[CANCER RESEARCH 47, 5361-5366, October 15, 1987] Potentiation of Halogenated Pyrimidine Radiosensitizers in Human Carcinoma Cells byß-Lapachone(3,4-Dihydro-2,2-dimethyl-2//-naphtho[l,2-£]pyran-5,6- dione), a Novel DNA Repair Inhibitor1 David A. Boothman, Sheldon Greer, and Arthur B. Pardee2 Division of Cell Growth and Regulation ¡D.A. B.] and Department of Pharmacology [A. B. P.], Harvard Medical School, Dana Farber Cancer Institute (D-8IOA), Boston, Massachusetts 021IS, and Department of Microbiology and Immunology (R-I3S), School of Medicine, University of Miami, Miami, Florida 33101 fS. G.J ABSTRACT 3,4-Dihydro-2,2-dimethyl-2H-naptho[l,2,-e]pyran-5,6-dione (/ï-lapa- chone) is a novel DNA repair inhibitor. It was tested for synergistic X- ray-induced lethality in combination with several halogenated pyrimidine radiosensitizers. Logarithmic-phase growing human epiderntoid laryn- geal carcinoma (HEp-2) cells were allowed to incorporate pyrimidine analogues for 48 h (approximately two cell doublings) and then were X- irradiated and subjected to various posttreatments. 0-Lapachone syner- gistically increased the dose enhancement ratios (DERs) of all analogues screened, with the exception of the 2'-chloro derivative of 5-bromode- oxyuridine. For example, following 5-bromodeoxycytidine sensitization an X-ray DER value of 1.87 ±0.04 at 1% survival was increased to 3.51 ±0.42 due to a4-h post-X-irradiation exposure to 4 MM0-lapachone. />„ and I),, values for halogenated pyrimidine-sensitized human epidermoid laryngeal carcinoma cells were decreased 1.4- to 5.4-fold and 1.4- to 4.0- fold, respectively. 0-Lapachone had little effect upon the cytotoxicities of unirradiated human epidermoid laryngeal carcinoma cells whether or not they were previously exposed to any of the halogenated pyrimidine radiosensitizers. /3-Lapachone treatment following X-irradiation of cells that had not incorporated a pyrimidine analogue exhibited DER values of 1.38 ±0.05 and 1.40 ±0.01 at 10and 1%survival levels, respectively. /J-Lapachone enhanced the radiosensitization of deoxycytidine ana logues to a greater extent than the structurally related deoxyuridine analogues. Greater DERs and lower M, and I),, values were found for deoxycytidine than for deoxyuridine analogue radiosensitizers following 0-lapachone treatment. This agent may improve presently used radiation therapies and enhance proposed strategies which utilize deoxycytidine analogue radiosensitization together with protection of normal tissues by tetrahydrouridine to achieve tumor-selective radiotherapy. INTRODUCTION A major problem in chemotherapy or radiotherapy against neoplastic disease is the ability of at least a minor subset of tumor cells to survive owing to repair of DNA lesions inflicted by oncolytic agents and/or X-rays (1, 2). Compounds which inhibit repair or misrepair of DNA lesions can increase the lethalities of oncolytic agents (3) and thus enhance the thera peutic indices of antineoplastic drugs (4). The influence of cellular repair processes upon the ability of a radiosensitized cell to respond to X-rays remains unexplored in detail. We propose that a novel radiotherapeutic approach is to use com pounds which inhibit PLDR3 to specifically modify the survival Received 3/31/87; revised 7/6/87; accepted 7/14/87. 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. 1Supported by Grant CA22427 to A. B. P. from the National Cancer Institute and. in part, by Grant CA33219 to S. G. from the National Cancer Institute. 1To whom requests for reprints should be addressed. 