Expression pattern of REIC/Dkk-3 in mouse squamous epithelia.

The REIC/Dkk (reduced expression in immortalized cells/Dickkopf-3) gene was originally identified as a tumour-suppressor gene with reduced expression in immortalized cells, cancer-cell lines and tumour tissues. Of the four members of the Dkk family, the REIC/Dkk-3 protein is unique in terms of DNA sequence, expression profiles and biological functions. In this study, we investigated and compared the expression patterns of the REIC/Dkk-3 protein in mouse squamous epithelia. Expression of REIC/Dkk-3 in the back skin was localized to the upper layer of the interfollicular epidermis, and the inner root sheath of hair follicles. Expression of REIC/Dkk-3 was detected in the ear skin, oral mucosa, tongue, oesophagus, uterine cervix, footpad and tail skin, but not in the cornea. Interestingly, expression was localized to the upper layers of these epithelial tissues. The physiological function of REIC/Dkk-3 is still unclear, but our detailed observation highlight its unique expression pattern in squamous epithelia.

Role of intraoperative enteroscopy for surgical decision making with Crohn's disease.

BACKGROUND: This study aimed to assess the role of intraoperative enteroscopy (IOE) in determining surgical treatment. METHODS: The IOE procedure was performed for 30 patients with Crohn's disease. The degree of stricture and the presence of active ulcer were examined. Preoperative diagnoses and intraoperative findings obtained by inspection and palpation were noted and compared with the IOE findings. RESULTS: Of the 78 intestinal strictures observed by IOE (42%), 33 were not found by preoperative examination. Of the 45 strictures confirmed by IOE to be severe (<15 mm in diameter), 8 were judged to be mild (15-25 mm in diameter) or were not even identified by intraoperative inspection and palpation. Active ulcer was found at 12 of 33 mild strictures, and all 12 strictures were surgically corrected. Of 11 severe strictures detected by IOE at previous surgical sites, 9 were found preoperatively, and 4 were judged to be mild on the basis of inspection and palpation. Stricture was found at the ileocecal valve by IOE in seven patients, but was not diagnosed preoperatively in two of these patients. CONCLUSION: Intraoperative enteroscopy provides useful information regarding the status of the lumen in patients with Crohn's disease.

Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model.

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator in tumor-stromal interactions through enhancing tumor invasion and metastasis. We found recently that NK4, a four-kringle fragment of HGF, functions as both an HGF-antagonist and an angiogenesis inhibitor. We have now determined whether blockade of the HGF-c-Met/HGF receptor pathway and tumor angiogenesis by administration of recombinant NK4 would inhibit growth, invasion, and metastasis of human pancreatic carcinoma implanted into the pancreas of nude mice. When treatment with NK4 or anti-HGF neutralizing antibody was initiated from the third day after orthotopic injection of SUIT-2 human pancreatic cancer cells, both NK4 and anti-HGF antibody suppressed the conversion of orthotopic pancreatic tumors from carcinoma in situ to aberrantly invading cancers during days 3-14. On the other hand, when the treatment was begun on day 10, a time when cancer cells were already invading surrounding tissues, NK4 but not anti-HGF antibody inhibited tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Antitumor effects of NK4 correlated with decreased microvessel density in pancreatic tumors thereby indicating that the antiangiogenic activity of NK4 may have mainly contributed to its antitumor effects. Moreover, although NK4-treatment was initiated from the end stage (day 24 after tumor inoculation), NK4 prolonged survival time of mice, and the suppression of peritoneal dissemination, ascites accumulation, and invasion of metastasized cancer cells into the peritoneal wall were remarkable. We propose that simultaneous targeting of both tumor angiogenesis and the HGF-mediated invasion-metastasis may prove to be a new approach to treating patients with pancreatic cancer.

Stepwise progression of centrosome defects associated with local tumor growth and metastatic process of human pancreatic carcinoma cells transplanted orthotopically into nude mice.

