bluuebunny/crossref_metadata_2025 huggingface dataset:
Incidence of Mutation and Deletion in Topoisomerase IIα mRNA of Etoposide and mAMSA–resistant Cell Lines
Authors: Matsumoto Yoshihito , Takano Hiroshi , Kunishio Katsuzo , Nagao Seigo , Fojo Tito
Published: August 2005

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. To further understand resistance to topoisomerase II inhibitors, 50 sublines were isolated as single clones from parental cells by exposure to VP–16 (etoposide) or mAMSA (m–amsacrine). Subsequently, a population of cells from each subline was exposed to three–fold higher drug concentrations allowing 16 stable sublines to be established at higher extracellular drug concentration. Finally, 66 sublines were picked up. The frequency and nature of mutations in the topoisomerase II gene in the drug–selected cell lines were evaluated. In order to screen a large number of cell lines, an RNAse protection assay was developed and mismatches were observed in 13.6% of resistant cell lines (12% of resistant cell lines exposed to lower drug concentrations and 18.8% of resistant cell lines exposed to higher drug concentrations). Some of these mutations are located in vital regions of topoisomerase II (phosphorylation sites in the C–terminal or N–terminal, and nuclear localizing signal of topoisomerase II). Our findings suggest that mutations of topoisomerase II gene are an important and frequent mechanism of resistance to topoisomerase II inhibitors.
Lack of Effect of Soy Isoflavone on Thyroid Hyperplasia in Rats Receiving an Iodine‐deficient Diet
Authors: Son Hwa‐Young , Nishikawa Akiyoshi , Ikeda Takako , Imazawa Takayoshi , Kimura Shuichi , Hirose Masao
Published: August 2005

We have reported a dramatic synergism between soy intake and iodine deficiency regarding induction of thyroid hyperplasia in rats. Because isoflavones are active constituents of soybeans, in the present study, their possible contribution was examined. Female F344 rats were divided into 8 groups, exposed to diet containing a 0.2% soy isoflavone mixture (SI), 0.2% SI+iodine deficiency (ID), 0.04% SI, 0.04% SI+ID, 20% defatted soybean (DS) alone, 20% DS+ID, ID alone or basal diet alone for 5 weeks. Thyroid weight was not influenced by SI, but was increased by the ID and DS diets with a further significant increment in the DS+ID group (P<0.01). Compared to the control value, serum T4 was significantly (P<0.01) increased by 20% DS alone and decreased in all groups given the ID treatment (P<0.001). Serum thyroid stimulating hormone (TSH) level was increased by ID, and further enhanced by DS (P<0.01) but not SI. Histopathologically, diffuse hypertrophy and/or hyperplasia of thyroid follicles were observed in the ID‐treated groups, the severity being enhanced by DS but not SI. Proliferating cell nuclear antigen labeling indices (%) were elevated in the ID diet groups and again enhanced by DS, but not SI. These results thus suggest that isoflavones may not be involved in the mechanisms underlying the synergistic goitrogenic effect of soybean with iodine deficiency.
Hexosaminidase‐altered Aberrant Crypts, Carrying Decreased Hexosaminidase α and β Subunit mRNAs, in Colon of 1,2‐Dimethylhydrazine‐treated Rats
Authors: Tsukamoto Tetsuya , Fukami Hiroko , Yamanaka Shoji , Yamaguchi Akira , Nakanishi Hayao , Sakai Hiroki , Aoki Ichiro , Tatematsu Masae
Published: August 2005

Aberrant crypt foci (ACF), consisting of morphologically irregular crypts, are thought to be precancerous lesions for colon cancers. For their molecular analysis, it is necessary to avoid contamination with adjacent normal crypts and stromal cells. Decreased hexosaminidase activity in ACF, which has been histochemically demonstrated, was used in the present study to classify isolated crypts in combination with morphological changes. The length, rim diameter, and width (average±SD, μm) of hexosaminidase‐positive (Hex+) crypts were 238.6±40.4, 89.5±22.9, and 57.6±14.0, respectively. For hexosaminidase‐negative (Hex‐) crypts, the values were 314.4±77.8, 140.3±45.7, and 97.3±34.7, the width being 1.69 tunes greater (P<0.0001). Crypts wider than 115 μm (approximately 2 tunes the average size of Hex+ crypts) were all from ACF, judging from hexosaminidase staining. To analyze transcription levels of Hex α and β subunits (Hexa and Hexb, respectively), real‐tune relative quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) analysis was performed using the LightCycler system. In aberrant crypts, both Hexa and Hexb were significantly down‐regulated to 0.266 (P<0.002) and 0.131 (P<0.001) units, respectively, compared with those in morphologically normal crypts, with β‐actin as the internal standard. This decrease could be a molecular marker for precancerous enzyme‐altered ACF.
Mutations of p53 in Morphologically Non‐neoplastic Mucosa of Long‐standing Ulcerative Colitis
Authors: Takaku Hideya , Ajioka Yoichi , Watanabe Hidenobu , Hashidate Hideki , Yamada Satoshi , Yokoyama Junji , Kazama Shinsuke , Suda Takeyasu , Hatakeyama Katsuyoshi
Published: August 2005

Two cases of ulcerative colitis (UC)‐associated carcinoma or dysplasia and morphologically non‐neoplastic mucosa with p53 protein overexpression (MNNM‐p53OE) were selected. DNA was extracted from the paraffin blocks of these lesions and exons 5‐8 of the p53 gene were analyzed by PCR and direct sequencing. In addition, mutations in K‐ras codon 12 were analyzed by PCR‐RFLP methods. MNNM‐p53OE was located surrounding and adjoining a coexisting carcinoma and/or dysplasia. A p53 mutation was detected in 12/22 (54.5%) MNNM‐p53OE samples, 4/8 (50%) dysplasia samples and 8/8 (100%) carcinoma samples. The/j53 mutations detected in MNNM‐p53OE were identical to those demonstrated in the adjoining carcinoma and/or dysplasia. No K‐ras codon 12 mutation was detected in any of the samples. These results indicate that MNNM‐p53OE may share an identical clonal linkage with a coexisting carcinoma and/or dysplasia, and may be an initial and submorphological form of UC‐associated neoplasia. Recognition of MNNM‐p53OE in biopsy specimens may help to identify patients with UC at risk of developing colorectal carcinoma.
Fusion of a Sequence from HEI10 (14q11) to the HMGIC Gene at 12ql5 in a Uterine Leiomyoma
Authors: Mine Nobuya , Kurose Keisuke , Konishi Hideki , Araki Tsutomu , Nagai Hisaki , Emi Mitsuru
Published: August 2005

