Growth Inhibition and Induction of Apoptosis in Mesothelioma Cells by Selenium and Dependence on Selenoprotein SEP15 Genotype

Growth inhibition and induction of apoptosis in mesothelioma cells by selenium and dependence on selenoprotein SEP15 genotype

Sinoula Apostolou1, Julian O Klein1, Yasuhiro Mitsuuchi1, Justin N Shetler1, Poulikos I Poulikakos1, Suresh C Jhanwar2, Warren D Kruger2 and Joseph R Testa*,1

1Human Genetics Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA; 2Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Malignant mesotheliomas (MMs) are aggressive tumors (Zhang et al., 1997; Ip, 1998; van Lieshout et al., derived from mesothelial cells lining the lungs, pericar- 1998). Chemoprevention studies in animal model dium and peritoneum, and are often associated with systems have shown that selenium is protective against occupational asbestos exposure. Suppression subtractive a variety of carcinogens (El-Bayoumy, 1991). An inverse hybridization was used to identify genes differentially association has also been shown between selenium expressed in MM cells compared to normal mesothelial intake or tissue levels and several human cancers cells. Agene, SEP15, encoding a 15-kDa selenium- (Hunter et al., 1990; van den Brandt et al., 1993). The containing protein was isolated using this approach and manner by which the development of tumors is reduced was subsequently shown to be downregulated in B60% of by selenium is not clear. However, some biological MM cell lines and tumor specimens. A SEP15 poly- actions of selenium are known to be characteristic of its morphic variant, 1125A, resides in the SECIS recognition role as a component of selenoproteins. Selenium exists element in the 30-UTR and may influence the efficiency of as the 21st amino-acid selenocysteine (Sec) in all known Sec incorporation into the protein during translation. mammalian selenium-containingproteins. This amino Since previous studies have implicated a potential role of acid is inserted into the growing polypeptide chain at the the trace element selenium as a chemopreventive agent in UGA codon within the ORF of the mRNA (Stadtman, animal models and in several types of human cancer, we 1996). In mammals, insertion of selenocysteine into the investigated the effect of selenium on MM cells and its selenoprotein duringtranslation, rather than termina- dependence on SEP15 genotype. Selenium was shown to tion of translation, requires the selenocysteine insertion inhibit cell growth and induce apoptosis in a dose- sequence (SECIS) recognition element within the 30- dependent manner in MM cells but had minimal effect UTR (Berry et al., 1993; Clark et al., 1996). SECIS on normal mesothelial cells. However, MM cells with elements are present in the mRNAs of all of the downregulated SEP15 or the 1125Avariant were some- selenoproteins and possess a stem-loop structure con- what less responsive to the growth inhibitory and apoptotic tainingan internal and apical loop of unpaired effects of selenium than MM cells expressing wild-type nucleotides and a conserved 4-bp domain within the protein. RNAi-based knockdown studies demonstrated stem. The functions of several selenium-containing that SEP15 inhibition makes sensitive MM cells more proteins are known, one of which (the 15-kDa resistant to selenium. These data imply that selenium may selenoprotein, Sep15) has recently been linked to certain be useful as a chemopreventive agent in individuals at high murine cancers (Kumaraswamy et al., 2000). risk of MM due to asbestos exposure, although those with Malignant mesothelioma (MM) is a highly aggressive the 1125Apolymorphism may be less responsive to the neoplasm of mesodermal origin often associated with protective benefits of dietary selenium supplementation. asbestos exposure and is characterized by a longlatency. Oncogene (2004) 23, 5032–5040. doi:10.1038/sj.onc.1207683 The length of the latent period suggests that multiple Published online 26 April 2004 genetic alterations may be required for tumorigenic conversion of a mesothelial cell. We used a modified Keywords: mesothelioma; selenium; chemoprevention; form of suppression subtractive hybridization (SSH) apoptosis; SEP15 genotype (Diatchenko et al., 1996) to identify genes differentially expressed in MM cells and their normal mesothelial cell counterparts. One gene identified by this approach encodes a human 15-kDa selenoprotein (SEP15). This Introduction gene maps to 1p31, a region of frequent (55%) loss of heterozygosity (LOH) (Apostolou et al., 1999) in MM, Selenium is an essential trace element in redox pathways which may implicate SEP15 as a candidate tumor and has been utilized as a chemopreventive agent suppressor gene (TSG). Previous studies have revealed two polymorphisms in the 30-UTR SECIS of SEP15,at *Correspondence: JR Testa; E-mail: [email protected] nucleotides 811 (C/T) and 1125 (G/A) (Gladyshev et al., Received 22 July 2003; revised 1 March 2004; accepted 1 March 2004; 1998). Nucleotide 1125 resides within the apical loop of published online 26 April 2004 the SEP15 SECIS element and may potentially influence Effects of selenium on mesothelioma cells and SEP15 status S Apostolou et al 5033

