bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow Cytometric Detection of Proliferative Cells in Leukemias
Authors: Wakita Atsushi , Nitta Masakazu , Mitomo Yasuharu , Takahashi Masaaki , Tanaka Masao , Kaneda Tsuguhiro
Published: August 2005

We studied the proliferative activity of leukemic cells obtained from the peripheral blood and bone marrow of 34 patients; 30 with acute leukemia and 4 with chronic myelogenous leukemia in blastic crisis. Flow cytometry was performed using monoclonal antibody against DNA polymerase α. Since fresh and frozen cells showed virtually identical DNA polymerase α‐positive populations and flow cytometric histograms, 52 cryopreserved samples (25 from peripheral blood and 27 from bone marrow) were used in this study. The DNA polymerase α‐positive population ranged from 20.4% to 84.7% in peripheral blood, and from 6.5% to 92.1% in bone marrow. A positive correlation (r=0.76, P≤0.01) was found between DNA polymerase α‐positive populations in peripheral blood and bone marrow from the same patient. This suggests that the DNA polymerase α‐positive population in the bone marrow can be estimated from that in peripheral blood. No relationship was observed between the positive population and the response to chemotherapy. Statistical analyses for all cases showed no relationship between the DNA polymerase α‐positive population and either the tumor cell count or time to reach a nadir. However, a negative correlation was observed between the positive population in bone marrow samples and the time to reach a nadir (r=−0.64, P≤0.05) in those patients who achieved a complete response. In addition, in the cases of acute non‐lymphocytic leukemia who did not respond to chemotherapy, a positive correlation was observed between the tumor cell count in bone marrow and the DNA polymerase α‐positive population (r=0.93, P≤0.01). Thus, the method described here provides a simple and time‐efficient means of detecting the proliferative activity of leukemic cells, which is a useful parameter in the treatment of leukemia.
A Case‐Control Study on Risk Factors for Uterine Endometrial Cancer in Japan
Authors: Inoue Masaki , Okayama Akira , Fujita Masami , Enomoto Takayuki , Tanizawa Osamu , Ueshima Hirotsugu
Published: August 2005

A case control study of 143 Japanese women with uterine endometrial cancer and 143 individually age‐matched controls was conducted to assess the risk factors for endometrial cancers in Japan. Among the characteristics studied, the following factors were significantly greater in the cases than in the controls: nulliparity (odds ratio for parity 1–3 and ≥4 versus nullipara are 0.40 and 0.02, respectively), obesity (odds ratio: 2.73), hypertension (odds ratio: 2.4), diabetes mellitus (odds ratio: 6.30), and a personal medical history of cancer (odds ratio: 3.06). The present study showed that Japanese women have the same risk factors for endometrial cancer as those reported in Western countries. The recent increase in the incidence of endometrial cancer in Japan may be largely attributed to the decrease in parity.
Identification and Purification of a Toxic Component to B Cell Hybridoma Cells in Fetal Calf Serum
Authors: Nonaka Noritaka , Hirai (deceased) Hidematsu
Published: August 2005

A component exhibiting toxicity to B cell hybridoma cells was isolated and purified from fetal calf serum (FCS) by immunoaffinity chromatography using a monoclonal antibody (mAb) which reacted with the high‐molecular‐weight glycoprotein (6B3·Ag) recognized by a mAb, 6B3, to human large cell lung carcinoma cells (HLC‐2). The component (FCS‐6B3·Ag) was a high‐molecular‐weight antigen (approximately 1,000,000), consisting mainly of 76,000 subunits. FCS‐6B3·Ag showed the same mobility in the pre‐β globulin region as that of 6B3·Ag on electrophoresis in 1.2% agarose gel. When FCS‐6B3·Ag was analyzed by double immunodiffusion, it reacted with anti‐6B3·Ag antiserum and the precipitin line fused partially with that formed between 6B3·Ag and anti‐6B3·Ag antiserum. FCS‐6B3·Ag was found to be toxic to hybridoma cells (anti‐6B3·Ag, anti‐α‐fetoprotein, anti‐carcinoembryonic antigen or anti‐C‐reactive protein mAb producing cells) specifically in vitro at 5 μg/ml. The antigen also strongly suppressed their growth. The toxic effect of FCS‐6B3·Ag appeared immediately after addition, and death of the target cells was complete only after 36–48 h. However, the antigen exhibited only weak suppression of Ig‐non‐secretory mouse myeloma (P3U1), thymic lymphoma (EL4) or mastocytoma (P815) cell growth. Five lots of FCS contained 2.1 to 4.1 μg/ml of FCS‐6B3·Ag.
Adenosquamous Carcinoma of the Gallbladder: A Clinicopathological, Immunohistochemical and Flow‐cytometric Study of Twenty Cases
Authors: Nishihara Kazuyoshi , Nagai Eishi , Izumi Yasuharu , Yamaguchi Koji , Tsuneyoshi Masazumi
Published: August 2005

Twenty patients (7.4%) with adenosquamous carcinoma of the gallbladder were selected from 271 surgically resected gallbladder cancers. The 20 patients were composed of 8 men and 12 women with a mean age of 66.9 years. Histologically, all twenty tumors showed an abrupt transition between the adenocarcinoma (AC) and squamous cell carcinoma (SCC) areas, and well differentiated AC was also found in the peripheral area of the tumor. A histochemical and immunohistochemical study using alcian blue, periodic acid‐Schiff, cytokeratins, involculin and tissue polypeptide antigen disclosed a different nature of the two components. DNA heterogeneity between the components was detected in 5 of 7 cases by flow cytometry. The positive rate of immunostaining for proliferating cell nuclear antigen in the SCC areas (mean 20.55%) was larger than that of the AC areas (mean 11.40%) (P=0.0029), which indicated that the SCC areas had a greater proliferative capacity than AC areas. These results suggest that the SCC component of adenosquamous carcinoma of the gallbladder arose by a stepwise molecular progression of the pre‐existing AC. Furthermore, the prognosis of adenosquamous carcinomas of the gallbladder (mean survival: 10 months) in the advanced stage (pTNM 2–4) was less favorable than those of papillary and well differentiated AC (mean survival: 99 months and 86 months) (p<0.0001).
Case‐Control Study for Lung Cancer and Cigarette Smoking in Osaka, Japan: Comparison with the Results from Western Europe
Authors: Sobue Tomotaka , Suzuki Takaichiro , Fujimoto Isaburo , Matsuda Minoru , Doi Osamu , Mori Takashi , Furuse Kiyoyuki , Fukuoka Masahiro , Yasumitsu Tsutomu , Kuwahara Osamu , Kono Keijiro , Taki Toshihiko , Kuwabara Masayoshi , Nakahara Kazuya , Endo Shozo , Sawamura Kenji , Kurata Masahiko , Ichitani Michio , Hattori Shoji
Published: August 2005

