Responses of Synchronized HeLa Cells to the Tumor-promoting Phorbol Ester 12-O-Tetradecanoylphorbol-13-acetate

[CANCER RESEARCH 41. 306-309. January 1981]
0008-5472/81 /0041-0000$02.00
Responses of Synchronized HeLa Cells to the Tumor-promoting Phorbol
Ester 12-O-Tetradecanoylphorbol-13-acetate as Evaluated by Flow
Cytometry
Volker Kinzel, James Richards, and Michael Stohr
Institute ol Experimental Pathology, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, Germany
ABSTRACT
A flow cytometric study was carried out on the effects of
tumor-promoting 12-O-tetradecanoylphorbol-13-acetate (TPA;
10~8 M) on HeLa cells synchronized by amethopterin for DNA
synthesis. Cells treated with TPA at the time of release from
the amethopterin block showed a delayed passage through S
phase and partially through G? in their immediate life span as
measured 24 hr after release. This late G2 delay was not
observed when TPA was added to cells during late S or G2
phase. In this case, however, a direct inhibition of cells in G2
became evident as observed about 15 hr after release from
block. None of these effects was caused by the nonpromoting
4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (10~6 M).
These data support observations obtained with asynchronous
cultures. The TPA effects resemble those reported after X-
irradiation of cell cultures.
INTRODUCTION
Earlier studies with HeLa cells as a model system for the
action of a variety of phorbol esters have shown that these
compounds elicit specific responses. Changes in the phospho-
lipid metabolism (2) as well as in the thymidine incorporation
(6) were well correlated with the capacity of the chemicals to
cause inflammation and promotion in the mouse skin system.
In a previous study (3), indications for a variety of transient
changes in the HeLa cell cycle caused by small doses of TPA1
were observed. These data obtained with asynchronous cul
tures pointed to differential sensitivities and pharmacokinetics
dependent on the cycle staging. In order to clarify further the
complex situation, experiments with synchronized cultures
were carried out, thus increasing target cell compartments for
the action of TPA in particular stages of the cell cycle. Analysis
was by high-speed FCM. The data support results and inter
pretation of experiments with asynchronous HeLa cells (3).
MATERIALS AND METHODS
The phorbol esters TPA and 4-O-Me-TPA were a generous
gift from Prof. Dr. E. Hecker (Institute of Biochemistry, German
Cancer Research Center, Heidelberg, Germany). Acetone,
DMSO (Merck, Darmstadt, Germany), thymidine, adenosine,
DAPI (Serva, Heidelberg, Germany), and amethopterin (Lederle
Laboratories, Munich, Germany) were obtained from the
quoted sources.
Cloned HeLa cells were cultivated routinely in Eagle's mini
mum essential medium containing Earle's salts supplemented
with 10% calf serum as described elsewhere (3). For experi
ments, cells were transferred to plastic Petri dishes (Falcon) 2
or 3 days in advance. For synchronization, cells were treated
with 1CT6M amethopterin and 5 x 10~5 M adenosine for 16 hr
in complete medium if not otherwise stated (5). The block was
released by addition of thymidine (10 /¿g/106cells). At the time
points indicated, the medium was replaced by fresh medium
containing acetone or DMSO (0.05% final concentration) with
or without phorbol ester and with thymidine as above. For cell
counting (Coulter counter) and FCM analysis, cells were re
moved with trypsin (0.06% in phosphate-buffered saline con
taining, per liter, 8g NaCI, 0.02 g KCI, 1.15 g Na2HPO4-2 H2O,
and 0.2 g KH2PO4) at 37°. For FCM measurement, the sus
pended cells were washed with 0.1 M Tris-CI (pH 7.5) contain
ing 0.1 M NaCI, fixed with 70% ethanol, and collected at this
stage at 4°. Prior to the measurements, the above buffer
containing DAPI (3 /xg/ml) was added to the cells after removal
of the ethanol for quantitative fluorescent staining of cellular
DNA (1). The suspensions were carefully checked for their
single-cell status. The FCM analysis was carried out with a
computerized cell sorter FACS II (Becton-Dickinson Co., Moun-
1The abbreviations used are: TPA, 12-O-tetradecanoylphorboM 3-acetate;
FCM. flow cytometry: 4-O-Me-TPA, 4-O-methyl-12-O-tetradecanoylphorbol-13-
acetate; DMSO, dimethyl sulfoxide; DAPI, 4',6-diamidino-2-phenylindol.
