bluuebunny/crossref_metadata_2025 huggingface dataset:
Prognostic Value of the Doubling Time of Serum C‐reactive Protein and Alkaline Phosphatase Levels in Primary Bone and Soft Tissue Tumors
Authors: Yudoh Kazuo , Matsui Hisao , Kamanori Masahiko , Ohmori Kazuo , Yasuda Taketosi , Tsuji Haruo , Tatezaki Shin‐ichiro
Published: August 2005

We investigated the clinical relevance of doubling time (DT) of serum laboratory data obtained in routine clinical examination of patients with primary bone and soft tissue tumors, in comparison with major clinical and pathological parameters (age at presentation, sex, tumor size, location, clinical stage and histologic grade) by uni‐ and multivariate analyses. In 64 patients with primary bone and soft tissue tumors (primary bone tumors: 39, primary soft tissue tumors: 25) and 68 cancer patients, the pretreatment DT values of serum C‐reactive protein (CRP), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), calcium (Ca), phosphate (P) levels were measured, as well as the erythrocyte sedimentation rate (ESR: mm/60 min); these values were then compared with overall survival, local recurrence‐free survival and metastasis‐free survival. Only DT of CRP and ALP (CRP‐DT, ALP‐DT) were found to be correlated with disease outcome in patients with primary bone and soft tissue tumors. In cancer patients, only CRP‐DT showed a relation with clinical stage and histologic grade, but the ALP‐DT in patients with bone metastasis was significantly shorter than that in patients with metastases at other sites or in those with no metastasis. Among all tumor patients, those with bone metastasis showed the shortest ALP‐DT compared with those with lung, liver and brain metastasis. Univariate analysis showed that shorter CRP‐DT and ALP‐DT are associated with poor overall survival, and the development of local recurrence and metastasis. These findings suggest that pretreatment CRP‐ and ALP‐DT could be additional prognostic parameters for disease outcome in patients with primary malignant bone and soft tissue tumors. However, in multivariate analysis, only ALP‐DT, but not CRP‐DT, was an independent prognostic parameter for these disease outcomes.
Establishment and Characterization of Human Gastric Carcinoma Lines with High Metastatic Potential in the Liver: Changes in Integrin Expression Associated with the Ability to Metastasize in the Liver of Nude Mice
Authors: Yasoshima Takahiro , Denno Ryuichi , Kawaguchi Satoshi , Sato Noriyuki , Okada Yojiro , Ura Hideki , Kikuchi Kokichi , Hirata Koichi
Published: August 2005

There is a need to establish animal models which are suitable for investigation of human gastric cancer metastasis to the liver. To this end, a human gastric carcinoma line, AZ521 was injected into the spleens of nude mice. Cells from the few liver metastatic foci of injected AZ521 were expanded in vitro and subsequently injected into the spleens of nude mice. By repeating these procedures three times, we were able to obtain a cell line, designated as AZ‐M3c, with high metastatic potential in nude mice. Liver metastasis developed in 15 of 21 (71%) animals injected with AZ‐H3c, but in only 14% of those injected with parental AZ521. Further, AZ‐H3c caused faster tumor development than did AZ521. However, the primary AZ‐H3c tumors and liver metastatic AZ‐H3c tumors showed essentially the same histological appearance. We also analyzed the cell surface expression of adhesion molecules. The data showed that the expression of VLA‐1, VLA‐2, VLA‐3, VLA‐4, VLA‐5 was enhanced in AZ‐H3c. In contrast, the expression of VLA‐6, αvβ3, E‐cadherin, ICAM‐1 and LFA‐1 was reduced in this high‐metastatic line. These results suggest that β1 mtegrins play an important role in the liver metastasis of human gastric carcinoma cells. Our high‐metastatic line should be useful for studies aimed at the prevention of liver metastasis.
Immunocytochemistry of Apoptosis Induced by Bromodeoxyuridine in Human Leukemic HL‐60 Cells
Authors: Kondo Kazumasa , Makita Takashi
Published: August 2005

Various antitumor drugs which target tumor cells in the S‐phase of the cell cycle induce apoptosis in HL‐60 cells. The present study shows that apoptosis is inducible in HL‐60 cells by continuous exposure to bromodeoxyuridine (BrdU), an S‐phase specific reagent and a thymidine analogue. The localization of BrdU in the apoptotic cells was visualized with immunogold particles using an antl‐BrdU monoclonal antibody. After 3 days of treatment with 20 μM BrdU, cells ceased to grow and their viability was significantly decreased. Morphologically, nuclear segmentation and cytoplasmic maturation were observed in many viable cells, and the dead cells showed typical features of apoptosis. The nuclear DNA of apoptotic cells was visualized by the immunocytochemical detection of BrdU with post‐embedding immunogold staining. The condensed chromatin of the apoptotic cells was highly labeled with immunogold particles, while the nucleolus was sparsely labeled. The chromatin could be traced with BrdU‐immunocytochemistry even after disappearance of the nuclear envelope. These results indicated that apoptosis in HL‐60 cells was induced by incorporation of BrdU into DNA of the S‐phase cells. Using BrdU‐immunocytochemistry, the localization of nuclear DNA in apoptotic cells was visualized and the changes of nuclear structures were followed during the progression of apoptosis.
Induction of Aberrant Crypt Foci and Flat‐type Adenocarcinoma in the Colons of Dogs by N‐Ethyl‐N'‐nitro‐N‐nitrosoguanidine and Their Sequential Changes
Authors: Sugiyama Kenji , Oda Yasushi , Otori Kazuhiko , Kato Shigeharu , Hasebe Takahiro , Fujii Takahiro , Tajiri Hisao , Esumi Hiroyasu
Published: August 2005

