Preliminary molecular analysis of the TK locus in L5178Y large- and small-colony mouse lymphoma cell mutants

Mutation Research, 226 (1989) 253-258

253

Elsevier MUTLET0235

Preliminary molecular analysis of the TK locus in L5178Y large- and small-colony mouse lymphoma cell mutants R o n a l d R. C o b b , J a m e s M a r t i n , E l i s a b e t h K o r y t y n s k i , L i n d a M o n t e i t h a n d T h o m a s J. H u g h e s Centerfor Life Sciences and Toxicology, Chemistry and Life Sciences, Research Triangle Park, NC 27709 (U.S.A.)

(Accepted6 April 1989)

Keywords: TK locus;Mouselymphomacells;Molecularanalysis

Summary Mouse lymphoma cells of the L5178Y TK+/--3.7.2C line were exposed to sidestream and mainstream cigarette smoke condensates (CSC). Ceils which survived the trifluorothymidine (TFT) challenge fell in 2 classes: large- and small-colony formers. Southern blot analysis of N c o I - d i g e s t e d DNA from mutant colonies yielded 2 distinct restriction fragment banding patterns when probed with the thymidine kinase (TK) cDNA clone pMtk4. One such pattern was composed of 4 bands at 6.4, 5.5, 4.7 and 2.9 kilobase pairs (kb) and was identical to that of TK ÷/- controls. A second pattern differed from the first only in the absence of the 6.4-kb band. The majority (83/95) of both large and small colonies derived from cells exposed to CSC exhibited restriction fragment banding patterns lacking the 6.4-kb band. The data from the present study suggest that there is no association between mutant colony size and the presence of the 6.4-kb N c o I restriction fragment at the TK locus in the mouse lymphoma mutants analyzed.

The mouse lymphoma assay (MLA), developed by Clive and coworkers (1972), is an in vitro mutagenicity test in mammalian cells. Mouse lymphoma L5178Y TK+/--3.7.2C clonal line cells are heterozygous at the TK locus (Fisher et al., 1971). Mutations that occur at the single functional TK gene in L5178Y ( T K + / - ~ T K -/-) cells result in loss of thymidine kinase activity, thus allowing the Correspondence: Dr. R.R. Cobb, Center for LifeSciencesand Toxicology, Chemistry and Life Sciences, Research Triangle Park, NC 27709 (U.S.A.).

selection of mutant cells which are resistant to the cytotoxic effects of trifluorothymidine (TFT) (Moore-Brown et al., 1981). Two classes of TFTresistant (TK-deficient) mutants are formed, based on the criterion of colony size in soft agar cloning medium. Both large and small TFT-resistant colonies are heritably TK-deficient (Moore et al., 1985). The TK locus in the mouse has been mapped to chromosome 11 (Kozak and Ruddle, 1977). Recently, Applegate et al. (1987, 1988) found a heteromorphism between the 2 chromosome 11

0165-7992/89/$ 03.50 © 1989Elsevier SciencePublishers B.V. (BiomedicalDivision)

254

homologs in the TK +/- progenitor cells. The homologs of this chromosome in mouse lymphoma cells have been shown to possess a centromeric heteromorphism which allows them to be identified as chromosome 1la or 1lb. Karyotype analysis of small-colony TK-deficient mutants revealed that the majority possess chromosome l i b abnormalities ranging from 2-band deletions to whole chromosome translocations (Hozier et al., 1982). In contrast, large-colony TK-deficient mutants appeared karyotypically indistinguishable from the parental TK÷/--3.7.2C cells (Hozier et al., 1985). Restriction enzyme analysis of the DNA from the TK +/- progenitor ceils with NcoI revealed a unique restriction fragment banding pattern following hybridization to a mouse thymidine kinase cDNA clone, pMtk4 (Lin et al., 1986). Previous work by Applegate (personal communication) had shown that a 6.4-kb fragment, along with a 4.7-kb TK-specific restriction fragment, corresponded to the region that contained TK gene sequences. Applegate et al. (1987) also showed that the only detectable difference between the wildtype and mutant TK alleles in lymphoma cells is the presence of the 6.4-kb NcoI restriction fragment in the wild-type allele. Absence of this band in mutant cells would indicate that the TK locus had been deleted or altered in a manner which would prevent hybridization with the probe. In the present experiment, L5178Y TK+/--3.7.2C cells were exposed to dimethyl sulfoxide (DMSO), the solvent control, and complex fractions of sidestream or mainstream cigarette smoke condensates (CSC). TK - / mutants were selected in soft agar medium containing TFT. Southern blot analysis of DNA from large- and small-colony mutants was used to investigate the correlation between colony size and NcoI restriction fragment banding pattern of TKspecific DNA sequences, specifically the presence or absence of the 6.4-kb band. It should be emphasized that only a comparison between mutant colony size and detectable alterations of the TK locus (presence or absence of the 6.4-kb band) rather than the molecular mechanism of mutagenicity is presented.

