doi:10.1006/mthe.2000.0064, available online at http://www.idealibrary.com on IDEAL ARTICLE Use of the Ornithine Decarboxylase Promoter to Achieve N-MYC-Mediated Overexpression of a Rabbit Carboxylesterase to Sensitize Neuroblastoma Cells to CPT-11 Cynthia A. Pawlik, Rekha V. Iyengar, Erik J. Krull, Stephen E. Mason, Ruchi Khanna, Linda C. Harris, Philip M. Potter, Mary K. Danks,1 and Sylvie M. Guichard2 Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 Received for publication February 7, 2000, and accepted in revised form March 30, 2000 Overexpression of specific transcription factors by tumor cells can be exploited to regulate expression of proteins that induce apoptosis or activate prodrugs, thereby producing tumor- selective toxicity. A majority of advanced-stage neuroblastomas overexpress the transcription fac- tor N-MYC, and this overexpression is associated with poor prognosis. This study describes reg- ulation of expression by N-MYC, via the ornithine decarboxylase (ODC) promoter, of a rabbit liver carboxylesterase (CE) that activates the prodrug CPT-11. Chloramphenicol acetyltransferase reporter assays and CE activity assays in transiently transfected neuroblastoma cell lines (SJNB-1, SJNB-4, NB-1691) and rhabdomyosarcoma cell lines (JR1neo20, JR1Nmyc6, JR1Nmyc9) support this approach as a potential method for sensitizing tumor cells to CPT-11. Clonogenic assays with IMR32 human neuroblastoma cells which express N-MYC and that had been stably transfected with a plasmid containing an ODC promoter/CE cassette corroborated results of enzyme activity assays. Specifically, IMR32.ODC.CE cells expressed approximately eightfold more CE activity than IMR32.CMV.neo cells; and 5 µM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 µM CPT-11 was required to produce the same effect with IMR32.CMV.neo cells. Current experiments focus on adenoviral delivery of an ODC promoter/CE cDNA cassette for potential virus-directed enzyme prodrug therapy applications. Key Words: irinotecan; gene therapy; prodrug; VDEPT; esterase; ornithine decarboxylase promot- er; N-MYC. INTRODUCTION tive therapy. Since viruses transduce both normal and tumor cells, selective expression of CE might be achieved We recently reported the isolation of a cDNA encoding a by a transcription factor/promoter combination unique rabbit liver carboxylesterase (CE) that efficiently con- to tumor cells. Protooncogene/transcription factor N- verts the prodrug CPT-11 to SN-38 and sensitizes human MYC and the ornithine decarboxylase (ODC) promoter tumor cells to CPT-11 both in vitro and in a preclinical represent such a combination. xenograft model (1–3). The level of expression of rabbit N-MYC is a transcription factor overexpressed in CE produces a parallel increase in sensitivity to CPT-11 ~40% of Grade III/IV neuroblastomas, and this overex- (1, 2). Our long-range goal is to use the rabbit CE in com- pression correlates with rapid tumor progression and bination with CPT-11 in a virus-directed enzyme pro- poor prognosis (4, 5). MYC family proteins (c-, N-, L-) drug therapy (VDEPT) approach to achieve tumor-selec- dimerize with MAX and bind to the consensus CACGTG E-box sequence (6–8), thereby activating several promot- ers including those that control expression of ODC, α- prothymosin, and p53 (9–12). Use of the ODC promoter 1To whom correspondence should be addressed at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 to achieve tumor-specific expression of CE and activa- North Lauderdale, Memphis, TN 38105. Fax: (901) 521-1668. E-mail: tion of CPT-11 in neuroblastoma cells that overexpress [email protected]. N-MYC is the focus of this study. No studies of this type 2Present address: Institut Claudius Regaud, Laboratoire de Pharmacologie, 20-24, rue du pont St Pierre 31052, Toulouse cedex, have been done with the ODC promoter, but several France. studies have been published that investigate the effect of MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 1 of 2 Parts 457 Copyright The American Society of Gene Therapy ARTICLE MYC-responsive E-box enhancer sequences (CACGTG) in combination with Simian virus-40 (SV40) or Herpes simplex virus thymidine kinase (HSVtk) promoters to upregulate expression of various prodrug-activating enzymes (13, 14). In those studies some degree of speci- ficity was achieved, but promoter/enhancer activity was dependent on the interaction of different transcription factors with the promoter and the enhancer sequences. The purpose of this study was to demonstrate specific ODC promoter-regulated expression of the CAT reporter gene and rabbit CE in several human tumor cell lines that overexpress N-MYC and to show that increased sen- sitivity to CPT-11 results from ODC promoter-regulated CE expression. MATERIALS AND METHODS Drugs and chemicals. CPT-11 was a generous gift from Dr. J. P. McGovren p∆ODC.CAT were repeated with