Figure 1 SEP15 expression in MM cell lines and tumor (T) samples. Real-time PCR analysis was performed on cDNA from three different normal mesothelial cell cultures and individual MM cell lines. The LOH and nucleotide 1125 status of each MM cell line is indicated. Expression of SEP15 is similar in the three mesothelial cell samples. SEP15 expression in five frozen MM tumor specimens (T) is also shown. Data are normalized to b-actin and are presented as fold-changes in SEP15 mRNA levels, relative to normal mesothelial cells the efficiency of Sec incorporation (Gladyshev et al., regulated in 14 of 23 (60%) of the cell lines by two-fold 1998). or greater, when normalized to b-actin and compared Since LOH at 1p31 is common in MM and the trace with the average of three normal mesothelial cell element selenium is a candidate chemopreventive agent samples (Figure 1). The range of SEP15 expression in several cancers, we investigated the possible role of was found to be similar in the three individual SEP15 in MM. We report that expression of this gene is mesothelial cell cultures. In total, 12 of the 20 MM cell frequently downregulated in human MM cell lines and lines with LOH at 1p31 showed downregulation of tumor specimens when compared with normal mesothe- SEP15. SEP15 expression was not detectable in either lial cells. We demonstrate differential effects of selenium normal mesothelial cells or MM cells by Northern blot on the growth of normal versus malignant mesothelial analysis. This was most likely due to the levels of SEP15 cells and between MM cell lines possessingdown- beinglow in MM. However, SEP15 transcripts were regulated SEP15, wild-type SEP15 or the 1125A detectable in cell lines derived from prostate, breast and variant. These data indicate that SEP15 genotype and colon tissues that were present on the same Northern expression alterations affect SECIS function. The blot. Expression of SEP15 was also downregulated in findings of this study suggest that SEP15 may be three of five MM tumor specimens when compared to involved in the development of MM and could influence that in normal mesothelial cells (Figure 1). The LOH response to the chemopreventive properties of selenium. status of these tumors could not be determined since their normal counterparts were not available for analysis. Results Differential effects of selenium on MM cell growth are Expression of SEP15 is frequently downregulated in a associated with genotype and expression of SEP15 subset of MM cells We first tested the effects of selenium on the growth of We performed SSH to identify genes downregulated in seven MM cell lines with varying SEP15 status and one MM cells compared to normal mesothelial cells. One of normal mesothelial cell culture (7086A1). Three MM the genes identified as being downregulated in MM was cell lines (Meso 8, 14 and 45) had downregulated SEP15 SEP15. Further analysis of the expression of SEP15 in expression, two (Meso 22 and 34) possessed the 1125A five MM tumor specimens and a panel of 23 MM cell allele and two (Meso 6 and 59) expressed only the wild- lines was performed by real-time quantitative PCR. In type 1125G allele. Cell growth was evaluated with total, 20 of these cell lines displayed LOH at 1p31, where increasingconcentrations of selenium in the tissue SEP15 is located. Expression of SEP15 was down- culture media. The cells were incubated in selenium

Oncogene Effects of selenium on mesothelioma cells and SEP15 status S Apostolou et al 5034 (SeMet, Sec or Na2SeO3) supplemented media at apoptosis in normal mesothelial cells. Similar results concentrations between 0 and 500 mM for 48 and 72 h, were obtained with Sec-treated cells, further demon- and cell proliferation was assessed. Na2SeO3 and Sec stratinga differential effect of selenium in normal versus were found to be consistently more potent growth malignant mesothelial cells and between MM cells with inhibitors than SeMet (data not shown), consistent with varyingSEP15 status. The proportion of apoptotic MM reports by other groups (Menter et al., 2000). cells treated with Na2SeO3 and Sec is summarized in Figure 2 illustrates the concentration-dependent Figure 3. effects of Sec observed at 48 h. Sec-induced growth inhibition was observed in all MM cell lines but had little or no effect on normal mesothelial cells. Growth Apoptosis is increased in cells transfected with wild-type inhibition was most pronounced in Meso 6 and 59 SEP15, but not the 1125A variant, following treatment cells, which possess wild-type SEP15. MM cell lines with selenium with the 1125A allele were less responsive to added We next examined the effect of selenium on an MM cell selenium than were the wild-type 1125G cells. MM cell line transiently transfected with two different SEP15 lines with reduced expression of SEP15 were least constructs, to test experimentally whether the differen- affected by increasingconcentrations of Sec. Similar, tial effects of selenium described above are mediated more marked, effects were observed at the 72-h time through SEP15. Meso 8 cells, which express low levels of point. Collectively, these findings suggest a selective SEP15 and are resistant to the effects of selenium, were effect of selenium on malignant mesothelial cells co-transfected with vector containingGFP and either compared to their normal counterparts, and that the wild-type SEP15 or 1125A variant SEP15 constructs. degree of drug response in MM cells is also influenced Cells were treated with 0, 5 or 25 mM of Sec for 72 h, and by SEP15 status. apoptotic levels were assayed by immunofluorescence usingAnnexin V-Cy3 and 200–250 cells per concentra- Induction of apoptosis by selenium varies depending on tion of Sec were counted. Transfected cells were green, SEP15 status apoptotic cells were red, and yellow cells indicated apoptotic, transfected cells. Untreated cells and cells We also examined the effects of selenium on apoptosis in treated with 5 mM Sec displayed similar levels of these same MM cell lines. MM cells were treated with 5 apoptosis (3–4%). However, at a concentration of and 25 mM of Na2SeO3 or Sec for 48 and 72 h, and the 25 mM Sec, the proportion of apoptotic cells was higher early event of apoptosis involvingthe translocation of in cells transfected with the wild-type SEP15 construct phospholipid phosphatidylserine from the inner part of (B16%) than in cells transfected with the 1125A SEP15 the plasma membrane to the cell surface was assayed. At construct (4–6%) (Figure 4). Thus, the 1125A variant both concentrations of Na2SeO3, induction of apoptosis was demonstrated to be less responsive to added was much higher in MM cells with wild-type SEP15 selenium than the wild-type protein. Expression of the protein (Meso 59) than in cells with either down- SEP15 protein in transfected cells from individual regulated SEP15 (Meso 8) or the 1125A variant (Meso constructs was shown to be similar by Western blot 34). Importantly, selenium induced only low levels of analysis (Figure 4).

