bluuebunny/crossref_metadata_2025 huggingface dataset:
Transgenic Rats Carrying Human c‐Ha‐ras Proto‐oncogene Are Highly Susceptible to N‐Nitrosomethylbenzylamine Induction of Esophageal Tumorigenesis
Authors: Asamoto Makoto , Toriyama‐Baba Hiroyasu , Ohnishi Takamasa , Naito Akihiro , Ota Tomonori , Ando Akira , Ochiya Takahiro , Tsuda Hiroyuki
Published: August 2005

A transgenic rat line carrying three copies of the human c‐Ha‐ras proto‐oncogene, including its own promoter region, has been established in our laboratory (Hras128 rats), and shown to be highly susceptible to induction of mammary and urinary bladder tumors. Mutation analysis of induced lesions indicated the majority to contain some but not all cells with transgene mutation. In the present study, the susceptibility of Hras128 rats to N‐nitrosomethylbenzylamine (NMBA) induction of esophageal tumors was examined with a similar mutation analysis of the transgenes. Male 6‐week‐old Hras128 and wild littermate rats were treated with NMBA, 0.5 mg/kg subcutaneously, 3 tunes a week for 5 weeks and then maintained for 5 weeks without any further treatment. Multiple esophageal tumors, squamous cell pappillomas and carcinomas, rapidly developed within this 10–week experimental period in Hras128 rats (11.05+7.83/rat). In contrast, wild‐type litter‐mates had only small numbers of mostly benign tumors (1.67+2.06/rat). The Hras128 rats had no other tumors or abnormalities. In their esophageal lesions, codon 12 GGC to GAC mutations of the transgene were frequently detected by restriction fragment length polymorphisms (RFLP) and subsequent direct sequencing analysis (19/25, 76%). In the endogenous rat c‐Ha‐ras gene they were less frequent (2/25, 8%), than in wild‐type rats (8/14, 57.1%). The densities of mutated bands in the RFLP analysis indicated that mutated cells were major populations in tumors, in contrast to the case with mammary and urinary bladder lesions. Furthermore, activated ras protein, detected by binding to raf protein, was clearly increased in tumors as compared to surrounding epithelium or the normal esophagus of untreated rats. The results show that Hras128 rats are highly susceptible to NMBA esophageal carcinogenesis, as well as induction of mammary and urinary bladder tumors, but that tissue‐specific characteristics exist for the roles of transgene ras mutations.
U‐shaped Effect of Drinking and Linear Effect of Smoking on Risk for Stomach Cancer in Japan
Authors: Kikuchi Shogo , Nakajima Toshifusa , Kobayashi Osamu , Yamazaki Tatsuo , Kikuichi Masahiro , Mori Kiyoshi , Oura Shinsuke , Watanabe Hideaki , Nagawa Hirokazu , Otani Reiji , Okamoto Naoyuki , Kurosawa Michiko , Anzai Haruyuki , Konishi Toshiro , Futagawa Shunji , Mizobuchi Noboru , Kobori Oichiro , Kaise Ruriko , Inaba Yutaka , Wada Osamu
Published: August 2005

A case‐control study was conducted to evaluate the relationship between smoking or drinking doses and risk for stomach cancer, and to clarify whether the relationship is dose‐dependent or U‐shaped. Smoking dose was categorized as 0,1–399, 400–799, or 800+ cigarette‐years, and drinking dose as 0, occasional/0.1–134.9, 135–1349.9, or 1350+ alcohol‐years (ml of pure alcohol intake per day multiplied by years of drinking). Helicobacter pylori status was determined by serology for adjustment. Using logistic regression, the adjusted effects of smoking and drinking doses on risk for stomach cancer were calculated for both genders. Among male subjects, the odds ratios (95% confidence intervals (CIs)) were 1.29 (0.76, 2.18) for 1–399, 1.71 (1.05, 2.80) for 400–799 and 2.46 (1.49, 4.07) for 800+ cigarette‐years compared with never‐smokers, and 1.89 (0.97, 3.69) for never‐drinkers, 2.82 (1.63, 4.86) for 135–1349.9 and 2.84 (1.97, 4.83) for 1350.0+, compared with occasional/0.1–134.9 alcohol‐years. Among female subjects, they were 0.44 (0.20, 1.00) for 1–399 and 2.471 (0.91, 6.68) for 400+ cigarette‐years compared with never‐smokers, and 1.54 (0.90, 2.63) for never‐drinkers and 1.39 (0.66, 2.93) for 135.0+ alcohol‐years. Smoking seems to exert a linear effect and drinking, a J‐ or U‐shaped effect on risk for stomach cancer, although there might be a dip of risk in light smokers among female subjects.
Appearance of Osteonectin‐expressing Fibroblastic Cells in Early Rat Stomach Carcinogenesis and Stomach Tumors Induced with N‐Methyl‐N′‐nitro‐N‐nitrosoguanidine
Authors: Maeng Hack‐Young , Choi Dong‐Kug , Takeuchi Motoi , Yamamoto Masami , Tominaga Michiyo , Tsukamoto Tetsuya , Tatematsu Masae , Ito Takashi , Sakaki Yoshiyuki , Furihata Chie
Published: August 2005

