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Met tyrosine kinase receptor, the receptor of hepatocyte growth factor/scatter factor (HGF/SF), is present in mouse tissues as two major isoforms differing by a 47-aminoacid segment in the juxtamembrane domain via alternative splicing of exon 14. We found that the smaller isoform of Met (Sm-Met) was highly transformable in both in vitro and in vivo tumorigenesis assays. In this report, close examination of the transforming activity of the Sm-Met showed that the expression of Sm-Met conferred the cells serum independence and anti- apoptotic property when treated with doxorubicin. These properties of Sm-Met seemed to be originated from its far longer maintenance of tyrosine kinase activity after the binding of HGF/SF. Interestingly, the longer maintenance of activated status was accompanied with more increase of tyrosine phosphorylation of Stat3 protein. Moreover, we have tried to find (an) animal tumorigenesis model(s) showing the increase in the expression of this transforming variant of Met. In gamma-ray-induced mouse thymic lymphoma model, the expression of the mRNAs for Sm-Met was significantly increased as well as those of wild type Met and HGF/SF, suggesting a possible role of the Sm-Met in tumorigenesis in vivo.

The chronic hepatotoxic effects of carbon tetrachloride (CCl(4)) in heat-shock protein (HSP) 70 knock out (HSP70-/-) mice were examined. After repeated intraperitoneal injections of CCl(4) for six weeks, the level of ALT and weight ratio of the liver to body were lower in HSP70-/- mice than in the control (WT) mice. The levels of HSP25 and HSP47 were lowered in HSP70-/- mice as compared with WT mice. The grades of hepatic necrosis and neutrophil infiltration were not significantly different between HSP70-/- and WT mice. The collagen content was not affected significantly by CCl(4) treatment.

To determine the methylation profile of multiple tumor-related genes during multistep hepatocarcinogenesis, we investigated the methylation status of CpG islands of 9 genes, using methylation-specific polymerase chain reaction for 60 paired hepatocellular carcinoma (HCC) and non-HCC liver tissue samples, 22 dysplastic nodule (DN), 30 liver cirrhosis (LC), 34 chronic hepatitis (CH) and 20 normal liver samples. The methylation status of 9 genes was correlated to the clinicopathological findings of HCC patients. All HCC samples showed methylation of at least one gene, whereas it was shown in 72.7% of DN and 40% of LC, but was not shown in CH and normal liver samples (P < 0.001). The number of genes methylated showed a stepwise increase with the progression of stages (0 for normal liver and CH, 0.5 for LC, 1.5 for DN, and 3.7 for HCC (P < 0.001)). The genes frequently methylated in HCC were APC (81.7%), GSTP1 (76.7%), RASSF1A (66.7%), p16 (48.3%), COX-2 (35%), and E-cadherin (33.3%). COX-2, p16, RASSF1A, and TIMP-3 were not methylated in LC and CH from patients without concurrent HCC. Chronic liver diseases with concurrent HCC showed higher methylation frequencies of the tested genes, and a higher number of methylated genes than those without concurrent HCC. HCC patients with methylation of E-cadherin or GSTP1 showed poorer survival than those without (P = 0.034 and 0.043, respectively). In conclusion, our results indicated that CpG island methylation of tumor-related genes is an early and frequent event, and accumulates step-by-step during a multistep hepatocarcinogenesis. CpG island methylation of E-cadherin or GSTP1 might serve as a potential biomarker for prognostication of HCC patients.

Hepatoblastoma is the most frequent pediatric liver tumor that develops mostly in young children. Abnormal regulation of cell cycle regulatory genes including p16 has been described, displaying no p16 mRNA and p16 protein in hepatoblastomas. The inactivation of p16, leading to the disruption of cell cycle control is involved in many types of human malignancies. However, the mechanism of the p16 inactivation in hepatoblastomas has not yet been elucidated. In this present study, we examined the methylation status of the p16 gene promoter by using methylation-specific PCR in 24 cases of hepatoblastomas and in 20 cases of corresponding non-neoplastic liver tissue. Aberrant methylation of 5' CpG islands of p16 was present in 12 of 24 (50.0%) cases of hepatoblastoma. Clinicopathologic parameters were not associated with the methylation status of p16. To correlate the methylation status of p16 with the expression of p16, immunohistochemical staining was done in tumors and non-neoplastic liver tissue. All non-neoplastic liver tissues displayed moderate, but heterogeneous immunoreactivity for p16. Eight of 12 (66.6%) methylation-positive hepatoblastomas showed a complete lack of immunoreactivity for p16. The other 4 methylation-positive hepatoblastomas had heterogeneous immunoreactivity. Nine of 12 (75.0%) unmethylated cases of hepatoblastoma displayed diffuse immunoreactivity, whereas 3 cases of unmethylated hepatoblastoma were not immunostained for p16. Our data indicate that the hypermethylation of p16 is a major mechanism of the transcriptional repression of p16 in hepatoblastomas, and we suggest that the inactivation of p16, leading to the lack of p16, may play an important role in the tumorigenesis of hepatoblastomas.