Methods

Cell culture and mutagenesis The mouse lymphoma TK+/--~TK - / - cell culture mutagenicity assay was performed as described by Turner et al. (1984). Sidestream and mainstream CSC were collected as described by Monteith et al. (1987). CSCs were assayed in cultures of L5178Y TK+/--3.7.2C cells in the presence of Aroclor-induced rat liver $9. DMSO (10 /~l/ml) with $9 was included as solvent and positive control. Following cloning of treated mouse lymphoma cells in soft agar with TFT, both large- (greater than 0.6 mm in diameter) and small(less than 0.6 mm in diameter) colony mutants were isolated. Single colonies were picked (by sight) from each of 6 plates containing cigarette smoke condensates (200/~g/ml). Each colony was placed into 2 ml of FloP in a single well of a 6-well tissue culture dish (Linbro), and incubated for 7 days at 37°C in 5% CO2. Wells indicating growth were transferred into 15 ml of FloP, incubated at 37°C, and shaken for 2 days in flasks to a density of 0.2-1.0 x 106 cells/ml. Cells were pelleted by centrifugation at 1200 rpm for 5 min at room temperature and DNA isolated as described below. DNA isolation High-molecular-weight genomic DNA was immediately isolated from each cell culture pellet by a modification of the technique for DNA isolation from mouse tails (Jenkins and Copeland, Frederick Cancer Institute, personal communication). Following isolation, each DNA sample was suspended in 400 #l sterile H20 and stored at - 70oC. Restriction enzyme analysis Approximately 10/~g (-20/zl) of each sample was digested with 20 units of the restriction enzyme NcoI or HindIII under conditions recommended by the supplier (Boehringer-Mannheim). The restriction fragments were separated by agarose gel electrophoresis and Southern-blotted onto Schleicher and SchueU BA85 nitrocellulose membranes (Southern, 1975). The immobilized DNA

255

was subsequently hybridized to nick-translated (Rigby et al., 1977) thymidine kinase cDNA probe pMtk4 (Lin et al., 1985) under the conditions recommended by Whitney et al. (1981). Autoradiography of the hybridized membranes was carried out with Kodak X-Omat AR X-Ray film and Kodak X-Omatic intensifying screens at - 7 0 ° C . Results

The mouse lymphoma assay was performed according to the procedures of Clive and Moore (Turner et al., 1984). It is clear from the data presented (Table 1) that several o f the endpoints o f the assay do not meet the criteria for an acceptable mutagenicity test. Specifically, the spontaneous mutation frequency was higher than expected (i.e., 178 × 10 -6 vs. 100 x 10-6). For these reasons, the treatment group o f each mutant colony selected for further investigation was not a factor in our interpretation o f the overall data, because in some cases the induced samples may contain a high proportion o f mutants o f spontaneous origin. However, the mutant colonies selected met the criteria of smallor large-colony mutants, as described by Clive and Moore (Turner et al., 1984), and this factor enabled us to evaluate the effect o f colony size. When HindIII-digested samples were analyzed, there were no differences between mutant and control cells in restriction fragment banding patterns. All such patterns were identical to those of T K ÷ / -

Fig. 1. Restriction enzyme analysis of large- and small-colony mutants. Approximately 10/Lg of genomic DNA was restricted with HindIII. The resulting restriction fragments were Southern-blotted and subsequently probed with 32p-labeled pMtk4. The resulting autoradiograph is shown. Samples 177, 124, 90, and 91 were taken from cells exposed to cigarette smoke fractions; samples 82 and 84 were taken from cells exposed to DMSO. TK +/- refers to the sample taken from the normal progenitor cell line L5178Y TK+/--3.7.2C. The numbers on the left-hand side refer to restriction fragment length sizes in kilobase pairs (kb). The (+) or ( - ) designation refers to the presence or absence of the 6.4-kb Ncol restriction fragment. )~ refers to )~ phage DNA digested with HindIII.

TABLE 1 MUTAGENIC ACTIVITY OF CONTROLS AND CSC IN THE MOUSE LYMPHOMA MUTAGENICITY ASSAY

Compound DMSOb CSC Bubbler, mainstream Bubbler, sidestream Sand, mainstream Sand, sidestream

Dose Relativeplating F (× 10-6) a (#g/ml) efficiency(070) 0

100

178

200 200 200 200

17 121 11 234

1,032 645 4,398 360

a Mutant frequency, average of 6 plates per dose. b DMSO exposure was 10/~l/ml.

