Download Cell Cycle-specific Effects of Tumor Necrosis

[CANCER RESEARCH 44, 83-90, January 1984]
Cell Cycle-specific Effects of Tumor Necrosis Factor1
Zbigniew Darzynkiewicz,2 Barbara Williamson, Elizabeth A. Carswell, and Lloyd J. Old
Sloan-Kettering Institute for Cancer Research, New York, New York 10021
ABSTRACT
The immediate effect of tumor necrosis factor (TNF) added to
cultures of L-cells is cytostasis, manifested as cell arrest in G2.
This effect prevails during the initial 4 hr when the number of G2
cells increases markedly in the absence of any significant cell
death in the culture. Shortly thereafter, the cytolytic effect be
comes apparent; extensive cell lysis can be detected after 7 hr
of exposure to TNF. After 24 hr nearly all cells are lysed. Results
of experiments in which the effects of TNF on the kinetics of cell
progression through various phases of the cell cycle were studied
indicate that in the presence of TNF cells do progress through
G2, although with considerable delay, and reach mitosis. Most
cells die (undergo lysis) specifically at late stages of mitosis
(telophase) or soon after cytokinesis. Sensitivity of cells to the
lytic effect of TNF is increased by cell arrest in mitosis with
Colcemid or vinblastine. TNF exerts little effect on rates of cell
progression through G1 or S phases of the cell cycle, although
few cells die during S phase. Nonlysed cells from cultures treated
with TNF do not show any changes in nuclear chromatin, sug
gesting that neither DNA nor nuclear proteins are the primary
targets of the TNF. The present data implicate metabolic changes
which occur during mitosis (cytokinesis), perhaps associated
with synthesis or assembly of cell membrane components, as
being responsible for increased cell sensitivity towards the cy
tolytic effect of TNF. The mechanism of the early cytostatic effect
of the factor is unknown.
INTRODUCTION
TNF3 is a protein found in the serum of mice, rats, or rabbits
presensitized with Bacillus Calmette-Guerin or Corynebacterium
parvum and challenged with bacterial endotoxin (4, 16, 19, 20,
22, 24). Its name derives from the hemorrhagic necrosis that is
observed in certain transplantable tumors of the mouse after
systemic administration of the TNF. Because bacterial endotoxin
elicits a similar antitumor reaction, it had to be proved that the
activity of TNF was not due to the presence of residual or
modified endotoxin. Evidence accumulated over the past several
years indicates that TNF and endotoxin are separate factors and
that TNF is involved in mediating the hemorrhagic necrotic effect
of endotoxin (for reviews, see Refs. 17,18,23, and 27). A major
distinction between endotoxin and TNF is that TNF has a direct
cytotoxic or cytostatic effect on a variety of different tumor cell
types in vitro, whereas endotoxin does not. Cloned lines of
mouse histiocytomas produce a factor with TNF activity, sug
gesting macrophages as at least one of the normal cellular
sources of TNF in vivo (21, 23). Although it was observed that
1Supported by Grants CA28704 and 23296 from the National Cancer Institute.
2To whom requests tor reprints should be addressed, at Sloan-Kettering Insti
tute for Cancer Research, Walker Laboratory,
145 Boston Post Road,
Rye.N.Y. 10580.
3 The abbreviations used are: TNF, tumor necrosis factor; DAPI, 4' ,6-diamidino2-phenylmdole; AO, acridine orange.
Received March 11,1983; accepted September 28,1983.
JANUARY
cell sensitivity to TNF correlates with rate of proliferation (27),
mechanisms by which TNF exerts its effect on the target cells,
especially as related to the cell cycle position, are unknown.
Flow cytometric techniques recently have been developed to
investigate the cell cycle distribution and kinetics in more detail
than was possible previously (for review, see Ref. 6). These
techniques, based on simultaneous differential staining of DNA
versus RNA (9,10) or analysis of nuclear chromatin as reflected
by sensitivity of DNA in situ to denaturation (8, 11), were ex
tended to studies on the mechanism of drug effects in relation
to the cell cycle (12, 13). Utilizing these methods, we have
investigated the cell cycle-specific effects of TNF. Staining of
DNA alone or simultaneous staining of DNA versus RNA enabled
us to observe 2 distinct effects of TNF, cytostasis followed by
cytolysis, as well as to measure the extent of RNA accumulation
during cytostasis. Effects of TNF on cell progression through Gì,
S, G2, and transition to M were studied in stathmokinetic exper
iments, in which recognition of cells at different phases of the
cycle, as well as living versus dead cells, was based on differ
ences in both DNA content and chromatin structure (11-13).
MATERIALS AND METHODS
TNF Preparation. A highly active TNF fraction was prepared from the
serum of C. pa/vum-primed (1 mg i.p.; Burroughs Wellcome Laboratories,
Triangle Park, N. C.) female CD-1 mice (Charles River Breeding Labora
tories, Inc., Wilmington, Mass.), which were given injections of endotoxin
on Day 10 (10 ^g i.V., lipopolysaccharide W from Escherichia coli
0127:B8; Difco Laboratories, Detroit, Mich.) and exsanguinated 1.5 hr
later (4, 16). After lipid was removed by differential centrifugaron (16 hr
at 110,000 x g), the serum was fractionated by batch method DEAEion exchange using a ratio of 10 g dry gel to 1 g protein and was further
purified by Sephadex G-100 and G-200 (Pharmacia Fine Chemicals,
Piscataway, N. J.) column chromatography and Affi-Gel Blue (Bio-Rad
Laboratories, Richmond, Calif.) affinity chromatography.4 The final prep
aration of TNF contained 355 /»gprotein per ml and was further diluted
for the experiments described in the text.
