Mutagenesis vol.13 no.3 pp.275-280, 1998
Structural and numerical chromosomal aberrations in a
metabolkally competent human lymphoblast cell line (MCL-5)
Candace Lippoli Dospkei-1-3, Gordon K. Livingston2,
Brenda L-Schumaran1 and Ashok ILSrivastava1
'Department of Environmental Health, Unhersity of Cincinnati, College of
Medicine, Cincinnati, OH 45267-0529 and 2DYNCORP of Colorado Inc.,
Health Effects Group, Golden, CO 80402-0464, USA
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 24—48 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
metaphase (Huang and Moore, 1969). The RPMI 1788 cells
were eventually acquired by the Gentest Corp. and determined
to be mycoplasma free (Freedman et al., 1979).
The RPMI 1788 cell line was genetically modified and
designated MCL-5 by Gentest Corp. for use in mutagenicity
studies. The genetic modification consisted of transfection with
five human cDNAs which encode drug metabolizing enzymes
of the P-450 superfamily (Crespi et al., 1991). Because these
enzymes are actively involved in xenobiotic metabolism
(Nebert and Gonzalez, 1987) this cell line provides a sensitive
screening tool for monitoring mutagenic damage induced by
procarcinogen exposure. Data from a number of studies have
been reported in which MCL-5 cells have been used for specific
^ o whom correspondence shojld be addressed at: Beauty Care Division, The Proctor and Gamble Co., SWTC, 11511 Reed Hartman Hwy, Cinncinnati,
OH 45241, USA. Tel: -M 513 626 5536; Fax: -"-1 513 626 1C05: Email: [email protected]
© UK Environmental Mutagcn Society/Oxford University Press 1998
275
C.L.Doepker et aL
locus (i.e. hprt locus) mutagenicity testing and induction of
micronuclei (MN) as biomarkers of chromosomal damage
(Crespi et aL, 1991; White etal., 1992; Crofton-Sleigh etal.,
1993; Styles etal., 1994).
A clear understanding of the karyotype and genomic stability
of the MCL-5 cell line is a prerequisite for successful use of
these cells in assays where cytogenetic changes are the endpoints of interest. To our knowledge there have been no
published reports describing the karyotype of the MCL-5 cell
line. The present study was therefore undertaken to provide a
detailed cytogenetic profile of the MCL-5 human lymphoblast genome.
Materials and methods
Cell culture conditions
The MCL-5 cell line was purchased from the Gentest Corp. (Wobum, MA)
Following the first set of MN experiments it was noted that our results differed
significantly from values reported in the literature (White et al, 1992; CroftonSleigh etal., 1993; Styles etal., 1994). We contacted the Gentest Corp. and
upon their recommendation a new culture of exponentially growing cells in
suspension was sent to ensure that results could not be a consequence of
Gentest's or our technical error in handling of the original frozen ampule of
cells. Thus we have designated the first stock culture (received frozen 10/267
92) stock A and the actively growing culture (received 6/28/93) stock B Cells
from both stocks were stored in liquid nitrogen The cells were cultured in
accordance with Gentest Guidelines (Gentest Corp., 1990). Tests were performed in replicate for both shipments. In each experiment a frozen ampule
containing 1 ml ~1X1O7 cells was quickly thawed The 1 ml cell suspension
was added to 10 ml prewarmed RPM1-1640 medium (Gentest) with 2 mM
/-histidinol, without /-histidine and L-glutamine. The medium was supplemented with 2 mM L-glutamine, 9% (v/v) horse serum (Gentest) and 100200 mg/ml hygromycin B. The cells were centrifuged at 40 g for 10 min (the
protocol for all centrifugations unless otherwise stated) and then resuspended
in 20 ml medium in a 75 cm2 flask. Cell number was enumerated using a
model ZM coulter counter (Coulter Electronics Inc ). The cells were incubated
for 48 h at 37°C, 5% CO2, 95% air. After 48 h the cell number was determined
daily and diluted every other day to maintain 1.5-2.0X105 cells/ml Cells
were carefully maintained in a volume of 10 ml in 25 cm2 flasks such that
concentrations never exceeded 1.2x 106 cells/ml. Successive subcultures were
designated Nx, where N is the number of passages since purchase and x is
the number of passages while in Gentest's possession. Doubling time (DT)
was monitored over a period of 48 h. Any cultures showing DT values 5»30 h
were discarded, as suggested in the Gentest guidelines (Gentest Corp., 1990).
