Baskaran K, Laconi S, Reddy M K
Department of Pathology, Northwestern University Medical School, Chicago, IL 60611-3008.
Carcinogenesis. 1994 Nov;15(11):2461-6. doi: 10.1093/carcin/15.11.2461.
The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen in laboratory animals. In the present study, in vitro transformation of spontaneously immortal hamster pancreatic duct cells following exposure to 20 mM NNK for 1,3,5 and 7 days is described. NNK imparted a dose-dependent and time-dependent toxicity to pancreatic duct cells in vitro. After NNK treatment, duct cells were grown either in complete duct medium (CDM) or in the absence of bovine pituitary extract, epidermal growth factor and Nu-serum (incomplete duct medium, IDM). Addition of NNK to the culture for 1 and 3 days did not affect the growth of the cells, whereas exposure of the cells for 5 and 7 days was inhibitory. One and 3 day NNK-treated cells were able to grow in the absence of growth factors and serum immediately after the treatment without any inhibition of growth. Untreated cells grew as a monolayer consisting of tightly packed polygonal cells with single nuclei. NNK treated cells also grew as a monolayer with numerous mitotic figures and multi-nucleated large cells. The doubling time between the untreated (16 h) and NNK-treated cells (14 h) was not significantly different prior to injection into the nude mice. NNK treated cells grown in IDM displayed anchorage independency in soft-agar. The tumorigenicity of the untreated and NNK treated cells (5 x 10(6)) was determined in nude mice. One and 3 day NNK-treated cells grown in CDM produced well-differentiated, mucinous tumors with a lower frequency (2/4 sites) and longer duration, but produced tumors at a higher frequency (4/4 sites) and shorter duration when grown in IDM. Five and 7 day NNK-treated cells grown in CDM did not produce any tumors; however, they produced tumors when grown in CDM followed by IDM (5/8 and 6/8 sites) with a shorter duration in nude mice. Analysis of DNA for k-ras mutation at codons 12, 13 and 61 showed G-A transition at codon 12 of the k-ras oncogene in tumor cells of 1 and 3 day NNK treatment. No mutation was detected in tumor cells from 5 and 7 day treatment.
烟草特异性亚硝胺4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)是实验动物中的一种强效致癌物。在本研究中，描述了自发永生化的仓鼠胰腺导管细胞在暴露于20 mM NNK 1、3、5和7天后的体外转化情况。NNK在体外对胰腺导管细胞具有剂量依赖性和时间依赖性毒性。NNK处理后，导管细胞在完全导管培养基(CDM)中或在无牛垂体提取物、表皮生长因子和Nu-血清的情况下(不完全导管培养基，IDM)生长。在培养物中添加NNK 1天和3天不影响细胞生长，而细胞暴露5天和7天则具有抑制作用。经NNK处理1天和3天的细胞在处理后立即能够在无生长因子和血清的情况下生长，且生长不受任何抑制。未处理的细胞生长为单层，由紧密排列的单核多边形细胞组成。经NNK处理的细胞也生长为单层，有许多有丝分裂象和多核大细胞。在注入裸鼠之前，未处理细胞(16小时)和经NNK处理细胞(14小时)之间的倍增时间没有显著差异。在IDM中生长的经NNK处理的细胞在软琼脂中表现出不依赖贴壁生长的特性。在裸鼠中测定了未处理细胞和经NNK处理细胞(5×10⁶)的致瘤性。在CDM中生长的经NNK处理1天和3天的细胞产生分化良好的黏液性肿瘤，频率较低(2/4个部位)且持续时间较长，但在IDM中生长时产生肿瘤的频率较高(4/4个部位)且持续时间较短。在CDM中生长的经NNK处理5天和7天的细胞未产生任何肿瘤；然而，当它们先在CDM中生长然后在IDM中生长时(5/8和6/8个部位)，在裸鼠中产生肿瘤的持续时间较短。对肿瘤细胞中k-ras基因第12、13和61密码子的DNA进行k-ras突变分析显示，在经NNK处理1天和3天的肿瘤细胞中，k-ras癌基因第12密码子发生了G-A转换。在经NNK处理5天和7天的肿瘤细胞中未检测到突变。
