bluuebunny/crossref_metadata_2025 huggingface dataset:
MeCP2 and promoter methylation cooperatively regulate E‐cadherin gene expression in colorectal carcinoma
Authors: Darwanto Agus , Kitazawa Riko , Maeda Sakan , Kitazawa Sohei
Published: August 2005

Reduced expression of E‐cadherin (E‐cad) owing to aberrant 5′CpG island hypermethylation has been regarded as one of the main molecular events involved in the dysfunction of the cell‐cell adhesion system. The molecular mechanisms providing diversity and heterogeneity of E‐cad expression in colorectal carcinoma were explored. In 29 cases of colorectal carcinoma in Indonesia, the expression of E‐cad was analyzed by immunohistochemical staining, the methylation status of the E‐cad promoter was determined by methylation‐specific PCR, and the expression of methyl‐CpG‐binding protein (MeCP) 2 was studied by in situ hybridization. E‐cad expression was strong, and no methylation was observed in normal colon mucosa and most of the well‐differentiated adenocarcinoma. In contrast, both signet‐ring cell carcinoma and mucinous adenocarcinoma showed fully methylated patterns and strong MeCP2 expression. In moderately‐ and poorly‐differentiated adenocarcinomas, however, E‐cad expression was rather heterogeneous, especially at the front of invasion and in the dissociated areas, where loss of MeCP2 expression correlated with E‐cad reexpression even in the presence of E‐cad promoter methylation. We conclude that both CpG methylation of the E‐cad promoter and significant MeCP2 expression cooperatively and epigenetically regulate E‐cad expression in colorectal cancer. (Cancer Sci 2003; 94: 442–447)
Parp‐1 deficiency implicated in colon and liver tumorigenesis induced by azoxymethane
Authors: Nozaki Tadashige , Fujihara Hisako , Watanabe Masatoshi , Tsutsumi Masahiro , Nakamoto Kentaro , Kusuoka Osamu , Kamada Nobuo , Suzuki Hiroshi , Nakagama Hitoshi , Sugimura Takashi , Masutani Mitsuko
Published: August 2005

Poly(ADP‐ribose) polymerase‐1 (Parp‐1) is activated by DNA strand breaks and functions in the maintenance of genomic integrity and cell death control. On the other hand, Parp‐1 is also involved in transcriptional regulation of various genes, and the relationship between Parp‐1 deficiency and susceptibility to tumorigenesis has not been fully elucidated. In the present study, Parp‐1−1− mice, harboring exon 1 disruption in Parp‐1, and Parp‐1+l+ animals were administered azoxymethane (AOM) at a dose of 10 mg/kg body weight once a week for 6 weeks. At 30 weeks after the first carcinogen treatment, mice were sacrificed. The incidence of animals bearing either adenomas or adenocarcinomas in the colon and the average number of colon tumors per mouse were significantly higher in Parp‐1−1− mice than in Parp‐1+l+ animals. β‐Catenin accumulation was observed in 43/44 of Parp‐1−1− tumors and 19/21 of the Parp‐1+l+ tumors and was not statistically different between the genotypes. This suggests that most tumors developed through a pathway involving the alteration of Wnt‐β‐catenin signaling in both Parp‐1−1‐ and Parp‐1+l+ mice. In the liver, where AOM is primarily activated, the incidence of animals bearing nodules and the average number of nodules per section were significantly increased in Parp‐1−1‐ mice compared with Parp‐1+l+ mice. Therefore, the results indicate that susceptibility to AOM‐induced tumorigenesis in the colon and also in the liver is enhanced in Parp‐1−1‐ mice, and Parp‐1 could have a substantial role in colon and liver tumorigenesis.
Transfection of MS‐36 melanoma cells with gef gene inhibits proliferation and induces modulation of the cell cycle
Authors: Boulaiz Houria , Prados José , Marchal Juan Antonio , García Ángel Miguel , Álvarez Luis , Melguizo Consolación , Carrillo Esmeralda , Ramos Juan Luis , Aránega Antonia
Published: August 2005

The gef gene, found in Escherichia coli DNA, encodes a small (50 amino acids) protein which is related to cell‐killing functions. We used the MS‐36 melanoma cell line as an experimental model to examine the usefulness of the gef gene as a new strategy for cancer therapy. We transfected MS‐36 cells using the pMAMneo vector, and induced gef gene expression with dexamethasone. This decreased the proliferation rate of MS‐36TG by as much as 85% in comparison with MS‐36 parental cells. The decrease in cell growth was accompanied with significant modifications of the cell cycle and morphology. The G1‐phase gradually disappeared, with accumulation in the S‐phase. However, studies with annexin V‐FITC and 7‐aminoactinomycin D failed to demonstrate induction of apoptosis. Morphological changes were an increase in cell size and the number of filopodia, and especially the appearance of pore‐like alterations in the cell membrane which were not seen in parental cells. Our results demonstrate that the gef gene, a system independent of the administration of a prodrug, significantly reduces the proliferation of MS‐36 cells. This gene may therefore be considered a new candidate for cancer gene therapy.
Cell adhesion system and human cancer morphogenesis
Authors: Hirohashi Setsuo , Kanai Yae
Published: August 2005