Uterine leiomyoma, a benign smooth‐muscle tumor of the myometrium, is the most commonly encountered neoplasm in women of reproductive age. Band q15 of chromosome 12 is often rearranged in benign mesenchymal tumors such as uterine leiomyomas, and the HMGIC gene, encoding a protein of the high‐mobility‐group (HMG), is present in that region. Using 3′ rapid amplification of cDNA ends (3RACE) experiments, we isolated an ectopic sequence that was fused to HMGIC in a uterine leiomyoma. Cloning of the fusion cDNA identified a gene termed “homo sapiens enhancer of invasion 10” (HEI10) as the fusion partner. Radiation hybrid mapping revealed that the normal location of HEI10 is at 14qll. In the fusion transcript the first two exons of the HMGIC gene, which encode DNA‐binding domains, were fused to the 3’portion of the HEI10 gene. This rearrangement implicates HMGIC in the tumorigenesis of uterine leiomyoma, and suggests that its fusion HMGIC product may play a role in mesenchymal differentiation.
Structure, Expression and Chromosome Mapping of MLZE, a Novel Gene Which Is Preferentially Expressed in Metastatic Melanoma Cells
Authors: Watabe Kenji , Ito Akihiko , Asada Hideo , Endo Yuichi , Kobayashi Toshiko , Nakamoto Ken'i , Itami Satoshi , Takao Sonshin , Shinomura Yasuhisa , Aikou Takashi , Yoshikawa Kunihiko , Matsuzawa Yuji , Kitamura Yukihiko , Nojima Hiroshi
Published: August 2005

We isolated a novel gene, termed MLZE, from a B16‐BL6 cDNA library after subtraction of B16‐F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1‐2, which contains the c‐myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze‐positive cases was significantly larger in Clark levels III, IV and V melanomas (6/11=55%) than in Clark levels I and II melanomas (2/15=13%). In two cases of hMlze‐positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.
Heterogeneity of p53 Mutational Status in Intramucosal Carcinoma of the Colorectum
Authors: Yamada Satoshi , Ajioka Yoichi , Watanabe Hidenobu , Hashidate Hideki , Takaku Hideya , Kazama Shinsuke , Yokoyama Junji , Nishikura Ken , Fujiwara Takato , Asakura Hitoshi
Published: August 2005

The aim of this study was to elucidate whether or not p53 genetic heterogeneity would occur while colorectal carcinoma was limited to the mucosa. Eight cases of endoscopically resected colorectal intramucosal carcinomas were analyzed to determine the p53 gene sequence (exons 5 to 8). Six out of 8 cases showed p53 gene mutations, and in all of them, the mutational status was heterogeneous. In 4 cases, mutated codons were heterogeneous as well. These data indicate that p53 gene alterations in colorectal carcinomas occur and diverge at the stage of intramucosal carcinoma, supporting our previously proposed hypothesis that colorectal carcinomas can be composed of various subclones as regards p53 gene mutation, while the carcinoma is limited to the mucosa, and one of these subclones commences invasion to the submucosa after clonal selection, thus generating a monoclonal invasive carcinoma.
Establishment of Cell Lines with High‐ and Low‐metastatic Potential from PC‐14 Human Lung Adenocarcinoma
Authors: Shindo‐Okada Nobuko , Takeuchi Kaori , Nagamachi Yasuhiro
Published: August 2005

This article reports the establishment of variant cell lines with high and low metastatic potential by repeated selection and the dilution plating technique. Five clones with high metastatic potential, Lu‐2, Lu‐7, Lu‐4, Lu‐1 and Lu‐5, and four clones with low metastatic potential, 3S, 7S, 8S and 13S, were established from PC‐14 human lung adenocarcinoma. The high‐metastatic cell lines produced enhanced lung metastases, but the low‐metastatic cell lines did not produce lung metastasis by injection into the tail vein of 5‐week‐old BALB/c nude mice. The high‐metastatic cell lines produced enhanced tumors on both visceral and parietal pleurae, and enhanced metastases to the mediastinum and contralateral pleural cavity. The low‐metastatic cell lines produced reduced tumors on both visceral and parietal pleurae and reduced metastases to the mediastinum and contralateral pleural cavity after injection into the left preceral cavity of the nude mice. When the nine variant cell lines and original PC‐14 cells were embedded in collagen gels, the PC‐14 cells and the low‐metastatic cell lines gave rise to colonies with a dendritic morphology, and cells were tightly associated. The high‐metastatic cell lines were more loosely associated and scattered into three‐dimensional colonies. These nine cloned cell lines originated from heterogeneous populations of the parental PC‐14 cells should be useful tools for studying the process of metastasis of lung cancer.
Different Susceptibilities of Postmitotic Checkpoint‐proficient and ‐deficient Balb/3T3 Cells to ICRF‐193, a Catalytic Inhibitor of DNA Topoisomerase II
Authors: Nishida Keiko , Seto Masao , Ishida Ryoji
Published: August 2005