Cells. Mouse L-M cells derived from the NCTC clone 929 (L) line were
obtained from the American Type Culture Collection and grown in Eagle's
minimum essential medium (Grand Island Biological Co., Grand Island,
N. Y.) supplemented with nonessential amino acids, penicillin (100 units/
ml), streptomycin (100 /ig/ml), and 8% heat-inactivated fetal calf serum
at 37°.A TNF-resistant L-cell line was derived from sensitive L-cells by
repeated passages in TNF-containing
medium (4, 16). The activity of
TNF was assayed (4) by adding 0.5 ml of medium containing serial
dilutions of TNF to wells (24-well Costar plates) seeded 3 hr previously
with 50,000 trypsinized L-cells (in 0.5 ml of medium), and determining
the amount of protein that resulted in 50% cell kill estimated at 48 hr by
phase microscopy. The TNF preparation used in this study had an activity
of 3.2'ng (50% cell kill) on the sensitive cell line but showed no cytotoxicity
on the resistant L-cell line up to 7 ¿>g
(the highest concentration tested).
Cells for cell cycle experiments were trypsinized from exponentially
growing cultures and inoculated into T-35 flasks (Falcon) 12 hr before
addition of various concentrations
of TNF. During exponential growth,
4 K. Haranaka, E. A. Carswell, B. D. Williamson, J. S. Prendergast, and L. J.
Old, manuscript in preparation.
1984
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83
Z. DarzynkÃ-ewicz et al.
the doubling time of these cells was 18 to 20 hr.
Stathmokinetic Experiments. Two sets of parallel cultures of expo
nentially growing cells were prepared in T-35 flasks (500,000 cells/flask).
At 0 time, all cultures received vinblastine (0.1 tiQ/m\, final concentration;
Eli Lilly, Indianapolis, Ind.). At the same time, one set of cultures was
treated with 0.88 >tg TNF per flask. Beginning from 0 time, individual
cultures were harvested by trypsinization at hourly intervals up to the
12th hr. Care was taken to collect both cells that were attached to dishes
(removed by trypsinization) and cells floating in the medium. The latter
consisted of either living cells detached during mitosis or dead cells. All
cells from each culture were pooled, rinsed with Hanks' salt solution,
and fixed as described previously (11-13). The Stathmokinetic experi
ment was repeated several times using different mitotic blockers (Colcernid, 0.05 ¿ig/ml,and vincristine, 0.01 ^g/ml) and different concentra
tions of vinblastine, with essentially similar results as reported presently.
Different concentrations of TNF (0.3 to 2.2 pg) were tested, and the
experiments were also performed on the TNF-resistant subline of L-cells.
Cell Staining. Several staining techniques were used for cell analysis.
To stain DNA in intact cells, 0.2-ml aliquots of cell suspensions (contain
ing approximately 2 to 5 x 105 cells in tissue culture medium) were
admixed with 2.0 ml of a solution containing DAPI (2 itg/m\), 0.1% (v/v)
Triton X-100 (Sigma Chemical Co., St. Louis, Mo.), 0.1 M piperazineN,N'-bis(2-ethanesulfonic
acid buffer (Calbiochem. La Jolla. Calif.) and 2
mm MgCI2, pH 6.4 and 0°.DAPI was synthesized and kindly provided
by Dr. Jan Kapuscinski of the Sloan-Kettering Institute. At 0.1% Triton
X-100 and in the presence of serum proteins (from the original suspen
sion), the cells do not lyse (unless subjected to mechanical stresses),
but they do become permeable and, thus, may be stained with DAPI. In
some experiments, only viable cells were stained. To this end, prior to
staining, cells were prei neu bated in the presence of a freshly prepared
mixture of enzymes containing DNase I (100 fig/ml; Worthington Bio
chemical Corp., Freehold, N. J.) and trypsin (2.5 mg/ml; Grand Island
Biological Co.) in Hanks' salt solution, at 37°for 30 min. As described
before (7, 25), such pretreatment removes from the suspension all dead
cells that cannot exclude the enzymes, leaving viable cells with un
changed DNA content. Nearly 103 cells were counted per DNA channel
at maximal cell frequency (Chart 1), and approximately 2 x 10* cells
were counted for each sample.
Simultaneous staining of DNA and RNA by AO (Polysciences, Inc.,
Wamngton, Pa.) was described previously (9, 10). The staining of perme
able cells was done at an AO concentration of 13 /¿M
and dye/DNA-P
molar ratio >2.0 in the presence of 1 rriM sodium EDTA. Under these
conditions, interaction of AO with DNA results in green fluorescence,
with maximum emission at 530 nm (FM(>)whereas binding to RNA gives
red metachromasia with maximum emission at 640 (F>«oo).
The intensity
of green fluorescence is proportional to DNA content of the cell, and the
intensity of red fluorescence correlates with RNA content (2). The spec
ificity of DNA and RNA staining was evaluated by preincubation of
permeable cells with DNase or RNase as described previously (9, 10).
Denaturation of DNA in situ by acid was described previously (9-11).