Sequential endoscopic observation of dog colons was performed during colon carcinogenesis. Two beagle dogs were given suppositories containing N‐ethyl‐N'‐nitro‐N‐nitrosoguanidine (ENNG) every day for five months. In month 3, aberrant crypt foci (ACF), a putative preneoplastic lesion, were found in the colons of both dogs, but not in an untreated dog. The frequency of ACF increased until month 10, and then decreased. In month 9, very small lesions, less than 1 mm in diameter, which were similar to human early flat tumors, were first noticed. One of these lesions grew to about 7 mm in size without a change in its shape for 10 months. There were more than ten flat‐type tumors in the two dogs, but such lesions were not found in the untreated dog. By biopsy, two of the lesions were proved to be well‐differentiated adenocarcinomas histologically. Four polypoid lesions were found in one of the carcinogen‐treated dogs. Thus, flat‐type adenocarcinomas were induced in the dog colon by ENNG, and their development was followed by magnifying endoscopy.
Mapping of a Breast Cancer Tumor Suppressor Gene Locus to a 4‐cM Interval on Chromosome 18q21
Authors: Yokota Takashi , Matsumoto Satoshi , Yoshimoto Masataka , Kasumi Fujio , Akiyama Futoshi , Sakamoto Goi , Nakamura Yusuke , Emi Mitsuru
Published: August 2005

DPC4 and DCC, putative tumor suppressor genes implicated in the genesis of several types of human cancer, lie on the long arm of human chromosome IS. We examined 200 primary breast cancers for allelic losses on chromosome IS, using 15 microsatellite markers distributed along the long arm. Allelic loss was detected most frequently (29–30%) at loci mapped to 18q21. Deletion mapping of the 34 tumors showing partial or interstitial deletions identified a commonly deleted region within the 4‐cM interval flanked by D18S474 and D18S487 at 18q21.1‐q21.3. Although this interval included the DPC4 and DCC genes, we excluded DPC4 from candidacy when polymerase chain reaction‐single‐strand conformation polymorphism analysis of each exon failed to detect abnormalities in any of the 54 breast cancers that exhibited loss of heterozygosity involving 18q. Allelic loss on 18q was found more frequently in tumors of the solid tubular histological type (24 of 55, 44%) than in other types (24 of 113, 21%) (P=0.0049). The results suggest that a tumor suppressor gene located within the 4‐cM region at 18q21, either DCC or another gene not yet identified, may play a role in the development of some sporadic breast cancers, particularly those of the solid tubular type.
Lifestyle and Anti‐Helicobacter pylori Immunoglobulin G Antibody among Outpatients
Authors: Hamajima Nobuyuki , Inoue Manami , Tajima Kazuo , Tominaga Suketami , Matsuura Akira , Kobayashi Seibi , Ariyoshi Yutaka
Published: August 2005

Since eradication of Helicobacter pylori (H. pylori) is thought to be a preventive measure against stomach cancer, several studies have examined factors associated with the infection. This paper reports the association of the infection with lifestyle factors observed in a hospital‐based case‐control study. Cases were 140 anti‐H. pylori IgG antibody‐positive outpatients (75 males and 65 females). Controls were 52 antibody‐negative outpatients (22 males and 30 females). Both groups had undergone gastroscopy at Aichi Cancer Center Hospital between February 1995 and February 1997, and lifestyle data collected on the first visit were linked to calculate odds ratios. A strong association was observed with smoking among males; age‐adjusted odds ratio (OR)=7.85, 95% confidence interval (CI), 2.03–30.4. Rice breakfast (OR=3.74; 95%CI, 1.30–10.8) and soybean paste soup (every day vs. occasionally, OR=5.24; 95%CI, 1.80–15.2) were also associated with antibody positivity in males, but not in females. In females, pickled Chinese cabbage (≥l/week vs. ≥3/month, OR=2.82; 95%CI, 1.06–7.48) and lettuce (≥ I/week vs. ≤3/month, OR=2.90; 95%CI, 1.09 7.76) were significantly associated with positivity. Multivariate analysis gave similar estimates for the above factors. Although the association between smoking and H. pylori infection has not been detected in past studies of a general population, except one recent one, this study on outpatients suggested a possible association. Smoking may work as a cofactor disturbing incidental eradication of H. pylori by antibacterial agents administered for other reasons.
Inhibition of Development of N, N′‐Dimethylhydrazine‐induced Rat Colonic Aberrant Crypt Foci by Pre, Post and Simultaneous Treatments with 24R,25‐Dihydroxyvitamin D3
Authors: Salim Elsayed I. , Wanibuchi Hideki , Taniyama Tetsuhide , Yano Yoshihisa , Morimura Keiichirou , Yamamoto Shinji , Otani Shuzo , Nishizawa Yoshiki , Morii Hirotoshi , Fukushima Shoji
Published: August 2005

It has recently been reported that new vitamin D3 derivatives can exert inhibitory effects on colon carcinogenesis in rats. In the present study the chemopreventive potential of 24R,25‐dihydroxyvitamin D3 (24ff,25(OH)2vitainin D3) was assessed in a murine model of colon carcinogenesis. In experiment 1, male 6‐week‐old F344 rats were administered N2N‐dimethylhydrazme (DMH) 20 mg/kg s.c. once a week 4 times. The rats were fed 24R,25(OH)2vitaniin D3 at 10 ppm in the diet prior to (pre), together with (simultaneous) or after (post) DMH treatment. Modifying effects were assessed using aberrant crypt foci (ACF), putative preneoplastic lesions, as the end point markers in this model of colon carcinogenesis. After 8 weeks, pre and more markedly simultaneous administration of 24R,25‐(OH)2vitamin D3 was found to have reduced the total numbers of ACF and significantly inhibited the development of foci. After 16 weeks, numbers of foci with ≥4 crypts, which are more likely to progress to tumors, were significantly reduced. The most pronounced inhibition of ACF development was noted in rats fed the 24R,25(OH)2vitaniin D3 after DMH administration. The reduction was particularly marked in the proximal colon. Blood levels of calcium were not significantly increased over the control levels in groups administered DMH and the vitamin. Immunohistochemical staining showed numbers of proliferating cell nuclear antigen‐positive cells to.be lower in the colonic epithelia of rats fed the vitamin D3 metabolite than in the controls. In experiment 2, the effect of 24R,25‐(OH)2vitamin D3 on the alterations in c‐fos, c‐myc and c‐jun oncogene expression in response to DMH administration was examined by northern blot analysis. The early increase in expression of ornithine decarboxylase (ODC) activity was not altered by 24R,25(OH)2vitamin D3. The results suggest that 24R,25(OH)2vitamin D3 is a cancer chemopreventive agent which may suppresses DMH induction of lesions and their subsequent development via an antiproliferative action.
Decrease in Class Pi Glutathione Transferase mRNA Levels by Ultraviolet Irradiation of Cultured Rat Keratinocytes
Authors: Nakano Hajime , Kimura Junya , Kumano Takayuki , Hanada Katsumi , Satoh Kimihiko , Hashimoto Isao , Tsuchida Shigeki
Published: August 2005

