Cell cycle regulation by the Wee1 inhibitor PD0166285, pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line.
BACKGROUND
Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 muM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line.


METHODS
Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed.


RESULTS
In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 muM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 muM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours.


CONCLUSIONS
We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription. These effects together suggest that Wee1 inhibitor may thus be a potentially useful anti-cancer therapy.
Hashimoto
Osamu
O
Liver Cancer Division, Research Center for innovatve cancer therapy and Center of the 21st century COE program for medical Science, Kurume University, Kurume, Japan. osamuhas@med.kurume-u.ac.jp
Shinkawa
Masako
M
Torimura
Takuji
T
Nakamura
Toru
T
Selvendiran
Karuppaiyah
K
Sakamoto
Masaharu
M
Koga
Hironori
H
Ueno
Takato
T
Sata
Michio
M
D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D016195:G2 Phase / Q000187:drug effects / Q000502:physiology; D008546:Melanoma, Experimental / Q000473:pathology*; D051379:Mice; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000494:pharmacology*; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D009687:Nuclear Proteins; C440869:PD 0166285; D011743:Pyrimidines; D012334:RNA, Neoplasm; D011505:Protein-Tyrosine Kinases; C499348:Wee1 protein, mouse