PMC:3583298 / 9131-12648
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3583298","sourcedb":"PMC","sourceid":"3583298","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3583298","text":"YAP expression promotes increased proliferation, survival and cell cycle checkpoints override in irradiated cerebellar neural precursors\nCGNPs are proposed to be the cell-of-origin for medulloblastomas associated with increased Shh pathway activity (Wechsler-Reya and Scott 1999). Consistent with this hypothesis, treatment of CGNPs in culture with exogenous Shh causes expression of many of the same proliferation markers seen in this class of medulloblastomas, including Gli1, N-myc and YAP. We wished to determine whether we could use in vitro primary CGNP cultures to mechanistically dissect the effects of YAP on proliferation and survival after irradiation. Cells prepared from post-natal day 4-5 mouse cerebella are maintained in serum free medium; addition of Shh sustains their proliferation. We transduced Shh-treated, proliferating CGNPs with retroviruses carrying GFP or YAP, then irradiated them (10 Gy) twenty-four hours later. As shown in Figure 2A, ectopic YAP expression is associated with increased CGNP proliferation as determined by cyclin D2 levels, and as we have previously reported (Fernandez et al 2009). At 20 hours post-irradiation, YAP-infected CGNPs had reduced levels of cleaved caspase 3 in comparison with GFP-infected CGNPs, and reduced numbers of apoptotic cells as determined by quantification of pyknotic nuclei (GFP: 62.5+/−0.5% vs YAP: 35.5+/−5.5%). YAP-transduced CGNPs were significantly more proliferative, as indicated by levels of cyclin D2, and by Ki67 and P-Hist3 staining (Figures 2A, B). Taken together, these results indicate that CGNP cultures recapitulate the in vivo effect of YAP expression on promoting survival and proliferation after irradiation. Moreover, they suggest that YAP-mediated radiation resistance may contribute to medulloblastoma recurrence by promoting increased tumor cell survival and proliferation.\nIonizing radiation (IR) causes the formation of double-strand DNA breaks (DSBs) that are responsible for the activation of the DNA Damage Response (DDR), a complex network of proteins required for cell-cycle checkpoint maintenance and DNA repair. The G1/S checkpoint prevents cells from entering S phase, while the G2/M checkpoint arrests cells with unrepaired DNA before entry into mitosis. We wanted to assess whether high YAP expression causes defects in cell cycle checkpoints, allowing cells to go through the cell cycle regardless of damage to their DNA. To examine the G1/S checkpoint, we exposed CGNPs to IR (10 Gy), and determined the percentage of cells in S phase 18 hours post-treatment by quantifying bromo-deoxyuridine (BrdU) incorporation during a one-hour pulse. Upon IR treatment both GFP and YAP-expressing CGNPs showed a strong reduction of the number of cells incorporating BrdU, however YAP-expressing cells have a significantly higher S-phase ratio (%BrdU-positive of treated/%BrdU-positive untreated), suggesting a mild defect in the G1/S checkpoint (Figure 2C). The infection efficiency was similar in both GFP- and YAP-infected cultures (representative images, Supplementary Figure 2). To determine whether the G2/M checkpoint was disabled in YAP-infected irradiated CGNPs, we evaluated P-Hist3 levels at an early time point (1 hour) after radiation. GFP-transduced cells showed an 80% reduction of the number of P-Hist3 positive cells as compared to untreated cells. In contrast, YAP-expressing CGNPs showed only a 50% reduction of cells undergoing mitosis, indicating that the G2/M checkpoint was defective (Figure 2C).","divisions":[{"label":"title","span":{"begin":0,"end":136}},{"label":"p","span":{"begin":137,"end":1870}}],"tracks":[{"project":"2_test","denotations":[{"id":"21874045-10027293-79138688","span":{"begin":274,"end":278},"obj":"10027293"},{"id":"21874045-10027293-79138688","span":{"begin":274,"end":278},"obj":"10027293"},{"id":"21874045-19952108-79138689","span":{"begin":1123,"end":1127},"obj":"19952108"},{"id":"21874045-19952108-79138689","span":{"begin":1123,"end":1127},"obj":"19952108"}],"attributes":[{"subj":"21874045-10027293-79138688","pred":"source","obj":"2_test"},{"subj":"21874045-10027293-79138688","pred":"source","obj":"2_test"},{"subj":"21874045-19952108-79138689","pred":"source","obj":"2_test"},{"subj":"21874045-19952108-79138689","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#a493ec","default":true}]}]}}