Mutagenesis, Vol. 14, No. 1, 57-61,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Dual-colour FISH analysis to characterize a marker chromosome in cytochrome P450 2B1 recombinant V79 Chinese hamster cells
1 Genetica e Biochimica Tossicologica, Istituto di Mutagenesi e Differenziamento, CNR, via Svezia 10, 56124 Pisa, Italy, 2 MGC, Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, Medical Centre, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands and 3 Institut fur Toxikologie und Umwelthygiene, TU Munchen, Lazarettstrasse 62, D-80636 Munchen, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Recombinant V79 Chinese hamster cell lines have been constructed for the expression of useful functions, e.g. cytochromes P450, in order to study metabolism-dependent toxicity. Recombinant cell lines are derived as single clones upon gene transfer and selection from the parental V79 cell line. It is of fundamental importance for a reliable application of the recombinant cell lines to show that the original biological characteristics of the parental line are maintained. As part of these efforts, fluorescence in situ hybridization with Chinese hamster chromosome-specific DNA libraries was performed in order to identify the origin of the chromosomes from which a marker chromosome present in the recombinant cell line V79MZr2B1 was derived, which could not be identified by standard cytogenetic techniques.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The V79 cell line was established from Chinese hamster lung cells (Ford and Yerganian, 1958
) and is being widely used for somatic cell genetics and mutagenesis studies (Colella et al., 1988; Swierenga et al., 1991; Ellard and Parry, 1993). Due to a low chromosome number (2n = 22 chromosomes), a short doubling time of <12 h and a cloning efficiency of >90%, the V79 cell line has also proved to be a suitable biological system for cytogenetic studies (Simi et al., 1988; Hermine et al., l997).
The V79 Chinese hamster cell line was chosen as host for heterologous expression of xenobiotic metabolizing enzymes, e.g. cytochromes P450, because in these cells no xenobiotic metabolizing cytochrome P450 has ever been detected (Doehmer et al., 1988). Several cytochromes P450 from human, rat and fish are being stably expressed in V79-derived cell lines comprising the V79 Cell Battery (Doehmer and Greim, 1993).
Recently we have used three cell lines genetically enginereed to express rat cytochromes CYP1A1 (V79MZr1A1), CYP1A2 (V79MZr1A2) and CYP2B1 (V79MZr2B1) to evaluate the clastogenicity of a class of anthraquinones (Simi et al., 1995). In the course of this study we also confirmed the presence of a marker chromosome, as reported elsewhere (Ellard et al., 1995). This marker was easily recognizable even without banding techniques, due to its anomalous morphology.
In this work a G-banding karyotype analysis of the engineered V79MZr1A1, V79MZr1A2 and V79MZr2B1 cell lines in comparison with the parental V79MZ cell line was carried out both to cytogenetically characterize these cell lines as well as to identify the V79MZr2B1 marker chromosome.
Since it was not possible to determine the origin of the marker chromosomes by standard cytogenetic techniques (i.e. G-banding), dual-colour fluorescence in situ hybridization (FISH) analysis with chromosome-specific painting probes generated for Chinese hamster (Xiao et al., 1996) was performed.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The parental V79MZ and the V79 Chinese hamster-derived cell lines were cultured in Dulbecco's modified Eagle's mimimal essential medium (DMEM) supplemented with 5% fetal calf serum. An aliquot of 400 µg/ml G 418 was also added to the DMEM for the V79-derived cell lines.
Metaphase chromosome spreads were prepared from exponentially growing culture by standard cytogenetic techniques. G-banding was obtained by a modification of the trypsin digestion procedure (Seabright, 1971). The slides were treated with 0.05% trypsin in 0.9% NaCl for 5–45 s at room temperature, washed in Sorensen buffer and then stained with 5% Giemsa in the same buffer for 3–5 min. The proposed nomenclature of Ray and Mohandas (1976) for Chinese hamster chromosomes was used for karyotyping.