Recent evidence indicates that loss of centrosome integrity may be a major cause of genetic instability underlying various human cancers. The aim of this study was to define the role of centrosome defects during the in vivo tumor progression of pancreatic carcinoma using an orthotopic implantation model. Injection of Suit-2 human pancreatic cancer cells into the pancreata of nude mice reproduced the pattern of local tumor growth and distant metastasis observed in humans. Pancreatic xenografts, peritoneal disseminations, and hepatic metastases were harvested, and tumor cells were examined for centrosomes by immunofluorescence microscopy. Centrosome abnormalities, characterized by increased numbers of centrosomes, were detected in only a small fraction of parental Suit-2 cells in culture, whereas the frequency was markedly increased in cells isolated from the pancreatic xenografts. Abnormal centrosome numbers were found at higher frequencies in metastatic foci than in pancreatic xenografts. A significant positive correlation existed between the fraction of cells with multiple centrosomes and that with multipolar mitotic spindles, suggesting a functional involvement of aberrant centrosomes in spindle disorganization and chromosome missegregation. In addition, the increased frequency of abnormal centrosomes was associated with an enhanced degree of chromosomal instability. These findings suggest a novel model of pancreatic tumor progression whereby a stepwise increase in the magnitude of centrosomal abnormalities confers an increased chance for aberrant mitotic events, thus accelerating genetic instability and causing the tumor to progress to a more advanced stage.

Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation.

The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that gamma-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to gamma-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication.

Correlation between centrosome abnormalities and chromosomal instability in human pancreatic cancer cells.

Chromosomal instability, characterized by abnormal numbers or structures of chromosomes, is a common feature of human cancers, but the mechanisms behind these changes are still unclear. Since centrosomes play a pivotal role in balanced chromosomal segregation during mitosis, we attempted to investigate the association between centrosome abnormalities and chromosomal instability in a large number of human pancreatic cancer cell lines. Immunofluorescence microscopy revealed centrosomes that were highly atypical with respect to their size, shape, and number in most cell lines. These abnormal centrosomes contributed to the assembly of multipolar spindles, resulting in defective mitosis and chromosome mis-segregation. Interestingly, a high frequency of centrosome defects inversely correlated with the growth rate of cells in culture. Fluorescence in situ hybridization revealed a dramatic variation of chromosome numbers in cell lines with the defective centrosome phenotype. Furthermore, a significant positive correlation existed between the level of centrosome defects and the level of chromosomal imbalances. These results indicate that centrosome abnormalities can lead to spindle disorganization and chromosome segregation errors, which may drive the accumulation of chromosomal alterations. Thus, defects in centrosome function may be an underlying cause of genetic instability in human pancreatic cancers.

NK4, a four-kringle antagonist of HGF, inhibits spreading and invasion of human pancreatic cancer cells.

Because of the highly aggressive behaviour, i.e. invasive, disseminative and metastatic properties, the outcome for patients with pancreatic cancer is morbid. A better understanding and interference with the malignant behaviour of pancreatic cancer may provide new directions for treatment. We report here the induction of highly motile and invasive properties in human pancreatic cancer cells by hepatocyte growth factor (HGF) and blockage of these properties by NK4, a newly identified antagonist for HGF. In all of eight human pancreatic cancer cell lines we used (AsPC-1, BxPC-3, H-48N, KP-1N, KP-2, KP-3, MIA PaCa-2 and SUIT-2 cells), the c-Met/HGF receptor was expressed at varying levels. Although weak mitogenic activity of HGF was seen only in SUIT-2 and KP-3 cells, HGF strongly stimulated migration and invasion of these pancreatic cancer cells, except for BxPC-3 and MIA PaCa-2 cells. In contrast, migration and invasion potently induced by HGF in KP-1N, KP-3 and SUIT-2 cells were inhibited by NK4. The invasion of SUIT-2 cells was also potently stimulated with the influence of cocultured pancreatic fibroblasts and by ascitic fluid obtained after pancreatic cancer resection, however, invasiveness of the cancer cells in such conditions was practically abolished by NK4. Consistently, the ascitic fluid in patients who had undergone pancreatic cancer surgery contained high levels of HGF. These findings mean that HGF is probably involved in invasion, dissemination, and metastasis of pancreatic cancer, particularly through tumour-stromal interaction and after resection of the pancreatic cancer. NK4, an effective antagonist of HGF, may prove to have the potential for anti-invasion/metastasis.

