mAFP基因转染树突状细胞免疫对小鼠诱发性肝癌发生的影响

目的:探讨用mAFP基因转染的树突状细胞(dendritic cells,DC)瘤苗免疫对小鼠诱发性肝癌发生的影响.方法:诱导、扩增C57BL/6J小鼠DC.将表达小鼠AFP基因(mAFP)的重组腺病毒转染DC.80只C57BL/6J雄性小鼠随机分为A、B、C、D组,每组20只.以单纯DC为A组;将表达mAFP基因的重组腺病毒转染DC(pAdBM5-mAFP-DCs)为B组;表达mAFP基因的质粒DNA(pAdBM5-mAFP)为C组;以单纯PBS(磷酸缓冲液)注射为对照组D组.免疫方法:实验组在每只小鼠的左胁部注射5×105个细胞(0.1ml/资),连续免疫3天,以后每7天接种疫苗1次,继续免疫4次.正常对照组,仅注射0.1ml PBS.在接种免疫的同时,给以二乙基亚硝胺(DEN)、四氯化碳(CCl4)和乙醇联合诱癌.诱癌20周后处死小鼠,检查成瘤情况.同时对肝脏标本进行组织病理学检查.结果:80只免疫小鼠中,A组肝细胞癌发生率为70%;B组肝细胞癌发生率仅为25%;C组肝癌发生率为65%;D组肝癌发生率为75%.B组与其它对照组比较有显著性差异(p＜0.05),其他各组比较无显著性意义.结论:转基因pAdBM5-mAFP-DC瘤苗免疫自然诱癌的C57BL/6J小鼠,能激发较强的抗肿瘤免疫反应,降低肝癌的发生率.     

mAFP基因转染树突状细胞免疫对小鼠诱发性肝癌发生的影响

目的:探讨用mAFP基因转染的树突状细胞(dendritic cells,DC)瘤苗免疫对小鼠诱发性肝癌发生的影响。方法:诱导、扩增C57BL/6J小鼠DC。将表达小鼠AFP基因(mAFP)的重组腺病毒转染DC。80只C57BL/6J雄性小鼠随机分为A、B、C、D组,每组20只。以单纯DC为A组;将表达mAFP基因的重组腺病毒转染DC(pAd-BM5-mAFP-DCs)为B组;表达mAFP基因的质粒DNA(pAdBM5-mAFP)为C组;以单纯PBS(磷酸缓冲液)注射为对照组D组。免疫方法:实验组在每只小鼠的左胁部注射5×105个细胞(0.1ml/次),连续免疫3天,以后每7天接种疫苗1次,继续免疫4次。正常对照组,仅注射0.1ml PBS。在接种免疫的同时,给以二乙基亚硝胺(DEN)、四氯化碳(CCl4)和乙醇联合诱癌。诱癌20周后处死小鼠,检查成瘤情况。同时对肝脏标本进行组织病理学检查。结果:80只免疫小鼠中,A组肝细胞癌发生率为70%;B组肝细胞癌发生率仅为25%;C组肝癌发生率为65%;D组肝癌发生率为75%。B组与其它对照组比较有显著性差异(p<0.05),其他各组比较无显著性意义。结论:转基因pAdBM5-mAFP-DC瘤苗免疫自然诱癌的C57BL/6J小鼠,能激发较强的抗肿瘤免疫反应,降低肝癌的发生率。     

