Ets1 in Squamous Neoplasia Figure 8. Reinduction of Ets1 in older mice leads to a less severe phenotype. (A) Schematic representation of a typical time course for the re-induction studies. Some BT mice underwent transgene induction at an early age (4–6 weeks) and were then rescued by restoring the Dox diet to suppress transgene expression. The same mice were later reinduced by withdrawing Dox supplementation for an extended time course. After prolonged transgene induction, these re-induced mice developed discrete lesions (papillomatous or ulcerative, white arrows). (B) Hematoxylin and eosin stained sections below the time course indicate typical epidermal morphology at each stage of the induction process. (C) Trichrome staining of the lesions obtained from reinduced BT mice demonstrates downgrowths into the dermis. Boxed areas show regions of thin chords of invading keratinocytes. doi:10.1371/journal.pone.0004179.g008 epithelium remained unaffected (Figure 8B). Microscopic exam- ination of biopsies obtained from these lesions revealed the presence of hyperproliferation, dysplasia and deep downgrowths similar to that seen in the primary induction (Figure 8C). Staining for the basement membrane component laminin demonstrated that in some samples there were regions of basement membrane discontinuity (Figure S2). Examination of the differentiation profile of re-induced lesions indicated that they shared properties seen in the epidermis of mice that had undergone an early induction, i.e., they exhibited greatly enhanced proliferation and a partial block to terminal differentiation (Figure S3). Overall, the lesions arising in mice that had undergone secondary induction were less extensive, but potentially more invasive as demonstrated by basement membrane discontinuities. Ets1 expression upregulates a panel of genes involved in tumor growth and invasion, including MMP13 To understand the molecular mechanisms that drive the dysplastic changes in the Ets1 BT animals, we analyzed the expression levels of potential Ets1 target genes. These target genes were chosen based on prior studies demonstrating that they were regulated by Ets1 or because they play a critical role in cellular transformation, epithelial-mesenchymal transition (EMT), cell cycle regulation, apoptosis or angiogenesis. For this purpose, we isolated RNA from skin samples of affected BT animals as well as control animals and performed real-time RT-PCR. These analyses allowed us to identify a number of genes whose expression was upregulated in BT epidermis as compared to wild-type or single- transgenic epidermis. The results of these analyses are summarized in Table 1. We detected increased expression of several of the target genes examined including those that are involved in cellular transformation such as Myc and AP-1 proteins (including Junb, Jun (c-Jun) and Fos), cell cycle regulators (including Cdkn1a (p21) and Cdkn2a (p16)) and the anti-apoptotic protein Bcl2l1 (Bcl-xL). In addition to these genes, we also examined a panel of genes that are involved in invasive processes, since the downgrowth of keratinocyte fronts into the dermis was a common feature in all the affected stratified epithelia. Several matrix metallo-proteases (MMPs) including collagenases (MMPs 8 and 13), gelatinase (MMP 9), stromelysins (MMPs 11 and 12), elastase (MMP12) and membrane associated MMPs (MMP 14 and 16) as well as their natural inhibitor Timp-1 were upregulated in BT samples (Table 1). Of the upregulated target genes, MMP13 was the most dramatically induced gene in Ets1 BT mice. PLoS ONE | www.plosone.org 8 January 2009 | Volume 4 | Issue 1 | e4179