Abstract
DNA-dependent protein kinase (DNA-PK) has an important role in the repair of DNA damage and regulates the radiation sensitivity of glioblastoma cells. The VCP (valosine-containing protein), a chaperone protein that regulates ubiquitin-dependent protein degradation, is phosphorylated by DNA-PK and recruited to DNA double-strand break sites to regulate DNA damage repair. However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma. Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP. VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase. As expected, this increase promoted the efficiency of DNA repair in several glioblastoma cell lines; in turn, this enhanced activity decreased the radiation sensitivity and prolonged the survival fraction of glioblastoma cells in vitro. Moreover, the VCP knockdown in glioblastoma cells reduced the survival time of the xenografted mice with radiation treatment relative to the control xenografted glioblastoma mice. In addition, the VCP protein was also downregulated in ∼25% of GBM tissues from patients (WHO, grade IV astrocytoma), and the VCP protein level was correlated with patient survival (R2=0.5222, P<0.05). These findings demonstrated that VCP regulates DNA-PKcs degradation and increases the sensitivity of GBM cells to radiation.
Highlights
Valosin-containing protein (VCP) has been reported to be recruited to DNA double-strand break (DSBs) sites by interacting with
To determine whether VCP is associated with DNA-PK, co-immunoprecipitation was performed using the DNA-PKcs antibody in several glioblastoma cell lines; the M059J cell is DNA-PKcs-deficient, and was used as a negative control
We first found that the DNA-PKcs is ubiquitinated and bound to VCP; the treatment with proteasome inhibitor (MG132), but not lysosome inhibitor, increased the protein level of DNA-PKcs; the VCP knockdown had a synergistic effect with the MG132 treatment on DNA-PKcs protein accumulation
Summary
VCP has been reported to be recruited to DNA double-strand break (DSBs) sites by interacting with. DNA-PK has a critical role in the DNA damage repair pathway, but it is involved in other important physiological or pathological processes,[20] such as radiation sensitivity, inflammatory responses and Received 26.1.13; revised 12.4.13; accepted 15.4.13; Edited by A Stephanou metabolic gene expression.[21,22,23] it is still unknown how DNA-PK protein degradation is regulated and whether VCP affects the radiation sensitivity. We investigated the association of VCP with DNA-PKcs and the effect of the regulation of VCP on the radiosensitivity of glioblastoma cells. Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP; the VCP knockdown resulted in the accumulation of DNA-PKcs protein in glioblastoma cells and increased DNA-PK activity, which eventually increased the survival fraction of GBM cells post-IR and shortened the survival time of orthotopic xenografted mice
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