No Role of Homologous Recombination in Dealing with β-LapachoneCytotoxicity in YeastOliver Quevedo,† Jonay García-Luis,† Isabel Lorenzo-Castrillejo, and Felix Machín*

Unidad de Investigacio n, Hospital Universitario Nuestra Sen ora de Candelaria, Carretera del Rosario 145, 38010 Santa Cruz deTenerife, Spain

*S Supporting Information

ABSTRACT: β-Lapachone (β-lap) is a promising antitumoral agent.DNA base oxidation and alkylation are among the expected damages by β-lap. Herein, we have explored the role that the homologousrecombination pathway (HR), a critical DNA repair process inSaccharomyces cerevisiae, has in the cytotoxic profile of β-lap. We havefurther compared β-lap to the closely related compound menadione andthe well-known alkylating agent methyl methanesulfonate (MMS).Surprisingly, we found that β-lap does not trigger HR, as seen for (i)the mutant sensitivity profiles, (ii) concentration-dependent arrestprofiles, (iii) absence of nuclear DNA repair factories, and (iv) frequency of recombination between direct repeats.

Primarily obtained from the bark of the lapacho tree(Tabebuia avellaneda), β-lapachone (β-lap) has remarkable

pharmacological properties, including antiviral, antibacterial,antifungal, antiprotozoan, and antitumor activities.1,2 β-lap iscurrently being used in clinical trials against cancer. Chemically,β-lap belongs to the naturally occurring naphthoquinone family,where we find the widely studied menadione (vitamin K3). β-lap and menadione cytotoxicity has been mainly attributed toboth their ability to generate reactive oxygen species (ROS)and the electrophilic potential of their quinone moiety.3,4 Ingeneral, the antitumor specificity of β-lap is considered to bedue to its bioactivation by the otherwise detoxifying enzymeNAD(P)H:quinone oxidoreductase (NQO1) since this enzymeis overexpressed in many tumors.5−7

Electrophilic attacks and ROS may direct or indirectly affectthe DNA, generating DNA damage. Quinones themselves canarylate DNA bases following a mechanism similar to that ofalkylating agents.4 Moreover, ROS can make other biomole-cules extremely reactive, which in turn can alkylate the DNA.8

Besides, ROS themselves or secondary species arising fromthem can oxidize the DNA bases.8 Thus, a plethora of chemicalmodifications on the bases of the DNA is expected afterincubating cells with naphthoquinones. In the case of β-lap, invitro antitopoisomerase I and II activities have been reported aswell.9,10 These antitopoisomerase activities could generatesingle and double strand breaks (SSB and DSB respectively).SSB and DSB are also expected during the repair of DNA basealkylation and oxidation through mechanisms such as the baseexcision repair (BER) pathway.11 Confounding matters, SSBscan be further converted to DSBs during DNA replication.Homologous recombination (HR) and nonhomologous endjoining (NHEJ) are the main repair pathways for DSBs. Theformer seems to be preferred in rapidly dividing cells (i.e.,cancer and yeast cells) when many DSBs arise during or after

chromosome replication.12,13 NHEJ plays a major role inhealthy mammalian cells since they normally rest in G1.To know whether a given drug with antitumor activities

generates DSBs in vivo and/or triggers HR is an important issuewith implications in the understanding of its mechanism ofaction and genotoxic safety. This is especially true for β-lap dueto its expected DNA damage profile against cancer cells.Therefore, we decided to assess whether this drug triggers HRin yeast, a model organism well suited for HR studies.12,13 Wedid so while further comparing β-lap to the closely relatednaphthoquinone menadione and the well-known DNAalkylating agent methyl methanesulfonate (MMS).We began our study by testing the relative cytotoxicity of

MMS, menadione, and β-lap in HR mutants. We employed asimple yet quantitative growth inhibition assay in the presenceof different drug concentrations (Table 1). We also includedthe ROS-generating agent hydrogen peroxide (H2O2) asanother control. Previously, all these compounds have beenshown to be cytotoxic against yeast.12−15 In our conditions, β-lap was about two times stronger than menadione in inhibitingthe growth of the wild type strain (BY4741); whereas MMSwas 10 times stronger than β-lap, and H2O2 was the weakest(Table 1). The selected set of single mutant strains for HR wasmre11Δ, rad50Δ, rad51Δ, rad52Δ, and rad54Δ. The first twomutants are at the root of the DNA damage response to DSBsand are shared between HR and NHEJ.13 The other threemutants are HR-specific, although rad51Δ does not disruptother HR-related repair mechanisms such as break-inducedreplication (BIR).13,16 HR mutants are highly hypersensitive toMMS,12,13 and as expected, MMS strongly inhibited growth inHR mutants (the growth inhibition was 10-fold in our

Received: October 26, 2011Published: November 17, 2011

Rapid Report

pubs.acs.org/crt

© 2011 American Chemical Society 2106 dx.doi.org/10.1021/tx2004618 | Chem. Res. Toxicol. 2011, 24, 2106−2108

conditions; Table 1). We found less inhibition when thenaphthoquinones were employed instead of MMS (Table 1).Interestingly, mutants shared by HR and NHEJ weremoderately hypersensitive to menadione (just 2-fold). Sincethis was not the case in HR-specific mutants; this mightindicate a possible role of NHEJ against menadionecytotoxicity. HR mutants were also slightly hypersensitive toH2O2 (but less than 2-fold). Strikingly, no mutants werehypersensitive to β-lap.Despite the surprising HR mutants profile for β-lap, we next

decided to study whether HR was activated anyway. DNAdamage that is repaired though HR triggers a checkpointresponse that arrests the cell cycle in G2/M.12,13 Accordingly,we would expect this arrest to take place under the presence ofdrugs that activate HR. Indeed, when we treated G1-synchronized cells with increasing concentrations of MMS weobserved a range of concentrations that resulted in G2/M arrest(Figure 1). Remarkably, this G2/M arrest was not seen foreither β-lap or menadione. The low percentage of cells thatseemed not to reach anaphase yet able to get out from G1 had abud size which was less than half of the mother in more than80% of the cases. This indicates that those cells stop growing involume rather than getting arrested (a G2/M arrest is seen bythe presence of yeast cells with a large bud and a single nucleus.This was the case for >80% of the S/G2/M cells in 1 μMMMS; Figure 1).Many HR proteins form nuclear factories when DNA

damage occurs.17 These factories are visible as fluorescentfoci when the proteins are tagged with GFP variants.17 We havealso used this approach to check for Rad52-YFP and Rfa1-YFPfoci after treatment with MMS, menadione, or β-lap. We havefurther employed different drug concentrations that cover therange that gave different arrest profiles in Figure 1. Wemonitored the formation of such factories for up to 3 h, takingsamples for microscopy every 10 min (data not shown). Asexpected, MMS gave an increment in the frequency of Rad52and Rfa1 foci. However, little foci (<5% of the cells) wereobserved under any condition of β-lap treatment, whereasmenadione led to an intermediate result (see Figure 2 forresults with Rfa1-YFP).Finally, we also tested whether β-lap would promote long-

term HR-dependent genetic products. Toward this goal, wemeasured the frequency of recombination between directrepeats in haploid yeast cells treated with the drug during ∼1cell cycle. Once more, we included MMS and menadione inthis analysis. We observed that in our model (SupportingInformation for details), the recombination frequency was 1.18× 10−4. This frequency barely changed after treatment with thevehicle DMSO (1.67 × 10−4). However, it did change when 3

μM MMS was used (1.5 × 10−3, ∼10-times higher than thebackground); whereas neither 100 μM menadione (6.65 ×10−5) nor 100 μM β-lap (6.67 × 10−5) increased the valuesseen with DMSO. Therefore, only MMS led to an increase inrecombination.Despite both naphthoquinones giving a much weaker HR

response than MMS, there was a slight response to menadionethat we did not observe for β-lap. We wondered whether β-lapcould in yeast produce less ROS than menadione and thereforeless DNA damage. Thus, we measured ROS production afterdrug treatment by using the fluorescent probe 2′,7′-dichloro-fluorescin diacetate (DCFH-DA).18 We found not only thatthere was more ROS production after β-lap treatment but alsothat ROS lasted for longer (Figure S1, Supporting Informa-tion), a result that fits well with β-lap entering the NQO1 futilecycle.5

In conclusion, we have measured the contribution of HR inovercoming cytotoxicity by MMS, menadione, and β-lap.

Table 1. Sensitivity Profiles of Homologous Recombination Mutants to MMS, Menadione, β-lap, and H2O2a

strain MMS menadione β-lap H2O2

BY4741 1.464 ± 0.556 18.99 ± 4.25 10.03 ± 5.69 3.46 ± 0.54mre11Δ 0.159 ± 0.041* 7.13 ± 3.05* 15.22 ± 1.30 1.92 ± 0.54*rad50Δ 0.110 ± 0.048* 5.58 ± 0.64* 9.21 ± 5.43 1.87 ± 0.32*rad51Δ 0.120 ± 0.027* 13.41 ± 8.97 6.78 ± 3.65 2.00 ± 0.42*rad52Δ 0.137 ± 0.005* 19.99 ± 8.85 7.33 ± 0.20 2.12 ± 0.53*rad54Δ 0.210 ± 0.045* 15.84 ± 7.18 7.63 ± 2.52 3.22 ± 0.39

aExperimental details can be found in Supporting Information. GI50 values (mean ± SD) are in μM except for H2O2 (mM). The number ofindependent experiments was 3 except for the reference strain BY4741 (n = 6 for MMS, n = 8 for menadione, n = 10 for β-lap, and n = 3 for H2O2).Statistical significance of mean differences between the mutants and the reference strain was estimated by one-way ANOVA followed by Tukey’spost-test (* denotes p < 0.05).

Figure 1. Cell cycle arrest profiles under increasing concentrations ofMMS, menadione, or β-lapachone. Strain FM149 (cdc15-2) was firstsynchronized in G1 for 3 h at 25 °C. Then, the culture was split, anddrugs were added at the indicated concentrations at the same time thatcells were allowed to enter a new cell cycle at 37 °C (i.e., cells areprevented to go beyond anaphase and perform cytokinesis because ofthe cdc15-2 thermosensitive mutation). After 4 h, samples were taken,observed under the microscope, and sorted according to the cell cyclestage they were in. Cells reached anaphase (budded and binucleatedcells) when unperturbed by the drug; whereas an S/G2/M phenotype(budded with a single nucleus) might indicate a G2/M arrest (see textfor more details). Very high concentrations of any drug precluded cellsfrom getting out of G1 (no new bud was seen).

Chemical Research in Toxicology Rapid Report

dx.doi.org/10.1021/tx2004618 | Chem. Res. Toxicol. 2011, 24, 2106−21082107

Altogether, we could correlate profiles of mutant sensitivities,cell cycle arrests, nuclear DNA repair factories, and HR-dependent genetic products to the important role that the HRhas in dealing with the MMS cytotoxicity, whereas this rolegreatly diminishes for menadione, and it seems nonexistent forβ-lap. This latter conclusion points out that the antitumoro-genic properties of β-lap are likely not related to the drug beingable to generate DSB, arrest cells in G2/M, and/or trigger HR.This is so despite the fact that β-lap is able to produce highlevels of ROS in yeast. Noticeably, a previous work in yeast alsotreated with β-lap reported that the drug is able to cause DNAdamage, although without activation of the G2/M check-points.14 Rather, β-lap triggered a G1/S checkpoint. Interest-ingly, we also observed a dose-dependent G1-arrest for β-lap(Figure 1) but no signs of a DNA damage response.Finally, our observations raise interesting questions of why

such a powerful oxidizing agent does not cause a strong DNAdamage response in yeast at concentrations sufficient forcytotoxicity. A similar scenario has been shown recently inhuman cells.19 It would be interesting to explore in the future ifthe presence of ROS (and/or the concomitant side effects ofthe β-lap futile cycle on the cell’s reducing power and energy)changes the way cells activate the DNA damage response.

■ ASSOCIATED CONTENT*S Supporting InformationExperimental procedures and supplementary Figures S1, S2,and Table S1. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +34 922 602 951. Fax: +34 922 600 562. E-mail:fmacconw@gmail.com.

Author Contributions†These authors contributed equally to this work.

FundingThis work was supported by grants from the Spanish Ministryof Science and “Instituto de Salud Carlos III” (PS 09/00106);Spanish Ministry of Education (AP2009-2511 to J.G.-L.) and“Agencia Canaria de Investigacion, Innovacio n y Sociedad de laInformacion” (BOC-A-2010-023-596 to O.Q.).

■ ACKNOWLEDGMENTSWe thank R. Rothstein for strains.

