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21世紀になって2017年までに，34件のノーベル生理学・医学賞と化学賞が出ているが，その約半数の研究に培養細胞（動物の細胞，あるいは，イースト）が使われている．すなわち，現代の生命科学の研究には，培養細胞がきわめて大きな役割を果たしているといえる．今回は，今から約100年前，細胞を培養して研究を進めようとした先見性に富んだ 2人のノーベル生理学・医学賞受賞者，Alexis Carrel（以下，本文ではカレルとする）と Peyton Rous（以下，ラウス）の経歴と業績を紹介し，彼等の培養細胞との係りについて述べる．

Hepatocellular carcinoma is the third most frequent cause of cancer-related death worldwide; and its incidence rate is increasing. Clinical and molecular medical analyses have revealed substantial information on hepatocarcinogenesis. Hepatocarcinogenesis is a stepwise process during which multiple genes are altered. Genetic changes and their biological consequences in human HCC can be divided into at least 4 groups: i) tumor suppressor genes (p53, retinoblastoma, phosphatase tensin homolog and runt-related transcription factor 3), ii) oncogenes (myc, K-ras, BRAF), iii) reactivation of developmental pathways (Wnt, hedgehog), and iv) growth factors and their receptors (transforming growth factor-α, insulin-like growth factor-2 receptor). An experimental model of human hepatocarcinogenesis such as in vitro neoplastic transformation of human hepatocytes has not been successfully achieved yet, but several immortalized human hepatocyte cell lines have been established. These immortalized human hepatocytes will become useful tools for the elucidation of hepatocarcinogenesis, especially for the initial step of multistep hepatocarcinogenesis.

Background: Interferon-λs (IFN-λs) are novel cytokines with multiple functions, like IFN-α and -β. We examined possible anti-tumour effects produced by adenoviruses bearing the IFN-λ1 or -λ2 gene (Ad/IFN-λ) with the type-35 fibre-knob structure. Methods: Proliferation of oesophageal carcinoma cells transduced with Ad/IFN-λ and mechanisms of the inhibited growth were investigated. Results: Transduction with Ad/IFN-λ upregulated the expression of the class I antigens of the major histocompatibility complexes and induced the growth suppression. Increased sub-G1 populations and the cleavage of caspase-3 and poly (ADP-ribose) polymerase were detected in IFN-λ-sensitive YES-2 and T.Tn cells. The cell death was accompanied by cytoplasmic cytochrome C and increased cleaved caspase-9 and Bax expression, suggesting mitochondria-mediated apoptosis. Adenovirus/IFN-λ-infected YES-2 cells subsequently reduced the tumourigenicity. Adenovirus/IFN-λ-infected fibroblasts, negative for the IFN-λ receptors, induced death of YES-2 or T.Tn cells that were co-cultured. Inoculation of YES-2 cells in nude mice, when mixed with the Ad/IFN-λ-infected fibroblasts, resulted in retardation of the tumour growth. The growth suppression was not linked with upregulated CD69 expression on natural killer cells or increased numbers of CD31-positive cells. Conclusion: Adenovirus/IFN-λ induced apoptosis, and fibroblast-mediated delivery of IFN-λs is a potential cancer treatment by inducing direct cell death of the target carcinoma.

現在，医学分野の多くの研究は培養細胞や培養組織を用いてなされている．それらの研究成果が報告されている論文の“材料と方法”の項で，ダルベッコ培地を使用して細胞を培養したといった記載を読んだ研究者は少なくないであろう．また，実際にダルベッコ培地を使用した研究者もいると思う．今回は，この培地を報告したDulbecco（1975年，がんウイルスの研究によってノーベル賞）の波乱万丈の経歴，彼の人脈（多くの人物がノーベル賞を受賞），彼の研究概略などについて紹介したい．そして，筆者のダルベッコ培地に対する見解とその培地を使用した経験について述べる．筆者の結論は，初代細胞培養にはダルベッコ培地が勧められること，また，培養細胞を用いての毒性検定に際しては，使用する培地によって結果が異なることがあるので注意する必要があることである．

Out of normal human fibroblasts OUMS-36 and three clones (T1, T2 and T3) of telomere reverse transcriptase gene ( hTERT )-transfectants, telomere length is in order: T3 >T2 >T1 >>OUMS-36 (young) >>OUMS-36 (old), and telomerase activity is in order: T2 >T3 >T1 >>OUMS-36 (young, old), suggesting that telomere length may be roughly governed by telomerase activity. Telomere-like oligonucleotides [5′- (TTAGGG) 1–3 -3′] (ON mono-/di-/trimer) and a mononucleotide (T:A:G=2:1:3, mol/mol) mixture (MN mix), as candidates for telomerase activators, maintained above 80% of cell viability at marginally higher doses of 2 μM for MN-mix or ON monomer, 1 μM for ON dimmer and 0.67 μM for ON trimer, respectively, in OUMS-36 and T2 cells, and administered for 4 weeks, resulting in no elongation of telomere length in both the cell lines. In contrast, telomerase activity was enhanced by administration with ON mono-/di-/trimer, but not MN mix, in a manner dependent on treatment periods, in T2 transfectants, whereas similar effects were not observed in OUMS-36 parents. The 4-week treatment with ON mono-/di-/trimers, but not MN mix, also suppressed cell-viability diminishment induced by the oxidative-stressor tert -butylhydroperoxide in T2 cells, but scarcely in OUMS-36 cells. Thus, the promoting effects of oligonucleotide [5′-(TTAGGG) 1-3 -3′] on both telomerase enhancement and oxidative-stress resistance can be exerted for telomerase-abundant T2 hTERT -transfectants, but not for telomerase-poor OUMS-36 parents.

