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The relationship between sensitivity to anti-cancer agents and EGF receptor expression on squamous cell carcinoma (SCC) cells was investigated. The cytotoxicity of peplomycin (PEP) was correlated with the number of the EGF receptors expressed on the cancer cells, but no correlations were found between the cytotoxicity of adriamycin and cisplatin and EGF receptors. Addition of TNFalpha increased the number of EGF receptors in the SCC cell lines 1.5- to 2-fold. The cytotoxic effect of combined administration of PEP and TNFalpha was correlated with the number of EGF receptors, and produced a 2- to 5-fold increase in IC50 compared with administration of PEP alone. These observations suggest that EGF receptor expression is closely associated with the cytotoxic effect of PEP on SCC cells.

Restriction landmark genomic scanning (RLGS) was utilized to identify novel genomic alterations in hepatocellular carcinoma (HCC). Thirty-one HCC samples were examined by RLGS. Two high intensity spots were common to several RLGS profiles of different HCCs. Nucleotide sequencing and homology search analysis showed that these spots represented repetitive sequences, Human tandem repeat sequence (Genbank, L09552) and centromeric NotI cluster (Genbank, Y10752). These intensified signals were attributable to the occurrence of demethylated areas in the recognition sequence of the NotI site of the corresponding fragments. The intensity of these spots in the RLGS profile reflects their degree of demethylation, which was significantly correlated with postoperative recurrence, even in patients regarded as belonging to the good prognosis group by conventional prognostic factors. Multivariate analysis showed that the intensities of the two spots retained independent prognostic value. This is a new type of predictive factor for HCC based on epigenetic changes in hepatocarcinogenesis, and in the future it is expected to be of great value in making preoperative diagnosis and selecting postoperative therapy.

Esophageal squamous cell carcinoma (ESCC) has high malignant potential with a poor outcome. Lymph node metastasis is the most useful indicator for predicting the outcome of ESCC. The p16/MTS1/CDKN2 gene and the cyclin D1/PRAD-1 gene cooperatively regulate CDK4-mediated phosphorylation of RB protein in the cell cycle. We immunohistochemically detected p16, cyclin D1, and RB expressions in both primary lesions and metastatic lymph nodes in ESCC. Among the 50 ESCC primary lesions, 24 (48%) were positive for p16, while 26 (52%) were negative for p16. Sixteen (32%) were p16-positive, 34 (68%) were p16-negative among the 50 ESCC metastatic lymph nodes. Eight cases (16%) were p16-positive in primary lesion and p16-negative in lymph node, however, no cases that was p16-negative in the primary tumor exhibited p16-positivity in metastatic lymph nodes (p < 0.0001). Seventeen (34%) of the 50 ESCC primary lesions were cyclin D1-positive, while 33 (66%) were cyclin D1-negative. Twenty-four (48%) were cyclin D1-positive, 26 (52%) were cyclin D1-negative among the 50 metastatic lymph nodes. Five cases (10%) were cyclin D1-positive in primary lesion and cyclin D1-negative in lymph node, and 12 cases (24%) were cyclin D1-negative in primary lesion and cyclin D1-positive in lymph nodes. Nine cases (18%) were RB-negative in 50 primary lesions, and the rate of loss of RB expression in metastatic lymph nodes was not markedly higher than in primary lesions. Thirty-nine (78%) of 50 primary lesions and 46 (92%) of 50 metastatic lymph nodes had altered expression of at least one of the three G1 control genes. Tumor cell with disruption of these cell cycle regulators can get a growth advantage and metastatic potential during tumor progression, especially p16/CDKN2 alterations may be associated with lymph node metastasis in ESCC. These results also suggest that tumor cells in metastatic lymph nodes may have more aggressive proliferation and higher malignant potential than tumor cells in primary lesions.

Recombinant human ribonuclease 1 (RNase 1) was chemically linked to recombinant human epidermal growth factor (EGF). The cytotoxicity of this conjugate was assayed using MTT assay. The EGF-RNase conjugate showed dose-dependent cytotoxicity against breast and squamous cell carcinomas overexpressing the EGF receptor (EGFR). The cytotoxicity of the conjugate correlated positively with the level of EGFR expression by each cell line. These results suggest that the EGF-RNase conjugate is a more effective anticancer agent with less immunogenicity and toxicity than conventional chimeric breast cancer toxins.

A hybrid human protein was produced in E. coli by fusing the genes encoding human pancreatic RNase1 (hpRNase1) and human IL-2 (hIL-2). The recombinant hpRNase1-hIL-2 inhibited protein synthesis in HTLV-1-infected, malignant T cells, which hyperproduce high affinity IL-2 receptors, with an IC(50)of 2x10(-8) M, whereas no inhibition was detectable in control cells with lower affinity receptors. HpRNase1 alone had an IC(50)of almost 10(-3) M. A molar excess of hIL-2 blocked the protein synthesis inhibition dose-dependently. In a human mixed lymphocyte culture, hpRNase1-hIL-2 inhibited the proliferation of responder cells with potency comparable to that of cyclosporine, while non-effective doses of FK506 importantly improved its potency. Despite its short half-life in animals, hpRNase1-hIL-2 rapidly enters cells in a few minutes and arrests the protein translation in less than 10 h. Thus, hpRNase1-hIL-2 may be useful to selectively eliminate activated lymphocytes hyperproducing high affinity IL-2 receptors, as in allograft rejection, graft-versus-host disease, autoimmune disorders, adult T cell leukaemia and other lymphoproliferative or retroviral malignancies including HIV infection, without inducing general immunosuppression. As an entirely human "immunotoxin analogue" it may alleviate the dose limiting toxicity and immunogenicity of conventional immunotoxins.