Aberrations of G1-S cell cycle arrest and TGF-beta/Smad pathway are critical events in human carcinogenesis. We studied alterations of both pathways by immunohistochemical staining for p53, p16, p27, cyclin D1, Rb and Smad4/Dpc4 in 42 intrahepatic cholangiocarcinomas (ICCs). Abnormal nuclear overexpression of p53 and cyclin D1 was noted in 15 (35.7%) and 26 (61.9%) cases, respectively. Total loss of p16, p27, Rb and Smad4 was detected in 15 (35.7%), 13 (31.0%), 5 (11.9%) and 19 (45.2%) cases, respectively. Forty cases (95.2%) showed aberrations of at least one of the pathways, of which 21 (50%) revealed abnormality in G1-S pathway only, 17 (40.5%) had abnormalities in both pathways and 2 (4.8%) had an abnormality in TGF-beta/Smad pathway only. Among the examined genes, loss of Smad4 was found to have a positive relationship with the pTNM stage (P < 0.05). The overall stage of the high-altered group (alterations in 2 to 5 of the genes, n = 29) was significantly higher than that of the low-altered group (alteration of one or no gene, n = 13) (P < 0.01). We also examined the expression of above genes in the accompanying biliary dysplasia and found out abnormal expression of p53, cyclin D1 or p16 in 7 out of 13 dysplastic lesions. Our data suggest that abnormal G1-S cell cycle and altered TGF-beta/Smad pathway are major events in cholangiocarcinogenesis. Moreover, there might be a possible cumulative effect of the alterations in the examined genes upon the clinical outcome of patients with resectable ICCs.

Background The incidence, prevalence, and mortality of intrahepatic cholangiocarcinoma (ICC) are increasing worldwide. Protein tyrosine kinase-7 (PTK7) is upregulated in many common human cancers. However, its expression in ICC has not been studied. The present study aimed to explore the underlying mechanism of PTK7 in ICC. Materials and Methods The role of PTK7 was studied in vitro by suppressing PTK7 expression in ICC cell lines. The in vivo effect of PTK7 was evaluated using a nude mouse model inoculated with a human ICC cell line. We also examined the role of PTK7 in human ICC samples. Results Cells with high PTK7 expression exhibited higher proliferation, DNA synthesis, invasion, and migration abilities than did cells with low PTK7 expression. The knockdown of PTK7 with small interfering RNA (siRNA) in high PTK7 expressing cells resulted in impairment of invasion, migration, and DNA synthesis through the regulation of several cell-cycle-related proteins. It also induced cell apoptosis and decreased phospho-RhoA expression. In a xenograft nude mouse model, PTK7 siRNA resulted in a reduction of the tumor size, compared with scrambled siRNA injection. PTK7 expression was higher in human ICC than in the normal bile duct. Patients with low expression of PTK7 had a longer disease-free survival and overall survival than those with high expression. Conclusions PTK7 expression plays an important role in the invasiveness of ICC cells and leads to a poor prognosis in ICC patients. Thus, PTK7 can be used as a prognostic indicator, and the inhibition of PTK7 expression could be a new therapeutic target for ICC.

Background/Aims Protein disulfide isomerase (PDI) has been implicated in the survival and progression of some cancer cells, by compensating for endoplasmic reticulum stress by upregulating the protein-folding capacity. However, its prognostic role in patients with hepatocellular carcinoma (HCC) has not been investigated. Methods We collected HCC tissues from 83 HCC patients who underwent surgical resection for an immunohistochemical study of PDI. Overall survival (OS) was measured from the date of surgical resection until the date of death from any cause. Radiological progression was evaluated using the modified Response Evaluation Criteria in Solid Tumors in an independent radiological assessment. Results PDI expression was found to be increased in human HCC compared to adjacent nontumor tissues. Increased immunopositivity for PDI was associated with a high Edmondson-Steiner grade (p = 0.028). Univariate analysis of patients who had undergone surgical resection for HCC showed that tumor PDI upregulation is a significant risk factor for poor OS (p = 0.016; hazard ratio [HR], 1.980) and time to progression (TTP; p = 0.007; HR, 1.971). Multivariate analyses revealed that high PDI expression was an independent predictor of a shorter TTP (p = 0.015; HR, 1.865) and poor OS (p = 0.012; HR, 2.069). Conclusions Upregulated PDI expression is associated with aggressive clinicopathological features of HCC; thus, PDI might serve as an independent prognostic factor and a potential therapeutic target for HCC patients.