約1世紀前に, 生きた組織や細胞を動物の体外に取り出した研究―組織培養あるいは細胞培養―が始まった．その後の研究の流れの中で, 1) 組織・細胞培養の特性を生かして行われた研究でノーベル賞に輝いた研究, 2) 組織・細胞培養の経験をもつノーベル賞受賞者, 3) ノーベル賞にはならなかったけれども特記できると私が考える細胞培養の研究，などについて取り上げてみたい.

本報告は、平成20年度より開始された「質の高い大学教育推進プログラム」に、これまでの本学の国際交流・国際貢献の取組をまとめて応募した申請書の内容を中心としたものである。本学の、オーストラリア研修旅行、アメリカ研修旅行、カンボジア・スタディツアー、新見英語サロン等の実践についての報告である。, This report is based on the application form for the Distinctive University Education Support Program (Good Practice [GP]) of the Ministry of Education, Culture, Sports, Science and Technology of Japan. We have overviewed the Study Tour to Australia, the Study Tour to the United States of America, the Study Tour to Cambodia, Niimi English Salon and so forth.

平成19年度特色ある大学教育支援プログラムの「教育方法の工夫改善を主とする取組」に応募したところ、全国から331件の応募があり、本取組が審査によって選定された。短期大学士課程では、70件中12件のみ選定された。そこで、看護学科全員で取組んでいる看護研究について申請内容を中心に報告する。, We applied for Support Program for Distinctive University Education in the category of improvement of teaching methods in 2007, and was selected as one of the "Good Practice (GP)" out of 331 programs nationwide. Only 12 programs were chosen out of 70 applicants in the junior college division. This is a report of the application about the nursing research in which all the faculty members of the Department of Nursing have been engaged

本学の学生が国際ボランティア活動について学ぶことができる研修制度の設立のため、我々は、2006年1月5日～1月9日、カンボジアのシェムリアップ市においてボランティア活動を継続している岡山県に本部を置くNGOを視察した。その団体の活動は、孤児・地雷障害者・母子家庭・エイズ患者の支援プロジェクト、井戸掘り・ジャックフルーツ植樹プロジェクト、および職業訓練施設(子供レストラン技能学校)の運営などである。帰国後、「新見公立短大カンボジア会」を設立して学生メンバーを募った。現在、我々は、支援物資の調達などの国内ボランティア活動、定例ワークショップの開催、開発途上国とそれらの国に係わるNGO (nongovernmental organization:非政府組織)のこと、国際協力に関する啓発・学習などを行っている。本稿では、カンボジアでの現地研修の報告と共に、今後の「新見公立短大カンボジア会」活動について検討する。, We are helping students to learn International Contributions on our own college program. At the beginning of that, we visited The Kingdom of Cambodia to inspect Japanese NGO (nongovernmental organization) for 4days. We have got much experience there and learned how participants in NGO could support a mother-and-child family, orphans and mine disorders. After we came back here, we have established an activity group in our college. We report here the possibility our Cambodia study tour, various activities with our students and how we can make our students understand matters of International Contributions.

本短大看護学科の地域貢献活動について、平成16年度から開始された文部科学省の現代的教育ニーズ取組支援プログラム(現代GP)において設定されたテーマのうち「地域活性化への貢献(地元型)」へ応募し、今回平成18年度の審査において選定され、大学改革推進等補助金が受けられることに決定した。本論文は、その申請内容を基に、看護学科の地域貢献活動と看護専門職養成との融合である「サービス・ラーニング」の取組を紹介する。, The Nursing Department of Niimi College entered the research paper contest in the field of "Contribution for Local Revitalization", which is one of the themes set in modern educational needs support programs (Modern GP) started by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in 2004. In 2006 our paper was selected as one of the winners, and MEXT are to award us the subsidy for the promotion of college reform. This study, based on the contents of the entry paper, shows "Service Learning" which is the combination of nursing training and local contribution activities of our nursing department.

本研究は一人でも多くの学生に読書習慣を身につけさせるための方法を考え出すことを目的として行った。そのために、2005年春休みに、看護学科1年生と2年生、地域福祉学科1年生、そして、5月の連休に、幼児教育学科1年生と2年生に、読書感想文を提出してもらい、その結果を分析した。この読書の課題は、正規の授業科目ではないので、提出ほ各自の裁量にまかせた。その結果、提出率は約87%であった。読まれた本を分類すると、ノンフィクションが多く(53%)、次いで、小説(23%)、実用書(17%)、随筆その他など(6%)、となった。もっともよく読まれた本は『五体不満足』であったが、281人中のわずか7人であった。このことは、学生が広範囲にわたって種々の本を読んでいることを示している。また、最近テレビで放映されたドラマを文字化した本や、新聞や雑誌で大きく宣伝されている本がよく読まれている傾向があった。このことは、歴史に耐えた良書が見のがされる危険性を示唆している。また、今回の感想文の分析より、学生に読書習慣を身につけさせるには、学生の目線にあった、あるいは、それより少し高いレベルの本を薦める必要があることが分かった。, We attempted to learn what measures could be useful for letting students of Niimi College read more books. For this purpose, we asked students of Nursing, Early Childhood Education, and Community Welfare Courses of this college to voluntarily submit their impressions of the book they read recently. Then we analyzed what books the students liked to read. As a result, more than 50% of them read books classified as non-fiction. About 30% of students of Early Childhood Education and Community Welfare Courses read novels, but only 16% of the students of Nursing Course did so. Interestingly, the books which have recently been dramatized on TV or strongly advertised in the newspapers or magazines showed a tendency to be read more frequently. These findings indicated that there are some problems for students to choose books worth reading. Whether recommendation of books from each teacher to the students can enhance their willingness to read and advance their selection power remains to be studied in the future.