We found no significant difference in DT between stocks A and B and their
respective mean doubling times were 24.5 and 27.3 h
Cytogenetics
Modal chromosome number and karyotype. Cells were routinely plated at
1.5-2.0X103 cells/ml (total volume 10 ml/25 cm2 flask). Forty six hours after
subculture colcemid (Gibco BRL) was added at a final concentration of 0.05
Hg/ml. After 2 h the cells were centrifuged and resuspended in prewarmed
75 mM KCL for 17 min. The cells were then centrifuged and treated with
freshly prepared fixative (methanol:glacial acetic acid 3.1 and 6:1 v/v) After
two washes in 3:1 fixative and one wash in 6.1 fixative the cells were dropped
onto clean chilled slides A 2% Giemsa solution was used for conventional
solid staining to evaluate quality and quantity of metaphases. Giemsa banded
chromosome preparations were prepared by the "G-bands by trypsin using
Giemsa' (GTG) method.
Mtcronucleus assay. Cells were plated at 1.5-2.0X106 cells/10 ml in 25 cm2
flasks. After 24 h in culture cytochalasin B (final concentration 3.0 Hg/ml;
Sigma) was added. At 48 h cells were centrifuged and resuspended in 1 X
Hank's balanced salt solution (HBSS) without MgCl2, MgSO4 and CaCl2
(Gibco BRL). Using a Shandon Cytospin-2 cytocentrifuge cells were harvested
directly onto slides. Cell monolayers were fixed in methanol and stained with
Wright's Giemsa (Harleco). In experiment 5 cells were stained with acndine
orange (Allied Chemical) at a working concentration of 45 ug/ml. A minimum
of 1000 binucleate cells were scored for the presence of MN. Criteria for
identifying and scoring MN included- (i) chromatin staining and texture
(comparable to main nuclei); (li) shape (round to oval with distinct boundary),
(iii) size (one third the diameter of main nuclei); (iv) location (within the
cytoplasm); (v) physically separate from the main nuclei.
276
Table L Distribution of chromosome number in MCL-5 cells*
Stock Passage No. of cells with chromosome nos
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
A
A
B
B
8x
7X
5X
5X
0 0 1 0
0 0 2 0
3 1 4 5
1 0 1 0
0
1
4
3
0 3
0 4
2 4
4
10
17
13
12
13
64
62
32
34
11
12
17
15
3
3
5
9
0
3
4
3
1
0
2
5
0
0
3
2
0
0
2
0
"No significant difference between replicate experiments (A, P = 0.85;
B, P = 0.17), n = 100 metaphase cells for each experiment.
Sister chromatid exchange. Cells were plated at 1.5-2.0X106 cells/10 ml in
25 cm2 flasks supplemented with 100 ]iM 5-bromodeoxyundine (5-BrdU) and
the cultures incubated in the dark. Cells were treated with 0.05 u.g/ml colcemid
(Gibco BRL) 2 h before harvesting Chromosomes were prepared as described
previously. The fixed cells were dropped onto slides and stained by the
'fluorescence plus Giemsa' technique (Wolff and Perry, 1974). A total of 100
metaphases from one experiment were analyzed for SCE frequency.
Microscope and statistical analyses
All metaphase cells were scored using an Olympus BHS bright field microscope
at a magnification of 1250X Cells stained with acndine orange for MN
counts were examined at a magnification of 500X using epifluorescence.
