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Selenomethionine induction of DNA repair response in human fibroblasts

Abstract

Selenium compounds have a long history in chemoprevention of mammary and colon cancers in rodent models. Selenium compounds are in current clinical trials, having shown promise in prevention of prostate and other human cancers. In human tissues, it has been estimated that each cell sustains approximately 10 000 potentially mutagenic (if not repaired) lesions per day due to endogenous DNA damage. Almost no studies have addressed the potential for selenium compounds to induce DNA repair, a potential mechanism for their cancer-preventive actions. We show that selenium in the form of selenomethionine induces a DNA repair response in normal human fibroblasts in vitro, and protects cells from DNA damage. We show a possible mechanism for the inducible DNA repair response, in which enhanced repair complex formation was observed in selenomethionine-treated cells.

Introduction

Selenium compounds

The cancer chemopreventive properties of selenium (Se) have been evidenced by studies conducted over the past 20 years, mainly with rodent models of mammary carcinogenesis (Ip, 1981). Both organic and inorganic forms of Se have been used. The latter, the prototype form being sodium selenite, suffer from limitations associated with metabolic conversion to hydrogen selenide, generation of DNA strand breaks, and cytotoxicity (Sinha et al., 1996; Lu et al., 1995; Ip et al., 1994a). In contrast, organic forms of Se are relatively nontoxic (Ip et al., 1994a; Lu et al., 1995; Sinha et al., 1996; Patterson and Levander, 1997; Stewart et al., 1999), and can be administered orally. A phase I clinical trial in the US (Patterson and Levander, 1997) indicated no harmful effects due to long-term intakes of organic (dietary) Se as mixed naturally-occurring compounds in excess of 200 μg/day, amounting to 3–4 times the US Food and Drug Administration (FDA) recommended daily allowance (RDA; Clark et al., 1996). Current clinical trials holds promise in applying Se to prostate cancer chemoprevention (Nelson et al., 1999).

The inhibition of mammary carcinogenesis in rodent models was shown by chronically feeding the animals Se-enriched diets. Brazil nuts represent a naturally rich source of dietary Se, containing 16–30 μg Se per gram, and when included in a diet consumed ad libitum were found to inhibit rat mammary carcinogenesis induced by dimethylbenz[a]anthracene (DMBA; Ip and Lisk, 1994). Dietary supplementation with Brazil nuts (Ip and Lisk, 1994) or with the organic compounds triphenylselenonium chloride (Ip et al., 1994b) or selenomethionine (Ip et al., 1999) at levels up to 200 p.p.m. Se had no adverse effects, and a Se dietary level of 30 p.p.m. reduced the total tumor yield by up to 70% when fed during the DMBA post-initiation phase (Ip et al., 1994b, 1998, 1999). These studies and others established organic Se compounds as effective anticancer agents, with a definite separation between preventive and toxic dose ranges (Ip et al., 1994a). We chose selenomethionine (SeMet) for our studies, being the major component of Se-enriched foods, relatively non-toxic, and can be applied to cells in culture (Redman et al., 1998). In addition to mammary carcinogenesis, organoselenium compounds have been applied to the prevention of colon carcinogenesis in rodents. Dietary SeMet supplementation of 2 p.p.m. resulted in a marked reduction of the number of aberrant crypt foci in an azoxymethane-induced model (Baines et al., 2000).

Despite the body of literature documenting the cancer preventing properties of Se, there is less knowledge of the mechanism(s) whereby this agent inhibits carcinogenesis. In this study, we show that SeMet induces a DNA repair response and protects normal human fibroblasts from DNA damage. The induction of DNA repair occurs at a concentration (10 μM) easily attainable in vivo by even modest dietary supplementation (Clark et al., 1996), and would be predicted to decrease the load of mutations associated with carcinogenesis initiation and progression.

