bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Water‐soluble Camptothecin Derivative, DX‐8951f, Exhibits Potent Antitumor Activity against Human Tumors in vitro and in vivo
Authors: Mitsui Ikuo , Kumazawa Eiji , Hirota Yasuhide , Aonuma Masashi , Sugimori Masamichi , Ohsuki Satoru , Uoto Kouichi , Ejima Akio , Terasawa Hirofumi , Sato Keiki
Published: August 2005

CPT‐11, a semisynthetic derivative of camptothecin, exhibited strong antitumor activity against lymphoma, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, and cervical cancer. CPT‐11 is a pro‐drug that is converted to an active metabolite, SN‐38, in vivo by enzymes such as carboxylesterase. We synthesized a water‐soluble and non‐pro‐drug analog of camptothecin, DX‐8951f. It showed both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The anti‐proliferative activity of DX‐8951f, as indicated by the mean GI50 value, was about 6 and 28 times greater than that of SN‐38 or SK&F 10486‐A (Topotecan), respectively. These three derivatives of camptothecin showed similar patterns of differential response among 32 cell lines, that is, their spectra of in vitro cytotoxicity were almost the same. The antitumor activity of three doses of DX‐8951f administered i.v. at 4‐day intervals against human gastric adenocarcinoma SC‐6 xenografts was greater than that of CPT‐11 or SK&F 10486‐A. Moreover, it overcame P‐glycoprotein‐mediated multi‐drug resistance. These data suggest that DX‐8951f has a high antitumor activity and is a potential therapeutic agent.
Localization of Urokinase‐type Plasminogen Activator, Plasminogen Activator Inhibitor‐1, 2 and Plasminogen in Colon Cancer
Authors: Naitoh Hiroyuki , Eguchi Yutaka , Ueyama Hisao , Kodama Masashi , Hattori Takanori
Published: August 2005

We examined the localization of urokinase‐type plasminogen activator (u‐PA), plasminogen activator inhibitors (PAI‐1 and PAI‐2) and plasminogen (plg) in 26 cases of colon cancer by immunohistochemical staining. The u‐PA antigen was detected in the cytoplasm of cancer cells (18/26) and stromal cells adjacent to cancer tissues (9/26). The localization of u‐PA mRNA examined by in situ hybridization was consistent with that of u‐PA antigen. The PAI‐1 antigen was detected in fibroblasts and endothelial cells (22/26), while PAI‐2 antigen was found in cancer cells (20/26). The plg antigen was seen in the extracellular matrix of the cancer stroma. The u‐PA expression in cancer cells was significantly more frequently detected in cases with lymph node metastasis than in cases without metastasis. In either PAI‐1‐ or PAI‐2‐expressing cases, lymph node metastasis seemed to be restrained. These findings indicate that cancer cells themselves produce u‐PA, and suggest that u‐PA converts plg into plasmin, which dissolves the extracellular matrix surrounding cancer cells, resulting in cancer invasion and metastasis. PAI‐1 and PAI‐2 may have inhibitory actions on cancer invasion and metastasis mediated by u‐PA.
A Limited Sampling Model for Estimating Pharmacokinetics of CPT‐11 and Its Metabolite SN‐38
Authors: Sasaki Yasutsuna , Mizuno Shoichi , Fujii Hirofumi , Ohtsu Tomoko , Wakita Hisashi , Igarashi Tadahiko , Itoh Kuniaki , Sekine Ikuo , Miyata Yoshinori , Saijo Nagahiro
Published: August 2005

The objective of this study was to develop a limited sampling model (LSM) to estimate the area under the curve (AUC) of 7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]carbonyloxycamptothecin (CPT‐11) and that of 7‐ethyl‐10‐hydroxycamptothecin (SN‐38) as predictive pharmacokinetic variables for leukopenia and episodes of diarrhea induced by CPT‐11 administration. The model was developed with a training set consisting of pharmacokinetic studies in 36 patients who received a 90‐min i.v. infusion of CPT‐11 at a dose of 100 mg/m2. A multiple regression analysis of CPT‐11 or SN‐38 concentrations observed at each time point in the training set was used to predict the AUC of CPT‐11 or SN‐38. The final sampling models using only two time points were:

AUCCPT‐11=3.7891★C2.5+14.0479*C13.5+1.5463

AUCSN‐38=0.5319★C2.5+19.1468*C13.5+72.7349

where C2.5 and C13.5 are the plasma concentration of CPT‐11 (μg/ml) or SN‐38 (ng/ml) at 2.5 and 13.5 h after the initiation of CPT‐11 infusion, respectively. The models were validated prospectively on a separate test data set of 12 patients receiving the same dose of CPT‐11 investigated in a previous study. Validation of the final LSM on the test data set gave values of root mean square error (RMSE) of 12.72% and 5.97% for the AUC of CPT‐11 and that of SN‐38, respectively. The model can be used to monitor the AUCs of both CPT‐11 and SN‐38 for the early prediction of toxicities and to establish a pharmacokinetically based dose modification strategy for safe administration of CPT‐11.
Transformation of BALB/c 3T3 Cells in vitro by the Fungicides Captan, Captafol and Folpet
Authors: Perocco Paolo , Colacci Annamaria , Ciello Carmela Del , Grilli Sandro
Published: August 2005

Cytotoxic and cell‐transforming activities of the three fungicides, captan, captafol and folpet, have been studied in an experimental in vitro model by exposing BALB/c 3T3 cells to the chemicals with or without S‐9 mix‐induced bioactivation. Cytotoxicity of the three compounds was reduced in the presence of the metabolizing system. Each assayed pesticide displayed cell‐transforming ability in the presence of the metabolizing system. The relative efficiency was: captafol > captan < folpet. Cell transformation was considered to be due to carcinogenesis‐promoting activity. These data, obtained in a medium‐term (6‐8 weeks) experimental model, contribute to a better understanding of the action of the three pesticides in the multistep carcinogenesis process and provide more information concerning the oncogenic risk of these xenobiotic compounds for humans.
Quantitative Detection of Ultraviolet Light‐induced Photoproducts in Mouse Skin by Immunohistochemistry
Authors: Qin Xiusheng , Zhang Shaomin , Oda Hideaki , Nakatsuru Yoko , Shimizu Seiichiro , Yamazaki Yukari , Nikaido Osamu , Ishikawa Takatoshi
Published: August 2005