TNNT3突变转基因小鼠筛选及肌钙蛋白表达的研究

目的：研究TNNT3（R69H）突变转基因小鼠快反应骨骼肌肌钙蛋白表达和组织结构的变化。方法：将杂合TNNT3（R69H）突变转基因小鼠与野生型C57BL/6J小鼠杂交,选同窝阳性杂合后代鼠进行交配,再用野生型C57BL/6J小鼠对后代进行检测,得到TNNT3（R69H）突变转基因纯合个体。取野生型C57BL/6J小鼠、杂合转基因小鼠、纯合转基因小鼠的快反应骨骼肌进行RT-PCR和WESTERN实验并制备透射电镜切片。结果：RT-PCR和WESTERN电泳条带从亮到暗依次为纯合转基因小鼠、杂合转基因小鼠、野生型C57BL/6J小鼠。电镜结果显示转基因小鼠快反应骨骼肌明暗带不清、出现肌纤维间水肿和线粒体空泡等现象。结论：TNNT3（R69H）突变纯合转基因小鼠快反应骨骼肌中TNNT3的mRNA和蛋白质量高于杂合转基因小鼠、杂合转基因小鼠高于野生型C57BL/6J小鼠,并且转基因的表达使快反应骨骼肌结构异常。     

TNNT3突变转基因小鼠筛选及肌钙蛋白表达的研究

目的:研究TNNT3(R69H)突变转基因小鼠快反应骨骼肌肌钙蛋白表达和组织结构的变化。方法:将杂合TNNT3(R69H)突变转基因小鼠与野生型C57BL/6J小鼠杂交,选同窝阳性杂合后代鼠进行交配,再用野生型C57BL/6J小鼠对后代进行检测,得到TNNT3(R69H)突变转基因纯合个体。取野生型C57BL/6J小鼠、杂合转基因小鼠、纯合转基因小鼠的快反应骨骼肌进行RT-PCR和WESTERN实验并制备透射电镜切片。结果:RT-PCR和WESTERN电泳条带从亮到暗依次为纯合转基因小鼠、杂合转基因小鼠、野生型C57BL/6J小鼠。电镜结果显示转基因小鼠快反应骨骼肌明暗带不清、出现肌纤维间水肿和线粒体空泡等现象。结论:TNNT3(R69H)突变纯合转基因小鼠快反应骨骼肌中TNNT3的mRNA和蛋白质量高于杂合转基因小鼠、杂合转基因小鼠高于野生型C57BL/6J小鼠,并且转基因的表达使快反应骨骼肌结构异常。     

肝癌干细胞表面标志物CK19在化学诱癌过程中的差异表达

目的检测CK19基因在化学诱发C57BL/6J小鼠肝癌过程中各阶段的表达差异。方法对50只C57BL/6J雄性小鼠通过化学法诱发肝癌,以50只正常C57BL/6J雄性小鼠为对照组。观察诱癌小鼠的生长状况,每4周处死一批小鼠获取组织标本进行病理学、荧光实时定量PCR(FQ-RT-PCR)法、Western blot等检测CK19基因及蛋白表达差异。结果在化学诱癌第16周后处死的小鼠出现明显的灰白色小结节,直径为2 mm大小,第20周时肝癌结节直径在5 ~25 mm之间,呈多发性。早期病理改变主要是肝细胞排列结构轻度紊乱,细胞轻度的异型增生。病理切片显示为中、高分化的肝癌细胞,可见瘤巨细胞和病理性核分裂,间质肝纤维组织增生,提示有肝硬化改变;RT-PCR,Western blot 结果显示,实验组小鼠在化学诱癌第16周开始CK19-mRNA表达升高,第20周时明显增强;荧光定量结果显示,CK19 mRNA在化学诱癌第4、8、12、16及20周的小鼠肝癌组织中的表达水平分别为(2.133±0.470)、(2.395±0.472)、(2.767±0.729)、(3.217±0.627)和(14.095±5.812),与同期对照组比较差异具有统计学意义（P<0.05）。结论肝癌干细胞标志物CK19参与了肝癌的发生发展,其表达量随诱癌时间的延长而逐渐增加,但其对肝癌发生发展的确切调控机制有待深入研究。     