The present study was designed to define molecular alterations in the initiation stage of rat stomach carcinogenesis. Groups of male Lewis rats, 6 weeks old, were given drinking water with or without N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG; 100 mg/liter). Total RNA was isolated from the stomach pyloric mucosa, and fluorescent differential display analysis was performed. A cDNA fragment of 125 bp encoding an extracellular matrix‐associated matricellular glycoprotein, osteonectin, was identified after 14 days of MNNG exposure. A severalfold increase in expression was observed after 14 and 27 days of MNNG exposure, as determined by northern blot and RT‐PCR. Immunohistochemistry revealed that osteonectin‐mAb‐stained flbroblastic cells appeared in interstitial tissue of pyloric mucosa. Additionally the gene expression of other extracellular matrix proteins, viz., collagen type III, fibronectin, osteopontin, proteoglycan NG2, laminin γ1 and S‐laminin, was also markedly increased, as determined by competitive RT‐PCR after 14 days of MNNG exposure. The gene expression of osteonectin and the six other extracellular matrix proteins was elevated in twelve stomach adenocarcinomas and adenomas induced by MNNG in Lewis and WKY rats. Osteonectin‐mAb‐stained flbroblastic cells were evident in interstitial tissue of stomach tumor. These results suggest that osteonectin‐expressing flbroblastic cells appear in the interstitial tissue of pyloric mucosa from the early initiation stage of rat stomach chemical carcinogenesis, and that this phenomenon probably plays a role in cancer development.
Invasion of Melanoma in Double Knockout Mice Lacking Tenascin‐X and Tenascin‐C
Authors: Matsumoto Ken‐ichi , Takahashi Kazuhisa , Yoshiki Atsushi , Kusakabe Moriaki , Ariga Hiroyoshi
Published: August 2005

The roles of extracellular matrix glycoproteins belonging to the tenascin family in the regulation of tumor cell proliferation, invasion, and metastasis are not known. To address this issue, we generated tenascin‐X (TNX) and tenascin‐C (TNC) double knockout mice and compared findings in these mice with those in single knockout (TNX+/+TNC‐/‐ and TNX‐/‐TNC+/+) mice. We investigated the proliferation and invasion of B16‐BL6 melanoma cells after these cells had been injected into the footpads of mice in each group. The primary tumor size and invasion to the ankle adjacent to the primary tumor site were examined at 35 days after injection of the melanoma cells. The primary tumor size in TNX‐/‐TNC+/+ mice was significantly larger than that in wild‐type mice, but those of TNX+/+TNC‐/‐ and double knockout mice were comparable to that in the wild‐type mice. On the other hand, invasion to the ankle was obviously promoted in TNX‐I‐ TNC+/+ and double knockout mice compared with that in the wild‐type mice, but invasion to the ankle in TNX+/+TNC‐/‐ mice was only slightly promoted. Gelatin zymography confirmed increased matrix metalloproteinase (MMP)‐9 activity in the dorsal skin of TNX‐/‐TNC+/+, TNX+/+TNC‐/‐ and double knockout mice. However, the amounts of MMP‐9 mRNA in the dorsal skins of all mice were almost the same, indicating that the increased activity of MMP‐9 in the single and double knockout mice is regulated at the MMP‐9 processing level. These results indicate that MMP‐9 is activated in all TN‐deficient mice, but that TNX exerts a greater effect on tumor invasion than does TNC.
Ras Farnesylation Inhibitor FTI‐277 Restores the E‐Cadherin/Catenin Cell Adhesion System in Human Cancer Cells and Reduces Cancer Metastasis
Authors: Nam Jeong‐Seok , Ino Yoshinori , Sakamoto Michiie , Hirohashi Setsuo
Published: August 2005

The E‐cadherin/catenin cell adhesion system is often down‐regulated in epithelial tumors. This is thought to play an important role in cancer invasion and metastasis, and restoration of this system may suppress metastatic spread of cancer. In this study, the effects of a Ras farnesylation inhibitor (FTI‐277) on E‐cadherin‐mediated cell‐cell adhesion and metastatic potential were examined. In cell aggregation assays, FTI‐277 stimulated aggregation of colon, liver and breast cancer cells. In vitro cultures of cancer cells showed that FIT‐277 induced strong cell‐cell contact. Immunoblotting analysis showed that FTI‐277 increased E‐cadherin/catenin (α, β and γ) expression and strongly stabilized E‐cadherin/catenin with the actin cytoskeleton. Northern blotting studies indicated that the observed increase in the E‐cadherin/catenin protein content was due to increased expression of their genes. After inoculation of the spleens of mice with severe combined immunodeficiency (SCID) with cancer cells, FTI‐277 treatment for 3 weeks markedly reduced splenic primary tumor growth and the rate of liver metastasis compared with control counterparts. Our data demonstrate that FTI‐277 can activate functioning of the E‐cadherin‐mediated cell adhesion system, which is associated with suppression of cancer cell metastasis. Therefore, selective inhibition of Ras activation may be useful for preventing cancer metastasis.
Reversal of P‐Glycoprotein‐mediated Paclitaxel Resistance by New Synthetic Isoprenoids in Human Bladder Cancer Cell Line
Authors: Enokida Hideki , Gotanda Takenari , Oku Shoichi , Imazono Yoshiharu , Kubo Hiroyuki , Hanada Toshikatsu , Suzuki Shigenori , Inomata Kouhei , Kishiye Takao , Tahara Yoshiyuki , Nishiyama Kenryu , Nakagawa Masayuki
Published: August 2005

We isolated a paclitaxel‐resistant cell line (KK47/TX30) from a human bladder cancer cell line (KK47/WT) in order to investigate the mechanism of and reversal agents for paclitaxel resistance. KK47/TX30 cells exhibited 700‐fold resistance to paclitaxel and cross‐resistance to vinca alkaloids and topoisomerase II inhibitors. Tubulin polymerization assay showed no significant difference in the ratio of polymerized α‐ and β‐tubulin between KK47/WT and KK47/TX30 cells. Western blot analysis demonstrated overexpression of P‐glycoprotein (P‐gp) and lung resistance‐related protein (LRP) in KK47/TX30 cells. Drug accumulation and efflux studies showed that the decreased paclitaxel accumulation in KK47/TX30 cells was due to enhanced paclitaxel efflux. Cell survival assay revealed that verapamil and cepharanthine, conventional P‐gp modulators, could completely overcome paclitaxel resistance. To investigate whether new synthetic isoprenoids could overcome paclitaxel resistance, we synthesized 31 isoprenoids based on the structure of N‐solanesyl‐N, N′‐bis(3,4‐dimethoxybenzyl)ethylenediamine (SDB), which could reverse multidrug resistance (MDR), as shown previously. Among those examined, trans‐N, N′‐bis(3,4‐dimethoxybenzyl)‐.N‐solanesyl‐l,2‐diaminocyclohexane (N‐5228) could completely reverse paclitaxel resistance in KK47/TX30 cells. N‐5228 inhibited photoaffinity labeling of P‐gp by [3H]azidopine, suggesting that N‐5228 could bind to P‐gp directly and could be a substrate of P‐gp. Next, we investigated structural features of these 31 isoprenoids in order to determine the structural requirements for the reversal of P‐gp‐mediated paclitaxel resistance, suggesting that the following structural features are important for overcoming paclitaxel resistance: (1) a basic structure of 8 to 10 isoprene units, (2) a cyclohexane ring or benzene ring within the framework, (3) two cationic sites in close proximity to each other, and (4) a benzyl group with 3, 4‐dimethoxy functionalities, which have moderate electron‐donating ability. These findings may provide valuable information for the development of P‐gp‐mediated MDR‐reversing agents.
Significance of Nuclear Glutathione S‐Transferase π in Resistance to Anti‐cancer Drugs
Authors: Goto Shinji , Kamada Kensaku , Soh Yoko , Ihara Yoshito , Kondo Takahito
Published: August 2005

