Potentiation of Halogenated Pyrimidine

Home , Pyrimidine analogue

[CANCER RESEARCH 47, 5361-5366, October 15, 1987] Potentiation of Halogenated Pyrimidine Radiosensitizers in Human Carcinoma Cells by ÃŸ-Lapachone(3,4-Dihydro-2,2-dimethyl-2//-naphtho[l,2-Â£]pyran-5,6- dione), a Novel DNA Repair Inhibitor1

David A. Boothman, Sheldon Greer, and Arthur B. Pardee2

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

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. ENHANCED RADIOSENSITIZATION BY /3-LAPACHONE passaged and routinely examined for Mycoplasma contamination as previously described (21). Cell Plating and Nucleoside Analogue Exposure. HEp-2 cells (5.0 x IO4)were seeded from subconfluent cultures onto 25-cm2 tissue culture flasks (Corning Glass Works, Corning, NY) and after growth for 48 h at 37Â°Cin a humidified 10% CO2-90% air atmosphere, microscopic examination demonstrated individual colonies of 4-8 cells. This pro cedure allowed for maximum PLDR of X-irradiated exponential-phase HEp-2 cells and is consistent with recent studies using HeLa hybrid cells (22). Media were then removed, the appropriate concentration of halogenated deoxynucleoside analogue dissolved in DME containing 10% dFCS was applied, and the cells were incubated in the dark at 37"C for 48 h. All flasks were letter coded. For experiments not utilizing deoxynucleoside analogues, 1.0 x 10s HEp-2 cells were plated and experiments were initiated following the first 48-h logarithmic-phase growth period. Irradiation of Cells and Colony-forming Assays. HEp-2 cells were grown for 96 or 48 h as described above and then prepared for X- irradiation. Media were aspirated, cells were washed with CMF-PBS, 24 6 8 10 12 Q-Lapachone Concentration (jjM) and all flasks were filled with radiation medium [2.1 ml penicillin (25 Fig. 1. Effect of/3-lapachone upon X-irradiated and unirradiated HEp-2 cells. units/ml), 150 ml 0.2% dFCS-DME, 341 ml sterile CMF-PBS, and HEp-2 cells (1.0 x 10s) were plated onto 25-cm2 tissue culture flasks. Half of the 6.7 ml glutamine (2.7 mM)]. Flasks were then cooled to 4Â°Con ice for cultures were X-irradiated 48 h later with a dose of 300 cGy using the procedure described in "Materials and Methods." Then media were aspirated and 0.2% l h to prevent repair between the interval of X-irradiation and ÃŸ- dFCS-DME with or without various /3-lapachone concentrations were added. lapachone addition. Cells were X-irradiated on ice at a distance of 50 Samples were incubated for 4 h at 37Â°Cin a humidified 10% CO2-90% air cm using a Phillips RT-250 X-ray generator with 0.35 mm Copper atmosphere (except for a pair of unirradiated samples that were plated immedi ately). Following the posttreatment period, cells were assayed for colony-forming filtration at 15 mA, 250 kV, and a dose rate of 128.8 cGy/min. Doses ability as described in "Materials and Methods." , surviving fraction of of 0, 100, 300, 500, 700, and 800 cGy were used to generate dose- X-irradiated HEp-2 cells given a posttreatment of 0.2% dFCS-DME for 4 h. response survival curves. An attenuation factor of 0.865 due to the , survival of X-irradiated cells given no posttreatment. The difference between polystyrene composition of the flask and radiation medium was used these two levels demonstrates the ability of HEp-2 cells to "repair" X-ray damage. in calculating the exposure times as previously described (16, 20). Plating efficiency was 79 Â±3.1%. Inset, chemical structure of/3-lapachone. Immediately after X-irradiation, media were aspirated and 0.2% dFCS-DME with or without /3-lapachone was added. Posttreatments RESULTS lasted 4 h at 37Â°Cunless otherwise indicated. Some cells received no Repair Inhibition Studies. Fig. 1 illustrates the effect of a posttreatment. Media were then aspirated and cells were washed with CMF-PBS and trypsinized for 1 min at 37Â°C.Complete 10% dFCS- range (1-11 /Â¿M)of/3-lapachone concentrations upon the sur vival of unirradiated or X-irradiated (300 cGy) HEp-2 cells. A