BACKGROUND. Iodine-nonreactive lesions of esophageal epithelium often are associated with dysplasia and carcinoma. The authors examined the usefulness of telomerase activity as an indicator for esophageal carcinogenesis in such lesions. METHODS. Telomerase activity was measured using the telomeric repeat amplification protocol assay in 18 samples of iodine-nonreactive lesions apart from the primary tumor in surgically resected specimens obtained from patients with esophageal squamous cell carcinoma (ESCC) and 55 endoscopic punch biopsies of iodine-nonreactive lesions obtained from 25 patients with ESCC and 30 patients who had undergone endoscopic examination for other reasons. RESULTS. Ten of 18 iodine-nonreactive samples (56%) obtained from surgically resected specimens showed telomerase activity. In all ten telomerase positive samples, carcinoma in situ (CIS) was observed in iodine-nonreactive mucosa by light microscopy. In eight telomerase negative samples, no tumor tissue was observed in iodine-nonreactive lesions. In a parallel study, telomerase activity was detected in 28 of 55 endoscopic punch biopsy specimens (51%). CIS was observed in 25 of 28 iodine-nonreactive lesions with positive telomerase activity (89%), and tumor tissue was not observed in the other 3 samples (11%), which included 2 cases of severe dysplasia and 1 case of moderate dysplasia. No tumor tissue was observed in any of the 27 telomerase negative samples. CONCLUSIONS. Positive and negative telomerase activity was found to be correlated with the presence and absence, respectively, of immortalized tumor cells in iodine-nonreactive lesions. The measurement of telomerase activity in iodine-nonreactive lesions independently contributes to the selection of an appropriate therapeutic strategy.

Pancreatic-type RNases are considered to have cytotoxic potential due to their ability to degrade RNA molecules when they enter the cytosol. However, most of these RNases show little cytotoxicity because cells have no active uptake mechanism for these RNases and because the ubiquitous cytoplasmic RNase inhibitor is considered to play a protective role against the endocytotic leak of RNases from the outside of cells. To study the cytotoxic potential of RNase toward malignant cells targeting growth factor receptors, the C-terminus of human RNase 1 was fused to the N-terminus of human basic fibroblast growth factor (bFGF). This RNase-FGF fused protein effectively inhibited the growth of mouse melanoma cell line B16/BL6 with high levels of cell surface FGF receptor. This effect appeared to result from prolongation of the overall cell cycle rather than the killing of cells or specific arrest in a particular phase of the cell cycle. Thus, human RNase 1 fused to a ligand of cell surface molecules, such as the FGF receptor, is shown to be an effective candidate for a selective cell targeting agent with low toxic effects on normal cell types.

The effect of exogenous atrial natriuretic peptide (ANP) on pulmonary injury induced by aspiration of hydrochloric acid (0.1 N HCl, 2.0 ml/kg) was examined in anesthetized pigs ventilated with 100% oxygen. Group-specific diuretic treatment was used 60 min after aspiration and followed for a further 120 min. No diuretics were used for Group 1, 0.1 microg/kg/min of ANP was infused intravenously for 60 min in Group 2, and 0.05 mg/kg of furosemide was injected (intravenous bolus) in Group 3. Total urine volume after diuretic treatment did not differ significantly between Groups 2 and 3. However, the increase in PaO2 (to 386.3 +/- 67.5 mm Hg) after infusion of ANP was significantly higher than the increase in PaO2 (to 275.9 +/- 63.3 mm Hg) after furosemide treatment, and in the no treatment control (to 171.1 +/- 31.5 mm Hg). Pulmonary hypertension induced by acid aspiration was significantly reduced (p < 0.01 versus Groups 1 and 3) during ANP infusion. ANP has a beneficial effect on acute lung injury, possibly through its diuretic and/or pulmonary vasodilating action.

Objective The purpose of this study was to establish a rapid and sensitive diagnostic method for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia in postoperative patients. Summary background data As a result of diffusion and abuse of third-generation cephalosporin antibiotics in the 1980s in Japan, an outbreak of MRSA infection has been posed. In the field of surgery, severe postoperative infections with MRSA such as MRSA bacteremia, which may lead to multiple organ failure, have emerged with a high mortality. Methods Thirty-five patients with high fever (above 38.5 C) or watery diarrhea or both within 2 weeks after gastrointestinal major surgery and 6 healthy volunteers were examined. Nested polymerase chain reaction was used to detect mecA and toxic shock syndrome toxin-1 (TSST-1) genes in blood specimens. Results The mecA and TSST-1 genes were not detected in the blood samples of any of the six healthy volunteers. In all 12 samples from which MRSA colonies were isolated by blood culture, mecA and TSST-1 genes were detected. Although it took at least 48 hours to identify MRSA by the blood culture method, the presence of mecA and TSST-1 genes was determined by nested polymerase chain reaction method within only 3 to 4 hours after blood sampling. Conclusions This method, as a sensitive and rapid monitoring system for MRS bacteremia, would be clinically beneficial for prevention of cross infection and for early determination of appropriate treatment for infected patients.

