[CANCER RESEARCH57, 4205-4209, October 1, 19971 Advances in Brief Virally Directed Cytosine Deaminase/5-Fluorocytosine Gene Therapy Enhances Radiation Response in Human Cancer Xenografts' Nader N. Hanna, Helena J. Mauceri, Jeffery D. Wayne, Dennis E. Hallahan, Donald W. Kufe, and Ralph R. Weichselbaum2 Department ofSurgery (N. N. H., J. D. W.J and Department ofRadiation and Cellular Oncology (H. J. M., D. E. H., R. R. WI, Pritzker School ofMedicine, University of Chicago, Chicago, Illinois 60637, and Division of Cancer Pharmacology, Dana-Farber Cancer institute ID. W. K.], Boston, Massachusetts 02115 Abstract Gene therapy combined with radiation therapy to enhance selectively radiation cytotoxicity in malignant cells represents a new approach for cancer treatment. We investigated the efficacy of adenoviral (AdS) directed cytosine deaminase/5-fluorocytosine (CD/5-FC) enzyme/prodrug gene therapy to enhance selectively the tumoricidal action of ionizing radiation in human cancer xenografts derived from a human squamous carcinoma cell line (SQ-20B). Tumor xenografts grown in hindlimbs of nude mice were transfected with an adenoviral vector (Ad.CMV.CD) containing the cytosine deaminase (CD) gene under the control of a cyto megalovirus (CMV) promoter. Mice were Injected i.p. with 800 mg/kg of S-FC for 12 days, and tumors were treated with fractionated radiation at a dose of 5 Gy/day to a total dose of 50 Gy. In larger tumors with a mean volume of 1069 mm3, marked tumor regression to 11% of the original tumor volume was observed at day 21 (P 0.01). The volumetric regres. sion of smaller tumors with a mean volume of 199 mini', which received the same combined treatment protocol, was significant at day 12 (P = 0.014). However, unlike large tumors, regression of the smaller tumors continued until day 36 (P = 0.01), with 43% cured at day 26. No cures or significant volumetric reduction in size was observed in tumors treated with radiation alone; AdCMV.CD with or without radiation; or with Ad.CMV.CD and 5-FC. These results suggest that the CD/S-FC gene therapy approach is an effective radiosensitizing strategy and may lead to substantial improvement in local tumor control that would translate into improved cure rates and better survival. Introduction Radiation therapy is frequently used as a primary or adjuvant treatment for many human cancers with curative intent or to improve local tumor control. For many large epithelial and mesenchymal tumors or tumors of certain histological types, such as glioblastoma, complete tumor eradication and local cure following radiotherapy are rare. Potential mechanisms of radiation resistance include a large population of clonogenic tumor cells, repopulation of tumor cells between doses, cellular repair of radiation damage, and inherent tumor radioresistance (1). Also microenvironmental factors such as tumor hypoxia may play an important role in clinical radioresistance. Attempts to improve the therapeutic efficacy of radiation include the concomitant administration of chemotherapeutic agents. However, the lack of differential effects on tumors and some normal tissues limits the potential therapeutic efficacy of combining chemotherapeu tic agents and radiotherapy due to associated toxicities. One of the most widely used antineoplastic agent, as well as a clinical radiation sensitizer, is 5-FU.@In addition to its antitumor effects, 5-FU has been shown in several prospective clinical trials to enhance radiation re sponse and local tumor control in several types of human malignan cies (2—6). The enhancement of radiation by 5-FU may involve inhibition of the mechanisms of cell repair as a result of depletion of cellular nucleotides and eradication of radioresistant cells in the S phase of the cell cycle and additive cytotoxic tumor killing effects. Gene therapy combined with radiotherapy represents a new ap proach for cancer treatment by selectively radiosensitizing malignant cells. We have previously described the concept of gene therapy targeted by ionizing radiation, whereby a cDNA encoding tumor necrosis factor-a, is ligated downstream from a radiation-inducible promoter (7—9). In this way, radiation acts as a â€oemolecular switch―to activate transcription of tumor necrosis factor-a, which modifies the cellular response to radiation. Another gene therapy approach with the potential to enhance the radiation killing of malignant cells is VDEPT (10—21).VDEPT incorporates a viral vector system to deliver a gene encoding a prodrug-activating enzyme to malignant cells. Following intracellular expression of the enzyme, a nontoxic prodrug is con verted to a toxic compound to sensitize selectively tumor cells to radiation. Normal cells not expressing the therapeutic gene are not affected. Also, because the toxic drug is only produced intratumorally, systemic toxicity is avoided. Although several enzyme/prodrug sys tems have been investigated for gene therapy (22, 23), VDEPT has concentrated mainly on two enzyme/prodrug combinations for clinical application. The first is the HSVtk/GCV combination. The HSVtk enzyme phosphorylates GCV to GCV triphosphate, which acts as a chain terminator in DNA synthesis, resulting in the death of dividing cells. The second is the CD/5-FC combination. CD converts nontoxic 5-FC to toxic 5-FU. 5-FU inhibits thymidylate synthetase, blocking the methylation reaction of dUMP to TMP. In this manner, it disrupts DNA synthesis and 5-fluorUTP formation, which is incorporated into RNA, resulting in cell deaths in both dividing and nondividing cells. In addition to its antineoplastic effects, 5-Ri also sensitize the tumor cells to irradiation. Furthermore, this approach is reported to result in tumor-specific immunity (24—26).The present in vivo experiments were undertaken to investigate the efficacy of virally directed CD/ 5-FC combination to enhance the tumoricidal action of ionizing radiation in a human cancer xenograft model. Received 7/15/97; accepted 8/14/97. 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. I This work was tupported by NIH Grant T32CA09516, The Daniel F. and Ada L. Rice Foundation, The Chicago Tumor Institute, The Center for Radiation Therapy, and Na tional Cancer Institute Grant CA41068. 2 To whom requests for reprints should be addressed, at Department of Radiation and Cellular Oncology, Box MC 9030, 5758 South Maryland Avenue, University of Chicago, Chicago, IL 60637. Phone: (773) 702-0817; Fax: (773) 834-7233; E-mail: rrw@rover.uchicago.edu. Materials and Methods Cell Lines. Studies were performedusing SQ-20B cells derived from a patient with squamous cell cancer of the larynx after recurrence following primary radiotherapy (27). The SQ-20B cell line forms undifferentiated squa 3 The abbreviations used are: 5-FU, 5-fluorouracil; VDEPT, virus-directed enzyme/ pro-drug therapy; HSVtk, herpes simplex virus thymidine kinase; GCV, ganciclovir; CD, cytosine deaminase; 5-FC, 5-fluorocytosine; CMV, cytomegalovirus. 4205 Research. on November 4, 2015. © 1997 American Association for Cancer cancerres.aacrjournals.org Downloaded from