In order to clarify the relation between cigarette smoking and lung cancer, a case‐control study was conducted. The case series consisted of 1,376 lung cancer patients (1,082 males and 294 females) who were newly diagnosed and admitted to eight hospitals in Osaka during 1986–88. Smoking histories were compared with those of 2,230 controls (1,141 males and 1,089 females) admitted to the same hospitals during the same period without established smoking‐related diseases. Odds ratios of current smoker versus nonsmoker were 18.1, 1.9, 21.4, and 3.8 for squamous, adeno, small, and large cell carcinoma, respectively, for males, and 9.7, 1.3, 12.1, 3.7, respectively, for females. Compared to the results from previous studies in Japan, the magnitude of the odds ratios for squamous and small cell carcinoma is approaching the level of Western Europe in the late 1970s. Population attributable risk of exsmokers has also been increasing to the level of Western Europe. Among male current smokers, smoking intensity, such as number of cigarettes per day or fraction smoked per cigarette, seemed to have a slightly greater influence on squamous cell carcinoma than adenocarcinoma, while factors associated with the spread of cigarette smoke, such as inhalation, seemed to have greater influence on adenocarcinoma. The difference in the distribution of these smoking characteristics between Japan and Western Europe could not fully explain the difference in lung cancer incidence and distribution of histologic types between the two areas.
Interaction of Interleukin‐1 and Interferon‐γ on Fibroblast Growth Factor‐induced Angiogenesis
Authors: Norioka Ken‐ichi , Mitaka Toshihiro , Mochizuki Yohichi , Hara Masako , Kawagoe Mitsuhiro , Nakamura Haruo
Published: August 2005

The interaction of interleukin‐1 (IL‐1) and interferon‐γ (IFN‐γ) actions on several aspects of angiogenesis in vitro and in vivo was studied. The proliferation and migration of human umbilical vein endothelial cells cultured with basic fibroblast growth factor (bFGF) were synergistically inhibited by cotreatment with IL‐1 and IFN‐γ. Endothelial cell adhesion to collagen was suppressed by IL‐1 and the effect was slightly enhanced by the combination of IL‐1 and IFN‐γ. Local administration of IL‐1 (10,000 U) and IFN‐γ (1,000 U) inhibited bFGF‐induced angiogenesis in the skin of mice, and synergistic inhibitory activity of the combination was demonstrated. Expression of FGF receptors was strongly downregulated by the combination, whereas expressions of epidermal growth factor (EGF) receptors, integrin β1 and integrin β3 were not. EGF partially abrogated the growth‐inhibitory effects of IL‐1 and IFN‐γ. These findings indicate that IL‐1 and IFN‐γ are each able to act an angiogenesis inhibitor in a situation where FGF plays a major role in angiogenesis, and the activity is synergistically enhanced when they are used in combination.
Biodistribution of Neocarzinostatin Conjugated to Chimeric Fab Fragments of the Monoclonal Antibody A7 in Nude Mice Bearing Human Pancreatic Cancer Xenografts
Authors: Otsuji Eigo , Yamaguchi Toshiharu , Yamaoka Nobuki , Taniguchi Katsunori , Kato Makoto , Kotani Tatsuya , Kitamura Kazuya , Takahashi Toshio
Published: August 2005

In this study, we conjugated chimeric Fab fragments of the monoclonal antibody (MAb) A7, which reacts with pancreatic cancers, to the antitumor drug neocarzinostatin (chA7Fab‐NCS) and intravenously injected 125I‐labeled chA7Fab‐NCS into nude mice bearing a human pancreatic cancer xenograft. We compared the tumor localization of 125I‐labeled chA7Fab‐NCS with that of conventional 125I‐labeled A7‐NCS, which was produced by conjugation of MAb A7 and NCS. 125I‐Labeled chA7Fab‐NCS accumulated in the tumor earlier than 125I‐labeled A7‐NCS, and significantly larger amounts of 125I‐labeled chA7Fab‐NCS had accumulated in the tumor 1 hour after injection. The results suggest that chA7Fab may be a suitable carrier for NCS in immunotargeting therapy against pancreatic cancer.
Inhibition of Topoisomerase II by a Novel Antitumor Cyclic Depsipeptide, BE‐22179
Authors: Yoshinari Tomoko , Okada Hiromasa , Yamada Akihiro , Uemura Daisuke , Oka Hirofumi , Suda Hiroyuki , Okura Akira
Published: August 2005

BE‐22179, a novel cyclic depsipeptide antibiotic having two 3‐hydroxyquinoline moieties, inhibited the DNA‐relaxing activity of L1210 topoisomerase II completely at 0.08 μM. This effect was far stronger than that of VP‐16. However, it did not show any marked effect on topoisomerase II‐mediated DNA cleavage. BE‐22179 was ineffective in inhibiting the DNA relaxation by topoisomerase I at concentrations up to 10 μM, but showed DNA‐intercalating ability (DNA unwinding) at 30 μM. The structure of BE‐22179 is quite novel for a topoisomerase II inhibitor. Echinomycin, a quinoxaline antibiotic structurally related to BE‐22179, interfered with DNA relaxation by topoisomerase II, though the effect was not due to inhibition of the catalytic activity of topoisomerase II but to conformational change of DNA based on its intercalation into DNA. Therefore, the potent inhibitory activity on topoisomerase II might not be a common activity of quinoxaline antibiotics, but might rather be specific to BE‐22179. BE‐22179 prevented DNA synthesis as well as RNA synthesis in L1210 cells and inhibited the growth of the cells. However, it remains unclear to what extent the topoisomerase II inhibition was responsible for the cytotoxicity of BE‐22179.
Subcutaneous Sarcomas of Probable Neuronal Origin in a Transgenic Mouse Strain Containing an Albumin Promoter‐fused Simian Virus 40 Large T Antigen Gene
Authors: Kawabuchi Beniyo , Nomura Kimie , Ohtake Keiko , Hino Okio , Aizawa Shinichi , Machinami Rikuo , Kitagawa Tomoyuki
Published: August 2005