Recent study has shown that nuclear glutathione S‐transferase (GST) π accumulates in cancer cells resistant to doxorubicin hydrochloride (DOX) and may function to prevent nuclear DNA damage caused by DOX (Goto et al., FASEB J., 15, 2702–2714 (2001)). It is not clear if the amount of nuclear GSTπ increases in response to other anti‐cancer drugs and if so, what is the physiological significance of the nuclear transfer of GSTπ in the acquisition of drug‐resistance in cancer cells. In the present study, we employed three cancer cell lines, HCT8 human colonic cancer cells, A549 human lung adenocarcinoma cells, and T98G human glioblastoma cells. We estimated the nuclear transfer of GSTπ induced by the anti‐cancer drugs cisplatin (CDDP), irinotecan hydrochloride (CPT‐11), etoposide (VP‐16) and 5‐fluorouracil (5‐FU). It was found that: (1) Nuclear GSTjt accumulated in these cancer cells in response to CDDP, DOX, CPT‐11, VP‐16 and 5‐FU. (2) An inhibitor of the nuclear transport of GSTπ, edible mushroom lectin (Agaricus bisporus lectin, ABL), increased the sensitivity of the cancer cells to DOX and CDDP, and partially to CPT‐11. Treatment with ABL had no apparent effect on the cytotoxicity of VP‐16 and 5‐FU. These results suggest that inhibitors of the nuclear transfer of GSTπ have practical value in producing an increase of sensitivity to DOX, CDDP and CPT‐11.
A Role for Survivin in Radioresistance of Pancreatic Cancer Cells
Authors: Asanuma Koichi , Kobayashi Daisuke , Furuya Daisuke , Tsuji Naoki , Yagihashi Atsuhito , Watanabe Naoki
Published: August 2005

Using gene‐transduced pancreatic cancer cells, we examined whether survivin expression is directly involved in regulation of radiosensitivity. Ordinarily radiosensitive MIAPaCa‐2 cells transduced with wild‐type survivin gene (MS cells) proliferated more rapidly than cells transduced with control vector. MS cells were significantly less radiosensitive than control vector‐transduced cells. Radiation‐induced activity of caspase‐3, but not caspase‐7, was significantly inhibited in MS cells. On the other hand, transduction of a dominant‐negative mutant survivin gene into radioresistant PANC‐1 cells augmented radiosensitivity. Further, the radiation‐induced increase in caspase‐3 activity was enhanced, indicating that survivin function was truly inhibited. These results indicate that survivin expression directly down‐regulates radiosensitivity.
Occupational and lifestyle factors and urinary 8‐hydroxydeoxyguanosine
Authors: Irie Masahiro , Tamae Kazuyoshi , Iwamoto‐Tanaka Naoko , Kasai Hiroshi
Published: August 2005

The amount of 8‐hydroxydeoxyguanosine (8‐OH‐dG) excreted in urine can be used not only as an indicator of DNA repair capacity, but also as a potential marker of oxidative DNA damage. To clarify the oxidation‐related factors, in consideration of cancer risk, this study investigated how urinary 8‐OH‐dG was associated with occupational and lifestyle factors in 372 healthy workers. The creatinine‐adjusted urinary 8‐OH‐dG level was significantly higher in male subjects, smokers and drinkers compared with their counterparts. There were significant positive correlations of the 8‐OH‐dG level with average number of working hours, involvement in work, average number of cigarettes smoked, average volume of alcohol consumed and serum cortisol level, and there were significant negative correlations of the 8‐OH‐dG level with body mass index (BMI) and consumption of soybean products, rice and light‐colored vegetables. Multiple regression analysis showed that average number of working hours and average number of cigarettes smoked were significant predictors of increased 8‐OH‐dG levels, whereas being female and BMI were significant predictors of decreased 8‐OH‐dG levels. Working hours, BMI and smoking were significant predictors of urinary 8‐OH‐dG in male subjects, whereas age and BMI were significant predictors in female subjects. We suggest that several occupational and lifestyle factors, particularly long working hours and cigarette smoking, are linked to the formation of 8‐OH‐dG in workers. (Cancer Sci 2005; 96: 600 – 606)
Conditional expression of a suicide gene by the telomere reverse transcriptase promoter for potential post‐therapeutic deletion of tumorigenesis
Authors: Painter Richard G. , Lanson Nicholas A. , Jin Zhengmin , Park Frank , Wang Guoshun
Published: August 2005