Briefly, cells, after fixation, were resuspended in 1 ml of Hanks' solution
containing 1000 units of RNase A (Worthington) and incubated at 37°
for 1 hr. Then, 0.2-ml aliquots (~4 x 105 cells) were admixed with 0.5 ml
of 0.2 M KCI/HCI buffer, pH 1.35, at 20°.Thirty sec later, the cells were
stained with AO solution (4 /ig/ml) in 0.1 M citric acid/0.2 M Na;.HPO4
buffer, pH 2.6. The red and green fluorescence of cells thus stained
represents the stainability of denatured and double-stranded DNA, re
spectively (8, 13). In this technique, the distinction between cells at
various phases of the cell cycle is based both on differences in DNA
content and in chromatin structure. Changes in chromatin structure,
especially during progression through Gìor transition between G2 and
M, are reflected in the extent of DNA that is sensitive to denaturation
(11-13).
Fluorescence
Measurements.
Fluorescence of individual cells
stained with DAPI was measured with an ICP-22 flow cytometer (Ortho
Diagnostics, Westwood, Mass.) using the UG 1 excitation filter and
emission filter transmitting between 450 and 510 nm. The coefficient of
84
variation of the mean value of the G, peak was approximately 2.0%.
Fluorescence of cells stained with AO was measured in an FC 200 flow
cytometer (Ortho Diagnostics) as described in detail previously (8, 29).
The red (F>«oo;
600 to 640 nm) and green (F-ot,; 515 to 575 nm) fluores
cence emission from each cell were measured simultaneously by sepa
rate photomultipliers, and the data were stored in the computer. The
pulse width values of nuclei fluorescence were recorded to determine
nuclear diameter and to eliminate cell doublets from the measurements
(29). The experiments were repeated at least twice, and 5,000 or 10,000
cells were measured per sample.
The number of cells in Gì,S, and G2 (or G2 + M) compartments was
obtained using a modified graphical method of Baisch ef al. (1) by folding
the left half of the G, peak and the right half of the G2 + M peak around
their modes and interpreting areas under the new, symmetrical d and
G2 (or GS + M) peaks as representing G, and Go (or G2 + M) cells,
respectively, and the cells in between these peaks as being in the S
phase. Following acid denaturation of DNA, cells in mitosis could be
distinguished as described previously (7) based either on single-param
eter (F .eoo)histogram analysis or by the gating analysis of the 2-parameter
(Fsaoversus F>«oo)
histograms, inasmuch as the M-cell clusters were
totally separated from the interphase cells.
In all experiments, parallel to the cultures exposed to TNF, the timematched untreated control cultures were analyzed to assess whether or
not changes in cell cycle distribution or viability occurred throughout the
experiment. In none of the time-matched control cultures was there ever
observed a higher than 2% change in the proportion of cells in any
particular phase of the cell cycle or in the proportion of nonviable cells
when these cells were stained with DAPI. In AO-stained samples, the
variability was higher but did not exceed 3.5%. Thus, the experiments
were performed on cells during the steady-state exponential phase of
their growth. The straight line representing accumulation of cells in
mitosis during the Stathmokinetic experiment (Chart 5) provides addi
tional evidence that the cells studied grow exponentially.
The reproducibility of the staining and analysis steps, tested by
staining 10 separate samples from a single culture, and expressed as
maximal variability in the percentage of cells in any particular phase of
the cell cycle from the mean value, was below 1% for DAPI-stained cells
and below 2.5% for cells stained with AO.
A technical note should be added on frequency histograms as shown
in Charts 1 and 3. In Chart 1, for comparison of the relative heights of
G, versus G2 + M peaks within and between the samples, the vertical
scale is constant (103 cells/channel), and G, peaks are of the same
height, with approximately 103 cells/channel at maximum. Consequently,
when cells are arrested in G2 + M, the total number of counted cells per
sample is proportionately higher because there are proportionately fewer
d cells. Therefore, although the percentage of cells in S phase may be
the same or even lower (e.g., Chart 1, A versus B or D). the outline level
of the S-phase distribution on the histograms may be elevated because
more S-phase cells have to be counted for each channel (between 2C
and 4C). and more cells have to be counted per sample. In contrast to
Chart 1, the frequency histograms generated by the computer in Chart
3 (insets) have a different vertical scale; thus, the relative heights of G,
versus G2 peaks can be compared only within the same sample.
RESULTS
Effects of TNF on Exponentially Growing Unperturbed Cul
tures
Staining of Cellular DNA with DAPI. Effects of TNF on
exponentially growing L-cells were analyzed by studying the
distribution of cells in the cell cycle and cell viability by 3 different
staining techniques. The representative data from one of the
experiments in which the cultures were treated with TNF for up
to 6 hr and the cells harvested at hourly intervals and their DNA
content analyzed after staining with DAPI are shown in Chart 1.
CANCER
RESEARCH
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VOL. 44
Effects of TNF on the Cell Cycle
10*AG,-50
S - 29
S -29
G2.M-21MB^JG,-47
G2.M-24MCJG,~45
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-36
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E
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(channel
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100)
Chart 1. Changes in the cell cycte distribution of L<ells growing in the presence of TNF. Frequency distribution histograms indicating DNA content (after staining with
DAPI) of individual cells from the control (0 time) culture (A) and from cultures treated with 4.1 pg/ml of TNF for 2 (B), 4 (C), and 6 (D) hr. E, cells from cultures treated
with TNF for 6 hr (as in 0) which, prior to staining, were preincubated with DNase I and trypsin (see 'Materials and Methods"). As described previously (7, 25) and
confirmed recently by Frankfurt (15), such treatment selectively removes nonviable cells which, having lost membrane integrity, cannot exclude the enzymes. As is
evident, an increase in the proportion of cells in G, + M, from 21 to 33%, occurs during cell treatment with TNF for 6 hr. After 6 hr of treatment with TNF at that
concentration, there is approximately 20% of nonviable cells. When nonviable cells are removed by trypsin plus DNase treatment (E), a decrease in proportion of G, and
an increase in S and 62 cells is apparent, indicating that among the nonviable cells there are disproportionally more cells with a G, DNA content and fewer with S or G?
