Document O3O6ke3rmoajz5OqvvMOaaJMe

SEP 16 2002 10:54 FR CISTI ICIST 613 998 4926 TO 12145201181 ELSEVIER PLAINTIFFS EXHIBIT SA-540 Mutation Research 361 (19%) 113-120 jm Environmental Mutagenesis Chrysotile asbestos fibers mediate homologous recombination in Rat2X fibroblasts: implications for carcinogenesis Kimberly Lezon-Geydaa, Cindy M. Jaime \ James H. Godbold b,c, Ernest F. Savransky \ Aluko Hope a, Samir A. Kheiri \ Zlatica M. Dzmura \ Hiroshi Uehara *u, Edward M. Johnson a,c d, Thomas M. Fasy a'c'* * Department ofPathology, Mount Sinai School ofMedicine, New York. NY 10029, USA b Department ofCommunity Medicine. Mount Sinai School ofMedicine, New York NY 10029, USA c Centerfor Environmental Health Sciences, Mount Sinai School ofMedicine. New York, NY 10029, USA J Brookdale Centerfor Molecular Biology, Mount Sinai School ofMedicine. New York NY 10029, USA Received 18 October 1995: revised 30 April 1996; accepted 10 May 1996 Abstract Asbestos fibers are widespread environmental carcinogens whose mutagenicity is now established. Nonetheless, the molecular nature of these mutations and die mechanisms by which they accelerate carcinogenesis remain poorly understood. We have assessed the ability of asbestos fibers to promote homologous recombination, a potent mechanism for generating intrachromosomal rearrangements, such as deledons, and mitotic recombination. For this, we have developed a new assay which determines the extent to which a marker gene present in DNA introduced by asbestos can recombine with homologous genes residing in a transfected cell, We have demonstrated that Calidria chrysotile fibers are mutagenic and are able to mediate transfection of molecularly marked mutant lacl genes in a manner that results in their preferential recombination with homologous wild-type genes in the transfected cell. Asbestos induced recombination events may play a significant role in asbestos mutagenesis and carcinogenesis, and promotion of recombination may undertie the welt-recog nized synergy of asbestos with other carcinogens. Keywords: Chrysotile asbestos; tael: Homologous recombination; Rat2\ cell 1. Introduction * Corresponding author. One Gustave L. Levy Place. Box 1194, New York. NY 10029, USA. TeL: (1) (212) 241-9155: Fax: (1) (212) 860-7851. ' Present address: Oncor, Inc.. Gaithersburg, MD 20877, USA. Several different types of asbestos fibers are car cinogenic in humans (Selikoff et al., 1964; Nichol son, 1986; Begin et al,, 1992) and experimental animals (Wagner et al., 1974). Asbestos fibers can damage DNA (Libbus ct al., 1989; Kamp et al., 1992, 1995), cause mutations (Fasy, 1991; Hei, 1991; 0165-1161 /96/S15.00 Copyright 1996 Elsevier Science B.V. All rights reserved. PH SO 165-1161(96)00029-5 RECEIVED TIME SEP. 16. 9:57AM PRINT TIME SEP. 16. 10:01AM SEP 16 2002 10:55 PR CISTI ICIST 613 998 4926 TO 12145201181 P. 04 114 K. Leztm-Geyda a al./Mutation Research J6t (1996) I/<-120 Hei et al.. 1992; Stankowski et al., 1992), induce aneuploidy (Jaurand, 1989; Walker et al.. 1992) and mediate transfection of exogenous DNA (Appel et al., 1988: .Gan et al., 1993). In addition, asbestos fibers have been proposed to act as tumor promoters (Selikoff et al., 1968; Jaurand, 1989; Fasy, 1991; Walker et a!., 1992). The relative contributions of these different mechanisms to asbestos carcino genesis remain undefined. Although asbestos-media ted mutagenesis has been confirmed in several dif ferent cell culture assays (Fasy, 1991; Hei, 1991: Hei et all; 1992; Stankowski et al., 1992), the molecular mechanisms have yet to be characterized. Moreover, the ability of asbestos fibers to synergistically en hance the effect of other carcinogens and mutagens (Selikoff et a!., 1968; Jaurand, 1989; Fasy, 1991; Hei, 1991; Walker et al,, 1992), although well recog nized and frequently studied, has for almost three decades, remained unexplained in molecular terms. Homologous recombination is one mechanism which might readily contribute to both the muta genicity and carcinogenicity of asbestos fibers. All types of asbestos fibers have been shown to mediate cell transfection (Appel et al., 1988; Gan et al., 1993) and transfection is frequently employed in the laboratory to generate homologous recombination (Waldman, 1992; Savransky et al., 1994). Homolo gous recombination can give rise to deletions and genomic rearrangements (Schiestl, 1989). Cells which have already sustained an inactivating muta tion in one allele of a tumor