DME was added to neutralize the trypsin and cells were serially diluted 4-h exposure to /3-lapachone concentrations of 4 ^M* or less in 10-fold increments with DME containing 20% dFCS. All dilutions were plated onto 60-mm tissue culture dishes (Falcon, Becton Dickin had little effect upon the survival of unirradiated cells. Above 7 son, Lincoln Park, NJ), letter coded, and incubated for 7-9 days at AIMa considerable reduction in the survival of unirradiated 37Â°Cin a humidified 10% CO2-90% air atmosphere. Cells were then HEp-2 cells was observed. HEp-2 cells damaged via X-irradia washed with a 0.85% NaCl solution and stained for 5 min with 2% tion and then exposed for 4 h to ÃŸ-lapachoneshowed a drastic crystal violet in 20% ethanol. Those plates containing 100-50 colonies reduction in survival (Fig. 1). These results are similar to those were scored for colony-forming ability using a dissecting microscope reported for methyl methane sulfonate-damaged cells (5). fitted with an ocular micrometer. A viable colony consisted of 50 or X-irradiated HEp-2 cells treated for 4 h with 0.2% dFCS- more normal-appearing cells and was considered to have arisen from a DME increased in survival from 31 to 80% compared to X- single viable cell. All conditions were completed in duplicate and all irradiated cells given no posttreatment (see area marked Repair experiments were performed at least twice. Surviving fractions were in Fig. 1). This PLDR capacity is consistent with previous determined and the clonogenic ability of treated compared to untreated studies using HeLa cells (22, 24). Exposure to 2 pM /3-lapa control conditions were compared (using the appropriate designation chone, however, completely abolished this ability to repair X- of drug treatments from decoded samples). Plating efficiencies ranged from 70-85% as determined for untreated control conditions by using irradiation damage. At concentrations above 2 /Â¿Manadditional the ratio of colony-forming ability to initial viability as measured via lowering of survival was observed (Fig. 1). A concentration of 4 UMÃŸ-lapachonewas chosen for use in screening halogenated trypan blue exclusion (23). Calculations. Survival curves were normalized to correct for drug pyrimidine radiosensitizers for possible synergistic action, since cytotoxicities and variations in initial viable cells plated. Linear regres this dose resulted in minimal toxicity and significantly inhibited sion analysis was utilized to determine /',, values (i.e., slopes) and the repair. ^-intercept or n extrapolation values of the linear portions of the The effect of radiation dose upon the survival of HEp-2 cells survival curves. Shoulder regions were not considered in these calcula subsequently given various 4-h treatments was examined (Fig. tions. D9 values were calculated using the following relationship: 2). When X-irradiated cells were incubated for 4 h with 0.2% dFCS-DME, the resulting surviving fraction was greater (due to PLDR) than that produced with a 4-h post-X-irradiation treatment in 10% dFCS-DME (Fig. 2). Posttreatment with 10% dFCS-DME gave statistically similar results to those DER values at 10 and 1% surviving fractions for treated relative to control conditions (i.e., 4-h post X irradiation incubation in 0.2% 1A recent shipment of (3-lapachone from Ciba Geigy resulted in significantly dFCS-DME at 37"C) were determined by linear regression analysis (16, greater cytotoxicity (i.e., 50% survival) to unirradiated HEp-2 cells at 4 Â»IMas compared to the 3-year-old /3-lapachone stock used in this study. When directly 20). These ratios are defined as the radiation dose of untreated cells compared the new /3-lapachone was relatively nontoxic (>80% survival) to unir divided by the dose of treated cells required to reduce the surviving radiated cells given at 3 MMfor 4 h. At this concentration, a similar enhancement fraction to equal levels, either 10 or 1%. of radiolethality was demonstrated as noted in the present study. 5362