Two distinct types of Balb/3T3 cells were isolated which exhibit either 4 N DNA or both 4 N and 8 N DNA after exposure to colcemid for 48 h. They were found to differ with respect to the postmi‐totic checkpoint, but not the mitotic checkpoint. Firstly, the checkpoint‐proficient and ‐deficient cells exhibited the same accumulation and subsequent decrease in the number of mitotic cells following exposure to microtubule inhibitors. Secondly, after exit from abnormal mitosis in the presence of ICRF (Imperial Cancer Research Fund)‐193, the checkpoint‐proficient cells were arrested in the next cycle Gl, while the checkpoint‐deficient cells progressed into S and G2 phase. When either mitotic or asynchronous cells were exposed to ICRF‐193, the checkpoint‐proficient cells proved more sensitive to the cytotoxic effect of this agent than the checkpoint‐deficient cells. The different susceptibilities of the two types of cells to ICRF‐193 were not caused by variation in topoisomerase (topo) II function since both the biochemical activity of this enzyme and chromosome segregation were inhibited by similar concentrations of ICRF‐193 in both checkpoint‐proficient and ‐deficient cells. We propose that the inhibition of chromosome segregation by ICRF‐193 is monitored by the next Gl checkpoint, resulting in an irreversible Gl block in the case of postmi‐totic checkpoint‐proficient cells. As the checkpoint‐deficient cells can escape this Gl block, these cells have an increased survival capacity. In summary, ICRF‐193 may prove to be a very useful drug for examination of the postmitotic checkpoint.
Involvement of the Multidrug Resistance Protein 3 in Drug Sensitivity and Its Expression in Human Glioma
Authors: Haga Sei , Hinoshita Eiji , Ikezaki Kiyonobu , Fukui Masashi , Scheffer George L. , Uchiumi Takeshi , Kuwano Michihiko
Published: August 2005

The multidrug resistance protein (MRP) family belongs to the ATP‐binding cassette superfamily (ABC) of transporters, which are involved in ATP‐dependent transport of hydrophobic compounds. One of the MRP family,MRP1, is partially associated with the multidrug resistance phe‐notype in brain tumors. In this study, we asked whether another MRP family gene,MRP3, could affect drug sensitivity to anticancer agents in human glioma cell lines and clinical glioma specimens. We first produced two antisense transfectants by introduction of antisenseMRP3cDNA into the glioma cell line NHG2, which endogenously expressesMRP3.The twoMRP3antisense transfectants showed 2‐ to 5‐fold increases in drug sensitivity to etoposide and cisplatin compared with NHG2 cells, but their sensitivity to vincristine or nitrosourea was not changed. TwoMRP3cDNA sense transfectants of pig kidney cell lines showed 4‐ to 6‐fold drug resistance to etoposide, but only 1.4‐ to 1.5‐fold to cisplatin. We next compared the mRNA levels of four ABC transporters, multi‐drug resistance 1 (MDR1),MRP1, MRP2andMRP3in clinical samples, including 34 patients with gliomas, by quantitative RT‐PCR analysis. In some of the clinical samples, increased expression ofMRP1andMRP3was apparent in malignant gliomas.In situhybridization revealed that glioma cells were stained withMRP3probe. MRP3 may modulate drug sensitivity to certain anticancer agents in human gliomas.
Phase I Study of Intravenous PSC‐833 and Doxorubicin: Reversal of Multidrug Resistance
Authors: Minami Hironobu , Ohtsu Tomoko , Fujii Hirofumi , Igarashi Tadahiko , Itoh Kuniaki , Uchiyama‐Kokubu Noriko , Aizawa Tetsushi , Watanabe Torn , Uda Yoshinori , Tanigawara Yusuke , Sasaki Yasutsuna
Published: August 2005

PSC‐833 reverses multidrug resistance by P‐glycoprotein at concentrations <1000 ng/ml. A phase I study of PSC‐833 and doxorubicin was conducted to determine the maximum tolerated dose and to investigate pharmacokinetics. PSC‐833 was intravenously infused as a 2‐h loading dose (LD) and a subsequent 24‐h continuous dose (CD). Doxorubicin was infused over 5 min, 1 h after the LD. The starting dose was 1 mg/kg for both LD and CD with 30 mg/m2 doxorubicin; these dosages were increased to 2 and 10 mg/kg and 50 mg/m2, respectively. Thirty‐one patients were treated. Nausea/ vomiting was controllable with granisetron and dexamethasone. Neutropenia and ataxia were dose limiting. Steady‐state concentrations of PSC‐833 >1000 ng/ml were achieved at a 2 mg/kg LD and a 10 mg/kg CD. Ex‐vivo bioassay revealed that activity in serum for reversing multidrug resistance was achieved in all patients; IC50 of P‐glycoprotein expressing 8226/Dox6; in patients’ serum was decreased from 5.9 to 1.3 μg/ml (P<0.0001) by PSC‐833 administration. Doxorubicin clearance was 24.3±13.7 (mean±SD) liter/h/m2, which was lower than the 49.0±16.9 liter/h/m2 without PSC‐833 (P<0.0001). The relationship between doxorubicin exposure and neutropenia did not differ between patients treated and not treated with PSC‐833. The recommended phase II dose of PSC‐833 was 2 and 10 mg/kg for LD and CD, respectively, which achieved a sufficient concentration in serum to reverse drug resistance, as confirmed by bioassay. The dose of doxorubicin should be reduced to 40 mg/m2, not because of the pharmacodynamic interaction between PSC‐833 and doxorubicin affecting hematopoiesis, but because of pharmacokinetic interaction.
Pharmacodynamic Modeling of the Entire Time Course of Leukopenia after a 3‐Hour Infusion of Paclitaxel
Authors: Minami Hironobu , Sasaki Yasutsuna , Watanabe Torn , Ogawa Makoto
Published: August 2005