Adriamycin (ADM) was chemically conjugated to a murine monoclonal antibody, A0011, which recognizes the c-erbB-2 product, via a disulfide bond using N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and 2-iminothiolane (2-IT). The molar ratio of ADM to the monoclonal antibody ranged from 15:1 to 25:1 and enzyme-linked immunosorbent assay (ELISA) showed that the binding activity of the conjugate was almost retained. We compared the efficacy of A0011 alone, ADM alone, the A0011-ADM conjugate, and a nonspecific murine IgM-ADM conjugate, against the human breast cancer cell lines SK-BR-3, MDA-MB-361, MCF-7, and BT-20. The A0011-ADM conjugate was observed to be ten times more cytotoxic to the cell lines overexpressing the c-erbB-2 product, namely, SK-BR-3 and MDA-MB-361, than free ADM, but it showed weak cytotoxicity against the cell lines with a low level of c-erbB-2 product expression, namely, MCF-7 and BT-20. However, free A0011 and nonspecific murine IgM-ADM conjugate showed no cytotoxicity toward any of the four cell lines, while the addition of a tenfold molar excess of A0011 inhibited conjugate cytotoxicity. These data suggest that conjugate cytotoxicity is antibody-mediated. Moreover, conjugate cytotoxicity at 10−6M was correlated with antigen volume, and the data were fitted to the regression equation y=−11.63logX+116.38 where the correlation coefficient =0.950. Our results indicate that targeting therapy aiming at the c-erbB-2 product may be useful in the treatment of breast cancers overexpressing the c-erbB-2 product.

To determine if there are changes in circulating concentrations of endogenous atrial natriuretic peptide and the physiologic role of this peptide in endotoxic shock.A prospective, randomized, controlled animal trial.University research laboratory.Anesthetized male Wistar rats, weighing 250 to 350 g.Six rats received 1.5 mg/kg body weight of lipopolysaccharide alone. Five rats received 1.5 mg/kg of lipopolysaccharide and 200 microL/100 g body weight of rabbit anti-atrial natriuretic peptide serum. Another five rats received 1.5 mg/kg of lipopolysaccharide and normal rabbit serum in the same volume as the antiserum.Plasma concentrations of atrial natriuretic peptide, arginine vasopressin, and aldosterone were measured, and changes in hemodynamic parameters and renal function were monitored in rats with endotoxic shock after catheterization of the right jugular vein. Urine volume, urine sodium excretion, urinary potassium excretion, and urine 3', 5'-cyclic guanosine monophosphate (cGMP) excretion were measured at 12-hr intervals. The plasma atrial natriuretic peptide concentration was slightly but significantly lower 30 mins after the lipopolysaccharide injection (114.8 +/- 9.0 pg/mL at 0 hr, 75.6 +/- 6.2 pg/mL at 30 mins, p.01) and then began to increase, peaking at 6 hrs (752.8 +/- 104.5 pg/mL, p.01 vs. 0 time) and remaining at higher concentrations than before the preinjection value, up to 24 hrs. In contrast, acute spike-like increases of arginine vasopressin and aldosterone concentrations were observed 30 mins after the lipopolysaccharide injection, preceding the increase of the plasma atrial natriuretic peptide concentration. Measurements of urine volume and urine sodium excretion showed oliguria during the initial 12 hrs after the lipopolysaccharide injection, followed by diuresis and natriuresis during the subsequent 12 hrs. In addition, injection with anti-atrial natriuretic peptide serum in the diuretic phase 12 hrs after the lipopolysaccharide injection significantly inhibited the diuresis, natriuresis, and urine cGMP excretion in this model. Furthermore, the plasma aldosterone concentration 24 hrs after the lipopolysaccharide injection was significantly increased by the administration of the antisera.These findings suggest that endogenous atrial natriuretic peptide increases in the acute phase of endotoxic shock and plays an important role in water and electrolyte balance by regulating diuresis.

p53 gene mutations in early and advanced colorectal cancer were detected by PCR-SSCP analysis. Early colorectal cancer was classified into de novo cancer and polyp-forming cancer, respectively, according to morphology and presence of adenomatous components. A total of 94 paraffin-embedded tissue specimens from patients with colorectal cancer were analysed. p53 gene mutations were detected in 40.0% (12/30) of de novo cancer, 36.7% (11/30) of polyp-forming cancer, and 44% (15/34) of advanced cancer. p53 mutations were detected at a high frequency in both types of early colorectal cancer as well as in advanced cancer. No correlations were found between p53 mutations and clinicopathological data. Our data suggest that the p53 gene mutations occurred in the early colorectal cancer stage of carcinogenesis regardless of the pathway.

Hepatitis B virus (HBV) envelope L proteins, when synthesized in yeast cells, form a hollow bionanocapsule (BNC) in which genes (including large plasmids up to 40 kbp), small interfering RNA (siRNA), drugs, and proteins can be enclosed by electroporation. BNCs made from L proteins have several advantages as a delivery system: Because they display a human liver-specific receptor (the pre-S region of the L protein) on their surface, BNCs can efficiently and specifically deliver their contents to human liver-derived cells and tissues ex vivo (in cell culture) and in vivo (in a mouse xenograft model). Retargeting can be achieved simply by substituting other biorecognition molecules such as antibodies, ligands, receptors, and homing peptides for the pre-S region. In addition, BNCs have already been proven to be safe for use in humans during their development as an immunogen of hepatitis B vaccine. This protocol describes the loading of BNCs and their use in cell culture and in vivo.