C57BL/6小鼠微小残留白血病模型的建立

目的 探讨构建C57BL/6小鼠FBL-3微小残留白血病(MRL)模型的方法.方法 将5×106个FBL-3红白血病细胞经尾静脉接种至C57 BL/6小鼠,接种后第3天经尾静脉注射不同剂量环磷酰胺(CTX),观察小鼠生存时间与药物剂量间的关系;根据白血病小鼠生存时间与CTX剂量和与接种FBL-3细胞数量的关系,确定构建小鼠MRL模型所需的化疗药物剂量.结果 C57BL/6白血病小鼠对CTX敏感,随CTX剂量增加白血病小鼠的生存时间逐渐延长,药物剂量与生存时间有较好的回归关系.移植生物试验显示,接种500个FBL-3细胞的小鼠生存时间与接受100 mg·kg-1 CTX化疗白血病小鼠相当.结论每只C57BL/6小鼠经尾静脉接种5×106个FBL-3细胞3d后给予100 mg·kg-1 CTX化疗可以建立MRL动物模型.     

抑癌基因SLC5A8在食管癌组织及血浆中的甲基化状态分析

目的:探讨食管癌组织中抑癌基因SLC5A8的甲基化状态和表达情况,以及食管癌术前和术后血浆中SLC5A8基因的甲基化状态.方法:采用甲基化特异性PCR(methylation-specific PCR,MSP)法检测45例食管癌、癌旁、切缘组织及30例食管癌术前、术后血浆中SLC5A8基因的甲基化状态.RT-PCR法检测组织中SLC5A8基因表达情况.结果:45例食管癌、癌旁及切缘组织中SLC5A8基因的甲基化率分别为68.8%、11.1%和4.4%,癌组织的甲基化阳性率显著高于癌旁及切缘组织(P＜0.01).RT-PCR检测结果显示,31例SLC5A8甲基化阳性的食管癌标本中仅有2例SLC5A8基因阳性表达,14例甲基化阴性的标本中有10例检测到基因表达,差异具统计学意义(P＜0.001).30例术前、术后血浆中SLC5A8基因的甲基化率分别为43.3%及16.7%,差异有统计学意义(P＜0.05).结论:SLC5A8基因甲基化是食管癌发生的重要早期分子事件.血浆中SLC5A8基因甲基化检测可作为术前诊断的重要参考指标,并有可能成为监测治疗反应与判断预后的一项生物学指标.     

甲胎蛋白基因转染树突状细胞瘤苗对诱发性小鼠肝癌发生发展的免疫抑制作用

目的 构建小鼠甲胎蛋白(mAFP)基因转染的树突状细胞(DC)瘤苗,评价其在C57BL/6J小鼠自然肝癌诱发过程中的免疫保护作用.方法 诱导、扩增DC.将表达mAFP的重组腺病毒颗粒转染DC,检测转染前后DC细胞表面分子MHC Ⅰ、MHC Ⅱ、CDl8a(LFA)、CD54(ICAM)、CD80(B7.1)和CD86(B7.2)等的变化.80只C57BL/6J雄性小鼠随机分为单纯接种DC组、接种转mAFP基因DC(pAdBM5-mAFP-DC)组、单纯接种重组质粒(pAdBM5-mAFP)组和正常对照组,每组20只.实验组在每只小鼠的左肋部注射5×105个细胞,连续免疫3 d,以后每7 d接种疫苗1次,继续免疫4次;正常对照组仅注射0.1 ml PBS.在接种免疫的同时,给以二乙基亚硝胺(DEN)、四氯化碳(CCl4)和乙醇联合诱癌.诱癌20周后处死小鼠,检查成瘤情况,并对肝脏标本进行组织病理学检查.结果成功构建了转基因pAdBM5-mAFP-DC瘤苗;mAFP基因转染后的DC表面高表达MHC Ⅰ、MHC Ⅱ、CD18a、CD54、CD80和CD86等共刺激分子,其分子表达率分别为69.3%、41.0%、42.1%、63.2%、39.4%和38.6%,与转染前差异有统计学意义(P＜0.05).单纯接种DC组、pAdBM5-mAFPDC组、pAdBM5.mAFP组和正常对照组的肝癌发生率分别为65.0%、25.0%、70.0%和75.0%,pAdBM5-mAFP-DC组与其他组比较,差异有统计学意义(P＜0.05).结论 pAdBM5-mAFP-DC瘤苗免疫自然诱癌的C57BL/6J小鼠,可降低肝癌的发生率,对肝癌的发生有免疫保护作用.     