The effect of ultraviolet (UV) B irradiation on pi class glutathione transferase (GST‐P) gene expression was examined in cultured rat keratinocytes. Immunoblotting demonstrated GST‐P to be the major GST form in the cells, and it was significantly decreased following irradiation. Northern blot analysis revealed that the mRNA decreased to 10–25% of the initial value 24 h after irradiation at a dose of 40 mJ/cm2. No remarkable changes were observed at earlier time points. Hydrogen peroxide treatment enhanced GST‐P mRNA expression, with a 70% increase at 250 μM concentration. Alterations in possible trans‐acting factors were examined to clarify the mechanism of repression by UV irradiation. c‐Jun mRNA was induced 3.5‐fold at 4 h after irradiation, but by 24 h fell to a lower level than that observed initially. c‐Fos mRNA was increased 10‐fold at 1 h but was completely suppressed at 12 and 24 h. Thus, the changes of c‐Jun and c‐Fos mRNA differed from that of GST‐P mRNA. The level of mRNA for silencer factor‐B was decreased to less than 10% at 12 h. UV irradiation of cells transfected with the chloramphenicol acetyltransferase (CAT) reporter gene containing enhancer (GPE I) or silencer regions of the GST‐P gene did not suppress CAT activity. Although basal expression of the GST‐P gene was mainly dependent on GPE I, altered expression of c‐jun, c‐fos and other genes coding for factors possibly trans‐acting on GPE I did not appear to be responsible for the decreased GST‐P mRNA levels.
A High Prevalence of Functional Inactivation by Methylation Modification of p16INK4A/CDKN2/MTS1 Gene in Primary Urothelial Cancers
Authors: Akao Toshiya , Kakehi Yoshiyuki , Itoh Noriyuki , Özdemir Enver , Shimizu Takashi , Tachibana Akira , Sasaki Masao S. , Yoshida Osamu
Published: August 2005

We analyzed the genetic and epigenetic alterations p16INK4A/CDKN2/MTSl gene (MTS1 gene) in 38 primary urothelial cancers. Genetic alterations of the MTS1 gene consisted of one base substitution mutation in exon 2(2.6%)and 6 homozygous deletions (16.2%). Hypermethylation of the 5’CpG island in exon 1 of theMTS1 genewas observed in 12 tumors (37.5%). Consequently, 19 of 38 tumors (50%) showed genetic alterations or epigenetic hypermethylation of the MTS1 gene. Retention of hypermethylated MTS1 gene(s) in 36% of the tumors showing loss of heterozygosity at the critical region indicates that the methylation modification could be an initial event followed by genomic rearrangements associated with total loss of MTS1 gene function. Immunohistochemical analysis of MTS1 expression revealed that all the tumors with genetic alterations of the MTS1 gene and 9 of 12 highly methylated tumors displayed an absence of MTS1 nuclear antigen. Genetic and epigenetic changes of theMTS1 gene were not correlated with the grade and stage of tumors, indicating that these alterations are early events in nrothelial carcinogenesis, in which functional inactivation by hypermethylation is a predominant mechanism.
Combination Therapy with Antibody and Interleukin–2 Gene Transfer against Multidrug–resistant Cancer Cells
Authors: Shinohara Tsutomu , Sugimoto Yoshikazu , Sato Shigeo , Sone Saburo , Tsuruo Takashi
Published: August 2005

In the present study, we examined the effect of intcrlcukin–2 (IL–2) gene transfer into multidrug resistance (MDR) cancer cells on the therapeutic efficacy of MRK16. Human MDR ovarian cancer cells, AD10, were transduced with a bicistronic IL–2 retrovirus, Ha–IL2–IRES–Neo. The G418resistant population, IL2–AD10, secreted IL–2 into the culture supernatant and did not form a tumor mass in nude mice. The IL2–AD10 cells were more susceptible to the cytotoxicity of murine spleen cells than AD10 cells in vitro. For examination of the effect of IL–2 gene transfer on the antitumor activity of MRK16 against P–glycoprotein–positlve tumors, IL2–AD10 cells were cotransplanted s.c. with AD10 cells into nude mice in a ratio of 1: 3, and the mice were treated with MRK16 on days 2 and 7. MRK16 markedly inhibited the growth of AD10 cells mixed with IL2 AD10 cells under conditions (0.3–1 jag/body) where it showed only marginal effects on the growth of AD10 tumors. These findings suggest that IL–2 gene transfer potentiates the antitumor activity of MRK16 against MDR tumors.
Overexpression of Integrin–associated Protein (CD47) in Rat Kidney Treated with a Renal Carcinogen, Ferric Nitrilotriacetate
Authors: Nishiyama Yasuyuki , Tanaka Tomoyuki , Naitoh Hiroshi , Mori Chisato , Fukumoto Manabu , Hiai Hiroshi , Toyokuni Shinya
Published: August 2005