OBJECTIVE To clarify how plasma atrial natriuretic peptide concentrations vary with the severity of acute lung injury. The influence of coexisting diseases which trigger acute lung injury was also examined. DESIGN Prospective study. SETTING Intensive care unit of a university hospital. PATIENTS Fifty patients who had standard risk factors for acute lung injury including sepsis syndrome, major surgery, prolonged hypotension, aspiration of gastric contents, and burns. Twenty-five of these patients had acute lung injury (group 3) caused by these disorders; the remaining 25 patients had risk factors only (group 2). Ten age-matched normal volunteers were selected as controls (group 1). INTERVENTION None. MEASUREMENTS AND MAIN RESULTS Plasma atrial natriuretic peptide concentration was measured in these patients and compared with the severity of acute lung injury. In group 3, a significant increase in the mean plasma atrial natriuretic peptide concentration was observed (188 +/- 78 pg/mL, p < .01) compared with group 2 (54 +/- 28 pg/mL) and the age-matched control group (30 +/- 8 pg/mL). This increase was related to the onset of acute lung injury and returned to control concentrations after recovery. Plasma atrial natriuretic peptide concentrations in group 3 correlated highly with a lung injury score representing the severity of acute lung injury (r2 = .45, p < .01), but did not correlate with other cardiopulmonary variables. CONCLUSION The results suggest that severity of lung injury, but not other predisposing disorders, may be the key factor leading to the increase in plasma atrial natriuretic peptide concentrations observed in these patients.

Nerve growth factor (NGF), a target-derived neurotrophic molecule, is required specifically by sympathetic and dorsal root ganglion cells for their survival and maturation during embryonic and early postnatal development. In the present study, the NGF expression was studied both at the protein and mRNA level in normal and aganglionic intestines of Piebald-strain mice and also in 10 human specimens using immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR) techniques. In the aganglionic intestines of the mice, immunoreactive NGF was found on the giant nerve fibers in the submucosal layer, but not found in the mucosal layer. In the mRNA study, the signal for NGFmRNA was less intense in the aganglionic rectum of the congenitally megacolonic mice than in the rectum of the normal mice. In contrast, the distal dilated colon of the congenitally megacolonic mice had a more intense signal for NGFmRNA than did the colon of normal mice. The results obtained from human specimens were compatible with the findings in the Piebald mice; the distal colons harvested from the patients with Hirschsprung's disease (or its allied disease) had a uniformly more intense signal for NGFmRNA than did the normal colons. The results of this study may indicate that NGF production is altered in the aganglionic intestines and also in the "transitional zone" in Hirschsprung's disease. The altered production of NGF may be useful in increasing the accuracy of diagnosis of Hirschsprung's disease.

The efficacy, safety and usefulness of murine anti-endotoxin monoclonal IgM antibody "E5, an intravenous dose of 2 mg/kg" were evaluated in 88 patients with suspected Gram-negative sepsis from 37 institutes in Japan. Out of these, 74 patients were evaluable for the efficacy, 85 for safety and 75 for clinical usefulness. In assessing the efficacy, the patients were divided into 3 groups based on the plasma endotoxin levels (Endospecy with new PCA treatment of plasma): H group with a level of above 9.8 pg/ml and M group with a level of 3.0-9.8 pg/ml and L group with a level of below 3.0 pg/ml. 1. The efficacy rates as assessed following administration of E5 were 73.1% in the H group, 70.4% in the M group and 38.1% in the L group being higher in the groups with significantly high plasma endotoxin levels. 2. In both the H and M groups in whom plasma endotoxin levels were significantly high, the majority of the patients showed rapid reduction of the levels after administration of E5. 3. In all groups, improvement in body temperature, pulse rate, blood TNF-alpha and blood IL-6 was observed after treatment with E5. In the H and M groups with an endotoxin level of > or = 3.0 pg/ml, improvement in platelet count as well as in CRP was noted. The H group showed also improvement in WBC. 4. Improvement in the shock score was noted in all the groups but was more outstanding in the H and M groups in the early stage of treatment. 5. Side effects were seen in 5 (5.9%) of 85 patients and all thought to be allergic in symptoms such as rash, itching, fever and flare. 6. The reaction to the prick test performed before administration of E5 was negative in all these 5 patients. For 3 of the 5 patients, anti-E5 IgE antibody was measured. In all of them, the IgE levels were higher than those of healthy controls. Also, in 47.6% of patients, an elevation of anti-E5 IgG antibody was noted two weeks after the administration. 7. Clinical laboratory abnormalities were observed in 3 (3.5%) of 85 patients. They were an elevation of S-GOT.S-GPT and lowering of BUN, increased Al-p and decreased CH50, increased neutrophilia (%) and were all slight in the degree of the changes. 8. The clinical usefulness of E5 was evaluated for 75 patients.(ABSTRACT TRUNCATED AT 400 WORDS)

In Japan, in order to clinically apply a bioartificial liver (BAL), the necessity of existing therapeutic conditions needs to be sufficiently understood. From a clinical perspective, the BAL would prove useful if it could be successfully applied in the treatment of post-operative and fulminant hepatic failure, in "partial" adult-to-adult living-donor liver transplantation, etc. In order to develop the BAL device in Japan, understanding of Japanese medical conditions is indispensable. We consider that Japanese BAL researchers should aim to develop a device that supplies the liver with regenerative factors. Thus, a Japan-originated BAL device will exceed its Western counterparts.

