The premalignant evolution of chemically induced mouse skin papillomas is characterized by dysplastic changes, aneuploidy, induction of gamma-glutamyl transpeptidase (GGT), and changes in the expression of keratins, especially differentiation-associated K1. This keratin, which is expressed in normal epidermis and early papillomas, is no longer present in more advanced dysplastic and aneuploid papillomas and in fully invasive carcinomas. More recently, it has been shown that K13, a keratin normally present in internal epithelia but not in epidermis, is aberrantly expressed in epidermal tumors. In the present study, the timing of expression of K13 and its correlation with other markers of premalignant evolution were investigated. Papillomas were induced by SENCAR mice by a single initiating dose of 20 nmol of 7,12-dimethylbenz[a]-anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) (2 micrograms twice a week). Tumors were randomly harvested at 10, 20 and 35 weeks of promotion. K13 and K1 expression in papillomas was studied using immunoblotting and immunostaining of consecutive sections, as previously described. As expected from previous studies, the distribution of K1 in papillomas collected at 10 weeks of promotion was restricted to differentiated cells and was uniform throughout the section of the papilloma. Conversely, K13 was expressed only as small foci in 10 out of 21 papillomas (48%). Papillomas of 20 weeks were also positive for K1. Staining for K13 was positive in these papillomas with the exception of only one that was essentially negative, presenting only one small positive focus. Some of the papillomas collected at week 35 were negative for K1, but immunostaining with K13 showed uniform staining of suprabasal cells in all the papillomas studied. In all cases, immunohistochemical results were confirmed by immunoblotting with proteins extracted from 7 microns sections from each paraffin block. These results indicate that keratins K1 and K13 are coexpressed in most papillomas from 10 to 35 weeks of promotion. However, analysis of adjacent sections showed that K13 positive areas are topographically located in the K1 negative areas of the papillomas, suggesting a shift in the differentiation program from epidermal to mucosal types of keratinization. Based on these and previous studies from our laboratory, we conclude that K13 is an early marker of papillomas progression, which occurs before gross chromosomal abnormalities are present in the stem line of the tumors, and precedes dysplastic changes and the onset of GGT expression, and is probably concomitant at the individual cell level with loss of K1.

译文

化学诱导的小鼠皮肤乳头状瘤的恶变前特征是发育异常改变,非整倍性,γ-谷氨酰转肽酶(GGT)的诱导以及角蛋白表达的变化,特别是分化相关的K1。这种角蛋白在正常表皮和早期乳头状瘤中表达,不再存在于更晚期的发育异常和非整倍性乳头状瘤以及全浸润性癌中。最近,已经显示出正常存在于内部上皮而不是表皮中的角蛋白K13在表皮肿瘤中异常表达。在本研究中,研究了K13的表达时机及其与恶变前其他标志物的相关性。 SENCAR小鼠通过单次起始剂量20 nmol的7,12-二甲基苯并[a]-蒽(DMBA)诱导并用12-O-十四烷酰phorbol-13-乙酸盐(TPA)促进(每周两次,每次2微克)诱导乳头状瘤。在促进的第10、20和35周时随机收集肿瘤。如先前所述,使用免疫印迹和连续切片的免疫染色研究了乳头状瘤中K13和K1的表达。正如先前研究所预期的,在促进10周后收集的乳头状瘤中K1的分布仅限于分化的细胞,并且在整个乳头状瘤切片中是一致的。相反,在21个乳头状瘤中,有10个中的K13仅表达为小灶(48%)。 20周的乳头状瘤也对K1呈阳性。在这些乳头状瘤中,K13染色呈阳性,只有一种基本上呈阴性,只有一个小的阳性焦点。在第35周收集的一些乳头状瘤对K1呈阴性,但用K13进行的免疫染色显示在所有研究的乳头状瘤中均均匀地染色了上基底细胞。在所有情况下,免疫组织化学结果均通过从每个石蜡块的7微米切片中提取的蛋白质进行的免疫印迹证实。这些结果表明,角质蛋白K1和K13在大多数乳头状瘤的生长10至35周内共表达。然而,对相邻切片的分析表明,K13阳性区域在地形上位于乳头状瘤的K1阴性区域,这表明分化程序已从表皮类型向角膜黏膜类型转移。根据我们实验室的这些研究和先前的研究,我们得出结论,K13是乳头状瘤进展的早期标志物,发生在肿瘤的干系中出现明显的染色体异常之前,并在发育异常改变和GGT表达发作之前发生,以及在单个细胞水平可能伴随K1丢失。

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