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Summary Previous studies have revealed that, at the initial step of carcinogenesis, transformed cells are often eliminated from epithelia via cell competition with the surrounding normal cells. In this study, we performed cell competition-based high-throughput screening for chemical compounds using cultured epithelial cells and confocal microscopy. PLX4720 was identified as a hit compound that promoted apical extrusion of RasV12-transformed cells surrounded by normal epithelial cells. Knockdown/knockout of ZAK, a target of PLX4720, substantially enhanced the apical elimination of RasV12 cells in vitro and in vivo. ZAK negatively modulated the accumulation or activation of multiple cell competition regulators. Moreover, PLX4720 treatment promoted apical elimination of RasV12-transformed cells in vivo and suppressed the formation of potentially precancerous tumors. This is the first report demonstrating that a cell competition-promoting chemical drug facilitates apical elimination of transformed cells in vivo, providing a new dimension in cancer preventive medicine., Graphical Abstract, Highlights • A cell competition-based high-throughput screen identifies chemical enhancers • PLX4720 enhances the apical elimination of RasV12 cells via ZAK inhibition • ZAK negatively modulates cell competition regulators • PLX4720 treatment promotes apical elimination of RasV12 cells in vivo, Chemistry; Biological Sciences; Cancer

2008年HLEG（High Level Expert Group on European Low Dose Risk Research）は低線量影響研究の意義とその重要性を提唱し，発がんも含めて低線量放射線影響研究は国際的関心事となってきている［1，2］．我々は長年動物実験を行っているが，放射線発がん実験は動物種の選択や，放射線の種類，量，照射方法など組合せが複雑で，解析もマクロからミクロ，分子生物学分野におよび，線量と効果の関係の目標を定めにくい領域である．我々の実験系では特異的自家発生がんが少ないマウスを選択し，目的の誘発がんとしてマウスで自家発生が希有な皮膚がんを選択した．また被ばくによる他臓器への影響を避け，かつ皮膚限局照射が可能なβ線を用いた．これにより照射部位の皮膚のみをがんの発生部位とし，他臓器の放射線影響を最小限にして長期反復被ばくを可能にし，放射線誘発腫瘍が生じる実験系を作った．照射は週3回反復照射で1回当りの線量を0.5～11.8 Gyまで段階的に線量を設定した．11.8～2.5 Gy線量域ではどの線量でも発がん時期と発がん率に変化はなかったが，この線量域から1.5～1 Gy線量域に1 回当りの線量を下げると発がん率に変化はみられず発がん時期の遅延が生じた．1回当りの線量0.5 Gyではマウスの生涯を通じ照射を続けてもがんは生じなかった．この結果はマウスでは，生涯低線量放射線被ばくを受け続けても生存中にがんが発生しない線量，つまりしきい値様線量が存在することを示している． We studied the carcinogenic effects caused by repetitive irradiation at a low dose, which has received attention in recent years, and examined the experimental methods used to evaluate radiation-induced carcinogenesis. For this experiment, we selected a mouse with as few autochthonous cancers as possible. Skin cancer was selected as the target for analysis, because it is a rare cancer in mice. Beta-rays were selected as the radiation source. The advantage of using beta-rays is weaker penetration power into tissues, thus protecting organs, such as the digestive and hematogenous organs. The benefit of our experimental method is that only skin cancer requires monitoring, and it is possible to perform long-term experiments. The back skin of mice was exposed repetitively to beta-rays three times a week until the occurrence of cancer or death, and the dose per exposure ranged from 0.5 to 11.8 Gy. With the high-dose range (2.5-11.8 Gy), the latency period and carcinogenic rate were almost the same in each experimental group. When the dose was reduced to 1-1.5 Gy, the latency period increased, but the carcinogenic rate remained. When the dose was further reduced to 0.5 Gy, skin cancer never happened, even though we continued irradiation until death of the last mouse in this group. The lifespan of 0.5 Gy group mice was the same as that of the controls. We showed that the 0.5 Gy dose did not cause cancer, even in mice exposed repetitively throughout their life span, and thus refer to 0.5 Gy as the threshold-like dose.