Data from these experiments were analyzed using the JMP statistical
analysis and visualization software program from the SAS Institute Inc. In
each experiment all data were found to be normally distributed using the
Shapiro Wilk W test. Either a Student's r-test or Tukey-Kramer test for
significant difference between means was utilized.
Results
Data showing evaluation of chromosome number are summarized in Table I. Chromosome counts were performed on a total
of 400 metaphase cells and no significant differences were
found within replicate experiments (stock A, P = 0.85; stock
B, P = 0.17) or between the A and B stocks (P = 0.29). The
modal chromosome number was found to be 48 and thus
hyperdiploid. Averaging values from replicate experiments
showed that for stock A 63% of 200 cells scored had 48
chromosomes/metaphase; whereas stock B showed 33% of
200 metaphases with a count of 48 chromosomes. It can be
seen from the data in Table I that stock B exhibits wider
variation around the modal number than stock A (i.e. more
metaphases with <48 and >48 chromosomes).
A total of 60 G-banded metaphase cells was carefully
examined and seven karyotypes were prepared, one of which
is illustrated in Figure la. The X sex chromosome and seven
autosomes were consistently found to exhibit structural abnormalities. Translocations accounted for six of these abnormal
chromosomes. Balanced reciprocal translocations were
observed to occur between the long arm (q) of chromosome
I and the short arm (p) of chromosome 2 [t( 1 ;2)(q23,p23)],
as well as between the long and short arms of chromosomes
5 and 6 [t(5;6)(q31;p23)]. In addition, a derived chromosome
II was consistently observed [der(ll)t(ll;13)(q23;ql2)]. The
translocation involving the X chromosome was only partially
identifiable [t(X,?)(p22.3;?)]. The two isochromosomes, one
derived from the long arm of chromosome 3 [i(3q)] and the
other from the short arm of chromosome 9 [i(9p)] accounted
for the two extra chromosomes, producing a hyperdiploid
modal number of 48. An interstitial deletion in the long arm
of chromosome 3 (ql2q21) was also detected. The karyotype
presented is from a B stock cell, but is representative of the
most common and frequent abnormalities seen in both stocks
of the MCL-5 cell line. Multiple examples of each of the
Karyotype of the MCL-5 cell line
a
deKX)t<X:?Mp22.3:?)
1
H
V
der<l)t(!:2)<q23;p23)
i) \\ n \%\ if i{
li
tr
13
14
••
9*
19
20
it
15
21
10
11
Ift
Ift
16
17
22
ii
K
12
il
X
<W(3Kql2q21)
I 1
dei<5)t<5;6Xq3I;p2J)
I t
Y
•
I
Fig. 1. (a) G-banded karyotype of a cultured MCL-5 cell (stock B) showing
eight structural abnormalities. 48,der(X)t(X;?)(p22.3;?)Y,t(l;2)(q23;
p23),del(3Xq 12q21),+i(3q),t(5;6)(q31 ;p23),+i(9p);der( 11 )t( 11; 13Xq23;q 12). (b)
Multiple examples of the eight structurally abnormal chromosomes.
Chromosomes were selected from both stock A and stock B G-banded
metaphase spreads.
I
drr(l 1)1(1 l;l3Mq23;ql2)
t
1
I
a
I
: I
»
19
Two isochromosomes in one metaphase
ant difference between the five replicate experiments (P =
0.35). It follows that no significant difference in MN frequency
between cell stocks was observed. In experiments 1-4 cells
were stained with Wright's Giemsa (Harleco), however, fluorescence analysis with acridine orange was utilized in experiment 5B. No significant difference was found between these
two staining procedures (P = 0.93).
One experiment was conducted to measure the spontaneous
sister chromatid exchange (SCE) frequency. This was determined for stock A and is depicted by the histogram in Figure 3.