0.01

0.01 -

0.001

0.0001

02Â»dFCS-DME 900 300 600 0.00001 0-LÂ«pachonÂ« IOUro-0 2Â» dFCS-DME

Dose (cGy) IdUrd+No PoÂ»lTrÂ«Â«lmÂ«nt(â€¢) Fig. 2. Ability of /j-lapachone to increase the radiolethality of cells regardless IdUrd*0-LapÂ«chonÂ« M of serum concentration. HEp-2 cells were seeded and X-irradiated at various doses as described in "Materials and Methods." Immediately following X-irradia- 900 tion, posttreatments were applied for 4 h at 37*C. Survival was determined by 300 clonogenic ability as described in "Materials and Methods." A plating efficiency Dose (cGy) of 82 Â±2.4% was observed. Post-X-irradiation treatments were: O, 0.2% dFCS- Fig. 3. Potentiation of UH'ni radiosensitization by /3-lapachone. HEp-2 cells DME; A, 10% dFCS-DME; â€¢,4nM 0-lapachone in 0.2% dFCS-DME; and A. 4 were plated and exposed, or not exposed, to 10 (At IdUrd at 37*C in a humidified Â«IM0-lapachonein 10% dFCS-DME (broken diagonal line). Error bars were not 10% COj-90% air atmosphere as described in "Materials and Methods." Follow drawn in instances where the standard errors of two or more conditions were ing X-irradiation, unsensitized and IdUrd-sensitized HEp-2 cells were exposed to similar. DME containing 0.2% dFCS with or without 4 (-M/3-lapachone for 4 h. Other cells were immediately assayed for clonogenic ability (i.e., no posttreatment) obtained when HEp-2 cells were plated immediately (data not following X-irradiation. Error bars were not drawn in instances where the stan dard errors of two or more conditions were similar. The plating efficiency of shown) following X-irradiation (i.e., no posttreatment). Post- control cells was 77 â€¢1.3%.The survival of unirradiated, sensitized or unsensi treatments in 0.2% dFCS-DME containing 0.02% DMSO re tized cells exposed to these post-X-irradiation treatments is included in Table 1. Conditions were: O, X-irradiated cells exposed to 0.2% dFCS-DME, 4 h; â€¢,X- sulted in cytotoxicities and PLDR responses which were iden irradiated cells exposed for 4 h to 4 (AI /3-lapachone; O, X-irradiated, UH ni tical to those found using the same medium lacking DMSO sensitized cells exposed to 0.2% dFCS-DME, 4 h; A, X-irradiated, Ml nl (data not shown). Postirradiation exposure to 0-lapachone (4 sensitized cells given no posttreatment (broken diagonal line); and â€¢.X-irradiated IdUrd-sensitized cells treated with 4 iiM /3-lapachone, 4 h. UM) and 0.02% DMSO caused a large reduction in survival irrespective of the concentration of dFCS (Fig. 2). MI\ radiosensitized HEp-2 cells with or without /3-lapachone. A We have investigated the timing of /3-lapachone addition post-X-irradiation treatment with 4 /IM /3-lapachone in 0.2% upon the survival recovery (i.e., PLDR) of HEp-2 cells follow ing X-irradiation at 300 cGy.5 For maximal PLDR inhibition dFCS-DME to unsensitized HEp-2 cells (i.e., cells not exposed of HEp-2 cells, /3-lapachone must be applied during and/or to IdUrd) enhanced cell killing (as also shown in Fig. 2); DER immediately following X-irradiation. When added during X- values were 1.38 Â±0.05 and 1.40 Â±0.01 for 10 and 1% survival levels, respectively (Table 1). Unirradiated cells exhibited only irradiation no significant difference in the surviving fraction a slight decrease in survival (i.e., 84% survival) when exposed was noted as compared to the posttreatments found in Figs. 1 to 0-lapachone (Table 1). or 2. When /3-lapachone was applied for 4 h and washed away HEp-2 cells similarly sensitized with IdUrd for 48 h and prior to X-irradiation, only slight radiosensitization occurred treated with 0.2% dFCS-DME for 4 h exhibited reduced sur with respect to a low serum holding period. The capacity of vival after X-irradiation; DER values were 2.00 Â±0.01 and 2.07 HEp-2 cells to carry out PLDR was found to have a half-life of roughly 2 h. Addition of ÃŸ-lapachone4 or more hours following Â±0.03 at 10 and 1% survival levels, respectively (Fig. 3 and Table 1). When 4 ^M /3-lapachone was added for 4 h to IdUrd- X-irradiation resulted in only a slight reduction in survival (roughly 15%) of HEp-2 cells. Thus far, several rodent (i.e., sensitized cells, DER values increased to 3.12 Â±0.04 and 3.26 Â±0.10 for 10 and 1% survival levels, respectively (Fig. 3, Table LI210, P388, and Lewis lung carcinoma) as well as human tumor cells in culture have shown similar increased radiole- 1). IdUrd-radiosensitized HEp-2 cells did not exhibit extensive thalities following /3-lapachone treatment. PLDR in 4 h following X-irradiation (Fig. 3). No comparative Nucleoside Analogue Radiosensitizer Studies. /3-Lapachone (4 difference in survival was seen in sensitized cells given no UM)was then examined for possible synergistic effects upon the posttreatment or a 4-h 0.2% dFCS-DME posttreatment (Fig. radiosensitization caused by various halogenated pyrimidines. Both deoxyuridine analogues (BrdUrd, IdUrd, CldUrd, and 2'- 3). A similar lack of PLDR in sensitized HEp-2 cells was found with all of the pyrimidine analogues tested, as evidenced by ClBrdUrd) and deoxycytidine analogues (BrdCyd, IdCyd, and CldCyd) were examined. HEp-2 cells were allowed to incorpo little change in /;. />â€žand/>â€žvalues. A series of experiments are summarized in which the effects rate the metabolites of these analogues into their DNA for 48 of /3-lapachone upon the radiosensitizing abilities of both hal h (roughly 2 cell doublings) at approximately equitoxic concen trations producing 40-70% survival of unirradiated cells (Ta ogenated deoxyuridine (Table 1) and deoxycytidine (Table 2) analogues were investigated. 0-Lapachone increased the X-ray bles 1 and 2). These cells were then X-irradiated as described in Fig. 2 and "Materials and Methods." sensitivities (in terms of increased DER values) of analogue- incorporated HEp-2 cells to levels exceeding those of cells given Fig. 3 illustrates typical dose-response curves of normal ver- no post-X-irradiation treatment. The effect of /3-lapachone ' D. A. Boothman and A. B. Pardee, unpublished data. upon analogue-radiosensitized HEp-2 cells was greater than the 5363

