Molecular alterations after Polo-like kinase 1 mRNA suppression versus pharmacologic inhibition in cancer cells.
Multiple roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. We have employed chimeric antisense oligonucleotides to investigate the molecular alterations after targeted interference with Plk1 in RKO human colon adenocarcinoma and PC3 prostate cancer cells. Suppression of Plk1 mRNA resulted in a dramatic increase of the mitotic index followed by the onset of apoptosis. Mitotically arrested cells displayed randomly separated condensed chromosomes and the occurrence of multiple spindle poles with well-formed asters. Induction of apoptosis was strictly dependent on cell cycle progression: Genetically engineered RKO cells with inducible expression of the cyclin-dependent kinase inhibitor p27(Kip1) were completely refractory to Plk1 depletion-induced apoptosis when they were arrested in the G1 phase of the cell cycle. Various mitotic markers, including MPM-2, cdc25c, cyclin B1, or phosphorylated histone H3, were investigated to explore the molecular consequences of Plk1 depletion. Whereas most marker proteins showed similar alterations compared with treatment with paclitaxel, cdc25c was fully phosphorylated solely in paclitaxel-treated cells but only partially phosphorylated in Plk1-depleted cells, although both treatments caused a profound mitotic arrest. This differential phosphorylation of cdc25c was used to test whether a pharmacologic inhibitor of Plk1 would exert the same cellular effects as interference with Plk1 on a mRNA level. It was found that the differential electrophoretic mobility of cdc25c can serve as a reliable molecular marker to track inhibition of Plk1 by small-molecule inhibitors within a cell.
Schmidt
Mathias
M
Therapeutic Area Oncology, ALTANA Pharma AG, Byk-Gulden-Str. 2, D-78467 Konstanz, Germany. mathias.schmidt@altanapharma.com
Hofmann
Hans-Peter
HP
Sanders
Karl
K
Sczakiel
Georg
G
Beckers
Thomas L
TL
Gekeler
Volker
V
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D017931:DNA Primers; D006801:Humans; D008938:Mitosis / Q000187:drug effects / Q000235:genetics; D008940:Mitotic Index; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics*; D013489:Suppression, Genetic / Q000187:drug effects; D014162:Transfection; D020687:cdc25 Phosphatases / Q000378:metabolism; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D020687:cdc25 Phosphatases; D000097869:Polo-Like Kinase 1; C517009:CDC25C protein, human