Cell‐cell adhesion determines the polarity of cells and participates in the maintenance of the cell societies called tissues. Cell‐cell adhesiveness is generally reduced in human cancers. Reduced intercellular adhesiveness allows cancer cells to disobey the social order, resulting in destruction of histological structure, which is the morphological hallmark of malignant tumors. Reduced intercellular adhesiveness is also indispensable for cancer invasion and metastasis. A tumor‐suppressor gene product, E‐cadherin, and its undercoat proteins, catenins, which connect cadherins to actin filaments, are located at lateral borders, concentrating on adherens junctions, of epithelial cells and establish firm cell‐cell adhesion. The E‐cadherin cell adhesion system in cancer cells is inactivated by various mechanisms that reflect the morphological and biological characteristics of the tumor. Silencing of the E‐cadherin gene by DNA hypermethylation around the promoter region occurs frequently, even in precancerous conditions. In diffuse infiltrating cancers, mutations are found in the genes for E‐cadherin and α‐and β‐catenins. At the invading front of cancers, the E‐cadherin cell adhesion system is inactivated by tyrosine phosphorylation of β‐catenin; an oncogene product, c‐erb B‐2 protein, is found to associate directly with β‐catenin. The E‐cadherin cell adhesion system cross‐talks with the Wingless/Wnt signaling pathway through β‐catenin, and expression of genes, which participate in cancer morphogenesis, may be regulated in conjunction with the Wingless/Wnt signaling pathway. Dysadherin, a newly identified cancer‐associated cell membrane glycoprotein, down‐regulates E‐cadherin and promotes cancer metastasis. In conclusion, inactivation of the E‐cadherin cell adhesion system by both genetic and epigenetic mechanisms plays a significant role during multistage human carcinogenesis.
Expression of short‐form caspase 8 correlates with decreased sensitivity to Fas‐mediated apoptosis in neuroblastoma cells
Authors: Kisenge Rodrick R. , Toyoda Hidemi , Kang Jian , Tanaka Shigeki , Yamamoto Hatsumi , Azuma Eiichi , Komada Yoshihiro
Published: August 2005

Disruption of apoptotic death signal transduction pathways may be responsible for tumor formation, progression and resistance to treatment in neuroblastoma. Caspase 8, one of the initiator caspases, plays an important role in the Fas‐Fas ligand pathway. This caspase signals through the formation of a death‐inducing signaling complex in response to Fas activation by its ligand. In this study, we evaluated the sensitivity of a series of human neuroblastoma cell lines to membrane‐bound Fas ligand induced‐cell death, as well as the expression of Fas, caspase 3 and caspase 8. Sensitivity to Fas‐mediated cell death did not correlate with the expression of Fas in neuroblastoma cells, but was directly associated with the pattern of caspase 8 protein expression. We found that the majority of neuroblastoma cell lines we evaluated lacked caspase 8 expression, and these cell lines were invariably resistant to Fas‐mediated cell death. In contrast, cell lines expressing normal caspase 8 protein were quite sensitive to Fas‐mediated cell death. More interestingly, a group of cell lines expressing a distinct short form of caspase 8 with splicing out of exon 3 consistently showed moderate sensitivity to Fas‐mediated cell death. These results indicate that the profile of caspase 8 expression is an important determinant of the response of neuroblastoma cells to Fas‐mediated cell death.
Synergistic decline in expressions of p73 and p21 with invasion in esophageal cancers
Authors: Masuda Norihiro , Kato Hiroyuki , Nakajima Takashi , Sano Takaaki , Kashiwabara Kenji , Oyama Tetsunari , Kuwano Hiroyuki
Published: August 2005

The significance of the p73 gene, a homologue of the p53 gene, in esophageal cancers is not fully understood. In order to clarify the role of p73 expression in esophageal cancers, p73 expression was immunohistochemically investigated in 106 surgically resected esophageal cancers and the results were compared with various clinicopathological factors. In normal esophageal epithelium, the expression of p73 was observed only in the nuclei of basal cells. In esophageal cancers, p73 immunoreactivity was observed in all intraepithelial lesions except one cancer, and was reduced with cancer invasion, to 78% and 64% at superficial invasion and deep invasion sites, respectively. However, p73 expression was not correlated with any other clinicopathological factor. The expressions of p53 and p21 were also investigated in esophageal cancer. To evaluate the status of the p53 gene mutation immunohistochemically, two monoclonal antibodies (DO7 and PAb240) were used. There seemed to be an inverse correlation between p73 expression and p53 mutation. Moreover, the expression of p21 was highly correlated with p73 expression irrespective of the p53 mutation status. In human esophageal cancers, p73 expression decreased with increasing degree of tumor invasion, and its decreased expression in local advanced tumor caused down‐regulation of p21 expression, which might reflect tumor progression.
Screening of stress enhancer based on analysis of gene expression profiles: Enhancement of hyperthermia‐induced tumor necrosis by an MMP‐3 inhibitor
Authors: Kato Naoki , Kobayashi Takeshi , Honda Hiroyuki
Published: August 2005

To improve the therapeutic benefit of hyperthermia, we examined changes of global gene expression after heat shock using DNA microarrays consisting of 12 814 clones. HeLa cells were treated for 1 h at 44°C and RNA was extracted from the cells 0, 3, 6, and 12 h after heat shock. The 664 genes that were up or down‐regulated after heat shock were classified into 7 clusters using fuzzy adaptive resonance theory (fuzzy ART). There were 41 genes in two clusters that were induced in the early phase after heat shock. In addition to shock response genes, such as hsp70 and hsp40, the stress response genes c‐jun, c‐fos and egr‐1 were expressed in the early phase after heat shock. We also found that expression of matrix metalloproteinase 3 (MMP‐3) was enhanced during the early response. We therefore investigated the role of MMP‐3 in the heat shock response by examining HeLa cell survival after heat treatment in the presence and absence of an MMP‐3 inhibitor, N‐isobutyl‐N‐(4‐methoxyphenyl‐sulfonyl)glycylhydroxamic acid (NNGH) or N‐hydroxy‐2(R)‐[[4‐methoxysulfonyl] (3‐picolyl)amino]‐3‐methylbutaneamide hydro‐chloride (MMI270). The number of surviving cells 3 days after heat treatment significantly decreased, reaching 3.5% for NNGH and 0.2% for MMI270. These results indicate that the MMP‐3 inhibitors enhanced heat shock‐induced cell death and behaved as stress enhancers in cancer cells. This valuable conclusion was reached as a direct result of the gene expression profiling that was performed in these studies.
Expression of HNFs and C/EBPα is correlated with immunocytochemical differentiation of cell lines derived from human hepatocellular carcinomas, hepatoblastomas and immortalized hepatocytes
Authors: Ishiyama Tadashi , Kano Junko , Minami Yuko , Iijima Tatsuo , Morishita Yukio , Noguchi Masayuki
Published: August 2005