Table 1 Influence of 0-lapachone upon the radiosensitizing abilities of various Halogenaled deoxyuridine analogues' of values'Of(cGy)213 Drug unirradiated exposure exposure cells*(%)100 h)DME (48 (4h)None Â±7.2 Â±3.6227 Â±0.08 Â±0.06 DME DME (0.2% dFCS) 100 239 Â±5.3 Â±2.8 44 DMEBrdUrd(lOpM) /3-Lapachone)None (4 ^ M 84.3 Â±1.164.6 182 Â±8.0172 167 Â±1.2155 1.38Â±0.051.75 1.40Â±0.011.83 448

Â±4.3 Â±14 Â±4.2 Â±0.04 Â±0.02 BrdUrd(lOMM) DME (0.2% dFCS) 67.0 Â±1.2 213 Â±6.7 147 Â±8.8 1.76 Â±0.02 1.87 Â±0.01 8 BrdUrd(lOMM)IdUrd(lOMM) /3-Lapachone!None (4 uv 61.6 Â±1.659.9 98 Â±4.5128 69 Â±7.2106 3.03 Â±0.032.15 2.92 Â±0.022.10 88

Â±1.4 Â±13 Â±3.3 Â±0.01 Â±0.05 IdUrd(lOMM) DME (0.2% dFCS) 65.4 Â±1.0 138 Â±1.4 109Â± 1.1 2.00 Â±0.01 2.07 Â±0.03 8 IdUrd(lOMM)( /3-LapachoneINone (4 Â«M 55.2 Â±3.754.9 96 Â±4.8180 59 Â±2.8132 3.12Â±0.041.67 3.26Â±0.101.69 844

Ml nllllÂ»Â«\n Â±1.8 + 0.6 Â±0.6 + 0.02 Â±0.04 CldUrd(IOMM) DME (0.2% dFCS) 59.3 Â±4.5 218 Â±2.3 154 Â±2.3 1.55 Â±0.10 1.63 Â±0.06 CldUrd(lOMM)2'-CIBrdUrd /3-LapachoneMM)None (4 52.0 Â±4.944.6 127Â±9.5193 107 Â±3.2153 2.41 Â±0.031.49 2.62 Â±0.041.40 44

(0.8 mM) Â±0.5 Â±0.1 Â±2.1 Â±0.12 Â±0.14 2'-CIBrdUrd (0.8 min) DME (0.2% dFCS) 51.8 Â±1.4 187 Â±2.8 169 Â±4.9 1.50 Â±0.11 1.34 Â±0.21 4 2'-CIBrdUrd (0.8 mM)Post-X-ray/3-Lapachone (4 /IM)Viability 52.3 Â±2.6DER 159 Â±1.6ooc(cGy)195146 Â±5.210%1.101.95 Â±0.101%1.091.83 Â±0.26n"16 4 ' HEp-2 cells were plated and exposed to various pyrimidine analogue radiosensitizers as described in "Materials and Methods." * Mean Â±SE. c Mean Â±SE. Values were derived from dose-response survival curves following drug cytotoxicity normalization. '' Number of experiments in which full dose-response radiation survival curves were conducted.