Deranged expression of cell cycle modulators has been reported to contribute to the development and progression of hepatocellular carcinoma (HCC). However, their expression patterns remain poorly understood in hepatitis B virus (HBV)-related HCC, which constitutes about 65-70% of HCC in Korea. The aims of this study were to evaluate the expressions of G1-S modulators in HBV-related HCCs and dysplastic nodules (DNs), and to correlate with the histopathologic features of HCCs. Immunohistochemical expressions of cyclin D1, cyclin E, p53, p27, p21, p16, Rb, and PCNA proteins were investigated in 80 HCCs and 22 DNs. Cyclin D1 overexpression showed positive relationships with advanced tumor stage, poor differentiation, larger tumor size, microvascular invasion, intrahepatic meta-stasis, no tumor capsule formation, infiltrative growth, aberrant p53 expression, and high PCNA labeling index (LI) of HCC (p

To analyze the aberrant expression of cell cycle-related proteins and their biological significance in relation to cirrhosis, we compared the cirrhotic patterns induced by two different types of cirrhotic agents, CCl4 and thioacetamide (TAA) in rats. CCl4 or TAA treatment was given to rats for 8 or 30 weeks, respectively, and the livers were removed at 9, 20, and 30 weeks after the experiment began. The TAA-induced fibrotic pattern was different from the CCl4-induced one, in terms of the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed severe cirrhotic changes at 9 weeks in CCl4-treated rats and at 30 weeks in TAA-treated rats. Immunoblotting for cyclin D1, E, A, B, and proliferating cell nuclear antigen (PCNA) and their counterpart protein kinases (CDK2, 4, and CDC2) showed significant overexpression in rats with severely cirrhotic livers. The p53 tumor suppressor protein increased dramatically in the CCl4-treated group, while it was not detected in the livers of TAA-treated rats. Upregulation of p21WAF1, a CDK inhibitory protein, was detected in TAA-treated rats, but not in CCl4-treated rats. Immunohistochemical data for cyclin D1, E, and PCNA were well correlated with immunoblotting data; these proteins were increased in hepatocytes surrounding the cirrhotic lesions, suggesting that hepatocyte regeneration is correlated with cell cycle-related protein expression in cirrhotic liver. In the TAA-treated rats, the expression of these proteins was increased both in hepatocytes and in ductule cells. Our data suggest that liver cirrhosis induced by CCl4 or TAA is associated with alterations in cell cycle-related proteins, and that the expression of these proteins is responsible for hepatocyte regeneration in the damaged liver and may be involved in liver carcinogenesis.

Tumor necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIL-R1) and tumor necrosis factor-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) are cell-surface receptors involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell-death signaling. TRAIL-R1 and TRAIL-R2 genes have recently been mapped to chromosome 8p21-22, which is a frequent site of allelic deletions in many types of human tumors, including non-Hodgkin's lymphoma (NHL). Because TRAIL/TRAIL receptor system plays an important role in lymphocyte homeostasis, we hypothesized that the mutations of TRAIL-R1 and TRAIL-R2 may be involved in the development of NHL and that such mutations may be responsible for the allelic losses of 8p21-22 in NHL. In this study, we analysed the entire coding region of TRAIL-R2 gene and the death domain region of TRAIL-R1 gene for the detection of the somatic mutations in a series of 117 human NHLs using polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis. Overall, eight tumors (6.8%) were found to have two TRAIL-R1 gene mutations or six TRAIL-R2 gene mutations. Interestingly, of the eight mutations, six missense mutations (two TRAIL-R1 and four TRAIL-R2) were detected in the death domains and one nonsense mutation of TRAIL-R2 was detected just before the death domain. Our data suggest that somatic mutations of TRAIL-R1 and TRAIL-R2 genes may play a role in the pathogenesis of some NHLs and that TRAIL-R1 and TRAIL-R2 genes might be the relevant genes to the frequent loss of chromosome 8p21-22 in human NHL.

Telomerase, an enzyme associated with cellular immortality and malignancy, is stringently repressed in most normal somatic cells but is reactivated in malignant tumor cells and immortal cell lines, indicating that activation of telomerase may play an important role in tumorigenesis and immortalization. The pattern of human telomerase RNA (hTR) expression during progression of gastric cancer was investigated by a radioactive in situ hybridization (ISH) assay. Paraffin-embedded sections of 85 archival samples from Korean patients with benign and various malignant stages of gastric carcinomas as well as normal and regenerative tissues were studied. In normal gastric mucosae and regenerative lesions such as chronic peptic ulcer and hyperplastic polyps, only a weak degree of hTR expression was noted, and the expression was limited to basal cells of the gastric glands. Also, a moderate degree of hTR expression was present in the germinal centers of lymphoid follicles present in the submucosa. In tubular adenomas, the degree of hTR expression was also generally weak, but, unlike normal gastric mucosa, the expression was rather diffuse and occasionally focal in distribution. However, moderate to intense and usually diffuse hTR expression was present in all cancerous tissues at different stages. Although some heterogeneity of hTR expression was noted, there was a tendency for intensity of hTR expression to increase gradually as the cancer progressed to a more advanced stage. Our results indicate that upregulation of telomerase expression is associated with gastric cancer development or plays some role in gastric carcinogenesis. Upregulation of hTR expression detected by ISH assay may be a useful marker or tool for the early detection of gastric cancer.

