Awwad Rana A, Sergina Natalia, Yang Haisu, Ziober Barry, Willson James K V, Zborowska Elizabeth, Humphrey Lisa E, Fan Robert, Ko Tien C, Brattain Michael G, Howell Gillian M
Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Cancer Res. 2003 Aug 1;63(15):4731-8.
Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.
生长因子非依赖性是恶性肿瘤的一个标志，这归因于癌细胞中自分泌生长因子环的形成。然而，依赖生长因子的正常细胞也表现出自分泌活性，因此引发了一个问题，即癌细胞内源性产生的活性是以何种方式不同从而导致生长因子非依赖性的。我们通过将不依赖生长因子的HCT116人结肠癌细胞与从同一患者肿瘤中分离出的称为HCT116b的依赖生长因子的恶性细胞亚群进行比较来研究这个问题。因此，不依赖生长因子表型的形成代表了体内肿瘤内的克隆进展。已表明HCT116细胞的生长因子非依赖性依赖于自分泌转化生长因子（TGF）-α活性，然而，当细胞处于指数生长时，同亲本的HCT116b亚群显示出与HCT116相似水平的TGF-α表达。当两种细胞系因营养剥夺而生长停滞时，HCT116b细胞重新启动DNA合成需要补充营养和生长因子，而HCT116细胞仅需要补充营养。与依赖生长因子的HCT116b细胞相反，HCT116细胞在生长停滞期间由于转录增强而出现TGF-α表达上调。静止的HCT116细胞中这种TGF-α表达的增加与生长停滞状态下组成型表皮生长因子受体（EGFR）激活相关，而生长停滞的HCT116b细胞未显示EGFR激活。HCT116细胞的TGF-α反义转染表明EGFR激活是由于TGF-α表达增加。用AG1478（一种EGFR酪氨酸激酶活性的选择性抑制剂）预处理生长停滞的HCT116细胞可阻断DNA合成的重新启动，这表明生长因子非依赖性是由于这些生长停滞细胞相对于依赖生长因子的HCT116b细胞中TGF-α表达增加和EGFR激活所致。重要的是，生长停滞的HCT116细胞中EGFR激活水平仅略高于指数生长细胞，这表明是生长停滞时不适当的EGFR激活而非激活幅度产生了生长因子非依赖性。