Integration of a therapeutic gene into the host cell genome permits stable expression of the gene product in the target cells and its progeny. However, non‐directional integration of any given gene can pose the risk of activating tumor genes or silencing tumor suppressor genes. Therefore, including a safety‐control element into integrating vector systems is an important advance towards safer human gene therapy. Here, we report on a gene expression cassette that can be potentially exploited in integrating vector systems to eliminate post‐therapeutic tumorigenesis. The Herpes simplex virus thymidine kinase (hsvTK) gene under the transcriptional control of the human telomere reverse transcriptase promoter (hTERTp) was incorporated into a self‐inactivating HIV‐based lentiviral vector. The hTERT promoter is silent in normal somatic cells and re‐activated in tumor cells. Therefore, normal gene‐corrected cells should not express hsvTK from the promoter. However, if some gene‐corrected cells subsequently become tumorigenic and the hTERT promoter is re‐activated, application of ganciclovir (GCV), a clinically used antiviral drug, will achieve selective deletion of the cancerous cells. Our experimental data indicated that the hTERTp‐hsvTK cassette in the lentiviral vector was sufficient to differentiate between tumor cells and normal cells, thus eradicating tumor cells selectively in vitro and in vivo. These results proved the principle of using the element in integrating vectors for safer gene delivery. (Cancer Sci 2005; 96: 607 –613)
Ribozymes targeting serine/threonine kinase Akt1 sensitize cells to anticancer drugs
Authors: Yanagihara Miyako , Katano Masayoshi , Takahashi‐Sasaki Noriko , Kimata Kiyonori , Taira Kazunari , Andoh Toshiwo
Published: August 2005

The serine/threonine kinase Akt is a key component of the cellular signaling pathway for survival and drug‐resistance in cancer cells. In the present study we confirmed this view by expressing an antagonist of Akt, a dominant negative form of Akt, in HCT116 colon carcinoma cells and observing apoptosis induction in cells in which expression of the mutant protein had been induced. Three isoforms of Akt have been found: Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ. However, the function of individual isoforms with respect to tumorigenicity and drug‐resistance of cancer cells is largely unknown. We designed ribozymes targeting the Akt1 protein in mammalian cells. Our data indicate that Akt1 ribozymes downregulate Akt1 expression to less than half that of control cells. Downregulation of Akt1 expression appears to sensitize HEK293 and HeLa cells to typical chemotherapeutic agents. However, Akt1 ribozymes had little effect on the proliferative activity of the cells. Thus, Akt as a whole and even just the Akt1 isozyme is an excellent target for chemotherapy. We further suggest a synergistic effect for combination therapy targeting Akt and other vital molecules such as tubulins, topoisomerases and protein kinases. (Cancer Sci 2005; 96: 620 – 626)
Involvement of a cell adhesion molecule, TSLC1/IGSF4, in human oncogenesis
Authors: Murakami Yoshinori
Published: August 2005

The occurrence of aberrations in cell adhesion is a critical phase in the invasion and metastasis of human cancer. A tumor suppressor gene, TSLC1/IGSF4, from chromosomal region 11q23 was identified in non‐small cell lung cancer (NSCLC) by its tumor suppressor activity in nude mice. TSLC1/IGSF4 is expressed in most tissues except for peripheral blood lymphocytes, but it is inactivated in 44% of NSCLC and 30–60% of various cancers, including liver, pancreatic, and prostate cancers, especially in those with invasion or metastasis. Inactivation occurs by two hits: through promoter methylation, and through loss of heterozygosity at the gene locus. TSLC1/IGSF4 encodes an immunoglobulin superfamily cell adhesion molecule and associates with an actin‐binding protein, DAL‐1/4.1B, and members of the membrane‐associated guanylate kinase homologue (MAGuK) group, providing a novel tumor suppressor cascade that is inactivated in more than 80% of NSCLC. TSLC1/IGSF4 appears to be involved in the formation of an epithelial cell structure with DAL‐1/4.1B and MAGuK. Furthermore, TSLC1/IGSF4 may act as a tumor antigen recognized by activated NK or CD8+ T cells. These two distinct mechanisms based on homophilic and heterophilic interactions would be responsible for tumor suppression by TSLC1/IGSF4. TSLC1/IGSF4 is ectopically expressed in adult T‐cell leukemia (ATL) cells, providing not only a diagnostic marker for ATL, but also a possible therapeutic target against its invasion. The distinct roles of TSLC1/IGSF4 in the oncogenesis of carcinomas and ATL could be due to tissue‐specific differences in the downstream cascades, and is a novel concept with respect to cell adhesion in human oncogenesis. (Cancer Sci 2005; 96: 543 –552)
Genetic polymorphisms of methylenetetrahydrofolate reductase and colorectal cancer and adenoma
Authors: Kono Suminori , Chen Kun
Published: August 2005

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating folate metabolism, which affects DNA methylation and synthesis. Two functional, common polymorphisms (C677T and A1298C) are known in the MTHFR gene. MTHFR activity is lowered in individuals with the 677TT genotype and is somewhat reduced in those with the 1298CC genotype. We reviewed the consistency of reported associations of these polymorphisms with colorectal cancer and adenoma with consideration of the effects of nutritional status. A total of 16 studies have addressed the association between MTHFR C677T polymorphism and colorectal cancer in 10 countries. Decreased risk of colorectal cancer associated with the 677TT genotype has fairly consistently been observed, with few exceptions. This decrease was observable in people with either high or low folate status. Alteration in the thymidylate pool associated with MTHFR activity is postulated as an underlying mechanism. Studies on the A1298C polymorphism are limited, and their results are variable. Almost all of seven studies of colorectal adenoma have found no association between C677T polymorphism and adenoma, but the 677TT genotype seems to be related to increased risk when folate status is poor. Reduced availability of methyl groups for DNA methylation might be more relevant to adenoma formation. Although the underlying mechanisms still remain to be clarified, epidemiological findings regarding MTHFR C677T polymorphism provide strong evidence that adequate folate status confers protection from colorectal cancer. (Cancer Sci 2005; 96: 535 –542)
Nitrative and oxidative DNA damage in oral lichen planus in relation to human oral carcinogenesis
Authors: Chaiyarit Ponlatham , Ma Ning , Hiraku Yusuke , Pinlaor Somchai , Yongvanit Puangrat , Jintakanon Darunee , Murata Mariko , Oikawa Shinji , Kawanishi Shosuke
Published: August 2005