Instability of chromosome 8 as an indicator of aggressive tumor phenotype in pancreatic cancer.

BACKGROUND AND OBJECTIVES: Chromosomal instability is a common feature of pancreatic carcinoma, but its biological significance remains unclear. In this study, we investigated the association between chromosomal instability and biological aggressiveness in human pancreatic cancer cells. METHODS: Fluorescence in situ hybridization was performed to examine changes in chromosomal numbers in a total of 13 pancreatic cancer cell lines. We also assessed the potential for tumor aggressiveness within cancer cells by in vitro migration and invasion assay and by subcutaneous implantation into nude mice. RESULTS: Chromosomal instability, characterized by numerical variations in copy numbers of chromosome 8, was observed in most cell lines, and the magnitude of instability was correlated well with both motility (P < 0.001) and invasion rate (P < 0.001) of these cells. Furthermore, a significant positive correlation existed between chromosome instability and tumor growth in vivo (P < 0.01). CONCLUSIONS: These results suggest that the increased level of chromosomal instability may play a critical role in the development of aggressive tumor phenotype during pancreatic cancer progression. J. Surg. Oncol. 2001;76:181-187.

Telomerase activity of cultured human pancreatic carcinoma cell lines correlates with their potential for migration and invasion.

BACKGROUND: Despite the recent clinical finding that high telomerase activity is an unfavorable prognostic marker for various human malignant tumors, there has been no experimental evidence supporting the link between telomerase and tumor aggressiveness. In the current investigation, the authors examined the relation between telomerase activity and potential for biologic aggressiveness in human pancreatic carcinoma cells. METHODS: Telomerase activity was measured in a poorly metastatic cell line HPC-3 and its highly metastatic variant HPC-3H4, as well as in many pancreatic carcinoma cell lines. Aggressive behavior of cancer cells was assessed by in vitro migration and invasion assay. RESULTS: Compared with parental HPC-3, HPC-3H4 displayed higher telomerase activity, which was associated with a scattered phenotype and enhanced migration activity. Furthermore, the authors found that relative telomerase levels correlated well with both motility (P = 0.0041) and invasion (P = 0.0114) in 13 pancreatic carcinoma cell lines. There was, however, no significant association between telomerase activity and cell proliferation. When telomerase activity of KP-1N cells was inhibited by transfection with antisense oligonucleotides, their motility and invasion rates were significantly decreased. CONCLUSIONS: The authors concluded that the magnitude of telomerase activation may reflect the potential for aggressive behavior within cancer cells. These findings support the clinical utility of telomerase activity as a prognostic indicator. Their results also suggest a therapeutic potential for telomerase inhibitors to prevent tumor invasion and possibly metastasis.

Up-regulation of telomerase activity in human pancreatic cancer cells after exposure to etoposide.

Telomerase plays a critical role in the development of cellular immortality and oncogenesis. Activation of telomerase occurs in a majority of human malignant tumours, and the relation between telomerase and vulnerability to drug-mediated apoptosis remains unclear. In this study, we demonstrate, for the first time, up-regulation of telomerase activity in human pancreatic cancer cells treated with etoposide, a topoisomerase II inhibitor. Exposure of MIA PaCa-2 cells to etoposide at various concentrations (1-30 microM) resulted in two- to threefold increases in telomerase activity. Up-regulation was detectable 24 h after drug exposure and was accompanied by enhanced expression of mRNA of the human telomerase reverse transcriptase. Telomerase activation was also observed in AsPC-1 and PANC-1 cells but not in KP-3 and KP-1N cells. Furthermore, we found a negative correlation between increased telomerase activity and the percentage of dead cells after etoposide treatment. These findings suggest the existence of an anti-apoptotic pathway through which telomerase is up-regulated in response to DNA damage. This telomerase activation pathway may be one of the mechanisms responsible for the development of etoposide resistance in certain pancreatic cancer cells.

