J. Cancer (1994),
(D MacmiUan Press Ltd., 1994
Etoposide-induced cell cycle delay and arrest-dependent modulation of
DNA topoisomerase H in small-cell lung cancer cells
P.J. Smith, S. Soues, T. Gottlieb, S.J. Falk, J.V. Watson, R.J. Osborne & N.M. Bleehen
MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge CB2 2QH, UK.
DNA topoisomerase II poison etoposide (VP-16), we have studied the dynamic changes occurring in small-cell
lung cancer (SCLC) cell populations during protracted VP-16 exposure. Cytometric methods were used to
analyse changes in target enzyme availability and cell cycle progression in a SCLC cell line, mutant for the
tumour-suppressor gene p53 and defective in the ability to arrest at the GI/S phase boundary. At concentra-
tions up to 0.25gmM VP-16, cells became arrested in G2 by 24 h exposure, whereas at concentrations 0.25-2iLM
G. arrest was preceded by a dose-dependent early S-phase delay, confirmed by bromodeoxyuridine incorpora-
tion. Recovery potential was determined by stathmokinetic analysis and was studied further in aphidicolin-
synchronised cultures released from GI/S and subsequently exposed to VP-16 in early S-phase. Cells not
experiencing a VP-16-induced S-phase delay entered G, delay dependent upon the continued presence of
VP-16. These cells could progress to mitosis during a 6-24 h period after drug removal. Cells experiencing an
early S-phase delay remained in long-term G2 arrest with greatly reducing ability to enter mitosis up to 24 h
after removal of VP-16. Irreversible G2 arrest was delimited by the induction of significant levels of DNA
cleavage or fragmentation, not associated with overt apoptosis, in the majority of cells. Western blotting of
whole-cell preparations showed increases in topoisomerase II levels (up to 4-fold) attributable to cell cycle
redistribution, while nuclei from cells recovering from S-phase delay showed enhanced immunoreactivity with
an anti-topoisomerase Ila antibody. The results imply that traverse of G,/S and early S-phase in the presence
of a specific topoisomerase II poison gives rise to progressive low-level trapping of topoisomerase IIE,
availability and the subsequent irreversible arrest in G2 of cells showing limited
DNA fragmentation. We suggest that protracted, low-dose chemotherapeutic regimens incorporating VP-16
are preferentially active towards cells attempting G,/S transition and have the potential for increasing the
subsequent action of other topoisomerase II-targeted agents through target enzyme modulation. Combination
modalities which prevent such dynamic changes occurring would act to reduce the effectiveness of the VP-16
As an approach to the rational design of combination chemotherapy involving the anti-cancer
VP-16 (VP-16-213, etoposide), a semisynthetic derivative of
the naturally occumrng antimitotic agent podophyllotoxin,
has become established as one of the most active agents in
the treatment of small-cell lung cancer (SCLC). Preclinical
studies have provided
evidence of schedule dependency
(Dombernowsky & Nissen, 1973; Wolfe et al., 1987) and the
suggested that continuous low concentrations of VP-16 are
required for optimal activity of the drug when administered
as a single agent (Clark et al., 1989), and very prolonged
schedules of oral VP-16 have been evaluated and found to be
effective (Hainsworth et al., 1989; Clark et al., 1990, 1991;
1990; Johnson et al.,
induced, persistent cytostasis is an important clinical goal for
the control of rapidly proliferating tumours, we have studied
the dose dependency and kinetics of processes leading to
irreversible cell cycle arrest of SCLC cells to VP-16 following
protracted exposure in vitro.
VP-16 appears to initiate its cytotoxic action by acting as a
specific poison for the cell cycle-regulated protein DNA
topoisomerase II is a nuclear enzyme that effects unknotting,
decatenation or relaxation of supercoiled DNA molecules by
process of introducing
transient double-strand breaks
through which the strands of an intact helix can pass (Wang,
1985). Topoisomerase poisoning results in the trapping of
enzyme molecules on DNA as cleavable complexes and the
subsequent generation of potentially lethal lesions (Glisson &
Ross, 1987; Liu, 1989). The majority of laboratory studies
out with VP-16 have involved the
exposures of cultured
Although this investigational approach may aid the study of
the immediate DNA-damaging effects of the agent and its
in man by Slevin et al. (1989a,b). It has been
1990). Since drug-
use of acute
to high doses of the drug.