One hundred metaphase spreads were analyzed and the mean
SCE frequency was found to be 7.32 ± 2.90.
abnormal chromosomes (from both stocks A and B metaphase
spreads) are shown in Figure lb and the overall frequency of
the eight abnormalities detected in seven karyotypes is shown
in Table II. Cells with >48 or <48 chromosomes generally
contained random abnormalities, including some structural but
predominantly numerical changes, such as del(7p),-8,+ 14,
-15-17.+21.
The results of five replicate MN assays are shown in
Figure 2. The mean frequency of spontaneous MN was 4.18
per 100 binucleate cells (n = 5000). The Tukey-Kramer
statistical means comparison indicates that there is no signific-
Discussion
Our original interest in the MCL-5 cell line was to assess the
cytogenetic effects of procarcinogen treatment. Because this
B lymphoid cell line contains foreign compound metabolizing
enzymes and is of human origin, results might have greater
relevance for human risk assessment. Proper interpretation of
data generated in cytogenetic assays requires careful evaluation
of the chromosomal constitution and intrinsic variability that
may exist in the cell line. We have investigated various
cytogenetic end-points in order to establish baseline data for
the MCL-5 cell line.
lable II. Description and frequency of chromosome abnormalities in the
MCL-5 cell line
Description
Frequency in seven karyotypes
der(X)t(X;?)(p22.3;?)
t(l;2)(q23;p23)
del(3Xql2q21)
+i(3q)
«5;6)(q31;p23)
+i(9p)
der(ll)t(ll;13)(q23;ql2)
7/7
in
6/7
8°/7
in
in
in
277
C.L.Doepker et aL
—
.
•
o
a.
Mi
o
z
—
n
n
n i 1
n
On
SCE / METAPHASE
Fig. 3. The spontaneous SCE frequency was determined for stock A A
total of 100 metaphase spreads were analyzed and the mean SCE frequency
was found to be 7.3 ± 2.9 per melaphase
EXPERIMENT
Fig. 2. The j:-axis indicates experiment number; A, old stock; B, new stock.
The mean MN frequency for the total sample is shown as the dotted line
For each experiment (n = 1000) a means diamond is displayed with a line
drawn at the mean MN/100 cells The upper and lower points of the
diamond span a 95% confidence interval The width of each diamond is
proportional to group size
Our data have demonstrated that the MCL-5 cell line
consistently possesses an aneuploid karyotype. The modal
chromosome number of 48 indicates that chromosome gain
has occurred since the time of establishment in 1968, when
the parent cell line, RPMI 1788, was determined to have 46
chromosomes (Huang and Moore, 1969). Our findings are in
agreement with the findings of Crespi and Thilly (1984). In
their report RPMI 1788 cells were determined to have 48
chromosomes, however, a karyotype analysis was not performed.
The modal chromosome number of 48 was characteristic of
both cell stocks, however, the frequency distribution about the
mode showed some difference between stocks (A and B) For
replicate experiments the mean modal count of 48 occurred in
63% of cells examined in stock A, but in only 33% of stock
B cells. This greater variance in chromosome distribution may
reflect a tendency toward clonal evolution, i.e. a trend towards
numerical changes in the chromosomal constitution resulting
from emergence of a new clonal population.
It has been shown in human lymphocytes cultured in vitro
that numerical changes in karyotype involving chromosome
loss occur more frequently than chromosome gain (Brown
etal., 1983; Richard et at.. 1993). Furthermore, there is an
inverse relationship with chromosome length, i.e. smaller
chromosomes tend to be lost more frequently. These results
are believed to be due to technical artifacts during preparation.
Hyperdiploidy. on the other hand, was concluded to be a nonrandom event and probably a consequence of non-disjunction.
However, in the case of MCL-5 cells we believe that hyperdiploidy is accounted for by duplication and not non-disjunction,
as indicated by the presence of two isochromosomes [i(3q)]
and [i(9p)]. This was clarified through analysis of the Gbanded metaphase spreads.