The entire time course of leukopenia after anticancer treatment is clinically more relevant than a singly measured nadir count. In order to identify factors associated with neutropenic fever, a mechanistic pharmacodynamic model with two compartments corresponding to leukocytes in bone marrow and peripheral blood was applied to describe the time course of leukopenia. Seventeen patients with breast cancer were treated with 210 mg/m2 of paclitaxel infused over 3 h as a single agent in a phase II study. Adequate fitting of the time course of leukopenia was achieved in all patients, and time‐dependent parameters, including the tune period during which leukocyte counts remained below 2000/μl and the area between the curve for time versus leukocyte counts and the line of a leukocyte count of 2000/μl (A <2000), were calculated in each patient. Leukopenia was not significantly correlated with pharmacokinetic parameters, including time above a threshold concentration or the area under the tune‐concentration curve. A negative correlation between age and the sensitivity parameter of the pharmacodynamic model was observed (r2=0.21, P=0.07). Patients who experienced neutropenic fever had a larger A<2000 than patients who did not experience fever (4512 vs. 6 days/μl, P=0.05), but fever was not significantly related to any pharmacokinetic parameter or the leukocyte nadir count. Febrile episodes were better associated with the time course of leukopenia than the singly measured nadir count, and the pharmacodynamic model presents a novel platform to analyze the entire tune course of leukopenia.
Effect of Smoking on Serum Pepsinogen I Level Depends on Serological Status of Helicobacter pylori
Authors: Tatemichi Masayuki , Kabuto Michinori , Tsugane Shoichiro
Published: August 2005

Serum pepsinogen (sPG) levels are used in gastric cancer screening programs. However, modification of sPG levels by smoking habit, according to the status of Helicobacter pylori (H. pylori) infection has been little investigated. This study investigated the effects of smoking on serum levels of pepsinogen I (PG I), pepsinogen II (PG II), and gastrin by IgG titer of antibody against H. pylori (Hp‐IgG titer) using the data from 356 current‐smokers and 262 non‐smokers (133 never‐smokers and 129 ex‐smokers) in a cross‐sectional study of 618 men aged 40 to 49 years. PG I, PG II, PG I/PG II ratio and gastrin were significantly associated with Hp‐IgG titer in never‐smokers [Spearman's correlation coefficient (95% confidence interval): 0.23 (0.07, 0.39), 0.52 (0.41, 0.63), ‐0.40 (‐0.54, ‐0.27), and 0.25 (0.10, 0.41), respectively]. However, the correlation coefficients of PG I and PG H decreased in current‐smokers, 0.02 (‐0.1, 0.13) and 0.32 (0.22, 0.42), respectively. In H. pylori seronegative and low titer cases, the mean PG I level was significantly (P<0.01) higher in current‐smokers, compared with non‐smokers. However, in high titer cases, the mean PG I level was lower in current‐smokers. Mean PG II and gastrin levels, and PG I/PG II ratio did not differ according to smoking habits by Hp‐IgG titer. The gastrin level was significantly correlated with PG H, but not PG I. These data indicate that current smoking influences the serum PG I level depending on Hp‐IgG titer and the associations between sPGs and Hp‐IgG titer. Gastrin is not involved in the modification of PG I levels by smoking.
Aberrant Expression of pRb, p16, p14ARF, MDM2, p21 and p53 in Squamous Cell Carcinomas of Lung
Authors: Xue Qi , Sano Takaaki , Kashiwabara Kenji , Oyama Tetsunari , Nakajima Takashi
Published: August 2005

Expression of cell cycle regulatory proteins in both the RB and p53 pathways was investigated in 50 cases of squamaous cell carcinoma (SCC) of the lung using immunohistochemical techniques.Abnormality of pRb and p16 expression was seen at the frequencies of 16% and 78%, respectively, and appeared to be in a reciprocal relationship. On the other hand, strong and diffuse p53 immunoreactivity was seen in 60% of SCCs. MDM2 and p14ARF expression. Moreover, strong p53 expression Moreover, strong p53 expression was not correlated with the expression of p21. In comparing clinicopathological status with immunohistochemical results, lack of p16 immunoreactivity was observed in the elderly group (over 65 years) as compared with the younger group (less than 65 years) Strong p53 expression was frequently observed in higher stages of SCC, with the developing tumor located in the central field of the lung. Similarly, the frequency of p14ARF expression was high in centrally developed SCC, but low in SCC developed in the periphery. These results suggest that disruption of the RB and p53 pathways is a frequent event in SCC, and that abnormal expression of p16 and p53 plays a more critical role than that of pRB, p14ARF and MDM2 in the development of SCC of the lung.
Successful Induction of Tumor‐specific Cytotoxic T Lymphocytes from Patients with Non‐small Cell Lung Cancer Using CD80‐transfected Autologous Tumor Cells
Authors: Takenoyama Mitsuhiro , Yoshino Ichiro , Eifuku Ryozo , So Tomoko , Imahayashi Satoru , Sugaya Masakazu , Yasuda Manabu , Inoue Masaaki , Ichiyoshi Yuji , Osaki Toshihiro , Nagashima Akira , Nomoto Kikuo , Yasumoto Kosei
Published: August 2005

Cytotoxic T lymphocytes (CTL) against human lung cancer cells are difficult to induce by a conventional method using tumor cell stimulation probably due to an insufficiency of tumor antigens (TA) or costimulatory molecules such as CD80. We, therefore, investigated the potential of CD80‐transfected tumor cells as stimulators of the in vitro induction of autologous tumor‐specific CTL from regional lymph node lymphocytes in patients with lung cancer. Five non‐small cell lung cancer cell lines (two adenocarcinomas, 1 squamous cell carcinoma, 1 large cell carcinoma and 1 adenosquamous cell carcinoma) were established from surgical specimens and were successfully transduced with a plasmid constructed with expression vector pBj and human CD80 cDNA, using a lipofection method. CD80‐transfected tumor cells (CD80‐AT) significantly augmented the proliferation of autologous lymphocytes from all cases as compared with non‐transfected tumor cells (AT). AT‐stimulated lymphocytes from 4 out of 5 cases did not show any cytotoxicity against AT; however, lymphocytes stimulated with CD80‐AT exhibited substantial cytotoxicity against parental AT in all 5 cases tested. AT‐stimulated lymphocytes derived from only one out of 5 cases showed major histocompatibility complex (MHC)‐class I‐restricted cytokine production in response to AT, while the MHC‐class I‐restricted responses were found in CD80‐AT‐stimulated lymphocytes from 4 out of 5 cases. These results indicate that CD80 on tumor cells could be a beneficial costimulatory molecule to elicit CTL against lung cancer, and also show that TA recognized by CTL was frequently expressed on lung cancer cells.
Autoantibody to Heat Shock Protein Hsp40 in Sera of Lung Cancer Patients
Authors: Oka Mikio , Sato Sho‐ichi , Soda Hiroshi , Fukuda Minoru , Kawabata Shigeru , Nakatomi Katsumi , Shiozawa Ken , Nakamura Yo‐ichi , Ohtsuka Kenzo , Kohno Shigeru
Published: August 2005