relationship with topoisomerase
reflect the pharmacodynamics of the clinical situation
which tumour cells typically undergo protracted exposure to
VP-16 (Miller et al., 1990). The intrinsic sensitivity of actively
appears to depend in part on the availability of the target
enzyme (Liu, 1989; Smith & Makinson, 1989). The major
type II enzyme, topoisomerase Ila, is cell cycle regulated, and
as such its availability increases as cells progress towards
mitosis (Heck et al., 1988). Thus, protracted VP-16 exposure
would be expected to modulate the availability of the target
enzyme as a result of changes in cell cycle progression. The
study is pertinent to the use of low levels of VP-16 in the
control of tumour growth since changes in the expression of
topoisomerase II may play a central role in the inhibition of
cell cycle transit (Lock & Ross, 1990), the development of
drug resistance associated with low levels of target enzyme
and in defining chemosensitivity to other agents used in
The responses of tumour cells to topoisomerase poisoning
alone. There appears to be a requirement for the p53 proto-
oncogene-encoded protein both in the efficient activation of
apoptosis and in cell cycle arrest following exposure to
DNA-damaging anti-cancer agents or acute irradiation (Liv-
ingstone et al., 1992; Clarke et al., 1993; Lowe et al., 1993).
The p53 protein appears to act as an element in the oper-
ation of a G1/S checkpoint (Kastan et al., 1992; Lane, 1992),
whereby the induction of DNA damage causes the half-life of
the protein to increase, preventing S-phase entry and block-
ing the replication of damaged DNA. However, somatic
mutation of the p53 locus is a frequent occurrence in human
tumours (HolUstein et al., 1991), with small-cell lung cancer
showing one of the highest rates of p53 mutation (Takahashi
et al., 1989, 1991; Levine et al., 1991). Cancers with p53
mutations tend to respond to chemotherapy more weakly
than those showing wild-type alleles (Callahan, 1992).
Here we have explored the cell cycle arrest responses of a
it does not
Correspondence: P.J. Smith.
Received 23 March 1994: and in revised form 4 July 1994.
Br. J. Cwtcer (1994), 70, 914-921
EFFECTS OF ETOPOSIDE IN SCLC
SCLC cell line, with a defined p53 mutation, to continuous
exposure to VP-16. The objective was to determine the effects
of drug exposure on target enzyme availability as cells evade
the GI/S checkpoint, in addition to investigating the dose
dependency and kinetics of processes leading to irreversible
Materials ad methods
Cell culture, synchronisaton and VP-16 treatments
The SCLC cell line NCI-H69/P (designated H69; originally
obtained from a patient with recurrent SCLC treated with
doxorubicin) was maintained in suspension culture in RPMI
medium supplemented with 10%
glutamine and antibiotics and incubated at 37C in an at-
mosphere of 5% carbon dioxide in air. Ancilary experiments
confirmed that the H69 cell line used in these studies carried
a mutation in exon 5 (G to T at amino acid 171,P. Rabbitts,
personal communication). VP-16 (Vepesid; Bristol Myers
Syracuse, NY, USA) was provided as
34mM stock solutions. Cells in exponential growth phase
were diluted (2 x I0 cell ml-') in fresh growth medium and
VP-16 added to cultures following a 24 h growth period.
Cultures were resuspended by aspiration using a Pasteur
pipette and cell concentrations determined using a Coulter
counter. Partial synchrony in early S-phase was achieved by
incubating cells with aphidicolhn (APC; Sigma) at I Lg ml1-
for 24 h. Cells were released from early S-phase block by
washing cultures in prewarmed fresh medium followed by
incubation in fresh medium. When releasing blocked cells
washed with medium supplemented with VP-16.
fetal calf serum,
Cells were plated in a 96-well microtitre plate (100gl per
well) in the presence of varying concentrations of VP-16 and
incubated for 1-5 days at 37C. Viable cell number was
assessed in triplicate by a non-separation, chemiluminometric
assay (Cytolite Assay; Packard Instrument Company, Meri-
den, CT, USA) based upon the ability of cells with intact
membranes to bind a probe which is activated to produce
luminescence. Probe activation occurs in response to the
intracellular generation of reactive oxygen species through
solution) was added to each well and luminescence was
generated by the addition of 25 l1 of a chemiluminogenic
probe (activator solution) and measured in a TopCount
Microplate Luminescence Counter (Packard Instruments). A
calibration was carried out using a serial dilution of cells
from a sister culture to ensure linearity between viable cell
number and luminescence measured in counts s-'.
