GSK3 regulates the expressions of human and mouse c-Myb via different mechanisms

<p>Abstract</p> <p>Background</p> <p>c-Myb is expressed at high levels in immature progenitors of all the hematopoietic lineages. It is associated with the regulation of proliferation, differentiation and survival of erythroid, myeloid and lymphoid cells, but decreases during the terminal differentiation to mature blood cells. The cellular level of c-Myb is controlled by not only transcriptional regulation but also ubiquitin-dependent proteolysis. We recently reported that mouse c-Myb protein is controlled by ubiquitin-dependent degradation by SCF-Fbw7 E3 ligase via glycogen synthase kinase 3 (GSK3)-mediated phosphorylation of Thr-572 in a Cdc4 phosphodegron (CPD)-dependent manner. However, this critical threonine residue is not conserved in human c-Myb. In this study, we investigated whether GSK3 is involved in the regulatory mechanism for human c-Myb expression.</p> <p>Results</p> <p>Human c-Myb was degraded by ubiquitin-dependent degradation via SCF-Fbw7. Human Fbw7 ubiquitylated not only human c-Myb but also mouse c-Myb, whereas mouse Fbw7 ubiquitylated mouse c-Myb but not human c-Myb. Human Fbw7 mutants with mutations of arginine residues important for recognition of the CPD still ubiquitylated human c-Myb. These data strongly suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner. Mutations of the putative GSK3 phosphorylation sites in human c-Myb did not affect the Fbw7-dependent ubiquitylation of human c-Myb. Neither chemical inhibitors nor a siRNA for GSK3β affected the stability of human c-Myb. However, depletion of GSK3β upregulated the transcription of human <it>c-Myb</it>, resulting in transcriptional suppression of <it>γ-globin</it>, one of the c-Myb target genes.</p> <p>Conclusions</p> <p>The present observations suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner, whereas mouse Fbw7 ubiquitylates human c-Myb in a CPD-dependent manner. Moreover, GSK3 negatively regulates the transcriptional expression of human <it>c-Myb </it>but does not promote Fbw7-dependent degradation of human c-Myb protein. Inactivation of GSK3 as well as mutations of Fbw7 may be causes of the enhanced c-Myb expression observed in leukemia cells. We conclude that expression levels of human and mouse c-Myb are regulated via different mechanisms.</p

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

<p>Abstract</p> <p>Background</p> <p>Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as <it>ERBB2</it>, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.</p> <p>Methods and Results</p> <p>A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including <it>ERBB2</it>) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the <it>CRKL</it> gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high <it>CRKL</it> copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence <it>in situ</it> hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The <it>CRKL</it> copy number was also examined in 360 primary gastric cancers using a FISH analysis, and <it>CRKL</it> amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with <it>CRKL</it> amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.</p> <p>Conclusion</p> <p>These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with <it>CRKL</it> amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.</p

Mutation Spectrum Induced by 8-Bromoguanine, a Base Damaged by Reactive Brominating Species, in Human Cells

To date, the types of mutations caused by 8-bromoguanine (8BrG), a major base lesion induced by reactive brominating species during inflammation, in human cells and the 8BrG repair system remain largely unknown. In this study, we performed a supF forward mutation assay using a shuttle vector plasmid containing a single 8BrG in three kinds of human cell lines and revealed that 8BrG in DNA predominantly induces a G → T mutation but can also induce G → C, G → A, and delG mutations in human cells. Next, we tested whether eight kinds of DNA glycosylases (MUTYH, MPG, NEIL1, OGG1, SMUG1, TDG, UNG2, and NTHL1) are capable of repairing 8BrG mispairs with any of the four bases using a DNA cleavage activity assay. We found that both the SMUG1 protein and the TDG protein exhibit DNA glycosylase activity against thymine mispaired with 8BrG and that the MUTYH protein exhibits DNA glycosylase activity against adenine mispaired with 8BrG. These results suggest that 8BrG induces some types of mutations, chiefly a G → T mutation, in human cells, and some DNA glycosylases are involved in the repair of 8BrG