Oral lichen planus (OLP) is a chronic inflammatory disease, which has been clinically associated with development to oral cancer. A double immunofluorescence labeling study found that 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) accumulated in oral epithelium in OLP and oral squamous cell carcinoma (OSCC) biopsy specimens, whereas little or no immunoreactivity was observed in normal oral mucosa. Colocalization of 8‐nitroguanine and inducible nitric oxide synthase (iNOS) was found in oral epithelium of OLP and OSCC. Immunoreactivity of 3‐nitrotyrosine, which is formed by protein tyrosine nitration and is considered to be a biochemical marker for inflammation, was also observed in oral epithelial cells and colocalized with 8‐nitroguanine. Accumulation of p53 was more strongly observed in oral epithelium in OSCC than OLP, whereas there was no p53 accumulation in normal oral mucosa. Our findings demonstrate that iNOS‐dependent DNA damage in OLP may lead to p53 accumulation in not only OLP but also OSCC. We conclude that the formation of potentially mutagenic DNA lesions including 8‐nitroguanine and 8‐oxodG may contribute to the development of oral cancer from OLP. (Cancer Sci 2005; 96: 553 –559)
Induction of multicellular 3‐D spheroids of MCF‐7 breast carcinoma cells by neutrophil‐derived cathepsin G and elastase
Authors: Yui Satoru , Tomita Kazutaka , Kudo Tomoya , Ando Shingo , Yamazaki Masatoshi
Published: August 2005

In tumor metastasis, multicellular aggregates of tumor cells form and disseminate into the blood or lymph vessels from the tumor mass, following the formation of tumor cell emboli in distant vessels. However, the mechanism by which aggregates form in the tumor mass is unknown. Neutrophils often exist in tumors and are considered to affect tumor development. We observed that neutrophils had the capacity to induce the aggregation of MCF‐7 human breast carcinoma cells adhering to culture substrates. When MCF‐7 cells were cultured with rat inflammatory neutrophils, the soluble fraction of their lysate, and the conditioned medium of neutrophils stimulated with N‐formyl‐Met‐Leu‐Phe plus cytochalasin B, multicellular aggregates formed within 16 h, and tightly aggregated 3‐D spheroids formed when the cultures were prolonged. The spheroid‐inducing reaction was reversible and energy‐dependent. The MCF‐7 cells induced to aggregate by the neutrophil extract showed growth potential, although the growth rate of the cells was slightly reduced. The aggregation was dependent on E‐cadherin, because the spheroids dispersed into isolated cells on incubation with EGTA or anti‐E‐cadherin antibody following pipetting. The aggregation‐inducing activity in neutrophils was completely inhibited by soybean trypsin‐chymotrypsin inhibitor. Moreover, the commercially available human neutrophil elastase and cathepsin G induced the aggregation of MCF‐7 cells and formation of spheroids. The proteases secreted by infiltrated neutrophils in tumors are implicated in the dissemination of tumor aggregates from primary tumor sites. (Cancer Sci 2005; 96: 560 –570)
Ubiquitin targeting of rat muscle proteins during short periods of unloading
Authors: Vermaelen M. , Marini J.‐F. , Chopard A. , Benyamin Y. , Mercier J. , Astier C.
Published: August 2005

Aim:  The ubiquitin‐proteasome system is known to be involved in many situations leading to skeletal muscle atrophy. However, the cellular mechanisms triggering the atrophic process initiation are still poorly understood. For short periods of rat hindlimb unloading, we assessed the specific ubiquitin targeting of sarcoplasmic or myofibrillar proteins in slow and fast rat muscle types.

Methods:  Adult Sprague Dawley rats were randomly assigned to three groups: control, hindlimb‐unloaded for 4 days (HU4) and hindlimb‐unloaded for 8 days (HU8). In fractionated extracts from soleus (SOL) and Extensor Digitorum Longus (EDL) muscles, the relative contents of free and conjugated ubiquitin were quantified by immunoblotting.

Results:  Hindlimb unloading of short durations resulted in a preferential atrophy of slow‐twitch fibres and bound ubiquitin levels were increased by 37 and 68% in the soleus myofibrillar fraction after respectively 4 and 8 days. The ubiquitin conjugation was shown to principally affect the high molecular weight proteins. Free and conjugated ubiquitin levels remained unchanged in sarcoplasmic fraction from SOL muscle after 8 days HU. For the fast muscle (EDL), ubiquitin contents were approximately twofold lower in control conditions, and did not significantly change during the hindlimb unloading periods considered.

Conclusion:  The postural SOL muscle was shown to contain higher constitutive sarcoplasmic ubiquitin levels than the phasic EDL. The high response to unloading of the slow twitch fibres rich SOL muscle was accompanied by a specific conjugation of its myofibrillar proteins that may participate in the initiation of skeletal muscle remodelling consequent to disuse.
Doublets and low‐frequency fatigue in potentiated human muscle
Authors: Bentley L. F. , Lehman S. L.
Published: August 2005

Aim:  To test the hypothesis that doublets compensate for low‐frequency fatigue. Doublets increase force output from muscles stimulated at low frequencies. Low‐frequency fatigue is a decline in the force elicited by low‐frequency stimulation.

Methods:  Human flexor carpi radialis muscles were stimulated with 20 Hz trains with and without an initial doublet and with and without low‐frequency fatigue and the resulting force response measured.

Results:  An initial doublet caused an increase in the maximum rate of force rise of 179.6 ± 27.9% in rested and 242.9 ± 37.7% in muscles with low‐frequency fatigue, and a substantial enhancement in force in the first three inter‐pulse intervals after the extra pulse. The magnitude and time course of the early doublet enhancement were very similar regardless of low‐frequency fatigue, consistent with current theories regarding the mechanisms of the doublet effect and of low‐frequency fatigue. By the end of the 1 s stimulus train, force enhancement was insignificant in rested muscles and was small and subject‐dependent in muscles with low‐frequency fatigue (17.3 ± 8.1% of force without a doublet), reducing the force deficit by 25.2 ± 5.5%.