Objective assessment of the differentiation grade of hepatocellular carcinomas (HCCs) is important for evaluation of the pathological diagnosis, prognosis and therapeutic treatment. Differentiation of hepatocytes is reflected by their expression of hepatic functional proteins in the mouse embryo, and liver‐enriched transcription factors (LETFs) have been shown to regulate hepatic functional genes strictly. Previous reports demonstrated that the level of LETF expression is altered in HCC or preneoplastic nodules compared with noncancerous tissues. Therefore, LETF expression levels might be useful as a measure of HCC maturation. In this study, to clarify the correlation between the expression of LETFs and the differentiation grade of HCCs, we performed a quantitative analysis of the mRNA expressions of HNFs and C/EBPα using real‐time reverse‐transcription PCR and immunocytochemical analysis for hepatic functional proteins in twelve cell lines. Furthermore, we examined orthotopic transplantations of the HCC cell lines in C.B‐17/Icrj‐scid/scid mice and characterized the histologic and cytologic differentiation of the tumors that developed. Our results showed that comprehensive expressions of HNF‐3β, HNF‐4α, HNF‐1α, and C/EBPα were specific to HCCs with well‐differentiated function and morphology. Furthermore, among these four transcription factors, HNF‐4α and HNF‐1α expressions showed synchronism and had a close relation with HCC differentiation. These in vitro results were confirmed in tumors developed in SCID mice in vivo. These findings suggested that HNF‐4α and HNF‐1α are useful markers to assess the degree of HCC differentiation, which we suggest could be evaluated objectively by the quantitative analysis of HNFs and C/EBPα in HCCs.
Identification of a mouse cytoskeleton‐associated protein, CKAP2, with microtubule‐stabilizing properties
Authors: Jin Yi , Murakumo Yoshiki , Ueno Kaoru , Hashimoto Mizuo , Watanabe Tsuyoshi , Shimoyama Yoshie , Ichihara Masatoshi , Takahashi Masahide
Published: August 2005

Microtubule dynamics is an important factor in cell proliferation and one of the main targets of cancer chemotherapy. Since microtubule‐associated proteins (MAPs) are known to influence microtubule stability, study of MAPs may contribute both to knowledge of cancer cell biology and to the production of new anti‐cancer drugs. In this study, we identified a new mouse gene which is a homolog of human cytoskeleton‐associated protein, CKAP2 gene, by differential display analysis. The level of expression of mouse CKAP2 (mCKAP2) was significantly higher in NIH3T3 cells expressing RET with a multiple endocrine neoplasia (MEN) 2A or MEN2B mutation than in parental NIH3T3 cells. Immunocytochemical analysis showed that mCKAP2 protein is localized in cytoplasm with a fibrillar appearance, and is co‐localized with microtubules throughout the cell cycle. Furthermore, overexpression of mCKAP2 in cells appeared to stabilize microtubules against treatment with nocodazole, a microtubule‐depolymerizing agent. In addition, levels of human CKAP2 were increased in some human tumor cell lines examined. These findings suggest that CKAP2 is a new MAP with microtubule‐stabilizing properties and may represent a new molecular target for cancer chemotherapy.
Distinct pattern of gene expression in pyothorax‐associated lymphoma (PAL), a lymphoma developing in long‐standing inflammation
Authors: Nishiu Mieko , Tomita Yasuhiko , Nakatsuka Shin‐ichi , Takakuwa Tetsuya , Lizuka Norishige , Hoshida Yoshihiko , Ikeda Jun‐ichiro , Luchi Keiji , Yanagawa Rempei , Nakamura Yusuke , Aozasa Katsuyuki
Published: August 2005

Pyothorax‐associated lymphoma (PAL) is a unique lymphoma developing in the pleural cavity after long‐standing pyothorax. They are diffuse large B‐cell lymphomas (DLBCLs), frequently with immunoblastic morphology, and show a strong association with Epstein‐Barr virus (EBV) infection. In this study, cDNA microarray analysis was performed in six cases with PAL and 12 with nodal DLBCL. Among 5516 informative genes, 348 displayed more than 2‐fold difference (higher or lower) of expression level between PAL and nodal DLBCL (P>0.001). These genes are known to be involved in apoptosis, interferon response, and signal transduction. One of the most differentially expressed genes, IFI27 (interferon‐α‐inducible protein 27) was subjected to quantitative RT‐PCR analysis, and increased expression of IFI27 was confirmed. Over‐expression of IFI27 was also found in cell lines derived from PAL, but not in other lymphoid cell lines. This study shows that PAL is a distinctive subtype of DLBCL not only in its clinical presentation, but also in its molecular profile.
Transforming growth factor‐β1‐induced apoptosis is blocked by β1‐integrin‐mediated mitogen‐activated protein kinase activation in human hepatoma cells
Authors: Zhang Hao , Ozaki Iwata , Mizuta Toshihiko , Yoshimura Toru , Matsuhashi Sachiko , Eguchi Yuichiro , Yasutake Tsutomu , Hisatomi Akitaka , Sakai Takahiro , Yamamoto Kyosuke
Published: August 2005

