Germ-line mutations of LKB1 (STK11) lead to Peutz-Jeghers syndrome characterized by gastrointestinal polyps and cancer of different organ systems. The mutations lead to loss or severe impairment of Lkb1 serine͞threonine kinase activity. Therefore LKB1 has been implicated as a tumor suppressor gene, but only a few mutations in the coding exons of LKB1 have been detected in sporadic tumors. Here, we have identified tumor cell lines with severely reduced mRNA levels and impaired Lkb1 kinase activity. Reintroducing Lkb1 into these cells suppressed cell growth. The Lkb1-mediated growth inhibition was caused by a G 1 cell cycle block and was not detected with several naturally occurring Lkb1 mutants. These results indicate that LKB1 has functional and specific growth-suppressing activity.
Germline mutations of the LKB1 tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS), with a predisposition to cancer. LKB1 encodes for a nuclear and cytoplasmic serine/threonine kinase, which is inactivated by mutations observed in PJS patients. Restoring LKB1 activity into cancer cell lines defective for its expression results in a G(1) cell cycle arrest. Here we have investigated molecular mechanisms leading to this arrest. Reintroduced active LKB1 was cytoplasmic and nuclear, whereas most kinase-defective PJS mutants of LKB1 localized predominantly to the nucleus. Moreover, when LKB1 was forced to remain cytoplasmic through disruption of the nuclear localization signal, it retained full growth suppression activity in a kinase-dependent manner. LKB1-mediated G(1) arrest was found to be bypassed by co-expression of the G(1) cyclins cyclin D1 and cyclin E. In addition, the protein levels of the CDK inhibitor p21(WAF1/CIP1) and p21 promoter activity were specifically upregulated in LKB1-transfected cells. Both the growth arrest and the induction of the p21 promoter were found to be p53-dependent. These results suggest that growth suppression by LKB1 is mediated through signaling of cytoplasmic LKB1 to induce p21 through a p53-dependent mechanism.
The observation that cyclin B1 protein and mRNAs are down-regulated in terminally dierentiated (TD) C2C12 cells, suggested us to investigate the transcriptional regulation of the cyclin B1 gene in these cells. Transfections of cyclin B1 promoter constructs indicate that two CCAAT boxes support cyclin B1 promoter activity in proliferating cells. EMSAs demonstrate that both CCAAT boxes are recognized by the trimeric NF-Y complex in proliferating but not in TD cells. Transfecting a dominant-negative mutant of NF-YA we provide evidence that NF-Y is required for maximal promoter activity. Addition of recombinant NF-YA to TD C2C12 nuclear extracts restores binding activity in vitro, thus indicating that the loss of NF-YA in TD cells is responsible for the lack of the NF-Y binding to the CCAAT boxes. Consistent with this, we found that the NF-YA protein is absent in TD C2C12 cells. In conclusion, our data demonstrate that NF-Y is required for cyclin B1 promoter activity. We also demonstrate that cyclin B1 expression is regulated at the transcriptional level in TD C2C12 cells and that the switch-o of cyclin B1 promoter activity in dierentiated cells depends upon the loss of a functional NF-Y complex. In particular the loss of NF-YA protein is most likely responsible for its inactivation.
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