Variation from normal diploid (heteroploidy) with time in
culture has been reported as 'typical' in EBV-transformed B
lymphoblastoid lines derived from healthy donors (Moore.
1972; Nilsson and Ponten, 1975; Glade and Beratis, 1976;
278
Steel etal., 1977, 1980; Shade et al.. 1980; Nilsson, 1992).
The work of Steel et al. (1977, 1980) and Shade etal. (1980)
supports the aforementioned work in cultured lymphocytes
(Crespi and Thilly, 1984; Richard etal.. 1993), indicating
chromosome gain as a non-random occurrence. The Steel and
Shade groups found that over extended periods of time in
culture emergence of aneuploid clones occurred frequently in
EBV-transformed lines, involving autosomes 3, 7-9 and 12.
To elucidate the structural and numerical changes we have
successfully prepared G-banded karyotypes. The X chromosome and seven autosomes (numbers 1-3, 5, 6, 9 and 11) were
observed to have undergone structural changes which remained
stable. This was indicated by consistency of these changes in
>85% of the karyotypes prepared. These structural modifications appeared to be independent of passage number and stock.
It is important to note that the G-banding technique was not
available at the time of the work of Huang and Moore (1969),
which identified the RPMI 1788 cells (parent cell line) as
having 46 chromosomes. Therefore, the possibility exists that
some of the structural abnormalities may have been overlooked.
Whether EBV itself could be responsible for the chromosomal
alterations appears improbable, considering that we observed
non-random abnormalities and the EB virus has been shown
to typically cause random aberrations (Minowada et al., 1981).
However, direct association between EBV and chromosome
change with time in culture remains unclear (Sutherland et al.,
1988; Risin et al., 1992). It is of interest to note that a few of
the abnormalities we report in the MCL-5 cell line have
been observed in other EBV-transformed lymphoid lines.
For example, Sutherland etal. (1988) found that in EBVtransformed cell lines a BrdU-enhanceable fragile site existed
in the long arm of chromosome 11 at position q23 1. We also
suspected 1 Iq23 to be a break point involved in a translocation
in all karyotypes. Also, as mentioned above, the Steel (1977,
1980) and Shade groups (1980) found that chromosomes 3
and 9 were consistently affected in EBV-transformed lymphoid
lines and contributed to aneuploidy. We also observed consistent abnormalities involving these two autosomes and found
that i(3q) and i(9p) accounted for the hyperdiploidy. Despite
these similarities, we believe this study reports the highest
number of non-random aberrations found in an EBV-transformed lymphoid line from a normal donor. Furthermore, the
X chromosome abnormality appears to be novel.
The SCE frequency for the MCL-5 cell line appears consistent with values generated from human PBLs and EBV-
Karyotype of the MCL-5 ceU line
transformed lymphoid lines (data not shown). In a previous
study of PBLs from 29 healthy male subjects we found a
mean of 7.7 ± 1.3 SCE/cell (Suruda etai, 1993). Tohda etal.
(1980) demonstrated that three EBV-transformed lymphoid
lines derived from healthy donors possessed a mean frequency
of 8.3 ± 0.3 SCE/cell. In this study the MCL-5 cell line was
found to have a mean of 7.3 ± 2.9 SCE/metaphase cell.
Although SCEs are not believed to represent mutations (Sorsa
et al, 1990), the SCE assay has been used extensively to study
exposure to mutagens. The concordance in SCE values between
MCL-5 cells and human PBLs indicates the potential use of
these cells for mutagenicity testing where SCEs are the endpoint of interest. However, because we investigated only two
cultures (100 metaphases), further studies of background SCE
levels are warranted.