2-(Allylthio)pyrazine (2-AP), a synthetic pyrazine derivative with an allylsulfur moiety, has hepatoprotective effects against toxicants. Effect of 2-AP on hepatic tumorigenesis in association with glutathione S-transferase (GST) induction was examined in rats exposed to aflatoxin B1 (AFB1). Both AFB1-DNA adduct formation in the liver and urinary elimination of 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-N7-guanine) adduct were also determined. Male Sprague Dawley rats were treated with 2-AP at the daily oral doses of 10, 25 and 50 mg/kg for 16 consecutive days, during which four repeated doses of AFB1 (1.0 mg/kg) were given to the animals. Rats were then subjected to two-thirds of hepatectomy, followed by administration of phenobarbital (PB). Focal areas of hepatocellular alteration were identified after 44 days and preneoplastic foci expressing the placental form of glutathione S-transferase P (GST-P) were quantified by immunostaining of liver sections. 2-AP reduced the volume of liver occupied by GST-P foci by 65-96%. Under these experimental conditions, 2-AP treatment resulted in significant elevations in GST activity in the liver. Levels of radiolabeled AFB1 covalently bound to hepatic DNA, RNA and proteins were significantly reduced in rats treated with 2-AP for 5 days. 2-AP pretreatment also caused a 45% reduction in the urinary elimination of AFB1-N7-guanine adduct over the 24-h postdosing period. The present findings demonstrated that 2-AP exhibited protective effects against AFB1-induced hepatocarcinogenesis in rats with a marked decrease in the level of AFB1-DNA adduct. Reduction of hepatic DNA adducts might result from elevations of activity of GST, which catalyzes detoxification of the carcinogen.

Abdominal lymphangiomas are uncommon angiomatous tumor occurring mainly in childhood. This is a retrospective clinicopathologic study of 17 cases of abdominal lymphangioma. The patients included are five children and 12 adults, with a mean age at initial presentation of 30.7 years (age ranges 3-63). The locations of the tumors were mesentery (5), retroperitoneum (4), colon (3), omentum (3), mesocolon (1) and gallbladder (1). Infiltrative growth was more common pattern than entirely circumscribed pattern. Masses were mostly multilocular cysts and contained chyle or serous fluid. On immunohistochemical staining, 16 cases were reactive for either CD31 or factor VIII-related antigen. These fact would suggest that intra-abdominal lymphangiomas simulate the immunohistochemical features of collecting lymphatics. Follow up was possible in 12 cases for 3-50 months (mean 19 months) and only one patient showed local recurrence. Although abdominal lymphangiomas are rare in adulthood and correct preoperative diagnosis is difficult, awareness of such a possibility in adulthood will contribute to make a correct preoperative diagnosis.

Experiments were conducted to determine whether gamma-ray-induced genetic damage in parental rats can lead to the development of cancer in their offspring rats using glutathione S-transferase-positive (GST-P+) hepatic foci with or without the addition of diethylnitrosamine (DEN), a carcinogen. A single 1 Gy whole-body exposure of gamma-rays was given to pregnant rats at day 14 and during postnatal week 3, DEN was intraperitoneally injected twice in 1 week. Female pups from irradiated maternal and paternal rats were also used. Twelve weeks after birth, the rats were sacrificed. GST-P+ foci in animals subjected only to radiation were not different to those of normal control pups, but the incidence of GST-P+ foci was 2.4 times higher in pups treated with DEN alone at 3 weeks after birth than in those irradiated after the onset of pregnancy. In DEN-combined groups, irradiation of post-pregnant or maternal and paternal rats with gamma-rays before mating significantly increased both the incidence and area of GST-P+ foci when compared to those of rats treated with DEN alone. The proliferating cell nuclear antigen (PCNA) labeling index was significantly higher in the offspring of rats subjected to radiation alone or radiation combined with DEN than in normal control pups. Using a rat-liver model, the results of this study indicate that although the dose did not induce phenotypic malformation, exposure to radiation during the embryonic or pre-embryonic stage increases susceptibility to carcinogens.

Programmed cell death (PCD) is a prominent feature of the development of the immune and nervous systems. In both systems, widespread PCD occurs in primitive progenitor cells during development. In this study, we demonstrated that Ewing's sarcoma (ES) cells, undifferentiated neural precursors, underwent apoptosis upon engagement of CD99 with anti-CD99 monoclonal antibody. Apoptosis via CD99 occurred only in the undifferentiated state of ES cells, but not in differentiated ES cells. CD99-induced apoptosis in ES cells appeared to require de novo synthesis of RNA and protein as well as caspase activation. Cyclosporin A, known to be a potent inhibitor of both calcineurin activation and mitochondrial permeability transition pore opening, inhibited CD99-mediated apoptosis, whereas FK-506, a specific calcineurin inhibitor, did not, indicating the induction of CD99-mediated apoptosis through a calcineurin-independent pathway. Furthermore, the dying cells displayed the reduction of mitochondrial transmembrane potential (delta psi m). These results suggest that CD99 engagement induce CsA-inhibitable mitochondrial permeability transition pore opening, followed by a reduction of delta psi m and caspase activation, thereby leading to apoptosis. Based on these results, we suggest the possible involvement of CD99 in the apoptotic processes that occur during nervous system development and also its application in immunotherapeutic trials for ES cases.