suppressor gene are particularly vulnerable to the procarcinogenic poten tial"of mitotic recombination (Passarge and Bartram, 1976; Cavenee et al., 1983; Knudson, 1986) which arises not from residual sequence changes at the point of recombination, but from the potential mal distribution of mutant tumor suppressor alleles. In light of these considerations, it is important to assess the capacity of asbestos fibers to modulate the fre quency of homologous recombination events. Fol lowing asbestos-mediated transfection of DNA, the introduced DNA is damaged and fragmented upon entry into the host cell (Appel et al., 1988; Gan et al., 1993). Such DNA fragments could be mutagenic by recombining with homologous sequences in the genome. Here we have employed a new rat cell mutagenesis assay which demonstrates that chrysotile asbestos promotes a high level of homologous recombination between exogenous plasmid DNA and specific genes within the host cell genome. 2. Materials and methods 2. /. Detection of mutations in the lad gene Exposures for mutagenicity testing were done as follows. Rat2\ cells (Wyborski et al., 1995) were obtained from Stratagene and grown at 37C and 5% C03 in 75 cm2 flasks (Coming) until 25-40% con fluent. CM3 plasmid DNA (50 p.g in 70 p.1 of TE, pH 7.8), and Calidria chrysotile (Gan et al., 1993) (500 p.g in I ml of 140 mM NaCl, 25 mM HEPES, pH 7.4), were incubated together at 37C for 30 min before adding to 9 ml of serum-free Ham's nutrient mixture FI2 (J.R.H. Biosciences). The medium on the cells was aspirated and replaced by the chrysotile mixture. After 3 h, the mixture was aspirated, and the cells were washed twice with Ham's F12. Cal cium phosphate transfections were done as described previously (Gan et al., 1993) with 50 jxg of CM3 plasmid DNA, either untreated or y-irradiated with a Co source (total dose, 999.7 Gy), per 10 ml of precipitate suspension. Other controls were exposure to either 4.7 mM ethyl methanesulfonate (EMS, Sigma) or Calidria chrysotile (50 p.g/ml; 6.7 (xg/cm2) without DNA. After the cells were treated, each flask was washed and cells propagated in Dulbecco's modified Eagle's medium (Bio Whittaker) containing 11% fetal bovine serum, penicillin and streptomycin (Gibco). Following exposure to test agents, cells were grown for 1.5 doublings and har vested. Cell pellets were stored at -- 70C for muta tional analysis. For the mutagenesis assay, genomic DNA was prepared from treated or untreated Rat2\ cell pellets thawed and uniformly dispersed in 7 ml of 0.25 M sucrose with 20 mM Tris HC1 and 10 mM EDTA with a Dounce homogenizer. The proteinase K diges tion and subsequent phenol: chloroform extractions were performed as described (Shephard et al., 1993). Lambda shuttle vectors were rescued as infectious phage by exposing the genomic DNA, dissolved in Tris/EDTA, pH 7.4, to Transpack X-packaging ex tracts as described by the supplier (Stratagene). `Plat ing group' specifies genomic DNAs which were RECEIVED TIME SEP. 16. 9:57AM PRINT TIME SEP. 16. 10:01AM SEP 16 2002 10:55 PR CISTI ICIST 613 998 4926 TO 12145201181 P. 05 K. Le:aii-Gtyilit et/. /Mutation Rexearch .16/ t/WW / L1-/20 115 packaged and plated on the same day. The phage were assayed for lad mutations using previously described procedures (Mirsalis et al., 1993; Tinwell et ah, 1994). Identified blue plaques were removed and the agar plugs were stored at 4C in 500 p.1 of SM buffer with 20 p.1 of chloroform. Plaques were confirmed and purified by replating at low density. A blue plaque from a low density plate was selected and stored for sequence analysis (below). A packag ing control experiment was done as described above except that a 5:1 ratio of untreated genomic Rat2X. DNA and CM3 plasmid DNA, respectively, was added to the \-packaging extracts. 2.2. Sequence analysis pLIZ plasmids were excised from isolated, repuri fied mutant (blue) phage plaques according to the manufacturer's instructions (ExAssist/SOLR Sys tem, Stratagene) using M13 helper phage and E. coli strains XL-1 Blue and SOLR. SOLR colonies con taining pUZ plasmids were selected by ampicillin resistance and grown overnight in 10 ml bacterial cultures from which plasmid mini preps were iso lated by alkaline lysis (Sambrook et al., 1989). Plas mids were redissolved in 200 p.1 of TE, pH 7.4, containing DNase-free pancreatic RNase (Sigma), 20 p,g/ml. Plasmids were then characterized by elec trophoresis in agarose gels from which DNA concen trations were estimated. In sequencing reactions, plasmids were used as DNA template and an oligo nucleotide complimentary to base pairs 848-865 of the lad gene, 5'-GAC GAT ACC GAA GAC ACC S', was used as a primer, together with Taq poly merase and chain terminating, dye-labeled deoxyribonucleoside triphosphates. Reaction products were analyzed on an Applied Biosystems 373A automated sequencer (Smith et al., 1986; Connell et al., 1987; Halloran et al., 1993) which typically yielded 300 350 bases of readable sequence. 