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. 2002 Apr;35(2):93-104.
doi: 10.1046/j.1365-2184.2002.00234.x.

G2-phase radiation response in lymphoblastoid cell lines from Nijmegen breakage syndrome

A Antoccia  1 , A di Masi, P Maraschio, M Stumm, R Ricordy, C Tanzarella
Affiliations

G2-phase radiation response in lymphoblastoid cell lines from Nijmegen breakage syndrome

A Antoccia et al. Cell Prolif. 2002 Apr.

Abstract

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.

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Figures

Figure 1
Figure 1
Percentage of G2/M accumulated cells harvested 24 h after irradiation. Bars: SE.
Figure 2
Figure 2
Kinetics of delay into mitosis following exposure to X‐rays (0.5 Gy (a); 1 Gy (b)) or calicheamicin‐γ1 treatment [4 pg/ml (c); 8 pg/ml (d)]. The mitotic index (MI) of treated cells is represented as a percentage of the mitotic index of sham‐treated cells. 1228 (open square), ZRA (open triangle), 96B453 (open circle, grey), AT (filled circle). Bars: SE.
Figure 3
Figure 3
G2‐phase accumulation in cells irradiated with 1 Gy X‐rays and harvested 4 h later (a). The increase in the fraction of cells in the G2 phase (y‐axis) is obtained as the ratio between the frequency of labelled cells in treated and untreated cells. Those cells showing a bright green signal in cytoplasm after staining with an anticyclin‐B1 antibody were classified as G2 cells. An example of two G2‐phase cells is shown in b; (c) the same field with interphase nuclei counterstained with DAPI. Bars: SE.
Figure 4
Figure 4
Accumulation of cyclin‐B1 in cytoplasmic extracts (a) and tyrosine 15‐phosphorylated p34CDC2 in nuclear extracts (b) in cells treated with 1 and 2 Gy X‐rays and harvested 4 h later. Alpha‐tubulin signals as evaluated after stripping cyclin‐B1 antibody and reprobing the membrane (c). Cytoplasmic and nuclear protein extracts were immunoblotted with an anti‐IkB antibody and PARP to ascertain the validity of the extraction procedures (d). Bars: SE.
Figure 5
Figure 5
Induction of cytoplasmic cyclin‐B1 as evaluated in Western blot of cells treated with 1 Gy (a) and 2 Gy (b) and harvested at 2 and 4 h. Induction of nuclear tyrosine 15‐phosphorylated p34CDC2 as evaluated in Western blot of cells treated with 1 Gy (c) and 2 Gy (d) and harvested at 2 and 4 h. Bars: SE.
Figure 6
Figure 6
Accumulation of tyrosine 15‐phosphorylated p34 CDC2 as evaluated in nuclear extracts (a) of cells treated with 2 Gy X‐rays and harvested 24 h later. Parallel samples were immunoblotted with an anti‐p34CDC2 antibody (b).
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