Received February 22, 1980; accepted October 6, 1980.
Chart 1. DNA histograms of logarithmically growing HeLa cultures 04), of
cultures treated for 16 hr with amethopterin (B). and of cultures 6.5 hr (C) and 24
hr (D) after release by thymidine; abscissa, relative fluorescence intensity equiv
alent to DNA content; ordinate, relative cell number. HeLa cells were kept in
complete medium containing 10% calf serum.
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Effects of TPA on Synchronized HeLa Cells
8hr 10 hr
AFTER RELEASE
24 hr
TPA
1CT8[M]
4-0-ME-TPA
10'6[M]
-16hrAMETHOPTERIN0Tdr
RELEASE
COMPOUND8
10AA24 hrA
Chart 2. Progression of HeLa cells through the cell cycle in the presence of
1CT8 M TPA or 10"" M 4-O-Me-TPA as visualized by DMA histograms. HeLa cells
were grown for 2 days in the presence of 10% calf serum before addition of
amethopterin. The medium was replaced 16 hr later (cell number at this time, 1.2
x 106/5-cm dish) by fresh medium containing thymidine (Tdr) and a phorbol
ester (solvent. 0.05% DMSO). Cells were prepared for and analyzed by FCM
measurements; abscissa, relative fluorescence intensity equivalent to DNA con
tent; ordinate, relative cell number.
8hr 10 hr
AFTER RELEASE
24 hr
TPA
4-0-ME-TPA
KT6[M]
-16hr
r
AMETHOPTERIN
-r -t
6.5 8 «
A A 24 hr
Tdr COMPOUND
RELEASE
Chart 3. Progression of HeLa cells through the cell cycle with 10 8 M TPA or
10~6 M 4-O-Me-TPA added with fresh medium 6.5 hr after the amethopterin block had been released by direct addition of thymidine (Tdr). For further details,
see Chart 2.
tain View, Calif.) which was equipped with a UV laser beam.
For fluorescent excitation of DAPI, the laser was tuned to 363
nm at 500 milliwatts. The fluorescent pulses were detected
above 390 nm. In order to check the G2-M fraction during the
measurements for cell doublets, probes were sorted directly
on glass slides and controlled by fluorescence microscopy.
JANUARY 1981 307
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V. Kinzel et al.
15 HR 2t HR
-16 HR
ANETHOPTERIN 0 HR
RELEASE
V
6 HR
TPA 15 HR 2t HR
00vvu
nTPA
8 HR
TU D
TPA10 HR
TDa
Chart 4. Progression of HeLa cells through the cell cycle in the presence of
1CT8 M TPA and/or 0.05% acetone added with a medium change 6, 8, or 10 hr
after release from the amethopterin block. HeLa cells were grown for 2 days in
the presence of 0.5% calf serum prior to starting synchronization (cell number at
this time, 1.4 x 106 cells/5-cm dish). The block was released by directly adding
thymidine. For further details, see Chart 2.
RESULTS AND DISCUSSION
A preceding study (3) with asynchronous HeLa cells indi
cated differential sensitivities of HeLa cells to TPA depending
on the cell cycle staging. It seemed to be important in which
phase the cells were first exposed to the phorbol ester. In order
to amplify the number of cells, particularly in S phase and the
subsequent phases, synchronization at very early S phase by
amethopterin (5) was chosen. TPA, 4-O-Me-TPA, or the solvent
were added in fresh medium at time of release of the block or
later as indicated. At certain intervals, a set of cultures was
checked microscopically, counted, and prepared for FCM anal
ysis. The coincidence of results obtained with different methods
had indicated already (3) that TPA itself does not interfere with
the binding of the fluorochrome to DMA.
The synchronization procedure itself is documented by DMA
histograms in Chart 1. Cultures in logarithmic growth (A) have
accumulated mainly in early S phase 16 hr after addition of
amethopterin (ß).After release from the block by thymidine,
cells have moved to late S phase after 6.5 hr (C) and showed
24 hr after release the distribution of an almost random culture
(D). Chart 2 shows results of the addition of TPA (10"8 M) or 4-
O-Me-TPA (10~6 M) in fresh medium together with thymidine
for release of the amethopterin block. It can be noted that TPA
does not interfere with the release of the block by thymidine.
This result is in agreement with the first experiment ever done
and published on the action of TPA in cell cultures (4). There
fore, the Gìblock seen in asynchronous HeLa cultures (3)
occurs earlier in the cycle. The DNA histograms taken 8 and
10 hr after release show, however, a delayed traverse of the
synchronized fraction through S phase only for TPA, whereas
cultures with DMSO (data not shown) or 4-O-Me-TPA accu
mulate DNA faster (compare, e.g., the small valley between the
G, fraction and the right DNA peak of 8-hr TPA with the broader
one of 8-hr 4-O-Me-TPA, or compare the shape of the right
DNA peak of 10-hr TPA with 10-hr 4-O-Me-TPA). These data
support the observation obtained from asynchronous cultures
that, under the influence of TPA, cells accumulate DNA or
traverse through S phase more slowly than those in control
cultures in the presence of DMSO or 4-O-Me-TPA. The TPA
group shows in addition a rather large G2-M peak 24 hr after
release from the amethopterin block. The microscopic exami
nation of these cultures revealed only a relatively small number
of cells in mitosis at this time in the TPA group, indicating that
the peak represents mainly G2 cells. The addition of TPA 6.5
hr after the cells have been released from the amethopterin
block (Chart 3) also causes a delayed traverse of the cells
through the rest of S phase as evident at 10 hr (compare, e.g.,
the broad base of the right DNA peak of 10-hr TPA with the
small base of the corresponding peak of 10-hr 4-O-Me-TPA).
The decreased mitotic activity, i.e., the immediate G2 block in
the presence of TPA, became evident 11 to 12 hr after release
from the amethopterin block. In the DMSO and 4-O-Me-TPA
groups, more cells had divided than in the TPA group and
appeared therefore in the Gìfraction. Unlike the preceding
experiment (Chart 2), the 24-hr TPA group does not show a
pronounced G2-M peak. This supports the data obtained with
asynchronous cultures. Cells first exposed to TPA in the early
part of S phase exhibit an accumulation in G2 in their immediate
life span. This must probably be interpreted as a delayed
traverse through G2 of a part of these cells. This TPA effect is
different from the immediate G2 block seen in asynchronous or
here in synchronous cells.
For further analysis of these effects, experiments were car
ried out in medium containing 0.5% calf serum. Under these
conditions, cells pass more slowly through S phase after re
lease from the amethopterin block. TPA (10~8 M) together with
fresh medium was added either 6, 8, or 10 hr after release by
thymidine (Chart 4). The 24-hr values reveal a steady decrease
of the G2 fraction the later TPA was added, thus confirming the
above interpretation. The immediate TPA effect on G2, how
ever, is more pronounced the later TPA was added (compare,
e.g., 15-hr acetone values with 15-hr TPA values), in other
words, when most cells were in the sensitive phase at the
addition of TPA. Although no pulse treatment with TPA was
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possible as discussed earlier (3), these data seem to indicate
that the TPA addition as such mimics a pulse treatment even
though the material was not removed afterwards. The obser
vations described here are similar to results obtained after
treatment of synchronous cultures with X-irradiation or antitu-
mor drugs as discussed in detail elsewhere (3).
ACKNOWLEDGMENTS
We thank Dr. J. Reed for critically reading the manuscript.
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Effects of TPA on Synchronized HeLa Cells
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JANUARY 1981 309
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1981;41:306-309. Cancer Res
Volker Kinzel, James Richards and Michael Stöhr
Evaluated by Flow Cytometry
-Tetradecanoylphorbol-13-acetate asOPhorbol Ester 12-
Responses of Synchronized HeLa Cells to the Tumor-promoting
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