Carcinogenesis Vol.3 No.8 pp.961 - 9 6 2 , 1982
Short Communication
Some factors influencing the behaviour of BHK 21 Cl 13 cells in soft
agar medium
M.R.O'Donovan
The Boots Co. PLC, Research, The Priory, Thurgarton,
Nottingham NG14 7GX, UK.
(Received on 17 April 1982; accepted on 18 June 1982)
Abstract
As part of an attempt to reproduce Styles' cell transformation
assay, the effect of serum concentration on the growth of
normal and 4-nitroquinoline-l-oxide (4-NQO)-treated BHK
21 Cl 13 cells in soft agar medium was examined. Using
medium with 10% newborn calf serum, dilution of control
cultures from 5 x 104 to 1.56 x 103 cells/ml caused little increase in the number of countable ( > 0.3 mm diameter) colonies, but 4NQO caused a marked dose-related increase. In
contrast, using 20% of the same batch of serum, 4NQOtreated groups and controls diluted to comparable viability
counts showed very similar increases in countable colonies.
Clones >0.3 mm diameter isolated from control cultures
with 20% serum did not appear to be transformed when
grown in agar with 10% serum. These data indicate that factors other than neoplastic transformation can influence the
growth of BHK 21 Cl 13 cells in soft agar medium.
Various factors have been reported to influence the behaviour of BHK 21 Cl 13 cells in soft agar and, hence, the
performance of Styles' transformation assay. Daniel and
Dehnel (1) showed that batches of serum differ in their ability
to support a limited number of cell divisions after plating in
soft agar, and agree with Ishii, et al. (2) that such division
following treatment with a chemical carcinogen is necessary
for expression of the transformed phenotype. Bootman and
Cowshall (3) reported that the proportion of untreated cells
growing to form colonies in agar that are scored as
transformed increases with decreasing density and suggested
that toxicity of test compounds should be paralleled by corresponding dilution of control cultures. The purpose of the
present communication is to record some pertinent observations made in this laboratory whilst attempting to reproduce
Styles' transformation assay.
A clone of BHK 21 Cl 13 cells isolated at ICI's Central
Toxicology Laboratory (kindly supplied by Dr.M.Danile,
Huntingdon Research Centre) was used throughout this
work; these cells have the ability to divide a limited number of
times in soft agar to form microcolonies. Stock cultures were
grown as monolayers in plastic tissue culture flasks (Nunclon
Delta) in Dulbecco's modification of Eagles minimum essential medium (DMEM*; Flow Laboratories) and supplemented with 10% newborn calf serum (NBCS; Sera Lab.);
the NBCS had been pre-selected to allow optimal
microcolony-forming ability and for optimal plating efficien*Abbreviations: DMEM, Dulbecco's modification of Eagles minimum essential medium; NBCS, newborn calf serum; MEM, minimum essential medium;
EBSS, Earle's balanced salt solution; 4NQO, 4-nitroquinoline-l-oxide.
© IRL Press Ltd., Oxford, England.
0143-3334/82/0308-O961$2.00/0
cy and growth-supporting ability on plastic. The medium had
a sodium bicarbonate concentration of 2.2 g/1 to permit
equilibration with 5% CO2 in air, and no antibiotic was used
for stock maintenance or in transformation assays. 0.01%
Worthington crystalline trypsin was used to passage cells.
Two methods of exposure to test compounds were used.
(i) Suspension method. This was as described by Styles (4)
except that: 5 x 105 cells were suspended in serum-free
minimum essential medium (MEM) for suspension; 60 mm
bacterial grade Petri dishes contained freshly poured and
chilled underlays of 1.5 ml 1% Difco noble agar in MEM
with 20 mm HEPES; 625, rather than 5000, cells were sampled for the toxicity assays; doses were at 2-fold intervals
selected from a preliminary toxicity assay; five replicates per
treatment group were examined. In addition, to ensure a
population of exponentially dividing cells, stock cultures were
plated at 5 x 106 per 75 cm2 flask 24 h before use.
(ii) Monolayer exposure. This method was devised to
remove the possibility of cell clumping whilst in suspension. 5
x 106 cells were plated in 75 cm2 flasks and, 24 h later, growth
medium removed, the monolayer washed twice with Earle's
balanced salt solution (EBSS), and 20 ml serum-free DMEM
added. Test compound in dimethylsulphoxide (final concentration 1%) was then added and incubated at 37°C for 1 h.
At the end of the exposure period, the cells were washed twice
with EBSS, trypsinised and suspended in 10 ml growth
medium. The control culture was counted, diluted to 5 x 104
cells/ml and five 10 ml aliquots dispensed into universal bottles; all test groups were diluted exactly as the control. A 12.5
ftl sample was removed from each tube to assess survival, 625
id 5% Difco Noble agar added, mixed and poured onto 1%
agar underlays as in the suspension method.
Two parameters were examined for their influence on the
behaviour of control and treated cells in soft agar: serum concentration was varied so that the agar growth medium contained 10% or 20% NBCS (DMEM10 or DMEM20); cell
density was varied so that control cultures paralleled the numbers of viable cells plated in treated cultures. In the Styles'
method, all controls were suspended at 5 x JO5 cells/ml and
dilutions performed subsequently.
