Suppression of Cdc2 dephosphorylation at the tyrosine 15 residue during nitrosourea-induced G2M phase arrest in glioblastoma cell lines.
We examined the mechanism of action of nitrosoureas as represented by 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) with respect to p53 and the G2M cell cycle checkpoint using two glioblastoma cell lines: U251MG and U373MG, with mutated p53. At log-phase cell growth, fresh medium containing ACNU (final concentration, 3, 10, or 30 microg/ml) was added. After 24 h of incubation, cells were harvested for flow cytometric or Western analysis. In both lines, cell numbers in the G0/G1 phase decreased with ACNU treatment. Cells accumulated in G2M and S phases, and the peak was shifted from G2M to the S phase in a concentration-dependent manner. In both cell lines, the amount of Cdc2 protein phosphorylated at the tyrosine 15 residue was increased 2- to 6-fold by treatment with ACNU compared with untreated control cells. Expression of cyclin B protein was suppressed in cells treated with 30 microg/ml ACNU. Protein abundance for total Cdc2, Cdc2 phosphorylated at the threonine 161 residue, Wee 1, Myt 1, Chk 1, and 14-3-3sigma was not affected by treatment with ACNU in either cell line. We suggest that a low concentration of ACNU should be used with adjuvant therapies that act upon cells in the G2M phase. A high concentration of ACNU should be used with adjuvant therapies that act upon cells in the S phase.
Nakamizo
Akira
A
Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. nakamizo@ns.med.kyushu-u.ac.jp
Inamura
Takanori
T
Inoha
Satoshi
S
Amano
Toshiyuki
T
Ochi
Hirofumi
H
Ikezaki
Kiyonobu
K
Fukui
Masashi
M
D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects*; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D015376:Nimustine / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014443:Tyrosine
D015376:Nimustine; D014443:Tyrosine; D016203:CDC2 Protein Kinase