G2-phase radiation response in lymphoblastoid cell lines from Nijmegen breakage syndrome.
The relationship between G2-phase checkpoint activation, cytoplasmic cyclin-B1 accumulation and nuclear phosphorylation of p34CDC2 was studied in Nijmegen breakage syndrome cells treated with DNA damaging agents. Experiments were performed on lymphoblastoid cell lines from four Nijmegen breakage syndrome patients with different mutations, as well as on cells from an ataxia telangiectasia patient. Lymphoblastoid cell lines were irradiated with 0.50-2 Gy X-rays and the percentage of G2-phase accumulated cells was evaluated by means of flow cytometry in samples that were harvested 24 h later. The G2-checkpoint activation was analysed by scoring the mitotic index at 2 and 4 h after treatment with 0.5 and 1 Gy X-rays and treatment with the DNA double-strand break inducer calicheamicin-gamma1. Cytoplasmic accumulation of cyclin-B1 was evaluated by means of fluorescence immunostaining or Western blotting, in cells harvested shortly after irradiation with 1 and 2 Gy. The extent of tyrosine 15-phosphorylated p34CDC2 was assessed in the nuclear fractions. Nijmegen breakage syndrome cells showed suboptimal G2-phase checkpoint activation respect to normal cells and were greatly different from ataxia telangiectasia cells. Increased cytoplasmic cyclin-B1 accumulation was detected by both immunofluorescence and immunoblot in normal as well as in Nijmegen breakage syndrome cells. Furthermore, nuclear p34CDC2. phosphorylation was detected at a higher level in Nijmegen breakage syndrome than in ataxia telangiectasia cells. In conclusion, our data do not suggest that failure to activate checkpoints plays a major role in the radiosensitivity of Nijmegen breakage syndrome cells.
Antoccia
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A
Università 'La Sapienza', Roma, Italy, Università degli Studi di Roma 'Roma Tre', Italy. antoccia@bio.uniroma3.it
di Masi
A
A
Maraschio
P
P
Stumm
M
M
Ricordy
R
R
Tanzarella
C
C
D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002460:Cell Line; D019457:Chromosome Breakage; D019926:Cyclin B / Q000032:analysis; D056744:Cyclin B1; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D016195:G2 Phase / Q000502:physiology / Q000528:radiation effects*; D006801:Humans; D008214:Lymphocytes / Q000166:cytology* / Q000201:enzymology / Q000528:radiation effects*
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D016203:CDC2 Protein Kinase