ANTICANCER RESEARCH 30: 291-294 (2010)

Selenomethionine or Methylseleninic Acid Inhibits Mutagenesis of a Reporter Gene in Mouse Bone Marrow

MA SURESH KUMAR1, KAREN E. POLLOK2 and MARTIN L. SMITH1

1Indiana University School of Medicine, Department of Microbiology and Indiana University Simon Cancer Center, Indianapolis, IN 46202 U.S.A.; 2Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.

Abstract. Recent laboratory and clinical studies have utilized now a subject of human clinical trials (1). Secondly, in mice, selenium in the form of pure seleno-L-methionine (SeMet) in the maximum tolerated dose (MTD) of drugs combination with DNA-damaging cancer chemotherapy drugs. was doubled and even tripled when combined with SeMet In mice, the selenium protected bone marrow and other tissues (2). Thirdly, distinct anticancer effects are attributed to from dose-limiting toxicity. In fact, because of the protection SeMet either alone or in combination with chemotherapy from dose-limiting toxicity, a doubling or even tripling of the drugs in both human and mouse studies (1-4). maximum tolerated dose (MTD) was enabled. Previously we Cells possess a baseline capacity to repair DNA damage showed that SeMet protects bone marrow by a DNA repair such as that caused by or carboplatin, important mechanism that requires the XPC DNA repair protein. XPC is chemotherapy drugs used in the treatment of many types of rate-limiting and is required for repair of cisplatin or cancer. In each of the experimental conditions referred to carboplatin adducts. Herein we used a mouse strain that above, selenium pretreatment was required in order to carries a lambda phage reporter gene in the genome that modulate the effects of subsequent chemotherapy drugs. We serves as a mutagenesis target. SeMet protects from reported previously that SeMet enhanced DNA repair of carboplatin mutagenesis in mouse bone marrow. carboplatin-induced DNA damage in mouse bone marrow Methylseleninic acid (MSA), a metabolite of SeMet, also and fibroblasts. SeMet did not enhance DNA repair in cancer protected against mutagenesis in mouse bone marrow. cell lines. Thus, the SeMet promoted a selective DNA repair response that favored cell survival in bone marrow (5, 6). Agents which enhance the selectivity of cancer Here, we used a mouse strain that carries a lambda phage chemotherapy have been eagerly sought. Typically, dose- reporter gene in the genome. One can treat cells with a limiting toxicity to bone marrow and other tissues is an DNA-damaging agent, prepare genomic DNA, then rescue impediment to successful chemotherapy. Numerous wild-type or mutant lambda phage with the use of in vitro approaches have included agents that protect bone marrow, phage packaging extracts. The frequency of mutant phage or agents that sensitize cancer cells. An agent that did both, can be used to calculate mutant frequency. Pretreatment with protect bone marrow and sensitize cancer cells would be SeMet or MSA decreased the mutant frequency in mouse highly valuable. Studies in mice have shown that seleno-L- bone marrow cultures. methionine (SeMet) holds promise as such an agent. Firstly, it is well-tolerated and does not cause toxicity by itself. It is Materials and Methods

Mouse-derived bone marrow and fibroblast cultures. All mouse Abbreviations: XPC, protein encoded by the xeroderma treatments were in accordance with protocols approved by the pigmentosum Xpc gene; NER, nucleotide excision DNA repair. Institutional Animal Care and Use Committee at Indiana University. Female Big Blue mice were purchased from Stratagene (La Jolla, Correspondence to: Martin L. Smith, Ph.D., Indiana University CA, USA). Bone marrow was harvested from mice at 10 weeks of Cancer Center, 980 W. Walnut Street, Room C547, Indiana age and placed in tissue culture in IMDM liquid medium (Sigma, University School of Medicine, Indianapolis, IN 46202, U.S.A. St. Louis, MO, USA), 20% fetal bovine serum, interleukin-6 (200 e-mail: [email protected] units per mL), and stem cell factor 100 ng per mL (Stem Cell Technologies, Vancouver, Canada). Bone marrow cultures were Key Words: DNA repair, cisplatin, carboplatin, nucleotide excision treated with selenium compounds 15 h, then with indicated doses repair, selenium. of carboplatin.

