High-resolution cytogenetic characterization of the L5178Y TK± mouse lymphoma cell line

Mutation Research, 214 (1989) 181-193 Elsevier

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MUT 04771

High-resolution cytogenetic characterization of the L5178Y T K +/- mouse l y m p h o m a cell line Jeffrey R. Sawyer 1,,, Martha M. Moore 2 and John C. Hozier 1 I Medical Genetics Laboratory, Department of Biological Science, Florida Institute of Technology, Melbourne, FL 32901 (U.S.A.) and 2 Mutagenesis and Cellular Toxicology Branch, Genetic Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (U.S.A.) (Received 15 September 1988) (Revision received 3 March 1989) (Accepted 6 March 1989)

Keywords: High-resolution chromosome banding; L5178Y TK +/- mouse lymphoma cells; Cytogenetic characterization

Summaff High-resolution chromosome preparations from L5178Y T K +/- 3.7.2C mouse lymphoma cells were obtained using acridine orange in the cell harvest procedure. With this technique it is possible to visualize over 500 bands in elongated mouse lymphoma cell chromosomes as compared to the approximately 230 bands visualized in metaphase preparations. High-resolution lymphoma cell chromosomes are described, and chromosome rearrangements carried in the cell line are characterized by ideograms representing the position, number, size, and relative staining intensity of the G-band patterns. Use of elongated chromosomes of mouse lymphoma T K +/- mutants should facilitate analysis of the cytogenetic effects associated with T K +/ ~ T K - / - mutagenesis.

Recently high-resolution chromosome-banding techniques have been applied to the study of human birth defects and neoplasias. Studies with high-resolution banding have allowed the detection of previously unresolved chromosomal aberrations such as small deletions and translocations, many of which are associated with oncogene

Correspondence: Prof. J.C. Hozier, Medical Genetics Laboratory, Department of Biological Sciences, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL 32901-6988 (U.S.A.). * Present address: Department of Pathology, College of Medicine, University of Arkansas for Medical Services and Cytogenetics Laboratory, Arkansas Children's Hospital, Little Rock, 72205 (U.S.A.).

activation and progressive steps in cancer (Yunis, 1983). Since small chromosome aberrations can have such adverse impacts on the phenotype of the individuals in which they occur, the study of these same types of events in mammalian mutational assay systems should prove informative. Cell lines used in mutational analysis have seldom been characterized cytogenetically at high resolution even though such analyses might aid significantly in understanding induced mutational events in vitro. An important example is the L5178Y T K +/ --* T K - / - mouse lymphoma assay which allows quantitation of forward mutations at the autosomal thymidine kinase locus (Clive et al., 1979; Moore et al., 1985a,b; Turner et al., 1984). To date, standard cytogenetic analysis has been useful in studying the relationship between mutant

0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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phenotype (large vs. small colony size in soft agar) and abnormalities of chromosome 11 (Hozier et al., 1981, 1982; Moore et al., 1985a,b), the known location of the mouse thymidine kinase gene (Kozak and Ruddle, 1977). We have shown that chromosome aberrations occur in small-colony (slow-growing) mutants but not in large-colony (normal-growing) mutants (Hozier et al., 1981; Sawyer et al., 1985; Moore et al., 1985a,b). These aberrations seem to be implicated in the slow growth kinetics of small-colony mutants and consistently involve mouse chromosome 11. Since the L5178Y T K +/- assay system may detect a variety of genetic lesions, potentially spanning the range, from chromosomal aberrations to point mutations, the chromosomal analysis of small and large colonies at the highest possible level of band resolution is important in characterizing such mutants (Hozier et al., 1981, 1982; Moore et al., 1985a). To monitor more precisely the chromosomal rearrangements occurring in mouse lymphoma cell T K +/ --* T K / mutagenesis we have incorporated the use of acridine orange into the cellharvesting technique to induce more elongated and highly banded chromosomes (Matsubara and Nakagome, 1983). Acridine orange intercalates into the backbone of the DNA molecule and inhibits the process of chromosome condensation (Sawyer and Hozier, 1986). With this technique we have been able to visualize over 500 bands in the late-prophase stage of chromosome condensation compared to the approximately 230 bands visualized in metaphase preparations. The use of this technique makes it possible to determine the character of chromosomal events associated with T K +/- ~ T K / mutagenesis that may be too small to resolve at the metaphase band level.