Figure 2 Growth of MM cells treated with Sec at a concentration range of 1–500 mM for 48 h. Dose-dependent growth suppression by Sec of MM cell lines possessingwild type (Meso 6 and 59), 1125A polymorphic (Meso 22 and 34) or downregulated (Meso 8, 14 and 45) SEP15 is presented. MM cells with the polymorphic variant of SEP15 or downregulated SEP15 did not respond to Sec as efﬁciently as MM cells with wild-type SEP15. Growth of normal mesothelial cells (7086A1) was not affected by selenium. Data are presented as the mean of triplicate cultures followingnormalization to untreated cells. Bars, s.d.

Oncogene Effects of selenium on mesothelioma cells and SEP15 status S Apostolou et al 5035

Figure 3 Sensitivity of MM cells to Sec and Na2SeO3. The proportion of apoptotic cells followingtreatment with 0, 5 and 25 mM Sec for 72 h was quantiﬁed by calculating the percentage of annexin V-positive cells. MM cells with downregulated SEP15 displayed levels of apoptosis comparable to that of cells with the 1125A polymorphism. The percentage of apoptotic cells was highest in MM cells with wild-type SEP15. Apoptosis induction by selenium was minimal in normal mesothelial cells

specific siRNA prior to exposure to selenium. To monitor expression changes following siRNA transfection, we used real-time PCR to determine the extent of SEP15 inhibition. As shown in Figure 5a, SEP15 expression was markedly diminished by SEP15 siRNA. Cells transfected with an SEP15-specific siRNA exhibited increased cell proliferation followingexposure to selenium compared to cells transfected with a nonspecific siRNA pool (Figure 5b). In addition, cells treated with SEP15 siRNA exhibited decreased apoptosis followingselenium treatment at a concentration of 25 mM Sec (Figure 5c). Collectively, these data suggest that SEP15 expression affects the response of MM cells to growth inhibition and apoptosis induced by selenium.

SEP15 genotype frequencies are similar in MM patients Figure 4 Co-transfected Meso 8 cells were examined for the and the general population expression of 1125A or wild-type SEP15 followingtreatment with 0–25 mM Sec for 72 h. The numbers of transfected, apoptotic cells We determined the frequencies of the SEP15 genetic were counted and are presented as percentages. Bars, s.d. Expression of the SEP15 protein in transfected cells from variations within the general human population, MM individual constructs was also shown to be similar by Western tumors and cell lines, and peripheral blood from MM blot analysis (insert) patients. We initially performed SSCP analyses to determine the prevalence of nucleotide 1125 G or A in 60 MM cell lines, 44 of which exhibited LOH at 1p31. A SEP15 inhibition by siRNA makes sensitive MM cells total of 17 samples displayed band shifts indicating more resistant to selenium nucleotide changes (Figure 6). DNA sequencing demonstrated that each of these variants was due to an 1125 We previously demonstrated that SEP15 is involved in nucleotide alteration. The frequencies of the 1125G, cell proliferation and apoptosis followingexposure to 1125 G/A and 1125A genotypes in MM cell lines were selenium in MM cells. To further verify that SEP15 72, 13 and 15%, respectively. plays a potentially signiﬁcant role in cell proliferation We also ascertained the distribution of the SEP15 and apoptosis subsequent to selenium treatment, sensi- allele frequencies in nonmalignant tissue (PBL) from tive MM cells with wild-type SEP15 (Meso 6) were MM patients and in the general human population transiently transfected with a pooled mixture of SEP15- usingSSCP or RFLP analysis. The frequencies of the

Oncogene Effects of selenium on mesothelioma cells and SEP15 status S Apostolou et al 5036