Frequent development of subcutaneous neurogenic sarcomas was observed in a hepatocellular carcinoma‐producing transgenic mouse strain harboring an albumin‐promoted simian virus 40 (SV40) large T antigen gene. Found unexpectedly in 19 out of 306 mice (6.2%) by 6 months of age, all the sarcomas were similar and were characterized as neurogenic on the basis of histological features including Homer‐Wright type rosette formation, the presence of dense core granules of 100–200 nm diameter under the electron microscope, expression of neuron specific enolase, S‐100 protein, and catecholamines, and nerve cell‐like differentiation in culture in response to But2cAMP, Immunohistochemical study revealed tiny clusters of SV40 T antigen‐expressing cells with neurogenic character in normal‐appearing adult mouse subcutis as candidate progenitors of the sarcomas. The tumor cells strongly expressed large T antigen but did not express albumin or albumin mRNA at the detection sensitivity used. Transient transfection assay (CAT assay), however, revealed the presence of transcriptional factor(s) acting on the albumin promoter in tumor cells. Thus, the present investigation suggested the presence of specifically differentiated neurogenic cells in the mouse subcutis with aberrant expression of the transgene.
Circumvention of Daunorubicin Resistance by a New Tamoxifen Derivative, Toremifene, in Multidrug‐resistant Cell Line
Authors: Urasaki Yoshimasa , Ueda Takanori , Nakamura Toru
Published: August 2005

The reversing effect of toremifene, a new tamoxifen derivative, on multidrug resistance in a K562 subline and its mechanism were studied. K562 cells were cultured in serially increasing concentrations up to 1.0 μM daunorubicin (DNR), and were found to be 28 times more resistant to DNR in comparison to the parent cells. In the resistant cell line (K562/D1‐9), intracellular accumulation of DNR was less than that of the parent cell line, and P‐glycoprotein was overexpressed. The resistance was reversed by addition of toremifene in a dose‐dependent manner in K562/D1‐9, while toremifene had no effect in K562. DNR accumulation was also reversed by toremifene in K562/D1‐9, but not in K562. However, there was no significant difference of toremifene retention between K562/D1‐9 and K562, and neither verapamil nor DNR increased toremifene accumulation in K562/D1‐9. Moreover, toremifene and verapamil did not show an additive effect on intracellular DNR accumulation. These results suggested that the reversing mechanism of toremifene is different from that of verapamil, and this compound could be a good candidate for overcoming multidrug resistance.
Case‐Control Study of Hepatocellular Carcinoma among Koreans Living in Osaka, Japan
Authors: Pyong Su Jun , Tsukuma Hideaki , Hiyama Tomohiko
Published: August 2005

Mortality rates from liver cancer among Koreans living in Osaka are 2–3 times higher than those among Japanese. Our previous study revealed that chronic hepatitis B virus (HBV) infection and excessive alcohol drinking are two major risk factors for hepatocellular carcinoma (HCC) among Koreans in Osaka, although more than 70% of the HCC cases were negative for hepatitis B surface antigen (HBsAg). Using a recently developed immunoassay for detecting serum hepatitis C virus antibody (HCV‐Ab), the role of HCV infection was evaluated in a case‐control study. The case group consisted of 90 Korean patients who were admitted to Kyowa Hospital in Osaka, and were newly diagnosed as HCC during the period from January 1989 to December 1992. The control group consisted of 249 Korean patients admitted to Kyowa Hospital during the same period and matched in age groups to the HCC cases. Seventy‐four and 16.7% of cases were positive for HCV‐Ab and HBsAg, respectively. Besides, 41.1% of cases were heavy drinkers. Multiple logistic regression analysis revealed that the adjusted relative risk was 92.4 for HCV‐Ab positive and 58.2 for HBsAg positive, as compared with both HCV‐Ab and HBsAg negative. Elevated risk was also demonstrated for males with a history of heavy drinking. There was no significant association between the risk of HCC and a history of blood transfusion or cigarette smoking. It was concluded that chronic HCV infection plays a major role in the etiology of HCC among Koreans living in Osaka, in addition to HBV and heavy drinking.
Increased Cell Proliferation of Azoxymethane‐induced Aberrant Crypt Foci of Rat Colon
Authors: Yamashita Naoyuki , Minamoto Toshinari , Onda Masahiko , Esumi Hiroyasu
Published: August 2005

Aberrant crypt foci (ACF) were induced in the colon of F344 rats by s.c. injection of azoxymethane (AOM) twice in a three day‐interval and examined after 4 and 12 weeks. The number and crypt multiplicity of ACF in each section of rat colon increased during this period. Histologically, aberrant crypts consisted of proliferating atypical epithelial cells. Cell proliferation of ACF consisting of 4 aberrant crypts [ACF(4)] and 2 aberrant crypts [ACF(2)], and normal crypts in the colon of rats treated with AOM [normal crypts/AOM(+)] or saline [normal crypts/AOM(‐)] was investigated by measurement of the mitotic index, proliferating cell nuclear antigen‐labeling index (PCNA‐LI), and 5‐bromo‐2′‐deoxyuridine‐labeling index (BrdU‐LI). All three parameters of the cell proliferative activity of ACF(4) were higher than those of normal crypts/AOM(+) and normal crypts/AOM(‐). The PCNA‐LI and BrdU‐LI in ACF(2) were the same as those in ACF(4). These findings suggest that ACF have increased cell proliferative activity. The correlation of these three parameters confirmed that the PCNA‐LI is also a useful parameter for evaluating cell proliferative activity in ACF. The presence of many cells stained by PCNA in the upper portion of ACF suggested that ACF have more G1 phase cells, which readily respond to mitogenic stimulation, than G0 phase cells, which are predominant in normal crypts.
Flow Cytometric Analysis of Early Steps in Development of Adriamycin Resistance in a Human Gastric Cancer Cell Line
Authors: Tanaka Shinsuke , Aizawa Kikuo , Katayanagi Norio , Tanaka Otsuo
Published: August 2005