UVB‐induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine‐pyrimidone (6‐4)photoproducts [(6‐4)photoproducts] in mouse skin DNA were quantitatively measured using an immunohistochemical approach with a computer‐aided color image analyzer. The skins of the C3H/HeN mice were irradiated with ultraviolet B (UV‐B, 280‐320 nm), and processed to give conventional formalin‐fixed, paraffin‐embedded histologic sections. Routine immunohistochemistry clearly demonstrated a dosedependent induction of both photoproducts. CPDs were detectable at doses ġ 125 J/m2, while for (6‐4)photoproducts, the minimal dose at which they were detectable was 250 J/m1 in the present study. A time course study showed that the repair of (6‐4)photoproducts was more rapid than that of CPDs, and that epidermal cells bad a higher capacity for their removal than dermal cells. About half of the (6‐4)photoproducts were excised within the first 24 h after the irradiation, and the process was essentially complete by 72 h. In contrast, there was no apparent removal (less than 10%) of CPDs in the first 24 h and they only completely disappeared from the epidermal cells at 120 h after irradiation. The effect of DNA dilution due to increased turnover of epidermal cells after UV‐B irradiation was evaluated by quantitative immunohistochemical measurement of the time course of bromodeoxyuridine (BrdUrd) incorporated into nuclei at 2 days post irradiation when the proliferation reaches a peak. The removal of photoproducts was more marked than the decrease in BrdUrd staining. Our results suggest that mouse skin cells can repair both (6‐4)photoproducts and CPDs, but with considerably lower efficiency, especially in the latter case, than human or monkey skin cells.
Intratumoral Injection of an Adriamycin Immunoconjugate against Human Pancreatic Cancer Xenografts
Authors: Kondo Yasuyuki , Chung Yong‐Suk , Sawada Tetsuji , Inui Akimasa , Yamashita Yoshito , Hirayama Koji , Nakata Bunzo , Ho Jenny J. L. , Kim Young S. , Sowa Michio
Published: August 2005

We have evaluated the effect of an adriamycin conjugate of monoclonal antibody Nd2 (ADM‐Nd2) on the growth rate of SW1990 xenografts grown subcutaneously in athymic nude mice. Intravenous or intraperitoneal administration of radiolabeled Nd2 resulted in a maximum tumor accumulation of approximately 45% of the initial dose/g of tumor 3‐7 days after administration. However, administration into the tumor produced retention of 1200%ID/g 1 day after, with 50% of this high value remaining even at 7 days after administration. In contrast, intratumoral administration of a nonspecific immunoglobulin showed a lower initial retention and rapid loss of label. Both intravenously and intratumorally administered ADM‐Nd2 reduced the growth rate of SW1990 xenografts. While a single intravenous administration arrested growth for about two weeks, a single intratumoral injection prevented any increase in tumor size even 45 days after administration. Xenografts treated with ADM‐Nd2 showed degenerative changes at the histological level. Neither Nd2 alone nor Adriamycin alone inhibited growth when administered at the same dose as the conjugate.
Non‐P‐glycoprotein‐mediated Atypical Multidrug Resistance in a Human Bladder Cancer Cell Line
Authors: Naito Seiji , Hasegawa Shuji , Yokomizo Akira , Koga Hirofumi , Kotoh Shuji , Kuwano Michihiko , Kumazawa Joichi
Published: August 2005

A human bladder cancer cell line resistant to adriamycin (ADM), T24/ADM9 has been established in vitro by exposing T24 parent cells to progressively higher concentrations of the drug over a period of 12 months. The T24/ADM9 cells were found to be 9 times more resistant to ADM than the T24 parent, and showed various degrees of cross‐resistance to an ADM derivative, vinca alkaloids and a DNA topoisomerase II (Topo ID‐targeting agent, etoposide. No significant difference was observed in the cellular accumulation of ADM between the T24/ADM9 and T24 parent cells. A Northern blot analysis showed an overexpression of multidrug resistance‐associated protein (MRP) mRNA, but no overexpression of multidrug resistance‐1 (MDR1) mRNA was observed in the T24/ADM9 cells. A flow cytometric analysis showed that the MDR1 gene product, P‐glycoprotein (Pgp), is not expressed on the T24/ADM9 cells. T24/ADM9 showed approximately the parental level of DNA Topo II catalytic activity. In Western blot and Northern blot analyses, however, the cellular level of DNA topo II was apparently much lower in T24/ADM9 than in the T24 parent. Thus, these results suggest that a decreased cellular level of DNA Topo II and an overexpression of MRP gene may be responsible for the expression of an MDR phenotype in the T24/ADM9 cells and that such non‐Pgp‐mediated, atypical MDR may develop in bladder cancer treated with chemotherapy including ADM.
Novel After‐loading Interstitial Photodynamic Therapy of Canine Transmissible Sarcoma with Photofrin II and Excimer Dye Laser
Authors: Hashimoto Yasuo , Hirano Toru , Yamaguchi Noboru
Published: August 2005

Novel after‐loading interstitial photodynamic therapy was performed in a canine transmissible sarcoma (CTS) model, utilizing photofrin II and an excimer dye laser. First, photofrin II was injected intravenously at a dose of 5 mg/kg, then 48 h later, laser‐proof plastic tubing was inserted into the CTS, followed by photoradiation of the tumor from the inside. The mean diameter of tumor necrosis rapidly increased in parallel with increase in total irradiation energy below 240 J/cm; the mean diameter of tumor necrosis was 20.7 mm at an energy of 120 J/cm, and 24.5 mm at 240 J/cm. Beyond 240 J/cm, the diameter gradually increased to 26 mm at 960 J/cm. As a side effect, cutaneous tissue showed a deep open ulcer at 240 J/cm, a shallow open ulcer at 180 J/cm, and a scar healing at 120 J/cm. The thermal effect of laser light is considered negligible below 480 J/cm.
A Radiation‐induced Murine Ovarian Granulosa Cell Tumor Line: Introduction of v‐ras Gene Potentiates a High Metastatic Ability
Authors: Yanagihara Kazuyoshi , Nii Makoto , Tsumuraya Masaru , Numoto Michitaka , Seito Tsutomu , Seyama Toshio
Published: August 2005