ALMA Observations of the Terahertz Spectrum of Sagittarius A*

We present Atacama Large Millimeter/submillimeter Array (ALMA) observations at 233, 678, and 870 GHz of the Galactic Center black hole, Sagittarius A*. These observations reveal a flat spectrum over this frequency range with spectral index α ≈ −0.3, where the flux density S ∝ ν α . We model the submillimeter and far-infrared spectrum with a one-zone synchrotron model of thermal electrons. We infer electron densities n = (2–5) × 106 cm−3, electron temperatures T e = (1–3) × 1011 K, and magnetic field strength B = 10–50 G. The parameter range can be further constrained using the observed quiescent X-ray luminosity. The flat submillimeter spectrum results in a high electron temperature and implies that the emitting electrons are efficiently heated. We also find that the emission is most likely optically thin at 233 GHz. These results indicate that millimeter and submillimeter wavelength very long baseline interferometry of Sgr A* including those of the Event Horizon Telescope should see a transparent emission region down to event horizon scales.Alexander von Humboldt foundation; NWO VICI grant [639.043.513]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

The immediately-early response gene 5 (IER5) has been reported to be induced by γ-ray irradiation and to play a role in the induction of cell death caused by radiation. We previously identified IER5 as one of the 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP)-induced transcriptional responses in AML cells, using microarrays that encompassed the entire human genome. However, the biochemical pathway and mechanisms of IER5 function in regulation of the cell cycle remain unclear. In this study, we investigated the involvement of IER5 in the cell cycle and in cell proliferation of acute myeloid leukemia (AML) cells. We found that the over-expression of IER5 in AML cell lines and in AML-derived ALDHhi (High Aldehyde Dehydrogenase activity)/CD34+ cells inhibited their proliferation compared to control cells, through induction of G2/M cell cycle arrest and a decrease in Cdc25B expression. Moreover, the over-expression of IER5 reduced colony formation of AML-derived ALDHhi/CD34+ cells due to a decrease in Cdc25B expression. In addition, over-expression of Cdc25B restored TMPP inhibitory effects on colony formation in IER5-suppressed AML-derived ALDHhi/CD34+ cells. Furthermore, the IER5 reduced Cdc25B mRNA expression through direct binding to Cdc25B promoter and mediated its transcriptional attenuation through NF-YB and p300 transcriptinal factors. In summary, we found that transcriptional repression mediated by IER5 regulates Cdc25B expression levels via the release of NF-YB and p300 in AML-derived ALDHhi/CD34+ cells, resulting in inhibition of AML progenitor cell proliferation through modulation of cell cycle. Thus, the induction of IER5 expression represents an attractive target for AML therapy

Abnormal Expressions of DNA Glycosylase Genes NEIL1, NEIL2, and NEIL3 Are Associated with Somatic Mutation Loads in Human Cancer

The effects of abnormalities in the DNA glycosylases NEIL1, NEIL2, and NEIL3 on human cancer have not been fully elucidated. In this paper, we found that the median somatic total mutation loads and the median somatic single nucleotide mutation loads exhibited significant inverse correlations with the median NEIL1 and NEIL2 expression levels and a significant positive correlation with the median NEIL3 expression level using data for 13 cancer types from the Cancer Genome Atlas (TCGA) database. A subset of the cancer types exhibited reduced NEIL1 and NEIL2 expressions and elevated NEIL3 expression, and such abnormal expressions of NEIL1, NEIL2, and NEIL3 were also significantly associated with the mutation loads in cancer. As a mechanism underlying the reduced expression of NEIL1 in cancer, the epigenetic silencing of NEIL1 through promoter hypermethylation was found. Finally, we investigated the reason why an elevated NEIL3 expression level was associated with an increased number of somatic mutations in cancer and found that NEIL3 expression was positively correlated with the expression of APOBEC3B, a potent inducer of mutations, in diverse cancers. These results suggested that the abnormal expressions of NEIL1, NEIL2, and NEIL3 are involved in cancer through their association with the somatic mutation load

The Greenland Telescope: Antenna Retrofit Status and Future Plans

Since the ALMA North America Prototype Antenna was awarded to the Smithsonian Astrophysical Observatory (SAO), SAO and the Academia Sinica Institute of Astronomy & Astrophysics (ASIAA) are working jointly to relocate the antenna to Greenland. This paper shows the status of the antenna retrofit and the work carried out after the recommissioning and subsequent disassembly of the antenna at the VLA has taken place. The next coming months will see the start of the antenna reassembly at Thule Air Base. These activities are expected to last until the fall of 2017 when commissioning should take place. In parallel, design, fabrication and testing of the last components are taking place in Taiwan