The results (see Table I) show that the method of exposure
has little or no influence on the subsequent behaviour of cells
in soft agar. In contrast to the usual practice, no attempt has
been made to express transformation frequencies, but observed countable colonies have been recorded; survival figures are
shown purely for comparative purposes. However, the
amount of serum in the agar medium does have a significant
effect. In DMEM20, increases in "transformed" colonies are
seen with 4NQO but, when toxicity is examined, these increases compare directly with control cultures diluted to similar
viable counts. Using DMEM 10, some increase in countable
colonies was seen with control dilution, but 4NQO produced
a much greater increase at comparable viable counts.
In order to investigate the nature of some of the clones forming colonies in agar, seven colonies >0.3 mm in diameter
were picked off control DMEM20 plates, cultured and plated
961
M.R.O'Donovan
Table I. BHK 21 Cl 13 cells: the effect of dilution and treatment with 4NQO
on transformation frequency using 10% and 20% new born calf serum.
Treatment group
Control
Control x 1/2
Control x 1/4
Control x 1/8
Control x 1/16
Control x 1/32
4NQO 0.025 /tg/ml
4NQO 0.05 ^g/ml
4NQO 0.1 pg/ml
4NQO
Mean transformed colonies/platea ± S.E.
(survival11)
Monolaver exposure
Suspension exposure
20% serum 10% serum 20% serum 10% serum
0.6 ±0.4
(100)
2.0 ±0.3
(72)
3.0 ±0.4
(64)
9.2 i 0.7
(22)
13.5±2.9
(10)
26.2±3.5
(7)
2.2 ±0.4
(68)
2.4 ±0.4
(48)
5.0 ±0.4
(32)
24.4 ±4.3
(9)
0
(100)
4.2 ±0.7
(63)
3.6±0.7
(34)
6.8 ±0.8
(19)
3.4 ±0.7
(10)
1.8 ±0.6
(5)
1.4±0.5
(100)
3.2 ±0.4
(81)
18.6±3.5
(39)
27.0 ±3.8
(11)
2.8±1.0
(100)
2.0 ±0.7
(63)
3.3±1.1
(37)
3.3±1.8
(19)
29.3 ±1.6
(9)
6.6 ±2.2
(5)
3.6 ±1.2
(68)
8.0±1.8
(39)
17.6±6.1
(13)
51.4±3.7
(4)
0.2 ±0.2
(100)
4.4 ±1.2
(61)
6.8 ±1.5
(35)
8.4 ±1.6
(22)
11.0±2.5
(11)
2.5 ±0.9
(6)
2.0 ±1.2
(73)
7.6 ±0.7
(49)
23.4 ±4.6
(23)
29.8 ±3.5
(2)
"Colonies >0.3 mm diameter (counted manually using a plate reader, x 8
magnification). bSurvival expressed as percentage of control plating efficiency. C5 x 105 cells inoculated/plate. S.E., standard error.
at 5 x 10* cells/ml in soft agar with DMEM10. All seven
clones behaved similarly to the parent line, showing a uniform background of microcolonies, but with no colonies
>0.3 mm diameter.
Two main conclusions can be drawn from these findings.
(i) Factors other than chemical transformation can influence the behaviour of BHK 21 Cl 13 cells in soft agar
medium. As suggested by Bootman and Cowshall, control
dilutions using an inappropriate amount of serum can result
in apparent transformation. Colonies arising in such circumstances, i.e. dilution in DMEM20 appear not to show a
stable transformed phenotype.
(ii) Using an appropriate quantity of a suitable batch of
serum, a reproducible dose-related increase in numbers of
countable colonies could be included using 4NQO, and these
increases appear to be independent of any toxicity-induced
dilution effect;"" presumably this represents a genuine
transformation-related change.
In light of the above, the monolayer exposure protocol using agar medium with 10% of this batch of NBCS was
evaluated for its response to a number of compounds. Several
carcinogens, including benzidine, benzo[a]pyrene, 2-nitrofluorene and 2-aminofluorene (all incorporating an appropriate metabolic activation system), were examined but none
was found to produce any evidence of transformation. Because of the insensitivity of the modified protocol and the experience of workers in other laboratories (e.g., Bootman and
Cowshall loc. tit., Dr.M.Daniel, HRC, personal communication) it was decided that a cell transformation assay using
BHK 21 Cl 13 cells was unsuitable for routine use in this laboratory and all work on it has now been terminated.
962
Acknowledgement
I thank Mr.R.Durward for excellent technical assistance.
References
1. Daniel.M.R., and Dehnel.J.M. (1980), Factors affecting the performance
of the Styles cell transformation test, Carcinogenesis, 1, 657-667.
2. Ishii.Y., EUiott,J.A., Mishra.N.K., and Lieberman.M.W. (1977), Quantitative studies of transformation by chemical carcinogens and ultraviolet
radiation using a subclone of BHK 21 clone 13 Syrian hamster cells, Cancer
Res., 37, 2023-2029.
3. Bootman.J., and Cowshall.S. (1979), The BHK cell transformation
assay; problems of reproducibility and interpretation and the effect of cell
number on transformation rate, UKEMS meeting communication.
4. Styles.J.A. (1977), A method for detecting carcinogenic organic chemicals using mammalian cells in culture, Br. J. Cancer, 36, 558-563.