A non‐metastatic epithelial tumor cell line, OV3121, was established from ovarian granulosa cell tumor in B6C3F1 mouse irradiated with 60Co‐gamma rays. OV3121 cells showed an epithelial morphology and grew in monolayer with a population doubling time of 28–30 h. The production of estradiol and the expression of cytokeratin confirmed the epithelial origin of the line. No pulmonary metastasis was observed from solid tumors after subcutaneous (s.c.) injection or after intravenous (i.v.) injection of a clonal subline, OV3121–1 cells. We examined the experimental metastasis of individual clones of OV3121–1 cells, containing various introduced viral oncogenes: v‐Ha‐ras, v‐Ki‐ras, v‐fms, v‐mos, v‐raf, v‐src, v‐sis, v‐fos and v‐myc. Among them, only OV3121–1 cells with v‐Ha‐MuSV or v‐Ki‐MuSV produced lung colonies at high frequencies. In a more detailed analysis, the v‐Ha‐ras transfectants OV‐ras4 and OV‐ras7 were found to form colonies in various organs by metastasis from tumors after s.c. injection, as well as lung colonies after i.v. injection. Moderately metastatic OV‐ras7 cells showed high gelatinolytic activity at 72 kDa (MMP‐2) and 92 kDa (MMP‐9) as compared with the parental OV3121–1 and OV‐Neo control cells by zymographic analysis. However, more metastatic OV‐ras4 cells produced progressively weaker bands of 72 kDa gelatinolytic activity. No gross alterations in the expression of MMP‐1, MMP‐3, TIMP‐1 and TIMP‐2 transcripts were detected in these cell lines. These results suggest that this ovarian granulosa cell tumor line may provide a useful system for understanding the mechanisms by which oncogenes influence the occurrence of metastasis.
Enzymatic Basis for the Accumulation of Lewisb Antigen in Uterine Endometrial Cancer
Authors: Kubushiro Kaneyuki , Tsukazaki Katsumi , Sakuma Yuichi , Sakayori Motoko , Yazawa Shin , Nozawa Shiro
Published: August 2005

In order to clarify the mechanism of the abnormal expression of Lewisb antigen, which was specific for uterine endometrial cancer tissue, the activities of α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase in normal endometrial tissues and uterine endometrial cancer tissues were determined. Further, an immunocytochemical study of the expression of blood group‐related carbohydrate antigens in 6 cultured cell lines derived from various gynecologic malignant tumors was performed and the α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase activities of these cell lines were determined. Compared with normal endometrium, uterine endometrial cancer tissues showed significantly higher values of α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase activities. The specifically strong expression of type I carbohydrate chains, particularly the Lewisb antigen, was recognized in cultured cell lines derived from uterine endometrial cancer. Compared with those cell lines derived from uterine cervical cancer and ovarian cancer, the cultured cell lines derived from uterine endometrial cancer showed higher activities of α1→2fucosyltransferase and α1→4fucosyltransferase, which are enzymes related to the synthesis of Lewisb antigen. The cell lines derived from uterine endometrial cancer showed specifically high values of α1→4fucosyltransferase activity. These results suggest that the α1→2fucosyltransferase and α1→4fucosyltransferase activities, especially the α1→4fucosyltransferase activity, contribute to the abnormal expression of the Lewisb antigen in uterine endometrial cancer.
Inhibition of Tumor‐induced Angiogenesis by a Synthetic Lipid A Analogue with Low Endotoxicity, DT‐5461
Authors: Sato Katsuaki , Yoo Yung Choon , Mochizuki Mami , Saiki Ikuo , Takahashi Tsuneo A. , Azuma Ichiro
Published: August 2005

We investigated the effect of a synthetic lipid A analogue with low endotoxicity, DT‐5461, on the neovascularization induced by B16‐BL6 melanoma in syngenelc mice. A systemic single administration of DT‐5461 caused a marked decrease in the number of vessels oriented toward the tumor mass and in the tumor size during the early phase of vasculogenesis (on day 4 after tumor inoculation), with little or no inhibition in the following phases. Multiple i.v. administrations of DT‐5461 at intervals of 4 days (an effective schedule for inhibiting tumor metastasis) significantly reduced the number of capillary vessels and tumor growth over a period of 14 days after the tumor implantation. Multiple systemic administrations of DT‐5461 on days 1, 5 and 9 after tumor inoculation caused a high production of endogenous tumor necrosis factor‐α (TNF‐α) in tumor sites although this treatment modality induced a low production in serum of tumor‐bearing mice. Tumor homogenate from mice treated with DT‐5461 suppressed the proliferation of endothelium in vitro, whereas sera from animals given DT‐5461 had little effect. Furthermore, the antiproliferative effect of the tumor homogenate from mice treated with DT‐5461, was completely abrogated by anti‐mTNF‐α monoclonal antibody (mAb). The anti‐angiogenic effect of DT‐5461 was also completely abrogated by rabbit anti‐mouse tumor necrosis factor‐α (anti‐mTNF‐α) antiserum, whereas the inhibition of tumor growth by DT‐5461 was only slightly diminished. Tumor homogenate from mice treated with DT‐5461 suppressed the proliferation of endothelium in vitro, whereas sera from animals given DT‐5461 had little effect. Furthermore, the antiproliferative effect of the tumor homogenate from mice treated with DT‐5461 was completely neutralized by anti‐mTNF‐α mAb. Multiple i.v. administrations of DT‐5461 after s.c. implantation of B16‐BL6 cells significantly inhibited the growth of primary tumors measured at the time of tumor excision on day 21, and the lung metastasis of melanoma cells as compared with the untreated control in the spontaneous metastasis model. These results suggested that the suppressive effect upon tumor‐associated angiogenesis by DT‐5461 contributes in part to the inhibition of tumor metastasis.
High‐energy Underwater Shock Wave Treatment for Internal Iliac Muscle Metastasis of Prostatic Cancer: A First Clinicla Trial
Authors: Hoshi Senji , Orikasa Seiichi , Suzuki Ken‐ichi , Saitoh Toshinori , Takahashi Toshiko , Yoshikawa Kazuyuki , Kuwahara Masa‐aki , Nose Masato
Published: August 2005

