bluuebunny/crossref_metadata_2025 huggingface dataset:
A Tyrosine‐specific Protein Kinase Inhibitor, α‐Cyano‐3‐ethoxy‐4‐hydroxy‐5‐phenylthiomethylcinnamamide, Blocks the Phosphorylation of Tyrosine Kinase Substrate in Intact Cells
Authors: Shiraishi Tadayoshi , Owada M. Koji , Tatsuka Masaaki , Fuse Yoshihide , Watanabe Kiyoshi , Kakunaga Takeo
Published: August 2005

Inhibition by α‐cyano‐3‐ethoxy‐4‐hydroxy‐5‐phenylthiomethylcinnamamide (ST 638) of tyrosine‐specific protein kinase was examined using epidermal growth factor (EGF)‐treated A431 cells at the concentration of 25 to 100 μM. ST 638 had negligible effects on the growth and morphology of A431 cells and on EGF binding to its receptor, and subsequent down‐regulation of the receptor. ST 638 specifically inhibited EGF‐induced phosphorylation of tyrosine residues of whole cell proteins in a dose‐dependent manner without affecting the phosphorylation of serine and threonine residues. ST 638 greatly inhibited the EGF‐induced phosphorylation of lipocortin I at 25 μM, and yet had a negligible effect on the EGF‐induced phosphorylation of EGF receptor. Neither the amount of [35S]methionine‐labeled lipocortin I nor the serine/threonine phosphorylation level of fodrin β‐subunit was affected by the same concentration of ST 638. These results indicate that the phosphorylation of lipocortin I is not relevant to the transformation of A431 cells. In cell lines transformed by src or fgr oncogene encoding tyrosine kinase, ST 638 also inhibited phosphorylation of calpactin I (p36) without affecting that of the oncogene products. Two‐dimensional polyacrylamide gel electrophoresis showed that ST 638 specifically inhibited the EGF‐induced phosphorylation and dephosphorylation of cellular proteins in A431 cells.
Inhibition of Tumor Angiogenesis by a Synthetic Cell‐adhesive Polypeptide Containing the Arg‐Gly‐Asp (RGD) Sequence of Fibronectin, Poly(RGD)
Authors: Saiki Ikuo , Murata Jun , Makabe Takashi , Nishi Norio , Tokura Seiichi , Azuma Ichiro
Published: August 2005

We have investigated the anti‐angiogenic effect of a polymeric peptide based on the Arg‐Gly‐Asp (RGD) core sequence of fibronectin as a monomer unit, i.e., poly(RGD), in syngeneic mice and in vitro. Single intratumoral administration of poly(RGD) on day 0, 1 or 7 after tumor implantation achieved a significant reduction of B16‐BL6 melanoma colonization in the lungs, but did not affect the size of the primary tumor at the time of amputation. The number of capillary blood vessels oriented toward the tumor mass increased during the early growth phase after the intradermal inoculation of the tumor. Poly(RGD) significantly inhibited the formation of tumor neovascularization when co‐injected with the tumor cells or separately injected intratumorally or intravenously on day 1 or 3 after tumor inoculation. This inhibitory effect of poly(RGD) was dose‐dependent. Poly(RGD) was able to inhibit the haptotactic migration of endothelial cells along a gradient of substratum‐immobilized fibronectin but not laminin. Tumor‐conditioned medium (CM) by itself did not act as a chemoattractant when it was added in the lower compartment of a Transwell chamber, but promoted the endothelial cell migration to immobilized fibronectin or laminin. Poly(RGD) inhibited the enhanced cell migration to fibronectin but not to laminin in response to CM. Thus, poly(RGD)‐mediated inhibition of tumor metastasis may be partly due to the inhibition of tumor‐induced angiogenesis at primary and secondary sites.
Ribose‐transfer Activity from Uridine to 5‐Fluorouracil in Ehrlich Ascites Tumor Cells
Authors: Nabeya Yoshihiro , Isono Kaichi , Moriyama Yoichi , Fujimura Shinji
Published: August 2005

Anabolism of 5‐fluorouracil (5‐FU) in the presence of uracil was examined using the cell‐free extract of Ehrlich ascites tumor cells. FU‐nucleoside formation from 5‐FU with ribose 1‐phosphate (R‐1‐P) or 2′‐deoxyribose 1‐phosphate was not readily inhibited even by the addition of uracil at 100 times higher concentration than 5‐FU. FU‐nucleotide formation from 5‐FU with R‐1‐P and adenosine 5′‐triphosphate or with 5‐phosphorihosyl 1‐pyrophosphate was slightly reduced as the concentration of uracil was increased. It was also found that 5‐fluorouridine (5‐FUR) was produced by “nucleoside N‐ribosyltransferase,”transferring a ribose moiety from uridine (UR) to 5‐FU directly. This activity might play a role in the preferential formation of 5‐FUR. However, 5‐fluoro‐2′‐deoxyuridine was not produced by directly transferring a deoxyribose moiety. On the basis of several column chromatographies and characterization of kinetics, pH dependency, and response to inhibitors, the enzyme protein of the ribosyltransferase could not be distinguished from that of the phosphorylase.
Anthropometric Indicators of Breast Cancer Risk in Japanese Women in Fukuoka
Authors: Kyogoku Shinji , Hirohata Tomio , Takeshita Setsuko , Hirota Yoshio , Shigematsu Takao
Published: August 2005