Heat shock protein Hsp40 is a stress protein with chaperone activity and has a cooperative function with Hsp70 in mammalian cells. We examined the possible expression of Hsp40 in lung tumor tissues using immunoblotting and immunohistochemistry, and established an enzyme‐linked immuno‐sorbent assay (ELISA) method to detect IgG antibody to Hsp40 in the serum using purified human Hsp40. Sera were obtained from 130 normal subjects and 50 patients with lung cancer. Lung tumor tissues and cells specifically overexpressed Hsp40, and no such expression was detected in normal lung tissues. Compared with normal sera, significantly higher levels of auto‐antibody to Hsp40 were present in patients with lung cancer. The present study is the first to demonstrate overexpression of Hsp40 in human tumor tissue and the associated presence of autoantibody to Hsp40 in the serum. These results suggest that overexpression of Hsp40 in tumor cells may be recognized as a self‐antigen.
Germline Mutation of Dihydropyrimidine Dehydrogenese Gene among a Japanese Population in Relation to Toxicity to 5‐Fluorouracil
Authors: Yamaguchi Kensei , Aral Yoshiko , Kanda Yuzo , Akagi Kiwamu
Published: August 2005

5‐Fluorouracil (5FU) is most commonly used in chemotherapy for human malignancy. Over 80% of administered 5FU is metabolically degraded by dihydropyrimidine dehydrogenase (DPD), a primary and rate‐limiting enzyme in the 5FU metabolic pathway. A DPD‐deficient phenotype among cancer patients, which has posed a serious problem in 5FU‐based chemotherapy, was reported to be in part ascribed to germline mutations in dihydropyrimidine dehydrogenase (DPYD) gene. Therefore, we for the first tune examined the frequencies and types of germline mutations in the DPYD gene among a total of 107 Japanese cancer patients and healthy volunteers. Of 214 alleles examined among them, 181 alleles were of the same type, which was assigned as wild type; 21 alleles revealed a nucleotide substitution resulting in silent mutation; and the remaining 12 alleles showed five types of nucleotide deletion or substitutions resulting in one frameshift and four missense mutations. Three of them, A74G, 812delT and L572V, were novel mutations. None of the study subjects showed homozygous frameshift or missense mutated alleles. We also studied the association between toxic response to 5FU and heterozygous frame shift or missense mutation of the DPYD gene among eight cancer patients who had received 5FU‐based chemotherapy. These patients did not show any adverse effects higher than grade 3, suggesting that heterozygotes are not associated with increased toxicity to 5FU. Our results indicate that a very small percentage, about 0.2%, of the Japanese population seems to carry homozygous mutations in DPYD gene, mutations which possibly indicate genetically increased toxicity of 5FU‐based chemotherapy.
Overexpression of CDK2 Is a Prognostic Indicator of Oral Cancer Progression
Authors: Mihara Mariko , Shintani Satoru , Nakahara Yuuji , Kiyota Akihisa , Ueyama Yoshiya , Matsumura Tomohiro , Wong David T. W.
Published: August 2005

Cyclins and cyclin‐dependent kinases (CDKs) play key roles in cell cycle regulation, a process of which dysregulation can lead to uncontrolled cell growth and hence to cancer. We have already reported the alteration of CDK4 and cyclin Dl expression in oral cancer. In this study, we examined by immunohistochemistry the expression of CDK2, and cyclins A and E in 20 normal oral mucosa, 42 dysplastic epithelia, and 103 oral squamous cell carcinomas (SCCs). The expressions of CDK2, and cyclins A and E were not detected in the normal epithelium and significantly altered from epithelial dysplasia to SCC. While there were no significant correlations between the expression of cyclins A, E and the patients' survival, CDK2 expression was significantly correlated with lymph node involvement (P=0.025), tumor differentiation (P=0.032), mode of tumor invasion (P=0.017), and shorter survival period (P=0.0173). These results suggest that the elevated expression of CDK2 is a critical factor in oral cancer progression and can be used as a negative predictive marker of the patients' prognosis.
The Predictive Value of Vascular Endothelial Growth Factor and Nm23 for the Diagnosis of Occult Metastasis in Non‐small Cell Lung Cancer
Authors: Ohta Yasuhiko , Nozaki Zensei , Nozawa Hiroshi , Kamesui Tadashi , Tsunezuka Yoshio , Oda Makoto , Watanabe Go
Published: August 2005

We assessed the association of vascular endothelial growth factor (VEGF) and nml23 expression with occult micrometastasis in lung cancer. As destination sites for micrometastasis, we scrutinized lymph node (LN) and bone marrow (BM) specimens. For LN, 122 stage I patients who had received curative operations were studied. As regards BM, 203 patients in stage I‐IV who underwent operations were registered. Immunohistochemical anti‐cytokeratin staining was used to detect microdissemination of cancer cells. The VEGF and the nm23 expression at the primary sites were immunohistochemically studied in 285 cases in total. The percentages of the patients with micro‐dissemination were 28.7% for LN and 42.4% for BM. The outcome for the patients with LN or BM microdissemination was significantly worse than that for patients without it. The increased VEGF and the decreased nm23 expression within primary tumors were significantly associated with LN and BM microdissemination. The results indicate possible value of using these biological markers to predict the risk of systemic micrometastasis in non‐small cell lung cancer.
Psychosocial Factors as a Potential Trigger of Oxidative DNA Damage in Human Leukocytes
Authors: Irie Masahiro , Asami Shinya , Nagata Shoji , Ikeda Masato , Miyata Masakazu , Kasai Hiroshi
Published: August 2005