3The abbreviations used are: PLDR, potentially lethal damage repair, ,i- lapachone, 3,4-dihydro-2,2-dimethyl-2//-naphtho( 1,2-e]pyran-5,6-dione; BrdCyd, 5-bromo-2'-deoxycytidine; BrdUrd, 5-bromo-2'-deoxyuridine; cGy, centiGray; CMF-PBS, calcium- and magnesium-free phosphate-buffered saline; 2'-ClBrdUrd, 2'-chloro-5-bromodeoxyuridine; CldCyd, 5-chloro-2'-deoxycyti- dine; DER, dose enhancement ratio; dFCS, dialyzed fetal calf serum; DME, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; A,, dose re quired to reduce survival 37% on an exponential portion of a survival curve; D,, quasi-threshold dose (measure of sublethal damage repair); HEp-2, human epi dermoid laryngeal carcinoma; IdCyd, 5-iodo-2'-deoxycytidine; IdUrd, 5-iodo-2'- deoxyuridine. of X-irradiated neoplastic cells previously treated with tumor- selective radiosensitizing compounds. One very potent DNA repair inhibitor is 0-lapachone (see inset to Fig. 1 for structure). At very low concentrations of only 5 MMor less, /3-lapachone did not influence the metabolism of undamaged normal human fibroblast cells (5). At 4 juM, it greatly decreased the survival of methyl methane sulfonate- damaged cells. In these cells, unscheduled DNA synthesis in creased and the repair of single-stranded DNA lesions was inhibited (5). /3-Lapachone was originally isolated from lapacho! prepara tions (6) and was shown to have a variety of concentration- dependent activities. At high concentrations (i.e.. > 1 HIM), it inhibited retroviral reverse transcriptase and eukaryotic DNA polymerase-a and prolonged the survival time of chickens in fected with Rous sarcoma virus (7, 8). At concentrations rang ing from 100-10 A/M.¿f-lapachonedemonstrated antimicrobial (9), slight antitumor (7, 10, 11), and antitrypanosomal (12, 13) effects. Recent research involving radiosensitizers has focused on halogenated pyrimidine analogues (14-16), which in theory should sensitize hypoxic as well as nonhypoxic cells. These analogues rely upon incorporation into DNA (in place of thy- midine) for their radiosensitizing effects. Although clinical trials with two halogenated pyrimidines (i.e., BrdUrd and IdUrd) have revealed encouraging therapeutic gains, bone mar row suppression, rapid systemic catabolism, mutagenicity, der mal phototoxicity (with BrdUrd), and a general lack of tumor selectivity continue to limit their usefulness (17-19). The purpose of this study was 2-fold: (a) to examine the modifying effect(s) of /3-lapachone upon cellular repair proc esses so as to decrease overall survival following X-irradiation damage (i.e., by preventing PLDR); (b) to investigate the pos sible enhanced cytocidal effect(s) of /3-lapachone upon cells that have incorporated various halogenated deoxyuridine and de oxycytidine radiosensitizers. MATERIALS AND METHODS Chemicals. CldCyd, 2'-ClBrdUrd, and CldUrd were obtained and their radiosensitizing characteristics described in previous work (15, 16, 20). BrdUrd, BrdCyd, IdUrd, and IdCyd were purchased from Sigma Chemical Co. (St. Louis, MO). All halogenated nucleosideswere prepared as lOx solutions, stored at -20°C,and treated as photosen sitive compounds by aluminum foil enclosure, both as stock solutions and in cell treatments. 0-Lapachone (A/, 242.3) was a gift from Ciba Geigy and was initially prepared as a 40 /¿M stock solution by dissolving the compound in DMSO (5mg/ml, stored at -20°C)and diluting the mixture to volume with CMF-PBS (0.8% NaCl, 0.02% KC1, 0.02% KH2PO4,and 0.12% Na2HPO4).This stock solution was then used to prepare linai /3-lapachonemixtures in dFCS-DME. Cell Culture Techniques. HEp-2 cells were obtained from the Amer ican Type Culture Collection (Rockville, MD). They were maintained as attached monolayers in DME supplemented with 10% dFCS, peni cillin (100 units/ml), streptomycin (100 units/ml), and glutamine (3.2 HIM),and exhibited a doubling time of 18-22 h. HEp-2 cells were 5361 on March 13, 2017. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from