DNA content in comparison with viable cells.
In this experiment, the ceils were stained either directly after
harvesting or following incubation with a freshly prepared mixture
of trypsin (2.5 mg/ml) and DNase I (100 ng/rrA). The enzymatic
treatment, as reported previously (7, 25), results in hydrolysis of
DNA in dead cells, which, having lost their membrane integrity,
cannot exclude the enzymes, leaving the viable cells unchanged
for DNA analysis. A similar procedure was recently proposed by
Frankfurt (15) as a viability test for flow cytometry. The data in
Chart 1 clearly indicate that, in the presence of TNF, the propor
tion of cells with a G;> + M DNA content is increased. Thus,
during the 6-hr exposure to 4.1 ^g of TNF, there was an increase
from 21 to 33% of G2 + M cells and a decrease in the proportion
of Gìand S-phase cells, indicating that TNF arrests cells in G2 +
M.
In all cultures, both control and TNF-treated ones, the relative
proportion of cells in Gì,S, and G2 + M was measured before
and after incubation with trypsin plus DNase. Differences no
larger than up to 2% in cell cycle distribution, as a result of the
enzymatic treatment, were seen in all control cultures and up to
4 hr of exposure to TNF in TNF-treated cultures (not shown).
After 4 hr, however, the proportions changed, and as is evident
in Chart 1E, in TNF-treated and in trypsin-plus-DNase-incubated
cultures, a significant decrease in percentage of Gìand an
increase in percentage of S-phase cells were observed. These
data indicate that, among nonviable cells removed by the enzy
matic treatments, there was a higher proportion of cells with a
GìDNA content than among viable cells. When only viable cells
are compared during the experiment (Chart 1, A versus E), the
specific cytostatic effect of TNF in arresting cells in G2 + M is
even more pronounced, inasmuch as the respective heights of
the Gìversus G2 + M peaks at 0 time and after 6 hr of treatment
with TNF show an even greater difference. The experiment
reported in Chart 1 was run at hourly intervals. The results not
JANUARY
included in Chart 1, i.e., at 1, 3, and 5 hr of treatment with TNF,
agree with the included data, showing, e.g., an increase in the
percentage of G2 + M cells from 22 to 25 and 29% at 1, 3, and
5 hr, respectively. In this experiment, the parallel, time-matched
control cultures were analyzed and were found not to vary by
more than 2% in DNA distribution throughout the experiment
(not shown).
Simultaneous Staining of RNA and DNA. In this set of
experiments, the cultures were treated with different concentra
tions of TNF for various periods of time up to 24 hr, and the cells
were then stained with the metachromatic fluorochrome AO
under conditions in which cellular RNA and DNA stain differen
tially (9, 10). Description of the method, its applicability, and
specificity of staining are given elsewhere (6, 7, 9,10). With this
technique, it is possible not only to discriminate cells at different
phases of the cell cycle (based on DNA stainability) and to
estimate their relative RNA content but also to distinguish nonviable, lysed cells which have lost most of their cytoplasmic
constituents, including RNA (7). Results of these experiments
confirming the data already described are shown in Chart 2 and
Table 1. Namely, the effects of TNF were visible shortly after
addition of TNF to cultures; initially (up to 7 hr), the cytostatic
effect was most evident and was seen as an arrest of cells in G2
+ M. With time, the cytolytic effect became predominant, and
the lysed cells could be recognized easily on the scattergram
representing DNA versus RNA values (Chart 2). Their number
was markedly increased in cultures exposed to TNF for 7 hr or
longer. The population of lysed cells had a disproportionately
higher number of cells with a GìDNA content in comparison with
nonlysed cells. Nonlysed cells from TNF-treated cultures had
increased RNA content, which was especially evident for the G2
+ M population.
Table 1 lists quantitative changes in the cell cycle distribution,
1984
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85
Z. Darzynkiewicz et al.
mo
100
100
100
>eoo
Chart 2. Scattergrams representing green (F5M)and red (F>VM)fluorescence intensity (in arbitrary units equivalent to channel number) of individual L-ceiis from control
and TNF (2.2 (/g)-treated cultures. Following cell staining with AO, f saoand F^oo are proportional to DMA and RNA content, respectively (2,6). A separate cluster of cells
with minimal RNA content seen in TNF-treated cultures (7 hr) represents dead (lysed) cells; G, population of lysed cells has F>Ka values dose to 10 units. Nonlysed cells
from TNF-treated cultures, especially Gj + M, have increased RNA content. Thus, most cells of the G2 + M population in the TNF-treated cultures are located between
Channels 60 and 70 (F«oo)
compared with Channels 50 to 60 in the control. The ability of cells to accumulate RNA indicates that these cells are arrested in Gj rather than
in M, because in the latter phase the cells do not transcribe DNA. The quantitative data from these experiments are shown in Table 1. Parallel, time-matched control
cultures (up to 24 hr) did not show a change in the percentage of lysed cells or in the proportion of cells in G, versus S versus 62 + M greater than 3.5%; the variability
in mean values of f .«„
in these controls was below 3%.