Table 2 Influence of ÃŸ-lapachoneupon the radiosensitizing abilities of various Halogenaled deoxycytidine analogues'

of values'D,c Drug unirradiated exposure exposure cells*(%)50.0 (48h)BrdCyd (4h)None (cGy)150 (5 UM) Â±3.9 Â±4.2 Â±4.2 Â±0.09 Â±0.04 BrdCyd (5 MM) DME (0.2% dFCS) 53.2 Â±3.7 167 Â±9.4 103 Â±2.6 1.81 Â±0.14 1.60 Â±0.24 8 iIdCydBrdCyd (5 UM /3-LapachoneMM)None (4 48.5 Â±2.671.1 42Â±0.1114 31Â±0.292 3.41Â±0.212.04 3.51 Â±0.421.89 88

(5 MM) Â±1.872.2 Â±2.1 Â±0.1 Â±0.16 Â±0.24 IdCyd (5 MM) DME (0.2% dFCS) Â±1.7 132 Â±2.1 99 Â±1.037 1.88 Â±0.14 1.74 Â±0.05 8 IdCyd (5MM)CldCyd /3-LapachoneMM)None (4 68.6 Â±1.239.9 55Â±0.1160 Â±0.2137 3.93Â±0.141.72 3.67 Â±0.291.79 84

(5 MM) Â±2.6 Â±3.4 Â±2.1 Â±0.08 Â±0.14 CldCyd (5 MM) DME (0.2% dFCS) 41.3 Â±3.1 173 Â±1.2 147 Â±2.1 1.65 Â±0.08 1.70 Â±0.16 44 CldCyd (5 MM)Post-X-ray/3-Lapachone (4 MM)Viability39.8 Â±2.1DER 47 Â±3.2Ooc(cGy)10427 Â±1.010%1.922.81 Â±0.031%1.87 2.92 Â±0.04n"8 * HEp-2 cells were plated and exposed to pyrimidine analogues as described in "Materials and Methods." Control DME exposures were performed for each of the conditions and are presented as combined values in Table 1. * Mean Â±SE. ' Mean Â±SE. Values were derived from dose-response survival curves following drug cytotoxicity normalization. '' Number of experiments in which full dose-response radiation survival curves were conducted. product of the effects of /8-lapachone alone and halogenated following /3-lapachone exposure were greater for halogenated pyrimidine analogue alone. Only 2'-ClBrdUrd-sensitized HEp- deoxycytidine radiosensitization than for the corresponding 2 cells did not synergistically radiosensitize with /3-lapachone radiosensitization achieved using halogenated deoxyuridine an treatment (Table 1). /3-Lapachone did not greatly increase the alogues (compare values in Table 2 to those in Table 1). In cytotoxicities of unirradiated analogue-sensitized HEp-2 cells addition, the deoxycytidine analogues were found to be more (Tables 1 and 2). toxic to HEp-2 tumor cells than the corresponding deoxyuri Neither /3-lapachone nor any of the other post-X-irradiation dine analogues; relative concentrations needed to achieve equi treatments tested influenced the ^-intercept extrapolation num toxic effects were 1:2 (compare Table 1 to 2). ber; the mean value of n was 2.87 Â±0.21 and ranged from 2.52 to 3.22. /8-Lapachone did, however, decrease A> (1.4- to 5.3- fold) and Dâ€ž(1.4- to 4.0-fold) values, relative to analogue- DISCUSSION sensitized control cells, which received a post-X-irradiation This paper illustrates the potential usefulness of a DNA treatment of 0.2% dFCS-DME for 4 h (Tables 1 and 2). repair inhibitor, such as /3-lapachone, in radiation therapy. The In terms of survival kinetics, /3-lapachone seemingly influ data presented in Fig. 3 and Tables 1 and 2 demonstrate the enced the halogenated deoxycytidine analogues to a greater ability of /3-lapachone to enhance the radiolethality of HEp-2 extent than the deoxyuridine analogues. /3-Lapachone treatment cells with or without prior exposure to several halogenated (4 fiM for 4 h, post-X-irradiation) resulted in greater sensitiza- pyrimidine radiosensitizers. /3-Lapachone did not, however, tion in terms of X-ray response and decreased survival curve affect the survival of unirradiated. sensitized or unsensitized shoulder region for the deoxycytidine analogues as compared HEp-2 cells (Figs. 1 and 3). It is, therefore, reasonable to to the structurally related deoxyuridine analogues. Compara hypothesize that in vivo post-X-irradiation exposure with ÃŸ- tively lower Dq and D0 values were found for halogenated lapachone should potentiate the radiotherapy of tumors which deoxycytidine analogues than for the corresponding deoxyuri are proficient in repairing lethal damage (2, 25, 26). dine analogues. DER values at both 10 and 1% survival levels The mechanism by which /3-lapachone sensitizes cells to 5364