sists of 30 /~l of a 500 btg/ml solution of acridine orange (Matsubara and Nakagome, 1983), 120 /~l of a 10 /~g/ml solution of colcemid and 9 ml of 0.075 M KC1. The cells were hypotonically treated for 20 min at 37 ° C, after which they were spun at 100 x g for 10 min and fixed with 3 : 1 methanol : acetic acid. The fixative was added drop-wise with gently agitation to the cell suspension to avoid clumping. The cells remained in the first fixative for 20 min followed by 3 fixative changes to eliminate cell debris and to insure good spreading and staining of chromosomes. Slide preparations were made by dropping the cells, resuspended in 3 : 1 methanol : acetic acid fixative, onto alcoholcleaned slides wetted with room temperature distilled water. Chromosome preparations were banded by treatment with 1 N HC1 for 20 min followed by a 90-min incubation in 50% formamide and 2 x SSC. The slides were then rinsed in tap water and dehydrated in 95% ethanol for 30 rain. Following dehydration and a 2-h room temperature drying period, the slides were stained with a 3:1 (v/v) phosphate buffer (pH 6.8) : Wright stain mix. The chromosome ideogram presented here is related to D B A / 2 mouse chromosome ideograms which have been published elsewhere (Sawyer and Hozier, 1986; Sawyer et al., 1987). Certain homologous pairs of T K +/- 3.7.2C chromosomes show heteromorphic centromeres or other chromosomal distinctions; therefore a suffix (a or b) is used to distinguish the homologous chromosomes. Derivative chromosomes of unknown origin in the T K +/- 3.7.2C cell line are designated marker (m) chromosomes. These chromosomes are unrecognizable and morphologically distinct from normal, but appear consistent in their own banding patterns.

Materials and methods Results

The mouse lymphoma L5178Y T K +/ 3.7.2C cell line was maintained in our laboratory under conditions normally used in the mutagenesis assay (Turner et al., 1984). In the harvesting procedure developed for this analysis T K +/- cells (10-ml cultures at about 0.5 x 10 6 cells/ml) were spun at 100 x g for 10 min, the supernatant growth medium was removed and the cells were placed in a hypotonic solution. The hypotonic solution con-

High-resolution G-banding of mouse lymphoma cell chromosomes allowed the confirmation of chromosome assignments previously made at the metaphase stage of chromosome condensation (Sawyer et al., 1985). Elongation of the lymphoma chromosomes using the technique described here reveals sub-banding patterns that provide greater precision in the assignments of breakpoints in

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Fig. l(g) Fig. 1. Examples of G-banded mouse lymphoma chromosomes showing decreasing stages of mitotic condensation. From left to right are shown representative chromosomes from metaphase through late prophase. The solid line indicates centromere position. (a) Chromosomes la-3; (b) 4a-6a; (c) 6b-9a; (d) 9b-12; (e) t(12;13)-15; (f) 16a-19; (g) X-M3.

structural rearrangements. The high-resolution Gbanded chromosomes of L5178Y TK ÷/- 3.7.2C cells are presented in Fig. la-g. Fig. 2 illustrates the late-prophase ideogram for the mouse lymphoma cell chromosomes and assigns numbers to the sub-banding patterns related to landmark bands (Nesbitt and Franke, 1973). The comparison of the DBA/2 karyotype with that of the TK +/- 3.7.2C cell line for structural chromosome rearrangements at high resolution indicates the presence of numerous rearrangements. These will be discussed with the DBA/2 high-resolution karyotype considered as normal (Sawyer et al., 1987), since this mouse strain was the origin of the 3.7.2C mouse lymphoma cell line used in the assay. Chromosomes No. 1 at high resolution reveal the splitting of landmark band 1E1 into 2 and

