Structural and numerical chromosomal aberrations in a metabolically competent human lymphoblast cell line (MCL-5)

Mutagenesis, May 1998

MCL-5 cells are Epstein Barr virus-transformed human lymphoblasts which have been genetically engineered for use in mutagenicity testing. We have examined the modal chromosome number, karyotype and spontaneous micronucleus (MN) and sister chromatid exchange (SCE) frequencies of the cell line. Replicate experiments were conducted on two different shipments purchased from Gentest Corp.Although the modal chromosome number was 48 (range40-54, n = 400 metaphases) for both cell shipments, the second stock showed greater variation in chromosome number than the first.A total of 60 G-banded metaphase cells was analyzed and seven karyotypes were prepared.Consistent structural abnormalities (translocations, deletions and isochromosomes) were found involving the X chromosome and seven autosomes (1-3, 5, 6, 9 and 11).The karyotype typical of this cell line was: 48,der(X)t-(X;?)(P223;?)Y,t(l;2)(q23;p23),del(3)(ql2q21), + i(3q),t(5;6)(q31;p23), + i(9p),der(ll)t(ll;13)(q23,ql2).The mean MN frequency was 41.8 MN/1000 binucleate cells (n = 5000).When compared with our historical controls for primary lymphocyte cultures this number (41.8) is significantly(8.4-fold) higher. The mean SCE frequency was 13 per metaphase (n = 100).We observed a hyperdiploid chromosome number of 48 in the majority of metaphase spreads, indicating a significant deviation from the normal diploid number characteristic of the parent cells (RPMI 1788)established in 1969.The variation in chromosome number distribution observed between shipments suggests the potential for further changes. The elevated MN frequency suggests that evaluating mutagenicity using this cytogenetic end-point may require excessive dosing to produce a significant response over background.We conclude that careful interpretation of cytogenetic end-points is necessary when using MCL-5 cells in the light of the possibility of clonal evolution presented here.

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Structural and numerical chromosomal aberrations in a metabolically competent human lymphoblast cell line (MCL-5)

Candace Lippoli Dospkei- 1 Gordon K. Livingston 0 1 Brenda L-Schumaran 1 Ashok ILSrivastava 1 0 DYNCORP of Colorado Inc., Health Effects Group , Golden, CO 80402-0464 , USA 1 'Department of Environmental Health, Unhersity of Cincinnati, College of Medicine , Cincinnati, OH 45267-0529 MCL-5 cells are Epstein Barr virus-transformed human lymphoblasts which have been genetically engineered for use in mutagenicity testing. We have examined the modal chromosome number, karyotype and spontaneous micronucleus (MN) and sister chromatid exchange (SCE) frequencies of the cell line. Replicate experiments were conducted on two different shipments purchased from Gentest Corp. Although the modal chromosome number was 48 (range 40-54, n - 400 metaphases) for both cell shipments, the second stock showed greater variation in chromosome number than the first A total of 60 G-banded metaphase cells was analyzed and seven karyotypes were prepared. Consistent structural abnormalities (translocations, deletions and isochromosomes) were found involving the X chromosome and seven autosomes (1-3, 5, 6, 9 and 11). The karyotype typical of this cell line was: 48,der(X)t(X;?)(p223;?)Y,t(l;2)(q23;P23),del(3)(ql2q21), + i(3q),t(5;6) (q31;p23), + i(9p),der(ll)t(ll,-13)(q23,ql2). The mean MN frequency was 41.8 MN/1C00 binucleate cells (n = 5000). When compared with our historical controls for primary lymphocyte cultures this number (41.8) is significantly (8.4-fold) higher. The mean SCE frequency was 7.3 per metaphase (n = 100). We observed a hyperdiploid chromosome number of 48 in the majority of metaphase spreads, indicating a significant deviation from the normal diploid number characteristic of the parent cells (RPMI 1788) established in 1969. The variation in chromosome number distribution observed between shipments suggests the potential for further changes. The elevated MN frequency suggests that evaluating mutagenicity using this cytogenetic end-point may require excessive dosing to produce a significant response over background. We conclude that careful interpretation! of cytogezstfc era3"ip3imts is mecessary when using MCL-5 cells 5m the BigBitt of tt!?e possibility of clonal evolution presented Introduction Established human B lymphoblast cell lines are invaluable as research tools in the fields of genetics and cell biology (Nilsson, 1979). These cell lines have several advantages over the use of primary cells, such as peripheral blood lymphocytes (PBLs), for tissue culture studies: (i) transformed lymphoblasts are morphologically similar to phytohemagglutinin (PHA)-stimulated PBLs (Huang and Moore, 1969; Nilsson, 1979); (ii) the doubling time of 2448 h for most cell lines allows for growth of large quantities with relative ease; (iii) en immortal cell line eliminates the need to collect blood, which may introduce the possibility of inter-individual biological differences between lymphocyte donors; (iv) propagation of these cells in culture allows for studies over extended periods of time, unlike PBLs, which typically reach senescence after 4-5 days in culture. Epstein Barr virus (EBV)-transformed cell lines were first successfully propagated from malignant tissue in the early 1960s (Epstein and Barr, 1964; Pulvertaft, 1964) and from normal healthy hematopoietic tissue in 1967 (Moore, 1972). Cell lines of malignant origin have been shown to maintain the donors chromosomal constitution in vitro, however, the karyotype, although stable, may not have been normal when first established (Huang and Moore, 1969). EBV-transformed B lymphoid cell lines derived from normal donors generally retain a diploid or near diploid chromosome constitution following the first few months of establishment (Huang and Moore, 1969; Macek etal, 1971; Glade and Beratis, 1976; Nilsson, 1979). This cytogenetic stability implies that one can interpret any chemical- or radiation-induced chromosomal changes with a greater degree of confidence. However, after extended periods of continuous culture karyotypic evolution may occur (Saksela and Ponten, 1968; Huang and Moore, 1969; Glade and Beratis, 1976; Steel etal., 1977; Risin etal, 1992). The interpretation of assays utilizing lymphoid cell lines where cytogenetic end-points are of interest could be affected if changes in chromosomal constitution occur spontaneously. The metabolically competent B lymphoblastoid cell line MCL-5 (Gentest Corp. Woburn, MA; Gentest Corp., 1990) is an example of a spontaneously EBV-transformed B lymphoid cell line established from normal, healthy hematopoietic tissue. The origin of this cell line can be traced to lymphocytes which were isolated from a 33-year-old male and were found to be positive for the EBV genome (Minowada etal., 1974). These spontaneously transformed cells were designated RPMI 1788. Twelve passages after establishment the RPMI 1788 cells were reported to contain a normal diploid modal chromosome number of 46 based on the number of centromeres present per (...truncated)


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Candace Lippoli Doepker, Gordon K. Livingston, Brenda L. Schumann, Ashok k. Srivastava. Structural and numerical chromosomal aberrations in a metabolically competent human lymphoblast cell line (MCL-5), Mutagenesis, 1998, pp. 275-280, 13/3, DOI: 10.1093/mutage/13.3.275