2004年(平成16年)4月、本学では、将来構想委員会が「新見公立短期大学在学生アンケート(志望動向)」による在学生の受験志望動向に関する調査を行った。本報告は、その調査結果の集計をもとに、2002年度に本学で行われた「第1回学生生活実態調査」(学生生活委員会および自己点検・評価委員会による)集計と比較し、在学生の受験動向をまとめたものである。その結果、全学的に本学第一志望の学生の占める割合が増加していること、看護学科で国公立四年制大学志望者が増加していることが分かった。そして、公立・免許資格・専門職養成の三大動機が本学志望の特徴であることが明らかになった。, The planning committee of Niimi College investigated the entrance examination trends of our college in April, 2004. An investigation on students' life was done by the student affairs committee and the self evaluation committee in Niimi College in 2002. This report compares the two investigations, and reveals the students' trends of Niimi College's entrance examination. As a result of an investigation on entrance examination to Niimi College, we came to understand that the number of students who wanted to come to Niimi College as their first choice is increasing in all the departments, and that in the Department of Nursing, the number of students who wanted to go to a four-year national university is increasing. We have also clarified that there are the three major motives:1) being public, 2) providing license qualifications, and 3) being a professional training facility.

We examined possible application of a regulatory region of midkine (MK) gene, which is frequently upregulated in a number of human tumours but not in normal cells, to cancer gene therapy. We examined transcriptional activity of the MK genomic fragments in paired cell lines, immortalized cells and their parental normal fibroblasts, and found that the MK fragments activated a fused reporter or a suicide gene preferentially in the immortalized cells. Recombinant adenoviruses (Ad), in which the MK fragment was inserted upstream to the E1A gene (AdMK), replicated preferentially in the immortalized cells and were cytotoxie to them. Human hepatocellular carcinoma cells were significantly susceptible to AdMK compared with human normal fibroblasts in vitro and the replication of AdMK was less than that of wild-type Ad in the infected fibroblasts. Hepatocellular carcinoma cells infected with AdMK did not form tumours in immunocompromised mice and intratumoural injection of AdMK into the hepatocellular carcinoma developed in mice retarded the subsequent tumour growth. Expression of E1A and necrosis of tumours were detected in AdMK-injected but not control Ad-injected cases. The MK promoter-driven suicide gene therapy and -mediated replicative Ad can thereby produce cytotoxic effects to immortalized and tumour cells with minimal damage to normal cells.

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825-835). This paper addresses a question whether transforming growth factor beta (TGFbeta) shares the pathway with high Ca2+. On exposure of the cells to TGFbeta1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C alpha (PKCalpha) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFbeta1-induced growth inhibition was almost completely mitigated when PKCalpha activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCalpha-S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFbeta1.

An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at 10Thr and 94Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21CIP1/WAF1, a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21CIP1/WAF1. In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca2+-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

Cytotoxic free radicals generated during the metabolism of carbon tetrachloride by cytochrome P450 2E1 (CYP2E1) are thought to cause hepatotoxicity. Here, the cytotoxic effects of carbon tetrachloride in a liver cell line expressing CYP2E1 (HLE/2E1) are compared with those in the mother cell line (HLE). The effects of carbon tetrachloride on the gene expression of HSP70, a potential marker of oxidative stress, were also examined. The viability of HLE/2E1 cells after exposure to carbon tetrachloride was significantly decreased compared with that of HLE cells. Northern blot analysis revealed that the HSP70 mRNA level was significantly increased after carbon tetrachloride treatment in both cell lines, while the magnitude of its increase was much greater in HLE/2E1 cells than in HLE cells. These results suggest that the oxidative stress induced by CYP2E1 plays an important role in the increase in cytotoxicity of carbon tetrachloride in CYP2E1-overexpressing cells.

Summary. All-trans retinoic acid (ATRA) has been shown to induce differentiation of human acute promyelocytic leukaemia (APL) cells and eventual elimination of the malignant clone. Matrix metalloproteinase-9 (MMP-9) is produced by neutrophils and its expression appears to be linked with myeloid cell differentiation. We investigated effects of ATRA on MMP expression in two human myeloid leukaemia cell lines, PL-21 and NB4. Both cells could differentiate into neutrophils after exposure to ATRA. Both the activity and antigen levels of MMP-9 were much higher in NB4 cells than in PL-21 cells. Stimulation with ATRA significantly increased MMP-9 levels approximately three- to fivefold in both PL-21 and NB4-conditioned media. MMP-9 mRNA levels increased in ATRA-treated cells and was almost in parallel with the increase in MMP-9 activity, suggesting that ATRA induced MMP-9 by activating its gene expression. ATRA can induce interleukin 8 (IL-8) in APL cells. IL-8, chemokine for neutrophils and a potent inducer of MMP-9, was also induced by ATRA in PL-21 cells. However, recombinant IL-8 did not induce MMP-9 expression. In addition, a neutralizing antibody against IL-8 did not inhibit ATRA-induced MMP-9 expression in either cell type. These observations suggest that ATRA can induce both MMP-9 and IL-8, but IL-8 is not involved in ATRA-induced MMP-9 expression. As MMP-9 can truncate and activate IL-8, simultaneous induction of MMP-9 and IL-8 by ATRA could activate leucocytes excessively, causing the hyper-inflammatory events in retinoic acid syndrome.

Endostatin derived from collagen XVIII is a potent endogenous anti-angiogenic factor that induces regression of various tumors of epithelial origin. Endostatin has been shown to inhibit endothelial cell functions, however, its effect remains controversial. We first attempted here to apply the inhibitory effect of recombinant human endostatin on chondrosarcomas, which originate from the mesenchyme, in nude mice. Endostatin induced reduction of chondrosarcoma growth and tumor angiogenesis in vivo. However, endostatin showed no effect on the proliferation and migration of chondrosarcoma cells in vitro. Next, we investigated the interactions between endostatin and endothelial cells in detail. Endostatin inhibited the migration on and attachment to collagen I but did not affect the proliferation of endothelial cells. Although the migration of endothelial cells was stimulated by angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor, endostatin showed similar inhibitory effects on it in the presence and absence of the stimulants. Moreover, the inhibitory effect against endothelial cell attachment to collagen I was attenuated or modulated in the presence of neutralizing antibodies of alpha(2), alpha(5)beta(1), and alpha(V)beta(3) integrins but not that of alpha(1) integrin. Our results suggest that endostatin might suppress the alpha(2)beta(1) integrin function of endothelial cells via alpha(5)beta(1) or alpha(V)beta(3) integrin. We propose here that endostatin might be effective for anti-angiogenic therapy for human chondrosarcomas through the suppression of alpha(2)beta(1) integrin functions in endothelial cells.