Although numerous studies have been carried out on the stress‐cancer linkage, the results are still inconclusive. One of the useful, but rarely applied, methods to assess this linkage is to examine the relationship between psychosocial stress and cancer‐predisposing genetic alterations simultaneously. We investigated whether various psychosocial factors can be associated with the levels of 8‐hydroxydeoxyguanosine (8‐OH‐dG), a biomarker of cancer‐related oxidative DNA damage, in peripheral blood leukocytes in 362 healthy workers (276 males and 86 females). After adjustments for age, body mass index, cigarette smoking, and alcohol use, female subjects showed positive relationships between the amount of 8‐OH‐dG and the Tension‐Anxiety, Depression‐Rejection, Anger‐Hostility, Fatigue, and Confusion scores of the Profile of Mood States, respectively. The levels of 8‐OH‐dG also increased reliably in the female subjects who had poor stress‐coping behaviors, particularly wishful thinking strategy, in the NIOSH general job stress instrument. There were positive relationships of the 8‐OH‐dG levels to average working hours, a self‐blame coping strategy, and recent loss of a close family member in male subjects. These findings in a nonclinical sample of healthy adults not only provide evidence of a stress‐cancer linkage, but also suggest possible sex differences in the mechanisms of stress‐related cancer initiation.
Interleukin 1 Polymorphisms, Lifestyle Factors, and Helicobacter pylori Infection
Authors: Hamajima Nobuyuki , Matsuo Keitaro , Saito Toshiko , Tajima Kazuo , Okuma Katashi , Yamao Kenji , Tominaga Suketami
Published: August 2005

Associations between Helicobacter pylori (HP) infection and lifestyle factors have been reported by several authors, but little is known about the host factors associated with the infection. This study aims to examine the infection rate of HP according to gene polymorphisms of interleukin (TL)‐IA, IL‐1B, and IL‐1RN, and to investigate the interactions with lifestyle factors. Subjects were 241 non‐cancer outpatients who had participated in a HP eradication program. Polymorphisms at ‐889 (T to C) of IL‐1A, at ‐31 (C to T; T allele makes a TATA box) and ‐511 (C to T) of IL‐1B, and at intron 2 (86‐bp VNTR (variable number of tandem repeats)) of IL‐1RN were genotyped by PCR (polymerase chain reaction), PCR‐RFLP (restriction fragment length polymorphism) and PCR‐CTPP (PCR with confronting two‐pair primers). It was found that IL‐1B polymorphisms at ‐31 and ‐511 were near‐completely linked, but in the opposite way to that in Caucasians; ‐31C/ ‐511T and ‐31T/‐511C alleles were dominant in the present subjects. The HP infection rate was substantially different among the genotypes of IL‐1B C‐31T; 45.2% (19/42) for the C/C, 67.7% (90/133) for the C/T, and 63.6% (42/66) for the T/T. The age‐sex adjusted odds ratio (OR) relative to the C/C genotype was 2.32 (95%CI (confidence interval), 1.10‐4.92) for the T/C genotype and 2.46 (1.06‐5.74) for the T/T genotype. The OR for the T/T genotype was significantly modified by smoking status; interaction term=14.6 (1.12‐190). The polymorphisms of IL‐1A and IL‐1RN were not associated with the infection rate. The results suggested that the T allele of IL‐1B C‐31T is associated with vulnerability to persistent HP infection, and that the vulnerability is modified by smoking.
Synergistic Effects of High‐dose Soybean Intake with Iodine Deficiency, but Not Sulfadimethoxine or Phenobarbital, on Rat Thyroid Proliferation
Authors: Ikeda Takako , Nishikawa Akiyoshi , Son Hwa‐Young , Nakamura Hideaki , Miyauchi Makoto , Imazawa Takayoshi , Kimura Shuichi , Hirose Masao
Published: August 2005

The specificity and dose dependence of the synergistic effects of soybean intake with iodine deficiency on the induction of thyroid proliferation were investigated in female F344 rats. In the first experiment, rats were divided into 6 groups, each consisting of 5 annuals, and fed a basal diet containing 20% gluten, an iodine‐deficient basal diet alone or an iodine‐deficient diet containing 0.2%, 1.0%, 5.0% or 25% defatted soybean for 5 weeks. Soybean feeding synergistically induced thyroid hyperplasias with iodine deficiency only at the 25% dose. In the second experiment, rats were also divided into 6 groups, each consisting of 5 animals, and fed a basal diet, a diet containing 20% defatted soybean, 0.025% Sulfadimethoxine (SDM), 20% defatted soybean+0.025% SDM, 0.05% phenobarbital (PB) or 20% defatted soybean+0.05% PB for 5 weeks. The SDM treatments significantly (P<0.05‐0.01) increased the thyroid weights, but this increase rate was less prominent in the SDM+soybean group than in the SDM alone group. The PB treatment was also associated with a tendency for increase in thyroid weight, but again this was smaller in the PB+soybean group than in the PB alone group. Although the SDM or PB treatments reduced the serum triiodothyronine and thyroxine levels and consequently increased the serum thyroid‐stimulating hormone (TSH) levels, the soybean feeding did not affect or rather attenuated these changes. Our results clearly indicate that soybean feeding does not synergistically enhance the effects of SDM or PB on the rat thyroid. Thus it can be concluded that soybean intake specifically interacts with iodine deficiency in induction of thyroid proliferative lesions in rats, only at high doses.
A Ferulic Acid Derivative, Ethyl 3‐(4′‐Geranyloxy‐3‐methoxyphenyl)‐2‐propenoate, as a New Candidate Chemopreventive Agent for Colon Carcinogenesis in the Rat
Authors: Han Beom Seok , Park Cheol Beom , Takasuka Nobuo , Naito Akihiro , Sekine Kazunori , Nomura Eisaku , Taniguchi Hisaji , Tsuno Takuo , Tsuda Hiroyuki
Published: August 2005