Figure 5 Meso 6 cells transiently transfected with siRNA against SEP15.(a) SEP15 expression in siRNA transfected Meso 6 cells determined by real-time PCR analysis. SEP15 expression is decreased in cells transfected with SEP15-specific siRNA in comparison to cells transfected with a nonspecific siRNA pool. (b) Growth of Meso 6 cells treated with Sec at a concentration range of 1–25 mM for 48 h. MM cells transfected with SEP15-specific siRNA exhibited increased cell proliferation followingexposure to selenium compared to cells transfected with a nonspecific siRNA control pool. Data are presented as the mean of triplicate cultures following normalization to control-treated cells. Bars, s.d. (c) Noticeable inhibition of apoptosis (red cells) was observed in cells transfected with SEP15 siRNA compared to control cells followingSec treatment at a concentration of 25 mM. Cells stained with DAPI are shown in blue

MM cases (61%) is due to LOH in malignant cells from MM patients. LOH analysis was performed on tumor biopsies from 24 of these patients, and similar patterns of allelic loss were observed in tumor tissue and derived MM cell lines (data not shown). Preferential loss of one allele versus the other did not appear to occur in tumor Figure 6 SSCP mutation analysis of SEP15 in MM cell lines and cells from MM patients exhibitingLOH at 1p31. The tumors. SSCP was performed on cDNA and genomic DNA. Autoradiograph shows PCR products generated from the SECIS genotypes for all of the samples analysed are shown in element in the 30-UTR of SEP15 encompassingnucleotide 1125. Table 1. Lanes 1 and 6, wild-type 1125G; lane 2, 1125A variant; lanes 3–5, Finally, to determine if nucleotide changes occurred heterozygous 1125G/A in the codingregionof SEP15, we used SSCP to analyze cDNA from 20 MM cell lines displayingLOH at 1p31. cDNA from normal mesothelial cells was included as a 1125G, 1125 G/A and 1125A genotypes in the general control. Band shifts were not observed in any of the population were 46, 50 and 4%, respectively (Table 1). MM samples, indicatingno nucleotide alterations in the The frequencies in PBL from MM cases were 39, 61 and codingregionof SEP15. 0%. w2 analysis revealed that the difference in the distribution of 1125A and G alleles between MM blood samples and the general population was not statistically signiﬁcant, suggesting that the 1125A SEP15 variant Discussion does not predispose to the development of MM. The low frequency of the heterozygous 1125 genotype An abundance of data implicatinga potential role of in MM cell lines (13%) compared to that in PBL from selenium as a chemopreventive agent in animal models

Table 1 Allelic distribution of 1125G/A alleles in human DNA samples 1125G Heterozygous 1125A

Normal population (n ¼ 48) 22 (46%) 24 (50%) 2 (4%) Blood from MM cases (n ¼ 36) 14 (39%) 22 (61%) 0 (0%) MM tumors/cell lines (n ¼ 60) 43 (72%) 8 (13%) 9 (15%) MM tumors/cell lines without LOH (n ¼ 16) 8 (50%) 8 (50%) 0 (0%) MM tumors/cell lines possessingLOH ( n ¼ 44) 35 (80%) 0 (0%) 9 (20%)

