Pagano for sharing results of their Skp2 studies before publication. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-RasG12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors. Progression of mammalian cells from quiescence to proliferation involves the activation of G1-specific cyclinCcyclin-dependent protein kinase PNPP (Cdk) complexes, the inactivation of the retinoblastoma tumor suppressor protein (pRb), and accumulation of E2F transcription factors. Various studies have shown that the disruption of components of this control pathway, either by the activation of positive acting components such as cyclin D1 and Cdk4 or by the inactivation of negative components such as the tumor suppressor proteins pRb and p16, can lead to the loss of growth control underlying the development of various forms of human cancer (1). Recent developments also emphasize a central role for timely changes in protein stability at the hands of selected Rabbit Polyclonal to DDX51 E3 ubiquitinCprotein ligases during the transition from quiescence to proliferation and the F-box protein Skp2, the substrate recognition subunit of the SCFSkp2 ubiquitinCprotein ligase complex, appears to play a particularly important role in this regard (2, 3). Skp2 displays S-phase-promoting function and has been implicated in the ubiquitin-mediated proteolysis of the Cdk inhibitor p27 (4C6). Moreover, the targeted disruption of Skp2 leads to the accumulation of p27 and cell cycle arrest in G1 (7). SCFSkp2 has also been implicated in the ubiquitination of other cell cycle regulatory proteins, including cyclin E (7) and E2F-1 (8). Although Skp2 mediates PNPP the turnover of both positive and negative regulators of cell cycle progression, its overexpression leads to cell cycle progression. These apparently conflicting findings can be easily reconciled when one appreciates that these two classes of SCFSkp2 substrates differ in how their stability is influenced by cell cycle stage. p27 is stable throughout G0/G1 but becomes unstable as cells exit G1 and enter S phase. In contrast, cyclin E and E2F-1 proteolysis is constitutive and their rapid turnover during G1 and S phase progression may simply entrain the abundance of cyclin E and E2F-1 to their transcription rates. Numerous clinical studies have shown that reduced p27 PNPP protein levels correlate with poor patient prognosis in human epithelial cancers arising in diverse sites, including the breast (9C11) and colon (12). We previously reported reduced p27 protein expression in oral precancerous lesions and carcinomas compared with normal epithelial controls (13). Oral carcinomas have a significantly higher proliferative index than normal epithelium (14C20), and increased cell proliferation is also observed in precancerous oral epithelial dysplasia compared with nondysplastic epithelium (14C16, 20C22). Therefore, a progressive dysregulation of cell proliferation appears early in oral carcinogenesis. p27 appears to belong to a recently recognized class of tumor suppressors in which reduced protein expression is usually not caused by genetic change (23). The observation that Skp2 can promote the ubiquitin-mediated proteolysis of p27 prompted us to examine the relationship between the expression of Skp2 and p27 proteins during the progression from oral epithelial dysplasias to carcinoma and to test whether unregulated Skp2 function is linked to the acquisition of a neoplastic phenotype. Materials and Methods Tissues. Mucosal biopsies of 49 epithelial dysplasias and pretreatment biopsies of 24 squamous cell carcinomas (SCC) from the floor of the mouth were obtained.