Results

Enhancement of DNA repair measured by alkaline comet assays

The assay relies on the persistence of unligated repair patches that are alkali-labile, and are eluted from nuclei by alkaline electrophoresis (Seo et al., 2002). The relative number of incompletely repaired sites is reflected in the mean fragment length of genomic DNA eluted from nuclei under alkaline conditions. The tail moment is a relative measure of the mean fragment length, that we have determined through the use of confocal image analysis and NIH software (Seo et al., 2002; and Figure 1a). Treatment of human fibroblasts with SeMet alone (up to 100 μM) did not produce detectable DNA damage in the comet assay (Figure 1b). This was important to ascertain, because some Se compounds, in contrast to SeMet, can by themselves produce DNA damage (discussed in Introduction). In cells treated with SeMet, then challenged with UV-radiation to induce DNA damage, SeMet was found to decrease the number of UV-damaged sites (Figure 1b).

Figure 1
figure5

SeMet enhanced repair of UV-damage as assayed by alkaline comet electrophoresis. Cells (normal human fibroblasts) were treated or not with SeMet, then treated or not with UV-radiation at doses indicated. The number of damage-associated strand breaks is reflected in the electrophoretic mobility of genomic DNA eluted from each nucleus, which is linear over a wide range of UV-doses. The amount of DNA in the comet tails was calculated using digital analysis software. UV-induced DNA damage was decreased in cells treated with SeMet, reflected enhancement of DNA repair. Cells not treated with UV-radiation showed little or no DNA damage, irrespective of the presence or absence of SeMet, indicating that SeMet did not by itself cause any detectable DNA damage. The data are expressed relative to the initial amount of UV-damage, which was determined by treatment immediately after UV-irradiation with E. Coli endonuclease III or Schizosaccharomyces pombe UV-cutter enzymes (Trevigen, Inc.) which cleave UV-damaged sites independently of the cellular DNA repair capability. (a) Illustration of the methodology; (b) Quantification of the data, derived from human fibroblasts treated or not with SeMet, and damaged or not by UV-irradiation. Each bar of the bar graph represents 600 or more individual cells, in which data were averaged from three independent determinations of 200 cells per determination, and a subset of 50 cells were analysed by NIH software. P<0.01 for the third and fourth bars of the graph (t-test using SigmaPlot software)

Enhancement of DNA repair measured by host-cell reactivation (HCR)

An independent assay was used in order to substantiate the observed DNA repair response in SeMet-treated cells. We have used HCR assays previously, in which a UV-irradiated reporter plasmid encoding chloramphenicol acetyltransferase (CAT) is transfected into the host cell line, then transient expression of the CAT reporter gene is assayed (Smith et al., 1995; Ganesan et al., 1999). The reporter gene contains approximately 20 UV-induced cyclobutane pyrimidine dimers, that block transcription by DNA polymerase II (Smith et al., 1995). Consequently, the plasmid is fully inactive in DNA repair-defective cells, e.g. those of xeroderma pigmentosum patients (Smith et al., 1995). When introduced into normal cells, its reactivation is dependent upon DNA repair by the host cell line (Ganesan et al., 1999). Cells were first transfected with the UV-damaged reporter plasmid, then treated or not with SeMet. Treatment with SeMet resulted in an approximate threefold enhancement of DNA repair (Figure 2), consistent with our other findings (Figure 1).

Figure 2
figure4

SeMet enhancement of DNA repair as measured by host-cell reactivation of a UV-damaged reporter gene. Cells were transfected with the UV-damaged CAT reporter plasmid, incubated for 24 h under normal growth conditions, then treated with 10 μM SeMet for 15 h. Cells were grown for an additional 24 h prior to determination of CAT enzyme activity. Host-cell reactivation was enhanced by 2–3-fold in SeMet-treated cells compared to untreated cells; (a) raw data derived from ELISA of UV-damaged CAT reporter assay, in cells treated with 0–40 μM SeMet; (b) mean±s.d. from five independent experiments. P<0.01 by Wilcoxon rank-sum test

Protection of cells from DNA damage

Given evidence for SeMet induction of a DNA repair response, we tested whether SeMet would protect human fibroblasts from DNA damage. Cells were treated or not with SeMet, then treated or not with DNA-damaging agents UV-radiation or hydrogen peroxide. Cell survival was measured in 12-day clonogenic assays (Smith et al., 1995). SeMet protected cells from DNA damage induced by either agent (Figure 3). SeMet protected cells from DNA damage induced by either agent (Figure 3). Similar results were obtained with 4-nitroquinoline-1-oxide (4NQO; results not shown).