Oncogene Effects of selenium on mesothelioma cells and SEP15 status S Apostolou et al 5037 and humans (Hunter et al., 1990; El-Bayoumy, 1991; fact that the nucleotide at position 1125 does indeed van den Brandt et al., 1993; Zhang et al., 1997; Ip, 1998; influence SECIS function (Figures 3 and 4). MM cells van Lieshout et al., 1998) prompted our study of the with the 1125A variant were less responsive to the protective effects of selenium in MM cells in connection antiproliferative and proapoptotic effects of selenium with a gene encoding a selenium-containing protein, compared to cells with the wild-type allele. Tissue SEP15. In this investigation, SEP15 was found to be culture media is generally believed to be deficient in frequently underexpressed in MM cells relative to selenium when compared with the levels observed in normal mesothelial cells, suggesting a possible role of human tissues and plasma (Diplock, 1993). Thus, the SEP15 in the pathogenesis of MM. We have demon- effects we detected in our in vitro studies are attributed strated that selenium has differential effects on cell to selenium supplementation. We also observed that growth and survival in MM cells compared to normal different chemical forms of selenium can vary somewhat mesothelial cells. In addition, MM cells with a with respect to their effect on cell proliferation and polymorphic variant of SEP15 or loss of expression of apoptosis. Since tumor cells with the 1125A allele were SEP15 did not respond to selenium as efficiently as MM less sensitive to the effects of selenium, this allele may cells with wild-type SEP15. Importantly, our data result in a defect in SEP15 function or production of less suggest that SEP15 is involved in inducing apoptosis of the protein. While our data suggest a correlation in MM cells, since its inhibition by siRNA resulted in between selenocysteine sensitivity and SEP15 expres- increased proliferation and decreased apoptosis in Meso sion, it is also possible that other unknown genetic 6 cells that possess wild-type SEP15. alterations differentially present in MM cell lines may Since mutations were not detected in the coding influence selenocysteine sensitivity and SEP15 expres- region of SEP15, downregulation of this gene in MM sion. However, RNAi-based knockdown studies with cells may be caused by epigenetic mechanisms, such as Meso 6 cells, which contain wild-type SEP15 and are aberrant promoter hypermethylation associated with sensitive to selenium, revealed that SEP15 downregula- inappropriate gene silencing (Jones and Baylin, 2002). tion makes sensitive MM cells more resistant to the Such epigenetic changes are common in cancer and are compound (Figure 5). Moreover, transfection with only thought to affect multiple steps in tumor progression. the wild-type SEP15 construct induced an increased To our knowledge, this is the first report of altered apoptotic response to selenocysteine, whereas the SEP15 expression of SEP15 in a human tumor. However, variant construct or vector alone did not (Figure 4). aberrant expression of SEP15 has previously been These data imply that at least some effects of selenium reported in two rodent tumor types. Expression of are mediated through SEP15 and that this gene has a Sep15 was undetectable in mouse prostate adenocarci- significant role in selenium sensitivity in MM tumor noma cells and liver tumors, whereas normal mouse cells. Importantly, normal mesothelial cells containing prostate and liver tissues exhibited high levels of the the wild-type allele were not affected by selenium at the Sep15 protein (Kumaraswamy et al., 2000). concentrations used in this study. A similar differential Attempts to confirm SEP15 expression in MM by in vitro effect of selenium on growth inhibition and northern analysis demonstrated that SEP15 was not apoptosis has been observed in prostate cancer cells detectable in either normal mesothelial cells or MM cells compared with normal prostate cells (Menter et al., by northern blot analysis. This was most likely due to 2000). the levels of SEP15 beinglow in MM. However, SEP15 The SEP15 gene maps to human chromosome band transcripts were detectable in cell lines derived from 1p31. LOH involvingthe short arm of chromosome 1 is prostate, breast and colon tissues that were present on often observed in MM, with most cases showingallelic the same Northern blot. losses in the 1p21–31 region (Lee et al., 1996; Apostolou Overexpression of SEP15 was also observed in four of et al., 1999). Recurrent LOH at a discrete chromosomal 23 MM cell lines. This may be due to the fact that MMs region is generally considered an indication of the have a great deal of genetic variability. Although presence of a TSG, whose loss/inactivation contributes recurrent genetic alterations exist in MM, individual to tumorigenesis (Gray and Collins, 2000). Owing to its cell lines can also show considerable variability. Thus, it location at 1p31, we considered the possibility that was not unexpected that SEP15 was not downregulated SEP15 is a TSG. However, several facts argue against in all MM cases. Additionally, the number of primary this possibility. First, LOH analysis of overlapping1p and tumor samples (Figure 1) are too few to draw firm deletions from a large series of MMs indicated that the conclusions regarding SEP15 expression in primary minimally deleted (common) region, that is, 1p22, is tumors and will require further study in the future. proximal to the SEP15 locus (Lee et al., 1996). Second, However, the situation from the MM cell lines could be no SEP15 mutations were observed in any of the MM the same as in the MM tumors. cell lines we tested. Third, we did not observe a Previous work has demonstrated that the SEP15 statistically significant correlation between SEP15 nucleotide at position 1125, within the apical loop of the downregulation and LOH at 1p31 in our MM cases. SECIS element, can influence the efficiency of transla- These findings imply that SEP15 is not the critical target tion of the in-frame UGA codon (Kumaraswamy et al., of 1p allelic loss in MM. However, the frequent 2000). Our cell proliferation and apoptosis data on downregulated SEP15 expression observed in MM selenium-treated MM cell lines possessingeither wild- suggests that this change may contribute to the type SEP15 or the 1125A variant are consistent with the pathogenesis of MM.

