Gimenez-Conti I B, LaBate M, Liu F, Osterndorff E
University of Texas, M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957, USA.
Mol Carcinog. 1996 Aug;16(4):197-202. doi: 10.1002/(SICI)1098-2744(199608)16:4<197::AID-MC3>3.0.CO;2-D.
To confirm that the hamster cheek-pouch carcinogenesis model reflects development of human squamous cell carcinoma (SCC), we determined if and when p53 mutations occur in the development of SCC in this model by using immunohistochemical staining and polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis plus direct DNA sequencing. Twenty-four hamster cheek-pouches were treated with a solution of 0.5% 7,12-dimethylbenz[a]anthracene in mineral oil three times a week for 16 wk. The malignant endophytic and exophytic tumors induced with this protocol are preceded by a sequence of premalignant lesions such as hyperplasia with or without dysplasia and carcinoma in situ, similar to the development of this cancer in humans. For this study, p53 protein accumulation was evaluated by immunostaining of various hamster cheek-pouch exophytic and endophytic SCCs as well as flat dysplastic hyperplasia and carcinomas in situ. A moderate percentage (33.3%) of exophytic lesions and most endophytic carcinomas (90%) showed positive p53 staining. In addition we also found p53-positive staining in a number of preneoplastic lesions, including areas of focal hyperplasia, dysplastic hyperplasia, and carcinomas in situ. To determine whether the alterations in p53 staining were due to p53 gene mutation, we used PCR-SSCP analysis and direct sequencing. PCR products corresponding to exons 5a, 6, 7, and 8 from 40 tumors with the highest percentage of p53-stained cells were analyzed. We detected shifted bands in 17 lesions. Direct sequencing of eight selected shifted bands revealed four mutations, including two G-->T transversions in codons 216 (tumor #1) and 252 (tumor #2) and one G-->C transversion in codon 282 (tumor #3). Tumor #4 contained a frameshift mutation in codon 251. These mutations are consistent with those reported in many human cancers. Therefore, we concluded that in the hamster cheek-pouch model, p53 protein accumulation occurs frequently and early in carcinogenesis, as it does in human SCCs, and some of these p53 alterations are due to p53 gene mutations. These findings may help us better define the mechanisms of carcinogenesis in the hamster cheek-pouch model, and p53 alterations may be an early biomarker of progression for chemoprevention studies.
为了证实仓鼠颊囊癌发生模型反映了人类鳞状细胞癌（SCC）的发展过程，我们通过免疫组织化学染色以及聚合酶链反应（PCR）-单链构象多态性（SSCP）分析并结合直接DNA测序，来确定在该模型的SCC发展过程中p53突变是否发生以及何时发生。每周三次用含有0.5% 7,12-二甲基苯并[a]蒽的矿物油溶液处理24个仓鼠颊囊，持续16周。用此方案诱导产生的恶性内生性和外生性肿瘤之前会出现一系列癌前病变，如伴有或不伴有发育异常的增生以及原位癌，这与人类该癌症的发展过程相似。在本研究中，通过对各种仓鼠颊囊外生性和内生性SCC以及扁平发育异常增生和原位癌进行免疫染色来评估p53蛋白的积累情况。中等比例（33.3%）的外生性病变以及大多数内生性癌（90%）显示p53染色呈阳性。此外，我们还在一些癌前病变中发现了p53阳性染色，包括局灶性增生区域、发育异常增生区域和原位癌。为了确定p53染色的改变是否是由于p53基因突变所致，我们采用了PCR-SSCP分析和直接测序。对来自40个p53染色细胞百分比最高的肿瘤中对应于外显子5a、6、7和8的PCR产物进行了分析。我们在17个病变中检测到了条带迁移。对8个选定的迁移条带进行直接测序发现了4个突变，包括密码子216（肿瘤#1）和252（肿瘤#2）中的两个G→T颠换以及密码子282（肿瘤#3）中的一个G→C颠换。肿瘤#4在密码子251处含有一个移码突变。这些突变与许多人类癌症中报道的突变一致。因此，我们得出结论，在仓鼠颊囊模型中，p53蛋白积累在致癌过程中频繁且早期发生，就像在人类SCC中一样，并且这些p53改变中的一些是由于p53基因突变所致。这些发现可能有助于我们更好地定义仓鼠颊囊模型中的致癌机制，并且p53改变可能是化学预防研究中进展的早期生物标志物。