Figure 3
figure3

SeMet treatment protected human fibroblasts from DNA damage. (a) UV-radiation; and (b) hydrogen peroxide. Cells were treated with 10 μM SeMet for 15 h, then treated with the indicated doses of DNA-damaging agents. Cell survival was determined by clonogenic survival assays. Shown is the mean±s.d. from two or more independent experiments. P<0.01 by Wilcoxon rank-sum test determined for IC50 doses

SeMet enhancement of PCNA binding to damage sites in cellular DNA

One potential mechanism by which SeMet could lead to enhanced DNA repair is by augmentation of repair complex formation. PCNA has long been known to bind tightly to damaged sites of genomic DNA after UV-irradiation (Aboussekhra and Wood, 1995). PCNA that is associated with repair complexes is resistant to extraction with Triton-X100 detergent (Aboussekhra and Wood, 1995). We found that as expected, PCNA binding to damaged chromatin was observed in UV-irradiated cells (Figure 4a–c). The damage-specific binding of PCNA was enhanced 1.5–2-fold in the presence of SeMet. Shown is the mean s.d. from five independent experiments (Figure 4d).

Figure 4
figure2

Effects of SeMet on DNA repair protein localization. (a–c) Enhancement of PCNA binding to damage sites in UV-irradiated human fibroblasts. Although the abundance of total PCNA protein was unaffected by SeMet treatment, one can use Triton-X extraction to discern PCNA that is localized to repair sites within nuclei of UV-irradiated cells. PCNA that is associated with repairosomes is tightly bound, and hence resistant to Triton extraction. First UV-irradiation of cells markedly increased PCNA staining in Triton-treated cells a well-known characteristic of the PCNA response to UV-radiation (Aboussekhra and Wood, 1995). Second, pretreatment of cells with 10 μM SeMet for 15 h before UV-irradiation, further enhanced the amount of Triton-resistant PCNA detected by immunostaining (bar graph). (a) unirradiated cells; (b and c) UV-irradiated cells; (c) 100×magnification; (d) bar graph showing mean±s.d. of five experiments. P<0.01 by t-test for third and fourth bars of the graph (Sigmaplot software)

Immunoprecipitation/immunoblotting of repair proteins in SeMet-treated cells

As a corollary to the immunostaining experiments (Figure 4), we studied repair complex formation in the presence/absence of SeMet, in isolated immune complexes of repair proteins (He and Ingles, 1997). Immunoprecipitation of DNA polymerase epsilon (pol-ε), a protein required for excision DNA repair, was conducted in cells treated or not with SeMet (Figure 5). In addition to the target antigen pol-ε, we probed the immune complexes for the presence of PCNA, predicted by the data in Figure 4 to be enhanced in SeMet-treated cells. We found that PCNA was enriched in pol-ε immune complexes collected from SeMet-treated cells (Figure 5). Because pol-ε is involved not only in DNA repair, but also replication, we assayed additional repair proteins. We immunoprecipitated XPA/XPG proteins (He and Ingles, 1997), and found enhanced PCNA binding in SeMet-treated cells, consistent with the findings for pol-ε. With regard to potential complications owing to replication proteins, it should be noted that cells were either serum-starved (Figure 4) or density-arrested (Figure 5) to achieve quiescence, hence, replicative synthesis was minimal. Reciprocal immunoprecipitations with PCNA were uninformative, probably because only about 10% of PCNA is associated with repair complexes at any given time.