Table 1
Changes in viability, cell cycle distribution, and FINA content of L-cells treated
with TNF
Exponentially growing cells were treated with 2.2 or 8.8 t¡gof TNF for various
periods of time, stained with AO, and analyzed by flow cytometry. as shown in
Chart 2. Proportions of cells at different compartments, as listed, were estimated
using the computer-interactive programs. Lysed cells having minimal RNA formed
separate clusters which made possible their identification. Respective proportions
of G,, S, and G., + M cells were calculated among the nonlysed (high RNA) cell
population, except as shown in Line 8. These cells were viable as judged by their
progression through the cycle (cumulative arrest in Gz + M) and increase in RNA
content (F .«„.
last column). Approximately 5% increase in RNA content (F.«»)
of
Gì-and S-phase cells in TNF-treated cultures was also observed (not shown).
CÃœis
Treatment
TNF fcg
protein)
Time
(hr)
Dead
(lysed)
(%)
G, (%)
S (%)
Gz + M 62 + M
(%)
(F>«»)
RNA. Parallel, time-matched, untreated control cultures did not
show a change greater than 3.5% in cell viability or in the
percentage of cells in Gìversus S versus G2 + M during the
experiment for up to 24 hr. The results shown in Table 1,
indicating the early cell arrest in G2 + M and subsequent cell
lysis, were confirmed in 3 other experiments in which TNF was
used at different concentrations (0.22 to 19.8 /ig)Distinction between Cell Arrest in G2 versus M. It is clear
from the data obtained by cell staining with DAP) (Chart 1) and
AO (Table 1) that the TNF induces a transient cytostatic effect,
arresting cells in G2 + M. An increase in RNA content of the
viable G2 + M population suggests that the arrest is in G2 rather
than in M because during mitosis cells do not synthesize RNA.
To obtain direct evidence, however, as to whether the arrest is
in G2 or in M, experiments were done in which the TNF-treated
Control2.
1.
TNF3.
cultures were stained according to another technique utilizing
TNF4.
AO, which makes it possible to discriminate between G2 and M
TNF5.
TNF6.
(7, 8, 11). The results shown in Chart 3 indicate that among
TNF7.
viable cells there is an increased proportion of cells in G2.
TNF8.
TNF(None)2.28.82.28.8a2.28.88.84477242473.06.38.624.432.296.8100100a49.446.339.440.539.076.127.225.125.726.425.515.223.428.634.933.135.58.754.461.563.162.264.122.9
The results of several experiments in which 3 different tech
'' Selected population of dead (lysed) cells, obtained from a culture treated with
TNF (8.8 /jg protein) for 7 hr (une 5); there is a predominance of G, cells among
lysed cells.
proportion of dead (lysed) cells, and RNA content as a result of
exposure to 2.2 and 8.8 ng of TNF. The cytostatic effect is most
evident at 4 hr in culture with 8.8 ^9 TNF. At that time, the
number of cells in G2 + M is increased from 23.4 to 34.9%,
concomitant with a decrease in Gìfrom 49.4 to 39.4%. Thus,
nearly total cell arrest in G2 + M is observed under these
conditions at a time when there are only few (8.6%) dead cells.
After 7 hr, the number of cells arrested in G2 + M is similar at
both concentrations of TNF and is not significantly different from
that seen at 4 hr. Cell lysis, however, is already extensive. At 7
hr, one-fourth and one-third of the total cell population is lysed
in cultures with 2.2 or 8.8 ^g TNF, respectively. Surviving cells
arrested in G2 + M have their RNA content increased by 14 to
18%, while cells in Gìor S phase show only a 5% increase in
86
niques of cell staining were used all agree that TNF induces a
transient cell arrest in G2 + M, lasting for approximately 4 to 6
hr, which is followed by cell death (lysis). Among dead cells,
there is a disproportionally high percentage of cells with a G,
DNA content in comparison to that of viable cells. In these
studies, 3 different techniques were used to distinguish nonviable
cells, namely, cell ability to exclude the enzymes, loss of the
cytoplasmic constituents, and high denaturability of DNA: The
first technique detects the loss of cell membrane integrity; the
second, cell lysis; and the third, nuclear chromatin changes,
perhaps leading to pyknosis (11-13). Thus, the population of
nonviable cells detected by each of these methods may not be
identical and may represent different stages of cellular changes
associated with cell death.
Stathmokinetic Experiment
The experiments
described
above, in which exponentially
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RESEARCH
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VOL. 44
Effects of TNF on the Cell Cycle
Vinblastine
TNF
CONTROL
Vinblastine+TNF
DEAD
'CELLS
F>600
Chart 3. Discrimination of cells in G,, S, G2, and M phases of the cell cycle in
the exponentially growing culture of L-cells and in the culture treated with 4.1 pg
TNF for 6 hr. The distinction between the phases is based on differences in DMA
content (G, versus S versus G2) and chromatin structure (G2 versus M; viable
versus dead). In this technique of cell staining, RNA is specifically removed, and
cells are stained with AO following partial denaturatoli of DMA (8-10). Computerdrawn 2-parameter frequency histograms indicate cell distribution with respect to
FMOand F>eoo,which represent proportions of double-stranded and denatured DNA,
respectively (6-9). The cell number is proportional to the heights of the peaks,
scaled arbitrarily. The broken diagonal line separates G,, S, and G¡cells from the
M and dead cells. The M and dead cells were gated out, and only G,, S, and G2
cells (upper left) were projected on the F^y» coordinate to obtain the singleparameter histograms. It is apparent from the respective heights of the G, versus
G2 peaks on these histograms that there is an increase in proportion of G2 cells
among viable cells in the TNF-treated cultures. A large number of dead cells are
characterized by low DNA content. It should be stressed, however, that in this
staining procedure the fluorescence distribution among dead cells is not an accurate
reflection of their DNA content because the DNA stainability of dead cells may be
affected by DNA degradation or chromatin changes (pyknosis) related to cell death.