control DNA regardless o f mutagen exposure or colony size (see Fig. 1). However, Southern blot analysis using NcoIdigested progenitor cell DNA produced a restriction fragment . attern composed o f bands o f 6 4 kb, 5.5 kb, 4 7 kb ~nci 2.9 kb in length (Fig. 2). When restriction fragment banding patterns of genomic DNA derived from TK - / - colonies differed from the TK ÷/- pattern, they did so only by the presence or absence of the 6.4-kb band. 80°70 of the 109 TFT-resistant colonies analyzed

256 TABLE 2

lllltlll

SOUTHERN BLOT ANALYSIS OF THE TK LOCUS IN LARGE AND SMALL COLONIES OF MOUSE LYMPHOMA CELLS a $9

Origin DMSO +

Large colonies + 6.4 kb band - 6.4 kb band Small colonies + 6.4 kb band - 6.4 kb band Total colonies analyzed

Cigarette smoke fractionsb +

Totals

1 4

3 22

4 26

9 0 14

9 61 95

18 61 109

a L5178Y TK+/--3.7.2C cells selected for the forward mutation TK + / - ~ TK - / - . NcoI digests probed with the cDNA clone pMtk4. All colonies scored yielded bands at 5.8, 4.7, 2.9 kb. b Contains both mainstream and sidestream fractions.

Fig. 2. Restriction enzyme analysis of large- and small-colony mutants. Approximately 10 #g of high-molecular-weight genomic DNA was digested with NcoI. Following Southern transfer of the restriction fragments, the membranes were hybridized to 32p-labeled pMtk4 and placed against X-ray film for 5 days. Samples 13, 15, 16, 20 and 21 were taken from cells exposed to cigarette smoke fractions; samples 5, 6 and 7 were taken from cells exposed to DMSO. The numbers on the righthand side refer to restriction fragment length sites in kilobase pairs (kb). The (+) or ( - ) designation refers to the presence or absence of the 6.4-kb restriction fragment. The TK ÷/- sample is from normal progenitor strain cells.

in the present experiment lacked this 6.4-kb band; the remaining 22 colonies possessed the banding pattern characteristics of TK ÷/- controls (see Fig. 2 and Table 2). Of the total colonies analyzed, 79 (72%) were small-colony mutants; of these, 61 (77%) lacked the 6.4-kb band. The 30 large-colony mutants yielded a slightly larger proportion (87%) of samples without the 6.4-kb band (Table 2). The proportion of colonies which contained the 6.4-kb band varied with compound exposure. Among CSC-induced mutants only 13% (12/95) were found to have the 6.4-kb band, while those mutants obtained after DMSO treatment yielded a much higher proportion (73 %) of mutants with the 6.4-kb band. It should be noted that mutants of all

possible classes (large colonies with and without the 6.4-kb band and small colonies with and without the 6.4-kb band) were found among the CSCexposed cell group (Table 2). The majority, however, 87% (83/95), lacked the 6.4-kb band. Discussion It is clear from the data presented on the mouse lymphoma assay, from which the mutant colonies were selected, that this assay did not meet the criteria for acceptance as a mutagenicity test. Due to the high background seen in this assay along with the high plating efficiency in some samples, it is highly possible that a number of the mutant colonies selected from each group are of spontaneous origin. For these reasons a discussion of the mutagenic spectrum and mechanism of mutagenicity of CSC would not be appropriate. The restriction fragment banding pattern at the TK locus was analyzed in mouse lymphoma mutant cells. These data indicated that the forward mutation TK +/- --, TK - / - does not always result in easily detectable changes at the TK locus. Evidence was presented that both large- and small-colony TK - / mutants harbored deletions of a portion of the structural TK gene. The data also indicated that in

257

some large and small TK - / - mutants, there were no detectable deletions of the active TK allele. Recently, Applegate et al. (1987) reported that chromosomal aberrations occurred in small-colony mutants but not in large-colony mutants in the mouse lymphoma assay. The aberrations seemed to be implicated in the slow-growth kinetics associated with small-colony mutants and consistently involved chromosome 11. The process of evaluating this hypothesis would include the testing of clastogens and an estimation of the relative proportions of the small- and large-colony mutant frequencies. However, to determine specifically whether or not the TK locus was involved in the clastogenic event requires additional molecular studies. The data presented in this report (small colonies that possess the 6.4-kb NcoI fragment) lead to the conclusion that other loci, possibly on other chromosomes, have been altered resulting in the small-colony phenotype. From the data presented here, a correlation between mutant colony size and the TK-specific DNA restriction fragment banding pattern was not detected. Examples of small-colony mutants that did not show any major rearrangement of the TK locus were detected. In addition to the TK locus, other genes involved in growth rate may have been affected by exposure to mutagens. However, the data do not allow a definitive conclusion to be drawn regarding the correlation between chromosomal damage (cytogenetically detectable) and colony size. Further investigations into possible correlations between mutant colony size, chromosomal aberrations, and TK-specific restriction fragment banding pattern alterations are currently being planned. These and other studies will allow definitive conclusions regarding the nature of mutant colony growth and the extent of damage to the genome in mouse L5178Y TK+/--3.7.2C cells.