An iron chelate, ferric nitrilotriacetate (Fe–NTA), induces renal proximal tubular necrosis, a consequence of free radical–associated damage, that ultimately leads to a polycystic change of the renal cortex and a high incidence of renal cell carcinoma (RCC) in rodents. The differential display technique was used to search for inducible genes in the kidney of male Wistar rats treated with Fe–NTA and in the induced RCCs. Six fragments were selected that showed specific quantitative changes in mRNA. Two of them exhibited similar patterns in northern blots as well. One fragment showed a high limnology (89%) to murine integrin–associated protein (IAP; CD47). We thus cloned rat IAP cDNA including the entire coding region for use in further analysis. Rat IAP cDNA showed a 21–amino–acid deletion that was also observed in human, but not in mouse. Northern blots revealed that IAP was consistently overexpressed in non–tumorous parts of the kidney (2.4‐fold increase, n=9, P< 0.0001) as compared with matched controls 1to 2 years after Fe–NTA treatment. IAP Overexpression of more than 2.9–fold was found in 25% (2/8) of RCCs studied, and was limited to cases of a high histological grade and lung metastasis. Unexpectedly, IAP expression was higher in the non–tumorous part of the kidney after Fe–NTA treatment (2.8–fold) than in RCC (1.5–fold) in each case (n=4, P<0.05). Abundant expression of IAP mRNA in the renal tubular epithelium after Fe‐ ‐NTA treatment and RCC cells was observed by in situ hybridization. The results suggest that IAP Overexpression may he associated with Fe–NTA–induced renal cortical tubular damage and regeneration that lead to a polycystic state, and with tumor progression and metastasis of the induced RCCs.
Gene Mutation Analysis and Quantitation of DNA Topoisomerase I in Previously Untreated Non‐small Cell Lung Carcinomas
Authors: Takatani Hiroshi , Oka Mikio , Fukuda Minoru , Narasaki Fumihiko , Nakano Reiji , Ikeda Koki , Terashi Kenji , Kinoshita Akitoshi , Soda Hiroshi , Kanda Tetsuro , Schneider Erasmus , Kohno Shigeru
Published: August 2005

To elucidate whether gene alterations of topoisomerase I (topo I) exist in untreated non‐small cell lung carcinomas (NSCLC), polymerase chain reaction‐single strand conformation polymorphism analysis was performed in forty‐four NSCLC tissue samples. Gene alterations of topo I were sought in three regions, near codons 361 and 363, 533, and 722 and 729, where point mutations have been found in resistant tumor cell lines selected by chronic camptothecin exposure. In addition, nuclear topo I contents were determined by immunoblotting. No mobility shifts were observed compared to the pattern observed in a normal control at any of the three regions in any sample, whereas topo I levels showed an approximately 12‐fold variation. The variation is remarkably large compared to those seen in previous in vitro and in vivo studies. The results suggest that mutations of topo I may not contribute to intrinsic resistance of NSCLC to camptothecins, but low topo I levels may account, at least in part, for the resistance.
Human Papillomavirus Infection and Risk Determinants for Squamous Intraepithelial Lesion and Cervical Cancer in Japan
Authors: Sasagawa Toshiyuki , Dong Yu‐zhen , Saijoh Kiyofumi , Satake Shin‐ichirou , Tateno Masaya , Inoue Masaki
Published: August 2005

A case control design was used to investigate human papillomavirus (HPV) prevalence and risk factors associated with development of cervical squamous intraepithelial lesion (SIL) and cervical cancer (CC) in Japan, One hundred and twenty‐three women with histologically confirmed SIL or CC were compared to a control group of 778 cytologically normal women. With the use of a polymerase chain reaction (FCR)‐hased method for detection of low‐risk (types 6 and 11) and high‐risk (types 16, 18, 31, 33, 35, 52 and 58) HPVs, a high prevalence of HPV infection was observed in smokers among the controls. Logistic regression analysis demonstrated that high‐risk HPV infection was the most significant risk determinant for LSIL (OR=9.4, 95% CI=4.5–19), HSIL (OR=77, 95% CI=28–217) and CC (OR=97, 95% CI=35–269). It also showed that unmarried women, women married for 5 to 19 years and smokers represented high risk groups for SIL, while smokers and women with a history of many pregnancies/parities had increased risk for CC. Smoking was the only HPV infection‐independent factor for CC, suggesting that smoking may have a carcinogenic effect on the cervix. Since neither history of other cancer nor family cancer history was associated with SIL or CC, genetic factors appear to play little role in cervical carcinogenesis. The risk for cervical neoplasia due to HPV infection increased after marriage in Japan, suggesting a role for husbands as carriers of HPV transmission. Protection from high‐risk HPV infection may be of greatest importance for prevention of cervical cancer.
Auraptene, a Citrus Coumarin, Inhibits 12‐0‐Tetradecanoylphorbol‐13‐acetate‐induced Tumor Promotion in ICR Mouse Skin, Possibly through Suppression of Superoxide Generation in Leukocytes
Authors: Murakami Akira , Kuki Wataru , Takahashi Yasuo , Yonei Hiroshi , Nakamura Yoshimasa , Ohto Yoshimi , Ohigashi Hajime , Koshimizu Koichi
Published: August 2005

Coumarin‐related compounds, auraptene and umbelliferone, have been isolated from the cold‐pressed oil of natsumikan (Citrus natsudaidai HAYATA), and tested as inhibitors of tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced Epstein‐Barr virus activation in Raji cells. The 50% inhibitory concentration (IC50) of auraptene (18μM)was almost equal to that of genistein. Umbelliferone, which lacks a geranyloxyl group present in auraptene, was less active (IC50=450 μM). In a two‐stage carcinogenesis experiment with 7, 12‐dimethylbenz[α]anthracene (topical application at 0.19 μmol) and TPA (topical application at 1.6 nmol) in ICR mouse skin, topical application of auraptene (at 160 nmol) significantly reduced tumor incidence and the numbers of tumors per mouse by 27% (P < 0.01) and 23% (P < 0.05), respectively. Auraptene at a concentration of 50 μM markedly suppressed superoxide (O2−) generation induced by 100 nM TPA in differentiated human promyelocytic HL‐60 cells. Having no O2− ‐scavenging potential, auraptene may inhibit the multicomponent NADPH oxidase system. Inhibition of intracellular hydroperoxide formation in differentiated HL‐60 cells by auraptene was also confirmed by flow‐cytometric analysis using 2′,7′‐dichlorofluorescein diacetate as a fluorescence probe. Quantitative analyses using high‐performance liquid chromatography showed the occurrence of auraptene not only in both the peels and sarcocarps of natsumikan, but also in those of hassaku orange (C. hassaku) and grapefruit (C. paradisi,) and even in their bottled fresh juice form. These results indicate that auraptene is a chemopreventer of skin tumorigenesis, and implies that suppression of leukocyte activation might be the mechanism through which it inhibits tumor promotion.
A Novel Somatic Mutation in the RET Proto‐oncogene in Familial Medullary Thyroid Carcinoma with a Germline Codon 768 Mutation
Authors: Miyauchi Akira , Egawa Shin‐ichi , Futami Hitoyasu , Kuma Kanji , Obara Takao , Yamaguchi Ken
Published: August 2005