We have established a low‐level adriamycin (ADM)‐resistant human gastric cancer cell line (MKN45R) from the parental cell line (MKN45) by exposure to step wise increases of ADM concentration (final concentration, 0.026 μg/ml). The purpose of this study was to identify the early steps in the development of ADM resistance in MKN45R by flow cytometric (FCM) analysis. Comparison of the concentration required for 50% growth inhibition, determined by a tetrazolium‐based colorimetric assay, showed that MKN45R was about 2.6‐fold more resistant to ADM than MKN45. However, the inhibition index values were 89.5% for MKN45 and 86.4% for MKN4SR, respectively, showing that ADM was judged to be “effective” against both cell lines. On the other hand, cell kinetic analysis by FCM revealed that the increase of the ratio of G2M accumulation induced by ADM treatment was significantly lower (p0.01) in MKN45R. Moreover, the efflux of ADM estimated by FCM analysis was significantly increased (P<0.05) in MKN45R even though there was no significant increase of P‐glycoprotein expression. These results suggest that although ADM was still effective based on a standard drug sensitivity test, the cancer cells were already acquiring resistance to ADM as judged from FCM analysis. Moreover, the mechanism of this ADM resistance is considered to be independent of P‐glycoprotein expression. Thus, FCM analysis is useful for detecting the early steps in the development of drug resistance of cancer cells.
Induction of Synthesis of Heat Shock Protein 72 in Tumor Necrosis Factor Gene‐transduced Cells
Authors: Watanabe Naoki , Akiyama Shinichiro , Tsuji Naoki , Sasaki Hiroyoshi , Yamauchi Naofurai , Okamoto Tetsuro , Kobayashi Daisuke , Niitsu Yoshiro
Published: August 2005

Heat shock protein (HSP) and endogenous tumor necrosis factor (enTNF) both act as resistance factors against the cytotoxicity of various cellular stresses. To clarify the relationship between these two stress response systems, we investigated whether or not enTNF is capable of inducing HSP72. Without heating, no difference was found in HSP72 synthesis between enTNF‐nonexpressing L‐M cells and cells expressing L‐R or L‐M (pcDV‐TNF). After initiation of heat treatment, however, a remarkable increase in HSP72 synthesis was noted in enTNF‐expressing cells compared to enTNF‐nonexpressing L‐M cells. These findings indicated that enTNF augments heat‐inducihle HSP72 synthesis.
Gene Expression for Tumor Necrosis Factor a and Its Production in Gastric Cancer Patients
Authors: Ohno Masakazu , Kato Michio , Nakamura Takeshi , Saitoh Yoichi
Published: August 2005

To determine the gene expression for tumor necrosis factor (TNF) a and its main site of production in gastric cancer patients, serum levels in the peripheral venous blood of 50 patients and the portal blood from 15 of these 50 patients were measured by enzyme‐linked immunosorbent assay (ELISA). TNF gene expression in the peripheral blood mononuclear cells (PBMC) and in the surgically resected tissues was then studied in 16 patients by reverse transcription‐polymerase chain reaction (RT‐PCR) assay. Whereas TNF mRNA expression was detected in the PBMC from 13 of 16 gastric cancer patients (81.3%), it was detected in only one tumor tissue (6.3%). Preoperatively, TNF was detected in the serum from 13 of 50 patients (26.0%). In the portal blood sampled immediately after laparotomy, TNF was positive in 4 of 15 patients (26.7%). TNF gene expression was much more frequently detected in PBMC than in other resected tissues, and its expression was higher than in the serum. Various clinicopathological factors for gastric cancer were not related to the prcopcrative detection of TNF in the serum. It appears that TNF is produced mainly in PBMC but not in the cancer regions or the regional lymph nodes of gastric cancer patients. It is suggested that TNF is not always secreted even when TNF mRNA is expressed, and its preoperatlve production is not related to tumor progression.
Role of Increased DNA Synthesis Activity of Hepatocytes in Multicentric Hepatocarcinogenesis in Residual Liver of Hepatectomized Cirrhotic Patients with Hepatocellular Carcinoma
Authors: Tarao Kazuo , Hoshino Hiroshi , Shimizu Akio , Ohkawa Shinichi , Nakamura Yoshiyasu , Harada Masaoki , Ito Yoshihiko , Tamai Setsuo , Akaike Makoto , Sugimasa Yukio , Takemiya Shoji , Okamoto Takashi , Okamoto Naoyuki
Published: August 2005

To examine whether the marked increase in DNA synthesis of hepatocytes in cirrhotic liver might elicit multicentric hepatocarcinogenesis, we examined the relationship between new development of hepatocellular carcinoma (HCC) and the bromodeoxyuridine (BrdU) labeling index (LI) of hepatocytes in the residual liver of hepatectomized patients with liver cirrhosis (LC) and HCC, Eighteen hepatectomized patients with LC and HCC, whose resected liver revealed neither portal nor hepatic vein invasion by histologic examination, were studied (to exclude cases with intrahepatic metastasis). DNA synthesis activity of hepatocytes from the residual cirrhotic liver was measured by a BrdU/ anti‐BrdU in vitro method. The incidence of HCC recurrence was studied during a 3‐year follow‐up period. Among 18 patients, 9 patients had recurrence and 9 did not. The average BrdU LI in the recurrent patients was 2.6 ±1.3% and was significantly higher than that in patients without recurrence (1.4+0.5%, P<0.05). All five patients who had a BrdU LI of 2.4% or above showed recurrence within 3 years, as compared with 4 of 13 (30.8%) patients with BrdU LI of less than 2.4% (P 0.05). Our data indicate that abnormally high DNA synthesis in hepatocytes in the background cirrhosis might lead to the development of multicentric carcinogenesis in human cirrhotic liver, and in the residual cirrhotic liver of hepatectomized patients with HCC and LC, it may be a predictor of new development of HCC.
Association of Vascular Endothelial Growth Factor Expression with Tumor Angiogenesis and with Early Relapse in Primary Breast Cancer
Authors: Toi Masakazu , Hoshina Seigo , Takayanagi Toshiaki , Tominaga Takeshi
Published: August 2005