Using 2-dimensional gel electrophoresis, we previously demonstrated that the S100C protein remarkably decreased after immortalization of normal human fibroblasts, and that this protein caused growth inhibition of human tumor cells when forcibly expressed in these cells, suggesting that S100C plays a significant role in tumor suppression. The present study was carried out to determine what type of human tissues express S100C protein, and, subsequently, whether the S100C content in these tissues changes after normal cells have been transformed into cancer cells. We found that ductal cells in various tissues were positively stained with the S100C protein. In comparison, epithelial cells in digestive organs such as the stomach, small intestine, and colon were not stained as strongly. When 14 pairs of human normal and cancerous tissues were stained with the antibody, decreases in the staining levels of S100C were observed in 6 kinds of cancerous tissues--from the bronchus, mammary duct, renal tubule, prostate, uterus, and testis--in comparison with staining in their normal counterparts. These results suggest that S100C is a new tumor marker protein, the expression of which significantly decreases after malignant transformation of human tissues.

The human REIC gene is a recently found mortalization-related gene and a candidate tumor suppressor gene expression of which is largely attenuated in many immortalized and tumor-derived cell lines (Biochem. Biophys. Res. Commun. 268 (2000) 20-24). To gain insight into the mechanisms of the down-regulation, we investigated the genomic structure and promoter activity of the human REIC gene. The gene, identical with the DKK-3 gene, resides on chromosome 11p15.1, consists of nine exons, and has two promoters. Methylation in the main promoter region was detected in 11 out of 21 cell lines tested (52%) derived from a variety of human tumors, in which the expression of the REIC gene was decreased. In ten of these 11 cell lines the minor promoter was also methylated. Similarly, the REIC gene expression was decreased in 14 of 24 fresh non-small cell lung cancer specimens (58%) compared to that in corresponding non-cancerous tissue, though allelic loss and tumor-specific mutation were rare. Of these 14 tumors, at least five tumors exhibited heavy methylation of the REIC promoter region. These results indicate that the down-regulation of the REIC gene expression is ascribed to the aberrant promoter hyper-methylation at least in a subset of human tumors. The expression was restored upon treatment of SQ5 cells with 5-aza-deoxycytidine, confirming DNA methylation as the mode of downregulation. A notable single nucleotide polymorphism in the coding region (cSNP) with an amino acid substitution of glycine (GGG) to arginine (AGG) was found at codon 335 of the REIC gene. However, the distribution of the cSNP showed no significant difference between lung cancer patients and healthy population.

We recently reported the cloning of the REIC/Dkk-3 gene, whose expression was shown to be down-regulated in many human immortalized and tumor-derived cell lines [T. Tsuji et al. (2000) Biochem. Biophys. Res. Commun. 268, 20-24]. In the present study, we demonstrated that expression of the exogenous REIC/Dkk-3 gene in tumor cells inhibited cell growth. Furthermore, the level of REIC/Dkk-3 mRNA in normal human cells was lowest in the late G(1) phase during the cell cycle. Then we found that the expression of REIC/Dkk-3 was significantly down-regulated in surgically resected non-small-cell lung carcinomas. We determined the REIC/Dkk-3 locus on chromosome 11p15, where loss of heterozygosity has frequently been observed in human tumors. These findings indicate that REIC/Dkk-3 may function as a tumor suppressor.

Many lines of evidence indicate that the immortalization step is critical for the neoplastic transformation of normal human cells. Once normal human cells have been immortalized, they are relatively easily transformed into neoplastic cells. In order to understand these phenomena, patterns of protein phosphorylation in proliferating normal human fibroblast cell strains and their immortalized cell lines were compared by using two-dimensional polyacrylamide gel electrophoresis. It was found that the expression and phosphorylation levels of the human heat shock protein 27 (HSP27) were predominantly downregulated in the immortalized cells compared with those in their normal counterparts. In the normal cells, HSP27 expression and phosphorylation were markedly increased by physiological and nonphysiological stresses, such as serum addition, treatment with a carcinogenic agent like 4-nitroquinoline-1-oxide, and a high osmotic pressure. This may be a normal defense against acute changes of cellular environment and cytotoxic effects. However, these stresses had no effects on the expression and phosphorylation of HSP27 in the immortalized cells. These results suggest that an abnormal regulation of HSP27 expression and phosphorylation may be one of the reasons for easy neoplastic transformation of the immortalized cells by the treatment with carcinogenic agents.

By transfection of an expression vector of human cytochrome P450 2E1 (CYP2E1) into a human hepatoma cell line (HLE), a new cell line (HLE/2E1) that stably expresses activity of CYP2E1 has been established. The HLE/2E1 cell line expressed a higher level of CYP2E1 messenger ribonucleic acid than did the mother HLE cell line. CYP2E1 enzyme activity determined by a p-nitrophenol oxidation assay was also higher in HLE/2E1 cells than in HLE cells. In addition, the enzyme activity of the HLE/2E1 cells was increased by ethanol treatment. Exposure to acetaminophen (APAP) or buthionine sulfoximine (BSO) caused a greater decrease in viability of the HLE/2E1 cells than that of the HLE cells, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The cytotoxicity of APAP or BSO to HLE/2EI cells was inhibited by the addition of ethanol or vitamin E. However, the cytotoxicity of both APAP and BSO was enhanced by 24-h preincubation of HLE/2E1 cells with ethanol. These results show that this cell line provides a useful model for studying catalytic properties of CYP2E1 and cytotoxic mechanisms of chemicals metabolized by CYP2E1.