0250-7005/2010 $2.00+.40 291 ANTICANCER RESEARCH 30: 291-294 (2010)

Figure 1. XPC is critical in protection of bone marrow from carboplatin. Bone marrow primary cultures of wild-type or Xpc–/– mice were used. Figure 2. Effects of SeMet or MSA on mutagenesis of a reporter gene Cultures were pre-treated with SeMet, 15 μM, 15 hrs, then with in mouse bone marrow. Primary mouse bone marrow was cultured in carboplatin as indicated for 3 hrs. Colonys formed in methylcellulose cytokine-containing medium and treated with SeMet, 15 μM, 15 h, medium were counted 10 days later. SeMet protected wild-type bone immediately after harvest from Big Blue mice. Where indicated, marrow (p=0.05), but not Xpc–/– bone marrow. As Xpc–/– bone marrow is carboplatin was added to the medium, 100 μM, 3 h. Cells were grown 10-fold more sensitive to carboplatin, irrespective of SeMet, we adjusted an additional 48 h to ensure replication and hence fixation of . the carboplatin concentration to be approximately equitoxic in both After 48 h, genomic DNA was prepared from the cultures and the phage genotypes. SeMet still failed to protect Xpc–/– bone marrow. Actual rescued by a lambda packaging extract. After plating on a lawn of E. numbers of colonies are shown. coli G1250, duplicate plates were incubated at 24˚C and 37˚C, and plaques counted manually by a colony counter. The relative mutant frequency is the number of plaques formed at the non-permissive temperature of 24˚C, divided by the number of plaques formed at the Selenium and carboplatin treatments. In mice and in human patients permissive temperature of 37˚C (p<0.01 comparing lanes 2 and 4, or receiving SeMet, a serum selenium concentration of 15 μM was lanes 2 and 6). obtained (1). A fraction of SeMet is however metabolized to methylselenol, that fraction being variable among tissues (2). Serum selenium concentrations do not allow the identification of biochemical form after SeMet metabolism. Therefore, we used either 15 μM treatment (Figure 1). The relevance of XPC was shown by SeMet, to parallel the in vivo studies, or 1 μM MSA, to mimic the the use of bone marrow derived from Xpc–/– mice (6). Xpc–/– metabolite. Both selenium compounds are water soluble and were bone marrow was sensitive to carboplatin and was not kept frozen as 15 mM stock solutions in sterile water. Cell cultures rescued by SeMet, even when the carboplatin dose was were treated 15 h with SeMet or MSA. For some experiments as corrected to be approximately equitoxic in wild-type and indicated, SeMet or MSA treatment was followed by carboplatin to –/– induce DNA damage, at concentrations and duration indicated. Xpc bone marrow (Figure 1). Thus, Figure 1 shows that XPC is important to the SeMet protective mechanism. Xpc–/– Big Blue mutagenesis assays. The Big Blue mouse carries a lambda bone marrow lacked the protective response. LIZ phage reporter gene integrated in the genome. After treatments A prediction that follows is that if a bona fide DNA repair of Big Blue cell cultures, genomic DNA was prepared using Qiagen response is induced, then SeMet or its metabolite MSA would columns with reagents supplied by the manufacturer (Qiagen, inhibit mutagenesis. As our interest is in protecting bone Valencia, CA, USA) and packaged into lambda phage using marrow, we tested if SeMet or MSA would inhibit mutagenesis Stratagene Gigapack packaging extracts. Plaques were visible on a lawn of E Coli G1250 (Stratagene) after culture at 37˚C overnight. in bone marrow, using carboplatin as a DNA-damaging agent. The lambda LIZ system carries a temperature-sensitive allele of the As is shown in Figure 2, SeMet (15 μM, 15- h treatment) lambda cII gene. As such, all plaque-forming units can form plaques or MSA (1 μM, 15-h treatment) significantly inhibited at the permissive temperature of 37˚C. However, only mutant plaques mutagenesis in bone marrow (Figure 2). can grow at the non-permissive temperature of 24˚C. Therefore, the ratio of the number of plaques at 24˚C to the number of plaques at Discussion 37˚C provides a measure of mutant frequency of the cII gene as a target. Plaques were counted manually using a colony counter. In mice, SeMet has been shown to protect bone marrow Results and gut epithelium from cancer chemotherapy drugs. In fact, the MTD for chemotherapy drugs was increased by The DNA repair protein XPC is relevant to the protective 2-3-fold in SeMet-treated mice (2). At the same time, mechanism. SeMet treatment of bone marrow cells of wild- xenograft tumors were cured in SeMet-treated mice, type mice resulted in increased cell survival after carboplatin tumors that did not grow back even months post-treatment.