Angiogenesis is an independent prognostic indicator in breast cancer. In this report, the relationship between expression of vascular endothclial growth factor (VEGF; a selective mitogen for endothelial cells) and the microvessel density was examined in 103 primary breast cancers. The expression of VEGF was evaluated by immunocytochemical staining using anti‐VEGF antibody. The microvessel density, which was determined by immunostaining for factor VIII antigen, in VEGF‐rich tumors was clearly higher than that in VEGF‐poor tumors (P<0.01). There was a good correlation between VEGF expression and the increment of microvessel density. Furthermore, postoperative survey demonstrated that the relapse‐free survival rate of VEGF‐rich tumors was significantly worse than that of VEGF‐poor tumors. It was suggested that the expression of VEGF is closely associated with the promotion of angiogenesis and with early relapse in primary breast cancer.
In vitro and in vivo Effects of Cisplatin and Etoposide in Combination on Small Cell Lung Cancer Cell Lines
Authors: Kondo Hideki , Kanzawa Fumihiko , Nishio Kazuto , Saito Shiro , Saijo Nagahiro
Published: August 2005

The effects of cisplatin (CDDP) and etoposide (ETP) in combination were evaluated in vitro and in vivo using small cell lung cancer cell lines. The combination effects in vitro were investigated using isohologram analysis. Used together, CDDF and ETP showed a synergistic effect against cell growth on only 1 cell line (SBC‐3), additive effects on 6 (SBC‐2, SBC‐5, Lul30, Lul34AH, Lul35T and H69) and an antagonistic effect on 1 (SBC‐1). In the in vivo experiment, nude mice were inoculated with SBC‐1, SBC‐3 and SBC‐5 cells. Two or 5 mgAg CDDP and 10 or 30 mgAg ETP were administered intraperitoneally alone and simultaneously in combination to nude mice. The in vivo effects of the combination were determined by comparing the observed growth ratio in mice treated with the combination with the expected value of this ratio calculated based on the assumption that the effects of the drugs were simply additive. According to this definition, synergistic effects were observed against all 3 tumors. Thus, the in vivo and in vitro effects differed. The toxicity of the combination therapy, which was analyzed by estimating the body weight change of mice, was no higher than that of CDDP or ETP alone. These results suggest that the excellent clinical effects of CDDP and ETP combination therapy may he attributable not to drug interaction at the cellular level hut to the feasibility of combined use of them at full doses without overlapping side effects.
Epstein‐Barr Virus‐associated Post‐transplant Non‐Hodgkin's Lymphoma: Establishment and Characterization of a New Cell Line
Authors: Kawashima Kunihiro , Hayashi Kyoichi , Ohnoshi Taisuke , Teramoto Norihiro , Kimura Ikuro
Published: August 2005

A new human lymphoma cell line derived from pulmonary non‐Hodgkin's lymphoma that developed in a renal transplant recipient was established from the patient's pleural effusion and designated PTLC‐1. PTLC‐1 grew aggressively in suspension, forming very loose clamps with a doubling time of about 18.9 h. The morphological, chromosomal, and immunophenotypic characteristics of the patient's tumor cells and PTLC‐1 cells were very similar. PTLC‐1 showed a monoclonal rearrangement of IgH gene and was highly tumorigenic in athymic nude mice. In situ hybridization, Southern blot hybridization and polymerase chain reaction demonstrated the presence of Epstein‐Barr virus (EBV) genome in the patient's tumor and PTLC‐1. PTLC‐1 has been maintained in culture for over 60 months. Since EBV has been implicated in the pathogenesis of post‐transplant lymphoma, this new cell line should serve as a useful experimental model for studying the etiology and biology of lymphoma developing in organ transplant recipients
Characteristics of Human Hepatocellular Carcinoma Cell Lines (Hep‐KANO) Derived from a Non‐hepatitic, Non‐cirrhotic Hepatitis B Virus Carrier
Authors: Baba Masaru , Hasegawa Hiroshi , Nakayabu Masahiro , Tamaki Shigenori , Watanabe Shozo , Shima Teruo , Suzuki Shiro , Kusano Itsuo , Kamada Nanao
Published: August 2005

We have established two cell lines of hepatocellular carcinoma [Hep‐KANO, clone 1 (CL‐1) and clone 2 (CL‐2)] from tissue obtained at autopsy of a hepatitis B virus (HBV) carrier without histological signs of hepatitis or liver cirrhosis. These cell lines differed considerably from each other in morphology, proliferation pattern, α‐fetoprotein secretion, albumin synthesis, cytokine secretion, modal chromosome number and transplantability to nude mice. Histologic examinations also revealed differences between them. Amplification of N‐myc, L‐myc, H‐ras, K‐ras, N‐ras, c‐erb‐B and c‐erb‐B‐2 and rearrangement of p53 were not found in either of the cell lines. However, CL‐1 and CL‐2 showed an identical HBV‐DNA integration pattern. A 4‐fold amplification of c‐myc was observed in CL‐1, but not in CL‐2. Hep‐KANO cell lines, CL‐1 and CL‐2 may be useful in clarifying the question of whether hepatocarcinogenesis is directly caused by HBV infection.
Improvement of Macrophage Dysfunction by Administration of Anti‐transforming Growth Factor‐β Antibody in EL4‐bearing Hosts
Authors: Maeda Hiroaki , Tsuru Sumiaki , Shiraishi Akio
Published: August 2005

