Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway

B-Myb, a highly conserved member of the Myb oncoprotein family, is a 110 kDa sequence-specific DNA binding protein expressed in virtually all proliferating cells. B-myb expression reaches its maximum at the G1/S phase boundary and during the S phase of the cell cycle. We have previously shown that B-Myb activity is cell cycle regulated and it is controlled by the antagonistic effects of cyclin D1 and A. Here we show that ectopic expression of cyclin A causes a pronounced reduction of B-Myb protein level. We provide evidence that in addition to triggering B-Myb activity an important effect of cyclin A is to facilitate multiple ubiquitination of B-Myb. The C-terminal domain of B-Myb is of key importance in mediating this effect of cyclin A. Contrary to full-length B-Myb, a C-terminal deletion mutant displays activity irrespective of cyclin A expression, does not undergo ubiquitination, and its half-life is not affected by cyclin A. Ectopic expression of either Cdc34 or the F-box protein p45Skp2, respectively the E2 and E3 components of a ubiquitination pathway that regulates the G1/S transition, accelerates degradation of B-Myb. We show that B-Myb physically and functionally interacts with components of the Cdc34-SCFp45Skp2 ubiquitin pathway and propose that B-Myb degradation may be required for controlling the correct alternation of events during progression through the cell division cycle. Oncogene (2000).     

Loss of LIN9, a member of the DREAM complex, cooperates with SV40 large T antigen to induce genomic instability and anchorage-independent growth

The DREAM complex is an important regulator of mitotic gene expression during the cell cycle. Here we report that inactivation of LIN9, a subunit of DREAM, results in premature senescence, which can be overcome by the SV40 large T (LT) antigen. Together with the observation that p16(INK4a) and p21(Waf1) are upregulated upon loss of LIN9, these results indicate that senescence is triggered by the pRB and p53 tumor suppressor pathways. We also find that LIN9-null cells that escape senescence are chromosomally instable because of compromised mitotic fidelity. SV40 LT-expressing cells that adapt to the loss of LIN9 can grow anchorage-independently in soft agar, a hallmark of oncogenic transformation. Taken together, these results suggest an important role of mitotic gene regulation in the maintenance of genomic stability and tumor suppression.     

Characterization of epithelial senescence by serial analysis of gene expression: identification of genes potentially involved in prostate cancer

Evasion of cellular senescence is required for the immortal phenotype of tumor cells. The tumor suppressor genes p16(INK4A), pRb, and p53 have been implicated in the induction of cellular senescence. To identify additional genes and pathways involved in the regulation of senescence in prostate epithelial cells (PrECs), we performed serial analysis of gene expression (SAGE). The gene expression pattern of human PrECs arrested because of senescence was compared with the pattern of early passage cells arrested because of confluence. A total of 144,137 SAGE tags representing 25,645 unique mRNA species was collected and analyzed: 157 mRNAs (70 with known function) were up-regulated and 116 (65 with known function) were down-regulated significantly in senescent PrECs (P < 0.05; fold difference >2.5). The differential regulation of an exemplary set of genes during senescence was confirmed by quantitative real-time PCR in PrECs derived from three different donors. The results presented here provide the molecular basis of the characteristic changes in morphology and proliferation observed in senescent PrECs. Furthermore, the differentially expressed genes identified in this report will be instrumental in the further analysis of cellular senescence in PrECs and may lead to the identification of tumor suppressor genes and proto-oncogenes involved in the development of prostate cancer.