3. Results and discussion The Rat2\ assay differs from conventional mam malian cell mutagenesis assays in three important respects: (1) Rat2X cells (Wyborski et al., 1995) bpS7T Fig. I. The X LIZ shuttle vector, modified from Wyborski et al. (1995), which contains the target for the mutagenesis assay. The X. LIZ construct (lower bar) contains a central cassette which can be excised as a phagemid, pl.lZ (upper bar). The Rat2X cell line (Big Blue rat cell line, Stratagene) carries 50-70 copies per cell of the modified X LIZ vector stably integrated into the chromosomes ar two distinct sites on separate chromosomes (Wyborski et al., 1995). The upper bar, flanked by fl filamentous phage origins, represents that pan of the vector which is excised (Mirsalis et al,, 1993) as a phagemid. pUZ, with ExAssist helper phage (Stratagene). The length of the DNA inserted into the X arms was 4480 bp. Upon excision, however, the length of the excised pLIZ plasmid is 4334 bp (Wyborski, D.L., personal communication). The mutational target gene contained in pLIZ is the Lac repressor protein gene. lacl. Also contained in pLIZ is atacZ, an ampicillin-resistance geae and a colEl origin of replication. Control mutant 3 (CM3), provided by Stratagene as a reference standard, forms dark blue plaques on X-gai indicator plates and carries a point mutation of C -* T at bp 977 (vertical arrow) in the lad gene. This mutation converts a CAG codon (glutamine) to % TAG codoa (stop). contain multiple copies of the target gene, lad (Fig. l); (2) determinations of mutant frequencies are made in a prokaryotic format (a bacteriophage plaque as say) using DNA isolated from cells exposed to test mutagens in cell culture; and (3) mutants are identi fied not by metabolic selection, but by visual screen ing for mutant blue plaques amidst wild-type white plaques. The assay for homologous recombination is based on the following principle: Rat2X. cells are transfected with CM3 plasmid DNA (Fig. 1), which is a pLIZ plasmid containing a marker amber muta tion in the lad gene. The introduced mutant lad gene can be subsequently recovered from the ge nomic DNA of the transfected, cells by in vitro ^-packaging if, and only if, the introduced plasmid sequences have undergone recombination with ho mologous X sequences residing in the Rat2\ genome (Kohler et al.. 1991; Wyborski et al., 1995). In the absence of such recombination, the CM3 plasmid RECEIVED TIME SEP. !6. 9:57AM PRINT TIME SEP. 16. 10:01AM SEP 16 2002 10:55 FR CISTI ICIST 613 998 4926 TO 12145201181 P. 116 K. Lezon-CeyJa et at. /Mutation Research 36! t IV96) !33-120 Qinnot be recovered by in vitro packaging because it is too short and lacks X. cost sites. The region of homology between newly introduced and genomically integrated sequences extends over the entire length (4334 bp) of the CM3 plasmid, except for bp 977 of lad (Fig. 1). If a fragment containing the marker mutation stop codon undergoes homologous recombination with one of the lad genes in the Rat2X genome, then that genomic lad gene will subsequently produce dark blue plaques on agar containing X-gai. Once such a blue plaque is iso lated, pLIZ can be excised and its lad gene se quenced to determine if the CM3 marker mutation is present. Rat2\ cells treated with chrysotile and CM3 plas mid DNA have a mutant frequency more than 3-fold higher than media or untreated controls (Table 1) and nearly half as high as cells treated with EMS, a potent mutagen which preferentially induces G:C A:T transitions (Pienkowska et al., 1993). The media control and untreated cells show spontaneous mutant .frequencies not significantly different from those repotted previously (Wyborski et al., 1995). DNA from EMS-treated cells have a 6-fold increase in mutant frequency over the media control. Cells treated with chrysotile asbestos alone show a 2.5-fold increase over the media control. This value is signifi cantly (p < 0.05) higher than the spontaneous mu tant