An experimental therapy for improvement of macrophage dysfunction caused by transforming growth factor‐β (TGF‐β) was tried in EL4 tumor‐bearing mice. TGF‐β was detected in cell‐free ascitic fluid from EL4‐bearers, but not in tbat from normal mice, by western blot analysis. The ascites also showed growth‐suppressive activity against MvlLn cells, and the suppressive activity was potentiated by transient acidification. To investigate whether the functions of peritoneal macrophagcs were suppressed in EL4‐bearers, the abilities to produce nitric oxide and tumor necrosis factor‐α (TNF‐α) upon lipopolysaccharide (LPS) stimulation were measured. Both abilities of macrophages in EL4‐bearing mice were suppressed remarkably on day 9, and decreased further by day 14, compared with non‐tumor‐bearing controls. TGF‐β activity was abrogated by administration of anti‐TGF‐α antibody to EL4‐bearing mice. While a large amount of TGF‐β was detected in ascitic fluid from control EL4‐bearers, little TGF‐β was detectable in ascites from EL4‐bearers given anti‐TGF‐β antibody. Furthermore, while control macrophages exhibited little or no production of nitric oxide and TNF‐α on LPS stimulation in vitro, macrophages from EL4‐bearers administered with anti‐TGF‐β antibody showed the same ability as normal macrophages. These results clearly indicate that TGF‐β contributes to macrophage dysfunction and that the administration of specific antibody for TGF‐β reverses macrophage dysfunction in EL4‐bearing hosts.
High Detection Rate of Breast Cancer by Mass Screening Using Mammography in Japan
Authors: Morimoto Tadaoki , Sasa Mitsunori , Yamaguchi Tetsuo , Haráda Kunihiko , Sagara Yasunobu
Published: August 2005

A trial of mass screening for breast cancer using both mammography and physical examination (independently) was carried out in women over 50 years of age in Tokushima Prefecture. Breast cancer was detected in eight of a total of 950 examinees by mammography alone, and no cases of breast cancer were detected by physical examination. The detection rate of breast cancer was 0.84%, which is very much higher than that obtained by conventional mass screening using physical examination alone. The detection rate increased especially in the sixth and seventh decades of life. All eight detected breast cancers were in the early stage. Based on these results, it is recommended to employ mammography in breast cancer screening for asymptomatic women aged over 50 years.
Prognostic Significance of p53 and ras Gene Abnormalities in Lung Adenocarcinoma Patients with Stage I Disease after Curative Resection
Authors: Isobe Takeshi , Hiyama Keiko , Yoshida Yasuhiro , Fujiwara Yasuhiro , Yamakido Michio
Published: August 2005

We investigated the prognostic significance of p53 gene abnormalities and ras gene mutations in patients with curatively resected stage I lung adenocarciiioma. Formalin‐fixed and paraffin‐embedded tissues were obtained from 30 patients who had undergone curative resection for stage I lung adenocarciiioma. Abnormalities of the p53 gene were detected using polymcrasc chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) analysis and immunohistochemistry and ras mutations were detected using PCR‐restriction fragment length polymorphism (RFLP) analysis. Both univariate and multivariate analyses were performed to assess the relationship between the presence of abnormalities of these genes and the patients’disease‐free survival. Eleven tumors (37%) had mutated p53 sequences and 11 (37%) showed p53 overexpression. A total of 15 tumors (50%) had p53 gene abnormalities and the concordance rate was 73%. Seven tumors (23%) showed mutated ras sequences. The univariate analysis revealed that the disease‐free survival of patients with any p53 abnormality was shorter than that of those without abnormalities (P=0.02, generalized Wilcoxon test), and survival of those with p53 protein overexpression was more significantly shorter (P=0.003, generalized Wilcoxon test). Multivariate analysis using the Cox proportional hazards model indicated that the presence of p53 abnormalities was a significantly (P=0.01) unfavorable prognostic factor. There was no significant correlation between the presence of ras mutation and survival. These results suggest that analysis of the p53 gene may be helpful for the selection of high‐risk patients for clinical trials of adjuvant therapy for stage I lung adenocarcinoma.
Differential Effect of Duocarmycin A and Its Novel Derivative DU‐86 on DNA Strand Breaks in HeLa S3 Cells
Authors: Okamoto Akihiko , Asai Akira , Saito Hiromitsu , Okabe Masami , Gomi Katsushige
Published: August 2005

Duocarmycin A (DUMA) and DU‐86, a semisynthetic derivative of duocarmycins (DUMs) and a possible active form of KW‐2189, both showed potent cell growth‐inhibitory and cell‐killing activities against human uterine cervix carcinoma HeLa 83 cells. Both drugs showed similar profiles of inhibition of macromolecular synthesis and influence on cell‐cycle distribution. Namely, they inhibited [3H]thymidine uptake at lower concentrations than [3H]uridine or [3H]Ieucine uptake, suggesting that the inhibition of DNA synthesis is the primary site of their actions. Furthermore, they induced the accumulation of cells in early S phase. However, a significant difference was observed between these drugs in terms of DNA‐fragmentation activity against HeLa S3 cells by using two independent methods, pulse‐field gel electrophoresis and alkaline elution. DNA fragmentation was insignificant in the cells treated with DU‐86, in contrast to the cells treated with DUMA. The analysis of DNA adducts in the cells revealed that DU‐86 alkylated adenine quite selectively, while DUMA alkylated both adenine and guanine. These results suggest that the pyrrolidone ring of DUMA is responsible for its adduct formation with guanine and the subsequent DNA‐fragmentation and inhibition of DNA synthesis, while DU‐86 alkylated adenine and inhibited DNA synthesis through mechanisms other than DNA‐fragmentation.
N‐Nitroso‐2‐acetylaminofluorene: A Direct‐acting Carcinogen Inducing Hepatocellular Carcinoma in Sprague‐Dawley Rats
Authors: Ho Yuan‐Soon , Lin Jen‐Kun
Published: August 2005