Conclusions:  The time course of doublet force enhancement implies that an initial doublet may effectively compensate for the deficit in rate of force rise in muscles with low‐frequency fatigue, but may not compensate for force deficits beyond the first few inter‐pulse intervals.
Effects of 84‐days of bedrest and resistance training on single muscle fibre myosin heavy chain distribution in human vastus lateralis and soleus muscles
Authors: Gallagher P. , Trappe S. , Harber M. , Creer A. , Mazzetti S. , Trappe T. , Alkner B. , Tesch P.
Published: August 2005

Aim:  This investigation determined the effects of 84 days of bedrest on the composition of myosin heavy chain (MHC) in single skeletal muscle fibres with and without a resistance‐training countermeasure programme.

Methods:  Muscle biopsies were obtained from the m. vastus lateralis (VL) and m. soleus (SOL) before and after 84 days of bedrest. While control (BR) subjects (VL n = 9; SOL n = 3) refrained from exercise, BRE subjects (VL n = 8; SOL n = 3) performed knee extensor and plantar flexor resistance exercise every third day. Approximately 110 fibres per sample were analysed for MHC composition using SDS‐PAGE.

Results:  BR–VL had 16 and 14% decreases (P < 0.05) in MHC I and IIa fibres, respectively. There were 10% increases (P < 0.05) in MHC I/IIa, IIa/IIx, I/IIa/IIx, and a ∼30% increase (P < 0.05) in total hybrid fibres. BRE‐VL showed a 15% reduction (P < 0.05) in MHC I fibres, no change in MHC IIa fibres, and a 13% increase (P < 0.05) in total hybrids. BR–SOL had a 19% decrease (P < 0.05) in MHC I fibres with a 22% increase in total hybrids. BRE–SOL showed no change in MHC composition across all fibre types.

Conclusion:  These data suggest that the exercise countermeasures programme prevented MHC shifts in the SOL and mitigated MHC shifts in the VL. Furthermore, in the VL it appears that the resistance training programme employed in this investigation during bedrest, emphasized the use of MHC IIa phenotype muscle fibres.
Acute myocardial ischaemia induces specific alterations of ventricular mitochondrial function in experimental pigs
Authors: Zoll J. , Ponsot E. , Doutreleau S. , Mettauer B. , Piquard F. , Mazzucotelli J. P. , Diemunsch P. , Geny B.
Published: August 2005

Aims:  As cardiac metabolic flexibility is crucial, this study examined whether acute ischaemia can induce specific qualitative alterations of the mitochondrial metabolic pathways as well as energy transfer systems.

Methods:  Left descending coronary artery ligation was performed after sternotomy in eight pigs and the heart was excised after 45 min of ischaemia. Maximal O2 uptake (Vmax, μmol O2 min−1 g−1 dry weight) of saponin‐skinned myofibres were measured from ischaemic and non‐ischaemic area of ventricular myocardium.

Results:  V
max decreased by ∼20% in ischaemic myocardium with both glutamate‐malate (18.1 ± 1.3 vs. 22.1 ± 1.7 in control, P < 0.05) and pyruvate substrates (19.3 ± 1.0 vs. 23.3 ± 2.0 in control, P < 0.05) whereas no difference was observed with palmitoyl carnitine (15.6 ± 1.8 vs. 16.6 ± 0.9 in control). The Km of mitochondrial respiration for ADP decreased in ischaemic heart by 24% (679 ± 79 vs. 899 ± 84 μm of ADP in control, P < 0.05). Moreover, the mitochondrial creatine kinase efficacy (Km without creatine/Km with creatine), representative of the coupling of oxidative phosphorylation process with the mitochondrial creatine kinase, was reduced in ischaemic heart (11.6 ± 2.5 in ischaemic vs. 18.0 ± 2.2 in control, P < 0.05).

Conclusions:  These findings argue for specific mitochondrial impairments at the level of pyruvate oxidation and creatine kinase channelling system after an acute period of in vivo ischaemia, whereas the lipid mitochondrial oxidation pathway seems to be preserved. Such a loss of metabolic flexibility following acute ischaemia could become an early feature of metabolic dysregulation of the heart.
Acute exercise causes an enhancement of tissue renin–angiotensin system in the kidney in rats
Authors: Maeda S. , Iemitsu M. , Jesmin S. , Miyauchi T.
Published: August 2005

Aims:  Initially, the renin–angiotensin system (RAS) produced through the classical endocrine pathway was well known for its regulation of blood pressure. However, it was revealed that a local autocrine and/or paracrine RAS may exist in a number of tissues (such as kidney). Exercise causes a redistribution of tissue blood flow, by which the blood flow is greatly increased in active muscles, whereas it is decreased in the splanchnic circulation (such as in the kidney). We hypothesized that exercise causes an enhancement of tissue RAS in the kidney.

Methods:  We studied whether exercise affects expression of angiotensinogen and angiotensin‐converting enzyme (ACE) and tissue angiotensin II level in the kidney. The rats performed treadmill running for 30‐min. Immediately after this exercise, kidney was quickly removed. Control rats remained at rest during this 30‐min period.

Results:  The expression of angiotensinogen mRNA in the kidney was markedly higher in the exercise rats than in the control rats. ACE mRNA in the kidney was significantly higher in the exercise rats than in the control rats. Western blot analysis confirmed significant upregulation of ACE protein in the kidney after exercise. Tissue angiotensin II level was also increased by exercise.

Conclusion:  The present study suggests that the exercise‐induced enhancement of tissue RAS in the kidney causes vasoconstriction and hence decreases blood flow in the kidney, which are helpful in increasing blood flow in active muscles, thereby contributing to the redistribution of blood flow during exercise.
Biliary secretion of moxifloxacin in obstructive cholangitis and the non‐obstructed biliary tract
Authors: SCHWAB D. , GRAUER M. , HAHN E. G. , MÜHLDORFER S.
Published: August 2005

Background : Biliary secretion of antibiotic agents into the bile is considerably compromised by biliary obstruction, a precondition of bacterial cholangitis. Moxifloxacin may be advantageous according to secretion and antimicrobial spectrum.