Therefore, the frequency distribution of the dead cell population cannot be consid
ered as representative of their cell cycle distribution.
growing cells were exposed to TNF, could detect major effects
of TNF such as cell arrest in G2 or cell death but could not reveal
minor changes in the cell cycle (if they did occur) such as a
slowdown in cell progression through Gì
or S phase, for instance.
To this end, the next series of experiments were done in order
to estimate in more detail the points in the cell cycle that may be
specifically sensitive to the action of TNF. The recently intro
duced method of cell cycle analysis based on the stathmokinetic
experiment (12,13) was used. In this technique, the investigated
drug and the stathmokinetic agent (vinblastine) are added to
cultures of exponentially growing cells at the same time; cell
samples are collected at hourly intervals and stained with AO in
such a way that individual phases of the cell cycle, including G2
versus M, may be distinguished (13). In pilot experiments, it was
observed that the cytotoxicity of TNF is increased in the presence
of vinblastine. Therefore, to investigate a similar range of TNFinduced cytostatic and cytotoxic effects as shown in Charts 1 or
2 and Table 1, a lower concentration of TNF (0.88 tig) was used
in stathmokinetic experiments. Histograms representing cell dis
tribution in cultures treated with vinblastine alone or with vinblas
tine and TNF are shown in Chart 4.
As cell progression through the cycle is halted in M during
stathmokinesis, several kinetic parameters may be estimated by
analyzing changes in cell number in Gì,S, G?, and M (12,13). It
is already evident from the raw data (Chart 4) that vinblastine
alone induces a transient (up to 4 hr) arrest of cells in G? prior to
blocking them in M. This was observed in numerous experiments
using different concentrations (0.05 to 0.5 ng/m\) of vinblastine
as well as of Colcemid and vincristine. Thus, the subline of Lcells used in this study, after addition of the mitotic blockers,
JANUARY
Chart 4. Progression of cells through various phases of the cell cycle and their
accumulation in mitosis, in vinblastine (left column)- and vinblastine plus TNF (right
co/umn)-treated cultures. Vinblastine and TNF (0.88 ng) were added at 0 time, and
cells were sampled hourly up to 12 hr; cell histograms after 0, 4, 8, and 12 hr are
shown. Cells at different phases of the cell cycle were distinguished as described
previously (12, 13). In addition, dead cells with low F;,VJvalues (increased DNA
denaturation) could be discriminated and quantified.
exhibits an unusually long delay before cells begin to accumulate
in M; during the lag phase the cells are blocked in G2. This
peculiarity of the cells does not interfere with the stathmokinetic
experiment, because, although transiently arrested in G2 instead
of in M, the cells do not "leak" through the mitotic block (see
below), and thus the transit rates through earlier phases can be
estimated.
Several conclusions may be drawn concerning the effect of
TNF on the cell cycle based on visual inspection of the histo
grams obtained from stathmokinetic experiments (Chart 4). It is
evident that TNF does not markedly affect the kinetics of cell
progression through the early phases of the cycle; the rates of
emptying of Gìand early S-phase compartments are quite simi
lar, with only a few cells remaining in Gìafter 12 hr in both sets
of cultures. Likewise, the proportion of cells in S phase (among
viable cells) is similar in both the control and TNF-treated cultures
throughout the experiment. The main effects of TNF are mani
fested as a deficit in M-cell population and in the appearance of
dead cells, both changes already clearly seen after 8 hr and very
1984
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87
Z. Darzynkiewicz et al.
1
2
4
i
of
S
6
7
8
9
10
11 12
1
Ti
s tat
h mokinesi
s
(HD
Time
2
3
of
4
5
6
7
stathmokinesis
8
9
10
11 12
(Hr>
Chart 5. The rates of ce«exit from GìW and entrance to GÌ
+ M (B, upper slopes) or M (8, tower s/opes) in control and TNF-treated cultures during stathmokinesis
induced by vinbiastine The number of cells remaining in G,, S, G?, and M was estimated in samples collected at hourly intervals during cell treatment with vinblastme
(control) or vinbiastine plus TNF, as described in Ref. 12 and Chart 4. The number of cells in G,, Gfe,or M is plotted against the time of stathmokinesis; tc. fraction of
cells in M or 62 + M, respectively. As is evident, there is a 4-hr delay in the accumulation of cells in M in control cultures, while the number of cells in G, + M increases
from the onset of the experiment.
pronounced after 12 hr. This observation alone implies that in
TNF-treated cultures cell death occurs quite specifically, either
in G;., during transition from G2 to M, or in mitosis. If cell death
were to occur at random during the cycle, this would result in
preservation of identical proportions of cells in all phases, includ
ing M, among surviving cells in treated cultures as compared
with the control. This, however, was not observed.