Growth factors and extracellular matrices cooperatively regulate cellular behavior. However, the interactions between transforming growth factor‐β1 (TGF‐β1) and integrins in hepatic cells are not fully understood. We investigated the effects of β1‐integrin on TGF‐β1‐regulated growth of hepatoma cells. Human hepatoma cell lines HepG2, Huh7, and Hep3B were stably transfected with β1‐integrin, and the parental, and mock‐ and β1‐integrin‐transfected hepatoma cells were treated with TGF‐β1. Modulation of apoptosis and pathways involved in the process were investigated. TGF‐β1 suppressed the growth of hepatoma cells, and apoptosis was observed in Hep3B and Huh7. Hepatoma cells transfected with β1‐integrin were protected from TGF‐β1‐induced apoptosis. Mitogen‐activated protein (MAP) kinase inhibitors, PD98059, SB203580, and SP600125, abolished this protective effect of β1‐integrin, but herbimycin A and wortmannin were ineffective. Hepatoma cells overexpressing β1‐integrin showed increased activities of MAP kinases, and TGF‐β1 induced sustained activation of MAP kinases in these cells, but only transient activation in mock‐transfected cells. These data suggest that MAP kinases activated by β1‐integrin provide a strong anti‐apoptotic signal during TGF‐β1‐induced apoptosis in human hepatoma cells. Therefore β1‐integrin‐mediated signals may contribute to the development and progression of hepatocellular carcinoma.
Analysis of CD8 T‐cell response by IFNγ ELISPOT and H‐2Ld/ pRL1a tetramer assays in pRL1a multiple antigen peptide‐immunized and RL male 1‐bearing BALB/ c and (BALB/c×C57BL/6) F1 mice
Authors: Takada Itsuro , Noguchi Yuji , Kenjo Akira , Wada Hisashi , Uenaka Akiko , Fujita Teizo , Inoue Hajime , Nakayama Eiichi
Published: August 2005

We previously identified an H‐2Ld‐binding peptide pRL1a (IPGLPLSL) on RL male 1 that is predominantly recognized by cytotoxic T‐lymphocytes (CTLs). MAP is a multibranched lysine core with antigenic peptides. Immunization of BALB/c mice with pRL1a MAP effectively induced pRL1a CTLs. Here, we demonstrate the presence of pRL1a‐recognizing CD8+ T‐cells in pRL1a MAP‐immunized and RL male 1‐bearing BALB/c and (BALB/ cxC57BL/6)F1 mice by using IFNγ ELISPOT and H‐2Ld/pRL1a tetramer assays. A few IFNγ ELISPOTs and no tetramer‐positive cells were detected ex vivo in spleen cells from BALB/c mice immunized with pRL1a MAP. After a single in vitro stimulation with RL male 1, 432 and 741 IFNγ ELISPOTs/105 cells were detected and tetramer‐positive CD8+ T‐cells occurred at relative frequencies of 5.7% and 30.8% in splenic CD8+ T‐cells from mice that had been doubly and triply immunized, respectively, against pRL1a MAP. Tetramer‐positive cells displayed two distinct cell populations, CD62Llow and CD62Lhigh. Secondary in vitro stimulation expanded CD62Lhigh cells more efficiently than CD62Llow cells. Furthermore, a higher frequency of IFNγ‐producing and tetramer‐positive CD8+ T‐cells was detected ex vivo in RL male 1‐bearing semi‐allogeneic (BALB/cxC57BL/6)F1 than in BALB/c mice on day 14 after tumor inoculation.
Proteasome inhibitor PS‐341 down‐regulates prostate‐specific antigen (PSA) and induces growth arrest and apoptosis of androgen‐dependent human prostate cancer LNCaP cells
Authors: Ikezoe Takayuki , Yang Yang , Saito Tsuyako , Koeffler H. Phillip , Taguchi Hirokuni
Published: August 2005

Proteasome inhibitor PS‐341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of nuclear factor KB (NF‐KB) in vitro. In addition, recent clinical studies of PS‐341 have demonstrated some objective responses in individuals with relapsed, refractory MM. However, the activity of PS‐341 against non‐hematological malignancies remains to be fully elucidated. In this study, we found that PS‐341 induced growth arrest and apoptosis of androgen‐dependent human prostate cancer LNCaP cells in conjunction with markedly up‐regulated levels of p21waf1 and p53. In addition, we found that PS‐341 down‐regulated both 5α dihydrotestosterone (DHT)‐ and interleukin‐6 (IL‐6)‐induced expression of prostate‐specific antigen (PSA) as measured by western blot analysis. PS‐341 down‐regulated basal levels of the androgen receptor (AR) in the nucleus; however, it did not affect DHT‐induced nuclear translocation of AR in these cells. Reporter assays using a series of promoters of the PSA gene showed that down‐regulation of PSA by PS‐341 was caused by inhibition of the transcriptional activity of the androgen receptor response element (ARE) in these cells. Taken together, the results indicate that PS‐341 induced growth arrest and apoptosis of LNCaP cells by blockade of the AR signaling pathway. The proteasome may be a molecular target for treatment of a variety of cancers including prostate cancer.
Prompt tumor formation and maintenance of constitutive NF‐κB activity of multiple myeloma cells in NOD/SCID/γCnull mice
Authors: Dewan Md. Zahidunnabi , Watanabe Mariko , Terashima Kazuo , Aoki Mizuho , Sata Tetsutaro , Honda Mitsuo , Ito Mamoru , Yamaoka Shoji , Watanabe Toshiki , Horie Ryouichi , Yamamoto Naoki
Published: August 2005