The development of chromosome-specific DNA libraries has been described in detail elsewhere (Balajee et al., 1995; Xiao et al., 1996)
Dual-colour FISH was performed as previously described (Xiao et al., 1996). Briefly, amplified DNA was directly labelled with digoxigenin-11-dUTP or with biotin-16-dUTP by conventional PCR. Aliquots of 100 ng of labelled probe were added to the hybridization buffer (50% formamide,10% dextran sulphate, 2x SSC, 400 ng hamster Cot-1 DNA), incubated at 37°C for 1 h and denatured together with the slides at 80°C for 3.5 min. Hybridization was carried out overnight at 37°C in a humid chamber. After three washes in 50% formamide, 2x SSC at 42°C and three washes in 2x SSC, the biotynilated probe was detected with avidin–FITC, biotin–avidin and avidin–FITC. The probe labelled with digoxigenin was detected with sequential mouse anti-digoxigenin, sheep anti-mouse–digoxigenin and sheep anti-digoxigenin–TRITC. Slides were counterstained with 4,6-diamidino-2-phenylindol in antifade.
Colour images were collected using a computer controlled Zeiss epifluorescence microscope equipped with a CCD camera (Photometrics) and IPlab Spectrum software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The G-banded karyotypes of genetically engineered V79MZr1A1, V79MZr1A2 and V79MZr2B1 together with the parental V79MZ cell line are shown in Figure 1
. V79MZr1A2 revealed an identical karyotype to the parental line. V79MZr1A1 was characterized by the presence of a deleted chromosome (Chr) 5q, originating from a normal Chr 5 which had lost its p arm. In all the metaphases analysed, V79MZr2B1 cells showed the presence of a marker chromosome easily recognizable even in non-banded metaphases: it is a sub-telocentric, medium sized chromosome, with a small p arm and a q arm variable in length. The banding pattern of this marker did not allow a precise identification of its origin.
|
The V79MZr2B1 karyotype was also characterized by the absence of chr 8der in all methaphases. Further chromosome instability of Chr 7 (absent in ~60% of the metaphases analysed) and Chr 9 (one copy of which was absent in 20–30% of the metaphases analysed) (Figure 1
) was observed. For this reason, dual-colour FISH was performed using a digoxigenin-labelled Chr 7 probe and a biotinylated Chr 9 probe, both obtained by flow sorting and found to be specific (Balajee et al., 1995; Xiao et al., 1996).
The hybridization patterns of these two probes were analysed first in the V79MZ parental line (Figure 2A). The hybridization signals from the Chr 7 probe (red) clearly showed the lack of a normal Chr 7, while positive signals were found in several chromosomes. Upon G-banding analysis, the signals were localized: (i) in the long arms of two acrocentric chromosomes identified as Chr 7 and Chr 8der, respectively; (ii) interstitially in the long arm of Chr 7q+; (iii) in the long arm of a small sub-metacentric chromosome identified as Chr 9; (iv) in the two small telocentric chromosomes Chr 5p and Chr 6p.
|
The hybridization signals from the Chr 9 probe (yellow) showed an intact Chr 9 and two additional signals which were located on the short arms of Chr 7 and Chr 8der. The long arms of these chromosomes were also labelled by the Chr 7 probe.
The hybridization patterns in V79MZr2B1 cells (Figure 2B) showed that six chromosomes were identical to those found in V79MZ (5p, 6p, 7, 7q+ and two Chr 9), while Chr 8der was absent and was replaced by a marker the origin of which remained the same as Chr 8der, but presented a very long q arm derived from amplification of Chr 9.
The hybridization pattern as a whole is coherent with the modal G-banded karyotype 21, –7, –8der, +marker observed in V79MZr2B1 cells (Figure 1).
In addition, the V79MZr2B1 marker chromosome showed a high degree of instability. Some of the abnormal configurations are depicted in Figure 3.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The V79 Chinese hamster cell line has been widely used as a test system in toxicology for many years. Therefore, it was worthwhile to genetically engineer V79 cells for xenobiotic metabolizing enzymes, e.g. cytochromes P450, and to apply these cell lines in cytotoxicity and genotoxicity studies. However, previous observations and the results obtained during this study emphasize the importance of a thorough check both of the parental and the recombinant cell lines before these cells are used in long-term studies.