DNA-damaging agents is unclear. However, some inhibition After nearly 2 decades of research, radiation therapy utilizing characteristics are known (5) and point to DNA repair as the halogenated pyrimidines continues to suffer from rapid sys target of its inhibition. As shown in Fig. 1, unirradiated cells temic catabolism and a general lack of tumor selectivity. Recent are relatively unaffected by /3-lapachone. Once cells are exposed work utilizing CldCyd + tetrahydrouridine (16, 20) has, how to a DNA-damaging agent their survival is greatly decreased ever, pointed to the utilization of a halogenated deoxycytidine (Fig. 1). The fact that /3-lapachone enhanced the radiolethalities analogue to avoid premature catabolism and provide enhanced of all of the pyrimidine analogues except 2'-ClBrdUrd (Table tumor-selective radiotherapy. This radiotherapeutic approach 1) is consistent with a DNA-level mechanism of action of ÃŸ- takes advantage of: (a) quantitatively higher enzymatic levels lapachone. Previous work (20) has suggested that 2'-chloro of cytidine and deoxycytidylate deaminases in tumor tissue (30, derivatives are potent ribonucleotide reducÃaseinhibitors and 31), which allows preferential metabolism and incorporation of behave more as ribonucleotides than as deoxyribonucleotides. deoxycytidine analogues into the DNA of neoplastic tissue; (b) Unlike the other halogenated pyrimidine analogues, only a an inherent resistance of deoxycytidine analogues for the ubiq minor fraction of 2'-ClBrdUrd seems to incorporate into DNA, uitous catabolic enzymes (i.e.. uridine and thymidine phospho- unless cells have received prior /V-(phosphonacetyl)-L-aspartate rylases); and (c) the protective influence of tetrahydrouridine to and 5-fluorodeoxyuridine treatments to decrease competing inhibit systemic deamination (32,33) when coadministered with pyrimidine pools (20). The inability of /3-lapachone to enhance a halogenated deoxycytidine analogue. In this and in previous the radiosensitivity of 2'-ClBrdUrd-sensitized cells, therefore, studies, HEp-2 cells, which contain elevated levels of cytidine probably stems from a lack of incorporation of the analogue and deoxycytidylate deaminases similar to those found in many into DNA, resulting in low levels of X-ray-induced, primary malignant human neoplasms (30, 31), have been utilized to DNA lesions. mimic in vivo tumor metabolism of deoxycytidine analogues The data presented in Fig. 3 and Tables 1 and 2 demonstrate (16, 20, 21). a lack of PLDR of logarithmic-phase HEp-2 cells following Radiotherapy utilizing CldCyd + tetrahydrouridine is, how halogenated pyrimidine radiosensitization and X irradiation. ever, limited in its ability to provide effective radiosensitization; One conclusion which might be drawn from these data is that DER values of 1.2-1.8 were generally obtained (15, 16, 20). In PLDR is inhibited by halogenated pyrimidine treatment. In the present study /3-lapachone enhanced the radiolethality of contrast, Iliakis et al. (27) have shown that PLDR in confluent CIdCyd-sensitized HEp-2 cells; DER values of 1.65 Â±0.08 and arrested C3H10T1/2 mouse embryo cells, which were grown in 1.70 Â±0.16 for 10 and 1% survival levels for CIdCyd-sensitized IdUrd or BrdUrd for several days, was not inhibited. It is, HEp-2 cells were increased to 2.81 Â±0.03 and 2.92 Â±0.04, however, difficult to compare the two experimental approaches respectively. Therefore, our data indicate that the use of ÃŸ- since completely different repair conditions and cell growth lapachone in combination with CldCyd + tetrahydrouridine states were used. PLDR time-course experiments with radi- could enhance the overall therapeutic index of this agent in osensitized logarithmic-phase cells are required to determine radiation therapy against resistant, repair-competent tumors. the basis for these different