Chromosomal and extrachromosomal synthesis of exfoliative toxin from Staphylococcus hyicus.

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by Staphylococcus hyicus (SHET) under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxins are distinct from each other is given. The molecular weights of SHET from plasmidless strain P-1 (SHETA) and from plasmid-carrying strains P-10 and P-23 (SHETB) were almost equal. Both of the serotypes of SHET exhibited exfoliation in 1-day-old chickens. The plasmid-cured (P(-)) substrains (P-23C1 and P-23C2) of S. hyicus P-23 did not cause exfoliation in 1-day-old chickens, whereas P(-) substrains (P-10C1 and P-10C2) of strain P-10 caused exfoliation, but they decreased their exfoliative activity. These findings suggest that SHETB was synthesized along with SHETA by strain P-10, whereas the P-23 strain synthesized SHETB alone. The plasmid-carrying strain (P-23) as well as the plasmidless strain (P-1) exhibited the typical clinical signs of exudative epidermitis in pigs. However, plasmid-cured (P(-)) substrains of P-23 (P23C1 and P23C2) did not exhibit the typical clinical signs of exudative epidermitis. These findings suggest that SHETA is synthesized under chromosomal control and SHETB is synthesized under plasmid control and that SHET-producing strains can be divided into three groups: SHETA-producing strains, SHETB-producing strains, and strains producing both toxins.

Cloning of the gene coding for Staphylococcus hyicus exfoliative toxin B and its expression in Escherichia coli.

The Staphylococcus hyicus exfoliative toxin B (SHETB) gene was cloned into pUC118 and expressed in Escherichia coli. The nucleotide sequence of the SHETB gene consists of a coding region of 804 bp specifying a polypeptide of 268 amino acid residues, which included a putative 20-residue signal sequence.

Establishment of a new human pancreatic cancer cell line, NOR-P1, with high angiogenic activity and metastatic potential.

We present here a new cell line, NOR-P1, established from a metastatic subcutaneous tumor of a patient with pancreatic cancer. The cells show rapid growth in culture with a doubling time of 16 h and high migration activity. Genetic and molecular analyses revealed high telomerase activity and a mutation in the K-ras oncogene. Of particular interest, the cells express markedly elevated mRNA levels of angiogenic factors, vascular endothelial growth factor and platelet-derived growth factor, as well as other tumor growth-related factors. Subcutaneous transplantation of the NOR-P1 cells into nude mice formed solid, hemorrhagic tumors which were histologically diagnosed as adenocarcinoma with dense blood vessels and severe extravasation of blood. Furthermore, when NOR-P1 cell suspension was injected directly into the pancreas of nude mice, the cells grew rapidly to form intra-pancreatic tumors associated with liver metastases and peritoneal dissemination that resulted in cachexia and subsequent death. These properties suggest that NOR-P1 is an aggressive pancreatic cancer cell line with a high metastatic potential and may serve as a useful experimental model for studying tumor angiogenesis and metastasis of pancreatic cancer.

Enhancement of drug-induced apoptosis by antisense oligodeoxynucleotides targeted against Mdm2 and p21WAF1/CIP1.

