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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Enhancement of Cisplatin [cis-Diammine Dichloroplatinum (II)] Cytotoxicity by O⁶-Benzylguanine Involves Endoplasmic Reticulum Stress

Departments of Molecular Genetics and Cell Biology (C.A.R.), Health Studies (K.K.), and Medicine (M.E.D.), University of Chicago, Chicago, Illinois; Department of Pediatrics, Herman B. Wells Center for Pediatric Research (M.L.F., M.R.K.) and Departments of Pharmacology and Toxicology and Biochemistry and Molecular Biology (M.R.K.), Indiana University School of Medicine, Indianapolis, Indiana; and Departments of Medicine, Pharmacology, and Cancer Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (J.L.H., M.J.E.)

O⁶-Benzylguanine (BG) enhances cisplatin [cis-diammine dichloroplatinum (II)]-induced cytotoxicity and apoptosis in head and neck cancer cell lines by an unknown mechanism. We investigated the effect of cisplatin with and without BG on two targets of damage: DNA and the endoplasmic reticulum (ER). We chose three cancer cell lines to ascertain the mechanism of BG-enhanced cytotoxicity: SQ20b head and neck and SKOV-3x ovarian cancer cell lines, where BG enhanced cisplatin cytotoxicity, and A549 nonsmall cell lung cancer line, where BG did not enhance cisplatin cytotoxicity. All three lines had an increase in DNA damage when BG was added to cisplatin treatment, as evidenced by increased platination and phosphorylated histone H2AX formation. The increase in cisplatin-induced DNA damage after treatment with BG plus cisplatin is not sufficient to increase cytotoxicity or apoptosis in A549 cells. We evaluated the effect of cisplatin on the ER and observed increased caspase 12 cleavage in SQ20b and SKOV-3x cells, but not in A549 cells, after treatment with BG plus cisplatin versus cisplatin alone. Growth arrest and DNA damage inducible (GADD) 153, an ER stress-response gene, is up-regulated after treatment with BG plus cisplatin compared with cisplatin alone in SQ20b and SKOV-3x cells, but not in A549 cells. ER stress-induced apoptosis is an integral part of the mechanism by which BG enhances cisplatin. Inhibition of ER stress in the SQ20b cell line by salubrinal, an inhibitor of eIF2 dephosphorylation, or GADD153 small interfering RNA, abrogated BG-enhancement of cisplatin cytotoxicity and apoptosis through caspase 3 and 12 cleavage. These data indicate GADD153 up-regulation plays an important role in BG-enhanced cisplatin cytotoxicity and apoptosis.

Address correspondence to: Dr. M. Eileen Dolan, 5841 S. Maryland Ave., Box MC2115, University of Chicago, Chicago, IL 60637. E-mail: edolan{at}medicine.bsd.uchicago.edu