Dlugosz A A, Cheng C, Williams E K, Darwiche N, Dempsey P J, Mann B, Dunn A R, Coffey R J, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892, USA.
Cancer Res. 1995 May 1;55(9):1883-93.
Autocrine epidermal growth factor receptor activation by transforming growth factor alpha (TGF alpha) has been implicated in growth stimulation during epithelial neoplasia. Using keratinocytes isolated from mice with genetic defects in TGF alpha expression, we tested whether TGF alpha is required for transformation by the v-rasHa oncogene. Introduction of v-rasHa into primary epidermal cultures using a retroviral vector stimulated growth of both control (TGF alpha +/+, BALB/c) and TGF alpha-deficient (TGF alpha -/-, wa-1) keratinocytes. Moreover, v-rasHa elicited characteristic changes in marker expression (keratin 1 was suppressed; keratin 8 was induced), previously shown to be associated with epidermal growth factor (EGF) receptor activation, in both TGF alpha +/+ and TGF alpha -/- keratinocytes. v-rasHa markedly increased secreted (> 10-fold) and cell-associated (2-3-fold) TGF alpha levels in keratinocytes from TGF alpha +/+ and BALB/c mice, but not TGF alpha -/- or wa-1 mice. Based on Northern blot analysis, v-rasHa induced striking up-regulation of transcripts encoding the additional EGF family members amphiregulin, heparin-binding EGF-like growth factor, and betacellulin in cultured keratinocytes from all four mouse strains. Interestingly, in addition to the normal 4.5-kilobase TGF alpha transcript, wa-1 keratinocytes expressed two additional TGF alpha transcripts, 4.7 and 5.2 kilobases long. All three transcripts were up-regulated in response to v-rasHa, as well as exogenous TGF alpha or keratinocyte growth factor treatment, and were also detected in RNA isolated from wa-1 brain and skin. In vivo, v-rasHa keratinocytes from control as well as TGF alpha-deficient mice produced squamous tumors when grafted onto nude mice, and these lesions expressed high levels of amphiregulin, heparin-binding EGF-like growth factor, and betacellulin mRNA, regardless of their TGF alpha status. These findings indicate that TGF alpha is not essential for epidermal neoplasia induced by the v-rasHa oncogene and suggest that another EGF family member(s) may contribute to autocrine growth stimulation of ras-transformed keratinocytes.
转化生长因子α（TGFα）自分泌激活表皮生长因子受体与上皮肿瘤形成过程中的生长刺激有关。我们使用从TGFα表达存在基因缺陷的小鼠中分离出的角质形成细胞，来测试TGFα对于v-rasHa癌基因转化是否必要。使用逆转录病毒载体将v-rasHa导入原代表皮培养物中，刺激了对照（TGFα +/+，BALB/c）和TGFα缺陷（TGFα -/-，wa-1）角质形成细胞的生长。此外，v-rasHa在TGFα +/+和TGFα -/-角质形成细胞中均引发了标志物表达的特征性变化（角蛋白1被抑制；角蛋白8被诱导），此前已证明这些变化与表皮生长因子（EGF）受体激活有关。v-rasHa显著增加了来自TGFα +/+和BALB/c小鼠的角质形成细胞中分泌型（超过十倍）和细胞相关型（两到三倍）TGFα水平，但未增加TGFα -/-或wa-1小鼠角质形成细胞中的TGFα水平。基于Northern印迹分析，v-rasHa在来自所有四种小鼠品系的培养角质形成细胞中诱导了编码额外的EGF家族成员双调蛋白、肝素结合EGF样生长因子和β细胞ulin的转录本的显著上调。有趣的是，除了正常的4.5千碱基TGFα转录本外，wa-1角质形成细胞还表达了另外两种TGFα转录本，长度分别为4.7和5.2千碱基。所有三种转录本在对v-rasHa以及外源性TGFα或角质形成细胞生长因子处理的反应中均上调，并且在从wa-1脑和皮肤分离出的RNA中也被检测到。在体内，来自对照以及TGFα缺陷小鼠的v-rasHa角质形成细胞移植到裸鼠上时会产生鳞状肿瘤，并且这些病变表达高水平的双调蛋白、肝素结合EGF样生长因子和β细胞ulin mRNA，无论其TGFα状态如何。这些发现表明TGFα对于v-rasHa癌基因诱导的表皮肿瘤形成并非必不可少，并提示另一种EGF家族成员可能有助于ras转化的角质形成细胞的自分泌生长刺激。