To compare the hepatotoxicity and hepatocarcinogenicity of N‐nitroso‐2‐acetylaminofluorene (NO‐AAF) and its parent compound, 2‐acetylaminofluorene (AAF), male Sprague‐Dawley rats were given intraperitoneal (i.p.) or subcutaneous (s.c.) injections of AAF or NO‐AAF (60 mg/kg body weight/week) for ten months. In the AAF group, morphological changes were produced which involved gross distortions of the liver with multiple nodule formations. The rat livers in the NO‐AAF group appeared to be smooth with a blunt‐thick superior segment of the lateral lobe. The serum γ‐glutamyl transpeptidase activity in both the AAF group and the NO‐AAF group was significantly elevated (P< 0.0005). The present study shows that i.p. and s.c. injections of NO‐AAF resulted in a high incidence of well‐differentiated hepatocellular carcinomas (HCC) (7/9 and 4/6, respectively), while poorly differentiated HCCs were induced by i.p. or s.c. administration of AAF (6/9 or 2/6, respectively). Subcutaneous lesions consisting of an inflammatory reaction and fibroadenoma formation were observed in the NO‐AAF‐treated rats, whereas no such skin lesions were detected in the AAF‐treated animals. These results suggest that NO‐AAF is a new direct‐acting carcinogen which may be useful for investigating hepatocarcinogenesis.
Mouse Anti‐human Interleukin‐6 Receptor Monoclonal Antibody Inhibits Proliferation of Fresh Human Myeloma Cells in vitro
Authors: Goto Hideo , Shimazaki Chihiro , Tatsumi Tetsuya , Yamagata Noboru , Hirata Toshiyuki , Ashihara Eishi , Oku Naritoshi , Inaba Tohru , Fujita Naohisa , Koishihara Yasuo , Ohsugi Yoshiyuki , Nakagawa Masao
Published: August 2005

Interleukin‐6 (IL‐6) is a major growth factor in multiple myeloma. We investigated the effect of mouse anti‐human IL‐6 receptor monoclonal antibody (anti‐IL‐6R mAb) on the in vitro proliferation of freshly isolated myeloma cells from 21 patients to evaluate the therapeutic potential. The addition of anti‐IL‐6R mAb inhibited more than 30% of the spontaneous proliferation of myeloma cells in 9 of 21 cases in a dose‐ (0.1 to 20 /μ/ml) and time‐dependent manner. The inhibitory effects of anti‐IL‐6R mAb did not differ significantly from that of anti‐IL‐6 mAb, and were correlated with the extent of the response of myeloma cells to IL‐6. Flow cytometric analysis showed that all myeloma cells expressed IL‐6R, whose intensity was not correlated with either the extent of response of myeloma cells to IL‐6 or the inhibitory effects of anti‐IL‐6R mAb on proliferation of myeloma cells. Although our study showed heterogeneity in the proliferative responses of myeloma cells to IL‐6 and anti‐IL‐6R mAb, these observations suggest the possibility of using anti‐IL‐6R mAbs for treating some patients with multiple myeloma whose growth depends on IL‐6.
Infrequent Replication Errors at Microsatellite Loci in Tumors of Patients with Multiple Primary Cancers of the Esophagus and Various Other Tissues
Authors: Shimada Mamoru , Horii Akira , Sasaki Shin , Yanagisawa Akio , Kato Yo , Yamashita Kenichi , Okagawa Kazuhiro , Yamasaki Keiji , Ishiguro Shingo , Inoue Masatoshi , Shiozaki Hitoshi , Nakamura Yusuke
Published: August 2005

Patients with esophageal cancer are at high risk of developing other primary tumors, especially squamous cell carcinoma in the head and neck. Heavy smoking and excessive consumption of alcohol are considered to he crucial environmental risk‐factors for development of these multiple primary cancers. To investigate whether any genetic background, such as defects in the DNA‐mismatch repair system, may influence the development of these multiple primary tumors, we examined replication errors (RER) at six microsatellite loci in DNAs of 46 tumors from 33 patients who had developed primary cancers in various tissues in addition to the esophagus. RER(+) (RER‐positive) phenotype was observed in three tumors in two patients of the 33 patients examined. Our results suggested that development of multiple primary tumors in these patients would not be affected by an abnormality in the DNA repair system(s) detected as the RER phenotype. However, it is noteworthy that a single patient who developed multiple cancers revealed RER(+) phenotypes at multiple microsatellite loci in both tumors, indicating that a defect in the DNA repair gene(s) may have played an important role in the development of the tumors in this patient.
Biological Effectiveness of Fractionated Dose of Pions in Microscopic SCCVII Tumors: Comparison between Tumor Control Dose and Tumor Growth Time Assays
Authors: Ohizumi Yukio , Lam Gabriel K. Y. , Pickles Tom , Chaplin David J.
Published: August 2005

The relative biological effectiveness (RBE) of fractionated pions for tumor growth time (TGT) assay changes with the endpoints, so it is essential to determine the RBE for tumor control dose (TCD) assay. For this purpose, the TCD50 of fractionated pions was compared with that of photons, and the RBEs for TGT and TCD assays were concurrently compared as a function of the effect level. A “convenient” RBE (cRBE) was substituted for the RBE when the comparison was made between similar fractionation schedules with different dose per fraction. SCCVII tumors (2 ×104 or 2 × 105 cells) were implanted into the feet of C3H mice and irradiated starting from 2 days after implantation at a total dose range of either 9.6–38.4 Gy pions (2.4–6.4 Gy per fraction) or 14.4–50.4 Gy photons (3.6–7.2 Gy per fraction) in 2–10 fractions over 5–6 days. The cRBE and the RBE at the iso‐effective level of 30 days TGT were 1.53–1.60 for 2.4–4.8 Gy pions and 1.50 for 4‐fracrionated pions, respectively: there were only small differences within these schedules used. However, the cRBE values decreased from 1.60 to 1.15 with increasing TGT from 30 to 75 days. In contrast, the cRBE values for TCD50 increased from 1.08 to 1.40 (95% confidence limits [CL]; 1.18–1.63) with increasing evaluation time from 60 to 100 days: pions significantly inhibited late tumor appearance. The TCD50 at 100 days was 28.7 Gy (CL; 25.0–32.5 Gy) for pions and 40.3 Gy (CL; 36.3–44.2 Gy) for photons. In conclusion, the RBE for TCD50 was not predictable from the RBE for TGT assay. The cRBE value of 1.4 for microscopic tumor control was in close agreement with the reported values for skin reaction.
Enhancement of Ca2+‐dependent Endonuclease Activity in L1210 Cells during Apoptosis Induced by 1‐β‐D‐Arabinofuranosylcytosine: Possible Involvement of Activating Factor(s)
Authors: Takauji Rumiko , Yoshida Akira , Iwasaki Hiromichi , Tohyama Kaoru , Ueda Takanori , Nakamura Toru
Published: August 2005