Many lines of evidence indicate that neoplastic transformation of cells occurs by a multistep process. For neoplastic transformation of normal human cells, they must be first immortalized and then be converted into neoplastic cells. It is well known that the immortalization is a critical step for the neoplastic transformation of cells and that the immortal phenotype is recessive. Thus, we investigated proteins downregulated in immortalized cells by two-dimensional gel electrophoresis. As a result, S100C, a Ca2+-binding protein, was dramatically downregulated in immortalized human fibroblasts compared with their normal counterparts. When the cells reached confluence, S100C was phosphorylated on threonine 10. Then the phosphorylated S100C moved to and accumulated in the nuclei of normal cells, whereas in immortalized cells it was not phosphorylated and remained in the cytoplasm. Microinjection of the anti-S100C antibody into normal confluent quiescent cells induced DNA synthesis. Furthermore, when exogenous S100C was compelled to localize in the nuclei of HeLa cells, their DNA synthesis was remarkably inhibited with increase in cyclin-dependent kinase inhibitors such as p16Ink4a and p21Waf1. These data indicate the possible involvement of nuclear S100C in the contact inhibition of cell growth.

Normal human cells stop proliferation after a certain number of cell divisions. This phenomenon is called cellular aging. The fact that the senescence phenotype is dominant and the immortal one is recessive indicates that immortalization of human cells may be caused by loss of functions of certain genes in normal cells. Based on this evidence, several cDNA clones whose expression was down-regulated during the immortalization process of human cells were isolated by the representative difference analysis (RDA) system in our laboratory. One of them, which was named REIC, was expressed to a lower degree in three human immortalized cell lines as compared with their parental normal counterparts. In addition, the expression of REIC was markedly lower in eight human tumor-derived cell lines (Hep3B and HuH-7 hepatocellular carcinomas, HuH-6 Clone 5 hepatoblastoma, HuCCT-1 cholangiocarcinoma, A549 lung cancer, HaCaT immortalized keratinocyte, HeLa cervical carcinoma, and Saos-2 osteosarcoma). In contrast, among the human tissues examined, the heart and brain, which contain a large number of post-mitotic cells, showed the highest expression of REIC. The full-length REIC cDNA revealed that the predicted protein is 350 amino acids in length and possesses coiled-coil tertiary structures in each of the amino- and carboxyl-termini. Furthermore, a search of the protein database revealed a match of this gene product with Dkk-3, which is a novel inhibitor of Wnt oncogene. These results indicate that the REIC cloned by us may function as a tumor suppressor.

Deregulation of cell cycle regulatory mechanisms leads to disruption of normal control of cell growth and may be associated with neoplastic transformation. To determine whether immortalized human cells have alterations in their cell cycle regulatory mechanisms, we analyzed cell cycle regulatory proteins in 2 immortalized human fibroblast cell lines, KMST-6 and OUMS-24/P6X, established by repeated irradiation with 60Co gamma rays alone or mutant p53 gene transfection plus X-ray irradiation, respectively. Both the immortalized cell lines had markedly enhanced activity of cyclin A-associated kinase as compared with their normal counterparts. The high activity of cyclin A-associated kinase was well correlated with the increased expression of cyclin A mRNA and its protein. In addition, the immortalized cell lines showed significantly reduced amounts of p21Cip1/Waf1/Sdi1, a potent inhibitor of cyclin dependent kinases. Furthermore, among the pRb family of proteins, p107 and p130, were hyperphosphorylated in both the immortalized cell lines, suggesting possible participation in upregulation of cyclin A associated kinase activity. These changes represent some important characteristics of immortalized cells. Int. J. Cancer 82:754–758, 1999. © 1999 Wiley-Liss, Inc.

Drinking alcohol sometimes causes cardiac arrhythmia, but the precise mechanism remains unknown. To study the mechanism, we investigated the effects of ethanol exposure on the beating rate of cultured chick cardiac myocytes. Primary cultures of cardiac myocytes were prepared from the ventricles of 14-day-old chick embryos and then treated with ethanol which, in the range of 0.3 to 1.5 vol%, increased the beating rate in a dose-dependent manner. Ethanol (0.6 vol%) caused an increase in the beating rate, but disopyramide (5 microg/ml) and procainamide (10 microg/ml), Na+ and K+ channel blockers, inhibited the increase in the beating rate significantly. Neither lidocaine (5 microg/ml) nor mexiletine (2 microg/ml), Na+ channel blockers, nor calcium antagonist verapamil (5 ng/ml) inhibited the increase. However, tetraethylammonium chloride (ranging from 15 to 30 mmol/l), a K+ channel blocker, inhibited the increase. These findings indicate that ethanol increases the beating rate of cultured chick cardiac myocytes via the activation of the K+ channel. This experimental model may be useful in studying the effect of ethanol on the K+ channel.

The actions of phenobarbital, a liver tumour promoter, on growth and differentiation of primary culture normal rat hepatocytes change biphasically as a function of its concentration. At low concentrations of 0.5-2 mmol/L, phenobarbital enhances DNA synthesis of normal adult rat hepatocytes in the presence of epidermal growth factor (EGF) and/or dexamethasone. This is also true for normal suckling (1-2-week-old) rat hepatocytes, without added growth factor(s), in serum-free primary culture. Contrarily, phenobarbital at high concentrations (3-4 mmol/L) suppresses DNA synthesis of suckling rat hepatocytes. Furthermore, phenobarbital inhibits DNA synthesis of transforming growth factor-a-stimulated primary hepatocytes from normal adult rats in a dose-dependent manner within a concentration range of 3-6 mmol/L. When normal adult rat hepatocytes are led to undergo multiple proliferative cycles upon stimulation with hepatocyte growth factor (HGF) and EGF in the chemically defined hepatocyte growth medium (HGM), 3 mmol/L phenobarbital also remarkably suppresses DNA synthesis. Phenobarbital at 3 mmol/L effectively keeps these hepatocytes morphologically differentiated and accelerates restoration of the expression of markers characteristic of differentiated cells after the initial cellular growth phase. In addition, phenobarbital efficiently supports prolonged survival of the hepatocytes.

