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Divisions of ¹ Hematology-Oncology and ² Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, CA, and Departments of ³ Pediatrics and ⁴ Pathology, University of Southern California, Keck School of Medicine, Los Angeles, CA

Requests for reprints: C. Patrick Reynolds, Division of Hematology-Oncology, MS#57, Childrens Hospital Los Angeles, 4620 Sunset Boulevard, Los Angeles, CA 90027. Phone: (323) 669-5646; Fax: (323) 664-9226 or 9455. E-mail: preynolds{at}chla.usc.edu

Green fluorescent protein (GFP) is employed as a selection marker for gene transduction and to track tumor cells. Transduction of enhanced GFP (eGFP) into human neuroblastoma cell lines via a lentiviral vector significantly sensitized CHLA-20 (wild-type and functional TP53), and to a lesser extent CHLA-90 cells (multidrug-resistant, mutant, and nonfunctional TP53) to carboplatin, doxorubicin, etoposide, or melphalan, relative to cells transduced using the cell surface antigen CD80 as a selection marker. Total glutathione (GSH) was significantly up-regulated (1.8- to 2.8-fold) after eGFP (but not CD80) transduction in cell lines with, but not in those lacking, functional p53. Cytotoxicity of GSH depletion by buthionine sulfoximine in CHLA-20 (but not in CHLA-20-eGFP) was diminished by hypoxia (2% O₂). Thus, oxidative stress produced by GFP selects for cells with up-regulated GSH in a p53-dependent manner, and also enhanced the cytotoxicity of anticancer drugs in neuroblastoma cell lines. Our data suggest caution when employing GFP-transduced cells to assess drug sensitivity and that using a cell surface antigen as a selection marker for gene transduction may perturb cells less than GFP.

Key Words: cancer chemotherapy • glutathione • reactive oxygen species • gene transduction • hypoxia

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Grant support: Neil Bogart Memorial Laboratories of the T.J. Martell Foundation for Leukemia, Cancer, and AIDS Research; National Cancer Institute Grants CA82830 and CA60104; and a fellowship from Hope Street Kids (to H. Goto).

¹ M. Barcova, A. Logan, and P. M. Cannon. Engineering the polyadenylation sequence of a minimal self-inactivating lentiviral vector reduces read-through transcription and enhances gene expression from integrated vectors, manuscript in preparation.

Received 1/29/03; revised 6/24/03; accepted 6/24/03.