The p53 tumor suppressor gene plays an important role in DNA damage-induced apoptosis and, in general, inactivation of p53 contributes to poor response to chemotherapy. Apoptotic activity of p53 may be negatively modulated by expression of its downstream mediators, including Mdm2 and p21WAF1/CIP1. Consequently, these cellular pathways also represent potential targets for cancer therapy. This study investigated the effect of antisense oligodeoxynucleotides (ODNs), targeted against Mdm2 and p21WAF1/CIP1 on drug-mediated cell killing. Exposure of U2-OS osteosarcoma cells to DNA damaging agents, cisplatin or mitomycin C, caused upregulated expression of Mdm2 and p21WAF1/CIP1. Transient transfection of cells with antisense ODNs to Mdm2 mRNA inhibited Mdm2 protein expression and markedly enhanced apoptotic cell death induced by these drugs. Moreover, when p21WAF1/CIP1 expression was blocked by antisense transfection, drug-mediated cell killing was further accelerated. These results suggest that the enhanced expression of Mdm2 and p21WAF1/CIP1 may inhibit p53-mediated apoptosis and render cells resistant to the effects of DNA damaging agents. Consequently, antisense ODNs targeted against Mdm2 and p21WAF1/CIP1 could be employed in a potential therapeutic strategy sensitizing tumor cells to certain antineoplastic agents.

New functional polar maps for estimating regional cardiac function using ECG-gated technetium-99m-tetrofosmin SPECT.

OBJECTIVE: The purpose of this work was to develop functional parameters to analyze regional cardiac function using ECG-gated 99mTc-tetrofosmin SPECT. Our goal was to develop a methodology that used slice thickness correction, the generation of a time-activity curve and a polar map. METHODS: Fourteen normal patients without evidence of coronary artery disease were studied. One hour after intravenous injection of 740-1110 MBq (20-30 mCi) 99mTc-tetrofosmin, ECG-gated SPECT data were acquired by dividing a cardiac cycle into 12 frames. The SPECT data were reconstructed from 11 of 12 frames into 3 views. The reconstruction of these images was repeated after performing slice thickness correction. Excluding the effect of different apex-to-base lengths at any frame during a cardiac cycle, 10 short-axis images with the same slice thickness were obtained. Each short-axis image was divided by 40 radii into 40 segments. The time-activity curve was generated from the total counts included in each segment plus both neighboring segments. Subsequently the curve fitting was performed using the second Fourier function. RESULTS: From fitted curves and their differentials, we calculated end-systolic count, end-diastolic count, percent count increase, uptake, peak contraction rate, peak distention rate and contraction time. CONCLUSION: The functional polar maps visually demonstrated regional myocardial function. This method is expected to be helpful for assessing regional cardiac function using 99mTc-tetrofosmin.

Diverse effects of 9-hydroxyellipticine on the chemosensitivity of human pancreatic cancer cells harboring p53 mutations.

Recently, it has been shown that 9-hydroxyellipticine (9-HE), an antitumor alkaloid has a unique property of restoring functional wild-type (wt) p53 activity via inhibition of mutant (mt) p53 protein phosphorylation. In the present study, we investigated the effect of 9-HE on the drug sensitivity of human pancreatic cancer cells. Exposure of cells to 9-HE at a relatively low concentration of 1 microM induced almost no cell death but was sufficient to restore wt p53 activity, as evidenced by an induction of endogenous p21WAF1/CIP1 concomitant with G1 and G2/M arrests in cell-cycle progression. Pretreatment with 1 microM 9-HE markedly enhanced cell killing when combined with cisplatin or mitomycin C. In contrast, 9-HE pretreatment protected cells from killing by 5-fluorouracil, VP-16, or vincristine. These effects of 9-HE were specific for several cell lines containing mt p53 and were not observed in p53-negative or wt p53 expressing cells. Taken together, these findings suggest that 9-HE may exert different effects on the drug sensitivity of pancreatic cancer cells displaying p53 mutations possibly through restoration of wt p53.

Inhibition of the production of rat cytokine-induced neutrophil chemoattractant (CINC)-1, a member of the interleukin-8 family, by adenovirus-mediated overexpression of IkappaBalpha.

