Quantitative determination of DNA synthesis in cell
cultures is now a routine procedure in many laboratories.
Protocols are available for various applications,
especially in cell culture systems. The effects of growth
factors, inhibition of cell division by exogenous factors,
stimulation/inhibition by cytokines, development of
serum-free media, influence of hormones and receptor
activity on proliferation, and selection advantages for
aneuploid cells in vitro are just a few examples where
the determination of DNA synthesis provides important
information.
In the past, radioactive tagging of newly synthesized
DNA with 3H-labeled thymidine was the most frequently
applied method, while today non-radioactive labeling
with BrdU, a thymidine analog, is finding increasing use.
Although the advantages of non-radioactive immune
techniques using BrdU or anti-BrdU are not disputed,
the scope and limitations of the available systems for
detecting BrdU are not always clearly defined. These
practical aspects have been examined in more detail in
the experiments described below. Besides enabling
microscopic detection at the cellular level, the ELISA
technique represents a method for routine screening.
Two different detection systems are available: a) the
conventional colorimetric test and b) a chemiluminescence
assay also based on the anti-BrdU technique.
One of these test methods will be suitable for use
depending on the problem and the available laboratory
facilities.
We are particularly interested in the cultivation of
human prostatic epithelial cells through several
passages. For this purpose, a special culture medium
containing exogenous growth factors and added
hormones was developed [1]. In the course of this work
we have examined the colorimetric and the chemiluminescence
variants of the BrdU ELISA (Roche Molecular Biochemicals) in a series
of parallel experiments.
Materials and Methods
Details of our special cell culture system and a short
review on current measurement techniques for cell
proliferation has been published [1-4]. We have investigated
the proliferation of two distinct human prostatic
carcinoma cell lines, P1/289 and P1/373 previously
established in our laboratory.
In preliminary experiments, we had already defined the relevant
cultivation and labeling intervals as well as the optimal
cell concentrations for these cells. We have now investigated
the influence of growth factors (epidermal growth
factor, EGF) as well as the effects of insulin (Ins) and
hydrocortisone (HC) on DNA synthesis in the cell lines
P1/289 and P1/373. For this experiment, 104 cells were
seeded in standard 96-well microtiter plates in a volume of
200 l medium/well and cultivated in complete medium
(DMEM/F12, Life Technologies) containing 10 % fetal calf
serum (FCS) supplemented with EGF (10 ng/ml), hydrocortisone
(10 ng/ml), cholera toxin (CT, 10 ng/ml), and
insulin (4 g/ml). To examine the effects of the additives
(DMEM/F12+) on cell proliferation, the cell lines were also
cultivated in parallel in their absence (DMEM/F12-).
A second application concerned another cell culture
model: To prevent the outgrowth of fibroblasts when
establishing cell cultures from heterogeneous tissue
samples, cultivation of epithelial cells in the presence of
cholera toxin is recommended. When added to normal cell
culture medium, this toxin selectively suppresses the
growth of fibroblasts. Accordingly, we cultured human
fibroblasts obtained from explants (ZF 07) in microtiter
plates. The rate of DNA synthesis in standard medium
(DMEM) was determined and compared with that in a
medium containing 10 ng/ml cholera toxin (Sigma) for
inhibition of fibroblast growth (DMEM + CT).
In a third set of experiments, we examined the advantages
of a serum-free, special medium (PromoCell) for
the cultivation of human umbilical vein endothelial cell
(HUVEC) in comparison with the standard medium
(RPMI + 10 % FCS).
In each case, 24 hours after seeding of the cells, BrdU
was added to a final concentration of 1 M. After incubation
for further 24 hours, DNA synthesis was assayed
with the Cell Proliferation ELISA, BrdU (Roche Molecular
Biochemicals) using colorimetric or chemiluminescence
detection, both according to the manufacturer's instructions.
Newly synthesized BrdU-DNA was determined
using an ELISA reader (BioRad, 450) or a luminometer
(Orion, Berthold Detection Systems).
Results
The results clearly show that, depending on the labeling
conditions or the proliferation behavior of the cells, widely
differing results are obtained with the two detection
systems, colorimetry and chemiluminescence (Figure 1).