The first clinical trial of high‐energy shock wave (SW) combined with chemotherapy to treat metastasis of prostate cancer in the internal iliac muscle was conducted. The patient, a 57‐year‐old man, diagnosed as having mucin‐producing, poorly differentiated adenocarcinoma of the prostate invading the bladder wall, had been treated by total cystoprostatectomy. Five months later, metastatic tumors were found in the left axillar subcutaneous tissue and the right internal iliac muscles. For the axillar metastasis we performed radiation and left subclavicular arterial infusion of cisplatin 70 mg, THP‐adriamycin (THP) 50 mg and methotrexate 50 mg. For the right internal muscular metastasis, 10,000 to 20,000 shots of SW and simultaneous intravenous injection of carboplatin 100 mg and THP 10 mg were carried out. Neither of the tumors decreased in size, but on magnetic resonance images, the SW‐treated tumor exhibited a central low‐intensity area. The SW‐treated tumor was resected and central necrosis and a collection of mucin in the central area were observed. Hormone‐resistant prostate cancer is well‐known to be a multidrug‐resistant tumor. It is noteworthy that SW and chemotherapy induced necrosis in such a refractory cancer without any significant side effects.
Intraluminal Epidermal Growth Factor Affects Growth of N‐Methyl‐N‐nitrosourea‐initiated Rat Bladder Carcinoma
Authors: Kawai Koji , Kawamata Hitoshi , Kameyama Shuji , Rademaker Alfred , Oyasu Ryoichi
Published: August 2005

To confirm our recent finding that epidermal growth factor (EGF) appeared to contribute to the tumor‐enhancing effect demonstrated by normal rat urine, we conducted 2 experiments using our heterotopically transplanted rat urinary bladder model. In experiment 1, after a single dose (0.25 mg) of N‐methyl‐N‐nitrosourea (MNU), we intravesically administered EGF (0.5 ml of 500 ng/ml phosphate‐buffered saline) once a week for 30 weeks. Instillation of EGF induced a significantly larger number of tumors than did instillation of the vehicle (P=0.03). EGF without MNU initiation did not induce tumors. In experiment 2, 2 groups received instillation of killed Escherichia coli (5 × 108 cells)/0.5 ml phosphate‐buffered saline once a week for 4 weeks to expand the MNU‐initiated cell population. Subsequent EGF treatment significantly increased the incidence of tumors (P=0.01). In the groups which did not receive killed E. coli, EGF treatment induced a significantly higher number of tumors than did vehicle treatment (P<0.001). All of the tumors were low‐grade, superficial transitional cell carcinomas. These observations indicate that EGF acts as a growth‐stimulating factor on dormant neoplastic cells and thereby increases the number of tumors.
Role of Point Mutation of the K‐ras Gene in Tumorigenesis of B6C3F1 Mouse Lung Lesions Induced by Urethane
Authors: Kawano Ryoji , Nishisaka Takashi , Takeshima Yukio , Yonehara Shuji , Inai Kouki
Published: August 2005

In order to elucidate the role of point mutation of the K‐ras gene in the tumorigenetic process of lung tumors, an experimental model of lung lesions in mice induced by the administration of urethane was used. A total of 135 B6C3F1 male mice, 6 weeks old, were given urethane in the drinking water at 0, 6, 60, 600 or 1200 ppm, and were then killed after varying periods of time. The lung lesions were histologically characterized as hyperplasia, adenoma and adenocarcinoma. Point mutations in codons 12 and 61 of the K‐ras gene were detected by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and confirmed by using dideoxy sequencing analysis. K‐ras gene mutation was identified in 9 (23.7%) of 38 lesions classified as hyperplasia, 31 (46.3%) of 67 adenomas, and 3 (50%) of 6 adenocarcinomas. The most frequent mutation was an AT‐to‐TA transversion at the second base of codon 61 and this pattern accounted for 65% of the three mutant forms observed. These results suggest that the point mutation of K‐ras gene is involved in all stages of mouse lung tumorigenesis, i.e., activation of this gene can also influence the later stages of lung lesions.
N‐Acetyltransferase Activity in the Urine in Japanese Subjects: Comparison in Healthy Persons and Bladder Cancer Patients
Authors: Ishizu Sumiko , Hashida Chise , Hanaoka Tomoyuki , Maeda Keiko , Ohishi Yukihiko
Published: August 2005

The activity of urinary N‐acetylamino‐transferase was determined by high‐performance liquid chro‐matographic assay of acetylisoniazid and isoniazid after administration of isoniazid to healthy Japanese male and bladder cancer patients in Japan. The healthy subjects were 47 college students and 44 company employees ranging from 18 to 64 years old (mean±SD=34.5±13.7). The bladder cancer group consisted of 58 male and 13 female patients, ranging from 28 to 82 years old (mean±SD =60.8 ±11.6), who were being treated at several hospitals. The slow phenotype, defined as an acetylation ratio (acetylisoniazid/isoniazid) of less than 2.0, was observed in 13 (14.3%) of the 91 healthy subjects, and in 20 (28.2%) of the 71 bladder cancer patients; the difference between the two groups is significant (P < 0.05). A histogram of the acetylation ratio values showed an overall leftward shift of the patient group, indicating low values of acetylation ratio in this group as a whole (P < 0.01).
Effects of K‐ras Gene Mutations in the Development of Lung Lesions Induced by 4‐(N‐ Methyl‐N‐nitrosamino)‐1‐(3‐pyridyl)‐1‐butanone in A/J Mice
Authors: Kawano Ryoji , Takeshima Yukio , Inai Kouki
Published: August 2005