In individuals who carry gcrmline mutations in tumor suppressor genes predisposing them to inherited cancer syndromes, occurrence of somatic mutations in the same genes contributes to tumorigenesis. Germline mutations in the RET proto‐oncogene predispose individuals to multiple endocrine neoplasia (MEN) type 2 syndromes. Since these mutations are oncogenic by themselves, somatic mutations in the same gene had been thought unnecessary. Recently, a somatic mutation at codon 918 of RET was reported in medullary thyroid carcinoma (MTC) and C‐cell hyperplasia in patients with MEN 2A or familial MTC (FMTC), suggesting its possible contribution to tumorigenesis. We describe here a novel somatic mutation at codon 919 in a patient with FMTC carrying a gcrmline mutation at codon 768 that may also be related to tumor progression.
Chemopreventive Efficacy of Piroxicam Administered Alone or in Combination with Lycopene and β‐Carotene on the Development of Rat Urinary Bladder Carcinoma after A N‐Butyl‐N‐(4‐hydroxybutyl)nitrosamine Treatment
Authors: Okajima Eijiro , Ozono Seiichiro , Endo Takehiro , Majima Toshimitsu , Tsutsumi Masahiro , Fukuda Tomokazu , Akai Hiroyuki , Denda Ayumi , Hirao Yoshihiko , Okajima Eigoro , Nishino Hoyoku , Nir Zohar , Konishi Yoichi
Published: August 2005

The effects of the non‐steroidal anti‐inflammatory drug (NSAID) piroxicam and the carotenoids lycopene and β‐carotcnc, alone or in combination, on the development of rat superficial urinary bladder carcinomas induced by N‐butyl‐N‐(4‐hydroxybutyI)nitrosamine (BBN) were studied. Male Fischer 344 rats, 6 weeks old, were given 0.05% BBN in the drinking water for 8 weeks followed by administration of piroxicam (0.0075% in the diet), lycopene (0.0025% in the drinking water) and/or β‐carotene (0.0025% in the drinking water) for 12 weeks, then killed for histological analysis of urinary bladder lesions. Cell proliferation potential was analyzed by immunohistochemical staining of the proliferative cell nuclear antigen (PCNA). Piroxicam alone, piroxicam+lycopene, and piroxicam +lycopene+β‐carotene all significantly decreased the incidences and numbers of transitional cell carcinomas (TCCs), but the combination of piroxicam with carotenoids did not result in a clear improvement in the preventive potential of piroxicam. Piroxicam +β‐carotene also caused a significant reduction and lycopene alone a slight but not significant reduction in the number of TCCs. In contrast, β‐carotene alone and lycopene +β‐carotene were without inhibitory influence on any of the lesion categories examined, and the latter significantly increased the proportion of high‐grade TCCs. Nevertheless, all of the chemopreventive agents, either alone or in combination, significantly decreased the TCC PCNA index, the effect extending to the surrounding epithelium in the piroxicam 4‐lycopene and piroxicam+lycopene+β‐carotene groups. These results indicate that the NSAID piroxicam may he a more effective chemopreventive agent than lycopene and β‐carotene for superficial urinary bladder carcinogenesis.
Microsatellite Instability in Lung Cancer Patients 40 Years of Age or Younger
Authors: Sekine Ikuo , Yokose Tomoyuki , Ogura Tsutomu , Suzuki Kenji , Nagai Kanji , Kodama Tetsuro , Mukai Kiyoshi , Nishiwaki Yutaka , Esum Hiroyasu
Published: August 2005

Lung cancer in the young, which has the characteristics of a higher incidence of adenocarcinoma, lower male‐to‐female ratio of the patients, and less frequent smoking history in the patients, may possibly be associated with genetic predisposition to cancers. We studied six microsatellite loci (D2S123, D3S659, D3S966, D5S346, WT1, and TP53) in 18 surgically treated lung cancer patients aged 25–40 years and nine control patients aged 62–74 to determine the presence of microsatellite instability (MSI) and to correlate its occurrence with clinicopathological characteristics. Of the 18 patients, 11 were female and seven were non‐smokers. There were 15 adenocarcinomas and three squamous cell carcinomas, 15 (83%) of which had vascular invasion. MSI was positive in seven (39%) of 18 young patients and one (11%) of nine control patients. Moreover, MSIs in a half or more of six loci examined were demonstrated in five (28%) young patients, whereas no control patients showed such a high frequency of MSI. We observed no significant differences in clinical or pathologic parameters between cases with and without MSI. This result suggests that genetic factors play an important role in the development of lung cancer in young adults.
Intracellular Hyperthermia for Cancer Using Magnetite Cationic Liposomes: Ex vivo Study
Authors: Yanase Mitsugu , Shinkai Masashige , Honda Hiroyuki , Wakabayashi Toshihiko , Yoshida Jun , Kobayashi Takeshi
Published: August 2005