Aim : To establish the secretion of moxifloxacin into obstructed and non‐obstructed bile.

Methods : Biliary excretion of moxifloxacin was determined in plasma and bile of 10 patients with biliary obstruction and cholangitis and 10 patients without biliary obstruction 30 min after administration of 400 mg of moxifloxacin intravenously.

Results : The plasma concentration of moxifloxacin was similar in both groups (4.45 ± 1.58 μg/mL; 4.33 ± 1.23 μg/mL). The concentration of moxifloxacin in the bile was significantly lower in patients with biliary obstruction than without (4.63 ± 3.94 μg/mL; range 0.71–14.40; vs. 16.90 ± 13.77 μg/mL; range 1.79–42.50; P = 0.043). Although significantly different, the penetration index was extensively high in those without biliary obstruction (4.41 ± 4.40; range 0.35–14.45) but still sufficient in those patients with obstructive cholangitis (1.02 ± 0.74; range 0.29–2.83; P = 0.035).

Conclusion : These findings are suggestive of an active secretion mechanism for moxifloxacin into the obstructed bile, producing a biliary concentration sufficiently above the minimal inhibitory concentrations for most of the expected bacteria.
Colonic spread and serum pharmacokinetics of budesonide foam in patients with mildly to moderately active ulcerative colitis
Authors: BRUNNER M. , VOGELSANG H. , GREINWALD R. , KLETTER K. , KVATERNIK H. , SCHROLNBERGER C. , EICHLER H.‐G. , BRUNNER H. , DUDCZAK R. , MÜLLER M.
Published: August 2005

Background : Local treatment with foams in patients suffering from ulcerative proctitis or proctosigmoiditis is considered a rational treatment option.

Aims : To investigate colonic spread, safety, tolerability and acceptance of a newly developed budesonide foam formulation.

Methods : Twelve patients (four females, eight males) with acute proctosigmoiditis or left‐sided ulcerative colitis were rectally administered a single dose of [99Tcm]‐labelled budesonide foam (Budenofalk; Dr Falk Pharma GmbH, Freiburg, Germany) containing 2 mg budesonide in 20 mL foam after diagnostic colonoscopy. Thereafter, the colonic spread was assessed by means of γ‐scintigraphy for 6 h. Serum samples were taken simultaneously.

Results : Budesonide foam spread with a maximum between 11 and 40 cm, thus reaching the sigmoid colon in all patients. In some patients, the foam even extended into the distal third and the middle of the descending colon with maximum radioactivity at 4 h. Systemic budesonide absorption was rapid and pharmacokinetic data were comparable with published data on marketed budesonide enemas, with mean serum Cmax and AUC0‐8h values of 0.8 ± 0.5 ng/mL and 3.7 ± 1.9 ng h/mL, respectively. The new formulation was well accepted by all patients, who could retain the foam for at least 4 h.

Conclusions : In the majority of patients, budesonide foam effectively spread up to the left‐sided colon and thus qualifies for the local treatment of proctosigmoiditis.
Autoantibody profile during short‐term infliximab treatment for Crohn's disease: a prospective cohort study
Authors: ATZENI F. , ARDIZZONE S. , SARZI‐PUTTINI P. , COLOMBO E. , MACONI G. , DE PORTU S. , CARRABBA M. , BIANCHI PORRO G.
Published: August 2005

Background : The potential clinical implications of autoimmunity during treatment with infliximab are unclear.

Aim : To determine the frequency and correlation of autoantibody formation in patients with Crohn's disease treated with infliximab in a routine clinical setting.

Methods : Sixty‐three patients with refractory/inflammatory (31) and/or fistulising Crohn's disease (32), received an infliximab infusion at a dose 5 mg/kg in weeks 0, 2 and 6, and were evaluated for the development of antinuclear, anti‐double‐stranded DNA, anti‐Sm, anti‐RNP, anti‐SSA, anti‐SSB and antihistone antibodies. The correlates with pharmacological treatments, the response to infliximab and adverse events were evaluated.

Results : Antinuclear antibodies were found in five of the 63 patients (8%) at baseline and in 26 (42%) after 10 weeks (P < 0.001). Of the 26 antinuclear antibody‐positive patients who were further subtyped, nine of 63 (17%) had anti‐double‐stranded DNA (P = 0.003), and 1.5% were extractable nuclear antigen (ENA) and antihistone‐positive. Five patients were initially positive for anticardiolipin antibodies and two more patients became positive during infliximab treatment. New autoantibody formation was more frequent in the patients with inflammatory/refractory disease than in those with fistulising disease (17 vs. 7; P = 0.02). One patient developed drug‐induced lupus without major organ damage.

Conclusions : Autoantibody formation occurs in 42% of patients (8% of these patients were positive before infliximab treatment) with Crohn's disease receiving induction treatment with infliximab, but the clinical significance of this remains to be determined.
Effect of tegaserod on work and daily activity in irritable bowel syndrome with constipation
Authors: REILLY M. C. , BARGHOUT V. , MCBURNEY C. R. , NIECKO T. E.
Published: August 2005

Background : Tegaserod is a promotility agent with proven efficacy and safety in patients with irritable bowel syndrome with constipation.

Aim : To assess tegaserod's effect on work productivity and daily activity.

Methods : Women, 18–65 years old and meeting Rome II criteria for irritable bowel syndrome with constipation, were randomized to a double‐blind, placebo‐controlled, multicentre study of tegaserod 6 mg b.d. or placebo. Productivity loss and daily activity impairment because of irritable bowel syndrome were measured with the Work Productivity and Activity Impairment questionnaire for irritable bowel syndrome, modified to exclude diarrhoea as a symptom. Assessments were made at baseline, weeks 2 and 4.