This study was based on a case‐control study of breast cancer risk in Fukuoka, which was initiated to identify risk factors for the disease. We re‐analyzed the data of the study to evaluate the association of breast cancer with anthropometric variables in postmenopausal women. Data were obtained through interviewing 121 cases with historically confirmed breast cancer and an equal number of age‐matched hospital controls as well as neighborhood controls. The variables used for analysis were; height, weight at various ages, shoe size, and brassiere size and body size indices (body surface area and various body build indices) computed from the height and weight data. In a multiple covariance analysis, the results showed that cases were slightly lighter in weight and smaller for some of the indices, but the differences were not statistically significant. The odds ratios, estimated by multiple logistic regression analysis, suggested that the cases were slightly shorter in height and smaller in shoe size without any consistent dose‐response gradients. The effect of changes in weight and body size indices with aging were also assessed, but no significant findings were observed. The results reported here do not support the hypothesis of the positive association between obesity or large body size and breast cancer.
Modulation by Indomethacin or Prostaglandin E2 of the Incidence of Diethylnitrosamine‐induced γ‐Glutamyltranspeptidase‐positive Foci in Rat Liver
Authors: Taton Gerard , Servais Patricia , Galand Paul
Published: August 2005

We investigated the effect of a pretreatment with indomethacin (IMC, ip 3.6 mg/kg body weight (bw)) or dimethylprostaglandin E2 (PGE2, ip 10 μg/kg bw) on the incidence and development of γ‐glutamyl‐transpeptidase (GGT)‐positive foci of altered hepatocytes, scored 8 or 14 weeks after ip injection of diethylnitrosamine (DENA, 50 mg/kg bw) to rats submitted to two‐thirds hepatectomy (PH) or sham operation (Sh). IMC reduced by about 4 times the incidence of DENA‐induced GGT‐positive foci per cm3 of liver tissue in sham‐operated as well as in two‐thirds hepatectomized rats, compared to the respective unpretreated controls. In contrast, PGE2 pretreatment increased the incidence of DENA‐induced foci in both groups, this effect, in terms of absolute numbers of foci, being additive to that of PH alone. IMC pretreatment resulted in foci with lower average size in the Sh but not in the PH animals, whereas with PGE2 pretreatment the mean volume of the foci was increased in the two groups of rats. At the dose used, IMC did not modify the proliferative response of hepatocytes to PH, and PGE2 did not stimulate proliferation in the sham‐operated animals. Altogether, these results indicate that: 1, the incidence of DENA‐induced foci can be negatively modulated by interfering with the prostaglandins pathway through a mechanism that does not involve an action either on proliferative activity or on any other process that would be specific to the post‐hepatectomy regenerative state; 2, positive modulation of the incidence of DENA‐induced foci does not necessarily require stimulation of proliferation.
Cellular Differentiation and Histogenesis of Rat Glandular Stomach Cancers
Authors: Tatematsu Masae , Katsuyama Tsutomu , Furihata Chie , Fukushima Shoji , Shirai Tomoyuki , Kato Toshio , Ito Nobuyuki
Published: August 2005

The gastric and intestinal phenotypic expressions of tumor cells in 18 adenomatous hyperplasias, 33 well‐differentiated adenocarcinomas, and 16 undifferentiated adenocarcinomas (4 poorly differentiated adenocarcinomas, 10 signet‐ring cell carcinomas and 2 mucinous adenocarcinomas) induced by N‐methyl‐N’‐nitro‐N‐nitrosoguanidine or 4‐nitroquinoline‐I‐oxide in the rat glandular stomach were studied by histochemical stainings for mucin and immunohistochemical staining for pepsinogen isozyme 1 (Pg 1). By histochemical staining for mucin [by the paradoxical concanavalin A method, the modified method with labeled peanut lectin, the galactose oxidase‐Schiff (GOS) reaction, and the sialidase‐GOS reaction] and immunohistochemical staining of Pg 1, gastric cancer cells of each histological group could be clearly classified into a gastric type, including mucous neck cell pyloric gland cell, and surface mucous cell subtypes, and an intestinal type, including goblet‐cell, and intestinal absorptive cell subtypes. All tumors examined in this work consisted mainly of gastric‐type cells but intestinal‐type tumor cells were occasionally found among the gastric‐type tumor cells. The incidences of intestinal‐type cells in adenomatous hyperplasias (11.1%) and small well‐differentiated adenocarcinomas (28.6%) were significantly less (P<0.05) than that in large well‐differentiated adenocarcinomas (68.4%). The incidence of intestinal‐type cells in small undifferentiated adenocarcinomas (25.0%) was also less than that in large ones (58.3%). The present results suggest the occurrence of change of phenotypic expression of tumor cells from the gastric type to the intestinal type during growth of tumors.
Fv‐1 Restriction of Endogenous Feline C‐Type RD114 Virus Genome Phenotypically Mixed with Ecotropic Murine Leukemia Viruses
Authors: Kakimi Kazuhiro , Kishida Yoshiko , Higuchi Ikuko , Kiyomasu Takahiro , Sakai Hiroyuki , Shibata Riri , Yanagawa Shin‐ichiro , Adachi Akio , Ishimoto Akinori
Published: August 2005

Endogenous feline leukemia RD114 virus genome rendered capable of infecting mouse cells by phenotypic mixing with an ecotropic murine leukemia virus (MuLV) exhibited the Fv‐1 restriction pattern of the ecotropic murine virus. However, RD114 genomes phenotypically mixed with ecotropic MuLV showed one‐hit dose‐response kinetics, even when titrated with murine cells with the restricted Fv‐1 phenotype.
Antitumor Activity and Pharmacokinetics of a Morpholino‐anthracycline Derivative (KRN8602) against Human Breast Carcinoma Xenografts Serially Transplanted into Nude Mice
Authors: Kubota Tetsuro , Suto Akihiko , Josui Kazuya , Ishibiki Kyuya , Abe Osahiko , Yamada Yoshinori , Asanuma Fumiki , Kawamura Eiji , Koh Jun‐ichi , Shiina Eiichi , Inada Takao , Ogata Yoshiro
Published: August 2005