Heating properties of magnetite cationic liposomes (MCL) were investigated in ex vivo experiments using implanted cell pellets. The cell pellets, which consisted of rat glioma T9 cells into which MCL had been incorporated in a petri dish, were implanted subcntaneously in the left femoral region of female F344 rats. The rats were placed in a magnetic field generating coil and irradiated with an alternating magnetic field (384 Oe, 118 kHz) for 60 min. The cell pellets were heated to over 43°C by MCL in the magnetic field, hut other body parts of the rats were not heated. After 3 cycles of magnetic heating, all glioma cells were killed and no tumor take was observed.
Retroviral Introduction of the p16 Gene into Murine Cell Lines to Elicit Marked Antiproliferative Effects
Authors: Ikeda Hiroshi , Yoshida Jun , Yamada Hitoshi , Yoshimatsu Tadanori , Ikenaka Kazuhiro
Published: August 2005

The pl6 gene is a candidate tumor suppressor, because mutation of the gene has been reported in many transformed cell lines and some primary tumor tissues. We have examined this possibility in murine cell lines (NIH3T3 and RSV‐M) which lack pl6 gene expression. Full‐length human p16 cDNA was obtained from a HeLa cell line using polymerase chain reaction amplification. We constructed two separate retrovirus vectors carrying this pld cDNA. First, we transduced the p16 cDNA into the murine cell lines using a retrovirus vector harboring the neomycin‐resistance gene. The p16 genetransduced cells formed no colonies after selection with G418, in contrast to the vector‐transduced cells. Next, we used another retrovirus vector that expresses both the p16 cDNA and the Lac Z gene, which enabled us to distinguish affected cells from unaffected ones. Proliferation of the p16 gene‐transduced cells was markedly inhibited and morphological change in the cells was also observed. Thus, we concluded that the p16 gene has an antiproliferative effect on the cell cycle and that the loss of its function may play a major role in dysregulated proliferation of the cells.
The Induction of Cytotoxic T Lymphocytes against HLA‐A Locus‐matched Lung Adenocarcinoma in Patients with Non‐small Cell Lung Cancer
Authors: Yoshino Ichiro , Takenoyama Mitsuhiro , Fujie Hiroshi , Hanagiri Takeshi , Yoshimatsu Takashi , Imabayashi Satoshi , Eifuku Ryouzo , Ogami Akira , Yano Koichi , Osaki Toshihiro , Nakanishi Ryoichi , Ichiyoshi Yuji , Nomoto Kikuo , Yasumoto Kosei
Published: August 2005

To induce cytotoxic T lymphocytes (CTL) against non‐small cell lung cancer (NSCLC) efficiently, the induction of CTL was attempted using HLA‐A locus‐shared allogeneic NSCLC cells. T cells derived from either tumor tissue specimens or the regional lymph nodes of patients with NSCLC were stimulated twice or three times with an HLA‐A2/A24‐positive NSCLC cell line (PC‐9), and thereafter the cytotoxic activity was examined by 51Cr‐release assay. In patients with HLA‐A24/ adenocarcinoma, anti‐PC‐9 cytotoxicity was induced in all 6 patients tested. Anti‐PC‐9 cytotoxicity was induced in 2 out of 5 patients with HLA‐A2 (A24−)/adenocarcinoma, in 2 out of 4 patients with HLA‐A24/squamous cell carcinoma, and 1 of 2 patients with HLA‐A2/squamous cell carcinoma. The cytotoxic activity was observed to kill PC‐9 selectively, not other NSCLC lines, and the activity was substantially blocked by anti‐MHC class I antibody, but not by anti‐MHC class II antibody. The PC‐9‐specific CTL produced γ‐interferon in response to autologous tumor cells. These results indicated that the anti‐PC‐9 cytotoxicity was mediated by cytotoxic T lymphocytes that may recognize the T cell epitope(s) shared and presented by HLA‐A2 and/or HLA‐A24‐positive NSCLC.
Augmentation in Chemosensitivity of Intratumor Quiescent Cells by Combined Treatment with Nicotinamide and Mild Hyperthermia
Authors: Masunaga Shin‐ichiro , Ono Koji , Akaboshi Mitsuhiko , Kawai Ken‐ichi , Suzuki Minoru , Kinashi Yuko , Takagaki Masao
Published: August 2005

C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5‐bromo‐2′‐deoxyuridine (BrdU) for 5 days using implanted mini‐osmotic pumps to label all proliferating (P) cells. Nicotinamide was administered intraperitoneally before cisplatin injection and/or tumors were locally heated at 40°C for 60 min immediately after cisplatin injection. The tumors were then excised, minced and trypsinized. The tumor cell suspensions were incubated with cytochalasin‐B (a cytokinesis‐blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from tumors that had not been pretreated with BrdU labeling. The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). In both tumor systems, the MN frequency in Q cells was lower than that in the total cell population. Nicotinamide treatment elevated the MN frequency in total SCC VII cells. Mild heating raised the MN frequency more markedly in Q cells than in total cells. The combination of nicotinamide and mild heat treatment increased the MN frequency more markedly than either treatment alone. In total SCC VII cells, nicotinamide increased 195mPt‐cisplatin uptake. Mild heating elevated 195mPt‐cisplatin uptake in total EMT6/KU cells. Cisplatin‐sensitivity of Q cells was lower than that of total cells in both tumor systems. Nicotinamide sensitized tumor cells including a large acutely hypoxic fraction, such as those of SCC VII tumors, through inhibition of the fluctuations in tumor blood flow. Tumor cells including a large chronically hypoxic fraction such as Q cells were thought to be sensitized by mild heating through an increase in tumor blood flow.
Relationship between the Nature of Mucus and Crypt Multiplicity in Aberrant Crypt Foci in the Rat Colon
Authors: Uchida Kazumi , Kado Shouichi , Onoue Masaharu , Tohyama Kiyoshi
Published: August 2005

