we analyzed the AID expression and its correlation with mutation frequencies of the p53 gene in liver tissues from 51 patients who underwent resection of primary HCCs. The specific expression, inducibility by cytokine stimulation and mutagenic activity of AID were investigated in cultured human hepatocytes. Only trace amounts of AID transcripts were detected in the normal liver; however, endogenous AID was significantly upregulated in both HCC and surrounding noncancerous liver tissues with underlying chronic hepatitis or liver cirrhosis (p < 0.05). Most liver tissues with underlying chronic inflammation with endogenous AID upregulation already contained multiple genetic changes in the p53 gene. In both hepatoma cell lines and cultured human primary hepatocytes, the expression of AID was substantially induced by TGF‐β stimulation. Aberrant activation of AID in hepatocytes resulted in accumulation of multiple genetic alterations in the p53 gene. Our findings suggest that the aberrant expression of AID is observed in human hepatocytes with several pathological settings, including chronic liver disease and HCC, which might enhance the genetic susceptibility to mutagenesis leading to hepatocarcinogenesis.

Activation-Induced Cytidine Deaminase Links Between Inflammation and the Development of Colitis-Associated Colorectal Cancers

BACKGROUND & AIMS Activation-induced cytidine deaminase (AID) was originally identified as an inducer of somatic hypermutations in the immunoglobulin gene. We recently revealed that ectopic AID expression serves as a link between the cellular editing machinery and high mutation frequencies, leading to human cancer development. In the current study, we investigated whether AID might contribute to the development of colitis-associated colorectal cancers. METHODS The expression and regulation of AID in association with proinflammatory cytokine stimulation were investigated in cultured colonic cells. Genotoxic activity of AID in colonic cells was analyzed using retroviral system. Immunohistochemistry for AID was carried out on various human colonic tissues specimens. RESULTS Tumor necrosis factor-alpha induced aberrant AID expression via IkappaB kinase-dependent nuclear factor (NF)-kappaB-signaling pathways in human colonic epithelial cells. Moreover, AID expression was also induced in response to the T helper cell 2-driven cytokines interleukin-4 and interleukin-13, which are activated in human inflammatory bowel disease. Aberrant activation of AID in colonic cells preferentially induced genetic mutations in the TP53 gene, whereas there were no nucleotide alterations of the APC gene. Immunohistochemistry revealed enhanced expression of endogenous AID protein not only in the inflamed colonic mucosa of ulcerative colitis patients but also in tumor lesions of colitis-associated colorectal cancers. CONCLUSIONS Our findings indicate that proinflammatory cytokine-mediated aberrant expression of AID in colonic epithelial cells is a genotoxic factor linking inflammation, somatic mutations, and colorectal cancer development.

MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice

MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33−/−Srebf1+/− mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33−/− mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo.