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No Role of Homologous Recombination in Dealing with β-Lapachone Cytotoxicity in Yeast Oliver Quevedo,† Jonay García-Luis,† Isabel Lorenzo-Castrillejo, and Félix Machín* Unidad de Investigación, Hospital Universitario Nuestra Señora de Candelaria, Carretera del Rosario 145, 38010 Santa Cruz de Tenerife, 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 βlap. Herein, we have explored the role that the homologous recombination pathway (HR), a critical DNA repair process in Saccharomyces cerevisiae, has in the cytotoxic profile of β-lap. We have further compared β-lap to the closely related compound menadione and the 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 arrest profiles, (iii) absence of nuclear DNA repair factories, and (iv) frequency of recombination between direct repeats.
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chromosome replication.12,13 NHEJ plays a major role in healthy 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 issue with implications in the understanding of its mechanism of action and genotoxic safety. This is especially true for β-lap due to its expected DNA damage profile against cancer cells. Therefore, we decided to assess whether this drug triggers HR in yeast, a model organism well suited for HR studies.12,13 We did so while further comparing β-lap to the closely related naphthoquinone menadione and the well-known DNA alkylating agent methyl methanesulfonate (MMS). We began our study by testing the relative cytotoxicity of MMS, menadione, and β-lap in HR mutants. We employed a simple yet quantitative growth inhibition assay in the presence of different drug concentrations (Table 1). We also included the ROS-generating agent hydrogen peroxide (H2O2) as another control. Previously, all these compounds have been shown to be cytotoxic against yeast.12−15 In our conditions, βlap was about two times stronger than menadione in inhibiting the growth of the wild type strain (BY4741); whereas MMS was 10 times stronger than β-lap, and H2O2 was the weakest (Table 1). The selected set of single mutant strains for HR was mre11Δ, rad50Δ, rad51Δ, rad52Δ, and rad54Δ. The first two mutants are at the root of the DNA damage response to DSBs and are shared between HR and NHEJ.13 The other three mutants are HR-specific, although rad51Δ does not disrupt other HR-related repair mechanisms such as break-induced replication (BIR).13,16 HR mutants are highly hypersensitive to MMS,12,13 and as expected, MMS strongly inhibited growth in HR mutants (the growth inhibition was 10-fold in our
rimarily 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 is currently 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). βlap and menadione cytotoxicity has been mainly attributed to both their ability to generate reactive oxygen species (ROS) and the electrophilic potential of their quinone moiety.3,4 In general, the antitumor specificity of β-lap is considered to be due to its bioactivation by the otherwise detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1) since this enzyme is overexpressed in many tumors.5−7 Electrophilic attacks and ROS may direct or indirectly affect the DNA, generating DNA damage. Quinones themselves can arylate DNA bases following a mechanism similar to that of alkylating agents.4 Moreover, ROS can make other biomolecules extremely reactive, which in turn can alkylate the DNA. 8 Besides, ROS themselves or secondary species arising from them can oxidize the DNA bases.8 Thus, a plethora of chemical modifications on the bases of the DNA is expected after incubating cells with naphthoquinones. In the case of β-lap, in vitro antitopoisomerase I and II activities have been reported as well.9,10 These antitopoisomerase activities could generate single and double strand breaks (SSB and DSB respectively). SSB and DSB are also expected during the repair of DNA base alkylation and oxidation through mechanisms such as the base excision repair (BER) pathway.11 Confounding matters, SSBs can be further converted to DSBs during DNA replication. Homologous recombination (HR) and nonhomologous end joining (NHEJ) are the main repair pathways for DSBs. The former seems to be preferred in rapidly dividing cells (i.e., cancer and yeast cells) when many DSBs arise during or after © 2011 American Chemical Society
Received: October 26, 2011 Published: November 17, 2011 2106
dx.doi.org/10.1021/tx2004618 | Chem. Res. Toxicol. 2011, 24, 2106−2108
Chemical Research in Toxicology
Rapid Report
Table 1. Sensitivity Profiles of Homologous Recombination Mutants to MMS, Menadione, β-lap, and H2O2a strain BY4741 mre11Δ rad50Δ rad51Δ rad52Δ rad54Δ
MMS 1.464 0.159 0.110 0.120 0.137 0.210
± ± ± ± ± ±
0.556 0.041* 0.048* 0.027* 0.005* 0.045*
menadione
β-lap
18.99 ± 4.25 7.13 ± 3.05* 5.58 ± 0.64* 13.41 ± 8.97 19.99 ± 8.85 15.84 ± 7.18
10.03 ± 5.69 15.22 ± 1.30 9.21 ± 5.43 6.78 ± 3.65 7.33 ± 0.20 7.63 ± 2.52
H2O2 3.46 1.92 1.87 2.00 2.12 3.22
± ± ± ± ± ±
0.54 0.54* 0.32* 0.42* 0.53* 0.39
a
Experimental details can be found in Supporting Information. GI 50 values (mean ± SD) are in μM except for H2O2 (mM). The number of independent 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’s post-test (* denotes p < 0.05).
conditions; Table 1). We found less inhibition when the naphthoquinones were employed instead of MMS (Table 1). Interestingly, mutants shared by HR and NHEJ were moderately hypersensitive to menadione (just 2-fold). Since this was not the case in HR-specific mutants; this might indicate a possible role of NHEJ against menadione cytotoxicity. HR mutants were also slightly hypersensitive to H2O2 (but less than 2-fold). Strikingly, no mutants were hypersensitive to β-lap. Despite the surprising HR mutants profile for β-lap, we next decided to study whether HR was activated anyway. DNA damage that is repaired though HR triggers a checkpoint response that arrests the cell cycle in G2/M.12,13 Accordingly, we would expect this arrest to take place under the presence of drugs that activate HR. Indeed, when we treated G1synchronized cells with increasing concentrations of MMS we observed a range of concentrations that resulted in G2/M arrest (Figure 1). Remarkably, this G2/M arrest was not seen for either β-lap or menadione. The low percentage of cells that seemed not to reach anaphase yet able to get out from G1 had a bud size which was less than half of the mother in more than 80% of the cases. This indicates that those cells stop growing in volume rather than getting arrested (a G2/M arrest is seen by the 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 μM MMS; Figure 1). Many HR proteins form nuclear factories when DNA damage occurs.17 These factories are visible as fluorescent foci when the proteins are tagged with GFP variants. 17 We have also used this approach to check for Rad52-YFP and Rfa1-YFP foci after treatment with MMS, menadione, or β-lap. We have further employed different drug concentrations that cover the range that gave different arrest profiles in Figure 1. We monitored the formation of such factories for up to 3 h, taking samples for microscopy every 10 min (data not shown). As expected, MMS gave an increment in the frequency of Rad52 and Rfa1 foci. However, little foci (