化学诱导小鼠肝癌模型中CD133的动态变化

目的:检测肝癌干细胞标志CD133在化学诱导C57BL/6J小鼠肝癌过程中各时期的动态变化.方法:通过化学法[二乙基亚硝胺(diethylnirtosamine,DEN)/四氯化碳(carbon tetrachloride,CC14)/乙醇]诱发50只C57BL/6J雄性小鼠肝癌,对照组为45只正常C57BL/6J雄性小鼠.观察小鼠成瘤情况和生长状态,对每2周定期处死小鼠获得的组织标本分别进行病理学切片观察,并采用实时荧光定量PCR、免疫组织化学法、蛋白质印迹法和FCM法分别检测CD133 mRNA和蛋白的表达情况.结果:化学诱导20周后,成功诱发出小鼠肝癌.实时荧光定量PCR和FCM检测结果显示,第8周后诱癌组小鼠肝组织中CD133的表达高于正常对照组,差异有统计学意义(P＜ 0.05或P＜0.001);随着诱癌时间的增加,CD133的表达量呈上升趋势,第16周时诱癌组CD133的表达明显高于前期诱癌组(P＜0.001).蛋白质印迹法检测结果显示,诱癌组CD133蛋白从第4周起出现弱表达,随着诱癌时间的递增,CD133蛋白表达量逐渐增多.免疫组织化学检测结果显示,诱癌组第8周CD133出现弱阳性,第16周出现中等阳性,第20周出现强阳性;而正常对照组无表达.结论:肝癌干细胞标志CD133参与了肝癌的发生、发展全过程,随着诱癌进展其表达量逐渐增加.     

移植性C57BL/6小鼠FBL-3红白血病模型的建立及其生物学特性

目的探讨利用国内传代培养的FBL-3细胞建立H-2相合C57BL/6小鼠FBL-3红白血病模型的可行性,并观察模型的生物学特性。方法把FBL-3小鼠红白血病细胞经尾静脉接种C57BL/6小鼠,观察小鼠的生存时间和染色体变化,并对濒死小鼠的肝、脾、肺和肾进行病理检查,部分进行电镜检查。结果静脉接种1&#215;10^3-1&#215;10^7个白血病细胞,C57BL/6小鼠发病率分别为100%;接种细胞的数量与存活时间呈线性关系;白血病细胞主要侵及肝、脾、骨髓、肺和肾组织,糖原染色阳性、氯醋酸染色部分阳性,过氧化物酶、碱性磷酸酶、丁酸染色均阴性。电镜下观察到细胞内病毒样颗粒。脾脏和骨髓细胞染色体多数为非二倍体,只表达H-2b。结论经尾静脉接种FBL-3细胞可建立C57BL/6小鼠红白血病模型。     

A novel rasH2 mouse carcinogenesis model that is highly susceptible to 4-NQO-induced tongue and esophageal carcinogenesis is useful for preclinical chemoprevention studies