The antitumor activity and pharmacokinetics of (7R, 8S, 10S)‐10‐((3‐deamino‐3‐(4‐morpholino)‐2,3,6‐trideoxy‐α‐l‐lyxo‐hexopyranosyl)oxy)‐8‐ethyl‐7,8,9,10‐tetrahydro‐1,6,7,8,11‐pentahydroxy‐5,12‐naphthacenedione hydrochloride (KRN8602) were evaluated using five human breast carcinoma xenografts in nude mice. The maximum non‐toxic dose of KRN8602 was 2 mg/kg by q4d×3 intraperitoneal and peroral administration. KRN8602 showed significant antitumor activity against MX‐1, which is less sensitive to adriamycin, with the chemotherapeutic indices of 13.0 for po administration and 9.5 for ip injection. Although KRN8602 also inhibited the growth of T‐61 significantly, the antitumor activity of this agent against the other three breast carcinoma xenografts was limited. To elucidate this discrepancy, pharmacokinetic analysis and MTT assay were conducted using the KRN8602‐sensitive MX‐1 and KRN8602‐insensitive R‐27. While no differences were observed in the area under the curve and the peak concentration of KRN8602 for each tumor, a difference in the sensitivity of the tumor strains was obvious in MTT assay. The efficacy of this agent seemed to depend on the sensitivity of each type of tumor cell rather than the concentration of agent in tumor tissues. If it were possible to select patients with sensitive tumor cells to this agent by the MTT assay, the phase II trial might be completed within a short period by reducing the number of studied patients.
Synergistic Effect of Cyclosporin A and Verapamil in Overcoming Vincristine Resistance of Multidrug‐resistant Cultured Human Leukemia Cells
Authors: Ishida Yasushi , Shimada Yasuhiro , Shimoyama Masanori
Published: August 2005

Reversal of vincristine (VCR) resistance by cyclosporin A (CyA) or the combination of CyA and verapamil (VER) was investigated by using four P‐glycoprotein (P‐gp)‐associated human multidrug‐resistant (MDR) cell lines (K562/ADM, KYO‐1, HEL and CMK). Drug sensitivity was expressed as 50% inhibitory concentration (IC50). The degree of reversal of resistance was expressed as x‐fold decrease by dividing the IC50 value without modifier(s) by that with modifier(s). CyA overcame P‐gp‐associated MDR significantly in all four MDR cell lines. Reversal of VCR resistance by CyA appeared to be dose‐dependent. In the case of low‐grade MDR cell lines (KYO‐1, HEL and CMK), CyA at the low concentration of 0.5 μg/ml was still effective. The degree of reversal of VCR resistance in this condition was greater (6.3‐ to 16‐fold decrease) in the low‐grade MDR cell lines than in a high‐grade MDR cell line (K562/ADM) (2.9‐fold decrease). At a high concentration (5 μg/ml) of CyA, however, it was greater (240‐fold decrease) in the high‐grade MDR cell lines than in the low‐grade MDR cell line (20‐ to 100‐fold decrease). This indicates that concentration of CyA required for overcoming drug resistance in MDR cells was dependent on the degree of drug resistance. CyA overcame VCR resistance more efficiently than VER. The combination of CyA and VER enhanced reversal of VCR resistance in a supra‐additive or at least an additive manner and overcame VCR resistance at low concentrations of both modifiers that are clinically achievable with safety.
Menogaril, an Anthracycline Compound with a Novel Mechanism of Action: Cellular Pharmacology
Authors: Wierzba Konstanty , Sugimoto Yoshikazu , Matsuo Ken‐ichi , Toko Toshiyuki , Takeda Setsuo , Yamada Yuji , Tsukagoshi Shigeru
Published: August 2005

Menogaril, an anthracycline compound possessing a significant antitumor activity after both po and iv administration, has been introduced into clinical trials. However, its mechanism of action has not been clarified yet. This study revealed that its cytotoxicity correlated very well with the inhibition of macromolecular synthesis, indicating the involvement of interaction with DNA. The spectrophotometric study showed a weaker binding of this compound to calf thymus DNA when compared to that of doxorubicin (adriamycin). Despite the lower binding affinity of menogaril to DNA, pronounced DNA cleavage was observed in an intact cell system, indicating that the character of the interaction with DNA is different from intercalation. In contrast to doxorubicin, menogaril is extensively localized in the cytoplasm. The cytoplasmic localization prompted us to study its effect on cytoskeleton proteins. It was found that menogaril inhibited the initial polymerization rate of tubulin, indicating a possible contribution of this process to the overall cytotoxicity of menogaril.
Neoplastic Alteration of a Membrane‐associated Sialidase of Rat Liver
Authors: Miyagi Taeko , Konno Kimio , Sagawa Junji , Tsuiki Shigeru
Published: August 2005

Rat liver participate fraction contains two types of membrane‐associated and gangliosides‐hydrolyzing sialidase, which have been shown to be identical to two membrane‐associated sialidases of rat brain (I and II) chromatographically, immunologically and in substrate specificity. Chromatography on AH‐Sepharose 4B of the membrane sialidases of rat primary hepatoma induced by 3′‐methyl‐4‐dimethylaminoazobenzene (MeDAB) further revealed that hepatocarcinogenesis induces a marked decrease in sialidase II but no decrease in sialidase I. Using antisera against sialidases I and II of rat brain, immunoprecipitation studies of the solubilized particulate fractions of rat liver and MeDAB‐hepatoma gave results similar to those obtained chromatographically. Using the same immunological technique, sialidase II but not sialidase I was found to be decreased in AH109 A hepatoma and in regenerating and fetal liver.
Production and Enhanced Anti‐tumor Activity of Tumor Necrosis Factor in Mice Treated with Cyclophosphamide
Authors: Noso Yoshihiro , Becker Jeanne , Riveau Gilles , Audibert Francoise , Chedid Louis
Published: August 2005