本稿ではHPV感染が子宮頸癌を引き起こす分子生物学的機序について概説する。

本稿では子宮体癌の大部分を占めるタイプ１およびタイプ２腫瘍について概説する。

Reactive oxygen species generated by environmental factors, such as radiation, UV and chemicals can cause sequence-specific DNA damage and play important roles in mutagenesis and carcinogenesis. We have investigated sequence specificity of oxidative stress-mediated DNA damage by using ^P-labeled DNA fragments obtained from the human c-Ha-ras-1, p53 and p16 genes. Free hydroxyl radicals cause DNA damage with no marked site specificity. Copper-hydroperoxo complex caused DNA damage at thymine, cytosine and guanine residues. ^1O_2 preferentially induces lesions at guanine residues. Benzoyloxyl radical specifically causes damage to the 5'-G in GG sequence ; this sequence is easily oxidized because a large part of the highest occupied molecular orbital of this radical is distributed on this site. Recently, we demonstrated that BP-7, 8-dione, a metabolite of carcinogenic benzo [a] pyrene (BP), strongly damaged the G and C of the 5'-ACG-3' sequence complementary to codon 273 of the p53 gene in the presence of NADH and Cu (II). BP-7, 8-dione also caused preferential double base lesion at 5'-TG-3' sequences. Since clustered DNA damage is poorly repaired, it is speculated that induction of the double base lesions in DNA might lead to activation of proto-oncogene or inactivation of the tumor suppressor gene. Therefore, oxidative DNA damage induced by BP-7,8-dione, especially double base lesions, may participate in the expression of carcinogenicity of BP in addition to DNA adduct formation. Here, we discuss the mechanisms of sequence-specific DNA damage including clustered DNA damage in relation to mutagenesis and carcinogenesis.

症例は79歳，男性．1991年6月の集団検診で胃ポリープを指摘され，7月当科を受診．上部消化管内視鏡検査にて，食道噴門接合部直下に5㎜大の山田Ⅲ型，表面発赤調の隆起性病変を認めた．組織学的には腺窩上皮の過形成性ポリープであった．以後，毎年内視鏡にて経過観察していたが，肉眼的には変化を認めなかった．2003年4月，同部位からの生検組織が高分化型腺癌であったため，内視鏡的粘膜切除術を施行．病理組織では過形成病変の一部に高分化型腺癌を認め，過形成性ポリープが癌化したものと考えられた．本症例は12年間にわたる経時的観察の後に，過形成性病変からの癌化がみられた稀な症例である．

Reactive oxygen species are capable of causing damage to various cellular constituents, such as DNA, proteins and lipids, leading to carcinogenesis, aging and a number of diseases. We have investigated the sequence specificity of oxidative stress-mediated DNA damage by using ^P-labeled DNA fragments obtained from the human c-Ha-ras-1, p53 and p16 genes. The sequence specificity of DNA damage plays the key role in the mutagenic process, and affects the mutation frequency. Therefore, investigation on sequence specificity of DNA damage would provide clues on the biological significance of DNA damage which in turn may be beneficial for cancer prevention strategy. Here we discuss the mechanisms and sequence specificity of DNA damage caused by various environmental chemicals and UVA-activated photosensitizers in relation to carcinogenesis and aging.

MN/CA IX is a carbonic anhydrase (CA) isoenzyme expressed in normal alimentary tract in a tissue-specific manner. This antigen is activated in the majority of renal cell carcinomas (RCC) but not in normal kidney tissues. Although the exact role of CA activity in carcinogenesis and metastasis has not been established, MN/CA9 has been suggested to be implicated in acidification of extracellular milieu surrounding the cancer cells and thus create a microenvironment conductive to tumor growth and spread. Mutations in the von Hippel-Lindau (VHL) gene cause the familial syndrome and are also found in the majority of sporadic RCC. Wild-type VHL was recently described to down-regulate MN/CA9 in RCC cell lines, and the molecular mechanism of MN/CA9 and VHL in renal carcinogenesis is of interest. To investigate the mechanism of MN/CA9 activation in RCC, we examined the methylation status of this gene in RCC cell lines and human tissue samples and found that hypomethylation in the promoter region may play an important role in the expression of MN/CA9. RT-PCR analysis of blood samples from RCC patients revealed the presence of circulating MN-positive cells in the blood. This antigen may be a potential therapeutic target as well as diagnostic marker for RCC. Therefore, we are currently investigating whether or not MN/CA IX peptide could be an appropriate molecule for use antigen specific immunotherapy on RCC patients.