Acknowledgements The secretarial assistance of Teresa Erexson and Donna Martin is deeply appreciated.

References Applegate, M., and J.C. Hozier (1988) On the complexity of mutagenic events at the mouse lymphoma thymidine kinase locus, in: M. Moore, B. Demarini, F.J. de Serres and K. Tindall (Eds.), Banbury Reports, No. 28, Mammalian Cell Mutagenesis, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, in press. Applegate, M., C. Broden, A. Wadhams, K. Kasweck, J. Hozier, M. Moore, D. Clive and A. Burrell (1987) In situ and molecular analysis of mutation at the TK locus in mouse LSI78Y/TK+/--3.7.2.C cells, Environ. Mutagen., 9 ($8), 5. Clive, D., W.G. Flamm, M.R. Machesco and N.J. Bernheim (1972) A mutational assay system using the thymidine kinase locus in mouse lymphoma cells, Mutation Res., 16, 77-87. Clive, D., A.G. Batson and N.T. Turner (1980) The ability of L5178/TK +/- mouse lymphoma cells to detect single gene and viable chromosome mutations: evaluation and relevance to mutagen and carcinogen screening, in: G.M. Williams et al. (Eds.), The Predictive Value of in Vitro Short Term Screening Tests in Carcinogenicity Evaluation, Elsevier/North Holland, Amsterdam, pp. 103-123. Evans, H.H., J. Mence, M.F. Horng, M. Ricanati, C. Sanchez and J. Hozier (1986) Locus specificity in the mutability of L5178Y mouse lymphoma cells: the role of multilocus lesions, Proc. Natl. Acad. Sci. (U.S.A.), 83, 4379-4382. Fisher, G.A., S.Y. Lee and P. Calabresi (1971) Detection of chemical mutagens using a host-mediated assay (L5178Y) mutagenesis system, Mutation Res., 26, 501-511. Hozier, J., J. Sawyer, D. Clive and M. Moore (1982) Cytogenetic distinction between the TK + and TK- chromosomes in the L5178Y/TK+/--3.7.2C mouse lymphoma cell line, Mutation Res., 105,451-456. Hozier, J., J. Sawyer, D. Clive and M. Moore 0985) Chromosome 11 aberrations in small colony L5178Y TK - / - mutants early in their clonal history, Mutation Res., 147, 237-242. Kozak, C.A., and F.H. Ruddle (1977) Assignment of the genes for thymidine kinase and galactokinase to Mus musculus chromosome 11 and the preferential segregation of this in Chinese hamster/mouse somatic cell hybrids, Somatic Cell Genet., 3, 121-133. Lin, P.-F., H.B. Lieberman, D.-B. Yeh, T. Xu, S.-Y. Zhao and F.H. Ruddle (1985) Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences, Mol. Cell. Biol., 5, 3149-3156. Monteith, L.G., D.M. Simmons, C.G. Myers, T.J. Hughes and L.D. Claxton (1987) Comparative genotoxicity of sidestream and mainstream cigarette smoke, Environ. Mutagen., 9 ($8), 75.

258 Moore, M.M., D. Clive, J.C. Hozier, B.E. Howard, A.G. Batson, N.T. Turner and J. Sawyer (1985) Analysis of trifluorothymidine-resistant (TFT) mutants of L5178Y/TK ÷/- mouse lymphoma cells, Mutation Res., 151, 161-174. Moore-Brown, M.M., D. Clive, B.E. Howard, A.G. Batson and K.O. Johnson (1981) The utilization of trifluorothymidine (TFT) to select for thymidine kinase-deficient (TK - / - ) mutants from L5178Y/TK+/--mouse lymphoma cells, Mutation Res., 85, 363-378. Rigby, P.W.J., M. Diekmann, C. Rhodes and P. Berg (1977) Comparative genotoxicity of sidestream and mainstream cigarette smoke, J. Mol. Biol., 113, 237-246. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98, 503-517.

Turner, N.T., A.G. Batson and D. Clive (1984) Procedures for the L5178Y TK +/- ~ TK - / - mouse lymphoma assay, in: B.J. Kilbey et al. (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 239-268. Whitney, J.B. III, J. Martinell, R.A. Popp, L.B. Russell and W.F. Anderson (1981) Deletions in the a-globin gene complex in a-thalassemic mice, Proc. Natl. Acad. Sci. (U.S.A.), 78, 7644-7647. Yandell, D.W., T.P. Dryja and J.B. Little (1986) Somatic mutations at a heterozygous autosomal locus in human cells occur more frequently by allele loss than by intragenic structural alterations, Somat. Cell Mol. Genet., 12, 255-263. Communicated by R.J. Preston