Recently, we described phorbol ester-induced expression of the brain and skin serine proteinase Bssp/kallikrein 6 (Klk6), the mouse orthologue of human KLK6, in mouse back skin and in advanced tumor stages of a well-established multistage tumor model. Here, we show KLK6 up-regulation in squamous skin tumors of human patients and in tumors of other epithelial tissues. Ectopic Klk6 expression in mouse keratinocyte cell lines induces a spindle-like morphology associated with accelerated proliferation, migration, and invasion capacity. We found reduced E-cadherin protein levels in the cell membrane and nuclear translocation of B-catenin in Klk6-expressing mouse keratinocytes and human HEK293 cells transfected with a KLK6 expression plasmid. Additionally, HEK293 cells exhibited induced T-cell factor-dependent transcription and impaired cell-cell adhesion in the presence of KLK6, which was accompanied by induced E-cadherin ectodomain shedding. Interestingly, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 interfere with KLK6-induced E-cadherin ectodomain shedding and rescue the cell-cell adhesion defect in vitro, suggesting the involvement of matrix metalloproteinase and/or a disintegrin and metalloproteinase (ADAM) proteolytic activity. In line with this assumption, we found increased levels of the mature 62-kDa ADAM10 proteinase in cells expressing ectopic KLK6 compared with mock controls. Finally, enhanced epidermal keratinocyte proliferation and migration in concert with decreased E-cadherin protein levels are confirmed in an in vivo Klk6 transgenic mouse model. [Cancer Res 2007; 67(17):8198-206]

Polyomavirus (Py) large and small tumorantigens together are competent to induce S phase in growth-arrested mouse fibroblasts. The capacity of the large tumorantigen to bind the pocket proteins, pRB, p130 and p107, is important for the transactivation of DNA synthesis enzymes and the cyclins E and A, while the interference of small tumorantigen with protein phosphatase PP2A causes a destabilization of the cdk2 inhibitor p27, and thus leads to strong cyclin E-and cyclin A-dependent cdk2 activity. Py small tumorantigen, in addition, is able to transactivate cyclin A. Hence, this protein might have a much wider effect on gene expression in arrested mouse fibroblasts than hitherto suspected. This may have a profound part in the known capacity of Py to form tumors in mice. Therefore, it was interesting to gain an insight into the spectrum of transcriptional deregulation by Py tumorantigens. Accordingly, we performed microarray analysis of quiescent mouse fibroblasts in the absence and presence of small or large tumorantigen. We found that the viral proteins can induce or repress a great variety of genes beyond those involved in the S phase induction and DNA synthesis. The results of the microarray analysis were confirmed for selected genes by several methods, including real-time PCR. Interestingly, a mutation of the binding site for pocket proteins in case of LT and for PP2A in case of ST has a variable effect on the deregulation of genes by the viral proteins depending on the gene in question. In fact, some genes are transactivated by LT as well as ST completely independent of an interaction with their major cellular targets, pocket proteins and PP2A, respectively.

Polyomavirus large and small T antigens cooperate in the induction of S phase in serum-deprived Swiss 3T3 cells. While the large T antigen is able to induce S phase-specific enzymes, we have recently shown that both T antigens contribute to the production of the cyclins E and A and that the small T antigen is essential for the induction of cyclin A-dependent cdk2 activity (S. Schüchner and E. Wintersberger, J. Virol. 73:9266-9273, 1999). Here we present our attempts to elucidate the mechanisms by which the large and the small T antigens transactivate the murine cyclin A gene. Using Swiss 3T3 cells carrying the T antigens and various mutants thereof under the hormone-inducible mouse mammary tumor virus promoter, as well as transient-cotransfection experiments with the T antigens and cyclin A promoter-luciferase reporter constructs, we found the following. The large T antigen activates the cyclin A promoter via two transcription factor binding sites, a cyclic AMP responsive element (CRE), and the major negative regulatory site called CDE-CHR. While an intact binding site for pocket proteins is required for the function of this T antigen at the CDE-CHR, its activity at the CRE is largely independent thereof. In contrast, an intact J domain and an intact zinc finger are required at both sites. The small T antigen also appears to have an influence on the cyclin A promoter through the CRE as well as the CDE-CHR. For this an interaction with protein phosphatase 2A is essential; mutation of the J domain does not totally eliminate but greatly reduces the transactivating ability.The induction of S phase of the cell cycle is a complex reaction usually initiated at the cell surface through binding of ligands to receptors. Signal transduction pathways lead to the synthesis of S phase-specific enzymes and regulators, including the cyclins E and A. DNA tumor viruses require cells in S phase because they heavily depend on the cellular DNA synthesis machinery for the replication of their own DNA. Since they frequently infect differentiated cells, they have to interfere with the cellular mechanisms of growth regulation in order to drive cells out of the G 0 phase and into the S phase (reviewed in reference 19). This is accomplished by viral proteins which interact with various intermediates of the signal transduction pathway downstream of events taking place at the cell surface. One class of target for such viral proteins are the pocket proteins: pRB and its relatives p107 and p130. They negatively regulate members of the transcription factor family E2F (reviewed in reference 8). Adenovirus E1A, human papillomavirus (HPV) E7 protein, and the large T (LT) antigens of simian virus 40 (SV40), and polyomavirus (Py) bind to pocket proteins and cause a dissociation of the repressive complexes, which results in the transactivation of E2F responsive genes (19). Another region of the pleiotropic T antigens which is essential for this reaction is the J domain (reviewed in references 4 and 16), a sequence present in many chaperones capable ...

DNA tumor viruses are capable of driving quiescent cells into the cell cycle. In case of polyomaviridae, two viral proteins, the large and the small (ST) T antigens are responsible for this outcome. ST interacts with the protein phosphatase PP2A and with chaperons of the dnaK type and leads to the transactivation of several genes, which play a role in S-phase induction. One of these is the transcription factor myelocytomatosis (MYC), which by itself is an important regulator of growth. Microarray analysis has revealed several ST-induced genes, which are also targets of MYC; hence, ST may induce these genes via MYC. Experiments shown here are in line with this assumption. MYC-regulated genes are induced by ST at later times than MYC and a MYC responsive promoter is stimulated by ST. Regulation of MYC occurs through signal transduction pathways, which are co-ordinated by PP2A suggesting that they may be targets of ST. Here, we show that this is the case as important kinases involved in these pathways appear in the active phosphorylated form in the presence of ST. Inhibition of these kinases interferes with MYC induction and inhibition of MYC activity blocks ST-mediated transactivation.