Relationship between induction of phosphorylated H2AX and survival in breast cancer cells exposed to 111In-DTPA-hEGF.
The Auger electron-emitting radiopharmaceutical 111In-diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF) binds the epidermal growth factor receptor (EGFR), is internalized, and translocates to the nucleus. The purpose of this study was to investigate the relationship between EGFR expression, DNA damage, and cytotoxicity in cells exposed to 111In-DTPA-hEGF.


METHODS
Breast cancer cell lines with a range of EGFR expression levels were exposed to 111In-DTPA-hEGF or gamma-radiation. The cell lines (followed by number of EGFR per cell in parentheses) were MDA-MB-468 (1.3 x 10(6)), MDA-MB-231 (1.3 x 10(5)), and MCF-7 (1.5 x 10(4)). The proportion of radioactivity partitioning into the nucleus was measured by cell fractionation. DNA double-strand breaks were evaluated using the gamma-H2AX assay. Clonogenic survival assays were used to measure cytotoxicity.


RESULTS
All data are presented as mean +/- SD. The amount of 111In-DTPA-hEGF that translocated to the nucleus (in mBq/nucleus) in MDA-MB-468, MDA-MB-231, and MCF-7 cells incubated with 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h was 131 +/- 6, 8.1 +/- 0.1, and 1.1 +/- 0.9, respectively. The number of gamma-H2AX foci per nucleus was 35 +/- 15, 19 +/- 10, and 1.7 +/- 0.3, respectively. A reduction in the surviving fraction (SF) in MDA-MB-468 (0.013 +/- 0.001) and MDA-MB-231 (0.5 +/- 0.1) but not in MCF-7 cells after exposure to 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h has been demonstrated. The SF of MDA-MB-468 cells after exposure to DTPA-EGF (43 nM) and 111In-acetate (5.2 MBq/mL) for 20 h was 0.5 +/- 0.1 and 0.53 +/- 0.05, respectively. MDA-MB-468 was the most sensitive of the cell lines to gamma-irradiation, with an SF after 2 Gy of 0.45 +/- 0.04, compared with 0.7 +/- 0.1 and 0.8 +/- 0.1 for MCF-7 and MDA-MB-231, respectively. The number of gamma-H2AX foci per nucleus in MDA-MB-468 cells correlated with the concentration, specific activity, and incubation time of 111In-DTPA-hEGF.


CONCLUSIONS
DNA damage caused by 111In-DTPA-hEGF correlates with the EGFR expression level of the exposed cells and with concentration, specific activity, and incubation time of 111In-DTPA-hEGF. The gamma-H2AX assay may be a useful biomarker to predict and monitor the outcome of treatment with 111In-DTPA-hEGF.
Cai
Zhongli
Z
Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
Chen
Zhuo
Z
Bailey
Kristy E
KE
Scollard
Deborah A
DA
Reilly
Raymond M
RM
Vallis
Katherine A
KA
D021581:Active Transport, Cell Nucleus; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology*; D066246:ErbB Receptors / Q000096:biosynthesis; D005260:Female; D005720:Gamma Rays; D006657:Histones / Q000096:biosynthesis*; D006801:Humans; D007205:Indium Radioisotopes; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D019275:Radiopharmaceuticals / Q000378:metabolism / Q000494:pharmacology*
C478252:111In-DTPA-human epidermal growth factor; C468783:H2AX protein, human; D006657:Histones; D007205:Indium Radioisotopes; D019275:Radiopharmaceuticals; D004815:Epidermal Growth Factor; D004369:Pentetic Acid; D066246:ErbB Receptors