Gauchos and ochos: a Wee1-Cdk tango regulating mitotic entry

The kinase Wee1 has been recognized for a quarter century as a key inhibitor of Cyclin dependent kinase 1 (Cdk1) and mitotic entry in eukaryotes. Nonetheless, Wee1 regulation is not well understood and its large amino-terminal regulatory domain (NRD) has remained largely uncharted. Evidence has accumulated that cyclin B/Cdk1 complexes reciprocally inhibit Wee1 activity through NRD phosphorylation. Recent studies have identified the first functional NRD elements and suggested that vertebrate cyclin A/Cdk2 complexes also phosphorylate the NRD. A short NRD peptide, termed the Wee box, augments the activity of the Wee1 kinase domain. Cdk1/2-mediated phosphorylation of the Wee box (on T239) antagonizes kinase activity. A nearby region harbors a conserved RxL motif (RxL1) that promotes cyclin A/Cdk2 binding and T239 phosphorylation. Mutation of either T239 or RxL1 bolsters the ability of Wee1 to block mitotic entry, consistent with negative regulation of Wee1 through these sites. The region in human somatic Wee1 that encompasses RxL1 also binds Crm1, directing Wee1 export from the nucleus. These studies have illuminated important aspects of Wee1 regulation and defined a specific molecular pathway through which cyclin A/Cdk2 complexes foster mitotic entry. The complexity, speed, and importance of regulation of mitotic entry suggest that there is more to be learned

Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

<p>Abstract</p> <p>Background</p> <p>Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts.</p> <p>Results</p> <p>We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells.</p> <p>Conclusions</p> <p>In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.</p

Transgenic Expression of VEGF in Intestinal Epithelium Drives Mesenchymal Cell Interactions and Epithelial Neoplasia

Background & Aims: Vascular endothelial growth factor (VEGF) is expressed robustly in human colon neoplasia and is a major new "rational" target of therapy for cancers of the colon and other organs. Nonetheless, the mechanism(s) of action of VEGF-targeted therapies and the biologic roles of VEGF in tumorigenesis have not been well defined. We used a transgenic approach to directly test the hypothesis that augmented VEGF expression can drive progression of intestinal neoplasia. Methods: Transgenic mouse lines were generated with moderate (vilVEGF1) and high (vilVEGF2) VEGF expression from the intestinal epithelium. vilVEGF1 mice were bred to Min mice (adenomatous polyposis coli [APC] +/-). Colon epithelial cells from an APC patient were cocultured with endothelial cells and fibroblasts. Results: viIVEGF mice were generally healthy but displayed red small intestines. Vessels were larger and more numerous in the submucosa but not the mucosa. The mucosa showed striking stromal and epithelial hypercellularity, with increased epithelial proliferation. Many crypts formed cysts composed of relatively undifferentiated epithelial cells surrounded by cells with endothelial and myofibroblast markers. Compared with Min controls, vilVEGF1-Min mice developed 6-fold more intestinal adenomas of all sizes, with more advanced histologic features. Polycystic masses were also observed. Coculture of human colonocytes with endothelial cells and fibroblasts directly stimulated colonocyte proliferation. Conclusions: Augmented VEGF expression from intestinal epithelium potently stimulated cross talk with mesenchymal cells and proliferation of normal and neoplastic epithelium. These effects of VEGF, largely occurring prior to the canonical angiogenic switch in tumors, may be in part independent of angiogenesis

Wnt therapy for bone loss: golden goose or Trojan horse?

The Wnt pathway has been found to play a role in the development of many tissues and to spur growth and differentiation of adult osteoblasts, sparking interest in its potential clinical application for bone growth. However, when deregulated, this pathway can be oncogenic in some tissues. In this issue of the JCI, Kansara and colleagues reveal that Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcomas and that its absence augments osteosarcoma formation in mice (see the related article beginning on page 837). These observations suggest the need for caution in stimulating the Wnt pathway for therapeutic bone growth