The sequential changes of natural killer cell (NK) activity and prostaglandin E 2 (PGE 2 ) during hepatocarcinogenesis induced by diethylnitrosamine (DEN) in male Fischer 344 rats were investigated. DEN at a concentration of 40 ppm was administered in drinking water for 10 weeks. At weeks 5, 10, 20 and 30, rats were autopsied and the development of glutathione S-transferase placental form positive foci (GST-P + foci) at weeks 5 and 10 and hepatocellular tumors at weeks 20 and 30 were examined. The labeling index of bromodeoxyuridine (BrdU) an indicator of DNA synthesis, was also sequentially checked. GST-P + foci were found to increase with age. Hepatocellular nodules increased until week 20, but by week 30 when the incidence of hepatocellular carcinoma was 100%, the incidence of nodules had decreased. BrdU positive cells also increased with age, and by week 30 when the incidence of hepatocellular carcinoma was 100%, the number of BrdU-positive cells had decreased. NK cell activity increased until week 10, but at week 20, was less than in the untreated control group. The level of PGE 2 increased until week 5, but at week 10, levels were not significantly different from those seen in the untreated control group. On the basis of these results, we concluded that NK activity is closely related to the progression of hepatocarcinogenesis, but PGE 2 levels show no significant change.

Gastric adenocarcinoma is the most prevalent cancer in South Korea, and Helicobacter pylori (H. pylori) infection is also common. This study was performed to examine the association between H. pylori infection and gastric cancer, taking into account various other factors. To investigate the association between gastric adenocarcinoma and H. pylori infection, determined by urease-positive reaction in the CLO test, a total of 175 paired specimens (175 tumor and 175 tissues adjacent to tumor) of stomach cancer patients and a total of 113 control specimens were obtained. The positive H. pylori infection rates were 78.9% (138/175) among the patients in specimens of tumor or tissues adjacent to the tumor and 41.6% (47/113) among controls in the CLO test. A positive correlation between H. pylori infection and gastric cancer was observed (age-adjusted odds ratio, 7.0; MH chi2=34.5 with P

BPDE-I-DNA and oxidative DNA adducts (8-OHdG) were investigated in stomach tissues (tumor and tumor-adjacent) of patients (N = 211) with gastric adenocarcinoma and in normal stomach tissues (N = 113). In each stomach specimen, the levels of BPDE-I-DNA adducts were quantitatively measured by enzyme linked immunosorbent assay (ELISA) and oxidative DNA damage was measured by HPLC-ECD. Higher levels of total BPDE-I-DNA adduct were observed in tumor (4.20 +/- 0.59 fmol/microg DNA) and tumor-adjacent (3.68 +/- 0.62 fmol/microg DNA) tissues than in normal stomach tissues (2.80 +/- 0.53 fmol/microg DNA) but were not significant. In males, BPDE-I-DNA adduct was significantly higher in tumor tissues (4.25 +/- 0.42 fmol/microg DNA) than in normal tissues (2.83 +/- 0.59 fmol/microg DNA) (P0.05). In smokers, BPDE-I-DNA adduct was slightly higher in tumor tissues (4.92 +/- 0.82 fmol/microg DNA) than in tumor-adjacent tissues (3.99 +/- 0.92 fmol/microg DNA). Gastric cancer patients had significantly higher levels of 8-OHdG in their tumor-adjacent (7.54 +/- 0.43 residues/10(5) dG) and tumor tissues (6.29 +/- 0.39 residues/10(5) dG) than in normal tissues (2.86 +/- 0.11 residues/10(5) dG) (P0.001). Smokers showed higher levels of 8-OHdG in both tumor (6.44 +/- 0.62 residues/10(5) dG) and tumor-adjacent (8.12 +/- 0.68 residues/10(5) dG) tissues than in non-smokers (5.80 +/- 0.47 and 7.11 +/- 0.57 residues/10(5) dG, respectively). 8-OHdG levels were significantly increased in positive tissues with Helicobacter pylori (H. pylori) infection compared with negative tissues (P0.01). Also, the frequency of H. pylori infection was higher in tumor-adjacent tissues (73%) than in tumor (42%) or normal tissues (44%). These results demonstrate that there are higher levels of 8-OHdG and BPDE-I-DNA adducts in tumor and tumor-adjacent tissues than in normal tissues and that these higher levels might be related to gastric tumorigenesis, although benzo[a]pyrene could be a minor contributing component in the environment.

Although protein kinase C (PKC), a family of 12 related isoforms, plays an important role in carcinogenesis, little is known about the specific role of each isoform in the initiation stage of hepatocarcinogenesis. The subcellular distribution of PKC isoforms in the early stages of diethylnitrosamine (DEN)-initiated hepatocarcinogenesis was therefore examined. Three-week-old female Sprague–Dawley rats were intraperitoneally injected twice in 1 week with DEN; all animals were sacrificed at 1, 2 and 24 h and 3 and 7 days after the second injection. PKC α and - β expression in both cytosolic and particulate fractions decreased as a result of 1 h of DEN treatment and this effect lasted for 7 days. In both fractions, PKC ϵ expression showed a marked increase by DEN treatment, while the expression of PKC δ and - ζ was almost unchanged. These results suggest that differential expression of PKC isoforms may play an important role in the early stage of DEN-initiated hepatocarcinogenesis in rats.