Results : A total of 2660 women were randomized and, of these, 1675 [tegaserod (n = 1363), placebo (n = 312)] were employed and completed Work Productivity and Activity Impairment for irritable bowel syndrome questionnaires. Compared with placebo, tegaserod significantly reduced work and daily activity impairment at weeks 2 and 4. Tegaserod reduced absenteeism by 2.6% (P = 0.004), presenteeism by 5.4% (P < 0.0001), overall work productivity loss by 6.3% (P < 0.0001), and activity impairment by 5.8% (P < 0.0001) at week 4 (vs. baseline). Assuming a 40‐h workweek, tegaserod reduced work productivity loss by 2.5 h/week.

Conclusions : Tegaserod significantly reduced work productivity loss and daily activity impairment at 2 weeks, and this benefit was maintained at 4 weeks.
A probiotic mixture alleviates symptoms in irritable bowel syndrome patients: a controlled 6‐month intervention
Authors: KAJANDER K. , HATAKKA K. , POUSSA T. , FÄRKKILÄ M. , KORPELA R.
Published: August 2005

Background : Irritable bowel syndrome is a gastrointestinal disorder of unknown aetiology. The effect of probiotics in this syndrome remains unclear.

Aim : To investigate whether a probiotic mixture containing Lactobacillus rhamnosus GG, L. rhamnosus LC705, Bifidobacterium breve Bb99 and Propionibacterium freudenreichii ssp. shermanii JS is effective in alleviating irritable bowel syndrome symptoms.

Methods : A total of 103 patients fulfilling the Rome I or II criteria took part in this 6‐month, randomized, double‐blind placebo‐controlled trial. The patients received a probiotic capsule or a placebo capsule daily. Gastrointestinal symptoms and bowel habits were recorded.

Results : At the end the total symptom score (abdominal pain + distension + flatulence + borborygmi) was 7.7 (95% CI: −13.9 to −1.6) points lower in the probiotic group (P = 0.015). This represents a median reduction of 42% in the symptom score of the probiotic group compared with 6% in the placebo group. In individual symptoms, borborygmi was milder in the probiotic group (P = 0.008), and for the rest of the symptoms there was a non‐significant trend.

Conclusions : The results indicate that this probiotic mixture is effective in alleviating irritable bowel syndrome symptoms. Considering the high prevalence of irritable bowel syndrome and the lack of effective therapies, even a slight reduction in symptoms could have positive public health consequences.
Long‐term follow‐up in atrophic body gastritis patients: atrophy and intestinal metaplasia are persistent lesions irrespective of Helicobacter pylori infection
Authors: LAHNER E. , BORDI C. , CATTARUZZA M. S. , IANNONI C. , MILIONE M. , DELLE FAVE G. , ANNIBALE B.
Published: August 2005

Background : Long‐term outcome of atrophic body gastritis has not yet been defined.

Aim : To investigate at long‐term follow‐up the behaviour of atrophy and intestinal metaplasia and the occurrence of neoplastic lesions in atrophic body gastritis patients.

Methods : Overall 106 atrophic body gastritis patients with ≥ 4‐year follow‐up were studied; 38 were Helicobacter pylori‐positive at histology + serology and cured of infection (group A), 36 were positive at serology and not treated (group B) and 32 were H. pylori‐negative (group C). Patients underwent gastroscopy with antral (n = 3) and body (n = 3) biopsies for histology according to the Sydney System.

Results : At 6.7‐year follow‐up body atrophy and intestinal metaplasia remained unchanged in all 106 patients irrespective of H. pylori status. Antral atrophy was significantly increased at follow‐up only in group C, whereas antral intestinal metaplasia was unchanged in all three groups. During follow‐up eight (8%) patients developed neoplastic lesions (one adenocarcinoma, one adenoma with low‐grade dysplasia and six low‐grade dysplasia without endoscopic lesions). Antral atrophic gastritis was present at baseline in all but one (88%) of the eight patients with neoplastic lesions, but only in 15 (15%) of the 98 patients without (P < 0.0001, RR = 26.7).

Conclusions : Atrophy and intestinal metaplasia persist at 6.7‐year follow‐up and atrophic body gastritis patients with panatrophic gastritis are at increased risk of developing neoplastic lesions.
Disagreement between acute and chronic haemodynamic effects of nadolol in cirrhosis: a pathophysiological interpretation
Authors: MERKEL C. , BOLOGNESI M. , SACERDOTI D. , BOMBONATO G. , CAVASIN L. , GATTA A.
Published: August 2005

Background : The acute effects of β‐blockers may be different from chronic; mechanisms underlying this difference are poorly elucidated.

Aim : To assess portal pressure and its pathophysiological determinants after acute and chronic administration of nadolol.

Methods : In 24 patients with cirrhosis and portal hypertension hepatic venous pressure gradient, portal blood flow and resistance to portal blood flow were measured before, 60–90 min after acute administration of nadolol, and after 1 month. Patients were good‐responders if hepatic venous pressure gradient was ≤12 mmHg, or decreased by at least 20%.

Results : Eleven and 13 patients were good‐ and poor‐responders to acute administration, respectively. Acute poor‐responders showed a lower decrease in portal blood flow (P = 0.04) and a less evident decrease in mean arterial pressure (P < 0.001). Eleven and 13 patients were good‐ and poor‐responders to chronic administration, respectively. Chronic poor‐responders showed a larger increase in resistance to portal blood flow compared with good‐responders (P = 0.01).

Disagreement between acute and chronic effects was seen in 12 patients: six were acute good‐responders chronic poor‐responders and six were acute poor‐responders chronic good‐responders. Acute good‐responders chronic poor‐responders patients had the smallest decreases in portal blood flow and in mean arterial pressure after acute administration, while acute poor‐responders chronic good‐responders showed the largest (P = 0.05 and 0.01).

Conclusions : Disagreement between acute and chronic effects of nadolol on hepatic venous pressure gradient is common. The mechanism responsible is complex, the acute effect being mainly modulated by arterial hypotension and the chronic effect by changes in portal resistance.