Cytokine-induced neutrophil chemoattractant (CINC)-1, a counterpart of the human growth-regulated gene product (GRO) of the interleukin-8 family, has been suggested to play critical roles as a mediator of inflammatory reactions with neutrophil infiltration in rats. NF-kappaB has been speculated to be involved in the production of CINC-1, since the NF-kappaB-binding domain is important for the CINC-1 promoter activity in several of our reporter assays. In the present study, we examined the effects of an overexpression of IkappaBalpha, a specific natural inhibitor of NF-kappaB, on CINC-1 production. For this purpose, we constructed two recombinant adenoviruses, AxCAIkappaBalpha and AxCAmutantIkappaBalpha, which express respectively wild IkBa and a mutated nondegradable IkappaBalpha in which serine residues 32 and 36 are replaced by alanine residues. Transfecting wild and mutant IkBa by these adenovirus vectors inhibited NF-kappaB activation and CINC-1 production, which were both caused by IL-1beta stimulation in the normal rat kidney epithelial cell line NRK-52E. We also showed that the nondegradable mutant IkappaBalpha was approximately 30 times more potent than the wild type in inhibiting CINC-1 production. These findings demonstrate that CINC-1 production with NF-kappaB activation is primarily regulated by non-phosphorylated IkBa in the cytoplasm.

Centrosome abnormalities in human carcinomas of the gallbladder and intrahepatic and extrahepatic bile ducts.

During mitosis, 2 centrosomes ensure accurate assembly of bipolar spindles and fidelity of the chromosomal segregation. The presence of more than 2 copies of centrosomes during mitosis can result in the formation of multipolar spindles, unbalanced chromosome segregation, and aneuploidy. Recent studies have provided evidence that centrosome hyperamplification plays a pivotal role in carcinogenesis. Using immunofluorescence analysis with gamma-tubulin and pericentrin antibodies, paraffin-embedded sections from 40 malignant biliary diseases including gallbladder cancers (GC; n = 13), intrahepatic cholangiocellular carcinoma (CCC; n = 19), and extrahepatic bile duct cancers (BDC; n = 8) were examined. Thirty-seven benign biliary diseases including chronic cholecystitis, gallbladder adenoma, hepatolithiasis, and choledochal cyst were included as benign controls. The frequencies of the centrosome abnormalities were 70% for GC, 58% for CCC, and 50% for BDC, respectively. The frequencies of centrosome abnormalities in malignant biliary diseases were significantly higher than in their benign counterparts (GC, CCC, BDC; P =.001,.002, and.001, respectively). The results of current study also indicated that biliary malignancy in the advanced stage (III-IV) displayed a higher frequency of centrosome abnormalities than in the early stage (I-II) (P <.001). We conclude that abnormalities in size, number, and shape of the centrosome are frequently observed in biliary tract malignancy. Centrosome abnormalities started to occur in the early stage of biliary malignancy and became very frequent in the advanced stage. This implies that centrosome abnormality might relate to the transition from early to advanced malignancy in biliary malignancy.

New exfoliative toxin produced by a plasmid-carrying strain of Staphylococcus hyicus.

A new serotype of Staphylococcus hyicus exfoliative toxin (SHET), serotype B, was isolated from the culture filtrate of a plasmid-carrying strain of S. hyicus. The new SHET was purified by precipitation with 70% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column, column chromatography on DEAE-Cellulofine A-500, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The new SHET caused exfoliation of the epidermis as determined by the so-called Nikolsky sign when inoculated into 1-day-old chickens. The new SHET was serologically different from Staphylococcus aureus exfoliative toxins (ETs) (ETA, ETB, and ETC) and from the SHET from the plasmidless strain but showed the same molecular weight as the other serotypes of toxins on SDS-PAGE. It was thermolabile and lost its toxicity after being heated at 60 degrees C for 30 min. We propose that the new SHET be designated SHETB and that the SHET produced by the plasmidless strain be designated SHETA.