Both the Ku subunit of the
DNA-dependent protein kinase (
DNA-PK) and the facilitator of
chromatin transcription (FACT) complex reportedly bind
cisplatin-
DNA adducts. For this study, we developed an immunocytochemical assay based on
detergent extraction allowing unveiling nucleolar subpopulations of
proteins present in both the nucleoplasm and the nucleolus. Immunofluorescence analysis in various human
cancer cell lines and immunoblotting of isolated nucleoli show that
DNA-PK catalytic subunit (
DNA-
PKcs), Ku86, the
Werner syndrome protein (WRN), and the structure-specific recognition
protein 1 (SSRP1) subunit of FACT colocalize in the nucleolus and exit the nucleolus after
cisplatin treatment. Nucleolar localization of Ku is also lost after gamma or UV irradiation and exposure to
DNA-damaging drugs, such as
actinomycin D,
mitomycin C,
hydroxyurea, and
doxorubicin. Ku86 and WRN leave the nucleolus after exposure to low (>1 microg/mL) doses of
cisplatin. In contrast, the SSRP1 association with the nucleolus was disrupted only by high (50-100 microg/mL) doses of
cisplatin. Both
cisplatin-induced loss of nucleolar SSRP1 and
DNA-PK activation are suppressed by pretreatment of the cells with
wortmannin or the
DNA-PK inhibitor
NU7026 but not by the
phosphatidylinositol 3-kinase inhibitor
LY294002. In the same conditions,
kinase inhibitors did not alter the exit of
DNA-
PKcs and WRN, suggesting that different mechanisms regulate the exit of
DNA-PK/WRN and FACT from the nucleolus. Furthermore, RNA silencing of
DNA-
PKcs blocked the
cisplatin-induced exit of nucleolar SSRP1. Finally, silencing of
DNA-
PKcs or SSRP1 by
short hairpin RNA significantly increased the sensitivity of
cancer cells to
cisplatin.