Figure 5
figure1

PCNA repair complex formation enhanced by SeMet. Immune complexes were collected from SeMet-treated or –untreated cells, using an antibody to DNA polymerase epsilon (pol-ε) a known DNA repair protein required for excision repair. Complexes were probed for PCNA content, which was increased in SeMet-treated cells. We could not however detect XPA protein in the pol-ε complex. XPA/XPG complexes were probed for PCNA in SeMet-treated or untreated cells, and PCNA was detected in the complex. ERCC1, a distinct repair protein, was not detected in either complex (not shown). We did observe induction of ERCC1 protein itself by SeMet treatment (open arrow). Immunoglobulin heavy chains were detected in some immune complexes, positions indicated by asterisks. PCNA is indicated by arrows

Discussion

Se compounds have a long history in cancer chemoprevention. Interestingly, most studies have focused only on induction of apoptosis by Se compounds, as a possible mechanism for their chemopreventive actions. Indeed, elimination of damaged cells by apoptosis is probably an important mechanism of cancer prevention. If however, estimates are correct that each cell sustains as many as 10 000 potentially mutagenic (if not repaired) lesions per day arising from endogenous sources of DNA damage (Ames and Gold, 1997), then the capacity for DNA repair becomes a key determinant of cancer predisposition (Cheo et al., 1999). Indeed, the human population exhibits a range of DNA repair capabilities that correlate with cancer-predisposition (Parshad et al., 1996; Grossman, 1997). Very few studies have explored enhancement of DNA repair as an alternative mechanism by which Se and other known cancer chemopreventive agents might exert their anticancer properties. We conducted flow cytometric measurements, in consideration of the possibility that SeMet might cause cell cycle arrest and/or apoptosis. No cell cycle differences were observed in comparing SeMet-treated and untreated cells, nor was there any evidence of a sub-G1 apoptotic peak by flow cytometry (results not shown). No apoptotic laddering was detected (results not shown). Thus, at the concentrations used, SeMet induced DNA repair but not cell cycle arrest or apoptosis. It remains possible that cell cycle arrests or apoptosis may play a role in SeMet chemoprevention, particularly at higher concentrations. Our results are consistent with others showing lack of cell cycle arrest or apoptosis below 45 μM SeMet (Redman et al., 1998).

The experiments herein employed UV-radiation as the DNA-damaging agent, thereby implicating the nucleotide excision repair (NER) pathway, which is responsible for repair of UV-damage as well as bulky carcinogen adducts. We considered that UV-radiation also causes release of reactive oxygen species (ROS) by damaged mitochondria (Gniadecki et al., 2000). Because thioredoxin reductase and glutathione peroxidase are seleno-enzymes, that can directly scavenge free radicals, SeMet induction of these enzymatic activities may also play a role in protection from DNA damage (Ganther, 1999; Berggren et al., 1999; Allan et al., 1999). On the other hand, the HCR assays (Figure 2) employed only a UV-damaged reporter plasmid, that is, the cells received no DNA damage to generate ROS (Ganesan et al., 1999). The HCR assay provides strong evidence for DNA repair induction by SeMet. Moreover, any contribution by free-radical scavenging would be predicted to decrease the initial amount of DNA damage, which would be likely to decrease PCNA-dependent repairosome formation, rather than the observed increase (Figure 4). The enhancement of PCNA-dependent repair complex formation (Figure 4) argues for an inducible DNA repair response, coincident with induction of some DNA repair proteins (Figures 4 and 5). Thus, although we cannot completely exclude any contribution to cell protective effects by free-radical scavenging, a predominant DNA repair response is in clear evidence, and the contribution by free-radical scavenging may be minimal under these conditions.

Importantly, PCNA-dependent complex formation is required not only for NER, but also for repair of base damage by the base excision repair (BER) pathway (Karmakar et al., 2001). Repair complex formation (Figures 4 and 5) may imply also an upregulation of BER (Gary et al., 1999). This raises the important point that a number of DNA-damaging agents associated with carcinogenesis produce more than a single type of lesion. For example, UV-radiation, although long regarded as a prototype NER-inducing agent, also produces a fraction of base damage that is repaired by BER (Doetsch et al., 1995). Likewise, hydrogen peroxide, which serves as a prototype agent for endogenous DNA damage, likely critical in human carcinogenesis, induces largely BER but also NER (Saito et al., 1993). Our data suggests that SeMet might augment the repair of both classes of DNA damage. Indeed, pol-ε is a mediator of NER, but also of an alternate BER pathway (Stucki et al., 1998). Hence, SeMet may enhance repair of DMBA and other adducts, as well as decrease the load of secondary mutations attributed to oxidation.