The inhibitory influence of ferulic acid (FA), a rice germ component, and its geranylated derivative 3‐(4′‐geranyloxy‐3‐methoxyphenyl)‐2‐propenoate (EGMP) on the post‐initiation stage of azoxy‐methane (AOM)‐induced colon carcinogenesis was studied in male F344 rats given two s.c. injections of AOM (15 mg/kg body weight) during week 1. Diets containing EGMP or FA at doses of 0.1 or 0.2% were then fed for 3 weeks from week 2 to 5, when the animals were sacrificed. The numbers of aberrant crypt foci (ACF) and aberrant crypts (AC) per rat in the group given 0.2% FA were significantly decreased (P<0.001) as compared to the AOM alone group. Furthermore, the numbers of ACF and AC per rat fed the 0.2% and 0.1% EGMP were significantly reduced (P<0.001 and P<0.01, respectively). Colonic epithelial cells in S‐phase, as measured by bromodeoxy‐uridine (BrdU) labeling, in rats fed EGMP were significantly decreased in the 0.2 and 0.1% EGMP groups as compared to the AOM alone group (P<0.05). BrdU labeling indices in rats fed FA and EGMP assessed by a test using a coefficient for linear contrast were also significantly decreased as compared to the AOM alone value (P<0.05, P<0.01, respectively). The results indicate that FA and EGMP have inhibitory effects on ACF and AC development, EGMP being more potent, possibly due to stronger suppressive effects on cell proliferation. No toxic effects were observed in rats given either compound in terms of body and organ weights, and liver or kidney histology. The findings thus suggest that EGMP and FA, especially the former, might have potential as chemopreventive agents against colon tumor development.
Enhanced Vascular Permeability in Solid Tumor Involving Peroxynitrite and Matrix Metalloproteinases
Authors: Wu Jun , Akaike Takaaki , Hayashida Kazuyuki , Okamoto Tatsuya , Okuyama Akira , Maeda Hiroshi
Published: August 2005

Peroxynitrite (ONOO ‐), which is generated from nitric oxide (NO) and superoxide anion (O2‐) under pathological conditions, plays an important role in pathophysiological processes. Activation of matrix metalloproteinases (MMPs) contributes to tumor angiogenesis and metastasis. NO mediates the enhanced vascular permeability and retention (EPR) effect in solid tumors, and ONOO‐ activates proMMP to MMP in vitro. In this study, we examined the role of ONOO‐ in the EPR effect in solid tumors and normal tissues as related to MMP activation. Authentic ONOO‐, at 50 nmol or higher concentrations, induced the enhanced vascular permeability in normal dorsal skin of mice. ONOO‐ scavengers ebselen and uric acid significantly suppressed the EPR effect in mouse sarcoma 180 (S‐180) tumors. Indirect evidence for formation of ONOO‐ in S‐180 and mouse colon adenocarcinoma (C‐38) tumors included strong immunostaining for nitrotyrosine in the tumor tissue, predominantly surrounding the tumor vessels. MMP inhibitor BE16627B (66.6 mg/kg i.v., given 2 tunes) or SI‐27 (10 mg/kg i.p., given 2 times) significantly suppressed the ONOO‐induced EPR effect in S‐180 tumors and in normal skin. Soybean trypsin inhibitor (Kunitz type), broad‐spectrum proteinase inhibitor ovomacroglobulin, and bradykinin receptor antagonist HOE 140 also significantly suppressed the ONOO‐induced EPR effect in normal skin tissues. These data suggest that ONOO‐ may be involved in and promote the EPR effect in tumors, which could be mediated partly through activation of MMPs and a subsequent proteinase cascade to generate potent vasoactive mediators such as bradykinin.
Expression of Multidrug Resistance‐related Transporters in Human Breast Carcinoma
Authors: Kanzaki Atsuko , Toi Masakazu , Nakayama Kentaro , Bando Hiroko , Mutoh Masato , Uchida Takafumi , Fukumoto Manabu , Takebayashi Yuji
Published: August 2005

The expression levels of mRNA for multidrug resistance 1 (MDR1) gene, multidrug resistance protein 1 (MRP1), lung resistance‐related protein (LRP) and breast cancer resistance protein (BCRP), which confer multidrug resistance in vitro, were examined in 43 untreated breast carcinoma patients, of whom 38 subsequently received doxorubicin‐based chemotherapy after surgery, in order to elucidate the roles of these genes in drug resistance in vivo. The mRNA levels were determined using a semi‐quantitative reverse‐transcription polymerase chain reaction method in breast carcinoma tissues including at least 80% carcinoma cells. The expression level of BCRP gene was low and did not vary markedly in comparison with that of MDR1, MRP1 or LRP gene. The expressions of MDR1 and MRP1 genes were correlated with each other, but the expression of BCRP or LRP gene did not correlate with that of other genes. These four gene expressions were independent of age, TNM categories and the status of progesterone or estrogen receptor. The expression levels of these four genes were not related to the relapse or prognosis of the 38 patients treated with doxorubicin‐based chemotherapy. P‐glycoprotein (P‐gp)/MDRl, MRP1 and LRP may play more important roles than BCRP in chemotherapy of human breast carcinoma.
Chitosan Induces Apoptosis via Caspase‐3 Activation in Bladder Tumor Cells
Authors: Hasegawa Masumi , Yagi Keiko , Iwakawa Seigo , Hirai Midori
Published: August 2005

Recently, because of its low toxicity and biological effects, chitosan has been widely used in the medical and pharmaceutical fields, e.g., for nasal or oral delivery of peptide or polar drug delivery. Here, we report a growth‐inhibitory effect of chitosan on tumor cells. The growth inhibition was examined by WST‐1 colorimetric assay and cell counting. We also observed DNA fragmentation, which is characteristic of apoptosis, and elevated caspase‐3‐like activity in chitosan‐treated cancer cells. The findings suggest that chitosan may have potential value in cancer therapy.
Strain‐dependency of Chromosomal Abnormalities in Lymphomas Developed in Eμ‐myc Transgenic Mice
Authors: Akagi Kiwamu , Yamamura Ken‐ichi
Published: August 2005