Perhaps the most perplexing finding in our experiments was
related to the MN assays. Results of five different MN
assays indicated an overall mean frequency of 41.8 MN/1000
binucleate (BN) cells. No significant difference was observed
between stocks, replicate cultures or individual scorers, thus
the reproducibility of our data was considered good. Historical
values from our laboratory (Suruda etal., 1993) showed a
mean MN frequency of 4.9 ± 1.7/1000 binucleate PBLs from
29 healthy male subjects (n = 58 000 cells). Thus our studies
show an 8.4-fold difference in the spontaneous MN levels
between the MCL-5 cells and human PBLs.
The MCL-5 mean MN frequency was also found to differ
from other EBV-transformed lymphoblastoid cell lines. Honma
etal (1995) found that the TK-6 and WTK-1 lymphoid lines
possessed mean MN frequencies of 10.5 MN/1000 cells and
12.5 MN/1000 cells respectively. Thus the mean of 41.8 MN/
1000 for MCL-5 cells was 3.9-and 3.3-fold greater when
compared with these respective cell lines.
Previous studies have been published on the topic of MCL5 MN frequencies. All of these reports were from a group of
individuals located at the Institute of Cancer Research and the
MRC Toxicology Unit in the UK (White et al, 1992; CroftonSleigh etal, 1993; Styles etal, 1994; Doherty etal, 1996).
Interestingly, when we compared our data with their published
data our reported mean of 41.8 MN/1000 BN cells was
significantly higher (almost 3-fold). The mean MN frequency
found by these groups was consistent and reproducible and in
the range 9.0-16.0 MN/1000 BN cells. This mean value was
based on analysis of MN using predominantly fluorescence
microscopy (acridine orange staining). To rule out a difference
attributable to staining technique we set up MN assays identical
to the experiments of Crofton-Sleigh etal. (1993). The ~ 3 fold difference in MN counts was still apparent. We found no
significant difference between staining procedures, i.e. the use
of Wright's Giemsa versus acridine orange (see Results). We
believe that the clonal evolution alluded to may help explain
these reported differences in MN. We observed variation
between MCL-5 stocks in terms of the distribution of cells
with various chromosome numbers, especially in stock B, and
this finding suggests chromosomal instability. Therefore, in
the stocks we used numerical chromosomal changes may be
related to the elevated MN frequency. This is further supported
by the finding that < 3 % chromosomal aberrations were
observed when scoring 400 metaphase spreads to obtain the
modal chromosome number. We would thus predict that the
content of MN would more often contain whole chromosomes
rather than chromosome fragments. This prediction could be
tested using kinetochore painting techniques.
In conclusion, we have established the hyperdiploid Gbanded karyotype of the MCL-5 cell line. Further, we have
found this cell line to show chromosome structural rearrangements that are consistent. The SCE frequency appears to be
normal and the MN frequency is significantly elevated when
compared with human PBLs and other EBV-transformed
lymphoid lines. There have been reports describing successful
use of this metabolically competent cell line in specific
locus mutagenicity tests such as the hypoxanthine guanine
phosphoribosyltransferase (hprt) and thymidine kinase (tk)
assays (Crespi and Thilly, 1984; Crespi etal, 1991). It should
be noted that our findings cannot be compared with these
studies since cytogenetic end-points do not necessarily correlate
with mutagenicity assayed at the gene locus. Our results do,
however, call into question the use of this cell line as a tool
for mutagenicity testing where cytogenetic end-points are of
interest Because of the differences in spontaneous MN reported
here and elsewhere (White etal, 1992; Crofton-Sleigh etal,
1993; Styles et al, 1994; Doherty et al, 1996) we suggest that
MCL-5 cells be used with caution where cytogenetic endpoints are to be assessed.
Acknowledgements
Thanks are due to Dr Shirley Soukup and James Doepker U for their help in
preparing this manuscript, Dr Rakesh Shukla for statistical advice and Joyce
Long and Pete Jeremich for photographic assistance. We also wish to thank
both the Eastman Kodak Co. (C.L.D) and the Smokeless Tobacco Research
Council Inc. (G.JCL) for grants which provided financial support for this study.
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Received on August 13, 1997; accepted on October 10, 1997
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