Se is a required trace element. The US Food and Drug Administration recommended daily allowance for Se is 50 μg per day (Combs, 1997). Phase I clinical trials showed no adverse effects from 200 μg per day, a degree of supplementation that achieved a serum Se concentration in excess of 10 μM (Clark et al., 1996). Cancer chemopreventive studies in rodents have suggested that serum Se concentrations below 10 μM are cancer-preventive. We chose SeMet, being a major component of Se-enriched (and cancer preventive) foods, e.g. Brazil nuts (Ip and Lisk, 1994). We also intentionally chose a Se compound that is by itself relatively nontoxic and does not by itself cause detectable DNA damage (Figure 1b). Moreover, we preferred a Se compound that is applicable to animal models of cancer prevention, and to ongoing clinical trials (Patterson and Levander, 1997). The reported lack of toxicity in vivo accords well with lack of toxicity in vitro observed by us and others (Redman et al., 1998).

Implications

Beyond the implication that modest dietary Se supplementation may help prevent carcinogenesis in healthy individuals, it is conceivable that individuals that exhibit modest DNA repair-defects may be functionally rescued by dietary Se. As mentioned, the human population exhibits a spectrum of DNA repair capacities (Grossman, 1997). On the extreme low end is the cancer-prone genetic disease xeroderma pigmentosum (XP) which itself falls into several complementation groups differing in severity (Aboussekhra and Wood, 1995). The cancer-prone phenotype of XP demonstrates clearly the importance of DNA repair in carcinogenesis and its prevention (Parshad et al., 1996; Lindahl and Wood, 1999; Cheo et al., 1999). The intriguing possibility that Se compounds might rescue some of the more modest repair defects e.g. XPC, XPE, or Li-Fraumeni syndrome, warrants further study.

Materials and methods

Alkaline comet assays (Conducted as in Seo et al., 2002)

IMR90, GM-08399, or GM-01389 human fibroblast cells (Coriell Cell Repository, Camden, NJ, USA) were treated or untreated with SeMet and UV-radiation. Cells that received SeMet treatment were pretreated with 10 μM SeMet for 15 h. After UV-irradiation (254 nm UV light source, 10 Jm−2), cells (104–5×104) were directly embedded in 0.5% agarose (Sigma, MO, USA) and directly spread on a frosted slide (Trevigen, Inc., Gaithersburg, MD, USA) pre-coated with 1% agarose. The cells were lysed with cold lysing solution (2.5 M NaCl, 1% Triton X-100, 100 mM Tris base) for at least 2 h and placed in a horizontal electrophoresis box. Subsequently, cells were exposed to alkaline solution (300 mM NaOH, 10 mM EDTA, pH 13.0) for 20 min in order to allow for DNA unwinding, and then electrophoresis was performed at 12 V (250 mA) for 30 min. After electrophoresis, slides were neutralized in Tris buffer (0.4 M, pH 7.4), and stained with ethidium bromide (20 μg/ml). For quantification, we used first a visual scoring system (Collins et al., 1995) under fluorescence microscopy (Carl Zeiss, Germany). Two hundred comets on each slide are classified according to the relative intensity of fluorescence in the tail: I (none, <5%); II (low, 5–20%); III (medium, 20–40%), IV (high, 40–95%); V (total, >95%). For evaluation of DNA damage (arbitrary units), each classified comet is given a value of 0–4 (from undamaged [I], 0, to maximally damaged [V], 4). Thus, the total score for 200 comets could range from 0 (all undamaged) to 800 (all maximally damaged). Second, we used an image analysis system (Morris et al., 1998). Images of stained nuclei in random and non-overlapping fields were captured using confocal microscopic imaging system (Zeiss). Images were then analysed with Version 6.1 of NIH Image (available from: http://rsb.info.nih.gov/nih-image) and a macro written for the NIH Image program (Morris et al., 1998). For evaluation of DNA damage, comet tail moment was computed as the integrated density in the comet tail multiplied by the distance from the center of the nucleus to the center of mass of the tail.