We demonstrated recently that the production of tumor necrosis factor (TNF) is induced in normal mice and in the immunosuppressed nude mouse model by the administration of muramyl dipeptide (MDP) derivatives followed by endotoxin (lipopolysaccharide). In the present study, the ability of this treatment to induce the production of TNF in mice receiving cyclophosphamide (CY) was examined. Two days following treatment with high‐dose CY (250 mg/kg), mice exhibited leukocytopenia and drastically reduced splenic weight. However, these animals remained capable of producing TNF, albeit at lower levels, when treated with MDP derivatives and lipopolysaccharide (LPS), particularly when the lipophilic analogue MDP‐dipalmitoyl glycerol (GDP) was utilized. TNF was also induced by the administration of MDP‐GDP and LPS to Meth A sarcoma‐bearing mice treated with this dose of CY. Furthermore, in all animals receiving this combination therapy, sarcoma necrosis and complete regression were obtained without any sign of tumor regrowth. A dose of 100 mg/kg CY was not effective for inhibiting tumor regrowth under the same experimental conditions. These results demonstrated that the anti‐tumor activity of endogenously induced TNF is potentiated by combined therapy with a high dose of CY.
In situ Localization of the Human Multidrug‐resistance Gene mRNA Using Thymine‐Thymine Dimerized Single‐stranded cDNA
Authors: Sugawara Isamu , Koji Takehiko , Ueda Kazumitsu , Pastan Ira , Gottesman Michael M. , Nakane Paul K. , Mori Shigeo
Published: August 2005

In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T‐T dimerized single‐stranded DNA (300–400 bases) so that they could penetrate well into the cytoplasm. The hybridized T‐T DNA was detected immunohisto‐chemically using rabbit anti‐T‐T DNA antibody (Ab) and peroxidase‐labeled goat anti‐rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug‐resistant cell lines K562/ADM, CEM/VLB, 2780ad, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene.
Treatment of Experimental Tumors with a Combination of a Pulsing Magnetic Field and an Antitumor Drug
Authors: Omote Yoshiharu , Hosokawa Masuo , Komatsumoto Masashi , Namieno Tsutomu , Nakajima Susumu , Kubo Yoshihiko , Kobayashi Hiroshi
Published: August 2005

We investigated the effects of a combination treatment involving a pulsing magnetic field (PMF) and an antitumor drug, mitomycin C (MMC), on two experimental tumors (fibrosarcoma KMT‐17 and hepatocellular carcinoma KDH‐8) in WKA rats, paying attention to possible potentiation of the therapeutic effect of the antitumor drug. PMF was obtained using a system generating a pulsed current in a solenoid coil. On day 7 after tumor implantation into the right thighs of rats, the region of the tumor was exposed to PMF (frequency 200 Hz, mean magnetic flux density 40 gauss) for 1 h immediately after iv injection of MMC at a dose of 1 mg/kg. Survival rates at day 90 of KMT‐17 implanted rats were 0% (0/10) in the non‐treated group, 34% (4/12) in the MMC‐treated group, 47% (6/13) in the PMF‐treated group and 77% (10/13) in the MMC/PMF combination group. The increase of life span (ILS) of KDH‐8‐implanted rats in the combination therapy group was significantly prolonged (%ILS 17.6%) compared with that in the MMC‐treated (%ILS 3.4%) and PMF‐treated (%ILS 7.6%) groups. By using cultured cells of the above two lines of tumor, the therapeutic effects of MMC and PMF were also determined from the cell colony‐forming efficiency in soft agar. The colony‐forming efficiencies of both cell lines were significantly suppressed in the combination therapy group compared with those in the other single therapy groups. The present results indicate that PMF exhibited a potentiation of the antitumor effect of mitomycin C.
Detection of Proteins that Recognize Platinum‐modified DNA Using Gel Mobility Shift Assay
Authors: Fujiwara Yasuhiro , Kasahara Kazuo , Sugimoto Yoshikazu , Nishio Kazuto , Ohmori Tohru , Saijo Nagahiro
Published: August 2005

Using a gel mobility shift assay, we have identified proteins, in the nuclear extracts of a human lung cancer cell line, that recognize cis‐diamminedichloroplatinum(II) (cis‐DDP, CDDP)‐modified DNA. A 158‐base‐pair double‐stranded DNA fragment, derived from pBR322 plasmid DNA, was modified by either CDDP, tetrachloro(dl‐trans)‐1,2‐diaminocyclohexaneplatinum(IV) (tetraplatin) or trans‐DDP (the stereoisomer of CDDP and clinically ineffective). These platinum drug‐modified probes were incubated with nuclear extracts and analyzed by gel mobility shift assay. Proteins in the extracts selectively recognized the clinically active platinum‐modified DNA fragment. No binding to the trans‐DDP‐modified DNA fragment was observed. These proteins may play a role in the cytotoxicity or in a DNA repair process.
Promoting Effect of the Peroxisome Proliferator, Clofibrate, but Not Di(2‐ethylhexyl)phthalate, on Urinary Bladder Carcinogenesis in F344 Rats Initiated by N‐Butyl‐N‐(4‐hydroxybutyl)nitrosamine
Authors: Hagiwara Akihiro , Tamano Seiko , Ogiso Tadashi , Asakawa Emiko , Fukushima Shoji
Published: August 2005