[Introduction] Many cancer related genes in human cancer have been isolated by recent development of techniques in molecular biology, however, the existence of unknown genes still have been expected. Restriction landmark genomic scanning (RLGS) is one of the most powerful methods for the identification of a DNA methylation which correlate with protein expression level. This DNA analysis method is 2-dimensional gel electrophoresis of genomic DNA fragment digested with specific restriction enzyme. We have evaluated clinical samples such as breast cancer, hepatocellular carcinoma (HCC), glioma and colorectal cancer for more than 10 years. In this study, we evaluated RLGS to find genetic alterations relative to human colorectal cancer and cloned some of altered spots. We have found that one of these clone codes a part of dynein axonemal heavy chain 1 (DNAH1). Dyneins are large motor protein complexes that generate the force for the movement of eukaryotic cilia and flagella. [Materials and Methods] High molecular weight genomic DNAs were extracted from 25 colorectal cancerous tissues and corresponding normal mucosae. To analysis alteration spot, each DNA was cleaved with the restriction enzyme Not I, size fractionated by 1st-dimensional electrophoresis using Pvu II, and the DNA fragments were then cleaved by Pst I as the third enzyme in gel and separated by 2-dimensional electrophoresis. Various cell lines, such as A431, AsPC-1, TE10, MCF7, and WiDr were used for in vitro analysis. To examine gene expression of DNAH1, quantitative RT-PCR was performed using TaqMan® Gene Expression Assays. Cell migration was measured using Boyden chamber migration assay for vertical migration and EZ-TAXIScan (GE Healthcare Japan) for horizontal migration. AsPC-1 cells and AsPC-1 shRNA DNAH1 stable cells were used for xenograft model to evaluate function of DNAH1 in vivo. [Results] 19 of 25 (76.0%) colorectal cancer cases showed a spot with the same alteration, which never appeared in normal mucosae. A 410-bp DNA fragment corresponding to this spot was cloned. This sequence was found to be 98% homologous to part of dynein axonemal heavy chain 1 (DNAH1). Gene expression level of DNAH1 was correlated to cell motility in vitro migration assays. In addition, DNAH1 expression level and cell motility were altered by treatment of 5-azacytidin (a protein inhibitor of DNA methyltransferase) or siRNA of DNAH1. Histological and histopathological evaluation of xenogragt model showed that AsPC-1 is INF beta and shRNA DNAH1 stable AsPC-1 is INF alpha. [Conclusion] We have identified the genomic alteration specific in human colorectal cancer by RLGS. The expression of homologpus gene to DNAH1 is closely related to colorectal cancer. Moreover, DNAH1 might not be express only in colorectal cancer but also other cancers. Expression level of DNAH1 related to cancer cell motility and metastasis in vivo and in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2011-4814

Background and Objective: The NF-kB/Rel family of transcription factors is involved in the development of cancer. This family is constitutively activated in many human pancreatic cancers and pancreatic cell lines. DHMEQ inhibits NF-κB activity by preventing nuclear translocation and has been shown anti-tumor effect. However, because of its hydrophobic property, DHMEQ has to be administered intraperitoneally or subcutaneously every day. Therefore, it is necessary to develop effective drug delivery system (DDS) of DHMEQ for clinical application. In this study, we have used PMBN polymer, which forms a stable polymer aggregate in water that can enhance the solubility of hydrophobic substance such as DHMEQ. The active ester group of PMBN can be replaced by proteins such as anti-epidermal growth factor receptor (EGFR) antibody. It is expected that a selective increased accumulation of PMBN-DHMEQ antibody conjugate could lead to a reduction of its dose and/or numbers of administration. The aim of this study is that investigate active enhancement of anti-cancer effect by DHMEQ using PMBN. Methods: The human pancreas adenocarcinoma cell lines, AsPC-1, BxPC-3, MIAPaCa-2, which showed high expression level of EGFR were used in vitro study. DHMEQ, PMBN and mouse monoclonal anti-human EGFR antibody (528) were mixed at the weight ratio of 1:25:5 and sonicated to prepare PMBN-DHMEQ antibody conjugate in advance. Control DHMEQ was dissolved in 0.5% chrolomethylcellulose (CMC-DHMEQ). The effect of cell growth inhibition by each preparation was evaluated by WST assay in vitro. AsPC-1 cells which expressing Gaussia luciferase (AsPC-1-Luc) was used to investigate anti-tumor effect in vivo. 5×105 of AsPC-1-Luc was injected into nice portal vein to develop liver metastases model of pancreatic cancer. Mice were given PMBN-DHMEQ antibody conjugate (intravenously) or CMC-DHMEQ (intraperitoneally) every three days at a dose of 12mg/kg DHMEQ. Metastases level of each group was compared by measuring luciferase activity of mice whole liver homogenate using Renilla Luciferase Assay System (Promega). Result: PMBN-DHMEQ antibody conjugate showed higher cell growth inhibition than CMC-DHMEQ in vitro WST assay. Also it showed stronger growth inhibition effect than CMC-DHMEQ in vivo. Quantitative luciferase assay showed that PMBN-DHMEQ antibody conjugate have 49.2 times higher effect compare to CMC-DHMEQ. Conclusion: Our results demonstrate that the PMBN-DHMEQ-anti-EGFR antibody conjugate can efficiently decrease cancer development of cells overexpressing EGFR by an active enhancement effect. This result suggest that increased accumulation of PMBN-DHMEQ anti-EGFR antibody conjugate increased tumor concentration of DHMEQ and the therapeutic response. PMBN-mediated DDS, which in turn reduce its dose and/or numbers of administration of DHMEQ. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3223. doi:10.1158/1538-7445.AM2011-3223

