Fusenig N E, Boukamp P
Division of Carcinogenesis and Differentiation, German Cancer Research Center (DKFZ), Heidelberg.
Mol Carcinog. 1998 Nov;23(3):144-58. doi: 10.1002/(sici)1098-2744(199811)23:3<144::aid-mc3>3.0.co;2-u.
An in vitro carcinogenesis model of human skin keratinocytes has been developed based on the spontaneously immortalized keratinocyte cell line HaCaT. Immortalization, the initial stage in human carcinogenesis in vitro, was induced by ultraviolet-type mutations in the p53 gene followed by further genetic alterations leading to the loss of senescence genes, in particular on chromosome 3p. Despite multiple genetic changes, the HaCaT cell line sustained its genomic balance up to high passage levels and maintained a non-tumorigenic phenotype. Tumorigenic transformation was induced by ras oncogene transfection but also by culture stress and elevated temperature, resulting in benign and malignant tumorigenic clones. Malignant conversion was associated with the loss of a copy of chromosome 15, leading to a decrease in thrombospondin-1 (TSP-1) expression. Heat-induced malignant conversion was associated with a gain of material on chromosome 11, including the cyclin D1 gene. The microenvironment plays a major role in tumorigenic transformation and the control of malignant cells. Overexpression of platelet-derived growth factor in HaCaT cells caused mesenchyme activation and formation of benign tumors. Halting tumor angiogenesis completely prevented invasion of malignant cells and induced a benign tumor phenotype. Transfer of a normal chromosome 15 or TSP-1 transfection into a skin carcinoma line resulted in tumor suppression due to TSP-1-blocked tumor vascularization. Because of the reduced TSP-1 expression, blood vessels infiltrated the tumor, and it expanded. Progression to more aggressive tumor phenotypes required the in vivo environment and was caused by selection of a subpopulation and further genetic modifications. The improved autonomous growth of these cells was associated with new expression of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which acted in an autocrine manner to stimulate proliferation and migration. With this in vitro skin carcinogenesis model we were able to demonstrate multiple stages in the transformation process that were associated with different genetic and phenotypic characteristics. In addition, we documented that modulation of the tumor stroma plays an important and decisive role in tumor development and progression. From this we hypothesize that the growth restraints of the microenvironment are increasingly lost with advancing stages of carcinogenesis but can be restored by modulation of the tumor stroma.
基于自发永生化的角质形成细胞系HaCaT，已建立了一种人皮肤角质形成细胞的体外致癌模型。永生化是体外人类致癌作用的初始阶段，由p53基因中的紫外线型突变诱导，随后发生进一步的基因改变，导致衰老基因丧失，特别是3号染色体短臂上的衰老基因。尽管发生了多种基因变化，但HaCaT细胞系在传代至较高代数时仍维持其基因组平衡，并保持非致瘤表型。致癌转化可通过ras癌基因转染诱导，也可通过培养应激和升高温度诱导，从而产生良性和恶性致瘤克隆。恶性转化与15号染色体一个拷贝的缺失有关，导致血小板反应蛋白-1（TSP-1）表达降低。热诱导的恶性转化与11号染色体上物质的增加有关，包括细胞周期蛋白D1基因。微环境在致癌转化和恶性细胞的控制中起主要作用。HaCaT细胞中血小板衍生生长因子的过表达导致间充质激活和良性肿瘤形成。完全阻断肿瘤血管生成可防止恶性细胞侵袭并诱导良性肿瘤表型。将正常的15号染色体转移或TSP-1转染到皮肤癌系中，由于TSP-1阻断肿瘤血管生成，导致肿瘤抑制。由于TSP-1表达降低，血管浸润肿瘤并使其扩大。进展为更具侵袭性的肿瘤表型需要体内环境，并且是由亚群选择和进一步的基因修饰引起的。这些细胞自主生长的改善与粒细胞集落刺激因子和粒细胞-巨噬细胞集落刺激因子的新表达有关，它们以自分泌方式刺激增殖和迁移。通过这个体外皮肤致癌模型，我们能够证明转化过程中的多个阶段，这些阶段与不同的基因和表型特征相关。此外，我们记录到肿瘤基质的调节在肿瘤发生和进展中起重要且决定性的作用。由此我们推测，随着致癌作用进展，微环境的生长限制逐渐丧失，但可通过调节肿瘤基质恢复。