The modifying potential of clofibrate and di(2‐ethylhexyl)phthalate (DEHP) on second stage, N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐initiated urinary bladder carcinogenesis was investigated in male F344 rats, using a uracil‐accelerated transitional cell proliferation model. Six‐week‐old animals received 0.05% BBN in their drinking water for 4 weeks and then clofibrate (1.0, 0.5, and 0.25%) and DEHP (1.2, 0.6, and 0.3%) were given during experimental weeks 5–8 and weeks 12–20. Uracil was administered during weeks 9–11 at a dietary level of 3.0%. Control rats were treated with BBN and uracil without peroxisome proliferator. Surviving animals were killed at the end of week 20 of the experiment, when the densities of putative preneoplastic, papillary or nodular (PN) hyperplasias (numbers per 10 cm of basement membrane) were significantly increased in all clofibrate‐treated, but not the DEHP groups. The incidences of PN hyperplasia were similar in both treated animals and controls. In a second experiment, rats fed diets containing 1.0% clofibrate or 1.2% DEHP were assessed for levels of DNA synthesis in urinary bladder epithelium by 5‐bromo‐2′‐deoxyuridine immunohistochemistry. Numbers of labeled nuclei remained within normal levels, and no proliferative changes were evident. Thus, the present experiments indicated that while clofibrate, but not DEHP, exerts weak enhancing effects on BBN‐initiated urinary bladder carcinogenesis in rats this is not associated with increased levels of DNA synthesis in the affected epithelium.
Identification of Members of the Protein Phosphatase 1 Gene Family in the Rat and Enhanced Expression of Protein Phosphatase 1α Gene in Rat Hepatocellular Carcinomas
Authors: Sasaki Kazunori , Shima Hiroshi , Kitagawa Yoshinori , Irino Shozo , Sugimura Takashi , Nagao Minako
Published: August 2005

We isolated four kinds of cDNA clones of isotypes of catalytic subunits of protein phosphatase 1 (PP‐1) from rat liver and testis cDNA libraries. For the cloning, cDNA fragments of dis2ml and dis2m2, which encode mouse PP‐1 catalytic subunits, were used as probes. Two of the four isotypes were thought to be derived from the same gene and produced by alternative splicing. Based on the comparative study of their nucleotide and deduced amino acid sequences with those reported, these cDNA clones were named rat PP‐1α, PP‐1γ1, PP‐1γ2 and PP‐δ. The deduced amino acid sequences of these four cDNA clones showed about 90% identity. Their amino‐terminal regions were highly conserved, and their differences were mainly in the carboxy‐terminal regions. Furthermore, several amino acids located in the middle regions of the peptides were conserved in all the isotypes of the catalytic subunits of PP‐1, PP‐2A, PP‐2B and PP‐2C. These conserved regions are suggested to be the functional domains of the catalytic subunits of protein phosphatases. Rat hepatocellular carcinomas induced by a food mutagen, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline showed increased expression of PP‐1α, but no increased expression of PP‐1γ1, PP‐1γ2 or PP‐1δ. Involvement of PP‐1α in hepatocarcinogenesis or in hepatic cell proliferation was suspected.
Circulating Hematopoietic Progenitors in Patients with Primary Lung Cancer
Authors: Shimizu Eiji , Mukai Jun‐nosuke , Takaue Yoichi , Ogura Takeshi
Published: August 2005

The levels of circulating hematopoietic progenitors were measured in 28 patients with primary lung cancer. The average numbers of progenitors per milliliter of blood were 33 (range 0–360) for colony‐forming unit‐granulocyte macrophage (CFU‐GM), 23 (range 0–140) for burst‐forming unit‐erythrocyte (BFU‐E), and 4 (range 0–50) for colony‐forming unit‐mixed lineages (CPU‐mix). No significant influence of age, sex, histological type, or clinical stage of the tumor on the progenitor levels was detected. After cytoreductive chemotherapy of the patients by treatment with cisplatin plus etoposide, the cells showed 6‐ to 50‐fold rebound overshoots, but no rebound was observed after treatment with cisplatin alone, cisplatin plus mitomycin C or cisplatin plus vindesine plus mitomycin C, or in 4 of 5 patients treated with cyclophosphamide plus adriamycin plus vincristine. Peripheral blood hematopoietic progenitors should be useful as an alternative source of stem cells for lung cancer patients treated with marrow ablative chemotherapy.
Antitumor Effect of PSK at a Distant Site: Inductions of Interleukin‐8‐like Factor and Macrophage Chemotactic Factor in Murine Tumor
Authors: Ebina Takusaburo , Murata Kazuko
Published: August 2005

The antitumor effect of PSK, a Coriolus preparation, at a distant site was analyzed with the use of a double grafted tumor system in which male BALB/c mice received simultaneous intradermal inoculations of Meth‐A tumor in the right (106 cells) and the left (2 × 105 cells) flanks and were then injected with PSK in the right tumor on the third day thereafter. The antitumor effect of intratumoral administration of PSK in the right tumor on days 3, 4 and 5 was compared with the effect of surgical resection of the right tumor on day 5. Three out of 8 mice given PSK intratumorally became tumor‐free whereas no mouse tumor‐free in the left flank was found among the surgically resected mice. As regards sinecomitant immunity, tumor inoculation into the right flank followed by intratumoral administration of PSK on days 3 and 5 and surgical excision of the primary tumor on day 6 resulted in complete rejection of a tumor challenge in the left flank on day 21. The combination of presurgical intratumoral injections of PSK (more than 2 times) and postoperative oral administration of PSK appeared to be most effective in eradicating secondary tumors. Isolated TILs (tumor‐infiltrating lymphocytes), obtained from the right tumor (treated with PSK) and the left tumor on day 10 in the double grafted tumor system were cultured in RPMI1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor (NCF) and macrophage chemotactic factor (MCF) activities were detected in the culture media from PSK‐treated TILs that had been cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated TILs. NCF and MCF activities were also detected in the culture supernatant from PSK‐treated tumor tissue on day 6. PSK‐induced NCF in the murine tumor was neutralized by treatment with anti‐human IL‐8 IgG, and might be murine IL‐8‐like factor. Therefore, neutrophil and macrophage infiltrations of tumors following intratumoral injections of PSK are probably mediated by inductions of IL‐8‐like factor and MCF.
Metabolism and Toxicity of Electroporated 1‐β‐d‐Arabinofuranosylcytosine Triphosphate in a Human Leukemia Cell Line
Authors: Kubota Masaru , Yorifuji Tohru , Hashimoto Hisako , Shimizu Tsunehiro , Mikawa Haruki
Published: August 2005

