γ-H2AX kinetics as a novel approach to high content screening for small molecule radiosensitizers.

PLoS One 2012 29;7(6):e38465. Epub 2012 Jun 29.

Department of Radiation Oncology, Mount Sinai School of Medicine, New York, New York, United States of America.

Background: Persistence of γ-H2AX after ionizing radiation (IR) or drug therapy is a robust reporter of unrepaired DNA double strand breaks in treated cells.

Methods: DU-145 prostate cancer cells were treated with a chemical library ±IR and assayed for persistence of γ-H2AX using an automated 96-well immunocytochemistry assay at 4 hours after treatment. Hits that resulted in persistence of γ-H2AX foci were tested for effects on cell survival. The molecular targets of hits were validated by molecular, genetic and biochemical assays and in vivo activity was tested in a validated Drosophila cancer model.

Results: We identified 2 compounds, MS0019266 and MS0017509, which markedly increased persistence of γ-H2AX, apoptosis and radiosensitization in DU-145 cells. Chemical evaluation demonstrated that both compounds exhibited structurally similar and biochemical assays confirmed that these compounds inhibit ribonucleotide reductase. DNA microarray analysis and immunoblotting demonstrates that MS0019266 significantly decreased polo-like kinase 1 gene and protein expression. MS0019266 demonstrated in vivo antitumor activity without significant whole organism toxicity.

Conclusions: MS0019266 and MS0017509 are promising compounds that may be candidates for further development as radiosensitizing compounds as inhibitors of ribonucleotide reductase.

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038465PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387170PMC
November 2012
15 Reads

Publication Analysis

Top Keywords

persistence γ-h2ax
16
biochemical assays
8
ribonucleotide reductase
8
ms0019266 ms0017509
8
γ-h2ax
5
compounds
5
ms0019266 decreased
4
validated molecular
4
hits validated
4
targets hits
4
molecular targets
4
genetic biochemical
4
activity tested
4
tested validated
4
vivo activity
4
assays vivo
4
survival molecular
4
molecular genetic
4
cell survival
4
immunocytochemistry assay
4

References

(Supplied by CrossRef)
High throughput screening : methods and protocols. Totowa, N.J.: Humana Press.
WP Janzen et al.
2001
On the path to seeking novel radiosensitizers.
D Katz et al.
Int J Radiat Oncol Biol Phys 2009
Cellular imaging in drug discovery.
P Lang et al.
Nat Rev Drug Discov 2006
High content screening : a powerful approach to systems cell biology and drug discovery.
DL Taylor et al.
2006
Cellular response to DNA damage.
J Kao et al.
Ann N Y Acad Sci 2005
Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks.
JP Banath et al.
Cancer Res 2003
Maintaining integrity.
Y Shiloh et al.
Nat Cell Biol 2004

Similar Publications