Internucleosomal DNA fragmentation and morphological changes in nuclei typical of apoptosis were observed in L1210 cells incubated with 1.0 μg/ml of 1‐β‐D‐arabinofuranosylcytosine (ara‐C). To investigate the mechanisms involved, we examined the activities of endogenous endonucleases in nuclei and cytoplasm. Both fractions of control cells contained Ca2+ ‐dependent endonuclease which was capable of mediating internucleosomal DNA fragmentation. The assay system using two kinds of target substrates, i.e., nuclear chromatin of CCRF‐CEM cells and naked DNA purified from the same cells, revealed that the activity of Ca2+‐dependent endonuclease was enhanced in the crude nuclear extracts of cells treated with 1.0 μg/ml of ara‐C for 24 h or 48 h. The activity was extracted more easily from ara‐C‐treated cells than control cells without sonication of the nuclear fraction. On the other hand, in the cytoplasmic fraction of the cells, the activity towards naked DNA was unchanged, whereas that towards nuclear chromatin was clearly enhanced. These results suggest that internucleosomal DNA fragmentation induced by ara‐C treatment is associated with enhancement and activation of constitutively expressed Ca2+ ‐dependent endonuclease in L1210 cells.
Flat Serrated Adenomas and Flat Tubular Adenomas of the Colorectal Mucosa: Differences in the Pattern of Cell Proliferation
Authors: Rubio Carlos A. , Rodensjö Margareta
Published: August 2005

In the present work we have investigated the cell proliferation pattern of flat serrated adenomas and flat tubular adenomas. For this purpose tissue sections from 23 consecutive flat serrated adenomas and 22 consecutive flat tubular adenomas of the colorcctal mucosa were challenged with MID1, a monoclonal antibody directed against a proliferation‐related antigen. The results (including semi‐quantitative studies) demonstrated that, whereas flat serrated adenomas had a high cell proliferation at the lower part of the crypts, flat tubular adenomas had a high cell proliferation in the upper part of the crypts. In serrated adenomas with invasive adenocarcinoma, high cell proliferation was demonstrated both at the lower portion of the crypts and in the subjacent submucosa. This suggests that the cells of the lower portion of the crypts in serrated adenomas are truely neoplastic, with the capacity to evolve into invasive growth. The difference in cell proliferation betweeen the two types of flat lesions reported here is a new argument in favor of the classification of flat serrated adenomas as a novel and independent type of neoplastic change of the colorectal mucosa.
Differing Distribution of Hepatocyte Growth Factor‐positive Cells in the Liver of LEC Rats with Acute Hepatitis, Chronic Hepatitis and Hepatoma
Authors: Nakayama Nobuaki , Kashiwazaki Haruhiko , Kobayashi Narumi , Hamada Jun‐ichi , Matsumoto Kunio , Nakamura Toshikazu , Takeichi Noritoshi
Published: August 2005

Using anti‐rat hepatocyte growth factor (HGF) antibody, we investigated the distribution of HGF‐positive cells in the liver tissues of LEC rats at various phases of liver diseases. During the phase of fulminant hepatitis, HGF‐positive cells increased remarkably, and many of them were localized at the portal triads; these cells were identified from their shape as non‐epithelial cells. A reduced number of HGF‐positive cells was observed during the phase of chronic hepatitis, while no HGF‐positive cells were seen in the tissue of cholangiofibrosis. During the phase of carcinoma, staining revealed that both the hepatocellular carcinoma cells and the non‐epithelial cells in cancerous liver tissue were HGF‐positive. These results suggest that, in LEC rats, HGF may play an important role in the regeneration of hepatocytes as well as in the development of hepatocellular carcinoma.
In vitro Induction of Cytotoxic T Lymphocytes against HTLV‐I‐infected T‐Cells from Adult T‐Cell Leukemia Patients, Asymptomatic HTLV‐I Carriers and Seronegative Healthy Donors
Authors: Katahira Yoshihiko , Yashiki Shinji , Fujiyoshi Toshinobu , Nomura Kouichiro , Tara Mitsutoshi , Mori Miyuki , Setoyama Mitsuru , Kanzaki Tamotsu , Shida Hisatoshi , Sonoda Shunro
Published: August 2005

We investigated an in vitro method to produce cytotoxic T lymphocytes (CTLs) against HTLV‐I‐infected T‐cells using peripheral blood mononuclear cells (PBMC) of adult T‐cell leukemia (ATL) patients, asymptomatic HTLV‐I carriers (AC) and seronegative healthy donors. The PBMC were restimulated repeatedly for 4 weeks with HLA‐matched HTLV‐I‐infected T‐cells which had been pretreated at 56deg;C for 30 min to inactivate infectious HTLV‐I. The culture medium included 10–100 units/ml of recombinant lymphokines (rIL‐1, rIL‐2, rIL‐4, rIL‐6 and rIL‐7) and 10% fetal calf serum in RPMI‐1640 medium. The cytotoxic activity was measured against HLA‐matched HTLV‐I‐infected T‐cell lines after CD4+ or CD8+ cells were positively panned from the cultured PBMC. The PBMC of ATL, AC and healthy donors were able to produce either CD4+ or CD8+ CTLs against HTLV‐I‐related antigens (env, gag, p21x, p27rex and p40tax) as well as the antigen(s) of as‐yet unknown specificity expressed on HTLV‐I‐infected T‐cells. All the CTLs recognized the specific antigens in the context of either class I or class II HLA types. These results indicated that ATL patients, AC and healthy donors were immunocompetent to generate CTLs against HTLV‐I‐infected T‐cells and probably against HTLV‐I‐transformed T‐cells.