Oncogene Effects of selenium on mesothelioma cells and SEP15 status S Apostolou et al 5038 Genotype analyses indicated that the frequency of the AGG GCG TGG TGC GGA GGG CGG T-30;50-GAT CAC 1125A allele is similar in MM patients and in the general CGC CTT CCG-30). population. While most of our MM cases showed LOH at the SEP15 locus, there did not appear to be a Suppression subtractive hybridization selection for either allele. However, the sample size was SSH was used to identify genes downregulated in MM cell small. A difference in 1125G/A allele frequencies was lines compared to normal mesothelial cells. Two sets of recently reported in tumors from patients with either subtractive hybridizations were performed: 40 ngof tester breast cancer or head and neck tumors when compared (containingadapters 1a/1b or 2a/2b) cDNA was combined with cancer-free African-American controls (Hu et al., with 2 or 4 mgof driver cDNA and purified. The ethanol 2001). In these tumor types, the 1125A allele was precipitated mixture was then washed with 80% ethanol and associated with increased cancer risk. air dried before beingresuspended in 4 ml of hybridization In summary, selenium has substantially different buffer (50 mM HEPES, pH 8.3/0.5 M NaCl/0.02 mM EDTA, pH 8.0/10% PEG 8000). The mixtures were subsequently effects on normal and malignant mesothelial cells, denatured and then hybridized for 8 h at 681C. Two of the first exhibitingsignificant antiproliferative and proapoptotic hybridization reactions between normal tester/MM cDNA activity against MM cells. In tumor cells, differences driver were combined and mixed with an excess of each driver. in SEP15 genotype or expression are associated with This mixture, comprisingthe second hybridization, was further variation in response to selenium. Collectively, if our incubated for 20 h at 681C. in vitro findings are confirmed in an in vivo model, Finally, the cDNAs were PCR-amplified usingprimers 1 these may provide a rationale for the possibility of and 2 (50-GTA TAC GAC TCA CTA TAG GGC-30;50-TGT usingselenium as a chemopreventive agentin asbestos AGC GTG AAG ACG ACA GAA-30). To increase the amount of final difference product, nested PCR usingnested 1 workers or other groups occupationally exposed to 0 these carcinogenic mineral fibers, who are at high and 2 primers (5 -TCG AGC GGC CGC CCG GGC AGG T-30;50-AGG GCG TGG TGC GGA GGG CGG T-30) was risk of developingMM. Moreover, SEP15 genotyp- performed. ing may be useful in these high-risk groups, since individuals with the wild-type (GG) genotype might Product analysis be more likely to benefit from dietary selenium supplementation. Radiolabeled final difference products, representative of the mRNA present in normal mesothelial cells, but missingin the MM cell lines, were generated by PCR-amplification with nested 1 and 2 primers, then separated in 6% polyacrylamide/ Materials and methods 8 M urea gels. Following autoradiography, 40 bands, mostly common to each difference product were excised from the gel. Cell lines and RNA preparation The DNA was eluted then cloned into pGEM-T (Promega). The inserts were PCR-amplified usingnested 1 and 2 primers, Normal human mesothelial cells (including7086A1, Coriell then purified and sequenced. Cell Repositories; NM23 and WTU3) and MM cell lines Reverse northern hybridization (Hufton et al., 1999) was established from surgically explanted primary tumors were used to examine the differential expression of the identified grown under the same conditions in RPMI 1640 media clones. PCR products were electrophoresed on agarose gels. In (Invitrogen) supplemented with 2 mM glutamine, 100 U of addition, actin was loaded as a hybridization control. Replica penicillin and streptomycin, and 10% FBS. For RNA blots were prepared then probed individually with radiolabeled extraction, cell lines were grown to 70–80% confluence prior DpnII cut cDNA from tester and each driver. Verification of to harvest. Total RNA was isolated from cell lines using the differential expression of a number of these clones in MM TRIzol (Invitrogen). cell lines as compared to normal mesothelial cells was performed by RT–PCR. Tester and driver preparation Real-time quantitative PCR of SEP15 RNA utilized for the SSH procedure was purified usingthe Fast Track mRNA isolation kit (Invitrogen). cDNA subtrac- First-strand cDNA synthesis was performed with 2 mgof total tion was performed usinga modification of the protocol RNA and 500 ng of oligo-dT primer (Invitrogen), according to described by Roberts et al. (2002). Briefly, double-stranded the manufacturer’s instructions. Real-time PCR analysis was cDNA (5 mg) synthesized (Copy Kit cDNA kit, Invitrogen) performed on cDNA from MM samples usingprimers SEP15- from MM cell lines was digested with DpnII (New England F(50-GCA GCT CTT GTG ATC TTC TCG-30) and SEP15-R Biolabs). cDNA restriction digests were purified and precipi- (50-CTT GGA CTT GAG GGA ACC TTC-30) to amplify the tated prior to beingligatedto the R- Bgl-12/24 (50-GAT CTG cDNA together with the 50-FAM-TCA ACC TGC TTC AGC CGG TGA-30;50-AGC ACT CTC CAG CCT CTC ACC TGG ATC CTG-BHQ1-30 probe (Biosource International). GCA-30) adapters. Ligation reactions were then diluted and b-actin primers and probes were obtained from Perkin-Elmer/ PCR-amplified (951C, 1 min, 721C, 3 min for 35 cycles) using Applied Biosystems. Amplification reactions were performed the R-Bgl-24 primer to obtain B30 mg/cell line. PCR products with 1 ml of cDNA, 3 ml of each of the specific primers (3 mM) were purified, precipitated and digested with DpnII, prior to and 4 ml of the probe (2 mM) added to Ready-To-Go beads separation by electrophoresis. Products correspondingto 200– (Amersham). All reactions were performed with the Smart 2000 bp were gel purified to remove the adapters. Then 1.2 mg Cycler System (Cepheid), and the thermal cyclingconditions of tester cDNA was religated to a new set of adapters (adapter were as follows: 5 min at 941C, followed by 45 cycles of 941C 1a/1b: 50-GTA TAC GAC TCA CTA TAG GGC TCG AGC for 15 s, 521C for 30 s and 681C for 30 s. To quantitate the GGC CGC CCG GGC AGG T-30;50-GAT CAC CTG CCC amount of specific mRNA in the samples, a standard curve G-30; or 2a/2b: 50-TGT AGC GTG AAG ACG ACA GAA was generated for each run using 10-fold serially diluted