Clinically and biologically relevant animal models are indispensable to evaluate both the pathophysiology and strategies for diagnosis and treatment of multiple myeloma (MM). We examined the tumorigenicity of MM cell lines KMM‐1 and U‐266 in an in vivo cell proliferation model using NOD/SCID/γCnull (NOG) mice. Two cell lines were inoculated either subcutaneously (s.c.) in the post‐auricular region or intravenously (i.v.) in the tail of NOG mice. The KMM‐1 cell line produced a progressively growing large tumor with infiltration of the cells expressing human λ‐chain in various organs of all NOG mice, while the U‐266 cell line failed to do so. Tumor cells grown in NOG mice maintained the original histomorphology, as well as expression patterns of tumor markers human λ, Ig light chain and VEGF. Tumor progression in mice also correlated with elevation of serum human soluble IL‐6R and gp130. Tumor cells sustained a strong NF‐κB activity in vivo and induced NF‐κB components were indistinguishable from those in cells cultured in vitro. The rapid and efficient engraftment of the MM cell line in NOG mice suggests that this is a very useful animal model which could provide a novel system in which to clarify the mechanism of growth of cancer cells, as well as to develop new therapeutic regimens against MM.
HTLV‐1 provirus load in peripheral blood lymphocytes of HTLV‐1 carriers is diminished by green tea drinking
Authors: Sonoda Junichiro , Koriyama Chihaya , Yamamoto Shinako , Kozako Tomohiro , Li Hong Chuan , Lema Carolina , Yashiki Shinji , Fujiyoshi Toshinobu , Yoshinaga Mitsuhiro , Nagata Yukihiro , Akiba Suminori , Takezaki Toshiro , Yamada Katsushi , Sonoda Shunro
Published: August 2005

Human T‐cell lymphotropic virus type 1 (HTLV‐1) is causatively associated with adult T‐cell leukemia (ATL) and HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since a high level of HTLV‐1 provirus load in circulating lymphocytes is thought to be a risk for ATL and HAM/TSP, diminution of HTLV‐1 provirus load in the circulation may prevent these intractable diseases. Our previous study (Jpn J Cancer Res 2000; 91: 34–40) demonstrated that green tea polyphenols inhibit in vitro growth of ATL cells, as well as HTLV‐1‐infected T‐cells. The present study aimed to investigate the in vivo effect of green tea polyphenols on HTLV‐1 provirus load in peripheral blood lymphocytes on HTLV‐1 carriers. We recruited 83 asymptomatic HTLV‐1 carriers to examine HTLV‐1 provirus DNA with or without administration of capsulated green tea extract powder. Thirty‐seven subjects were followed up for 5 months by measuring HTLV‐1 provirus load after daily intake of 9 capsules of green tea extract powder per day (equivalent to 10 cups of regular green tea), and 46 subjects lived ad libitum without intake of any green tea capsule. The real‐time PCR quantification of HTLV‐1 DNA revealed a wide range of variation of HTLV‐1 provirus load among asymptomatic HTLV‐1 carriers (0.2‐200.2 copies of HTLV‐1 provirus load per 1000 peripheral blood lymphocytes). Daily intake of the capsulated green tea for 5 months significantly diminished the HTLV‐1 provirus load as compared with the controls (P=0.031). These results suggest that green tea drinking suppresses proliferation of HTLV‐1‐infected lymphocytes in vivo.
Induction of DNA‐adducts and increase of 8‐hydroxy‐2‐deoxyguanosine, but no development of preneoplastic lesions in offspring liver with transplacental and trans‐breast milk exposure to 2‐amino‐3,8‐dimethylimidazo [4,5‐f]quinoxaline (MelQx) in rats
Authors: Ichihara Toshio , Wanibuchi Hideki , Totsuka Yukari , Morimura Keiichirou , Wei Min , Nakae Dai , Fukushima Shoji
Published: August 2005

Humans may be exposed to 2‐amino‐3,8‐dimethylimidazo[4,5‐flquinoxaline (MelQx) at low doses during the period of gestation and lactation, and thereafter throughout life. The current study was designed to examine the possibility that early exposure may increase the risk of liver tumor development and related genetic changes. Male and female F344 rats were therefore administered MelQx in diet (1, 10 and 100 ppm) for 4 weeks before mating and also during gestation and lactation. We also examined the carcinogenic risk of low‐dose maternal and post‐weaning exposure (MelQx at doses of 1 and 10 ppm). Surviving male F1 rats were sacrificed under ether anesthesia at 19 weeks of age for analyses of glutathione S‐transferase placental form‐positive foci in the liver and aberrant crypt foci in the colon, as putative preneoplastic lesions. Transplacental and trans‐breast milk exposure to MelQx did not enhance development of the lesions, and levels of cell proliferation in the liver also did not differ from control values. However, excretion of MelQx into breast milk and transfer to the fetus and offspring were observed with resultant hepatic MelQx‐DNA adducts and 8‐hydroxy‐2′‐deox‐yguanosine formation. Thus, our data suggest that maternal exposure to MelQx during the period of pregnancy and lactation may not increase the risk of hepatocarcinogenesis in male offspring, despite causing genetic damage. If this result can be extrapolated to humans, exposure to MelQx may not increase carcinogenic risk in offspring at usual human exposure levels.
Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib (“Iressa”)‐sensitive and resistant xenograft models
Authors: Taguchi Fumiko , Koh Yasuhiro , Koizumi Fumiaki , Tamura Tomohide , Saijo Nagahiro , Nishio Kazuto
Published: August 2005