frequency. None of the samples exposed to calcium phosphate precipitation show a significant difference in mutant frequency indicating that trans fection of the CM3 plasmid into the Rat2X cells is not, in itself, sufficient to increase mutant frequency. Sequences of mutant lad genes from recovered phage reveal the extent of asbestos recombinogenicity. More than half (53.8%) of the lad sequences from mutant phage recovered from cells exposed to chrysotile + CM3 plasmid contain the CM3 marker mutation (C -> T at bp 977, Fig. 1). Fig. 2 shows sequences of plasmids recovered from the mutagene sis assay. One plasmid has a wild-type sequence and the second plasmid contains the CM3 marker muta^ tion. This point mutation is not seen in any sequence ' from other treatment groups. This indicates that as bestos promotes the recombination of the lad gene bearing the marker mutation with the X constructs residing in the Rat2X genome. The results summa rized in Table I demonstrate that a region of the aaoqgcaatCagctgttg I I 1. I I Fig. 2. Sequences of loci (bp 968-985) from * wild-type plasmid (a) and a bp-977 C -*T mutant plasmid (b). The plasmids were recovered from blue plaques identified in the mutagenesis assay. In this assay, identification of the marker mutation (C -T at bp 977) in a mutant plasmid is taken as evidence that a homologous recombination event occurred in the Rat2X celt from which the mutant vector was recovered. Atrows indicate bp 977. The se quencing reaction products were analyzed on an automated se quencer (see Materials and methods). CM3 plasmid containing the marker mutation has undergone homologous recombination in 7 of the 13 mutants derived from cells treated with chrysotile + CM3 plasmid. It is important to raise the question of whether or not the seven mutants with the CM3 marker muta tion reflect independent mutational events and to consider the possibility that they might have resulted from the expansion of a single mutant clone. In this experiment, following the 3-h exposure to test agent, the treated cultures were allowed to undergo 1.5 doublings; consequently, it is not possible to attribute all seven mutants to the postexposure expansion of a single mutant clone. The possibility that these seven mutants arose by a clonal expansion which occurred prior to exposure is effectively precluded by the failure to detect the marker mutation in any of the parallel control cultures all of which were seeded from the same starter culture shortly before exposure to test agents (Table 1). RECEIVED TIME SEP. 16. 9:57AM PRINT TIME SEP. 16. 10:01AM SEP 16 2002 10:56 FR CISTI I CIST 613 998 4926 TO 12145201181 P. 07 K. Letoti-Crula el til./Mutation Research 3611/91)6) 113-120 -a ts u 2I ^ r f" r~ T OOf-OOOOO 10U 6U >> -a i| i oooooooo idodddod VVVAAAAA OOO c o d o' d o V V V | AI A I &p9--<0<n0,e^as ^ vi oom r* pvcjtrt*-r^\ooC6-^~oodP~drc^4s^rinn r^oof^*A2PC*'gf2 r^c\cJc>J--o4^tvr> OSRgoo Sc - CS N 2 -* N rJ - s QJOiWiOn"nitn0jfvl(o}SOOOQ'OrntCd$ nwhQvi^-jN JSi t3 h Chrys., Cslidria chryxotiki; C M 3, the introduced plasmid D N A . 1 CuP04, cntcium phosphate precipitation. ` CM 3 (y-irrad.), y-irndiated C M 3 plasmid DNA. k Packaging control experiment. tn n < <s a rOt *--- rp^* O'f^O--^i'ONO <fflUQ 5 <<flmUK.ILQ s -a a a = I4I1 m o su .8 5 + *3 + 8* +- uC 1113 5=> sillSISi UOUDOUOi ' RECEIVED TIME SEP. 16. 9:57AM PRINT TIME SEP. 16. 10:01AM SEP 16 2002 10:56 FR CISTI ICIST 613 998 4926 TO 12145201181 P. i IK K. Lczon-Geythi et ai. / Mutation Research 361! 1996) 113-120 To test that the CM3 plasmid cannot be recovered by in vitro X-puckaging (i.e., that contaminating CM3 plasmids do not contribute to our results), the packaging reaction was performed on a mixture of CM3 plasmid DNA and genomic DNA from un treated cells. Of the fourteen mutant lad genes isolated and sequenced, none have the marker muta tion at bp 977 (Table I). The result of this control is consistent with known requirements for in vitro Xpackaging and demonstrates that residual contami nating CM3 plasmids are not incorporated into Xphage in the packaging reaction to any appreciable extent. A total of 3240 base substitution mutations may be made in the 1080 base lad gene. If we assume that 1800 of these base substitutions (55.5% of the total) can give rise to a detectable mutant and that all 1800 mutations occur at similar frequencies, then we may estimate the likelihood that the results obtained were due to chance. The probability