■ ABBREVIATIONSβ-lap, β-lapachone; BER, base excision repair; BIR, break-induced replication; DAPI, 4′,6-diamidino-2-phenylindole;DCFH-DA, 2′,7′-dichlorofluorescin diacetate; DMSO, dimethylsulfoxide; DSB, double strand break; G1, Gap1 cell cycle stage;G2/M, Gap2/Metaphase cell cycle stage; GFP, greenfluorescent protein; H2O2, hydrogen peroxide; HR, homolo-gous recombination pathway; MMS, methyl methanesulfonate;NHEJ, nonhomologous end joining; NQO1, NAD(P)-H:quinone oxidoreductase; ROS, reactive oxygen species; S/G2/M, S-phase/Gap2/metaphase cell cycle stages; SSB, singlestrand break; YFP, yellow fluorescent protein

■ REFERENCES(1) Pardee, A. B., Li, Y. Z., and Li, C. J. (2002) Curr. Cancer DrugTargets 2, 227−242.(2) Go mez Castellanos, J. R., Prieto, J. M., and Heinrich, M. (2009) J.Ethnopharmacol. 121, 1−13.(3) Powis, G. (1987) Pharmacol. Ther. 35, 57−162.(4) Brunmark, A., and Cadenas, E. (1989) Free Radical Biol. Med. 7,435−477.(5) Pink, J. J., Planchon, S. M., Tagliarino, C., Varnes, M. E., Siegel,D., and Boothman, D. A. (2000) J. Biol. Chem. 275, 5416−5424.(6) Reinicke, K. E., Bey, E. A., Bentle, M. S., Pink, J. J., Ingalls, S. T.,Hoppel, C. L., Misico, R. I., Arzac, G. M., Burton, G., Bornmann, W.G., Sutton, D., Gao, J., and Boothman, D. A. (2005) Clin. Cancer Res.11, 3055−3064.(7) Bey, E. A., Bentle, M. S., Reinicke, K. E., Dong, Y., Yang, C.-R.,Girard, L., Minna, J. D., Bornmann, W. G., Gao, J., and Boothman, D.A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 11832−11837.(8) Marnett, L. J., Riggins, J. N., and West, J. D. (2003) J. Clin. Invest.111, 583−593.(9) Li, C. J., Averboukh, L., and Pardee, A. B. (1993) J. Biol. Chem.268, 22463−22468.(10) Frydman, B., Marton, L. J., Sun, J. S., Neder, K., Witiak, D. T.,Liu, A. A., Wang, H. M., Mao, Y., Wu, H. Y., Sanders, M. M., and Liu,L. F. (1997) Cancer Res. 57, 620−627.(11) Demple, B., and Harrison, L. (1994) Annu. Rev. Biochem. 63,915−948.(12) Paques, F., and Haber, J. E. (1999) Microbiol. Mol. Biol. Rev. 63,349−404.(13) Symington, L. S. (2002) Microbiol. Mol. Biol. Rev. 66, 630−670.(14) Menacho-Marquez, M., and Murguía, J. R. (2006) Cell cycle 5,2509−2516.(15) Castro, F. A. V., Mariani, D., Panek, A. D., Eleutherio, E. C. A.,and Pereira, M. D. (2008) PloS One 3, e3999.(16) Kraus, E., Leung, W. Y., and Haber, J. E. (2001) Proc. Natl. Acad.Sci. U.S.A. 98, 8255.(17) Lisby, M., Barlow, J. H., Burgess, R. C., and Rothstein, R. (2004)Cell 118, 699−713.(18) LeBel, C. P., Ischiropoulos, H., and Bondy, S. C. (1992) Chem.Res. Toxicol. 5, 227−231.(19) Cavalcanti, B. C., Barros, F. W. A., Cabral, I. O., Ferreira, J. R.O., Magalhaes,, H. I. F., Junior, H. V. N., da Silva Junior, E. N., deAbreu, F. C., Costa, C. O., Goulart, M. O. F., Moraes, M. O., andPessoa, C. (2011) Chem. Res. Toxicol. 24, 1560−1574.

Figure 2. Formation of Rfa1 DNA repair factories after treatment withMMS, menadione, or β-lapachone. Strain W3775-12C (RFA1-YFP)was treated with the indicated drugs while asynchronously growing at25 °C. Representative photos of samples taken after 1 and 3 h areshown. MMS led to a massive Rfa1-YFP nuclear signal in all buddedcells. Menadione led to about 50% of the budded cells with at least onenuclear focus by 3 h. Less than 5% of foci were observed aftertreatment with β-lapachone.

Chemical Research in Toxicology Rapid Report

dx.doi.org/10.1021/tx2004618 | Chem. Res. Toxicol. 2011, 24, 2106−21082108