Proteomic analysis of nuclei isolated from cancer cell lines treated with indenoisoquinoline NSC 724998, a novel topoisomerase I inhibitor.
The indenoisoquinoline NSC724998 is a novel topoisomerase I (Top1) inhibitor entering Phase I clinical trials at the National Cancer Institute, USA. In this study, 2-D PAGE analysis was performed on nuclear lysates prepared from HCT-116 and A375 cells treated with 1 microM NSC724998 for 24 h and the differentially regulated spots identified by LC-MS/MS. One-hundred fourteen protein spot differentials were identified, 66 from A375 cells and 48 from HCT-116 cells. Proteins related to apoptosis changed specifically in A375 cells, whereas proteins involved in the ubiquitin-proteasome system were highly enriched in treated HCT-116 cells. Importantly, 12 differentially expressed proteins (ETFA, HCC1, HNRCL, KAP1, NPM, NUCL, PRDX1, PRP19, PSB6, RAE1L, RU2A, and SFRS9) were common to both cell lines. Western blotting and immunocytochemistry confirmed significant nuclear upregulation of both the proteasome subunit PSB6 and the transcriptional repressor KAP1. Interestingly, increased KAP1 polypeptide was accompanied by enhanced phosphorylation at Ser824. Similar to gammaH2AX, KAP1 phosphorylation was consistently enhanced in a panel of 12 cell lines and in A375 xenografts following NSC 724998 treatment. In summary, these data enhance our understanding of protein dynamics in the nucleus following DNA damage and provide an alternate marker (pKAP1) with potential for monitoring clinical responses to Top1 poisons.
Developmental Therapeutics Program, SAIC-Frederick Inc., NCI-Frederick, Frederick, Maryland 21702, USA.
Bingnan
B
Han
Luke H
LH
Stockwin
Chad
C
Hancock
Sherry X
SX
Yu
Melinda G
MG
Hollingshead
Dianne L
DL
Newton
D052117:Benzodioxoles / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002853:Chromatography, Liquid; D004249:DNA Damage; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D007546:Isoquinolines / Q000494:pharmacology*; D013058:Mass Spectrometry; D040901:Proteomics; D012097:Repressor Proteins / Q000378:metabolism; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology*; D000076183:Tripartite Motif-Containing Protein 28
D052117:Benzodioxoles; D007546:Isoquinolines; C553898:NSC 724998; D012097:Repressor Proteins; D059004:Topoisomerase I Inhibitors; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28