Quantitative analysis of changes in cell number in G1t G2 + M,
and M during the stathmokinetic experiment, allowing estimation
of the rates of cell progression through various points of the
cycle (12) is provided in Chart 5. A slowdown in the rate of cell
exit from Gìis evident after 5 hr of exposure to TNF. The effect
is rather minor and therefore escapes detection by visual inspec
tion of the histograms (Chart 4). The slope representing cell
accumulation in G2 + M or M during stathmokinesis permits one
to estimate the duration of the cell cycle (12, 26); based on this
slope, the duration of the cycle in L-cells is 18 hr. This value is
in agreement with the doubling time of these cells (18 to 20 hr)
as estimated from the growth curves. In the presence of TNF, a
decrease in the rate of cell entrance into G2 + M is already
observed by 3 hr. The most pronounced differences between
control and TNF-treated cultures is expressed in the rate of cell
accumulation in M (Chart 5). In the presence of TNF, fewer cells
are arrested in mitosis between 4 and 12 hr. The difference
increases progressively with time, and after 12 hr there are more
than twice as many mitotic cells in the control cultures than are
in the TNF-treated cultures.
Comparison of the slopes representing accumulation of cells
in M versus G2 + M in the TNF-treated cultures indicates that at
later stages (6 to 12 hr) the deficit in cell number in M is higher
than in G2 + M, with respect to control values. This indicates
that there is a higher proportion of G2 cells in TNF-treated than
88
in control cultures, compensating for the deficit in M cells. As a
consequence, the change in G2 + M attributed to TNF (in
absolute cell number) is less pronounced than the change in M.
At the end of the experiment (12 hr), among viable cells there
were 64.4% G2 cells in TNF-treated cultures, compared with
48.9% in controls (not shown). This finding is consistent with the
observation that TNF induces cell arrest in G2 + M (see Charts
1 and 3 and Table 1). However, the staining method used in the
stathmokinetic experiment, which discriminates G2 versus M
cells, makes it possible to localize the point of cell arrest in the
cell cycle with greater accuracy than does single-parameter DNA
analysis (Chart 1) or simultaneous staining of RNA and DNA
(Table 1). These data also confirm the observation on exponen
tially growing cells, as shown in Chart 3.
Changes in proportions of all cells, including dead (lysed) cells,
are shown in Chart 6. In control cultures, a continuous decrease
in cell number in G, is paralleled at first by an increase in G2 (up
to the 4th hr) and then by an increase in M, with cells in G2
remaining at a plateau during the later stage. A decrease in Sphase cells becomes evident after 8 hr, and there are few dead
cells throughout the experiment. In cultures treated with TNF, a
continuous decrease in cell number in Gìis also observed.
However, there is a progressive increase in the G2 cell population
without reaching a clear plateau. The increase in M-cell popula
tion is minor, and the number of dead cells begins to increase at
4 hr and shows a dramatic rise after 9 hr, reaching over 30% by
12 hr.
The cytostatic and cytolytic effects of TNF, although less
pronounced than described above, were also observed at a
lower concentration of TNF (0.03 pg/culture). No perturbations
of the cell cycle were observed when the TNF-resistant subline
of L-cells was treated with TNF (up to 8.8 ^g/culture) under
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VOL. 44
Effects of TNF on the Cell Cycle
O
Time
of
stathmokinesis
11
12
(Hr)
Chart 6. Changes in viability and in the proportion of cells at various phases of the cell cycle, in cultures treated with vmblastme alone, added at 0 time (left panel),
and with vinblastine, added together with 0.88 ^9 TNF (right panel).
similar conditions. The control, time-matched untreated cultures
did not show changes greater than 3.5% in the cell cycle distri
bution throughout the experiment, up to 12 hr.
Although it is rather difficult to estimate distribution of the cell
cycle among dead cells based on chromatin staining (Charts 3
and 4), it appears that in the stathmokinetic experiment most
dead cells are characterized by a G2 + M DNA content, as their
position on the histograms is close to G2 and M clusters (Chart
4; 8 and 12 hr). By contrast, in the absence of vinblastine, dead
cells in TNF cultures have a predominantly GìDNA content
(Chart 1; Table 1). These data, together with other evidence (see
"Discussion"), indicate that in the absence of vinblastine the TNFtreated cells die preferentially either late in M (telophase) or in
Gì;vinblastine, however, arrests them in metaphase, precluding
transit to Gì.In the presence of vinblastine, therefore, cells die
preferentially either in metaphase or earlier in M during transition
from G2. It should be noted that in the presence of vinblastine
(or other mitotic inhibitors), the cells become more sensitive to
the cytotoxic action of TNF.
DISCUSSION
Based on the results of experiments described above, the
following sequence of events leading to death of cells exposed
to TNF can be constructed. The first effect is a transient cell
arrest in G2. The arrested cells show signs of unbalanced growth,
characterized by elevated RNA content. Some increase in RNA
also occurs in cells that progress through S or Gt. Few cells,
however, die in Gì,S, or G2 during the first 0 to 8 hr following
addition of TNF. Although delayed, the cells enter and progress
through mitosis. Cell death (lysis) occurs specifically at the late
stages of mitosis (telophase) or shortly following cytokinesis. As
a consequence, most lysed cells have a Gìcontent of DNA. In
summary, either telophase or some very early postmitotic stage
appears to be the specific point in the cell cycle at which TNF
treated cells undergo lysis. This conclusion is also supported by
the earlier phase-contrast and time-lapse cinematography ob
servations of cells in culture, indicating that in the presence of
TNF many cells enter mitosis and lyse.5
The stathmokinetic
experiment was designed to analyze in
5 J. Hlinka, B. Williamson, S. Green, and L. J. Old. Tumor necrosis factor-cycle
dependent cytotoxicity
JANUARY
by TNF on L929 cells, unpublished observations.
more detail the cell cycle-specific effects of TNF, as was done
previously for several antitumor drugs (13, 29). The initial exper
iments brought to light 2 findings that require discussion. The
first was the observation that the subline of L-cells used in this
study responded to mitotic inhibitors such as Colcemid, vincristine, or vinblastine by a long delay prior to entering M phase.