The metabolism and toxicity of 1‐β‐d‐arabinofuranosylcytosine triphosphate (ara‐CTP) directly injected into cells by electroporation was studied in human leukemia cell lines. The intracellular accumulation of ara‐CTP (ara‐CTP‐Ep) was dependent on the cell type, extracellular ara‐CTP concentration and pulse voltage on electroporation. In a promyelocytic leukemia cell line, HL‐60, ara‐CTP‐Ep revealed a cytotoxic effect in a dose‐dependent manner, although electroporation alone did not have any significant toxicity. Furthermore, simultaneous injection of dCTP, or continuous exposure to deoxycytidine, but not to other deoxyribonucleosides, immediately after electroporation rescued the cells from the toxicity of ara‐CTP‐Ep. The degradation of ara‐CTP‐Ep consisted of an early rapid phase followed by a slower phase with a half life of 1.5 h. The addition of dipyridamole (10 μM), an inhibitor of nucleoside transport, retarded this degradation process. These data indicate that transfer of ara‐CTP by electroporation is a useful method for the study of ara‐CTP metabolism.
Action Site of the Gene Determining Susceptibility to Propylnitrosourea‐induced Thymic Lymphomas in F344 Rats
Authors: Shisa Hayase , Suzuki Michiyo
Published: August 2005

To clarify whether the determining effect of the thymic lymphoma susceptible‐1 (Tls‐1) gene is on putative N‐propyl‐N‐nitrosourea (PNU) target cells among T‐lineage cells or on other host factors, we investigated the PNU‐induced lymphomagenesis in transplantation chimeras between susceptible F344 and resistant LES strain rats. Administration of PNU to lethally irradiated (F344 X LES)F1 rats reconstituted with bone marrow cells from either F344 or LES parental rats invariably led to development of donor‐origin thymic lymphomas. On the other hand, thymic lymphomas were induced in thymectomized Fl rats grafted with neonatal LES thymtis, of which 4 out of 8 were of the donor origin. These observations indicate that the target cells of thymic lymphomagenesis of F344 and LES rats are equally susceptible to PNU provided they are in susceptible hosts and the LES thymus seems capable of supporting thymic lymphomagenesis, although this capability wanes with aging of the thymus. The effect of the Tls‐1 gene, therefore, is neither on PNU susceptibility of the target cells nor on the capability of the thymus to support lymphomagenesis, but on other host factors either in or out of the thymus.
Characterization of Heterogeneous Distribution of Tumor Blood Flow in the Rat
Authors: Hori Katsuyoshi , Suzuki March , Tanda Shigeru , Saito Sachiko
Published: August 2005

Angioarchitectures of ascites hepatoma AH109A and Sato lung carcinoma (SLC) were quantitatively compared by measuring the following morphometric parameters: vascular density, vascular length, distance hetween tissues and their nearest blood vessel, and total length of microvascular network per unit area. When the vascular networks in these two types of tumors were compared in the initial stage, the morphological parameters were almost identical. Correlations between tumor size and the number of starting vessels and between enlargement of the tumor and the ensuing increase in pressure of the starting vessel were also evaluated with a microcomputer and an apparatus for measuring micro–vascular pressure. The total length of tumor vascular network to which one starting vessel supplied blood increased exponentially as the tumor increased in size exponentially. There was a positive correlation between tumor size and the number of starting vessels. The range of the blood supply from one starting vessel was evidently limited. The pressure of the starting vessel increased with enlargement of the tumor size. As soon as the pressure of the starting vessel reached a plateau, however, there was a rapid increase in low–flow or no–flow areas in regions within the tumor. From the results obtained, we consider that low–flow or no–flow areas, resistant to delivery of anticancer drugs, inevitably appear with the progression of tumor growth.
Effects of Methylthiodeoxyadenosine and Its Analogs on in vitro Invasion of Rat Ascites Hepatoma Cells and Methylation of Their Phospholipids
Authors: Kido Jun‐ichi , Ashida Yoshiyuki , Shinkai Kiyoko , Akedo Hitoshi , Isoai Atsushi , Kumagai Hiromichi , Hideo Hideo
Published: August 2005

The relationship between tumor invasiveness in vitro and methyiation of plasma membrane phospholipids was investigated. For this purpose, two hepatoma cell lines, Cl‐30 and LC‐AH, were used which show specific penetration to below cultured monolayers of mesothelial cells from rat mesentery and endothelial cells from calf pulmonary artery, respectively. Methylthiodeoxyadenosine (MTA) and five of its analogs, difluoro‐MTA, deoxyadenosine, sinefungin, phenylthiodeoxyadenosine and fluorophenylthiodeoxyadenosine, inhibited the invasion of the tumor cells without affecting their proliferation. This inhibition was associated with reduction in the incorporation of radioactivity of [methyl‐3H]methionine into cellular phosphatidylethanolamine derivatives without changes in the labelings of RNA and DNA and carboxylmethylation of protein. These compounds also decreased the membrane fluidity of the tumor cells, measured by a steady‐state fluorescence polarization method. Three other MTA analogs (fluorodideoxyadenosine, fluoroazidodideoxyadenosine and flnoroamino‐dideoxyuridine) did not affect the invasiveness of the tumor cells or alter their phospholipid methyiation or membrane fluidity at concentrations that did not inhibit proliferation. These results suggest that the decrease in invasiveness of tumor cells by MTA and its analogs is due to alterations in the phospholipid composition and fluidity of the tumor cell membranes.
Adhesion Molecules on Murine Lymphokine‐activated Killer Cells Responsible for Target Cell Killing: A Role of CD2
Authors: Kato Kazunori , Tanabe Toshifumi , Yagita Hideo , Agatsuma Toshinori , Hojo Hiroshi , Hashimoto Yoshiyuki
Published: August 2005