We investigated the susceptibility of 4-nitroquinoline 1-oxide (4-NQO)-induced tongue carcinogenesis in male CB6F1-Tg-rasH2 @Jcl mice (Tg mice). The Tg mice were administered 4-NQO (20 p.p.m. in drinking water) for 2, 4, 6 or 8 weeks, and thereafter they were untreated up to week 24. At week 24, a higher incidence (80%) of tongue neoplasm with dysplasia was noted in the mice that received 4-NQO for 8 weeks in comparison with the other groups (20% incidence for each) treated with 4-NQO for 2, 4 and 6 weeks. Esophageal tumors also developed in the Tg mice were 4-NQO. Immunohistochemical observation revealed that the EP receptors, especially EP(1) and EP(2), expressed in the tongue and esophageal lesions induced by 4-NQO, thus suggesting the involvement of prostaglandin (PG) E(2) and EP(1,2) receptors in the tongue and esophageal carcinogenesis. Using this animal model, we investigated the potential chemopreventive ability of pitavastatin (1, 5 and 10 p.p.m. in diet for 15 weeks), starting 1 week after the cessation of 4-NQO-exposure (20 p.p.m. in drinking water for 8 weeks). Dietary pitavastatin at 10 p.p.m. significantly reduced the incidence and multiplicity of the tongue, but not esophageal neoplasms by the modulation of prostaglandin E2 biosynthesis, EP(1) and EP(2) expression and proliferation. Our results thus suggest that a rasH2 mouse model of 4-NQO-induced tongue and esophageal carcinogenesis can be utilized for investigating the pathogenesis of cancer development in these tissues and may well prove to be useful for identifying candidate cancer chemopreventive agents for the upper digestive organs.     

博安霉素对人食管癌等抑瘤作用的实验研究

目的：研究博安霉素（boanmycin)在体内外对人食管癌细胞等的抑瘤作用。方法：采用MTT法检测博安霉素在人体外对5种人癌细胞的细胞毒作用。同时用裸鼠异种移植人食管癌细胞模型观察博安霉素对人食管癌的抑瘤作用。结果：博安霉素在体外对5种肿瘤细胞均有明显的细胞毒作用。博安霉素在10．0mg/kg和15．0mg/kg剂量下对裸鼠异种移植人食管癌HEC2的抑制率分别为85．2％，90．6％；在7．5mg/kg和15．0mg/kg剂量下对裸鼠异种移植人食管癌Eca-109的抑制率分别为81．2％，92．0％，结论：博安霉素在体内外对人食管癌细胞等具有显著的抑瘤作用。     

Green Tea Polyphenol Protection Against 4-Nitroquinoline 1-Oxide-Induced Bone Marrow Lipid Peroxidation and Genotoxicity in Wistar Rats

4-Nitroquinoline 1-oxide (4-NQO) a potent oral carcinogen, widely used for induction of oral carcinogenesis,has been found to induce lipid peroxidation in vivo and in vitro. Green tea contains a high content of polyphenols,which are potent antioxidants. Thus green tea polyphenols (GTP) might be expected play a protective role against4-NQO induced lipid peroxidation and bone marrow toxicity. In the present study, a dose of 200 mg of GTP/kgb.wt/day was given orally for a week, simultaneously animals received 0.2 ml of 0.5% 4-NQO in propylene glycol(5 mg/ml) injected intramuscularly for three times/week. Oxidants and antioxidants such as malendialdehyde(MDA) and thiols, glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) andcatalase (CAT) were significantly decreased in 4-NQO induced animals except MDA, and these parameters werebrought back to near normalcy on treatment with GTP. The results suggest that GTP treatment offers significantprotection against 4-NQO induced lipid peroxidation and bone marrow toxicity and might be a promisingpotential candidate for prevention of mutations leading to cancer.     

Organ-specific, carcinogen-induced increases in cell proliferation in p53-deficient mice