These agents induced a prolongation of G2 lasting approximately
4 hr. Following this delay, cells entered M and became arrested
in M as expected. Other sublines of L-cells do not show this
effect (3). The second finding was an increased sensitivity to
TNF in the presence of the mitotic inhibitors. Thus, in stathmo
kinetic experiments utilizing vinblastine, the concentration of TNF
had to be lowered 10-fold to obtain a range of cytotoxicity
comparable to the experiment in which vinblastine was not used.
The mechanism of the synergistic action of the mitotic blockers
and TNF is unknown. One may assume, however, that if M is
the most sensitive portion of the cell cycle to TNF action,
prolongation of M by the blockers could enhance the cytotoxic
effect of TNF.
The stathmokinetic experiment also provided evidence that
cells in G! are resistant to TNF. Cell exit from Gìwas affected
very little by TNF, and also, few G1 cells died during exposure to
TNF. Likewise, TNF exerted little effect on cell transit through S
phase, although some cell death occurred during S. Analysis of
the relative proportions of cells at various cell cycle phases
during the stathmokinetic experiment and the kinetics of pro
gression suggests that the observed deficit in M-cell population
in TNF-treated cultures is a result of both cell death and their
transient arrest in G2. Two mechanisms may account for this
observation: (a) TNF retards cell progression through G2 and
selectively kills cells that advance to, and progress through, M;
(b) TNF arrests cells in G2, and some cells die during arrest while
others progress to M. In light of the data discussed above, the
first mechanism is more likely.
The method of cell staining used in the stathmokinetic exper
iment (8,11-13) makes it possible to observe changes in chro
matin structure. Recognition of the cell cycle phases is based
both on differences in DNA content and chromatin structure; the
latter is reflected by in situ DNA sensitivity to denaturation. Dead
cells that have markedly increased DNA sensitivity to denatura
tion could be recognized and quantified as described previously
(11,13). Viable cells from TNF-treated cultures did not show any
differences in chromatin structure in comparison with control
1984
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89
Z. Darzynkiewicz et al.
cells. Since drug-induced changes in DNA or nuclear proteins
are immediately reflected in altered sensitivity of DNA to denaturation (12,13,30), this observation suggests that neither DNA
nor nuclear proteins are the primary targets of TNF.
The increased sensitivity of mitotic cells to the cytotoxic action
of TNF bears certain implications. Because the cytotoxicity of
TNF is enhanced in combination with the mitotic blockers, per
haps as a result of prolongation of the TNF-sensitive phase of
the cell cycle, further enhancement might be expected if highly
synchronized mitotic populations (e.g., obtained by sequential
synchronization by hydroxyurea or thymidine, followed by vinblastine) were used. On the other hand, noncycling cells should
be resistant to TNF and their recruitment, and progression
through the cycle would increase sensitivity. Indeed, decreased
sensitivity to TNF was observed in L-929 cultures proliferating
at lower rates as a consequence of reduced serum concentration
(28).
Similar to many antitumor agents, especially these affecting
DNA structure (e.g., intercalating drugs), TNF induces cell arrest
in G2. In contrast to these drugs, however, the arrest is of rather
short duration and is followed by cell lysis occurring at quite
specific point of the cell cycle, i.e., during or shortly after cyto
kinesis. The exact point(s) of the cell cycle at which other natural
antitumor substances produced by eukaryotic cells (lymphokines, Interferon) affect the cells are not well established. In the
case of Interferon, transition of cells from quiescence into the
cell cycle was reported to be the most sensitive phase (5). Thus,
combination of TNF and interferon with TNF affecting cells in the
cycle and interferon affecting quiescent cells which undergo
recruitment to the cycle appears to offer, at least in theory, a
very attractive approach to tumor cell killing. The presence of
quiescent cells resistant to most radio- or chemotherapeutic
regimens is believed to be responsible for common failures in
tumor therapy (14).
The mechanisms by which TNF exerts it cytostatic or cytolytic
effects are unknown. TNF causes an early increase in RNA
synthesis as measured by the rate of [3H]deoxyuridine incorpo
ration (24) and accumulation of RNA above the control levels
has been observed in the present study (Chart 2; Table 1). In
view of these observations, and the evidence that actinomycin
D or cycloheximide markedly enhances sensitivity to TNF (21,
24), the role of ongoing or even accelerated protein synthesis as
a mechanism repairing the potentially lethal effects of TNF has
been postulated (24, 27). The maximal sensitivity to TNF coin
cides in time with maximal expansion of cell surface area (cyto
kinesis), i.e., with the highest rates of production and assembly
of the cell membrane components. Thus, the possibility that
components of cell membrane may be primary targets of TNF,
as suggested by Ruff and Gifford (27, 28), should be considered.
ACKNOWLEDGMENTS
The authors wish to thank Robin Nager for her assistance in the preparation of
the manuscript.
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VOL.
44
Cell Cycle-specific Effects of Tumor Necrosis Factor
Zbigniew Darzynkiewicz, Barbara Williamson, Elizabeth A. Carswell, et al.
Cancer Res 1984;44:83-90.
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