Aberrant crypt foci (ACF)induced in the distal colon of F344 male rats, 4, 8, 12 and 35 weeks after the first administration of 1, 2‐dimethylhydrazine‐2HCl (DMH) were examined to determine whether a correlation exists between the nature of goblet cell mucin and the number of crypts (crypt multiplicity) comprising the ACF. According to the ACF score calculated from the results of the qualitative observation of sulfomucins (SuMs) and sialomucins (SiMs), the ACF in the 4th week showed a weak correlation between the nature of the mucus and crypt multiplicity, and the ACF of each class showed similar mucous profiles. From the 8th week, a significant difference (P<0.01) was recognized between the ACF consisting of 3 crypts or less and those consisting of 4 crypts or more. The proportion of crypts with SiM predominance showed a decrease in the 8th.week in the ACF consisting of 1 crypt and in the 12th week in the ACF consisting of 2 or 3 crypts, implying a recovery tendency. The ACF consisting of more than 4 crypts showed little change over time, retaining the tendency of SiM predominance. Ulex europaeus agglutinin‐I (UEA‐I) lectin‐positive crypts appeared in the ACF. This finding was significantly more prominent (P< 0.001) in the ACF with SiM predominance than in the ACF with SuM predominance at each experimental period, and in the 12th week after the first administration of DMH, the incidence of ACF with UEA‐I‐reactive mucin was decreased in the ACF groups consisting of 3 crypts or less, compared with the ACF groups consisting of 4 or more crypts.These results suggest that the biological quality of mucus in ACF consisting of 4 or more crypts is different from that in ACF consisting of 3 crypts or less. This difference should be considered when ACF are used as an intermediate biomarker of colon cancer.
Chemoprevention of Azoxymethane‐induced Rat Colon Carcinogenesis by a Xanthine Oxidase Inhibitor, 1′‐Acetoxychavicol Acetate
Authors: Tanaka Takuji , Kawabata Kunihiro , Kakumoto Mikio , Makita Hiroki , Matsunaga Kengo , Mori Hideki , Satoh Kumiko , Kara Akira , Murakami Akira , Koshimizu Koichi , Ohigashi Hajime
Published: August 2005

In our studies to find natural compounds with chemopreventive efficacy in foods, using azoxymethane (AOM)‐induced colonic aberrant crypt foci and colonic mucosal cell proliferation as biomarkers, a x an thine oxidase inhibitor, 1′‐acetoxychavicol acetate (ACA), present in the edible plant Languas galanga from Thailand was found to be effective. This study was conducted to test the ability of ACA to inhibit AOM‐induced colon tumorigenesis when it was fed to rats during the initiation or post‐initiation phase. Male F344 rats were given three weekly s.c. injections of AOM (15mg/kg body weight) to induce colonic neoplasms. They were fed diet containing 100 or 500 ppm ACA for 4 weeks, starting one week before the first dosing of AOM (the initiation feeding). The other groups were fed the ACA diet for 34 weeks, starting one week after the last AOM injection (the post‐initiation feeding). At the termination of the study (week 38), AOM had induced 71% incidence of colonic adenocarcinoma (12/17 rats). The initiation feeding with ACA caused significant reduction in the incidence of colon carcinoma (54% inhibition by 100 ppm ACA feeding and 77% inhibition by 500 ppm ACA feeding, P=0.03 and P=0.001, respectively). The post‐initiation feeding with ACA also suppressed the incidence of colonic carcinoma (45% inhibition by 100 ppm ACA feeding and 93% inhibition by 500 ppm ACA feeding, P=0.06 and P=0.00003, respectively). Such inhibition was dose‐dependent and was associated with suppression of proliferation biomarkers, such as ornithine decarboxylase activity in the colonic mucosa, and blood and colonic mucosal polyamine contents. ACA also elevated the activities of phase II enzymes, glutathione S‐transferase (GST) and quinone reductase (QR), in the liver and colon. These results indicate that ACA could inhibit the development of AOM‐induced colon tumorigenesis through its suppression of cell proliferation in the colonic mucosa and its induction of GST and QR. The results confirm our previous finding that ACA feeding effectively suppressed the development of colonic aberrant crypt foci. These findings suggest possible chemopreventive ability of ACA against colon tumorigenesis.
Isolation and Characterization of Invasive and Noninvasive Variants of a Rat Bladder Tumor Cell Line
Authors: Nishi Nozomu , Inui Masashi , Kishi Yukiko , Miyanaka Hiroshi , Wada Fumio
Published: August 2005

We isolated, in vitro, spontaneous variants of the rat bladder tumor NBT‐II cell line with a distinctive morphology. Of five sublines obtained, three (NBT‐L1, L2a and L2b) exhibited an elongated shape and moderate to high invasive activity in vitro. The other two sublines (NBT‐T1 and T2) formed tight colonies and exhibited very low or negligible invasive activity. The contents of mRNAs coding for E‐cadherin and cadherin‐associated molecules (α‐catenin and β‐cutenin) were not correlated with the invasive activity of the cells. However, the expression level of the E‐cadherin protein, but not those of catenins, was lower in invasive cells (NBT‐L1, L2a and L2b) than in noninvasive cells (NBT‐T1 and T2). Analysis of mRNAs coding for several growth factors and their receptors showed that the transforming growth factor α mRNA content in invasive cells was higher than that in noninvasive cells, and that the content of epidermal growth factor receptor mRNA was low in NBT‐T2. Although NBT‐II is known to acquire a fibroblastic appearance and cell motility in response to several growth factors, the conditioned media of the invasive sublines hardly affected the morphology or motility of noninvasive cells. These results indicate that the decreased E‐cadherin expression is closely associated with the transition from the noninvasive to the invasive phenotype of the bladder tumor cells, and that a post‐transcriptional process is important in the control of E‐cadherin expression in the cells. These sublines may be useful as models for studies on the progression of bladder tumors.
Expressions of Cell Cycle Regulators in Human Colorectal Cancer Cell Lines
Authors: Tominaga Osamu , Nita Marcelo Eidi , Nagawa Hirokazu , Fujii Shin , Tsuruo Takashi , Muto Tetsuichiro
Published: August 2005