The relationship between the development of peripheral lung lesions induced by tobacco‐specific 4‐(N‐methyl‐N‐nitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and K‐ras gene mutation in A/J mice, and the correlations between histological alterations and the course of lung lesion development after NNK treatment and K‐ras gene mutation were investigated. The acquisition of a selective growth advantage by the lung lesions with mutations was also examined using immunohistochemical labeling with bromodeoxyuridine. Thirty female 5 weeks old A/J mice were each injected intraperitoneally with a single dose of NNK (100 mg/kg body weight) and subdivided into 6 groups according to the time after NNK treatment. The lung lesions were characterized histologically as alveolar/bronchiolar hyperplasia, adenoma and adenocarcinoma, and point mutations in codons 12 and 61 of the K‐ras gene were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) and dideoxy sequencing methods. K‐ras gene mutations were identified in 7 (58.3%) of 12 hyperplasias, 42 (75.0%) of 56 adenomas and 3 (75.0%) of 4 adenocarcinomas. The most frequent K‐ras gene mutation was a G‐to‐A transition at the second base of codon 12 and this accounted for 86.5% of all the mutations detected. Neither the frequency of activation of this gene nor the specific mutation was affected by the time after NNK treatment and there was no positive correlation between the proliferative activity of lung lesions and the presence of K‐ras gene mutations. Thus, K‐ras gene mutation is closely associated with the development of NNK‐induced peripheral lung lesions in A/J mice, but it plays no role in the selective growth advantage of these lesions.
Usefulness of Ultrasonography Combined with Conventional Physical Examination in Mass Screening for Breast Cancer: A Retrospective Study of Yamanashi Health Care Center Results from 1989 to 1994
Authors: Okamoto Hirotaka , Ogawara Tadahiko , Arihara Fumio , Kobayashi Kazuhisa , Inoue Shingo , Nagahori Kaoru , Yamamoto Masayuki , Sekikawa Takayoshi , Matsumoto Yoshiro
Published: August 2005

We retrospectively analyzed the records for 34,474 women who participated in mass screening for breast cancer by physical examination with or without ultrasonography (US) at Yamanashi Health Care Center between April, 1989 and March, 1994 to evaluate the usefulness of US in mass screening. In one group (15,935 women) conventional physical examination with inspection and palpation alone had been performed, and in another (18,539 women) both conventional physical and US examinations were performed. Breast cancer was detected in 27 of the women (0.08% of the total group screened), 22 of whom were in the group examined by US; moreover, 16 of these 22 women had early breast cancer, which was a non‐palpable tumor in 13. Half of the 22 women were examinees under the age of 50 years. Of the 22 tumors detected in the groups examined by US, 16 (73%) were early breast cancer. The overall detection of early breast cancer (0.09%) in the US group was significantly higher than that (0.01%) in the group examined by conventional methods (P < 0.05). Of the tumors detected in the US group, 59.1% were non‐palpable. These results suggest that early and non‐palpable breast cancer can be detected using US, and the incidence of detection of such tumors in women under the age 50 years is increased in mass screening including US examination. This examination is effective in mass screening for breast cancer, especially for early and non‐palpable breast cancer tumors.
Inhibition of 2‐Amino‐3, 8‐dimethylimidazo[4,5‐f]quinoxaline‐mediated DNA‐adduct Formation by Chlorophyllin in Drosophila
Authors: Sugiyama Chitose , Shinoda Aki , Hayatsu Hikoya , Negishi Tomoe
Published: August 2005

The effect of chlorophyllin on 2‐ammo‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx)‐mediated DNA‐adduct formation in Drosophila was studied. Third‐instar larvae of Drosophila were fed MeIQx at 1 mg/6.5 g‐feed/bottle, with or without chlorophyllin (100–300 ing). After a 6 h feeding exposure to MeIQx, the larvae were divided into 2 groups. The first group was examined for covalent DNA adducts by 32P‐postlabeIing assay. The second group was assayed for DNA damage by allowing the larvae to develop to adults and measuring the male/female ratio (males, DNA repair‐deficient; females, DNA repair‐proficient). The 32P‐postlabeling results indicated a significant decrease in DNA adduct levels in larvae treated with MeIQx and 300 mg chlorophyllin (1.7±0.7 adducts/107 nucleotides) as compared with MeIQx‐treated larvae (6.5±2.1 adducts/107 nucleotides). The results on male/female sex ratios also indicated a chlorophyllin‐indnced decrease in DNA damage by exposure to MeIQx. The suppressive effect of chlorophyllin on the genotoxic actions of a polycyclic mutagen, MeIQx, may be a result of complex formation between chlorophyllin and the mutagen.
p53 Accumulation in Colorectal Cancer with Hepatic Metastasis
Authors: Maruyama Keiji , Tanaka Tatsuo , Baba Shozo , Nakamura Satoshi , Endo Yutaka , Sugimura Haruhiko
Published: August 2005

The prevalence of immunoreactive p53 and argyrophilic nucleolar organizer region (AgNOR) numbers were compared between colorectal cancers with (n=44) and without (n=51) hepatic metastasis for at least 5 years. At the same time, the distribution of p53‐positive cells in primary, metastatic, and xenografted tumors from the same individuals were studied. Overall, p53 positivity was found more frequently in the cases with hepatic metastasis than in non‐metastatic controls, regardless of the distribution pattern (P<0.05), whereas AgNOR counts were not different between the two groups. Significant heterogeneity in the distribution of p53 immunoreactivity was noted in both the primary and metastatic lesions. The intratumor distribution patterns of p53 immunoreactive cells in the primary (n=33), metastatic (n=33), and xenografted (n=7) tumors of the same individuals were consistent in the majority of cases. There were a few cases in which the p53 immunoreactive cells were more dominant in the metastatic tumor cells. Our observations suggest that p53 accumulation in colorectal cancer is associated with increased risk for hepatic metastasis, while cell proliferation as represented by AgNOR numbers is not. In addition, heterogeneity of abnormal p53 accumulation in the tumor is maintained during the course of metastasis and even after implantation in nude mouse. p53‐Immunoreactive cells in the population of colorectal cancer cells do not necessarily have higher metastasizing potential.
Shortened Telomere Length in Hepatocellular Carcinomas and Corresponding Background Liver Tissues of Patients Infected with Hepatitis Virus
Authors: Ohashi Kazuo , Tsutsumi Masahiro , Kobitsu Kunihiko , Fukuda Tomokazu , Tsujiuchi Toshifumi , Okajima Eijiro , Ko Saiho , Nakajima Yoshiyuki , Nakano Hiroshige , Konishi Yoichi
Published: August 2005