Lymphokine‐activated killer (LAK) cells were induced from C57BL/6 mouse spleen cells and the effects of culture time on the expression of cell surface phenotypes and cytotoxic activity of LAK cells were determined. The expression of CD2 remarkably decreased after culture of LAK cells for 30 days, while LFA‐1, a principal adhesion molecule in LAK cells, and CD3 were not changed by the culture. LAK cells cultured for 90 days completely lost CD2. In accordance with the decrease of CD2, the cytotoxic activity of LAK cells declined but a certain leven was retained even after the complete loss of CD2. The established LAK cell clones were also strongly positive for the expression of LFA‐1 but negative for CD2. When the LAK cell clones were transfected with the CD2 cDNA, they started to express CD2 on their cell surface and to show greater binding ability and stronger cytotoxicity to target tumor cells. These results indicated that CD2 plays a role as an adhesion molecule responsible for target cell killing in murine LAK cells.
Identification and Characterization of Three DNA‐binding Proteins on the Promoter of the Human MDRl Gene in Drug‐sensitive and ‐resistant Cells
Authors: Ogura Mitsuo , Takatori Toshiro , Sugimoto Yoshikazu , Tsuruo Takashi
Published: August 2005

In multidrug‐resistant cell lines (K562/ADM), MDR1 was amplified and transcriptionally activated. But the mechanism of MDR1 expression is unknown in K562/ADM cells. A 131 bp DNA fragment upstream from the major cap site of MDR1 contained CAAT‐like and GC‐box‐like motifs and showed promoter activity in a CAT expression assay, resulting in a 3.5‐fold enhancement of MDR1 promoter activity in K562/ADM as compared with that in K562 cells. In the CAT assay using deletion mutants we found that sequences containing the CAAT‐like motif and the GC‐box‐like motif were required for MDR1 proximal promoter activity. To understand the details of MDR1 transcription in K562/ADM cells we analyzed the interaction between the proximal promoter and DNA‐binding protein(s). We have identified three DNA‐binding proteins on the MDR1 promoter from K562/ADM and parental K562 cells. The first binding protein to the MDR1 promoter was NF‐R1, which recognized the sequences containing the CAAT‐like motif and the GC‐box‐like motif as revealed by using a gel mobility shift assay. The second protein was NF‐R2, which bound to the sequence containing the CAAT‐like motif. The third protein, which bound to the sequence containing the GC‐box‐like motif was designated NF‐R3. NF‐R2 and NF‐R3 in the resistant cells formed different bands in the gel mobility shift assays as compared with those in the sensitive cells, respectively. The observed difference might be related to the transcriptional activation of MDR1 in K562/ADM cells.
Long‐lasting Accumulation of Vinblastine in Inostamycin‐treated Multidrug‐resistant KB Cells
Authors: Kawada Manabu , Umezawa Kazuo
Published: August 2005

Inostamycin, a novel polyether compound, reverses multidrug resistance in KB cells. The mechanism of its action was studied by use of radioactively labeled vinblastine. Inostamycin dose‐dependently increased the accumulation of [3H] vinblastine in multidrug‐resistant KB‐C4 cells at 0.5‐2 μg/ml, while it did not enhance accumulation in the drug‐sensitive KB‐3‐1 cells. At a concentration of 1 μ/ ml inostamycin inhibited active [3H] vinblastine efflux from KB‐C4 cells, but not from KB‐3‐1 cells, and inhibited [3H] vinblastine binding to KB‐C4 membranes with an IC5O of 0.94 μg/ml (1.3 μM). Furthermore, [3H] vinblastine accumulated by treatment with 1 /μg/ml of inostamycin was resistant to efflux from KB‐C4 cells, even after the removal of inostamycin.
Induction of Signet Ring Cell Carcinomas in X‐Irradiated Hypocatalasemic Mice (C3H/Ob8)
Authors: Watanabe Hiromitsu , Ogundigie Peter Osa , Takahashi Tadateru , Ishimoto Tatsuro , Ito Akihiro
Published: August 2005

The stomach region of hypocatalasemic mice of both sexes was X‐irradiated once with a dose of 20 Gy. Thirteen months after the irradiation, 3 out of 13 (20%) males and 2 out of 9 (15%) females were observed to have developed signet ring cell carcinomas in the glandular stomach. This finding was statistically significant (P < 0.01 in males and P < 0.05 in females) compared to a total absence of similar tumors in the non‐irradiated controls. Local invasion of malignant tumors into muscle and subserosal layers was observed, but no metastatic tumors were found in distant organs.
Transformation of CD8 + T‐Cells Producing a Strong Cytopathic Effect on CD4+ T‐Cells through Syncytium Formation by HTLV‐II
Authors: Miyamoto Kanji , Kamiya Takashi , Minowada Jun , Tomita Noriko , Koichi Koichi
Published: August 2005

Human T‐cell leukemia virus type II (HTLV‐II) is thought to play an important role in the development of CD8 + T‐cell malignancies resembling hairy cell leukemia. In this study, dramatic cytopathic effects characterized by syncytium formation in various CD4+ T‐cell lines were observed upon their cocultivation with HTLV‐II infected T‐cells. The HTLV‐II infected T‐cells, however, did not die as a result of syncytium formation. HTLV‐II also transformed CD4 + T‐cells and CD8 + T‐cells at various coculture ratios. Furthermore, sera from antiHTLV‐II antibody‐positive specific carriers inhibited syncytium formation in the CD4+ T‐cells. These results suggest that HTLV‐II infection may contribute to the pathogenesis of associated CD8 + T‐cell malignancies.
Presence of Human Papillomavirus Type 18 DNA in a Pharyngeal and a Laryngeal Carcinoma
Authors: Ogura Hajime , Watanabe Shuichi , Fukushima Kunihiro , Masuda Yu , Fujiwara Tazuko , Yoshiro Yoshiro
Published: August 2005