ZD6474 is a novel, orally available inhibitor of vascular endothelial growth factor (VEGF) receptor‐2 (KDR) tyrosine kinase, with additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. ZD6474 has been shown to inhibit angiogenesis and tumor growth in a range of tumor models. Gefitinib (“Iressa”) is an selective EGFR tyrosine kinase inhibitor (TKI) that blocks signal transduction pathways. We examined the antitumor activity of ZD6474 in the gefitinib‐sensitive lung adenocarcinoma cell line, PC‐9, and a gefitinib‐resistant variant (PC‐9/ZD). PC‐9/ZD cells showed cross‐resistance to ZD6474 in an in vitro dye formation assay. In addition, ZD6474 showed dose‐dependent inhibition of EGFR phosphorylation in PC‐9 cells, but inhibition was only partial in PC‐9/ZD cells. ZD6474‐mediated inhibition of tyrosine residue phosphorylation (Tyr992 and Tyr1045) on EGFR was greater in PC‐9 cells than in PC‐9/ZD cells. These findings suggest that the inhibition of EGFR phosphorylation by ZD6474 can contribute a significant, direct growth‐inhibitory effect in tumor cell lines dependent on EGFR signaling for growth and/or survival. The effect of ZD6474 (12.5–50 mg/kg/day p.o. for 21 days) on the growth of PC‐9 and PC‐9/ZD tumor xenografts in athymic mice was also investigated. The greatest effect was seen in gefitinib‐sensitive PC‐9 tumors, where ZD6474 treatment (>12.5 mg/kg/day) resulted in tumor regression. Dose‐dependent growth inhibition, but not tumor regression, was seen in ZD6474‐treated PC‐9/ZD tumors. These studies demonstrate that the additional EGFR TKI activity may contribute significantly to the anti‐tumor efficacy of ZD6474, in particular in those tumors that are dependent on continued EGFR‐signaling for proliferation or survival. In addition, these results provide a preclinical rationale for further investigation of ZD6474 as a potential treatment option for both EGFR‐TKI‐sensitive and EGFR‐TKI‐resistant tumors.
Rapid analysis of gene expression changes caused by liver carcinogens and chemopreventive agents using a newly developed three‐dimensional microarray system
Authors: Hokaiwado Naomi , Asamoto Makoto , Tsujimura Kazunari , Hirota Takeshi , Ichihara Toshio , Satoh Takatomo , Shirai Tomoyuki
Published: August 2005

We investigated changes of gene expression in livers of rats treated with carcinogens and tumor promoters using a novel three‐dimensional microarray system developed by Olympus Optical Co., Ltd., to assess the feasibility of predicting modifying effects on hepatocarcinogenesis on the basis of changes in the patterns. For this purpose, two genotoxic carcinogens, two nongenotoxic carcinogens (promoters) and seven candidate chemopreventive agents were examined. Six‐week‐old male F344 rats were treated for 2 weeks with the 11 chemicals (0.05% phenobarbital, 0.3% clofibrate, 0.01% N‐diethylnitrosamine (DEN), 0.01% 2‐amino‐3, 8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx), 1% catechol, 1% caffeic acid, 0.05% nobiletin, 0.05% garcinol, 0.05% auraptene, 0.05% zermbone and 0.05% 1′‐acetoxychavicol acetate (ACA). Test chemicals were mixed in food with the exception of DEN, which was administered in drinking water. RNAs from liver were then analyzed using two kinds of customized microarrays (PamChip® microarray A spotted for 28 genes of drugmetabolizing enzymes in duplicate, and PamChip® microarray B spotted for 131 genes which are known to be up‐ or down‐regulated in hepatocarcinoma cells). Hybridization and subsequent analysis were usually completed within 2 h and the data obtained were highly reproducible. Carcinogens were classified into genotoxic and nongenotoxic substances by clustering analysis. We could also divide test chemicals into carcinogens and chemopreventive agents from their effects on gene expression. In this study, we have thus shown that it is feasible to predict the modifying effects of chemicals on the basis of changes of gene expression patterns after only 2 weeks of exposure, using our novel three‐dimensional microarrays.
Correlation of EPHA2 overexpression with high microvessel count in human primary colorectal cancer
Authors: Kataoka Hideki , Igarashi Hisaki , Kanamori Masao , Ihara Megumi , Wang Jian‐Dong , Wang You‐Jie , Li Zhong‐You , Shimamura Takahiro , Kobayashi Toshihiko , Maruyama Keiji , Nakamura Toshio , Arai Hajime , Kajimura Masayoshi , Hanai Hiroyuki , Tanaka Masamitsu , Sugimura Haruhiko
Published: August 2005

Evidence suggests that the erythropoietin‐producing hepatocellular (EPH) receptor tyrosine kinases (RTKs) and their ephrin (EFN) ligands are involved in human carcinogenesis. Expression of two of them, EFNA1 ligand and its receptor, EPHA2, has been proposed to contribute to tumor‐induced neovascularization. Colorectal cancers were examined for expressions of EPHA2 and its ligand EFNA1 by semi‐quantitative RT‐PCR, and double‐immunostained for EPHA2 and CD34. Microvessels in the tumors were counted. Double‐staining was also performed in 25 cases of adenoma with focal cancer for comparison. Trends of overexpression of both EPHA2 and EFNA1 was found in tumor tissue compared to the corresponding normal tissue in the same specimen [22/37 (59.5%) and 25/37 (67.5%), respectively; P=0.100 for EPHA2 and P=0.009 for EFNA1]. Overexpression of EPHA2 and EFNA1 was noted more frequently in the early stage than in the late stage [EPHA2, 15/21 (71.4%) vs. 7/16 (43.8%), P=0.007; EFNA1, 15/21 (71.4%) vs. 10/16 (62.5%), P=0.007]. Both EPHA2 and EFNA1 were more frequently overexpressed in smaller tumors (less than 5 cm) than in larger tumors [EPHA2, 15/21 (71.4%) vs. 7/16 (43.8%), P=0.017; EFNA1, 16/21 (76.2%) vs. 8/16 (50%), P=0.001]. Tumors less than 5 cm in diameter and in stages I and II were significantly more likely to overexpress EPHA2 and EFNA1 (P=0.001 for EPHA2, P=0.001 for EFNA1). Microvessel counts (MVCs) after immuno‐staining for CD34 were significantly correlated (r=0.343, P=0.037) with overexpression of EPHA2. EPHA2‐expressing focal cancer also surrounded microvessels in adenomas with focal cancers. These findings suggest an involvement of EPHA2 in colon carcinogenesis, mainly in stages I and II, and probably through their effect on microvessel induction.
Proteasome inhibitor PS‐341 induces growth arrest and apoptosis of non‐small cell lung cancer cells via the JNK/c‐Jun/AP‐1 signaling
Authors: Yang Yang , Ikezoe Takeyuki , Saito Tsuyako , Kobayashi Makoto , Koeffler H. Phillip , Taguchi Hirokuni
Published: August 2005