Oncogene Effects of selenium on mesothelioma cells and SEP15 status S Apostolou et al 5039 plasmid containing SEP15 cDNA sequence (dilutions ranging siRNA transfection from 1 fg to 1 ng). In addition, a standard curve was generated SMARTpools of siRNA specific for SEP15 (M-007249-00-50), for b-actin ranging from 1 fg to 1 ng. This enabled standardi- or nonspecific control RNA duplexes (D-001206-13-05) were zation of the initial RNA content of a tissue relative to the purchased from Dharmacon Inc. (Lafayette, CO, USA). Meso amount of b-actin. 6 and Meso 59 cells were plated into 10 cm dishes. After 24 h, cells were transfected with Oligofectamine reagent (Invitrogen) Cell proliferation assay and gene-specific or nonspecific control siRNAs at a final MM cells were plated into 96-well tissue culture plates (Costar) concentration of 100 nM. After 12 h, cells were trypsinized and and allowed to establish monolayers (B48 h). Various plated into 96-well plates for cell proliferation assays or into concentrations of selenomethionine (SeMet), sodium selenite four-well chamber slides for apoptosis assays. (Na2SeO3) or Sec were added to selected wells and incubated for 24, 48 or 72 h. Followingincubation, the medium was DNA analysis of SEP15 removed and fresh medium with CCK-8 solution (Alexis Biochemicals), an indicator of proliferatingcells, was added to For single-strand conformation polymorphism (SSCP) analy- the cells and incubated for 1 h. Absorbance was measured at sis, genomic DNA and cDNA from MM cell lines were 450 nm usinga microplate reader. studied. The entire codingsequence of the SEP15 gene was amplified with the followingsets of primers (SEP15-1: F, 5 0- AGT GGG TGT CTG GTG CCG GCG TTT-30 and R, 50- Apoptosis assay TTG GAC TTG AGG GAA CCT TCC C-30; SEP15-2: F, 50- Normal mesothelial cells and MM cell lines were plated into TAT GCA GGA GCT ATT CTT GAA G-30 and R, 50-AAG four-well poly-D-lysine-coated microscope slide glass chamber GTA ACA AAA GGA TAG GAC-30). In total, 20 nggenomic slides (Costar) and allowed to establish monolayers. Cells were DNA or 1 ml cDNA, 75 ngof each primer and 0.2 ml a32P-dCTP treated with 5 and 25 mM Na2SeO3 or Sec for 48 and 72 h. (800 Ci/mmol) (DuPont NEN) were used in a reaction of 10 ml. Followingincubation the medium was removed and fresh After an initial denaturation at 941C for 5 min, 35 cycles were medium with Annexin V-Cy3 (Medical and Biological carried out at: 941C for 1 min, 531C for 1 min and 721C for Laboratories), an indicator of the induction of apoptosis by 1 min. detectingphosphatidylserine on the cell surface, was added to For each primer pair utilized, genomic DNA from two the cells and incubated for 5 min. The cells were subsequently peripheral blood lymphocyte (PBL) samples from normal stained with diamidino-2-phenylindole (DAPI) and visualized individuals or normal mesothelial cells were routinely included under a Zeiss Axiophot fluorescence microscope using as controls in each gel. Samples were analyzed in both 10% rhodamine and DAPI filters. Digital images of DAPI staining nondenaturing acrylamide gels containing 5% glycerol and and fluorescein signals were captured with a cooled CCD 0.5 Â mutation detection enhancement acrylamide (FMC camera and merged using Quips software. Products) gels. Band shifts were considered indicative of a potential mutation or polymorphism when they were repro- Generation of SEP15 constructs for functional analysis of the ducible on at least two separate occasions. Direct sequencing SECIS element of PCR products displayingband shifts was performed followingpurification with the Wizard PCR Product Purifica- 0 Oligonucleotide primers 5 -AGC GAT GGC GGC TGG GCC tion Kit (Promega), according to the manufacturer’s instruc- 0 0 GAG TGG-3 and 5 -GAT TTT TGA AAC TTT TTA TTT tions. 0 ATA TTT TGG-3 complementary to cDNA of human SEP15 Restriction fragment length polymorphism (RFLP) analysis (Accession number NM_004261) were designed with terminal was used to study nucleotide 1125 in MM cell lines, tumors BglII and XhoI sites, respectively. An entire cDNA encoding and blood samples, as described above. The SECIS element in human wild-type (wt-SEP15cHA) or the 1125A polymorphic the 30UTR of SEP15 was amplified with primers (SECIS-F 50- variant (poly-SEP15cHA) of SEP15 was amplified by RT– ATA AGA TAT ACT GAG CCT CAA-30 and SECIS-R 50- PCR utilizingtotal RNA from mesothelial cells. The nucleo- TGG TCT TAC AA TGA TCA CTT-30). PCR amplification 0 tide sequence encoding the hemagglutinin (HA) epitope (5 - of a 474-bp product from genomic DNA of PBL controls was 0 TAC CCT TAT GAT GTG CCA GAT TAT GCC TCT-3 ) achieved with the followingprimers (SEPUTR-F, 5 0-CAG was inserted into both constructs at the carboxyl terminus, ACT TGC GGT TAA TTA TGC-30 and SEPUTR-R, 50-TGG nucleotide 491, usingPCR with the primers listed above. TCT TAC AAA TGA TCA-30). PCR was performed at 951C Nucleotide sequences of the constructs were validated by for 3 min, followed by 35 cycles of 951C for 1 min, 521C for sequence analyses. 1 min and 721C for 1 min 30 s. Amplified DNA was digested with BfaI (New England Biolabs), recognition sequence Transient transfection of SEP15 in MM cells (50-CTAG-30), to identify the nucleotide at position 1125. Nucleotide G at position 1125 results in the sequence 50- Transient transfections were carried out with the MM cell line, 0 Meso 8, usingFuGENE6 transfection reagent(Roche TTTCTAGCCTAA-3 , which is cleaved by BfaI, whereas an A Molecular Biochemicals), accordingto the manufacturer’s results in a sequence not recognized by this enzyme. DNA instructions. MM cells were plated onto six-well culture plates digestion was evaluated by gel electrophoresis in 2.5% agarose. and incubated for at least 24 h prior to DNA transfection. Cells were then transfected with 1 mgof wt-Sep15cHA or Acknowledgements poly-Sep15cHA expression vector. To monitor transfection, Supported by the Mesothelioma Applied Research Founda- cells were co-transfected with a GFP-containingvector at a tion, National Institutes of Health Grants CA-45745 and CA- ratio of 1 : 2 and then incubated for 24 h. To quantitate 06927, a gift from the Local #14 Mesothelioma Fund of the apoptosis, cells were treated with Sec at concentrations International Association of Heat and Frost Insulators & of 0–100 mM for 72 h, followed by the Annexin V-Cy3 Asbestos Workers in memory of Hank Vaughan and Alice apoptosis assay. A total of 200 to 250 cells per concentration Haas, and by an appropriation from the Commonwealth of of Sec were counted. Pennsylvania.