肝内結石症では, 結石部胆管およびその近傍に胆管上皮の過形成がみられ, しばしば異形成を伴い, 少数例では偶発的に非浸潤癌がみられる. 一方, 肝内胆管癌合併肝内結石症の多くの例で, 浸潤癌の近傍では非浸潤癌領域と異形成を認める. さらに, 細胞動態的, 免疫組織化学的所見を考慮すると, 肝内結石に合併する肝内胆管癌は, 細菌感染を伴う慢性かつ活動性の炎症を背景に, 肝内結石近傍の大型胆管の被覆上皮より発生し, その発癌過程は過形成→異形成→非浸潤癌→浸潤癌というプロセスを経ると思われる. 前癌病変である異形成では, CEAの免疫反応性があり, 病変の同定に役立つ. なお, 癌抑制遺伝子p53は癌進展後期(浸潤癌)にその発現がみられた. しかし, 肝内結石症に合併する肝内胆管癌の発癌過程, 浸潤, 転移の機序は未だ不明で, 癌遺伝子, 癌抑制遺伝子の変化, Carcinogens, Promotorsの同定, 細胞接着分子, 成長因子, 基質分解酵素の生物学等, 残された課題が多い.

A method of DNA-RNA in situ hybridization （ISH） with a nonradioactive photobiotin-labelled DNA probe was established for identification of H-ras mRNAs in fresh frozen tissues of nude mouse tumor and human cervical tumor. In order to elucidate the role of ras onco-gene in the process of carcinogenesis of human uterine cervical cancer, we studied the expression of ras oncogene mRNA in histological specimens of various lesions of human uterine cervix. The fre-quency of enhanced H-ras mRNA expression was increased in accord with the grade of malignancy from 25% in mild dysplasia and 33% in moderate dysplasia to 60% in severe dysplasia and 64% in carcinoma in situ. In invasive squamous cell carcinoma, 53% of the tumors showed an increased H-ras mRNA expression. It was suggested that, in conjunction with our previous immunohistochemical study by anti-p21 monoclonal antibody （Sagae et al. Cancer 66 : 295-301, 1990）, the expression of ras oncogene correlates with the early phase of carcinogenesis of squamous cells of the uterine cervix.

In an earlier report, we demonstrated overexpression of a short isoform of Helios, Hel-5, which lacks three of four N-terminal zinc fingers, in patients with adult T-cell leukemia/lymphoma. Here, we characterized Hel-5 using immunoprecipitation, and gel shift and luciferase promoter assays, and found that Hel-5 lacks the repressor function observed with a full-length isoform of Helios. Moreover, Hel-5 associates with the full-length isoforms of the Ikaros gene family, Ikaros, Aiolos and Helios, and inhibits their DNA binding activity when present in excess, leading to dominant-negative effects on the full-length isoforms of the Ikaros gene family. Our results suggest a critical role for Helios in the mechanism of leukemogenesis.

BACKGROUND/AIMS: The hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, the HBV X gene, which encodes the pleiotropic transactivator HBx, has also been associated with the development of HCC. In this study, we investigated whether nucleotide changes in the X gene of genotype C are associated with the development of HCC. METHODS/RESULTS: We sequenced the X gene in age- and sex-matched 39 HBV-infected patients with HCC and 36 HBV-infected patients without HCC. A novel nucleotide change that resulted in a proline to serine substitution at codon 38 in HBx (codon-38 change) was preferentially found in patients with HCC. Then, sera were collected from a new group of age- and sex-matched 52 patients with HCC and 51 patients without HCC. In this cohort also, the codon-38 change was associated with HCC. Multiple logistic regression analysis showed the prevalence of the codon-38 change was significantly associated with HCC in all patients (P=0.001, odds ratio: 4.89). CONCLUSION: The codon-38 change in genotype C is an independent risk factor for the development of HCC and may serve as a useful molecular marker for predicting the clinical outcomes in patients infected with HBV.

We investigated the therapeutic potential of a newly developed antifibrotic agent, pirfenidone, to regulate airway remodeling and the development of allergic airway inflammation and airway hyperresponsiveness after chronic allergen challenge. Administration of pirfenidone after sensitization but during the period of ovalbumin challenge significantly prevented the development of airway hyperresponsiveness and prevented eosinophil and lymphocyte accumulation in the airways. IL-4, IL-5, and IL-13 levels in bronchoalveolar lavage fluid and ovalbumin-specific serum IgE antibody levels were also significantly reduced. Treatment with pirfenidone significantly reduced transforming growth factor-beta1 and platelet-derived growth factor levels in bronchoalveolar lavage fluid. Pirfenidone reduced the expression of transforming growth factor-beta1, the development of goblet cell hyperplasia and subepithelial collagenization, and the increases in contractile elements in the lung. These data indicate that pirfenidone may play an important role in the treatment of asthma and has the potential reduce or prevent airway remodeling.