Proteasome inhibitor PS‐341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of NF‐κB in vitro and has afforded some objective responses in individuals with relapsed, refractory MM. However, the activity of PS‐341 against non‐hematological malignancies remains to be fully elucidated. In this study, we found that PS‐341 induced growth arrest and apoptosis of NCI‐H520 and ‐H460 non‐small cell lung cancer (NSCLC) cells in conjunction with markedly up‐regulated levels of p21waf1 and p53, and down‐regulation of bcl‐2 protein in these cells. Also, PS‐341 caused phosphorylation of c‐Jun NH2‐terminal kinase (JNK) and c‐Jun, and enhanced AP‐1/DNA binding activities in these cells as measured by western blotting and enzyme‐linked immunosorbent assay (ELISA), respectively. Interestingly, when the JNK/ c‐Jun/AP‐1 signal pathway was disrupted by the JNK inhibitor SP600125, the ability of PS‐341 to inhibit the growth of NSCLC cells and to up‐regulate the levels of p21waf1 in these cells was blunted, but the expression of p53 was sustained at a high level, suggesting that the JNK/c‐Jun/AP‐1 signal pathway might mediate the anti‐lung cancer effects of PS‐341, with p21waf1 playing the central role. Thus, PS‐341 might be useful for the treatment of individuals with NSCLC.
Development of gene therapy to target pancreatic cancer
Authors: Yoshida Teruhiko , Ohnami Shumpei , Aoki Kazunori
Published: August 2005

Pancreatic cancer remains one of the most difficult cancers to treat. Its high propensity to infiltrate and metastasize early from a small primary focus necessitates development of a new therapy which can track down the disseminated cancer cells in vivo. Gene therapy may offer new opportunities for a variety of targeting strategies, and we review here some of our work related to the development of targeted gene therapy: 1) Targeting by specific molecular abnormality: Many pancreatic cancer cells show “addiction” to K‐as mutation, while normal cells appear resistant to suppression of K‐ras‐mediated signaling by antisense K‐ras RNA expression adenoviral vector. 2) Targeting by in vivo tumor characteristics: In a peritoneal dissemination model, intraperitoneal lipofection/polyfection can deliver and express transgenes highly preferentially in tumor nodules. 3) Targeting by vector: An efficient protocol for construction of an adenovirus expression vector library has been developed, which will enable a direct functional selection of fiber knob‐modified targeting vector species for given cells. 4) Targeting by tumor immunity: Several cytokines not only induce direct cytotoxicity, but are also expected to activate specific immunity to achieve targeted suppression of cancer cells in vivo. Unlike parenteral administration of short‐lived recombinant interferon protein, local interferon gene transfer can provide a target tissue‐restricted distribution and sustained expression, which may improve the efficacy/safety balance of cytokine therapy. Cancer gene therapy development is, in general, at the stage of proof of principles and safety. However, it is an art of integrated science. The recent rapid progress of related sciences and technologies will expand the potential and consolidate the clinical reality of gene therapy.
Identification of differentially expressed molecules in adult T‐cell leukemia cells proliferating in vivo
Authors: Koga Hikari , Imada Kazunori , Ueda Maki , Hishizawa Masakatsu , Uchiyama Takashi
Published: August 2005

HTLV‐I is the causative agent of adult T‐cell leukemia (ATL). However, the precise mechanism underlying the neoplastic cell growth of ATL remains unclear. In this study, we established a leukemic cell line, termed SYK‐11L(+), from tumor cells (S‐YU) in an in vivo cell proliferation model of ATL using severe combined immunodeficiency (SCID) mice. Unexpectedly, SYK‐11L(+) was found to have no tumorigenicity in SCID mice. Flow cytometric analysis showed that S‐YU expressed cell adhesion molecules including CD44, ICAM‐1 and OX40, whereas SYK‐11L(+) had lost the expression of these molecules. The administration of anti‐OX40 monoclonal antibody inhibited the engraftment of S‐YU cells into SCID mice, suggesting that OX40 is a potential target for immunotherapy. Significant differences in responsiveness to IL‐2 and IL‐15 were observed between the two cell types. To better understand the molecular basis of tumorigenicity, cDNA microarray analysis was performed using tumorigenic S‐YU and non‐tumori‐genic SYK‐11L(+) cells. We obtained several candidate genes differentially overexpressed in S‐YU compared with SYK‐11L(+). Interestingly, one such gene, regulator of G protein signaling 1 (RGS1), was shown to be overexpressed in most ATL patients. Further characterization of the differentially expressed molecules, such as OX40 and RGS1, would provide useful information not only to elucidate the mechanism of ATL cell growth in vivo, but also to develop novel molecularly targeted therapies.
Isoliquiritigenin, a flavonoid from licorice, reduces prostaglandin E2 and nitric oxide, causes apoptosis, and suppresses aberrant crypt foci development
Authors: Takahashi Tetsuyuki , Takasuka Nobuo , Iigo Masaaki , Baba Masaki , Nishino Hoyoku , Tsuda Hiroyuki , Okuyama Toru
Published: August 2005

