HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein (pVHL). We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe(2+), this protein modification may play a key role in mammalian oxygen sensing.

We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 ؉ 2] cycloaddition reaction (''click'' chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.BrdU ͉ click chemistry ͉ DNA replication ͉ EdU ͉ microscopy D etection of DNA synthesis in proliferating cells relies on the incorporation of labeled DNA precursors into cellular DNA during the S phase of the cell cycle (1-3). The labeled DNA precursors, usually pyrimidine deoxynucleosides, are added to cells during replication, and their incorporation into genomic DNA is quantified or visualized after incubation and sample staining. The same labeled deoxynucleosides can be injected into experimental animals to assay cellular proliferation in specific organs and tissues. The most common deoxynucleosides used for assaying DNA replication are [ 3 H]thymidine and 5-bromo-2Ј-deoxyuridine (BrdU). [ 3 H]Thymidine incorporated into DNA is usually detected by autoradiography, whereas detection of BrdU is accomplished immunologically, through specific anti-BrdU antibodies.Although [ 3 H]thymidine and BrdU labeling methods have been very useful for studying cell cycle kinetics, DNA replication, and sister chromatid exchange and for assessing cell proliferation of normal or pathological cells or tissues under different conditions, these methods exhibit several limitations. Working with [ 3 H]thymidine is cumbersome because of its radioactivity. Autoradiography is also labor-intensive and slow (detection often lasts several months), and thus not suitable for rapid high-throughput studies. Finally, microscopic images of [ 3 H]thymidine-labeled DNA suffer from poor resolution and low signal-to-noise ratios. In contrast to [ 3 H]thymidine autoradiography, BrdU immunostaining is both faster (although it still lasts several hours) and allows better microscopic imaging of the labeled DNA. One major disadvantage of BrdU staining is that the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits. To expose the BrdU epitope, cells and tissue samples are subjected to strong denaturing conditions such as concentrated hydrochloric acid or mixtures of methanol and acetic acid. These harsh staining conditions invariably degrade the structure of the specimen while also making the intensity of BrdU staining highly dependent on the conditions used for detection by each investigator (4). Finally, as with any immunohistologic...

Wnt signaling plays an important role in both oncogenesis and development. Activation of the Wnt pathway results in stabilization of the transcriptional coactivator β-catenin. Recent studies have demonstrated that axin, which coordinates β-catenin degradation, is itself degraded. Although the key molecules required for transducing a Wnt signal have been identified, a quantitative understanding of this pathway has been lacking. We have developed a mathematical model for the canonical Wnt pathway that describes the interactions among the core components: Wnt, Frizzled, Dishevelled, GSK3β, APC, axin, β-catenin, and TCF. Using a system of differential equations, the model incorporates the kinetics of protein–protein interactions, protein synthesis/degradation, and phosphorylation/dephosphorylation. We initially defined a reference state of kinetic, thermodynamic, and flux data from experiments using Xenopus extracts. Predictions based on the analysis of the reference state were used iteratively to develop a more refined model from which we analyzed the effects of prolonged and transient Wnt stimulation on β-catenin and axin turnover. We predict several unusual features of the Wnt pathway, some of which we tested experimentally. An insight from our model, which we confirmed experimentally, is that the two scaffold proteins axin and APC promote the formation of degradation complexes in very different ways. We can also explain the importance of axin degradation in amplifying and sharpening the Wnt signal, and we show that the dependence of axin degradation on APC is an essential part of an unappreciated regulatory loop that prevents the accumulation of β-catenin at decreased APC concentrations. By applying control analysis to our mathematical model, we demonstrate the modular design, sensitivity, and robustness of the Wnt pathway and derive an explicit expression for tumor suppression and oncogenicity.

Many aspects of cellular physiology remain unstudied in somatic stem cells. For example, there are almost no data on protein synthesis in any somatic stem cell. We found that the amount of protein synthesized per hour in haematopoietic stem cells (HSCs) in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24Bst/+ mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24Bst/+ cell-autonomously rescued the effects of Pten deletion in HSCs, blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.