Following G-banding analysis, V79MZ showed a slightly different karyotype when compared with other V79 Chinese hamster cell lines (Thacker, 1981; Simi et al., 1988; Slijepcevic et al., 1996). This is not unexpected, as it is known that sublines developed in various laboratories show differences in karyotype (Thacker et al., 1981; Simi et al., 1990; Koberle et al., 1993), response to mutagens (Speit et al., 1994) and metabolic activities (Glatt, 1994). A karyotype analysis of V79 cells with molecular probes also confirms this tendency: the hybridization pattern with a Chr 7 probe in V79MZ cells showed some differences to V79-4 cells (Balajee et al., 1995), while a similar hybridization pattern was found for Chr 9 (Xiao et al., 1996).
Previous findings of a high background of spontaneous micronucleus formation pointed to a genetic instability in the recombinant V79MZr2B1 cell line (Ellard and Parry, 1993). Chromosome instability was also related to amplification of the short arm of a so-called `n' chromosome, supposed to be derived from Chinese hamster Chr 8, giving rise to the V79MZr2B1 marker chromosome. The same study revealed that multiple copies of the transfected plasmid vector were present in the amplified region of this marker, suggesting chromosome destabilization upon chromosomal integration of plasmid DNA (Ellard et al., 1995).
Here it is unequivocally demonstrated, by molecular cytogenetic analysis, that the V79MZr2B1 marker chromosome originated from Chr 7 and Chr 9, together with amplification and related instability of the Chr 9-derived long arm. Cycles of breakage–fusion–bridge due to the presence of dicentric chromosomes (Figure 3) could further contribute to chromosome instability observed in this cell line.
It is worthwhile noting that a marker Chr 9q+ with a long arm variable in length has been reported to correlate with spontaneous progression to tumorigenicity in Chinese hamster cells (Simi et al., 1992), while amplified regions of Chr 9 were found during azacytidine-induced tumorigenesis (Gadi et al., 1984).
In conclusion, this study not only confirms the findings of Ellard et al. (1995), but explains in greater detail the origin of chromosome instability observed in the V79MZr2B1 cell line. Should the induction of chromosome instability by plasmid insertion prove to be a general phenomenon, then a careful analysis of the karyotype of any genetically engineered cell line is required before considering their application in cytogenetic studies, as recently discussed by Sawada and Kamataki (1998).
|
Acknowledgments |
|---|
This work was supported by a CNR short-term mobility grant to SS, who wishes to thank Prof. Natarajan for warm hospitality and critical reading of the manuscript. The construction of V79 cell lines is funded by the German Ministry for Education, Science and Technology (BMB), to JD.
|
Notes |
|---|
4 To whom correspondence should be addressed. Tel: +39 50 577834; Fax +39 50 576661; Email: s.simi{at}imd.pi.cnr.it
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Balajee,A.S., Dominguez,I. and Natarajan,A.T. (1995) Construction of Chinese hamster chromosome specific DNA libraries and their use in the analysis of spontaneous chromosome rearrangements in different cell lines. Cytogenet. Cell Genet., 70, 95–101.[Web of Science][Medline]
Colella,C.M. et al. (1988) Gene dosage mutants at adenine phosphoribosyltransferase locus induced by colcemid in Chinese hamster V79-AP4 cells. Somat. Cell Mol. Genet., 14, 593–604.[Web of Science][Medline]
Doehmer,J. and Greim,H. (1993) Cytochrome P450 in genetically engineered cell culture: the gene technological approach. In Schenkman,J.B. and Greim,H. (eds), Cytochrome P450. Springer-Verlag, Berlin, Germany, pp. 415–425.
Doehmer,J., Dogra,T., Friedberg,T., Monier,S., Adesnik,M., Glatt,H. and Oesch,F. (1988) Stable expression of rat cytochrome P450IIB1 cDNA in chinese hamster cells (V79) and metabolic activation of aflatoxin B. Proc. Natl Acad. Sci. USA, 85, 5769–5773.
Ellard,S. and Parry,E.M. (1993) Induction of micronuclei in V79 Chinese hamster cells by hydroquinone and econazole nitrate. Mutat. Res., 287, 87–92.[Web of Science][Medline]
Ellard,S., James,S.A., Parry,E.M. and Parry,J.M. (1995) A genetically engineered V79 cell line SD1 expressing rat CYP2B1 exhibits chromosomal instability at the integration site of the transfected DNA. Mutagenesis, 10, 549–554.