Host cell reactivation (HCR) experiments (Conducted as in Ganesan et al., 1999; Smith et al., 1995)

Plasmid pSV2CAT encoding the bacterial chloramphenicol acetyltransferase gene, was irradiated with 5000 Jm−2 254 nm UV light. The lesion frequency is estimated to be >20 cyclobutane pyrimidine dimers (CPDs) and 6–4 pyrimidine-pyrimidone photoproducts (6–4 p.p.s.) per plasmid molecule. Transfection of damaged plasmid into NER repair-defective xeroderma pigmentosum XP12BE (XPA cells; negative control) results in CAT activities less than 5% of the unirradiated plasmid (Smith et al., 1995). Plasmids were transfected into human breast cancer MCF7 cells by FuGene (Boehringer-Mannheim). SeMet was added to the dishes 24 h after transfection, and cells incubated for 15 h in the presence of 10 μM SeMet to induce DNA repair. Extracts were prepared and CAT activity determined 72 h after transfection. MCF7 cells were chosen for this set of experiments due to low transfection efficiency of the human fibroblasts.

Clonogenic cell survival (Conducted as in Smith et al., 1995)

IMR90 human fibroblast cells were maintained in RPMI 1640 medium, were seeded on 100 mm2 Petri dishes in serial 10-fold dilutions (10 000; 1000; 100 cells). Untreated dishes corresponding to each respective cell line served as controls. The plating efficiency was always greater than 50%. Cells were treated with hydrogen peroxide or UV radiation at doses indicated. Those that received SeMet treatment were pre-treated with 10 μM SeMet for 15 h. Medium was replenished and cells returned to their incubator for 12 days, at which time surviving colonies were counted after staining with crystal violet. Survival was calculated by comparison with untreated dishes, or appropriate dilutions thereof.

Immunostaining of fixed cells (Conducted as in Smith et al., 2000)

IMR90 or GM-01389 human fibroblasts were grown on the surface of glass cover slips, UV-irradiated, and were treated with 1% Triton X-100 for 15 min, then fixed in 100% methanol. Immunostaining of PCNA was conducted using Ab-PC10 (Santa Cruz) followed by FITC-goat anti-mouse IgG (Sigma, St. Louis, MO, USA). PCNA that is tightly bound to UV-damaged chromatin is resistant to Triton extraction, and it is the bound fraction that is detected by immunostaining. Determination of Triton-resistant PCNA is an established measure of UV-damage repair (Aboussekhra and Wood, 1995).

Immunoblotting of DNA repair proteins

Immunoprecipitations and immunoblots were conducted as in Smith et al., 1994, 1995. Cells were treated in the presence or absence of SeMet (15 h) as indicated, and then with 1 μM cisplatin for 3 h to induce repair complex formation. Antibodies were as follows: DNA polymerase epsilon (Ab93G1A, NeoMarkers, Fremont, CA, USA); PCNA (PC10, Santa Cruz Biotech, Santa Cruz, CA, USA); XPA (SC-853, Santa Cruz Biotechnology, CA, USA); XPG (Santa Cruz SC-10769); and ERCC1 (NeoMarkers clone 3H11). Immunoprecipitation lysis buffer consisted of 50 mM Tris/HCl pH 8, 150 mM NaCl, 1% Triton X-100, and protease inhibitors. Immune complexes were washed four times with lysis buffer, then boiled in SDS gel-loading buffer and subject to electrophoresis and transfer. Detection was with horseradish peroxidase conjugated secondary antibodies (Sigma, St. Louis, MO, USA) and enhanced chemiluminescence (Pierce Inc., Rockford, IL, USA).

Abbreviations

UV:

ultraviolet radiation

SeMet:

selenomethionine

XP:

xeroderma pigmentosum

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Acknowledgements

This work was supported by American Cancer Society RSG-02-028-01 CNE to ML Smith and by Indiana University Cancer Center.

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Correspondence to Martin L Smith.

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Seo, Y., Sweeney, C. & Smith, M. Selenomethionine induction of DNA repair response in human fibroblasts. Oncogene 21, 3663–3669 (2002). https://doi.org/10.1038/sj.onc.1205468

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Keywords

  • cancer-chemoprevention
  • selenium
  • nutrition
  • DNA-repair

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