Chen Y K, Hsue S S, Lin L M
Oral Pathology Department, School of Dentistry, Kaohsiung Medical University, Kaohsiung, Taiwan.
Oral Dis. 2003 Sep;9(5):227-34. doi: 10.1034/j.1601-0825.2003.02878.x.
Three isoforms of nitric oxide synthase (NOS) have been identified previously--endothelial NOS, neuronal NOS, and inducible NOS (iNOS). It has been reported previously that there may be a negative feedback loop existing between nitric oxide (NO) production and wild-type p53 tumor-suppressor gene, but the relationship has not previously been studied for oral experimental carcinogenesis. The purpose of the present study is to assess whether iNOS expression correlates with p53 expression at both protein and mRNA levels for 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch carcinomas.
Thirty-five out-bred, young (6 week old), male, Syrian golden hamsters (Mesocricatus auratus) were randomly divided into one experimental group (15 animals), and two control groups (10 animals each). Bilaterally, the pouches of a group of 15 animals from the experimental group were painted with a 0.5% DMBA solution three times a week for 12 weeks whilst each animal from one of the control groups was similarly treated with only mineral oil. Another control group of 10 animals remained untreated throughout the experimental procedure. Specimens obtained from the hamster buccal-pouch mucosa were evaluated using immunohistochemical assessment of iNOS and p53 protein and in situ reverse transcription-polymerase chain reaction (IS RT-PCR), as well as reverse transcription-polymerase chain reaction (RT-PCR) for iNOS and p53 mRNA.
Two of the 15 animals of the DMBA-treated group died during the experiment. Squamous-cell carcinomas with a 100% tumor incidence were apparent for all of the 15-week DMBA pouch-treated animals. Animals from the mineral oil-treated and untreated pouch groups revealed no obvious changes. Inducible NOS mRNA was identified as a band corresponding to a 499-bp PCR product and was observed for all 13 of the hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks. The p53 mRNA was found as a band corresponding to a 370-bp polymerase chain reaction (PCR) product and was noted for nine (9/13, 69%) of the 15-week DMBA-treated pouches. No such bands (iNOS and p53) were noted for the untreated animals, the mineral oil-treated tissues and the negative-control samples. Using IS RT-PCR, the proportional (percentage) expression of iNOS (13/13, 100%) and p53 (8/13, 62%) mRNA observed for the hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks was noted to be consistent with the findings using RT-PCR. Furthermore, the proportional expression of iNOS (13/13, 100%) and p53 (8/13, 62%) proteins for the 15-week DMBA-treated hamster buccal-pouch tissue specimens was noted to be consistent with the findings using RT-PCR and IS RT-PCR. A significant association between iNOS and p53 expression (at both protein and mRNA levels) was noted (Fisher's exact probability test, P < 0.05). Neither iNOS nor p53 activity (at both protein and mRNA levels) was found for any of the untreated and mineral oil-treated pouches.
Enhanced expression of iNOS and p53 at both protein and mRNA levels in DMBA-induced hamster buccal-pouch carcinomas compared with the untreated and mineral oil-treated counterparts, has been demonstrated in the current study. Furthermore, we report what is, to the best of our knowledge, the first identification of a significant association between iNOS and p53 expression (at both protein and mRNA levels) in this experimental model system for oral carcinogenesis, although their precise interactions remain to be clarified.
先前已鉴定出三种一氧化氮合酶（NOS）同工型——内皮型NOS、神经元型NOS和诱导型NOS（iNOS）。先前有报道称，一氧化氮（NO）生成与野生型p53肿瘤抑制基因之间可能存在负反馈环，但此前尚未针对口腔实验性致癌作用研究二者关系。本研究旨在评估在7,12 - 二甲基苯并[a]蒽（DMBA）诱导的仓鼠颊囊癌中，iNOS表达与p53表达在蛋白质和mRNA水平上是否相关。
35只远交、年轻（6周龄）、雄性叙利亚金黄仓鼠（Mesocricatus auratus）随机分为一个实验组（15只动物）和两个对照组（每组10只动物）。实验组的15只动物双侧颊囊每周用0.5% DMBA溶液涂抹三次，持续12周，而其中一个对照组的每只动物仅用矿物油进行类似处理。另一个由10只动物组成的对照组在整个实验过程中不做处理。从仓鼠颊囊黏膜获取的标本采用免疫组织化学法评估iNOS和p53蛋白，原位逆转录 - 聚合酶链反应（IS RT - PCR），以及用于iNOS和p53 mRNA的逆转录 - 聚合酶链反应（RT - PCR）进行评估。
DMBA处理组的15只动物中有2只在实验期间死亡。所有经15周DMBA处理颊囊的动物均出现了鳞状细胞癌，肿瘤发生率为100%。矿物油处理组和未处理组的动物未发现明显变化。诱导型NOS mRNA被鉴定为对应499 bp PCR产物的条带，在所有13个经15周DMBA处理的仓鼠颊囊组织标本中均观察到。p53 mRNA被发现为对应370 bp聚合酶链反应（PCR）产物的条带，在15周DMBA处理的颊囊中，有9个（9/13，69%）观察到该条带。未处理动物、矿物油处理组织和阴性对照样本均未观察到此类条带（iNOS和p53）。使用IS RT - PCR，在经15周DMBA处理的仓鼠颊囊组织标本中观察到的iNOS（13/13，100%）和p53（8/13，62%）mRNA的比例（百分比）表达与使用RT - PCR的结果一致。此外，经15周DMBA处理的仓鼠颊囊组织标本中iNOS（13/13，100%）和p53（8/13，62%）蛋白的比例表达与使用RT - PCR和IS RT - PCR的结果一致。注意到iNOS与p53表达（在蛋白质和mRNA水平上）之间存在显著关联（Fisher精确概率检验，P < 0.05）。未处理和矿物油处理的颊囊中均未发现iNOS和p53活性（在蛋白质和mRNA水平上）。
本研究表明，与未处理和矿物油处理的对照组相比，DMBA诱导的仓鼠颊囊癌中iNOS和p53在蛋白质和mRNA水平上均有增强表达。此外，据我们所知，本研究首次在该口腔致癌作用实验模型系统中鉴定出iNOS与p53表达（在蛋白质和mRNA水平上）之间存在显著关联，尽管它们的确切相互作用仍有待阐明。