Transgenic mice with germ-line p53 alleles disrupted by gene targeting are sensitive to the development of some spontaneous tumors and have provided researchers with much information with respect to cancer. In the present study, to cast light on the organ specificity of chemically induced carcinogenesis, we evaluated carcinogen-induced cell proliferation in target organs in heterozygote p53 knockout mice (p53-deficient mice). Groups of 9- or 10-week-old wild-type(+/+) and p53-deficient mice were respectively treated with one of the following carcinogens for 4 weeks: N-butyl-N-(4-hydroxybutyl)nitrosamine (0.0075% in drinking water); dimethylnitrosamine (0.001% in drinking water); dihydroxy-di-N-propylnitrosamine (0.1% in drinking water); 1,2-dimethylhydrazine (10 mg/kg body weight s.c. injection once a week); 4-nitroquinoline 1-oxide (4-NQO, 10 mg/kg b.w. s.c. injection once a week); or 7,12-dimethylbenz(a)anthracene (25 microg/kg body weight dermal application once a week). Cell proliferation was evaluated by measuring the 5-bromo-2'-deoxyuridine labeling indices in each target organ. The p53 and p21 statuses were evaluated by comparing the expressions of p53 protein, p21waf1/cip1 mRNA, and p21waf1/cip1 protein between the mice. 5-Bromo-2'-deoxyuridine labeling indices of the urinary bladder and the skin were significantly increased in p53-deficient mice as compared with the wild-type(+/+) mice. In the all organs examined, carcinogen-induced p21waf1/cip1 mRNA overexpression was detected with levels obviously lower in the p53-deficient animals. These data suggest that p53-deficient mice have an organ-specific increased sensitivity to the induction of cell proliferation in the urinary bladder and the skin. These are the same organs for which sensitivity to carcinogenesis has been reported. Because a decrease of p21waf1/cip1 protein overexpression was also observed in the organs in which cell proliferation did not appreciably differ from the level in wild-type(+/+) mice, this decrease might have no effect on sensitivity to cell proliferation and carcinogenesis. Alternatively, it might play an important role in the cell cycle regulation of only the sensitive organs.     

DICER基因多态性与食管癌遗传易感性研究

目的：研究中国汉族人DICER基因rs3742330A〉G多态性与食管癌易感性的关系。方法：本研究以病例一对照研究为基础,收集2008—10—01—20091130镇江第一人民医院和江苏大学附属人民医院380例食管癌患者和380例非肿瘤患者,采用质谱（MALDI—TOFMS）技术对DICER基因多态性位点rs3742330A〉G进行分型,计算不同基因型与食管癌的发生风险及95％CI。结果：DICER多态性位点rs3742330A〉G基因型AA、AG和GG在食管癌组频率分别为33．9％、51．6％和14．5％,在非肿瘤对照组的频率分别为38．1％、48．4％和13．5％。Logistic回归分析结果表明,相对于AA基因型的个体,AG基因型与食管癌的发病风险无明显相关,OR=1．19,95％CI=0．87～1．62;与AA＋AG基因型相比较,GG基因型与食管癌的发病风险无明显相关,OR=1．09,95％CI=0．72～1．65。结论：DICER基因rs3742330A〉G多态性可能不是食管癌发生的危险因素。     