We examined 81 cases of primary gastrointestinal lymphomas in Korea, including 64 gastric lymphomas and 17 intestinal lymphomas, for EBV expression by EBER-1 in situ hybridization (ISH) and EBNA-1 PCR. In EBER-1 positive cases, we performed immunohistochemistry for latent membrane protein-1 (LMP-1) and EBV diffuse early antigen (EA(D)) to compare EBV latent gene expression and lytic process. EBER-1 was detected in 15 of 81 cases of lymphomas. EBER-1 expression showed three different patterns on tumour cells; diffuse 4/81 (5%), localized 4/81 (8%), and a few scattered pattern 7/81 (9%). We regarded diffuse pattern and localized pattern as EBER-1 positive group (8/81: 10%). Diffuse pattern of EBER-1 was shown in all three T-cell lymphomas and one B-cell lymphoma. A localized pattern was seen all in B-cell lymphomas. The EBER-1 expression was 11% in the stomach (7/64) and 6% in the intestine (1/17). Five of the eight EBER-1 positive gastric lymphomas were histologically diffuse large B-cell lymphomas, and the other three were peripheral T-cell lymphoma, unspecified, one angiocentric lymphoma, and one intestinal T-cell lymphoma by REAL classification. Eight MALT type gastric B-cell lymphomas showed no EBV association. EBV nuclear antigen (EBNA-1) was detected in 15 of 45 resected cases (33%) by PCR. EBER-1 positive cases were all EBNA-1 positive. Twelve EBNA-positive/EBER-negative cases consisted of seven cases showing a few scattered EBER-1 positive lymphocytes. LMP-1 and diffuse early antigen (EA(D)) was detected in five and three cases, respectively. Although follow-up information in our series was incomplete, it seemed that there was no significant difference in their staging or prognosis between EBER-positive cases and EBER-negative group. It is concluded that EBV is associated with some lymphomas among Koreans without overt pre-existing immunodeficiency, especially in T-cell lymphomas.

MicroRNAs (miRNAs) participate in diverse biological functions and carcinogenesis by inhibiting specific gene expression. We previously reported that suppression of adenine nucleotide translocase 2 (ANT2) by using the short hairpin RNA (shRNA) approach has an antitumor effect in several cancer cells. We here examined the influence of ANT2 on expression of miRNAs in hepatocellular carcinoma (HCC) to further elucidate the tumor-suppressive mechanism of ANT2 shRNA. We first carried out screening for miRNAs, whose expression is regulated by ANT2 suppression in the Hep3B HCC cell line using miRNA microarrays. Validation of candidate miRNAs was done by incorporating clinical samples, and their effects on the tumorigenesis of HCC were studied in vitro and in vivo. miR-636 was one of the miRNAs whose expression was highly upregulated by ANT2 suppression in miRNA microarray analysis, as confirmed by real-time reverse transcription-polymerase chain reaction. Notably, miR-636 was markedly downregulated in HCC tissues compared with matched non-neoplastic liver in clinical samples. Restoration of miR-636 in Hep3B cells led to significant reduction of cell proliferation and colony formation. miR-636 restoration resulted in a decreased level of Ras, one of the putative targets of miR-636, and inactivation of its signaling pathway. Moreover, tumorigenesis was efficiently suppressed by miR-636 in an in vivo tumor xenograft model of HCC. The data suggest that miR-636 might function as a tumor suppressor miRNA affecting HCC tumorigenesis via downregulation of Ras, and that ANT2 suppression by shRNA could exert an anticancer effect by restoring miR-636 expression in HCC.

Background Hepatocellular adenoma (HCA) is a rare benign tumor of the liver. A subtype classification of HCA (hepatocyte nuclear factor 1α [HNF1α]-mutated, β-catenin-mutated HCA, inflammatory HCA, and unclassified HCA) has recently been established based on a single institutional review of a HCA series by the Bordeaux group. Methods We used histologic and immunohistochemical parameters to classify and evaluate eight cases from our institution. We evaluated the new classification method and analyzed correlations between our results and those of other reports. Results Seven of our eight cases showed histologic and immunohistochemical results consistent with previous reports. However, one case showed overlapping histologic features, as previously described by the Bordeaux group. Four cases showed glutamine synthetase immunohistochemical staining inconsistent with their classification, indicating that glutamine synthetase staining may not be diagnostic for β-catenin-mutated HCA. HNF1α-mutated HCA may be indicated by the absence of liver fatty acid binding protein expression. Detection of amyloid A may indicate inflammatory HCA. HCA with no mutation in the HNF1α or β-catenin genes and no inflammatory protein expression is categorized as unclassified HCA. Conclusions Although the new classification is now generally accepted, validation through follow-up studies is necessary.