We examined the presence of human papillomavirus (HPV) genome in pharyngeal and laryngeal squamous cell carcinomas by employing the polymerase chain reaction. We detected HPV 16 DNA in one of 11 pharyngeal carcinomas and in 3 of 28 laryngeal carcinomas, and HPV 18 DNA in a pharyngeal and a laryngeal carcinoma. In one of the laryngeal carcinomas, DNA of both HPV types 16 and 18 were detected. To our knowledge, this is the first report of detection of HPV type 18 in head and neck carcinomas.
Regulation of Human hst Expression by an Enhancer Element Residing in the Third Exon
Authors: Sasaki Akio , Kubo Mitsumasa , Hasan Shahid , Yano Yoke , Mitsuaki Mitsuaki
Published: August 2005

The hst gene is exclusively expressed in undifferentiated embryonal carcinoma cell lines and at a limited stage of embryonal development. Two DNase I‐hypersensitive sites were mapped in the 3’region (approximately 3.5 and 4,5 kb downstream of the translations) initiation site) of the human hst gene, irrespective of the presence or absence of hst mRNA in the cells. A DNA fragment containing one of these DNase I‐hypersensitive sites (at around 3.5 kb relative to the translational initiation site) showed enhancer activity when tested by chloramphenicol acetyltransferase (CAT) assay. These results strongly suggest that an enhancer element(s) exists in the third exon of the hst and that the expression of the hst may be regulated by the presence or absence of a putative protein factor(s) which binds to the enhancer.
Human Granulocyte‐Macrophage Colony‐stimulating Factor Is a Growth Factor Active on Human Ovarian Cancer Cells
Authors: Cimoli Guido , Russo Patrizia , Billi Giovanna , Mariani Gabriella Lucia , Rovini Elisabetta , Marco Marco
Published: August 2005

Human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is a regulatory glycoprotein that stimulates the production of granulocytes and macrophages from committed hematopoietic progenitor cells both in vitro and in vivo. In this report, we show that recomhinant human GM‐CSF enhances colony formation by nonhematopoietic human ovarian cancer cell lines, IGROV‐1, A2774, ME‐180, Pa‐1 and A2780. GM‐CSF also enhanced the colony formation by cells obtained from fresh ascites of a patient with ovarian mucinous cystadenocarcinoma and a patient with serous papillary ovarian carcinoma. Our observations were made with GM‐CSF concentrations between 0.1 to 1 ng/ ml; these concentrations are equivalent to the dosages generally used for bone marrow recovery after chemotherapy.
Assessment of New Macroscopical Classification of Gastric Malignant Lymphoma
Authors: Ishido Tatsuya , Nishizawa Mamoru , Nishimata Yoshito , Nakamura Kyoichi
Published: August 2005

We macroscopically classified gastric malignant lymphoma into ST type (submucosal tumor type), non‐ST type (non‐submucosal tumor type) and Combined type (combination of ST and non‐ST type) and assessed this new classification by examining 60 surgically resected cases. Of these, 18 cases (21 lesions) were classified into ST type, 38 cases (54 lesions) into non‐ST type and 4 cases (4 lesions) into Combined type. All the large cell, immunoblastic type cases were of ST type and most of the diffuse, small cleaved cell type cases and all the plasmacytoma cases were of non‐ST type. Diffuse, large cell type and diffuse, mixed type cases did not seem to be biased toward any of these three macroscopical types. The non‐ST type cases frequently showed an inflltrative growth pattern and reactive lymphoid cell hyperplasia adjoining the lesions, while the ST type cases frequently showed an expansive growth pattern without reactive lymphoid cell hyperplasia. The Combined type cases showed both infiltrative and expansive growth patterns in the same lesion. The macroscopical classification defined in this study can he considered useful in determining a range for surgical resection of gastric malignant lymphoma.
Murine Tumor Cells Metastasizing Selectively in the Liver: Ability to Produce Hepatocyte‐activating Cytokines Interleukin‐1 and/or ‐6
Authors: Takeda Kazuyoshi , Fujii Noriyuki , Nitta Yasutaka , Sakihara Hiroshi , Nakayama Katsutoshi , Rikiishi Hidemi , Katsuo Katsuo
Published: August 2005

Increasing evidence suggests that an intimate correlation may exist between the production of a cytokine, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and the ability to metastasize spontaneously in the lungs in murine transplantable tumors. In the present study, we further examined the cytokine production by tumor cells with the ability to metastasize in the liver. Four out of 8 test tumors, which produced metastasis in the lungs but not in the liver, exhibited the ability to produce GM‐CSF activity in culture. Three other tumors produced metastasis in the liver but not in the lungs. These tumor cells exhibited no ability to produce GM‐CSF, but two of them expressed an interleukin‐6 (IL‐6) mRNA and also produced IL‐6 activity in the culture fluids. One of the two IL‐6‐producing tumors and the remaining liver metastatic tumor produced interleukin‐1 (IL‐1) as revealed by bioassay and neutralization test. In the tumor cells producing pulmonary metastasis, neither IL‐6 gene expression nor IL‐1 production could be detected. The last test tumor, which produced no metastasis either in the lungs or liver, produced neither GM‐CSF, IL‐1 nor IL‐6. Furthermore, injection of antisera reactive to recombinant murine IL‐6 caused a marked decrease of the number of liver metastases of an IL‐6‐producing tumor, but not lung metastases of a GM‐CSF‐producing tumor, which could be markedly inhibited by injection of anti‐recombinant murine GM‐CSF sera. These results suggest the possibility that there may be a correlation between the cytokines produced by tumor cells and their organ specificity in spontaneous metastasis, and also indicate that these tumor models may provide a useful tool for studies on the role of cytokines in tumor metastasis.