Ford,D.K. and Yerganian,G. (1958) Observations on the chromosomes of Chinese hamster cells in tissue cultures. J. Natl Cancer Inst., 21, 393–425.
Gadi,I.K., Harrison,J.J. and Sager,R. (1984) Genetic analysis of tumorigenesis: XVI. Chromosome changes in azacytidine and insulin induced tumorigenesis. Somat. Cell Mol. Genet., 10, 521–529.
Glatt,H. (1994) Comparison of common gene mutation in mammalian cells in culture. Mutat. Res., 313, 7–20.[Web of Science][Medline]
Hermine,T., Jones,N.J. and Parry,J.M. (1997) Comparative induction of micronuclei in repair-deficient and proficient Chinese hamster cell lines following clastogen or aneugen exposures. Mutat. Res., 392, 151–163.[Web of Science][Medline]
Koberle,B., Roscheisen,C., Helbig,R. and Speit,G. (1993) Molecular characterization of methyl methanesulphonate-induced HPRT mutation in V79 cells. Mutat. Res., 301, 65–71.[Web of Science][Medline]
Ray,M. and Mohandas,T. (1976) Proposed banding nomenclature for the Chinese hamster chromosomes (Cricetulus griseus). Cytogenet. Cell Genet., 16, 83–91.[Web of Science][Medline]
Sawada,M. and Kamataki,T. (1998) Genetically engineered cells stably expressing cytochromes P450 and their application to mutagen assays. Mutat. Res., 411, 19–43.[Web of Science][Medline]
Seabright,M.A. (1971) A rapid banding technique for human chromosomes. Lancet, ii, 971–972.
Simi,S., Colella,C.M., Mariani,T., Piras,A. and Rainaldi,G. (1988) Qualitative analysis of chromosomal evolution in a colcemid-treated Chinese hamster population. Teratog. Carcinog. Mutagen., 8, 45–58.[Web of Science][Medline]
Simi,S., Vatteroni,L., Piras,A., Mariani,T. and Rainaldi,G. (1990) Folate-sensitive fragile sites in Chinese hamster cell lines. Cancer Genet. Cytogenet., 46, 209–216.[Web of Science][Medline]
Simi,S., Musio,A., Vatteroni,L., Piras,A. and Rainaldi,G. (1992) Specific chromosomal aberrations correlated to transformation in Chinese hamster cells. Cancer Genet. Cytogenet., 62, 81–87.[Web of Science][Medline]
Simi,S., Morelli,S., Gervasi,P.G. and Rainaldi,G. (1995) Clastogenicity of anthraquinones in V79 and in three derived cell lines expressing P450 enzymes. Mutat. Res., 347, 151–156.[Web of Science][Medline]
Slijepcevic,P., Xiao,Y., Dominguez,I. and Natarajan,A.T. (1996) Spontaneous and radiation-induced chromosomal breakage at interstitial telomeric sites. Chromosoma, 104, 596–604.[Web of Science][Medline]
Speit,G., Habermeier,B. and Helbig,R. (1994) Differences in the response to mutagens between two V79 sublines. Mutat. Res., 325, 105–111.[Web of Science][Medline]
Swierenga,S.H.H., Heddle,J.A., Sigal,E.A., Gilman,J.P.W., Brillinger,R.L., Douglas,G.R. and Nestmann,E.R. (1991) Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte culture. Mutat. Res., 246, 301–322.[Web of Science][Medline]
Thacker,J. (1981) The chromosomes of a V79 Chinese hamster line and a mutant subline lacking HPRT activity. Cytogenet. Cell Genet., 29, 16–25.[Web of Science][Medline]
Xiao,Y., Slijepcevic,P., Arkesteijn,G., Darroudi,F. and Natarajan,A.T. (1996) Development of DNA libraries specific for Chinese hamster chromosomes 3, 4, 9, 10, X, and Y by DOP-PCR. Cytogenet. Cell, Genet., 75, 57–62.[Web of Science][Medline]
Received on June 29, 1998; accepted on September 15, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||