Introduction: Esophagectomy is one of the most invasive surgery for digestive cancer, and the blood level of inflammatory cytokines such as IL-1, IL-6 and IL-8 arise several hours after surgery or in suffering postoperative complications. It is known that postoperative complications are negative predictors in long-term malignancy outcome. It has been reported that IL-8 receptor, one of the chemokine receptor, is expressed on several carcinomas and IL-8 signaling promotes proliferation of cancer cells, however, it is poorly understood in esophageal squamous cell carcinoma (ESCC). In this study, we demonstrate the significance of IL-8 receptor expression and validate the effect of IL-8 receptor expression and postoperative complications on long-term prognosis in ESCC using resected specimens and cancer cell lines. Materials and Methods: Eighty-two specimens were sectioned from archived, paraffin-embedded tumor tissues obtained from patients with ESCC underwent esophagectomy and extended lymphadenectomy to have no residual cancer in our institute between 1997 and 2002. These were stained using polyclonal antibody to IL-8 receptor, and correlation between stainability and disease-free survival as well as overall survival was examined. TE4, the human ESCC cell line with the high expression of IL-8 receptor at protein levels and TE11, the one with the low expression were cultured at 1×105cells/mL in medium. Cells were transferred to the 96-well microtitre plates containing diluted IL-8. Plates were incubated for indicated periods and WST assay was performed to evaluate the proliferation of ESCC cells. Four groups of 5 nude mice were injected subcutaneously in the dorsal flank with 3×106 cells (TE4, TE11), and 1 of 2 groups were received intraperitoneal (i.p.) injection of 300ng of IL-8 everyday. Tumor volume was measured twice weekly with a caliper in 2 weeks. Results: IL-8 receptor was expressed in 33 specimens of 82 (40.2%). The patients with IL-8 receptor expression in primary tumor had shorter disease-free survival compared to those with negative expression. Especially, the patients with IL-8 receptor expression who suffered postoperative complications such as pneumonia and ruptured suture had a poorer prognosis (p=0.013). TE4 cells in IL-8 administered group proliferated significantly more than those in control group (p Tumor growth of TE4 in i.p. group was significantly larger than that in control group (p=0.028). Conclusion: Our results suggest that the expression of IL-8 receptor is associated with the proliferation of ESCC. The patients with IL-8 receptor expression who suffered postoperative complications probably need strict follow-up and aggressive adjuvant therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1205. doi:10.1158/1538-7445.AM2011-1205

Squamous cell carcinoma is by far the most common neoplasm of the esophagus and the survival rate of its patients is generally low [1]. To treat this cancer more effectively, it is necessary to develop a strategy of targeted cancer therapy. Most squamous carcinoma cells hyperproduce epidermal growth factor (EGF) receptor [2, 3], and EGF in turn promotes the growth of EGF receptor-hyperproducing squamous carcinoma cells in vivo [4]. Moreover, an elevated EGF receptor level is a significant prognostic factor for patients with esophageal squamous cell carcinoma [5]. Therefore, since EGF and its receptor system play an important role in the growth of this cancer, EGF receptor is an ideal cell surface object for targeted therapy. We synthesized an immunotoxin consisting of murine monoclonal antibody (B4G7) against human EGF receptor and gelonin, a 60s ribosome-inactivating protein. We studied the cytotoxic effects of the immunotoxin on squamous carcinoma cells and tumors, and investigated the possibility of it as a new treatment for squamous cell carcinoma.

