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Suppression of Cdc2 Dephosphorylation at the Tyrosine 15 Residue during Nitrosourea-induced G2M phase Arrest in Glioblastoma Cell Lines

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Abstract

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 μg/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 μg/ml ACNU. Protein abundance for total Cdc2, Cdc2 phosphorylated at the threonine 161 residue, Wee 1, Myt 1, Chk 1, and 14-3-3σ 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.

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Authors and Affiliations

  1. Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Akira Nakamizo, Takanori Inamura, Satoshi Inoha, Toshiyuki Amano, Kiyonobu Ikezaki & Masashi Fukui

  2. Department of Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Hirofumi Ochi

Authors
  1. Akira Nakamizo
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  2. Takanori Inamura
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  3. Satoshi Inoha
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  4. Toshiyuki Amano
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  5. Hirofumi Ochi
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  6. Kiyonobu Ikezaki
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  7. Masashi Fukui
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Nakamizo, A., Inamura, T., Inoha, S. et al. Suppression of Cdc2 Dephosphorylation at the Tyrosine 15 Residue during Nitrosourea-induced G2M phase Arrest in Glioblastoma Cell Lines. J Neurooncol 59, 7–13 (2002). https://doi.org/10.1023/A:1016342013616

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