l of reduced coenzyme plus carrier (amplifier
Cell cycle analysis and detection ofmitotic subpopulations
Cells were stained with ethidium bromide (50igml'1) plus
0.125% Triton X-100 and ribonuclease (0.5pgml-') for
10 min prior to analysis. DNA fluorescence distributions
were analysed by a computer using a cell cycle phase-fitting
program, which assumes normal distributions for GI and
G2/M phase populations (Watson et al., 1987). A probability
function was calculated for the S-phase distribution based
upon the means and standard deviations of theGIand G2/M
phases. For stathmokinetic experiments, VP-16-treated and
control cultures were exposed to colmid (60 ngml') in
order to induce mitotic arrest and low-scatter mitotic popula-
tions were analysed as described previously (Epstein et al.,
DNA strand breakage in single cells with respect to cell cycle
The technique depends upon accurate measurements of the
fluorescence intensities (corresponding to cellular DNA con-
tent) and volumes (corresponding to the extent of DNA
damage-induced unwinding of nuclear DNA) of nuclei de-
natured in agarose gels (Smith & Sykes, 1992). An MRC-600
scanning confocal microscope (BioRad, Hemel Hempstead,
UK), operating at its minimal confocal aperture, was used to
optically section the spherical nucleoid bodies. Volumes were
determined from mean diameter measurements of the digi-
tised images accumulated under Kalman filtration to reduce
the signal-to-noise ratio. This process was aided by colour-
coding pixel intensity ranges above a selected threshold for
the scanned image. DNA content was estimated by correct-
ing the integrated fluorescence intensity of the nucleoid sec-
tion showing the greatest diameter by the factor 2.22 x
radius. This procedure was carried out on randomly selected
nulceoids for each treatment condition.
DNA synthesis detected by bromodeoxyuridine (BrdUrd)
Samples of VP-I6-treated (24h exposure) cells (1-10 x
106cells) were pulsed with 20 LM BrdUrd (Sigma) for
under normal growth conditions. Cells were washed twice in
phosphate-buffered saline (PBS) before fixing in cold 70%
ethanol for 30 min on ice. Fixed cells were treated with 4 N
hydrochloric acid for 30 min at room temperature, washed in
sodium borate (0.1 M, pH 8.5), resuspended in 20#1of 0.5%
Tween-20/PBS containing anti-BrdUrd antibody (Beckton
for 30 min
Antibody-treated samples were pelleted and resuspended in
0.5% Tween-20/PBS containing FITC-conjugated goat anti-
mouse IgG (Tago, Burlingame, CA, USA; 8plH + L chains)
and held for 30 min at room temperature. Finally, cells were
pelleted and resuspended in PBS containing propidium iodide
(at a final concentration Sjgml-') to stain nuclear DNA.
Subsequent analysis and the use of fluorescence controls has
been described previously (Karn et al., 1989).
at room temperature.
SDS polyacrylamide gel electrophoresis and Western blotting
Briefly, whole-cell lysates were prepared by direct lysis of
cells in hot (65'C) sample buffer (1.0 ml of 0.5 M Tris-Cl,
pH 6.8, 0.8 ml of glycerol, 1.6 ml of 10% SDS, 0.4ml of
2-mercaptoethanol, 0.2 ml of 0.05% bromophenol blue in
8 ml final volume) at a concentration of I x I0ocells 100#1l'.
Lysates were boiled for 10 min, forced through a 21-gauge
13,000r.p.m. in an Eppendorf centrifuge. The supernatant
was retained and 10#illoaded directly onto acrylamide gels
(7.5% acrylamide with a stacking gel of 4% acrylamide).
Gels were run at a constant 200 V in electrophoresis buffer
(25 mM Tris base, 0.2M glycine, 0.1% SDS) and either
stained with Coomassie blue to check for loading or soaked
for 30 min in transfer buffer [25 mM Tris base, 192 mM
glycine, 20% (v/w) methanol
lulose membranes (Schliecher & Schuell) using a Bio-Rad
mini transblot electrophoretic cell apparatus for
constant 100 V. Nitrocellulose blots were probed for 30 min
with rabbit polyclonal antibody raised against a C-terminal
peptide of topoisomerase
chemicals; 1:100 dilution in TBS). Blots were washed for
3 x 5 min with TTBS (0.1% Tween, 100mM Tris, 0.9%
sodium chloride, pH 7.5) and incubated for 30 min with a
1:200 dilution of biotinylated mouse anti-rabbit antiserum
(Vector Laboratories). The biotinylated second antibody was
detected using a Vectastain ABC immunoperoxidase kit (Vec-
tor Laboratories), with diaminobenzidine and nickel chloride
prior to transfer to nitrocel-
1 h at a
II (Cambridge Research
Single-cell analysis ofnuclear DNA topoisomerase HI content
Samples of VP-I6-treated (24 h continuous exposure) cells
were taken (approximately
nucleus buffer and permeabilised using the technique de-
scribed previously (Minford et al., 1986). Briefly cells were
resuspended in nucleus buffer supplemented with 0.35%
1 x 10' cells) and washed with
PJ. SMITH et al.