Activation of the nuclear transcription factor-kappaB (NF-κB) has been implicated in the progression and metastasis of pancreatic cancer. This study was designed to evaluate the effects of a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in the inhibition of liver metastasis of pancreatic cancer in a clinical liver metastasis-simulating mouse model in which cancer cells were intra-portal injected via small laparotomy. Moreover, synergistic effect of combination therapy with DHMEQ and gemcitabine was investigated. Male BALB/c nude mice were xenografted with intra-portal vein injection of AsPC-1. Mice were divided into four groups; 1) positive control (PC), without any treatment, 2) gemcitabine treated group (GEM), 3) DHMEQ treated group, and 4) combined treatment group (GEM+DHMEQ). Mice were fed for four weeks, and the number of liver metastases, induction of apoptosis and angiogenesis in metastasis foci were investigated after sacrifice. The number of liver metastases decreased significantly in the GEM group, DHMEQ group and GEM+DHMEQ group when compared with the PC group. Significant inductions of apoptosis were revealed in the GEM group and DHMEQ group compared to the PC group, in which apoptotic bodies in metastasis sections were counted by using the TUNEL method. Moreover, in the DHMEQ+GEM group, synergistic effect of combination therapy was observed in induction of apoptosis. Tumor vessels were indentified by immunohistochemical staining of tumor metastasis sections with antibody against CD31. There was a large reduction in the number of CD31-positive vessels in the tumor of DHMEQ or/and GEM-treated mice as compared with control group. Gene expression in metastasis sites was investigated by quantitative RT-PCR. DHMEQ down-regulated IL-8 and MMP-9 remarkably, while gemcitabine down-regulated VEGF moderately. The differences in down-regulated target genes and mechanisms of anti-tumor effects between DHMEQ and gemcitabine point towards the validity of combination therapy with DHMEQ and gemcitabine. In our intra-portal vein injection model, which mimics liver metastasis of pancreatic cancer very well, DHMEQ exhibited anti-tumor effect by inhibiting angiogenesis, invasiveness of tumor cells, and induction of apoptosis. Moreover, when DHMEQ and gemcitabine were administered simultaneously, our result suggested the possibility of a synergistic effect. DHMEQ is lipophilic agent and insoluble in common solvents. Thus, DHMEQ might be a potent drug for the treatment of pancreatic cancer, although in the case of clinical application, some difficulties remain to be solved, such as a dissolution method and an administration route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4058.

[Purpose] Previous reports suggest that chemokine receptor CCR7 may be involved in lymph node metastasis, but it remains unclear what an important role CCR7 expression plays in patients with esophageal squamous cell carcinoma (ESCC) because we have so far only a few reports available. Here, we investigated how CCR7-expressing cells behaved in response to its specific ligand CCL21/SLC in vitro and vivo and analyzed clinicopathological relationship in patients with ESCC. [Materials and Methods] Before starting experiments, we assessed CCR7 mRNA in 10 ESCC cell lines (TE series) by quantitative RT-PCR and subsequently adopted TE4 for further functional studies. Additionaly, we generated a modified TE4 cell line that overexpressed CCR7 (TE4CCR7+). First, we performed a cell migration assay using EZ-TAXIScan®, a cell mobility analyzer which enables us to observe migrating cells directly by a CCD camera. TE4 cells were seeded into a chamber containing serum-free medium with 300 ng/ml of CCL21/SLC and filmed every 3 minutes for 12 hours. Next, we examined how the CCR7-expressing cells behaved in vivo using lymph node metastatic model mice. The model mice were developed as follows: 1) subcutaneously growing tumors were established by injecting 1×107 tumor cells of native TE4 and TE4CCR7+ in nude mice, 2) the tumors were excised and a small piece of which was transplanted in the cubitals of other nude mice, 3) axillary lymph nodes were excised and examined three, four, five and six weeks after transplantation in which metastatic cells were detected by quantitative RT-PCR using a human ALU sequence. In a clinicopathological study, we analyzed 105 ESCC specimens with immunohistochemical staining, which specimens were obtained from patients who had undergone radical esophagectomy in our hospital during 1997 to 2002. [Results] The cell migration assay showed increased cell migration ability in CCR7-expressing cells. A VD plot analysis of the assay revealed a significant increase in cell motility. The mice transplanted with TE4CCR7+ cells developed lymph node metastases precociously compared with the mice with native TE4 cells. In the clinicopathological analysis, two researchers found CCR7 expression in 28 patients (27%), which positive expression was significantly associated with lymph node metastasis (p=0.04), five-year recurrence-free rate (positive 25% vs negative 52%, p [Conclusion] These results strongly suggest that CCR7, in part, plays a critical role in lymph node metastasis and subsequent poor prognosis in patients with ESCC. CCR7-targeted therapy can be one of promising treatments for lymph node metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3306.

Preclinical evidence has suggested that anticoagulants, including the unfractionated heparin (UFH), exert an anti-tumor effect, whereas the role of anticoagulants in tumor development and metastatic processes has not been fully elucidated yet. Heparin is the major inhibitor of heparanase, which cleaves heparan sulfate and hence participates in degradation of the extracellular matrix. This enzyme also releases a lot of biologically active substance including some growth factors, cytokines, adhesion molecules, and angiogenic factors. Thus, heparin can affect proliferation, migration, and invasion of cancer cells. Moreover, heparin can interfere with adherence of cancer cells to vascular endothelium. This study was designed to evaluate the effects of UFH in the inhibition of liver metastasis of pancreatic cancer. We made a clinical liver metastasis-simulating mouse model, in which cancer cells were intra-portal injected via small laparotomy. Moreover, synergistic effect of combination therapy with UFH and gemcitabine was investigated. Male BALB/c nude mice were xenografted with intra-portal vein injection of AsPC-1, a human pancreatic adenocarcinoma cell line. Mice were divided randomly into four groups; 1) positive control (PC) without any treatment, 2) gemcitabine treated group (GEM), 3) UFH treated group, and 4) combined treatment group (GEM+UFH). All mice were sacrificed on 28th day after transplantation of cells. The liver weight, the number of liver metastases, induction angiogenesis in metastasis foci were investigated. Heparan degrading enzyme, heparanase, were activated in liver metastasis, in which activity were measured by using the Heparan Degrading Enzyme Assay Kit. Gemcitabine inhibited tumor proliferation, whereas UFH little inhibited in vitro (WST assay). The number of liver metastases decreased significantly in the GEM (17.2±11.5) group, UFH (18.3±3.8) group and GEM+UFH (9.3±2.5) group when compared with the PC group (23.3±7.0). Liver/body weight ratio was lower in the GEM (7.44±1.36 %) group, UFH (7.56±1.47 %) group and GEM+UFH (6.98±0.63 %) group when compared with the PC group (7.99±0.94 %). Tumor vessels were identified by immunohistochemical staining of tumor metastasis sections with antibody against CD31. There was a large reduction in the number of CD31-positive vessels in the tumor of UFH or/and GEM-treated mice as compared with control group. UFH exhibited anti-tumor effect by inhibiting angiogenesis, and invasiveness of tumor cells. Furthermore, when UFH and gemcitabine were administered simultaneously, our results suggested the possibility of a synergistic effect. Thus UFH might be a potent drug for the treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4175.