pCAT3-Basic and pC3B.ODC.CAT. Both (Pharmacia–Upjohn Co., Kalmazoo, MI). A 10 mM drug stock was pre- sets of plasmids gave the same ratio after normalizing to the appropriate − pared in methanol and stored at 20°C. Drug was further diluted in water negative control. Importantly, CAT activities (raw data) for all cell lines immediately before use. transfected with pCAT3-Basic and for MYC-negative SJ-G3 cells transfect- Cell lines. The neuroblastoma cell lines SJNB-1 and SJNB-4 were ed with pC3B.ODC.CAT were in the 500 dpm range, equivalent to mock derived from patients at St. Jude Children’s Research Hospital (SJCRH) transfectants. (Memphis, TN). Tumor samples were obtained after informed consent Plasmids for CE activity. The ODC promoter and rabbit liver CE were was given, in accordance with the guidelines of the St. Jude Children’s ligated into the pCIneo plasmid (Promega) as follows: pCIneo was digest- Research Hospital Institutional Review Board (IRB). The embryonal rhab- ed with BglII and I-PpoI to remove the CMV promoter, leaving the domyosarcoma cell line JR1 was provided by Dr. P. Dias (Imgenex, San chimeric intron intact. Blunt ends were created at the BglII and I-PpoI Diego, CA). NB-1691 cells were obtained from the Pediatric Oncology sites. The ODC promoter was released from pODC.CAT by digestion with Group. These cell lines were grown in RPMI 1640 (BioWhittaker, SmaI and inserted into pCIneo at this site by blunt-end ligation. CE was Walkersville, MD) supplemented with 10% fetal calf serum (Hyclone, released from pIRESrabbit (1) by digestion with EcoRI and ligated into this Logan, UT) and 2 mM glutamine. The neuroblastoma cell line IMR32 was site of the plasmid to generate pCI.ODC.CE (Table 1). acquired from American Type Tissue Culture Collection (Rockville, MD) and grown in DMEM (BioWhittaker), 10% fetal calf serum, and 2 mM Establishment of stably transfected cell lines. JR1 cells were transfected glutamine. Multiple cell lines were used in this study to ensure that N- with pIRES.CMV.N-myc plasmid DNA by the calcium phosphate method MYC/ODC promoter-regulated protein expression was not unique to a (Stratagene, La Jolla, CA). Stable clones expressing different levels of N- single cell line. Pediatric glioblastoma cell lines SJ-G2 and SJ-G3 were MYC were selected in 200 µg/ml of geneticin (Gibco BRL, Gaithersburg, derived from tumors of patients at SJCRH. Tumor samples were obtained MD). Two clones expressing different levels of N-myc RNA were expand- in accordance with the guidelines of the SJCRH IRB. The cell lines were ed and used in this study: JR1Nmyc6 and JR1Nmyc9. The control maintained in DMEM, 15% fetal calf serum, and 2 mM glutamine. SJ-G2 JR1neo20 cell line was generated by transfecting JR1 cells with the parent cells express c-MYC but not N-MYC; SJ-G3 cells express neither c-MYC pIRESneo plasmid. In addition, IMR32 neuroblastoma cells were trans- nor N-MYC and were used as controls for specific experiments, as indi- fected by electroporation with either pIRESneo or pIRES.ODC.CE using cated in the text. conditions previously published (2), and stable transfectants were select- ed in 500 µg of geneticin/ml. IMR32.CMV.neo and IMR32.ODC.CE cell Plasmids for CAT reporter activity. The N-myc cDNA was ligated into lines were not cloned following transfection and geneticin selection, but the bicistronic pIRESneo plasmid (Clontech, Palo Alto, CA) in the fol- include all cells that express sufficient neomycin resistance protein to sur- lowing manner: The N-myc cDNA was excised from pCN64RX and insert- vive the selecting concentration of geneticin. Results with these IMR32 ed into the EcoRV and EcoRI sites of pIRESneo. This plasmid is referred to cell lines, therefore, represent the entire transfected population and are as pIRES.CMV.N-myc. For clarity, names of plasmids in this paper contain not the properties of a single clone. the name of the promoter (ODC, CMV) and the gene or cDNA (CAT, CE, N-myc, neo) in each construct (Table 1). Western analysis. For each sample, one flask of cells in log-phase The pODC∆CAT and pODC∆CAT∆S plasmids were provided by Drs. growth was washed twice with ice-cold PBS, and the cells were harvested J. L. Cleveland (St. Jude Children’s Research Hospital) and P. Coffino by scraping into 0.6 ml of ice-cold PBS–ST (1% SDS, 1% Triton X-100) (University of California, San Francisco, CA) and have been described in containing 100 µg/ml of each of the following protease inhibitors: apro- detail previously (9). These constructs are referred to in the text as tinin, leupeptin, and antipain (Sigma, St. Louis, MO). Loading buffer (0.2 pODC.CAT and p∆ODC.CAT, respectively. pODC.CAT contains the ml of 4ϫ) was added to 600 µl of the cell solution, and the cell lysate was murine ODC promoter which regulates transcription of the reporter gene passed through a 23-gauge needle 10 times to shear the DNA.