TGFb has both tumor suppressive and oncogenic activites in cancer development that may reflect stage specific responses of the tumor cells to this cytokine. In order to assess the effects of TGFb at different stages of premalignant progression in vivo, we have generated transgenic mice in which the tetracycline regulatable transactivators tTA and rtTA are targeted to the epidermis with a keratin 5 promoter. These mice have been crossed with a transgenic line containing a tetO regulated constitutively active TGFb. The expression of TGFb can be controlled in the double transgenic mice with doxycycline, and we have demonstrated reversible neonatal lethality and adult alopecia when TGFb is expressed during development or in the adult skin (Liu, et al., PNAS 93:9139-9144, 2001). To identify TGFb targets that might differ between benign and malignant epidermal tumors, we have generated tumors using a DMBA-TPA chemical carcinogenesis protocol. In double transgenic mice bearing either papillomas or squamous cell carcinoma (SCC) we have induced expression of the tetOTGFb construct with doxycycline. RT-PCR analysis of RNA isolated from whole skin showed that within 24 hrs of addition of doxycycline, TGFb1 transgene mRNA was induced to near maximal levels. Expression of TGFb1 in double transgenic papillomas caused a rapid reduction in cell proliferation, and regression within 1 week accompanied by a lymphocytic infiltrate. Induction of TGFb1 in bitransgenic squamous cell carcinoma did not reduce cell proliferation compared to single transgenic SCC. To identify stage specific transcriptional targets of TGFb1 we used cDNA microarrays to compare the pattern of gene expression in double transgenic papillomas and carcinomas 48 hrs after doxycycline addition with single transgenic papillomas and carcinomas, respectively. The filtered data was compared using a Two Group Wilcoxon Rank-Sum analysis to identify genes that were differentially regulated by TGFb1 between the papillomas and carcinomas. Of 117 genes identified with a p value<.02, 63% were upregulated in papillomas but downregulated in carcinomas. A significant fraction of the genes suppressed in the SCC expressing TGFb 1 were those regulated by, or important in the interferon signaling pathway such as Stat1, IRF-1, IRF-6, Ifi204, and H2 locus. We are currently verifying the array results in the tumor tissues and determining the significance of the downregulation of the interferon pathway by TGFb1 for tumor progression.
| Diamond, I; Owolabi, T; Marco, M et al. (2000) Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter. J Invest Dermatol 115:788-94 |
