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Relationship Between Induction of Phosphorylated H2AX and Survival in Breast Cancer Cells Exposed to 111In-DTPA-hEGF

Zhongli Cai, Zhuo Chen, Kristy E. Bailey, Deborah A. Scollard, Raymond M. Reilly and Katherine A. Vallis
Journal of Nuclear Medicine August 2008, 49 (8) 1353-1361; DOI: https://doi.org/10.2967/jnumed.108.051805

Article Figures & Data

Figures

  • FIGURE 1. 
    FIGURE 1. 

    Induction of γ-H2AX foci by 111In-DTPA-hEGF in breast cancer cell lines. (A) Cells were incubated with DTPA-hEGF (21 nM), 111In-acetate (3.2 MBq/mL), or 111In-DTPA-hEGF (3.2 MBq/mL, 21 nM) for 1 h; were exposed to γ-irradiation (1 Gy); or were untreated (control). (B) Cells were incubated with DTPA-hEGF (43 nM), 111In-acetate (5.2 MBq/mL), or 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h; were exposed to γ-irradiation (10 Gy); or were untreated (controls). (C) Confocal microscopy of MCF-7, MDA-MB-231, and MDA-MB-468 cells exposed to DTPA-hEGF (21 nM), 111In-acetate (3.2 MBq/mL), or 111In-DTPA-hEGF (3.2 MBq/mL, 21 nM) for 1 h; exposed to γ-irradiation (1 Gy); or untreated (control). Cells were immunostained for γ-H2AX. 4′,6-diamidino-2-phenylindole was used to visualize cell nucleus. Exposure to 111In-DTPA-hEGF caused increase in number of γ-H2AX foci in MDA-MB-468 and MDA-MB-231 cells but not in MCF-7 cells.

  • FIGURE 2. 
    FIGURE 2. 

    MDA-MB-468, MDA-MB-231, and MCF-7 cells were treated with 111In-DTPA-hEGF (3.2 MBq/mL, 21 nM) for 1 h (A) or 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h (B). Amount of 111In in cell membrane, cytoplasm, and nucleus was measured in cell fractionation experiments. Amount of 111In per cell decreased in order of MDA-MB-468 > MDA-MB-231 > MCF-7. (C) Amount of 111In that localizes in cytoplasm and nucleus is linearly dependent on amount of 111In that binds to cell membrane.

  • FIGURE 3. 
    FIGURE 3. 

    MCF-7, MDA-MB-468, and MDA-MB 231 cells were exposed to 111In-DTPA-hEGF (3.2 MBq/mL, 21 nM) for 1 h or 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h. Relationship between amount of nuclear 111In and induction of γ-H2AX foci is shown. Data were fitted to 1-site binding equation.

  • FIGURE 4. 
    FIGURE 4. 

    Clonogenic assays were performed using MDA-MB-468, MDA-MB-231, and MCF-7 cells incubated with DTPA-hEGF (21 nM), 111In-acetate (3.2 MBq/mL), or 111In-DTPA-hEGF (3.2 MBq/mL, 21 nM) for 1 h (A) or DTPA-hEGF (43 nM), 111In-acetate (5.2 MBq/mL), or 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h (B). After exposure to 111In-DTPA-hEGF, significant decrease in SF was observed in MDA-MB-468 (0.6 ± 0.1 at 1 h and 0.013 ± 0.001 at 20 h, P < 0.0001) and MDA-MB-231 cells (0.5 ± 0.1 at 20 h, P = 0.0001) but not in MCF-7 cells, compared with untreated cells. Error bars represent SD of mean SF, calculated from 3 experiments.

  • FIGURE 5. 
    FIGURE 5. 

    Dependence of SF on γ-absorbed dose. Clonogenic assays were generated for MDA-MB-468, MDA-MB-231, and MCF-7 cells. Curves are nonlinear regression fits to experimental data, using linear quadratic function for MDA-MB-468 and first-order exponential function for other cell lines.

  • FIGURE 6. 
    FIGURE 6. 

    Induction of γ-H2AX foci in MDA-MB-468 cells depends on concentration of 111In-DTPA-hEGF (specific activity, 20 MBq/μg) (A), specific activity of 111In-DTPA-hEGF (hEGF concentration, 17 nM) (B), and incubation time of 111In-DTPA-hEGF (1.9 MBq/mL, 11 nM) (C).

  • FIGURE 7. 
    FIGURE 7. 

    (A) Linear dependence of membrane-bound 111In on number of EGFR per cell. (B) Correlation of γ-H2AX foci formation (difference between foci number in 111In-DTPA-hEGF–treated and control cells) with EGFR number per cell. Data are fitted to 1-site binding equation. (C) Correlation of γ-H2AX foci formation with SF. Cells were treated with 111In-DTPA-hEGF (3.2 MBq/mL, 21 nM) for 1 h or 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h.

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