Investigation into the mechanism of tumorigenesis using p53-deficient epithelial cells

目的：从P53基因缺陷小鼠颌下腺建立永生化导管/基底细胞系(MSE)，探讨P53基因缺陷小鼠上皮细胞癌变的基本途径。最具挑衅性的暗示之一是，当MSE与NIH3T3(3T3)细胞和Matrigel细胞共同移植时，MSE显著形成肿瘤(MSA)。方法：从p53基因缺失的小鼠解剖的10块颌下腺，放置在包被I型胶原的细胞培养板上。生长后，进行有限稀释克隆，用于MSE的分选。同样，MSA也从MSE、Matrigel和3T3的异种移植瘤中亚克隆。进行了多种培养方法和异种移植实验。结果：与MSE相比，MSA在单层培养中表现出细胞多形性，并可见多个多核上皮细胞。MSA的增殖率是MSE的2倍。MSA的平均菌落数高于MSE，表现出明显的生长行为差异。然而，与高度侵袭的ADCC相反，这两种细胞系都缺乏侵袭能力。接种MSE和Matrigel的混合物重建极化导管，而将MSE与Matrigel和3T3细胞共同移植形成腺瘤和肉瘤的混合瘤。总体而言，MSA在体外获得了一些转化表型，但在体内明显缺乏恶性特性。结论：3T3肉瘤转化是MSE肿瘤发生的关键事件。P53缺失的涎腺上皮的固有致癌程序是通过3T3的肉瘤转化促进间质-上皮相互作用的。

Objective: To dissect the fundamental pathway governing neoplastic conversion of p53-deficient epithelial cells, we established an immortalized duct/ basal cell line (MSE) from the submandibular glands of p53-deficient mice. One of the most provocative implications is that MSE became dramatically tumorigenic (MSA), when co-transplanted with both NIH3T3 (3T3) cells and Matrigel.Methods: Ten pieces of minced submandibular glands dissected from p53 null mice were placed on cell culture plates coated with type I collagen. After outgrowth, limiting dilution cloning was performed for sorting MSE. Similarly, MSA were also subcloned from tumors derived from xenografts of MSE, Matrigel and 3T3. A variety of culture assays and xenograft experiments were conducted. Results: As compared with MSE, MSA showed cellular pleomorphism and several multinucleated epithelial cells were visible in the monolayer culture. The proliferation rate of MSA was 2-fold higher than MSE. The average number of colonies was higher in MSA than that in MSE, showing significant difference in growth behaviors. In contrast to a highly invasive AdCC, both cell lines, however, lacked invasive capacity. Inoculation of a mixture of MSE and Matrigel reconstructed polarized ducts whereas co-transplantation of MSE with both Matrigel and 3T3 cells developed mixed tumors of adenoma and sarcoma. Overall, MSA gained some transformed phenotypes in vitro, but apparently lacked malignant properties in vivo.Conclusion : Sarcomatous transformation of 3T3 is a key event in MSE tumorigenesis. The intrinsic tumorigenic programs of p53 null salivary epithelium are promoted by the stromal-epithelial interactions via sarcomatous transformation of 3T3.