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)