The telomere length in 20 surgically resected human hepatocellular carcinomas (HCCs) and adjacent non‐cancerous, livers with hepatitis virus infection were investigated. All the HCC samples examined demonstrated shorter telomere length than the corresponding non‐cancerous liver tissues, the respective average values being 5.4 kbp and 8.8 kbp (P < 0.001). The shortening of telomere length was most prominent in HCCs larger than 30 mm in diameter, and in both tumors and non‐cancerous livers it was more marked with hepatitis B virus as compared with hepatitis C virus infection. These results indicate that telomere shortening is associated with not only progression, but also development of HCC, and there is a possible difference in the nature of the association in patients with hepatitis viruses of B and C types.
Selective Reduction in α‐Hydroxypalmitic Acid‐containing Sphingomyelin and Concurrent Increase in Hydroxylated Ceramides in Murine Skin Tumors Induced by an Initiation‐promotion Regimen
Authors: Kitano Yukie , Iwamori Yuriko , Kiguchi Kaoru , DiGiovanni John , Takahashi Toshie , Kasama Kenji , Niwa Takashi , Harii Kiyonori , Iwamori Masao
Published: August 2005

The sphingomyelin cycle is activated to accumulate ceramides in the process of epidermal differentiation. We found that sphingomyelin in the epidermis of 4 different murine strains gave three bands on TLC, the lower band containing α‐hydroxypalmitic acid (C16h:0(α)). However, in the papillomas induced in the skin of SENCAR and SSIN mice by initiation with 7,12‐dimethylbenz[α] anthracene followed by promotion with 12‐O‐tetradecanoylphorbol acetate, the concentration of C16h:0(α)‐ containing sphingomyelin was selectively diminished with a concomitant increase in the concentrations of the ceramides containing α‐hydroxy fatty acids. These findings indicate a possible involvement of the selective hydrolysis of α‐hydroxy fatty acid‐containing sphingomyelin in the process of tumorigenesis in mouse skin.
Detection of K‐ras Point Mutations at Codon 12 in Pancreatic Juice for the Diagnosis of Pancreatic Cancer by Hybridization Protection Assay: A Simple Method for the Determination of the Types of Point Mutations
Authors: Watanabe Hiroyuki , Miyagi Chieko , Yamaguchi Yasushi , Satomura Yoshitake , Ohta Hideki , Motoo Yoshiharu , Okai Takashi , Yoshimura Tadashi , Tsuji Yasuhiro , Sawabu Norio
Published: August 2005

The present study was undertaken to detect K‐ras oncogene point mutations at codon 12 in pure pancreatic juice (PPJ) by the hybridization protection assay (HPA) method for the diagnosis of pancreatic cancer (PC). This assay can be carried out within 30 min and can determine not only the presence of a mutation, but also the mutational type of K‐ras at codon 12. The minimal ratio of mutant DNA detectable by the HPA was 5–10% of the total DNA. PPJ was collected through a cannula under duodenal fiberscope control from 20 patients with PC and 20 patients with chronic pancreatitis (CP). Analysis of PPJ by the HPA revealed that the incidence of K‐ras point mutations at codon 12 was 55% (11/20) in patients with PC and 0% (0/20) in those with CP. Mutational types of K‐ras at codon 12 in PC were aspartic acid (Asp) in nine cases, both Asp and cysteine in one case, and arginine in one case. Analysis of K‐ras point mutations at codon 12 in PPJ using the HPA method seems promising as a new genetic test for the diagnosis of PC, because the HPA method is simple, and can easily determine the mutational type.
Correlation between nm23 Protein and Several Cell Adhesion Molecules in Human Gastric Carcinoma
Authors: Ura Hideki , Denno Ryuichi , Hirata Koichi
Published: August 2005

The correlation between nm23 protein (nm23) expression and the expression of several cell adhesion molecules was studied immunohistochemically in 110 resected gastric carcinomas. Formalin‐fixed and paraffin‐embedded samples were serially sectioned and stained with antibodies against nm23, integrin β1 subfamily members (α2β1, α3β1 and α4β1), LFA‐1, ICAM‐1, sialyl Lewisx (sLex) and CD44h, ‐V3, and ‐V6. Primary carcinomas presenting with either lymph node involvement or liver metastasis expressed significantly reduced levels of nm23 compared to tumors without metastasis. The percent of tumors expressing each adhesion molecule was as follows: α2β1, 27.3%; α3β1, 20.0%; α4β1, 14.5%; LFA‐1, 14.5%; ICAM‐1, 12.7%; sLex, 67.3%; CD44h, 55.5%; CD44V3, 20.0%; and CD44V6, 4.5%. Expression of α2β1 integrin and high levels of sLex were significantly correlated with lymph node metastasis, and expression of α3β1 integrin and high levels of sLex were correlated with liver metastasis. Expression of ICAM‐1 was inversely correlated with liver metastasis. Comparing the expression of each cell adhesion molecule with nm23 immunoreactivity, expression of sLex was significantly associated with nm23 expression. Of tumors expressing high levels of sLex, 75% showed reduced nm23 expression, compared to 52% of tumors with weak or no sLex expression (P < 0.05). A similar tendency was also observed in the metastasized secondary tumors. These results suggest that reduced nm23 expression may promote the metastatic properties of cancer cells in concert with increased sLex expression.
Tumor Necrosis Factor‐α Gene Transfer Augments Anti‐Fas Antibody‐mediated Apoptosis in Human Glioma Cells
Authors: Mizuno Masaaki , Yoshida Jun
Published: August 2005