sometimes 3 sub-bands. Increased banding is found in the distal region H, but it is indistinct. Chromosome la is slightly shorter than chromosome lb, apparently due to a deletion of part of the centrometric region in la (Fig. la). The chromosome 2 show sub-banding of landmark bands 2G1 and in region 2C, and are apparently unchanged from DBA/2. In chromosomes 3 the band 3F subdivides and reveals 2 sub-bands F2.1 and F2.3. The chromosomes 3 also have remained unchanged from the normal mouse. The chromosomes 4 of the TK +/- 3.7.2C line include 1 normal (4a) homologue and 1 (4b) with a segment translocated onto the distal end. The translocated segment contains several G-positive sub-bands in this region, but its origin remains unknown (Fig. lb). The T K ÷ / - 3.7.2C cell line contains 1 normal

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Fig. 2. Schematic representation of the high-resolution chromosomes of L5178Y TK ÷ / - 3.7.2C mouse lymphoma cell line. The nomenclature used here follows that previously published for the mouse (Nesbitt and Franke, 1973; Sawyer and Hozier, 1986; Sawyer et al., 1987). Where the homologues are identical (i.e., chromosomes 2, 3, 8, 10, 17 and 19) only one ideogram is presented. The origin of the proximal portion of chromosomes 4b, 9b and 15 are in question and have not yet been assigned band designations.

191

chromosome 5a; chromosome 5b has an interstitial deletion from bands A2 through B1. Subbanding is apparent in region 5E, as band E3 subdivides. The one normal chromosome 6a shows a high degree of sub-banding. Major bands such as C1 and E1 clearly subdivide. Chromosome 6b, which had been tentatively identified as the deleted homologue of chromosome 6a (Sawyer et al., 1985) now reveals sub-bands that we believe confirm its assignment (Fig. lc). For chromosome 7, a normal homologue (7a) reveals sub-bands in region 7B while chromosome 7b contains a centromeric heteromorphism and a paracentric inversion with breakpoints involving bands 7D2 and 7F2. The heteromorphism shown by the chromosomes 7 is apparently a deletion of part of the centromeric region of chromosome 7a. Both chromosomes 8 in the mouse lymphoma 3.7.2C cell line appear normal at high resolution. Chromosome 9a reveals sub-banding in region 9El and an abnormal homologue (9b) contains a translocation of several G-positive bands onto the distal band (F3) of the chromosome. The chromosomes 10 reveal sub-banding in regions B and D and several sub-bands sometimes apparent in band D1. High-resolution banding reveals no structural changes (Fig. ld). The chromosomes 11 of the cell line reveal clear sub-banding in regions B, C and E. The only detectable difference in the chromosomes 11 is a centromeric heteromorphism in one of the homologues (11a) which makes it slightly shorter. This heteromorphism is probably a deletion of centromeric heterochromatin and has been discussed in detail elsewhere (Hozier et al., 1982). The chromosomes 12 are clearly sub-banded in region C and have an extra segment on the distal end of their long arms. This segment is on both chromosomes and appears to contain several Gpositive and G-negative bands at high resolution. Also, one of the chromosomes 12 is involved in a Robertsonian translocation (centromere to centromere joining) with one of the chromosomes No. 13. This fusion produces a very large metacentric chromosome which is the most conspicuous chromosome in the cell line. The chromosomes 13 are sub-banded in regions