findings. Recent alkaline and neu Further radiotherapeutic enhancement may result through N- tral elution studies with exponential IdUrd-sensitized V79 cells (phosphonacetyl)-L-aspartate and 5-fluorodeoxyuridine pre- have shown that repair does occur in 3 h (28). Both single and treatments to increase analogue incorporation as recently found double strand breaks did, however, persist for longer periods of with CldCyd -I-tetrahydrouridine (20). time than in unsensitized cells. It seems likely that the 4-h Previously ignored but potentially more effective deoxycyti holding period used in the present study may not have been dine analogues (for example, BrdCyd and IdCyd) could also be long enough to allow the repair of enough critical lesions utilized in similar radiotherapeutic approaches to that of required to increase survival. The apparent inhibition of PLDR CldCyd + tetrahydrouridine. Even though these deoxycytidine by halogenated pyrimidine exposure may be the result of lethal analogues have a low affinity for deoxycytidine kinase, their lesion saturation of the PLDR system; more lesions may be administration (in combination with low doses of tetrahydrour produced by radiosensitization than can be repaired by the idine to inhibit systemic deamination) could exploit elevated PLDR system in 4 h of holding. This hypothesis is strengthened cytidine deaminase levels in tumor tissue in a manner similar by the fact that a longer repair time was given to the confluent to that found with 5-trifluoromethy 1-2'-deoxycytidine (34). The arrested, IdUrd- and BrdUrd-sensitized cells (27). use of deoxycytidine analogues may be advantageous since ÃŸ- Importantly, 0-lapachone synergized with radiosensitized lapachone enhanced the radiosensitivities of these analogues to cells which could not carry out PLDR. We propose that /3- a greater extent than with the structurally related deoxyuridine lapachone converts lesions which are repaired very quickly and analogues. For example, /3-lapachone posttreatment lowered D0 do not usually result in cell death into lethal events. /3-Lapa- values 3.3-fold for BrdCyd-sensitized compared to 2.2-fold for chone may inhibit the repair of the majority of single strand BrdUrd-sensitized cells (compare Tables 1 and 2). breaks formed following X-irradiation which are repaired im mediately following damage (24, 28). We are currently investigating the tumor-selective potential ACKNOWLEDGMENTS of j3-lapachone. Like all other DNA repair inhibitors, however, The authors wish to thank Dr. Ed Bump, Joint Center For Radiation it is not clear that, by itself, a tumor-selective response can be Therapy, Harvard Medical School, for his assistance in dose-response achieved. Improved chemotherapy could, however, be achieved curve calculations and for allowing us unlimited time on the Phillips by combining halogenated pyrimidines, which are selectively X-ray machine. We also wish to thank Dr. Robert Schlegel for his metabolized and incorporated into tumor tissue, with 0-lapa- critical reviewof this manuscript. chone. For example, /3-lapachone might be used to enhance present radiation therapies [such as BrdUrd-aminopterin and REFERENCES radiation therapy (29)] which use Brdurd or IdUrd to exploit elevated iIn nudine kinase and DNA polymerase levels in tumor 1. Sieber, S. M., and Adamson, R. H. The clastogenic, mutagenic, teratogenic tissue. and carcinogenic effects of various antineoplastic agents. In: Pharmacological 5365

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. Potentiation of Halogenated Pyrimidine Radiosensitizers in Human Carcinoma Cells by β-Lapachone (3,4-Dihydro-2,2-dimethyl-2 H-naphtho[1,2-b]pyran-5,6-dione), a Novel DNA Repair Inhibitor

David A. Boothman, Sheldon Greer and Arthur B. Pardee

Cancer Res 1987;47:5361-5366.

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