that, among a set of thirteen spontaneously arising mutants, seven or more would carry the same specific base change at the same base is given by the expression: (1800), _ _____ P 180013 p => 2.91 X 10-20 where (1800), = 1800 (1799) -(1800 - i + 1). This equation describes the probability that any mu tation would occur seven or more times among a set of 13 independent mutants. It does not take into account the fact that the mutation recovered multiple times was indeed identical to the marker mutation present on the transfected lad gene. Nor does it account for the fact that the marker mutation at base 977 is situated in a 31-base zone (960-990) in which detectable mutations occur only sparsely. Lastly, this calculation considers only base substitution muta tions and neglects the contributions of small and large deletions and insertions to the totality of possi ble lad mutations. In light of this exceedingly low probability, we conclude that the seven marker mutations occurred, not by chance, but instead by a facilitated process, namely homologous recombination. Classical tumor promoters are operationally inef fective at tumor induction when applied alone or before an initiator (mutagen). This lack of productiv ity and apparent lack of permanence has fostered the conventional view that all tumor promoters must enhance carcinogenesis by epigenetic, non-genotoxic mechanisms. This view, however, has been difficult to reconcile with the strong association of tumor promotion with oxygen radical formation (Cerutti, 1985) and DNA strand breaks (Bimboim, 1983) which are potentially and explicitly genotoxic, re spectively. Any agent which can increase the fre quency of mitotic recombination events can cause the expression of recessive mutations at tumor sup pressor loci, and by this mechanism act as a tumor promoter (Kinsella and Radman, 1978). The ability of mitotic recombination events to act as tumor promoters is absolutely contingent on prior exposure to mutagen (initiator) which is required to create recessive mutations at tumor suppressor loci (Passarge and Bartram, 1976; Kinsella and Radman, 1978; Cavenee et a]., 1983; Knudson, 1986). This dependence on prior initiation is completely concor dant with the classic concepts of tumor promotion (Berenblum, 1975). Both et al. (1995) have recently reported that treatment of human mesothelioma cells in culture with crocidolite asbestos fibers induces an increased frequency of loss of heterozygosity (LOH) at two loci on chromosome 6. These authors attribute this asbestos-induced LOH to either of two mechanisms: mitotic recombination or very large deletions. Ho mologous recombination events arc likely to play a pivotal role in both of these mechanisms.The fre quently observed synergism of asbestos fibers with other carcinogens has prompted classification of these fibers as tumor promoters. Moreover, asbestos fibers display two additional stigmata of tumor promoters, namely, enhancement of oxygen radical formation (Kamp et al., 1992) and induction of DNA strand breaks (Libbus et al., 1989; Kamp et al., 1995). Our present results indicate an ability of asbestos fibers to increase the frequency of recombination events between homologous sequences within the nucleus and suggest a genetic mechanism for the promoter like synergism of asbestos with other mutagens (Hei, 1991) and carcinogens (SelikofF et al., 1968; Jaurand, 1989; Fasy, 1991; Walker et al., 1992). Most RECEIVED TIME SEP. 16. 9:57AM PRINT TIME SEP. 16. 10:01AM SEP 16 2002 10:57 FR CISTI ICIST 613 998 4926 TO 12145201181 K, Le:.an-G<?vtttf er at./Mutation Research 361 (1996) f 13-120 119 asbestos-induced mitotic recombination events would be inconsequential in the absence of prior mutations on the recombining chromosomes. Following expo sures to other mutagens, however, the recombinogenicity of asbestos may acquire enhanced muta genic, and carcinogenic, potential. Acknowledgements We thank Drs. Ainsley Weston, Jim Wetmur and Yasunosuke Suzuki for their critical reading of the manuscript. This study was supported by the As bestos Victims Special Fund Trust and by NIH Grants T32ES-0765, P30ES-00928 and HL-37130. - References Appel, J.D., T.M. Fasy, D.S. Kohtz, J.D. Kohtz and E.M. Johnson (1988) Asbestos fibers mediate transformation of monkey ceils by exogenous plasmid DNA, Proc. Natl. Acad. Sci., USA, 85. 7670-7674. Begin, R.. J.-J. Gaolthier. M. Desmeules and G. 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