In the cases of the prostatic carcinoma cells lines P1/289
and P1/373 treated with hormone, growth factor, and
toxin, an increase in the DNA labeling index of more
than 50 % is observed in the chemiluminescence test
whereas, for the same cells, the colorimetric BrdU assay
reveals an increase of only about 20 %. Growth inhibition by cholera toxin in fibroblast cultures
results in a significant reduction of BrdU incorporation
from 100 % in the control to values of 44 % and 35 % in
the presence of 10 ng/ml cholera toxin, irrespective of
the detection system used.
Comparable results for changes in proliferation behavior,
expressed as the amount of newly synthesized DNA, were
also obtained with the two detection systems in comparative
studies on cultivated human umbilical vein endothelial
cells. In this case, the serum-free medium from
PromoCell was clearly advantageous for proliferation,
showing an increase of almost 50 % in comparison with
the RPMI standard medium, irrespective of whether the
colorimetric assay (+50 %) or the chemiluminescence
assay (+49 %) was used.
Discussion
The results show that conventional colorimetric tests
exhibit a limited linear range. In general, this linearity is
found in the absorption range between 0.5 and 1.5.
Although most readers can also detect higher absorption
values (sometimes up to 3.0), linear relationships
between the measured absorption values and the actual
incorporation of BrdU do not exist in these ranges.
Accordingly, if one wants to document an increase in
proliferation or inhibition of a cell line by exogenous
growth factors, there are only two possibilities:
a) To perform the measurements in a region lying
within the limits of the linearity range. This
neccesitates appropriate preliminary trials.
b) To use a test exhibiting a linearity range extending
over several orders of magnitude.
Overall, the use of chemiluminescence test systems is
advantageous since the linearity region covers around
8-4 orders of magnitude and thus also provides the possibility
of detecting and documenting extreme changes of
the DNA synthesis index in one and the same experiment.
The use of an extensively supplemented medium
for the cultivation of human prostatic carcinoma cell
lines may, at first, seem to be unnecessary on the basis
of the BrdU colorimetric data as increases of merely
16 % and 20 % were recorded. However, the extent of the
cell proliferation already leads to values in the non-linear
region. Use of the chemiluminescence technique results
in different sets of data. In this case, depending on the
cell line, growth advantages of 55 % or 63 % are
achieved by supplementation of the medium. The alternative,
to choose the linear region of the colorimetric test
for the experiments, is in general not very practicable since the problems investigated with these test systems
usually involve examination of the effects of several
parameters in a single experiment. The positive or negative
changes in the proliferation index or incorporation of
BrdU may vary from parameter to parameter. Thus, it is
often necessary to repeat one or more of the experiments,
in some circumstances with differing cell numbers
or labeling times.
The colorimetric test can be employed without difficulty in
cell systems and experiments in which short labeling
times are chosen or in which changes in proliferation are
measured at the end of a growth curve, near confluence.
This is shown by the results with comparatively slowly
growing fibroblasts (ZF 07) and the endothelial cells
(HUVEC). In these cases, the two BrdU systems provide
almost the same information, namely inhibition of cell
division of fibroblasts upon use of cholera toxin or the
advantage of serum-free endothelial medium in comparison
with a standard cell culture medium. The absorption
values of the colorimetric BrdU test are in the linear
region; thus the differences in BrdU incorporation can be
reliably recorded by instruments and are comparable with
those from the chemiluminescence technique.
When measurements have to be made on several differently
proliferating cells under different conditions in one
experiment, the chemiluminescence test is the method of
choice because it enables linear results to be obtained
over a wide interval. When, on the other hand, proliferation
measurements are made on only a few or even just
one cell line over a limited period of time, the more easyto-
use colorimetric test can be employed. Using a
colorimetric assay one must always consider that this test
has only a limited linear range. In contrast to numerous
other ELISA kits, however, when the linear range of the
BrdU ELISA is exceeded, it is not possible to just dilute the sample because the latter is a cellular ELISA in which
the labeled, cultivated cells produce the signals. In such
cases, the only alternative is to perform a completely new
experiment with a shorter labeling time. Reducing the cell
number in order to bring the signal to the linear range is
not feasible because this would change the overall proliferation
behavior of the cell population in vitro.
These observations are of practical relevance. Not all
laboratories performing routine DNA synthesis determinations
possess the apparatus required for luminescence
measurements because it is considerably
more expensive than systems for measuring absorption.
The experiments described here may help in this
decision-making process.
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