title: Diversity of DNA damage response of astrocytes and glioblastoma cell lines with various p53 status to treatment with etoposide and temozolomide.
abstract: Phosphorylation of histone H2AX is a sensitive marker of DNA damage, particularly of DNA double strand breaks. Using multiparameter cytometry we explored effects of etoposide and temozolomide (TMZ) on three glioblastoma cell lines with different p53 status (A172, T98G, YKG-1) and on normal human astrocytes (NHA) correlating the drug-induced phosphorylated H2AX (gammaH2AX) with cell cycle phase and induction of apoptosis. Etoposide induced gammaH2AX in all phases of the cell cycle in all three glioblastoma lines and led to an arrest of T98G and YKG-1 cells in S and G(2)/M. NHA cells were arrested in G(1) with no evidence of gammaH2AX induction. A172 responded by rise in gammaH2AX throughout all phases of the cycle, arrest at the late S- to G(2)/M-phase, and appearance of senescence features: induction of p53, p21(WAF1/CIP1), p16(INK4A) and beta-galactosidase, accompanied by morphological changes typical of senescence. T98G cells showed the presence of gammaH2AX in S phase with no evidence of cell cycle arrest. A modest degree of arrest in G(1) was seen in YKG-1 cells with no rise in gammaH2AX. While frequency of apoptotic cells in all four TMZ-treated cell cultures was relatively low it is conceivable that the cells with extensive DNA damage were reproductively dead. The data show that neither the status of p53 (wild-type vs. mutated, or inhibited by pifithrin-alpha) nor the expression of O(6)-methylguanine-DNA methyltransferase significantly affected the cell response to TMZ. Because of diversity in response to TMZ between individual glioblastoma lines our data suggest that with better understanding of the mechanisms, the treatment may have to be customized to individual patients.
authors.affiliation: Department of Pathology, Iwate Medical University School of Medicine, Morioka, Iwate, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sato
authors.forename: Akira
authors.initials: A
authors.lastname: Kurose
authors.forename: Akira
authors.initials: A
authors.lastname: Ogawa
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Ogasawara
authors.forename: Frank
authors.initials: F
authors.lastname: Traganos
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
authors.forename: Takashi
authors.initials: T
authors.lastname: Sawai
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004249:DNA Damage; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005909:Glioblastoma / Q000378:metabolism / Q000473:pathology; D006657:Histones / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D000077204:Temozolomide; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; C468783:H2AX protein, human; D006657:Histones; D016159:Tumor Suppressor Protein p53; D005047:Etoposide; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D001616:beta-Galactosidase; D000077204:Temozolomide