To effectively induce apoptosis in human glioma cells, we tried to transfer the tumor necrosis factor (TNF)‐α gene into glioma cells to produce TNF‐α locally in these cells. The stable transfectants of three gliorna cells (U251‐SP, U251‐MG, and T98G) were resistant to exogenous TNF‐α, but their cell surface expression of the Fas antigen was dramatically enhanced by about 10 to 100‐fold as compared with untransfected glioma cells exposed to exogenous TNF‐α. The Fas antigen is a transmemhrane cytokine receptor protein of the nerve growth factor/TNF receptor superfamily. Although the untransfected glioma cells tested were resistant to anti‐Fas antibody‐mediated apoptosis, the TNF‐α gene‐transfected glioma cells exhibited high susceptibility to anti‐Fas antibody‐mediated apoptosis. Thus, TNF‐α gene transfer combined with anti‐Fas antibodies may be useful for the treatment of malignant glioma.
Effect of Heavy Alcohol Intake on Long‐term Results after Curative Resection of Hepatitis C Virus‐related Hepatocellular Carcinoma
Authors: Okada Shuichi , Ishii Hiroshi , Nose Haruhiko , Okusaka Takuji , Kyogoku Akiko , Yoshimori Masayoshi , Shimada Kazuaki , Yamamoto Junji , Kosuge Tomoo , Yamasaki Susumu , Sakamoto Michiie , Hirohashi Setsuo
Published: August 2005

We studied the effect of heavy alcohol intake (ethanol intake ≥80 g/day for ≥5 yr) on long‐term results in 53 patients with hepatitis C virus (HCV)‐related hepatocellular carcinoma (HCC) who had undergone curative hepatic resection. Cell proliferative activity in the tumor and non‐tumorous liver was also assessed by counting argyrophilic nucleolar organizer region‐associated proteins (Ag‐NOR) in the resected specimens. Twenty patients (20 males, 0 females) were positive for heavy alcohol intake [AI(+)] and 33 (28 males, 5 females) were not [AI(‐)]. All patients were positive for HCV antibody and negative for hepatitis B surface antigen. Carcinoma recurred within 3 to 51 postoperative months in 42 (79.2%) of the 53 patients. The median disease‐free survival time was 12.6 mo in the AI(+) group and 25.4 mo in the AI(‐) group (P<0.01). The AI(+) group also had significantly poorer survival than the AI(‐) group (P<0.05, 3‐year survival rate: 66.7% vs. 93.5%). HCC tumor in the AI(+) group showed significantly increased proliferative activity compared with that in the AI(‐) group (P<0.05, Ag‐NOR number: 2.3±0.8 vs. 1.9±0.4). However, there was no significant difference between the numbers of Ag‐NORs in non‐tumorous liver from these two groups (1.5±0.2 vs. 1.5±0.2). Patients with heavy alcohol intake should be followed particularly closely, even if they have received curative surgery, since heavy alcohol intake is closely related to a poor postoperative prognosis.
p53 Mutation in B‐Cell Lymphoid Neoplasms with Reference to Oncogene Rearrangements Associated with Chromosomal Translocations
Authors: Akasaka Takashi , Muramatsu Mikiko , Kadowaki Norimitsu , Ohno Hitoshi , Ishizaki Kanji , Yamabe Hirohiko , Fukuhara Shirou , Okuma Minoru
Published: August 2005

We investigated mutations of the p53 tumor suppressor gene in B‐cell lymphoid neoplasms with reference to oncogene rearrangements associated with specific chromosomal translocations. These included 15 patients with a BCL1/PRAD1 gene rearrangement and/or PRAD1 overexpression, 45 with a BCL2 rearrangement, 2 with a BCL3 rearrangement, 24 with a BCL6 rearrangement, and 6 with both BCL2 and BCL6 rearrangements. Thirty‐six patients lacked detectable oncogene rearrangements. Genomic DNA was isolated from involved tissues or leukemic cells obtained at diagnosis and/or at relapse, and established cell lines. Polymerase chain reaction‐mediated single‐strand conformation polymorphism analysis and direct sequencing were performed to analyze abnormalities of the p53 gene. We detected p53 gene alterations in 18 of 128 patients, representing 21 of the total 151 materials analyzed. In the total of 66 patients with an oncogene rearrangement studied at diagnosis, only one had a mutation; however, 6 of 37 patients studied at relapse showed p53 mutations. Sequential analysis revealed that the p53 mutation was closely associated with transformation from follicular lymphoma to large cell lymphoma, exclusively in BCL2‐positive lymphoma cases. Two of 13 mutations observed in oncogene rearrangement‐positive cases and cell lines were transitions at CpG dinucleotides. In contrast, the relationship between p53 mutations and clinical behavior in oncogene rearrangement‐negative cases was variable; 5 patients including one with indolent follicular lymphoma were positive for p53 mutation at initial presentation, and 2 of the 5 showed prolonged disease‐free survival. Our findings suggest that p53 alteration exhibits diverse functions in the development and progression of B‐cell tumors related to the presence or absence of oncogene rearrangement, and that chemotherapy‐related influences may be involved in the occurrence of progression‐associated p53 mutations.
Localization of Small‐cell Lung Cancer Xenografts with Iodine‐125‐, Indium‐111‐, and Rhenium‐188‐Somatostatin Analogs
Authors: Hosono Makoto , Hosono Masako N. , Haberberger Thomas , Zamora Paul O. , Guhlke Stefan , Bender Hans , Russ Knapp F. F. , Biersack Hans J.
Published: August 2005

