Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy
Waleed M. Abuzeid, Xiaoling Jiang, Guoli Shi, Hui Wang, David Paulson, Koji Araki, David Jungreis, James Carney, Bert W. O’Malley Jr., Daqing Li
Waleed M. Abuzeid, Xiaoling Jiang, Guoli Shi, Hui Wang, David Paulson, Koji Araki, David Jungreis, James Carney, Bert W. O’Malley Jr., Daqing Li
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Research Article Oncology

Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

Abstract

Platinum-based drugs that induce DNA damage are commonly used first-line chemotherapy agents for testicular, bladder, head and neck, lung, esophageal, stomach, and ovarian cancers. The inherent resistance of tumors to DNA damage often limits the therapeutic efficacy of these agents, such as cisplatin. An enhanced DNA repair and telomere maintenance response by the Mre11/Rad50/Nbs1 (MRN) complex is critical in driving this chemoresistance. We hypothesized therefore that the targeted impairment of native cellular MRN function could sensitize tumor cells to cisplatin. To test this, we designed what we believe to be a novel dominant-negative adenoviral vector containing a mutant RAD50 gene that significantly downregulated MRN expression and markedly disrupted MRN function in human squamous cell carcinoma cells. A combination of cisplatin and mutant RAD50 therapy produced significant tumor cytotoxicity in vitro, with a corresponding increase in DNA damage and telomere shortening. In cisplatin-resistant human squamous cell cancer xenografts in nude mice, this combination therapy caused dramatic tumor regression with increased apoptosis. Our findings suggest the use of targeted RAD50 disruption as what we believe to be a novel chemosensitizing approach for cancer therapy in the context of chemoresistance. This strategy is potentially applicable to several types of malignant tumors that demonstrate chemoresistance and may positively impact the treatment of these patients.

Authors

Waleed M. Abuzeid, Xiaoling Jiang, Guoli Shi, Hui Wang, David Paulson, Koji Araki, David Jungreis, James Carney, Bert W. O’Malley Jr., Daqing Li

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Figure 3

Mutant RAD50 protein interacts with endogenous RAD50 and downregulates the MRN complex.

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Mutant RAD50 protein interacts with endogenous RAD50 and downregulates t...
(A) Anti-MRE11 antibody coimmunoprecipitated wild-type RAD50 in all cells, suggesting a direct interaction between these proteins. Mutant RAD50 was only coimmunoprecipitated in infected cells treated with zinc, indicating that the interaction between mutant RAD50 and the wild-type MRN complex is mediated by zinc-dependent dimerization of RAD50 hook domains and not direct interactions between mutant RAD50 and MRE11. All lanes shown were run simultaneously on a single gel as contiguous lanes. (B) Multiple MRN nuclear foci are seen at sites of DNA damage in JHU012 cells treated with Ad-RAD50 and cisplatin. Original magnification, ×63. There is spatial overlap of the signals corresponding to wild-type RAD50 (blue) and wild-type MRE11 (red), highly suggestive of a binding interaction between these proteins. (C) Nuclear foci from JHU012 cells treated with Ad-RAD50 and cisplatin (original magnification, ×63) demonstrate spatial overlap of the wild-type (red) and mutant RAD50 (blue) proteins, further suggesting direct binding between these proteins. (D) Downregulation of wild-type MRE11 (P < 0.005), RAD50 (P < 0.001), and NBS1 proteins (P < 0.04) is seen after infection of JHU012 cells with Ad-RAD50 relative to noninfected controls. (Mean optical density of Western blot bands ± SEM is shown.) This is attributable to the demonstrated dimerization between mutant and wild-type RAD50 and inability of the mutant protein to directly bind MRE11. Consequently, only a single MRE112/RAD502 heterotetramer can form around the wild-type RAD50 with no complimentary heterotetramer assembling around mutant RAD50.