Wang D, Li W, Jiang W, Humphrey L E, Howell G M, Brattain M G
Department of Surgery, University of Texas Health Science Center at San Antonio, 78284-7840, USA.
J Cell Physiol. 1998 Dec;177(3):387-95. doi: 10.1002/(SICI)1097-4652(199812)177:3<387::AID-JCP2>3.0.CO;2-L.
Previously, we reported that unaggressive, growth factor-dependent FET human colon carcinoma cells downregulated their transforming growth factor alpha (TGFalpha) expression in a quiescent state (G0/G1) induced by growth factor and nutrient deprivation (Mulder, 1991, Cancer Res., 51:2256-2262). In contrast, highly aggressive, growth factor-independent HCT116 human colon carcinoma cells aberrantly upregulated this autocrine activity in the quiescent state (Mulder, 1991, Cancer Res., 51:2256-2262; Howell et al., 1998, Mol. Cell. Biol., 18:303-313). In this report, the role of autocrine TGFalpha and the mechanism of its regulation of expression during reentry into the cell cycle from a noncycling growth state were determined in FET cells. Optimal induction of DNA synthesis from a quiescent state in FET cells is dependent upon autocrine TGFalpha as well as exogenous transferrin and insulin. Reentry into the cell cycle resulting from treatment with exogenous transferrin and insulin resulted in approximately 3-fold induction of TGFalpha expression within 1 hr. TGFalpha induction was controlled at the transcription level, and the cis-controlling element was localized to the region between bp -370 - -201 relative to the translation start codon within the TGFalpha promoter. Thus neutralization of autocrine TGFalpha protein revealed that the induced TGFalpha autocrine activity was necessary for DNA synthesis and acted only in the early G1 phase of the cell cycle. Blockade of autocrine TGFalpha expression early in the cell cycle resulted in the reduction of DNA synthesis, whereas treatment with neutralization antibody at later times had no effect. This suggested that autocrine TGFalpha functions to initiate cell growth from noncycling states. This was further confirmed by the dependence of FET cells upon autocrine TGFalpha for colony formation in experiments where the plating density was sufficiently low to generate a lag phase in tissue culture. In contrast, TGFalpha autocrine activity was not required for exponential phase cells, as evidenced by the failure of TGFalpha neutralizing antibody to inhibit proliferation in this growth state. Taken together, these results suggest that autocrine TGFalpha acts primarily in the process of growth initiation by moving cells from a noncycling state back into the cell cycle, rather than supporting cell growth already initiated.
此前，我们报道了非侵袭性、生长因子依赖性的FET人结肠癌细胞在生长因子和营养物质剥夺诱导的静止状态（G0/G1）下会下调其转化生长因子α（TGFα）的表达（Mulder，1991，《癌症研究》，51:2256 - 2262）。相反，高度侵袭性、生长因子非依赖性的HCT116人结肠癌细胞在静止状态下会异常上调这种自分泌活性（Mulder，1991，《癌症研究》，51:2256 - 2262；Howell等人，1998，《分子细胞生物学》，18:303 - 313）。在本报告中，我们确定了自分泌TGFα在FET细胞从非循环生长状态重新进入细胞周期过程中的作用及其表达调控机制。FET细胞从静止状态最佳诱导DNA合成依赖于自分泌TGFα以及外源性转铁蛋白和胰岛素。用外源性转铁蛋白和胰岛素处理导致重新进入细胞周期，在1小时内TGFα表达诱导约3倍。TGFα诱导在转录水平受到控制，顺式调控元件定位于相对于TGFα启动子内翻译起始密码子的bp -370至 -201区域。因此，自分泌TGFα蛋白的中和表明，诱导的TGFα自分泌活性对于DNA合成是必需的，并且仅在细胞周期的早期G1期起作用。在细胞周期早期阻断自分泌TGFα表达导致DNA合成减少，而在后期用中和抗体处理则没有效果。这表明自分泌TGFα的功能是从非循环状态启动细胞生长。在接种密度足够低以在组织培养中产生滞后阶段的实验中，FET细胞对自分泌TGFα的集落形成依赖性进一步证实了这一点。相反，TGFα自分泌活性对于指数生长期细胞不是必需的，这由TGFα中和抗体在这种生长状态下未能抑制增殖所证明。综上所述，这些结果表明自分泌TGFα主要在通过将细胞从非循环状态带回细胞周期的生长起始过程中起作用，而不是支持已经启动的细胞生长。