蟾酥脂质微球注射液对四种荷瘤裸鼠肿瘤生长的抑制作用

目的:观察蟾酥脂质微球注射液对荷人肝癌Hep G2、人食管癌EC9706、人结肠癌HCT-8和人胃癌BGC 803瘤株裸鼠肿瘤生长的抑制作用.方法:在BALB/c-nu裸鼠右前肢腋下接种4种人癌细胞株建立荷瘤动物模型,移植10 d后采用均衡法随机分为5组,即蟾酥脂质微球注射液高剂量组(2.50 mg/kg)、中剂量组(1.25 mg/kg)、低剂量组(0.63 mg/kg)以及阳性药组(氟尿嘧啶注射液,1.25 mg/kg)和阴性对照组(等体积脂质微球空白注射液),每组7只裸鼠,给药体积0.2 ml.采用尾静脉注射方法给药,各组动物于分组后每周给药2次,连续给药6次.于给药后动态检测并计算各组肿瘤体积(Vt)、肿瘤相对体积(VRT)、肿瘤增殖率(T/C),末次给药后3 d检测并计算各组肿瘤质量和抑瘤率.结果:各组裸鼠给药前肿瘤体积均无显著性差异.荷人肝癌Hep G2细胞瘤裸鼠给药7 d后,各剂量组肿瘤体积和相对肿瘤体积均较阴性对照组明显降低(P<0.05),末次给药后中、高剂量组T/C值<40%,各剂量组的平均肿瘤质量均较阴性对照组明显降低(P<0.05),高剂量组的抑瘤率为74.07%.荷人食管癌EC9706瘤裸鼠末次给药后,各剂量组肿瘤体积和相对肿瘤体积均较阴性对照组明显降低(P<0.05),中、高剂量组T/C值<40%,各剂量组的平均肿瘤质量均较阴性对照组明显降低(P<0.05),高剂量组的抑瘤率为70.38%.荷人结肠癌HCT-8细胞瘤裸鼠末次给药后,各剂量组肿瘤体积和相对肿瘤体积均较阴性对照组明显降低(P<0.05),高剂量组T/C值<40%,各剂量组的平均肿瘤质量均较阴性对照组明显降低(P<0.05),高剂量组的抑瘤率为52.42%.荷人胃癌BGC 803细胞瘤裸鼠末次给药后,中、高剂量组的肿瘤体积和相对肿瘤体积均较阴性对照组明显降低(P<0.05),各剂量组T/C值>40%,各剂量组的平均肿瘤质量均较阴性对照组明显降低(P<0.05),高剂量组的抑瘤率为47.42%.结论:蟾酥脂质微球注射液具有确切的抑制人肝癌Hep G2、食管癌EC9706、结肠癌HCT-8和胃癌BGC 803瘤株增殖的作用,作用效果以抑制人肝癌Hep G2和人食管癌EC9706瘤株生长作用为最佳.     

Inhibitory Effects of the Natural Products Indole-3-carbinol and Sinigrin during Initiation and Promotion Phases of 4-Nitroquinoline 1-Oxide-induced Rat Tongue Carcinogenesis

The modifying effects of indole-3-carbinol (I3C) and sinigrin (SIN) on the initiation and post-initiation phases of tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4-NQO) were investigated in male ACI/N rats. Rats were divided into eight groups: group 1 was given 4-NQO (10 ppm) in the drinking watar for 12 weeks, starting at 7 weeks of age; groups 2 and 3 were given 4-NQO and fed the diets containing I3C (1,000 ppm) and SIN (1,200 ppm) for 14 weeks, respectively, starting at 6 weeks of age; groups 4 and 5 were given 4-NQO and then they were fed I3C and SIN containing diets for 23 weeks, respectively, starting one week after 4-NQO exposure; groups 6 and 7 were given I3C and SIN alone, respectively, during the experiment; group 8 served as an untreated control. At the termination of the experiment (week 37), the incidence of tongue neoplasms (squamous cell papilloma and carcinoma) in group 2 (1/15,7%), group 3 (1/15, 7%), group 4 (3/15, 20%) or group 5 (2/15, 13%) was significantly smaller than that in group 1 (12/17,71%) ( P = 0.0003, P=0.005 or P=0.002). No tongue carcinomas developed in rats of groups 2, 3, and 5. Similarly, the incidence of preneoplastic lesions (hyperplasia and dysplasia) of the tougue in group 2 (11/15, 73%), group 3 (10/15, 67%), group 4 (11/15, 73%) or group 5 (10/15, 67%) was significantly lower than that in group 1 (17/17, 100%) (P=0.04 or P=0.02). There were no tongue neoplasms in rats of groups 6, 7, and 8. Administration of I3C and SIN also caused significant decreases in the number and area of silver-stained nucleolar organizer regions protein (AgNORs), a new cell proliferation index, of tongue squamous epithelium. Thus, I3C and SIN inhibited rat tongue carcinogenesis in both the initiation and post-initiation phases, when administered in these respective phases together with, or following treatment with, 4-NQO.     