C and D. One of the chromosomes 13 is involved in a centromeric fusion with chromosome 12. The normal chromosome 14 subdivides distinctly in regions C and E. A centromeric heteromorphism in chromosome 14b and a terminal deletion at band D1 made this a tentative assignment previously (Sawyer et al., 1985). With the aid of the additional bands revealed by high resolution it has been possible to follow the splitting of landmark bands (ex: C1) and thus to confirm the assignment. One abnormal chromosome 15 is apparent. This chromosome 15 may be part of a complex rearrangement with 2 other chromosomes. The centromeric region is, based upon its high-resolution banding pattern, apparently from chromosome 5b; the area just below this segment is of unknown origin. The chromosome 15 centromeric region is deleted with regions B1 through the distal end remaining intact (Fig. le). The chromosomes No. 16 are sub-banded clearly in region B1. The absence of part of the centromere in one of the homologues (16b) makes it slightly smaller (Fig. lf). The chromosomes No. 17 subdivide in region E and clearly elongate almost uniformly through their length, probably due to the large number of G-negative bands. One normal chromosome, 18a, appears to clearly subdivide in regions B and D. The other chromosome, 18b, is involved in a translocation with the distal portion of chromosome 6. That is, the distal section of chromosome 6 (bands C through G) is translocated to the distal end of chromosome 18. The chromosomes 19 subdivide in region C from 3 bands in metaphase to 7 bands at high resolution. Both chromosomes 19 are apparently normal at high resolution. The X chromosome in the cell line subdivides in regions C and E, but in most cases is not distinctly banded. There are 3 marker chromosomes in the T K +/3.7.2C cell line, which cannot be assigned counterparts in the normal karyotype (Fig. lg). Even with the high-resolution banding these markers cannot be positively matched with normal chromosomal segments (Fig. lg).

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Discussion A m a j o r difficulty in a d o p t i n g existing high-resolution techniques for work on the L5178Y T K ÷/ ~ T K - / - m u t a g e n assay system was the fact that the D N A m e t a b o l i c a l t e r a t i o n in T K - / clonal m u t a n t s interferes with c o m m o n l y used techniques of cell s y n c h r o n i z a t i o n a n d chrom o s o m e elongation, which use m e t h o t r e x a t e a n d b r o m o d e o x y u r i d i n e (Yunis, 1983). Instead, a technique using acridine orange ( M a t s u b a r a a n d N a k a g o m e , 1983) was a d a p t e d which does not d e p e n d u p o n the m e t a b o l i c p a t h w a y c o n t a i n i n g t h y m i d i n e kinase. The results p r e s e n t e d here indicate a stable k a r y o t y p e for the T K + / - 3.7.2C cell line c o n t a i n ing n o r m a l m o u s e c h r o m o s o m e s , recognizable c h r o m o s o m e r e a r r a n g e m e n t s , a n d m a r k e r chromosomes. The high-resolution analysis has allowed the c o n f i r m a t i o n of c h r o m o s o m e assignm e n t s that were previously only tentative (Sawyer et al., 1985), such as those of c h r o m o s o m e s 6b a n d 14b. By high-resolution analysis there a p p e a r s to be at least 1 n o r m a l m o u s e h o m o l o g u e for each c h r o m o s o m e pair, with c e n t r o m e r i c h e t e r o m o r p h i s m s caused b y deletions or o t h e r a l t e r a t i o n s of c e n t r o m e r i c h e t e r o c h r o m a t i n occurring in 5 p a i r s of homologues. T h e detail offered b y high-resolution G - b a n d e d c h r o m o s o m e s allows the analysis of the m o u s e l y m p h o m a 3.7.2C k a r y o t y p e at a m u c h finer level. W i t h the technique d e s c r i b e d here it is p o s s i b l e to resolve c h r o m o s o m e a b e r r a t i o n s associated with T K ÷/ ~ T K / mutagenesis to a level of resolution of m o r e than twice that previously r e p o r t e d (Sawyer et al., 1985). A n initial analysis of chrom o s o m e a b e r r a t i o n s in the m o u s e l y m p h o m a assay by high-resolution b a n d i n g is p r e s e n t e d elsewhere ( H o z i e r et al., 1989), which c o m p l e m e n t s ongoing analysis of m u t a n t s at the m o l e c u lar genetic level.

