No abstract

Here we show that ectopic expression of a kinase-active Plk3 (Plk3-A) induced apoptosis. In response to DNA damage, the kinase activity of Plk3 was rapidly increased in an ATMdependent manner, whereas that of Plk1 was markedly inhibited. Recombinant Plk3 phosphorylated in vitro a glutathione S-transferase fusion protein containing p53, but not glutathione S-transferase alone. Recombinant Plk1 also phosphorylated p53 but on residues that differed from those targeted by Plk3. Co-immunoprecipitation and pull-down assays demonstrated that Plk3 physically interacted with p53 and that this interaction was enhanced upon DNA damage. In vitro kinase assays followed by immunoblotting showed that serine 20 of p53 was a target of Plk3. Furthermore, expression of a kinase-defective Plk3 mutant (Plk3 K52R ) resulted in significant reduction of p53 phosphorylation on serine 20, which was correlated with a decrease in the expression of p21 and with a concomitant increase in cell proliferation. These results strongly suggest that Plk3 functionally links DNA damage to cell cycle arrest and apoptosis via the p53 pathway.

Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and differentiation. Because microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using 2 spontaneous mouse MB models with specific initiating mutations, Ink4c؊/؊; Ptch1؉/؊ and Ink4c؊/؊; p53؊/؊. We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, 9 were encoded by the miR-17ϳ92 cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that 3 miR-17ϳ92 cluster miRNAs (miR-92, miR-19a, and miR-20) were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog (SHH) signaling pathway, but not in other forms of the disease. To test whether the miR-17ϳ92 cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6 Ink4c؊/؊; Ptch1؉/؊ mice. These, but not similarly engineered cells from Ink4c؊/؊; p53؊/؊ mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing miR-17ϳ92 lost expression of the wild-type Ptch1 allele. Our findings suggest a functional collaboration between the miR-17ϳ92 cluster and the SHH signaling pathway in the development of MBs in mouse and man.cerebellum ͉ microRNAs ͉ oncomiR1 ͉ granule neuron progenitors

Recurrent genetic alterations in human medulloblastoma (MB) include mutations in the sonic hedgehog (SHH)signaling pathway and TP53 inactivation (∼25% and 10% of cases, respectively). However, mouse models of MB, regardless of their initiating lesions, generally depend upon p53 inactivation for rapid onset and high penetrance. The gene encoding the cyclin-dependent kinase inhibitor p18 Ink4c is transiently expressed in mouse cerebellar granule neuronal precursor cells (GNPs) as they exit the cell division cycle and differentiate. Coinactivation of Ink4c and p53 provided cultured GNPs with an additive proliferative advantage, either in the presence or absence of Shh, and induced MB with low penetrance but with greatly increased incidence following postnatal irradiation. In contrast, mice lacking one or two functional Ink4c alleles and one copy of Patched (Ptc1) encoding the Shh receptor rapidly developed MBs that retained wild-type p53. In tumor cells purified from double heterozygotes, the wild-type Ptc1 allele, but not Ink4c, was inactivated. Therefore, when combined with Ptc1 mutation, Ink4c is haploinsufficient for tumor suppression. Methylation of INK4C (CDKN2C) was observed in four of 23 human MBs, and p18 INK4C protein expression was extinguished in 14 of 73 cases. Hence, p18 INK4C loss may contribute to MB formation in children.[Keywords: Cyclin-dependent kinase inhibitors; haploinsufficiency; sonic hedgehog signaling] Supplemental material is available at http://www.genesdev.org. Neuronal proliferation, differentiation, and migration are coordinated during cerebellar development, and disruption of these processes can lead to medulloblastoma (MB), the most common malignant pediatric brain tumor (Gilbertson 2004;Marino 2005). Unlike most organogenesis, the cerebellum is largely formed after birth. In the newborn mouse (postnatal day 0 [P0]), the cerebellum is only composed of a thin layer of granule neuronal precursor cells (GNPs) overlying the Purkinje cell plate. Purkinje neurons produce the mitogen sonic hedgehog (Shh) that drives a massive proliferation of GNPs between P1 and P15, thereby expanding the external germinal layer (EGL). GNPs then exit the cell division cycle, extend axons, and migrate inward through the underlying Purkinje layer to form the more deeply situated internal granule layer (IGL). Differentiation of granule neurons results in the elaboration of parallel fibers within the external molecular layer of the cerebellum and the formation of synapses on the dendritic arbors of Purkinje cells (Goldowitz and Hamre 1998;Wang and Zoghbi 2001;Hatten 2002).Recurrent genetic alterations in childhood MBs include lesions in components of the SHH and WNT signaling pathways, persistent expression of pro-proliferative cell cycle control genes such as MYCN, cyclin D1, and cyclin D2 (CCND1 and CCND2, respectively), and inactivation of the TP53 tumor suppressor (Gilbertson 2004;Marino 2005). While these molecular alterations might account for the development of certain subgroups of human MB, the geneti...

The physiologic function of BUBR1, a key component of the spindle checkpoint, was examined by generating BUBR1-mutant mice. BUBR1 ؊/؊ embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1 ؉/؊ blastocysts grew relatively normally in vitro, BUBR1 ؊/؊ blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1 ؉/؊ mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes, which was correlated with an increase in megakaryocytic, but a decrease in erythroid, progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However, enhanced megakaryopoiesis in BUBR1 ؉/؊ mice was not correlated with a significant increase in platelets in peripheral blood, which was at least partly due to a defect in the formation of proplateletproducing megakaryocytes. Together, these results indicate that BUBR1 is essential for early embryonic development and normal