We examined the potential of radiolabeled somatostatin analogs, 125I‐Tyr‐3‐octreotide (125I‐octreotide), 111In‐DTPA(diethylenetriaminepentaacetatic acid)‐d‐Phe‐1‐octreotide (111In‐octreotide), and 188Re‐octreotide for targeting small‐cell lung cancer (SCLC) in a mouse model. Tyr‐3‐octreotide was labeled with 125I by the chloramine T method, and 111In‐octreotide was obtained as a kit, while 188Re was eluted from a 188W/188Re generator, and octreotide was directly labeled with 188Re by reducing disulfide bonds. The 125I‐, 111In‐, and 188Re‐octreotides were injected i.v. into athymic mice bearing NCI‐H69 tumors, and the biodistributions were determined at 15 min, and 2, 4, 8, and 24 h. Tumor uptakes were 0.5±0.2, 0.3±0.1, 0.3±0.1 %ID/g, and tumor‐to‐blood ratios were 1.8, 11.9, 1.2 at 8 h for 125I‐, 111In‐, and 188Re‐octreotides, respectively. Accumulations of 111In‐octreotide in normal tissues were lower than those of 125I‐ and 188Re‐octreotides. 188Re‐octreotide can be used to localize SCLC lesions as efficiently as radioiodinated octreotide. However, 111In‐octreotide was the most suitable agent to obtain high tumor‐to‐normal tissue contrast for localizing SCLC.
Efficient Method for Rapid Induction of Aberrant Crypt Foci in Rats with 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine
Authors: Ochiai Masako , Nakagama Hitoshi , Watanabe Masatoshi , Ishiguro Yukiko , Sugimura Takashi , Nagao Minako
Published: August 2005

2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) induces aberrant crypt foci (ACFs) as well as colon cancer in F344 male rats. Conditions allowing rapid development of ACFs over a short period were investigated. F344 male rats were fed 400 ppm of PhIP‐HCl in a low‐fat diet (LF) for 2 weeks and then given a PhIP‐free, high‐fat diet containing PRIMEX (HF‐PR) or safflower oil, or PhIP‐free LF for 4 or 12 weeks. Rats fed HF‐PR for 4 weeks gave the highest number of ACFs/rat (3.3) and their size in terms of aberrant crypts/ACF(2.7) was much larger than that obtained with conventional continuous feeding of PhIP for 25 weeks in the CE‐2 diet. Therefore, 2 weeks of dietary exposure to 400 ppm of PhIP‐HCl, followed by HF‐PR for 4 weeks, is a practical and convenient method for obtaining ACFs. This protocol should be useful for studies of the early phase of colon carcinogenesis.
Increased Telomerase Activity in Hyperplastic Nodules and Hepatocellular Carcinomas Induced by a Choline‐deficient L‐Amino Acid‐defined Diet in Rats
Authors: Tsujiuchi Toshifumi , Tsutsumi Masahiro , Kido Akira , Kobitsu Kunihiko , Takahama Makoto , Majima Toshimitsu , Denda Ayumi , Nakae Dai , Konishi Yoichi
Published: August 2005

Activation of telomerase has been reported in several human cancers, including hepatocellular carcinomas (HCCs). We investigated telomerase activity during hepatocarcinogenesis induced by a choline‐deficient L‐amino acid‐defined (CDAA) diet in rats. Male F344 rats were given a CDAA diet or a choline‐supplemented L‐amino acid‐defined (CSAA) diet from 6 weeks of age for 75 weeks, and subgroups were killed 10 weeks, 50 weeks and 75 weeks after the beginning of the experiment. Hyperplastic nodules and HCCs were noted in rats fed a CDAA diet for 50 weeks and 75 weeks, respectively. Normal control liver specimens were obtained from 6‐week‐old rats. Telomerase activity was assessed by using a telomeric repeat amplification protocol (TRAP). Normal liver and background parenchyma of rats fed either of the diets for 10 weeks or 50 weeks showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver.
Analysis of Synergism in Hepatocarcinogenesis Based on Preneoplastic Foci Induction by 10 Heterocyclic Amines in the Rat
Authors: Hasegawa Ryohei , Yoshimura Isao , Imaida Katsumi , Ito Nobuyuki , Shirai Tomoyuki
Published: August 2005

The effects of simultaneous treatment with 5 or 10 heterocyclic amines at low dose levels on hepatocarcinogenesis in rats were investigated using a medium‐term liver bioassay protocol based on the two‐stage carcinogenesis hypothesis with diethylnitrosamine initiation (200 mg/kg, i.p.). Five carcinogenic heterocyclic amines in experiment 1 (Trp‐P‐1, Glu‐P‐2, IQ, MelQ, MelQx) and experiment 2 (Trp‐P‐2, Glu‐P‐1, MeAαC, AαC, PhIP) were administered together or individually in the diet at levels of 1/1, 1/5, or 1/25 careinogenic doses, and all 10 chemicals were given at 1/10 or 1/100 levels in experiment 3. Induction of preneoplastic glutathione S‐transferase placental form (GST‐P)‐positive foci in the liver was generally increased in the combination groups over the sums of the 5 or 10 individual effects. Thus, based on the heteroadditive concept, synergism was observed for each combination, being most obvious in the group given all 10 chemicals at the 1/10 dose levels. However, the values for the combined groups were generally close to the averages of the 5 or 10 data gained for the heterocyclic amines alone at the corresponding higher doses, indicating the possibility of isoadditivity. Based on these findings, we propose here a new statistical method for analysis of combined effects of multiple chemicals, and, using this, we demonstrated (true) synergism with some heterocyclic amine combinations. The importance of dose‐response curves for evaluation of combination effects is discussed.
Development of Aberrant Crypt Foci Involves a Fission Mechanism as Revealed by Isolation of Aberrant Crypts
Authors: Fujimitsu Yasunobu , Nakanishi Hayao , Inada Ken‐ichi , Yamachika Takasuke , Ichinose Masutaro , Fukami Hiroko , Tatematsu Masae
Published: August 2005

Morphological analysis of isolated colonic crypts in rats, postnatally, indicated that the crypts reproduce themselves by a fission mechanism, the division beginning at the crypt base and proceeding upwards until there are two separate crypts. Occasionally, before the separation is complete, a second fission process starts on one or both sides of a bifurcating crypt and a triple‐branched or quadruple‐branched crypt results. Analysis of isolated aberrant crypt foci (ACF) in rats treated with 1,2‐dimethylhydrazine revealed that the development of ACF consisting of multiple crypts is also due to a fission mechanism. Initially, an indentation appears at the base of a single ACF crypt, with subsequent formation of a bifurcation and eventual crypt division.