Background —Tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) modulate heart failure in part by provoking the hypertrophic response. Signal transduction pathways of those factors are implicated in reactive oxygen intermediates (ROIs). Therefore, we hypothesized that TNF-α and Ang II might cause myocyte hypertrophy via the generation of ROIs. Methods and Results —To test the hypothesis, we tested whether TNF-α and Ang II could induce the generation of ROIs and whether antioxidants such as butylated hydroxyanisole (BHA), vitamin E, and catalase might inhibit the hypertrophy in cultured neonatal rat cardiac myocytes. ROIs were measured by the ROI-specific probe 2′,7′-dichlorofluorescin diacetate in cultured cardiac myocytes. We demonstrated that TNF-α and Ang II induced the generation of ROIs in a dose-dependent manner. TNF-α (10 ng/mL) and Ang II (100 nmol/L) enlarged cardiac myocytes and increased [ 3 H]leucine uptake, and BHA (10 μmol/L) significantly inhibited both effects. Other antioxidants, such as vitamin E (1 μg/mL) and catalase (100 U/mL), also inhibited the enlargement of cardiac myocytes induced by TNF-α. Conclusions —These results indicate that TNF-α and Ang II cause hypertrophy in part via the generation of ROIs in cardiac myocytes.

To study the mechanisms of immortalization of human cells, an early step in cancer development, we compared the cellular proteins of normal and immortalized human fibroblasts. Two-dimensional gel electrophoresis showed that one spot with a molecular mass 20 kDa and an isoelectric point of 6.0, became significantly smaller after immortalization of human cells. Further, the spot was rarely observed in four human liver cancer cell lines. Investigation of the N-terminal amino acids revealed that the spot was a fragment of alpha-2-macroglobulin. Although the 20 kDa fragment contains methionine, the spot was not labeled with [35S]methionine. Thus we concluded that the spot might be derived from the culture medium. These results indicated that intracellular metabolism of a-2-macroglobulin, which is a multifunctional protease inhibitor, changed after the cells were transformed.

The 3' terminal region (3'-X tail) of hepatitis C virus (HCV) genomic RNA forms a stable stem-loop structure. The 3'-X tail consists of 98 nucleotides (nt) that are highly conserved among the HCV strains and supposed to function as a cis-acting region for replication of negative strand RNA and/or viral encapsidation. In the present study, by UV cross-linking assay we found two kinds of cellular proteins of approximately 87 and 130 kDa, which specifically bind to the full-length 3'-X tail (nt 1 to 98), but not the 3'- or 5'-truncated 3'-X tail, consisting of nt 1 to 50 or nt 51 to 98, respectively. These proteins were detected in human cell lines such as hepatic tumor cell lines and a T-lymphocyte cell line and also in a human embryonic lung fibroblast cell strain. In addition, human hepatocellular carcinoma tissues expressed these proteins regardless of infection or uninfection of HCV. Furthermore, these proteins were also detected in normal human tissues derived from the lung, heart, kidney, stomach, intestine, and colon. Thus, these cellular proteins, which are ubiquitously present in human tissues, might be involved in viral replication and/or encapsidation.

We examined a relationship between p21 WAF1/CIP1/SDI1 and cell-cycle-related proteins in 12 human liver tumor cell lines (JHH-1, -2, -4, -5, -6, -7; HLE; HuH-7; Hep3B; PLC/PRF/5; HuH-6; HepG2). Seven (JHH-1, -2, -5, -6, -7; Hep3B; HepG2) out of eight cell lines having p21 WAF1/CIP1/SDI1 protein overexpressed cyclin E protein, although one of them (JHH-5) overexpressed a reduced size of cyclin E. The rest (HuH-6) of the 8 cell lines with p21 WAF1/CIP1/SDI1 showed a decreased expression of cyclin E. Four cell lines (JHH-4; HLE; HuH-7; PLC/PRF/5) deficient of p21 WAF1/CIP1/SDI1 protein did not overexpress cyclin E protein. As to expression of the other cell-cycle-related proteins, cyclin A, cyclin D1, CDK2 or CDK4, no significant difference was detected among the 12 cell lines. These findings indicate that the human liver tumor cell lines which have the p21 WAF1/CIP1/SDI1 -inducible barriers of the cell cycle progression can go through the G1/S checkpoint by overexpressing cyclin E.

In a previous paper, we demonstrated that cultured human fibroblasts synthesize transferrin (Tf). Two types of Tf are present; one is produced by the cells and the other is internalized from the culture medium. To study the metabolism of intracellular Tf, we investigated the subcellular localization of the two types of Tf in human fibroblasts by immunocytochemical and fluorescence-labeling techniques. The internalized Tf was found to be localized in the perinuclear area, and the synthesized Tf was associated with microtubules, forming a fibrous structure in the cytoplasm. When the cells were treated with colchicine which depolymerizes microtubules irreversibly, the synthesized Tf lost its fibrous structure and spread out in cytoplasm, but the internalized Tf remained around the nucleus. These results suggest that the two types of Tf are regulated differently in the cells.

We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promoter in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth. p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30°C but not at 37°C. Four temperature-sensitive p53 mutations were isolated: CAT→CGT at codon 214 (H214R), TAC→TGC at codon 234 (Y234C), GTG→ATG at codon 272 (V272M), and GAG→AAG (E285K). Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization. Mol. Carcinog. 19:243–253, 1997. © 1997 Wiley-Liss, Inc.