292 Kumar et al: Selenium and Mutagenesis

While the molecular basis for the observed selectivity is Acknowledgements probably complex, we have focused on a selective DNA repair response involving XPC. This work was supported by NIH R01 HL086978 to M.L.S. We showed previously that p53 and a p53-regulated DNA repair protein XPC can contribute to the observed selectivity. Conflict of Interest Statement Specifically, bone marrow, gut epithelium, and other dose- limiting tissues have wild-type p53 genes and SeMet The Authors declare that there are no conflicts of interest. treatment results in elevated XPC protein and elevated DNA repair of cisplatin or carboplatin DNA damage (5). References Conversely, the majority of cancer cells carry somatically- acquired p53 mutations, rendering p53, and the elevation of 1 Fakih MG, Pendyala L, Brady W, Smith PF, Ross ME, Creaven XPC protein inactive. Thus, SeMet enhanced DNA repair in PJ, Badmaev V, Prey JD and Rustum YM: A phase I and pharmacokinetic study of selenomethionine in combination with bone marrow and gut, but did not enhance DNA repair in a fixed dose of irinotecan in solid tumors. Cancer Chemother cancer cells (5). Pharmacol 62: 499-508, 2008. The importance of the XPC protein in protecting bone 2 Cao S, Durrani FA and Rustum YM: Selective modulation of the marrow is evident by our experiments utilizing Xpc–/– mice. therapeutic efficacy of anticancer drugs by selenium-containing In wild-type mice, the bone marrow is protected by SeMet compounds against human tumor xenografts. Clin Cancer Res (Figure 1), but Xpc–/– bone marrow cannot be rescued by 10: 2561-2569, 2004. SeMet and is in fact quite sensitive to carboplatin 3 Sieja K and Talerczyk M: Selenium as an element in the treatment of ovarian cancer in woman receiving chemotherapy. irrespective of whether SeMet is used (6). Thus, cancer cells 93 –/– Gynecol Oncol : 320-327, 2004. that carry alterations in Xpc genes might, like the Xpc 4 Fakih M, Cao S, Durrani FA and Rustum YM: Selenium protects mouse, exhibit marked sensitivity to carboplatin. against toxicity induced by anticancer drugs and augments antitumor Surprisingly, this study is the first to test the hypothesis activity: a highly selective, new, and novel approach to the treatment that selenium would inhibit mutagenesis. The literature is of solid tumors. Clin Colorectal Cancer 5: 132-135, 2005. complicated by the use of different selenium compounds, but 5 Fischer JL, Mihelc E, Pollok KE and Smith ML: we used SeMet because that is the form widely used in Chemotherapeutic selectivity conferred by selenium: a role for p53-dependent DNA repair. Mol Cancer Ther 6: 355-361, 2007. clinical trials for both cancer prevention and in combination 6 Fischer JL, Kumar MA, Day TW, Hardy TM, Hamilton S, with chemotherapy (1, 3, 7). SeMet decreased Besch-Williford C, Safa AR, Pollok KE and Smith ML: The Xpc frequency in the lambda phage plaque-forming assay (Figure gene markedly affects cell survival in mouse bone marrow. 2). MSA, a metabolite of SeMet, likewise decreased Mutagenesis 24: 309-316, 2009. mutation frequency. Thus the mechanism is shared between 7 Reid ME, Diffield-Lillico AJ, Slate E, Natarajan N, Turnbull B, the two compounds. Jacobs E, Combs GF Jr, Alberts DS, Clark LC and Marshall JR: The mechanisms we have shown pertain to XPC protein The nutritional prevention of cancer: 400 μg per day selenium treatment. Nutr Cancer 60: 155-163, 2008. and global nucleotide excision DNA repair specifically. We 8 Ebert R, Ulmer M, Zeck S, Meissner-Weigl J, Schneider D, would caution against extrapolation to other classes of DNA Stopper H, Schupp N, Kassem M and Jakob F: Selenium damage. Our data pertain to cisplatin, carboplatin, nitrogen supplementation restores the antioxidative capacity and prevents mustards including melphalan, and possibly oxaliplatin. cell damage in bone marrow stromal cells in vitro. Stem Cells Although other authors have shown selenium protective 24: 1226-1235, 2006. effects in bone marrow against, for example (8), the mechanism we show here may not be Received January 15, 2010 applicable to those findings. Accepted January 22, 2010

293