DNA-dependent protein kinase (DNA-PK)-dependent cisplatin-induced loss of nucleolar facilitator of chromatin transcription (FACT) and regulation of cisplatin sensitivity by DNA-PK and FACT.
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.
Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Janna
J
Dejmek
J Dirk
JD
Iglehart
Jean-Bernard
JB
Lazaro
D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D034961:Antigens, Nuclear / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D002466:Cell Nucleolus / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002478:Cells, Cultured; D002843:Chromatin / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D053484:RecQ Helicases / Q000378:metabolism; D035602:Transcriptional Elongation Factors / Q000378:metabolism; D000071657:Werner Syndrome Helicase
D034961:Antigens, Nuclear; D000970:Antineoplastic Agents; D002843:Chromatin; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009687:Nuclear Proteins; C073537:SSRP1 protein, human; D035602:Transcriptional Elongation Factors; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D005090:Exodeoxyribonucleases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D002945:Cisplatin