According to the telomere hypothesis of senescence, the progressive shortening of telomeres that occurs upon division of normal somatic cells eventually leads to cellular senescence. The immortalisation of human cells is associated with the acquisition of a telomere maintenance mechanism which is usually dependent upon expression of the enzyme telomerase. About one third of in vitro immortalised human cell lines, however, have no detectable telomerase but contain telomeres that are abnormally long. The nature of the alternative telomere maintenance mechanism (referred to as ALT, for Alternative Lengthening of Telomeres) that must exist in these telomerase-negative cells has not been elucidated. It has previously been shown that abnormal lengthening of yeast telomeres may occur due to mutations in the yeast telomerase RNA gene. That this is not the mechanism of the abnormally long telomeres in ALT cell lines was demonstrated by the finding that seven of seven ALT lines have wild-type human telomerase RNA (hTR) sequence, including a novel polymorphism that is present in 30% of normal individuals. We found that two ALT cell lines have no detectable expression of the hTR gene. This shows that the ALT mechanism in these cell lines is not dependent on hTR. Expression of exogenous hTR via infection of these cells with a recombinant hTR-adenovirus vector did not result in telomerase activity, indicating that their lack of telomerase activity is not due to absence of hTR expression. We conclude that the ALT mechanism is not dependent on the expression of hTR, and does not involve mutations in the hTR sequence.

In vitro cell transformation is a valuable approach for studying the mechanisms of multistep carcinogenesis of human cells. Since immortalization is an essential step for in vitro neoplastic transformation of human cells, this study addresses the question of whether mutant p53 contributes to the immortalization process of human cells. The mutant p53 gene (mp53: codon273Arg-His) was introduced into normal human fibroblasts (OUMS-24 line) and a G418-resistant clone, OUMS-24/P6 line, was obtained. This clone showed an extended life span and chromosome abnormalities, but senesced at the 79th population doubling level (PDL). When these cells were subjected to intermittent X-ray treatment, they became an immortalized cell line (OUMS-24/P6X). Although these immortalized cells showed chromosome abnormalities, they were not tumorigenic. On the other hand, normal OUMS-24 cells into which mp53 had not been introduced were not immortalized by the same X-ray treatment. These results indicate that introduction and expression of mp53 alone were not sufficient for immortalization of human cells, and that mutations of the remaining wild-type p53 or other genes may have been necessary for immortalization. In fact, no expression of the wild-type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in an increase in cdk2 and cdc2 kinase activity. These findings indicate that the p53-p21 cascade may play some role in the immortalization of human cells. On the other hand, there was no significant difference in expression of proteins such as Rb, p16, cdk4, cdk6, cyclin A and cyclin D1 between the normal and immortalized human fibroblasts.

We examined the combinatory antitumor effects of adenoviruses expressing human mda-7/IL-24 gene (Ad-mda-7) and chemotherapeutic agents on nine kinds of human esophageal carcinoma cells. All the carcinoma cells expressed the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) receptor complexes, IL-20R2 and either IL-20R1 or IL-22R1, and were susceptible to Ad-mda-7, whereas fibroblasts were positive only for IL-20R2 gene and resistant to Ad-mda-7-mediated cytotoxicity. Sensitivity of these esophageal carcinoma cells to Ad-mda-7 was however lower than that to Ad expressing the wild-type p53 gene. We thereby investigated a possible combination of Ad-mda-7 and anticancer agents and found that Ad-mda-7 with 5-fluorouracil (5-FU), cisplatin, mitomycin C or etoposide produced greater cytotoxic effects than those by Ad-mda-7 or the agent alone. Half-maximal inhibitory concentration values of the agents in respective cells were decreased by the combination with Ad-mda-7. Cell cycle analyses showed that Ad-mda-7 and 5-FU increased G2/M-phase and S-phase populations, respectively, and the combination augmented sub-G1 populations. Ad-mda-7-treated cells showed cleavages of caspase-8, -9 and -3 and poly (ADP-ribose) polymerase, but the cleavage levels were not different from those of the combination-treated cells. Ad-mda-7 treatments upregulated Akt phosphorylation but suppressed IκB-α levels, whereas 5-FU treatments induced phosphorylation of p53 and extracellular signal-regulated protein kinases 1 and 2. Molecular changes caused by the combination were similar to those by Ad-mda-7 treatments, but the Ad-mda-7-mediated upregulation of Akt phosphorylation decreased with the combination. These data collectively suggest that Ad-mda-7 induced apoptosis despite Akt activation and that the combinatory antitumor effects with 5-FU were produced partly by downregulating the Ad-mda-7-induced Akt activation.

In vitro models of malignant transformation of human cells may provide considerable insight into the mechanisms of multistep carcinogenesis. It is well established that normal human cells must be immortalized before they can be malignantly transformed; however, they are stringently destined for aging and are rarely immortalized. The mechanism of cellular aging and immortalization is still unknown. We detected expression of only mutated p53 mRNA by direct sequencing of the reverse-transcribed mRNA in 3 human cell lines immortalized either with 4-nitroquinoline 1-oxide or with 60Co gamma rays. Consequently, only the mutated p53 protein was expressed in each immortalized cell line. The expression of sdiI/p21 and mdm2, both of which are positively regulated by wild-type p53, was significantly down-regulated in the immortalized cell lines, resulting in over-expression of cdk2 and cdk4. Introduction of the sdiI/p21 gene into these cells was followed by a remarkable decrease in their ability to synthesize DNA. These results indicate that the p53 cascade may play an important role in the immortalization of human cells. © 1996 Wiley-Liss, Inc.