To evaluate the prognostic significance of CD24 expression in patients undergoing adjuvant chemoradiotherapy for extrahepatic bile duct (EHBD) cancer.Eighty-four patients with EHBD cancer who underwent curative resection followed by adjuvant chemoradiotherapy were enrolled in this study. Postoperative radiotherapy was delivered to the tumor bed and regional lymph nodes up to a median of 40 Gy (range: 40-56 Gy). All patients also received fluoropyrimidine chemotherapy for radiosensitization during radiotherapy. CD24 expression was assessed with immunohistochemical staining on tissue microarray. Clinicopathologic factors as well as CD24 expression were evaluated in multivariate analysis for clinical outcomes including loco-regional recurrence, distant metastasis-free and overall survival.CD24 was expressed in 36 patients (42.9%). CD24 expression was associated with distant metastasis, but not with loco-regional recurrence nor with overall survival. The 5-year distant metastasis-free survival rates were 55.1% and 29.0% in patients with negative and positive expression, respectively (P = 0.0100). On multivariate analysis incorporating N stage, histologic differentiation and CD24 expression, N stage was the only significant factor predicting distant metastasis-free survival (P = 0.0089), while CD24 expression had borderline significance (P = 0.0733). In subgroup analysis, CD24 expression was significantly associated with 5-year distant metastasis-free survival in node-positive patients (38.4% with negative expression vs 0% with positive expression, P = 0.0110), but not in node-negative patients (62.0% with negative expression vs 64.0% with positive expression, P = 0.8599).CD24 expression was a significant predictor of distant metastasis for patients undergoing curative resection followed by adjuvant chemoradiotherapy especially for node-positive EHBD cancer.

We produced transgenic mice using SV40 Tag gene under the control of its own enhancer and promoter. Three transgenic lines (SNU-SVT125, 127, 248) consistently developed thymic carcinoma as well as choroid plexus carcinoma and dysplastic renal tubule. In SNU-SVT248 line, SV40 Tag transgene was expressed at thymus, spleen and kidney. Thymic epithelium showed high level expression of SV40 Tag in immunohistochemistry. Histopathological and electron microscopic analysis revealed that poorly differentiated carcinoma was derived from type 2 to 4 thymic epithelial cell. Our transgenic mice would provide a model for studies on the pathogenesis of thymic carcinoma and on the regulation of thymopoiesis by epithelial cells.

Objective Differentiation of invasive thymoma from thymic carcinoma is important because of their different clinical behaviors. Retrospectively, we evaluated the CT findings of invasive thymomas and thymic carcinomas to determine the differential points between them. Materials and methods We reviewed the CT scans of 12 patients with invasive thymoma and 10 patients with thymic carcinoma that were confirmed by surgery or percutaneous needle aspiration. We analyzed CT scans, paying special attention to homogeneity, attenuation, invasion of adjacent mediastinal structures, pleural implants, mediastinal nodes, and extramediastinal metastases. Results Most of the invasive thymomas and thymic carcinomas were homogeneous and isodense with chest wall muscle. Irregular infiltration into the adjacent organ suggesting invasion was seen in 11 patients (92%) with invasive thymoma and 8 patients (80%) with thymic carcinoma. Pleural implants were observed in four patients (33%) with invasive thymoma and one patient (10%) with thymic carcinoma. Mediastinal lymphadenopathy was seen in one patient (8%) with invasive thymoma and four patients (40%) with thymic carcinoma. Metastases to the lung, adrenal glands, or liver were observed in four patients (40%) with thymic carcinoma but none with invasive thymoma. Conclusion Despite the similarities of CT findings between invasive thymoma and thymic carcinoma, there are some differential points. Thymic carcinomas were infiltrating tumor and were more commonly associated with mediastinal nodes and extrathymic metastases, but less commonly associated with pleural implants than invasive thymoma.

p53 gene mutations have been known to be highly related to the particular stage of transformation in various types of human cancers. This study was conducted to investigate the p53 mutations at the protein level by an immunohistochemical method using anti-p53 antibody, NCL-p53-DO-7. Twenty-five cancer specimens were obtained surgically from patients with squamous cell cancer of the skin at the Korea Cancer Center Hospital. The cancers were classified according to the possible etiology into two groups, burn scar originated and UV-related cancers. Overexpression of p53 protein was detected in ten (40%) out of 25 cases tested: six (40%) of 15 cases associated with burn scar and four (40%) of ten cases related to UV exposure. In all normal skin cells in specimens, p53 protein was not stained at all. The stages and histological grades were evaluated for their relationship with the overexpression of p53 protein. No significant difference was found between the overexpression of p53 protein and the stages or histological grades. These results demonstrating that 40% of skin cancers were positive for p53 overexpression suggest that the alterations of the p53 gene may play a role and the exact role of p53 gene in the development of squamous cell carcinoma of the skin will be studied.

DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

The modifying action of experimentally induced chronic liver injury on diethylnitrosamine (DEN) hepatocarcinogenesis was investigated using a minimal treatment protocol. A single dose of DEN (15 mg/kg b.w.) was administered as a carcinogen to 1-day-old Sprague-Dawley rats. From 3 weeks of age rats received repeated intraperitoneal injections of carbon tetrachloride (CCl 4 ), or 10% ethanol or 5% acetaldehyde in the drinking water for 9 weeks. Combinations of CCl 4 and ethanol or acetaldehyde were also tested. Morphology, immunohistochemistry for glutathione S -transferaseplacental form, and incidence and quantity of preneoplastic lesions of the livers were studied. The chronic CCl 4 administration produced complete or incomplete liver cirrhosis and exerted a strong promoting effect on the development of neoplastic nodules. Ethanol alone revealed no cirrhogenous or tumor-promoting effect, but enhanced both actions of CCl 4 . Acetaldehyde increased only the cirrhogenous effect of CCl 4 .

Background/Aims P2/MS is known as a simple, accurate, and noninvasive marker for determination of the degree of hepatic fibrosis in patients with viral hepatitis. We aimed to validate P2/MS in patients with HCC. Methods Consecutive HCC patients who underwent surgical resection between June 2007 and March 2009 at Seoul National University Hospital were enrolled. Fibrosis stage was reviewed and assessed according to METAVIR scoring. P2/MS values [platelet count (109/L)]2/[monocyte fraction (%)×segmented neutrophil fraction (%)] and other noninvasive fibrosis scoring systems were calculated. Results A total of 171 patients were included; seven patients with METAVIR F1, 31 with F2, 41 with F3, and 92 with F4. The area under the receiver-operating characteristic curve of P2/MS was 0.804 [95% confidence interval (CI), 0.681~0.927] for detection of significant fibrosis (F2-F4) and 0.769 (95% CI, 0.698~0.839) for detection of histological cirrhosis (F4). At a value < 62, P2/MS detected significant fibrosis with a specificity of 85.7% (95% CI, 42.0~99.2) and a positive likelihood ratio of 4.268 (95% CI, 0.692~26.309); and at a value > 115, P2/MS ruled out significant fibrosis with a sensitivity of 90.2% (95% CI, 84.4~94.1) and a negative likelihood ratio of 0.34 (95% CI, 0.106~0.095). P2/MS had a superior efficacy for detection of hepatic fibrosis in patients with HCC compared to the other noninvasive panels. Conclusions P2/MS can accurately detect fibrosis in patients with HCC. Thus, P2/MS might be utilized as a noninvasive index reflecting the degree of hepatic fibrosis in HCC patients.

Liver fibrosis is a chronic liver disease and lots of people in Korea are suffered. There are many efforts to find candidates to suppress liver fibrogenesis and several chemical-induced model or bile duct ligation model have been used to research and develop hepatic fibrogenic suppressor. From the previous study about functional effects of turnip which cultivated in Kangha Island, we got the feasibility which turnip might be able to inhibit heptatic fibrogenesis. TAA is a representative hepatic fibrosis inducer, repeated 7- weeks i.p. injection of it results in hepatic fibrosis. We compared the level of hepatic fibrosis in TAAturnip group, TAA group, and vehicle control group. Nodules-formed by TAA were observed; they were rarely shown in vehicle control group, observed in most area in TAA group, but only shown in periportal regions in TAA-turnip group. These results were confirmed through Masson's trichrom stain; fibrous structures increased in TAA group (fibrosis score: 4) but significantly decreased in TAA-turnip group (fibrosis score: 2-3). In conclusion, we got the result that turnip water extract has a potency to protect TAA-induced hepatic fibrogenesis but it is necessary further study to find its mechanism.

Background/aims Cell cycle regulating proteins are known to have close relation with the proliferation of the mammalian cells. In injured liver, number of hepatic stellate cells is increased from proliferation. However, the expression of cell cycle proteins of hepatic stellate cells during proliferation remains unevaluated. Therefore, cell cycle protein profiles of hepatic stellate cells were studied in dimethylnitrosamine (DMN)-induced rat liver fibrosis model. Methods Six-week male Sprague-Dawley rats (n=19) were intraperitoneally injected with 10 μg/kg DMN and the animals were sacrificed at the end of 1st, 2nd, 3rd, and 4th week. Hepatic stellate cells were separated according to the original method of Alpini and the number of the cells in S phase was counted to evaluate the cell proliferation by flowcytometry. The expression of cyclin A, cyclin B, cyclin D1, cdk2, cdk4, cdc2, proliferating cell nuclear antigen (PCNA), p21, and p27 was examined with immunoblotting analysis. Results Portion of S-phase cells showed its peak on the 7th day. After 1st week, cyclin A, and PCNA showed significant increment in hepatic stellate cells compared to untreated hepatic stellate cells (114% and 116%, respectively, P Conclusion The increment of cyclin A, and PCNA and the decrement of p21 seem to play important roles in the proliferation of HSCs during the early period of DMN-induced rat hepatic fibrosis.