Acknowledgements The technical assistance of K e i t h Hoyle, J a n e t J u d d and D i a n e G o l d b e r g a n d the editorial assistance of D e b b i e L a F r a n c e , J a n c e Sims a n d L i n d a M a t h e w s are gratefully a c k n o w l e d g e d .

This w o r k was s u p p o r t e d in p a r t by C o o p e r ative A g r e e m e n t No. CR0813969-01 f r o m the U.S. E n v i r o n m e n t a l P r o t e c t i o n Agency. This m a n u s c r i p t has b e e n reviewed by the H e a l t h Effects R e s e a r c h L a b o r a t o r y , U.S.E.P.A., a n d a p p r o v e d for p u b l i c a t i o n . A p p r o v a l does not signify that the c o n t e n t s necessarily reflect the views a n d policies of this Agency, n o r does m e n tion of t r a d e n a m e s o r c o m m e r c i a l p r o d u c t s constitute e n d o r s e m e n t or r e c o m m e n d a t i o n for use.

References Clive, D., and J.F.S. Spector (1975) Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse [ymphoma cells, Mutation Res., 31, 17-29. Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M. Brown (1979) Validation and characterization of the L5178Y/TK +/ mouse lymphoma mutagen assay system, Mutation Res., 59, 61-108. HoNer, J., J. Sawyer, M. Moore, B. Howard and D. Clive (1981) Cytogenetic analysis of the L5178Y TK +/ --, T K - / - mouse lymphoma mutagenesis assay system, Mutation Res., 84, 168-181. HoNer, J., J. Sawyer, D. Clive and M. Moore (1982) Cytogenetic distinction between the TK + and TK chromosomes in the L5178Y TK +/ 3.7.3C mouse lymphoma cell line, Mutation Res., 105,451-456. HoNer, J., J. Sawyer and M. Moore (1989) High-resolution cytogenetic analysis of L5178Y TK +/ 3.7.2C cells: variation in chromosome 11 breakpoints among small-colony TK / mutants, Mutation Res., 214, 195-199. Kozak, C.A., and F.H. Ruddle (1977) Assignment of the genes for thymidine kinase and galactokinase to Mus musculm" chromosome 11 and the preferential segregation of this chromosome with Chinese hamster/mouse simatic cell hybrids, Somat. Cell Genet., 3, 121-13l. Matsubara, T., and Y. Nakagome (1983) High resolution banding by treating cells with acridine orange before fixation, Cytogenet. Cell Genet., 35, 148-151. Moore, M.M., D. Clive, B. Howard, A. Batson and N. Turner (1985a) In situ analysis of trifluorothymidine-resistance (TFT r) mutants of L5178Y/TK +/ mouse lymphoma cells, Mutation Res., 151, 147-159. Moore, M.M., D. Clive, J.C. HoNer, B.E. Howard, A.G. Batson, N.T. Turner and HJ. Sawyer (1985b) Analysis of trifluorothymidine-resistance (TFTr) mutants of L5178Y/TK +/ mouse lymphomas cells, Mutation Res., 151,161-174. Nesbitt, M.W., and U. Franke (1973) A system of nomenclature for band patterns of mouse chromosomes, Chromosoma, 41, 145-158. Sawyer, J., and J. Hozier (1986) High resolution of mouse chromosomes: Banding conservation between man and mouse, Science, 232, 1632-1635.

193 Sawyer, J., M.M. Moore, D. Clive and J. Hozier (1985) Cytogenetic characterization of the L5178Y TK +/- 3.6.2C mouse lymphoma cell line, Mutation Res., 147, 243-253. Sawyer, J., M.M. Moore and J. Hozier (1987) High resolution G-banded chromosomes of the mouse, Chromosoma, 95, 350-358. Turner, N.T., A.G. Batson and D. Clive (1984) Procedures for

the L5178Y/TK +/ ~ TK - / - mouse lymphoma assay, in: B.J. Kilbey, M. Ligator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenesis Test Procedures, Elsevier, New York, pp. 239-268. Yunis, J. (1983) The chromosomal basis of human neoplasia, Science, 221,227-236.