A human colon carcinoma cell line designated OUMS-23 has been established from metastatic pericardial fluid of a male familial adenomatous polyposis patient with colon cancer. Since 1984, the epithelial cells have been maintained in culture. Ultrastructural studies revealed the presence of numerous microvilli on the cell surface and desmosomes between the adjacent cells. The cells secreted carcinoembryonic antigen into the culture medium (15 ng/10(6) cells-1 24 h-1). The cells expressed heat-stable placental-type-like alkaline phosphatase, whereas the normal counterparts expressed tissue-unspecific alkaline phosphatase. Karyotypic analysis showed that the cell line was of human origin and that the chromosome number was broadly distributed between 53 and 118. Southern blot analysis of the APC gene revealed no abnormalities in OUMS-24 cells, while Northern blot analysis demonstrated that the expression of the gene was about one-half that of the normal human fibroblasts. No mutations at the "hot spots" of codons 12 and 61 of H-, K- and N-ras proto-oncogenes were detected in the cells. The cells could grow in soft agar at a cloning efficiency of 6.5%, and upon transplantation into nude mice the cells formed tumors, which were diagnosed as differentiated adenocarcinoma.

Many studies on in vitro transformation of human cells indicate that the cells must be immortalized before they can be neoplastically transformed, indicating that immortalization is a critical step in multistep neoplastic transformation of human cells. We immortalized three human cell lines by repeated treatment with either 60Co gamma rays or a chemical carcinogen, 4-nitroquinoline 1-oxide, and found that all three immortalized cell lines have mutations in the tumor suppressor gene, p53. Direct sequencing of the reverse-transcribed mRNA and immunoprecipitation of p53 protein revealed that mutant p53 is selectively expressed in all the immortalized cell lines, whereas the genomic fragments of the immortalized cells contain wild-type and mutated p53 alleles. Although the mutated p53 is selectively expressed in the immortalized cells, expression of the wild-type p53 was induced by treatment of the cells with a hypomethylating reagent, 5-azacytidine, indicating that the wild-type p53 allele might be inactivated by hypermethylation of DNA. Actually, the entire genomic locus including the promoter region of p53 is hypermethylated in all the immortalized cell lines. Expression and phosphorylation of Rb was normal in these three cell lines. Thus, inactivation of both wild type p53 alleles and selective expression of mutated p53 seem to be key factors in the immortalization of human fibroblasts.

Human fibroblasts (KMST-6/RAS cell line), which was malignantly transformed in vitro with 60Co gamma-rays and the c-Ha-ras oncogene, produced a large amount of granulocyte colony-stimulating factor (G-CSF). The production was greater during the logarithmic growth phase than during the stationary phase. cAMP and theophylline, alone or in combination, and butyrate significantly enhanced G-CSF production, but dexamethasone or 5-azacytidine did not. Enhanced production of G-CSF by these agents was regulated at the posttranscriptional level. Neither the expression of the ras oncogene nor the tumorigenicity of the cells correlated with the production of G-CSF.

The mutant p53 gene (mp53; codon 273Arg-His) was introduced into normal human fibroblasts (OUMS-24 strain), and a G418-resistant clone, OUMS-24/P6, was obtained. This clone expressed mp53 and showed an extended life span, but it senesced at the 79th population-doubling level (PDL). When these cells were subjected to eight intermittent treatments with 2 Gy of x-rays, they were immortalized, whereas normal fibroblasts (OUMS-24) into which mp53 had not been introduced were not immortalized by the same x-ray treatment. These results indicate that the introduction of mp53 alone is not sufficient for immortalization of human cells and that mutations of the remaining wild-type p53 or other genes are also necessary. © 1995 Wiley-Liss, Inc.

Reduced Expression in Immortalized Cell (REIC) was cloned by subtractive hybridization method as a gene whose expression is reduced in many human immortalized and neoplastic tumor cells. The REIC, when over-expressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on a wide variety of human cancers through a mechanism triggered by ER-stress-mediated JNK activation. In addition to this direct effect on cancer cells, Ad-REIC exerted another cytotoxicity on human cancers, an indirect host-mediated effect due to overproduction of IL-7 by mis-targeted normal cells. This "one-bullet two-arms" finding may lead to a powerful new therapeutic approach to the treatment of human cancers.

Reduced Expression in Immortalized Cell (REIC) was cloned by subtractive hybridization method as a gene whose expression is reduced in many human immortalized and neoplastic tumor cells. The REIC, when over-expressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on a wide variety of human cancers through a mechanism triggered by ER-stress-mediated JNK activation. In addition to this direct effect on cancer cells, Ad-REIC exerted another cytotoxicity on human cancers, an indirect host-mediated effect due to overproduction of IL-7 by mis-targeted normal cells. This "one-bullet two-arms" finding may lead to a powerful new therapeutic approach to the treatment of human cancers.

The effects of four barbiturates with or without liver-tumor-promoting activity were examined on survival and deoxyribonucleic acid (DNA) synthesis of suckling and adult rat hepatocytes in serum-free primary culture: Of the four barbiturates, two promoters, phenobarbital and barbital, enhanced DNA synthesis of suckling rat hepatocytes at low concentrations of 0.5-2 mM or 0.5 mM, but suppressed it at high concentrations of 3 mM or 1.5-4 mM. DNA synthesis of adult rat hepatocytes was, however, only suppressed by phenobarbital within the dose range tested of 1-3 mM. On the other hand, two remaining non-promoters, barbituric acid and amobarbital, did not increase but only suppressed DNA synthesis of suckling rat hepatocytes within the dose ranges of 0.5-4 mM and 0.05-0.5 mM respectively. Phenobarbital and amobarbital were effective for supporting survival and maintaining morphological features of suckling and adult rat hepatocytes at the relatively high concentrations of 3-4 mM and 0.5-0.75 mM respectively. However, barbital and barbituric acid were ineffective for maintenance of hepatocytes. The results show that the ability to support survival of primary cultured hepatocytes is not a common property of liver-tumor-promoter barbiturates but is a common property of some barbiturates with high lipophilicity, and that the maintenance of hepatocytes by phenobarbital or amobarbital is not due to a counterbalance of stimulated proliferation and death of the cells.