Isoliquiritigenin (ILTG), a flavonoid group compound, exists in some foodstuffs and herbal medicines such as licorice (Glycyrrhiza uralensis Fisher). Previously, we showed that ILTG can suppress azoxymethane (AOM)‐induced colon carcinogenesis in ddY mice. In the present report, we present evidence that ILTG markedly decreases both prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW264.7 mouse macrophage cells. The decrease of PGE2 was dependent on cyclooxygenase‐2 (COX‐2) expression and the decrease of NO appeared due to a decrease in inducible nitric oxide synthase (iNOS) protein expression. In mouse and human colon carcinoma cells, ILTG treatment suppressed cell growth and caused apoptosis. Furthermore, in vivo administration of ILTG inhibited the induction of preneoplastic aberrant crypt foci (ACF) in the male F344 rat colon. Our results suggest that ILTG is a promising chemopreventive agent against colon carcinogenesis.
c‐kit gene mutations in intracranial germinomas
Authors: Sakuma Yuji , Sakurai Shinji , Oguni Sachiko , Satoh Masaaki , Hironaka Mitsugu , Saito Ken
Published: August 2005

Gain‐of‐function mutations of the c‐kit gene and the expression of phosphorylated KIT are found in most gastrointestinal stromal tumors and mastocytosis. Further, almost all gonadal seminomas/ dysgerminomas exhibit KIT membranous staining, and several reports have clarified that some (10–25%) have a c‐kit gene mutation. But, whether intracranial germinomas also have a c‐kit gene mutation remains unsolved. To elucidate the presence, frequency, and location of c‐kit gene mutations in intracranial germinomas, we analyzed five mutational hot spots (exons 9, 10, 11, 13, and 17) in the c‐kit genomic DNA of 16 germinomas using polymerase chain reaction and direct sequencing. We found c‐kit gene mutations at exon 11 (W557C) or 17 (D816V, D820V, and N822Y) in four germinomas (25.0%), although no statistically significant difference in any clinicopathological factor was found between patients with or without mutations. These results are similar to those seen in gonadal seminoma/dysgerminoma patients, and confirm that intracranial germinomas are exact counterparts of gonadal seminomas/dysgerminomas, as would be expected on histological and immunohistochemical grounds. Moreover, molecular targeting drugs such as imatinib mesylate (STI571), which is a selective inhibitor of KIT, might be promising agents for the treatment of intracranial germinomas with c‐kit gene mutations.
Methylation‐associated silencing of the Wnt antagonist SFRP1 gene in human ovarian cancers
Authors: Takada Toshio , Yagi Yukiko , Maekita Takao , Imura Masayoshi , Nakagawa Shunsuke , Tsao Sai‐Wah , Miyamoto Kazuaki , Yoshino Osamu , Yasugi Toshiharu , Taketani Yuji , Ushijima Toshikazu
Published: August 2005

The SFRP1 gene on chromosome 8p11.2 encodes a Wnt signaling antagonist, and was recently demonstrated to be a new tumor suppressor that is inactivated by promoter methylation in human colon cancers. Here, we analyzed promoter methylation of the SFRP1 gene in human ovarian cancers, in which loss of heterozygosity in 8p is frequently observed and involvement of the Wnt signaling pathway has been suggested. Methylation‐specific PCR (MSP) analysis showed that four of 13 ovarian cancer cell lines and two of 17 primary ovarian cancers had methylated SFRP1, while an immortalized ovarian epithelial cell line, HOSE, and seven ovarian endometrial cyst samples did not. In the four ovarian cancer cell lines with the methylation, SFRP1 was not expressed at all as determined by quantitative RT‐PCR analysis. A cell line with SFRP1 methylation, MCAS, was treated with a demethylating agent, 5‐aza‐2′‐deoxycytidine, and demethylation of the promoter and re‐expression of SFRP1 were observed. These results show that SFRP1 is inactivated by promoter methylation in human ovarian cancers, as well as colon cancers.
Determinants of sensitivity and resistance to gemcitabine: The roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non‐small cell lung cancer
Authors: Achiwa Hiroyuki , Oguri Tetsuya , Sato Shigeki , Maeda Hiroyoshi , Niimi Takashi , Ueda Ryuzo
Published: August 2005

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT‐PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non‐small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r=‐0.6769, P=0.0005), whereas dCK expression levels were not. In a highly gemcitabine‐sensitive cell line, NCI‐H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI‐H23 cells to gemcitabine and subsequently established the drug‐resistant clone H23/GEM‐R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.
Obesity: basic science and medical aspects relevant to anaesthetists
Authors: Cheah M. H. , Kam P. C. A.
Published: August 2005

Obesity is becoming a major public health problem throughout the world. It is now the second leading cause of death in the United States and is associated with significant, potentially life‐threatening co‐morbidities. Significant advances in the understanding of the physiology of body weight regulation and the pathogenesis of obesity have been achieved. A better understanding of the physiology of appetite control has enabled advances in the medical and surgical treatment of obesity. Visceral or abdominal obesity is associated with an increased risk of cardiovascular disease and type 2 diabetes. Various drugs are used in the treatment of mild obesity but they are associated with adverse effects. Surgery has become an essential part of the treatment of morbid obesity, notwithstanding the potential adverse events that accompany it. An appreciation of these problems is essential to the anaesthetist and intensivist involved in the management of this group of patients.