Oncogene Effects of selenium on mesothelioma cells and SEP15 status S Apostolou et al 5040 References

Apostolou S, De Rienzo A, Murthy SS, Jhanwar SC and Testa Hufton SE, Moerkerk PT, Brandwijk R, de Bruine AP, Arends JR. (1999). Cell, 97, 684–686. JW and Hoogenboom HR. (1999). FEBS Lett., 10, 77–82. Berry MJ, Banu L, Harney JW and Larsen PR. (1993). EMBO Hunter DJ, Morris JS, Stampfer MJ, Colditz GA, Speizer FE J., 12, 3315–3322. and Willett WC. (1990). JAMA, 264, 1128–1131. Clark LC, Combs Jr GF, Turnbull BW, Slate EH, Ip C. (1998). J. Nutr., 128, 1845–1854. Chalker DK, Chow J, Davis LS, Glover RA, Graham GF, Jones PA and Baylin SB. (2002). Nat. Rev. Genet., 3, 415–428. Gross EG, Krongrad A, Lesher JL, Park HK, Sanders BB, Kumaraswamy E, Malykh A, Korotkov KV, Kozyavkin S, Smith CL and Taylor JR. (1996). J. Am. Med. Assoc., 276, Yajun Hu Y, Kwon S, Moustafa ME, Carlson BA, Berry 1957–1963. MJ, Lee BJ, Hatfield DL, Diamond AM and Gladyshev VN. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam (2000). J. Biol. Chem., 275, 35540–35547. F, HuangB, Lukyanov S, Lukyanov K, Gurskaya N, Lee WC, Balsara B, Liu Z, Jhanwar SC and Testa JR. (1996). Sverdlov ED and Siebert PD. (1996). Proc. Natl. Acad. Sci. Cancer Res., 56, 4297–4301. USA, 93, 6025–6030. Menter DG, Sabichi AL and Lippman SM. (2000). Cancer Diplock AT. (1993). Am. J. Clin. Nutr., 57, 256S–258S. Epidemiol. Biomarkers Prev., 9, 1171–1182. El-Bayoumy K. (1991). Cancer Prevention, De Vita VT, Roberts D, Williams SJ, Cvetkovic D, Weinstein JK, Godwin Hellman S and RosenbergSA (eds). J.B. Lippincott Co.: AK, Johnson SW and Hamilton TC. (2002). DNA Cell Biol., Philadelphia, PA, pp. 1–15. 21, 11–19. Gladyshev VN, JeangKT, Wootton JC and Hatfield DL. Stadtman TC. (1996). Annu. Rev. Biochem., 65, 83–100. (1998). J. Biol. Chem., 273, 8910–8915. van den Brandt PA, Goldbohm RA, van’t Veer P, Bode P, Gray JW and Collins C. (2000). Carcinogenesis, 21, Dorant E, Hermus RJ and Sturmans F. (1993). Cancer Res., 443–452. 53, 4860–4865. Hu YJ, Korotkov KV, Mehta R, Hatfield DL, Rotimi CN, van Lieshout EMM, Ekkel MPC, Bedaf MMG, Nijhoff WA Luke A, Prewitt TE, Cooper RS, Stock W, Vokes, Dolan and Peters WHM. (1998). Oncol. Rep., 5, 959–963. ME, Gladyshev VN and Diamond AM. (2001). Cancer Res., ZhangZ, Kimura M and Itokawa Y. (1997). Biol. Trace 61, 2307–2310. Element Res., 57, 147–155.

Oncogene