Increased E 2 F 1 activity induces skin tumors in mice heterozygous and nullizygous for p 53

The p16INK4a-cyclin D–retinoblastoma tumor suppressor pathway is disrupted in most human cancers, and it has been suggested that the subsequent release of E2F transcription factors from inhibitory complexes may be a key event in tumor development. We described recently the generation of transgenic mice with E2F1 gene expression targeted to squamous epithelial tissues by a keratin 5 (K5) promoter. In the present study, K5 E2F1 transgenic mice were crossed with p53 null mice to examine functional interactions between E2F1 and p53 in vivo. We find that E2F1-induced apoptosis of epidermal keratinocytes is reduced in K5 E2F1 transgenic mice lacking p53, whereas E2F1-induced hyperproliferation is unaffected by p53 status. We also find that K5 E2F1 transgenic mice heterozygous or nullizygous for p53 develop spontaneous skin carcinomas, which normally are rare in p53-deficient mice. The timing of tumor development correlates with the level of E2F1 transgene expression and the status of p53. In primary transgenic keratinocytes, the major change in E2F1 DNA-binding activity is the generation of a complex also containing the retinoblastoma tumor suppressor protein. Nevertheless, the expression and associated kinase activity of cyclin E, a known target for E2F transcriptional activity, is elevated significantly in K5 E2F1 transgenic keratinocytes. These findings firmly establish that increased E2F1 expression can contribute to tumor development and suggest that p53 plays an important role in eliminating cells with deregulated E2F1 activity. It has been demonstrated that loss of retinoblastoma tumor suppressor (Rb) function can lead to unchecked proliferation and tumorigenesis in a variety of model systems (1–6). In many cases, however, loss of Rb function leads to p53-dependent apoptosis (3–9). In the absence of functional p53, apoptosis as a consequence of Rb inactivation is suppressed. Thus, the status of p53 can determine whether loss of Rb results in apoptosis or deregulated proliferation. This finding likely explains why DNA tumor viruses such as simian virus 40, adenovirus, and human papilloma virus encode proteins that inactivate both Rb and p53. This ‘‘guardian of Rb’’ function for p53 also is consistent with the observation that in many human cancers with Rb mutations, p53 also is inactivated (10). The Rb protein has been shown to bind and regulate a large number of cellular proteins, including members of the E2F transcription factor family (11). It has been suggested that deregulation of E2F transcriptional activity as a consequence of Rb inactivation contributes to the development of cancer (11–13). This hypothesis is supported by the finding that several members of the E2F gene family can behave as oncogenes in cell culture transformation assays (14–17). However, increased E2F activity has not been shown directly to contribute to tumorigenesis in vivo. Deregulation of E2F transcription factors may also play a role in the induction of apoptosis as a result of Rb inactivation. At least one member of the E2F gene family, E2F1, has been shown to induce apoptosis in a p53-dependent manner (18–20). We described recently the generation of transgenic mice containing the human E2F1 gene under the control of the bovine keratin 5 (K5) promoter (21). Two lines were established (K5 E2F1 lines 1.0 and 1.1), both of which showed E2F1 overexpression in basal cells of the epidermis, hair follicles, and other squamous epithelial tissues. E2F1 overexpression results in epidermal hyperplasia and hyperproliferation but does not appear to block epidermal differentiation. The higher expressing line 1.0 transgenic mice also have a hair growth defect. Examination of the skin by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay suggested that the paucity of hair seen in this line is the result of aberrant apoptosis in developing hair follicles. In addition to promoting proliferation and inducing apoptosis, we also found that the K5 E2F1 transgene could cooperate with a v-Ha-ras transgene to induce benign skin papillomas. In this report we describe the phenotype of K5 E2F1 transgenic mice that are also deficient (heterozygous or nullizygous) for p53. We find that aberrant apoptosis in the epidermis of K5 E2F1 line 1.0 mice is reduced in a p53 null background. We also find that K5 E2F1 transgenic mice heterozygous or nullizygous for p53 develop spontaneous skin tumors. The majority of these tumors are squamous cell carcinomas (SCC) or basal cell carcinomas (BCC), which rarely develop in p53-deficient mice. Examination of transgenic keratinocytes suggests that tumor development may require only a modest increase in E2F1 DNA-binding activity and involve up-regulation of the cyclin E gene. MATERIALS AND METHODS Transgenic Mice. The generation of K5 E2F1 transgenic mice has been described (21). Briefly, the transgene contains the bovine keratin 5 promoter (22), the rabbit b-globin intron 2, the human E2F1 cDNA, and the simian virus 40 polyadenylation signal. Transgenic mice were genotyped by PCR amplification of sequences specific for the b-globin intron andyor the human E2F1 cDNA from tail genomic DNA. The genetic background of the founder mice was C57BLy6 3 SJL F2 hybrid. The original p53 heterozygous mice (23, 24) used for breeding were C57BLy6. All mice in this study were at least 75% C57BLy6. BrdUrd Incorporation. Mice were injected i.p. with 0.06 mg of BrdUrd per gram of body weight, allowed to incorporate for 30 min, and sacrificed. Skin samples were collected, fixed in formalin, and paraffin-embedded. Tissue sections then were subjected to immunohistochemistry as described below using antibody The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y958858-6$2.00y0 PNAS is available online at http:yywww.pnas. org. Abbreviations: Rb, retinoblastoma tumor suppressor; K5, keratin 5; SCC, squamous cell carcinoma; BCC, basal cell carcinoma; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick endlabeling. *To whom reprint requests should be addressed.