Abstract
The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding NIPA. NIPA-deficient animals are viable, but sterile due to a block of spermatogenesis. Moreover, our studies demonstrate that loss of NIPA has no substantive effect on the physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, NIPA checkpoint control can be unmasked by oncogenic transformation by c-Myc. Here we show that transformed focus formation assays revealed higly significant differences in c-Myc-induced transformation in NIPA-null and wild-type MEFs. c-Myc transduction caused a pronounced upregulation of cyclin-B in NIPA-null MEFs, which was completely reversible by ectopic NIPA expression. This increased cyclin-B1 expression after c-Myc transduction in the absence of NIPA has considerable functional consequences for the cells: Focus formation ability of c-Myc-infected Nipa−/− MEFs was greatly reduced in comparison to wild-type MEFs (24.6% vs. 100%). Moreover, c-Myc expression caused 12.8% apoptotic subG1 cells in wild-type MEFs, whereas Nipa−/− MEFs were more affected by c-Myc-induced apoptosis (22.45%). By contrast, transduction with other oncogenes like k-Ras in p53 knockdown Nipa−/− and Nipa+/+ MEFs showed no differences in various transformation and apoptosis assays pointing out the exclusive role of the G2/M checkpoint NIPA in c-Myc induced transformation. Furthermore, we investigated the impact of these findings for the pathogenesis of c-Myc induced tumorigenesis in vivo. Recipient mice transplanted with c-Myc transduced wild-type bone marrow rapidly developed an AML-like disease (median survival 33 days) characterized by bone marrow infiltration and expression of the myeloid lineage markers CD11b and Gr1. In contrast, animals transplanted with c-myc transduced NIPA knockout BM showed a substantially delayed onset of leukemia and survived significantly longer compared to the control group (median survival 52 days, p<0.01). Taken together, our data demonstrate that NIPA is required for efficient c-Myc transformation in vitro and and in vivo in a murine bone marrow transplantation model. Moreover, our results highlight the functional importance of NIPA in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis.
Dynamic changes in subcellular localization of cattle XLF during cell cycle, and focus formation of cattle XLF at DNA damage sites immediately after irradiation