To study the altered mechanisms of cell cycle regulation in colorectal cancer, the expressions of cyclins, cyclin‐dependent kinases (CDKs), CDK inhibitors, p53 and retinoblastoma (Rb) protein were analyzed by western blotting in a series of human colorectal cancer cell lines. The colorectal cancer cell lines exhibited various expression patterns of cell cycle regulators, which may reflect differences in the biological characteristics of cancer cells and in the genetic backgrounds of carcinogenesis. A correlation was found between p53 gene alteration and p21 expression, suggesting that p53 gene mutation usually suppresses p21 expression, though p21 expression could be induced via both ap53‐dependent and a p53 ‐independent pathway in colorectal cancer. None of the cell lines studied expressed p16 protein, suggesting that inactivation of p16 may be a common alteration in colorectal cancer. Moreover, all the D‐type cyclins, especially D2 and D3, were expressed at a high level in most of the cell lines. Loss of p16 expression and increased expression of D‐type cyclins promote CDK‐mediated Rb phosphorylation. All of the colorectal cancer cell lines studied herein expressed Rb protein, but the growth‐suppressive properties of Rb may be inactivated by the loss of p16 expression and increased expressions of D‐type cyclins. In view of the pivotal role of Rb in cell cycle regulation, loss of p16 expression and overexpression of D‐type cyclins may be critical alterations in colorectal cancer.
Vaccinations with T‐helper type 1 directing adjuvants have different suppressive effects on the development of allergen‐induced T‐helper type 2 responses
Authors: Trujillo‐Vargas C. M. , Mayer K. D. , Bickert T. , Palmetshofer A. , Grunewald S. , Ramirez‐Pineda J. R. , Polte T. , Hansen G. , Wohlleben G. , Erb K. J.
Published: August 2005

Background
Allergen‐induced T‐helper type 2 (Th2) responses can be inhibited with Th1 directing vaccines. However, studies comparing the efficacy of the different adjuvants have not been performed in detail.

Objective
For this reason we compare the effects of live Bacillus–Calmette–Guerin(BCG), heat‐killed (hk)‐BCG, CpG‐ODN (oligodeoxynucleotide) or PPD on the development of allergen‐induced Th2 responses in mice.

Methods
Ovalbumin (OVA)‐specific allergic responses were induced in C57BL/6 mice by two intraperitoneally (i.p.) applications of OVA/alum followed by the intranasal challenge with OVA. The different Th1‐inducing adjuvants were applied to the mice together with OVA/alum i.p. during the OVA‐sensitization period and, subsequently, different parameters of allergic immune responses were evaluated.

Results
All the adjuvants were effective in inhibiting the development of allergen‐induced airway eosinophilia, mucous production and, with the exception of PPD, also airway hyper‐reactivity, when they were applied together with OVA/alum. However, allergen‐specific IgG1 and IgE serum levels were only reduced in live BCG‐ and PPD‐treated mice. Suppression of airway eosinophilia was not observed in IFN‐γ‐ or IL‐12‐deficient mice (hk‐BCG, CpG‐ODN and PPD). Interestingly, live BCG was still able to suppress allergen‐induced Th2 responses in the absence of either IFN‐γ or IL‐12. When mice vaccinated with the different adjuvants together with OVA/alum were subjected to a second period of OVA/alum immunization, only live and hk‐BCG were able to efficiently suppress the development of airway inflammation. This effect could be adoptively transferred by splenic CD4+ T cells.

Conclusions
Taken together our data suggest that live BCG>hk‐BCG>CpG‐ODN >PPD are effective in suppressing allergen‐induced Th2 responses. The degree of suppression and the component of the Th2 response affected (airway inflammation vs. the production of allergen‐specific IgE and IgG1) were dependent upon the adjuvant used and how it was applied. Our results contribute to the design of novel vaccines protecting humans from developing allergic disorders.
SRD5A2 gene analysis in an Italian population of under‐masculinized 46,XY subjects
Authors: Nicoletti Annalisa , Baldazzi Lilia , Balsamo Antonio , Barp Lorella , Pirazzoli Piero , Gennari Monia , Radetti Giorgio , Cacciari Emanuele , Cicognani Alessandro
Published: August 2005

Objective  The differential diagnosis of male under‐masculinization, including a wide spectrum of phenotypes and a heterogeneous genetic basis, is crucial for the correct management of the patients. To characterize an Italian population of under‐masculinized males, we performed the molecular analysis of the SRD5A2 gene (2p23), encoding the 5α‐reductase‐2 enzyme that converts testosterone (T) to dihydrotestosterone (DHT), and is required for full masculinization of the male foetus.

Design and patients  Twenty‐six Italian patients with 46,XY kariotype and various degrees of ambiguous genitalia were retrospectively selected for this study. Twelve of these patients, 10 of whom were referred for partial androgen insensitivity syndrome (PAIS), were raised as females; 15 were raised as males and all had a severe hypospadias. For most of the patients, the case histories and hormonal findings were incomplete but all could be included in the clinical characteristics of under‐masculinization.

Measurement  For hormonal evaluation, T and DHT were measured by means of radioimmunoassay (RIA) and high‐performance liquid chromatography (HPLC)‐RIA methods, respectively. Genomic DNA of all patients and relatives was extracted from peripheral blood, the five exons of the SRD5A2 gene were amplified by polymerase chain reaction (PCR) and submitted to automatic sequencing.

Results  Five known mutations affecting the NADPH binding function and one new mutation affecting the enzyme C‐terminus were identified in a total of eight patients (two of whom were sisters). Five families were characterized, and in two patients only one affected allele was observed. The extension of the analysis to the regions flanking exons allowed the identification of a new polymorphism in intron 2, whose frequency was determined.

Conclusion  This first report of an Italian population underlines the importance of differential diagnoses in patients with under‐masculinization. The lack of precise genotype–phenotype correlation in some of the mutations highlights the necessity to improve knowledge about the biochemical aspects of steroid 5α‐reductase action and about the interactions of genetic and environmental factors.