Established gastric cancer cell lines transplantable into C57BL/6 mice show fibroblast growth factor receptor 4 promotion of tumor growth

Previously no mouse gastric cancer cell lines have been available for transplantation into C57BL/6 mice. However, a gastric cancer model in immunocompetent mice would be useful for analyzing putative therapies. N‐Methyl‐N‐nitrosourea (MNU) was given in drinking water to C57BL/6 mice and p53 heterozygous knockout mice. Only 1 tumor from a p53 knockout mouse could be cultured and the cells s.c. transplanted into a C57BL/6 mouse. We cultured this s.c. tumor, and subcloned it. mRNA expression in the most aggressive YTN16 subline was compared to the less aggressive YTN2 subline by microarray analysis, and fibroblast growth factor receptor 4 ( FGFR4) in YTN16 cells was knocked out with a CRISPR/Cas9 system and inhibited by an FGFR4 selective inhibitor, BLU9931. These transplanted cell lines formed s.c. tumors in C57BL/6 mice. Four cell lines (YTN2, YTN3, YTN5, YTN16) were subcloned and established. Their in vitro growth rates were similar. However, s.c. tumor establishment rates, metastatic rates, and peritoneal dissemination rates of YTN2 and YTN3 were lower than for YTN5 and YTN16. YTN16 established 8/8 s.c. tumors, 7/8 with lung metastases, 3/8 with lymph node metastases and 5/5 with peritoneal dissemination. FGFR4 expression by YTN16 was 121‐fold higher than YTN2. FGFR4‐deleted YTN16 cells failed to form s.c. tumors and showed lower rates of peritoneal dissemination. BLU9931 significantly inhibited the growth of peritoneal dissemination of YTN16. These studies present the first transplantable mouse gastric cancer lines. Our results further indicate that FGFR4 is an important growth signal receptor in gastric cancer cells with high FGFR4 expression.     

Translational strategies exploiting TNF-alpha that sensitize tumors to radiation therapy

TNFerade is a radioinducible adenoviral vector expressing tumor necrosis factor-alpha (TNF-alpha) (Ad.Egr-TNF) currently in a phase III trial for inoperable pancreatic cancer. We studied B16-F1 melanoma tumors in TNF receptor wild-type (C57BL/6) and deficient (TNFR1,2-/- and TNFR1-/-) mice. Ad.Egr-TNF+IR inhibited tumor growth compared with IR in C57BL/6 but not in receptor-deficient mice. Tumors resistant to TNF-alpha were also sensitive to Ad.Egr-TNF+IR in C57BL/6 mice. Ad.Egr-TNF+IR produced an increase in tumor-associated endothelial cell apoptosis not observed in receptor-deficient animals. Also, B16-F1 tumors in mice with germline deletions of TNFR1,2, TNFR1 or TNF-alpha, or in mice receiving anti-TNF-alpha exhibited radiosensitivity. These results show that tumor-associated endothelium is the principal target for Ad.Egr-TNF radiosensitization and implicate TNF-alpha signaling in tumor radiosensitivity.     

Carcinogenicity of ethylene oxide and 1,2-propylene oxide upon intragastric administration to rats.

Ethylene oxide and 1,2-propylene oxide were each administered intragastrically by gavage at 2 dosages (30 and 7.5 mg/kg body wt; 60 and 15 mg/kg body wt respectively) to groups of 50 female Sprague-Dawley rats twice weekly for a period of nearly 3 years using salad oil as the solvent. Both compounds induced local tumours, mainly squamous-cell carcinomas of the forestomach, dependent on the dosage. The first tumour occurred in the 79th week both in the group treated with ethylene oxide and in that treated with 1,2-propylene oxide. The following tumour rates resulted: ethylene oxide 62 and 16%; 1,2-propylene oxide 40 and 4%. In addition carcinomata in situ, papillomas and reactive changes of the squamous epithelium of the forestomach were observed in other animals, but neither ethylene oxide nor 1,2-propylene oxide induced tumours at sites away from the point of administration.