We previously showed that B and T cell lymphoma development in Eμ (immunoglobulin heavy chain enhancer)‐mye transgenic mice is dependent on the mouse strain. To determine whether any non‐random chromosomal abnormality that was present was caused by variations in the lymphoma cell type or by a different genetic background, we crossed C3H transgenic mice with other inbred strains of mice, C57BL/6 or BALB/c. Cytogenetic analysis showed a high frequency of non‐random chromosomal aberrations, namely, duplication or amplification of part of chromosome 5 containing the transgene and trisomy of chromosome 1, 6, or 12 in the genetic background of C3HXC57BL/6 mouse and C3HxBALB/c mouse, respectively, regardless of cell type of lymphoma. These results suggest that non‐random chromosomal abnormalities in lymphoma cells are dependent on the genetic background of mouse, not on the tumor cell type in Eμ‐myc transgenic
Overexpression of Latent Transforming Growth Factor‐βl (TGF‐βl) Binding Protein 1 (LTBP‐1) in Association with TGF‐β1 in Ovarian Carcinoma
Authors: Higashi Tomomi , Sasagawa Toshiyuki , Inoue Masaki , Oka Rie , Shuangying Leng , Saijoh Kiyofumi
Published: August 2005

Using the differential display method, latent transforming growth factor‐β (TGF‐β1) binding protein 1 (LTBP‐1) mRNA was identified as one of the enriched mRNAs in ovarian carcinoma tissues after isolation of genes responsible for the development of ovarian cancer. Semi‐quantitative reverse transcription (RT)‐PCR analysis showed that expression of LTBP‐1 and TGF‐β1 mRNAs was much higher in both serous and mucinous adenocarcinomas than in their benign counterparts, including serous and mucinous cystadenomas and cystadenomas of low malignant potential (LMPs). Immunohistochemical analysis demonstrated that only proliferating benign adenoma cells were immunoreactive for both LTBP‐1 and TGF‐β1 proteins. In contrast, most serous and mucinous adenocarcinoma cells and their surrounding stroma were intensely immunoreactive for LTBP‐1 and TGF‐β1. LTBP‐1 and TGF‐β1 proteins, and their complex forms were identified in ovarian carcinoma cell lines and in their culture media by western blot analysis, suggesting these products were produced in ovarian carcinoma cells. RT‐PCR analysis demonstrated that LTBP‐1L, one of the LTBP‐1 transcripts that has a strong activity in targeting the latent form of TGF‐β1 to extracellular matrix (ECM), was predominantly expressed in ovarian carcinomas. Taken together, the results suggest that upregulation of LTBP‐1 in ovarian carcinoma cells may have an important role in distributing TGF‐β1 in the stromal tissues surrounding carcinoma cells.
Urokinase Receptor and Vascular Endothelial Growth Factor Are Synergistically Associated with the Liver Metastasis of Colorectal Cancer
Authors: Konno Hiroyuki , Abe Jinro , Kaneko Takeshi , Baba Megumi , Shoji Atsushi , Sunayama Kenichi , Kamiya Kinji , Tanaka Tatsuo , Suzuki Shohachi , Nakamura Satoshi , Urano Tetsumei
Published: August 2005

Considering recent findings that the urokinase plasniinogen activation (PA) system is involved in invasion and vascular endothelial growth factor (VEGF) is involved in angiogenesis of colorectal cancer, we evaluated these factors in the liver metastasis of primary colorectal cancer. Cancer tissues from 71 colorectal cancer patients were assayed quantitatively for antigen levels of urokinase type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor‐1 and ‐2 (PAI‐1, PAI‐2), and were also assayed immunohistochemically for expression of VEGF protein. Among the PA system factors, both the levels of uPAR and PAI‐1 were significantly higher in larger tumors than in smaller ones, and were also significantly higher in tumors that invaded subserosa, serosa or adjacent organs than in mucosal, submucosal tumors or in tumors that invaded the muscle layer. The uPAR levels were significantly higher in tumors with liver metastasis than in those without. VEGF overexpression was significantly more frequent in tumors with lymph node involvement or liver metastasis than in those without. Among the PA system factors, the uPAR levels were significantly higher in tumors with VEGF overexpression and a multivariate analysis revealed that high uPA level and VEGF overexpression were independent risk factors for liver metastasis. The combination of high uPAR level and overexpression of VEGF was associated with the worst prognosis in patients with colorectal cancer. These results suggest that uPAR and VEGF might contribute Synergistically to the liver metastasis of colorectal cancer.
The Antitumor Mechanism of 1‐(2‐Deoxy‐2‐fluoro‐4‐thio‐β‐D‐arabinofuranosyl)‐cytosine: Effects of Its Triphosphate on Mammalian DNA Polymerases
Authors: Miura Shinji , Yoshimura Yuichi , Satoh Hiroshi , Izuta Shunji
Published: August 2005

The mechanism of action of the antitumor nucleoside analog l‐(2‐deoxy‐2‐fluoro‐4‐thio‐β‐D‐arabinofuranosyl)cytosine (4′‐thio‐FAC) was investigated. 4′‐Thio‐FAC inhibited cellular DNA synthesis, but not RNA and protein syntheses. We observed potent inhibitory action of the triphosphate of 4′‐thio‐FAC (4′‐thio‐FACTP) against DNA polymerase α, whereas it showed moderate inhibition of DNA polymerase P and little inhibition of DNA polymerase β. The kinetic analysis showed that the inhibition mode of 4′‐thio‐FACTP towards DNA polymerase a was mixed type, implying a chain‐terminating effect of 4′‐thio‐FACTP. The triphosphate of 2′‐deoxy‐2′,2′‐difluorocytidine (gemcitabine), a known antitumor nucleoside, did not show potent inhibition of these three DNA polymerases. Thus, the effect of the diphosphate of gemcitabine on ribonucleotide reductase was suggested to be more important for the antitumor action of gemcitabine. From these findings, the main target enzymes of 4′‐thio‐FAC and gemcitabine appear to be different. We found a synergistic effect of the two drugs in an in vitro model, which supports the above idea.