Equinatoxins were purified from the tentacles and bodies of the sea anemone Actinia equina by the use of acetone precipitation, as well as column chromatographies on Sephadex G-50 and CM-cellulose according to the modified method of Macek and Lebez (1988). The equinatoxins obtained, equinatoxin 1 and 2, were hemolytic glycoproteins with a relative molecular mass of 20000. Equinatoxin 2 was found to be rich in glycine, alanine and valine. The amino acid sequences of equinatoxins 1 and 2 were partially determined. A portion of the N-terminal amino acid sequence of equinatoxin 2 was similar to those of pyruvate kinase and sialidase.

Extensive resection of the bile ducts combined with hepatic resection is the procedure of choice for carcinoma of the main hepatic duct junction. Currently this procedure is done without great risk, and increasing long-term survival is now the issue. For this purpose, it is necessary to elucidate the biologic properties of the cancer to take reasonable measures. Autopsy findings of patients who died of recurrence may offer a reliable guide. Autopsy findings were studied in 14 patients: eight who had undergone curative resection and six who had received noncurative resection. Cancer recurred at the liver hilum with invasion into adjacent organs. Peritoneal dissemination and lymph node metastases were infrequent. These were common findings in both curative and noncurative resection groups. Cancer cells in the connective tissue of the hepatoduodenal ligament may play a major role in recurrence.

We report a case of a 74-year-old male with a trichilemmal tumor arising in a seborrheic keratosis on the buttock and the results of a cell kinetic study of this tumor using a BrdU staining method. The incidence of trichilemmal tumor arising in a seborrheic keratosis seems to be extremely rare. The labeling index of this tumor was 12.0%; this was a level intermediate between normal epidermis and a variety of hyperproliferative skin diseases such as squamous cell carcinoma, Bowen's disease, and psoriasis vulgaris. DNA replicating cells were present in the germinative layers in normal epidermis and the benign hyperproliferative skin diseases, psoriasis vulgaris. In contrast, DNA replicating cells were found throughout the entire epidermis in premalignant and malignant tumors such as in Bowen's disease and squamous cell carcinoma. In this case, DNA replicating cells were localized mainly in the basal and parabasal cell layers, but also seen in the upper squamous layers. These findings suggest that this trichilemmal tumor had a malignant tendency, though it was slow-growing and relatively benign in nature.

Hypovolemia, low cardiac output, and systemic vasoconstriction are major etiologic factors in acute renal failure occurring in the early postburn period, and elevated levels of stress-related hormones (catecholamines, angiotensin, aldosterone, and vasopressin) are implicated in the mechanism. By counteracting the effects of the hormones, atrial natriuretic polypeptide (ANP) regulates the renal response to burns. ANP was elevated after burns, protecting the kidneys by increasing renal blood flow and urine output. In pulmonary acid injury, increased ANP levels were associated with natriuresis which was reduced by administration of anti-ANP serum. Exogenous ANP given to dogs under constant norepinephrine infusion resulted in improvement of hemodynamic and renal parameters. To prevent tubular damage due to hemoglobinuria, a haptoglobin preparation is administered to patients with extensive third-degree burns. With sufficient fluid replacement, these new treatments will reduce the incidence of acute renal failure in the early postburn period.

summary The localization of DNA replicating cells, epidermal growth factor (EGF) receptor-expressing cells and ras oncogene product p21 (p-21ras) positive cells were examined in various skin tumours to elucidate the role of EGF receptor and p21ras in the epidermis. Normal skin, keratoacanthoma (KA), solar keratosis (SK), Bowen's disease (BD), squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and extramammary Paget's disease (PD) were studied. EGF receptors were seen in proliferating layers, where DNA replicating cells localize, but p21ras was found in the more differentiated layers. We conclude that EGF receptor expression is closely associated with cellular proliferation, but p21ras may play a role in the differentiation of cells in various skin tumours.

Correction to: Cancer Gene Therapy (2007) 14, 74–81. doi: 10.1038/sj.cgt.7700990 Since the publication of the above article, the authors have identified an error in the title of Figure 4d. The correct title, WiDr: PBS, is shown below. The authors would like to apologize for this mistake.