Triton X-100 and 0.1 mm phenyl methyl sulphonyl fluoride
(PMSF), and agitated for 20 min at 4C. Permeabilised cells
were fixed in 50% methanol (v/v) and agitated for 30 min at
4C. Fixed cells were washed once in PBS and resuspended in
20 1I of anti-topoisomerase antibody (see above; 1:4 dilution)
and held for I h at room temperature. Antibody-treated sam-
ples were washed once in PBS and resuspended in 20 tl of
whole molecule) and
temperature for 30 min. Finally, samples were pelleted and
resuspended in PBS containing ribonuclease and propidium
iodide (5 g ml-') to stain nuclear DNA. Samples were
analysed by flow cytometry. Controls samples were processed
as above but without the first anti-topoisomerase H antibody
treatment. The analysis of samples by flow cytometry has
been described previously (Smith & Makinson, 1989) pro-
viding dual-fluorescence analysis of cell populations gated for
the elimination of debris and cell clumps. The right-angle
fluorescence (RF) parameters monitor DNA content (630 nm
RF) and second antibody binding (530 nm RF).
Growth inhibition and cell cycle perturbations
Figure I shows the effects of VP-16 on H69 culture growth/
viability studied over a 5 day period of continuous drug
exposure. Doses of 0.25-0.5iLM produced cessation of cul-
ture growth within the first 48 h of exposure with no evidence
of loss of metabolic function up to 8 FLM. However after 5
days' exposure there was a dline in viability at high doses
commensurate with the loss of membrane integrity as deter-
mined by vital dye staining methods (data not shown).
The results for repetitive cell cycle analyses are shown in
Figure 2a and b, in which a value of unity for relative
frequency indicates no overall change, with respect to the
control, in the percentage of cells within a given cell cycle
phase. At both time points, the proportion of cells in GI
decreased as a function of VP-16 dose up to I iLM. At 24 h,
G, emptying at low doses (0.0625-0.25 FM VP-16) was
accompanied by delay of cells in G2. At higher doses
(0.5-2 FM VP-16), there was reduced G2 accumulation owing
to the dose-dependent collection of cells in S-phase. By 48 h
the cohort of cells initially delayed in S-phase by the higher
doses of VP-16 appeared to have progressed through to G2,
with this S-phase emptying effct being most apparent for the
0.5ylM VP-16 dose. Again at 48 h there was a reduced G2
accumulation at VP-16 doses >0.5iLM owing to the dose-
dependent trapping of cells in S-phase.
In the subsequent studies the effects of low (0.25jLM)and
high (2gM)dose levels of VP-16 were compared. At the low
dose level, maximum G2 arrest is observed at 24 h without
significant S-phase delay. On the other hand, 2 1M VP-16
allows maximal G, emptying but induces significant S-phase
delay during the first 24 h exposure.
Stathmokinetic analysis ofcell cycle delay and recovery
Using the mitotic spindle inhibitor colcemid, it was possible
to investigate further the cell cycle perturbations caused by
VP-16 without the complications of cell division and resupply
of G,. Analysis of bivariate plots of right-angle scatter and
DNA content of cells permitted the identification of a low
light scatter population (LSP) in G2 representing cells enter-
ing mitosis (Epstein et al., 1988). The colemid exposure of
H69 cells was staggered to follow the number of cells at-
tempting mitosis (i.e. escaping the G2 delay induced by VP-
16) in the presence of VP-16 or after release into VP-16-free
medium (Table I). The discrimination between G2 and M
populations is not absolute (Epstein et al., 1988), and the
average rate of cell cycle traverse may vary in multicellular
aggregates during the course of a 48 h incubation experiment
involving a centrifugation and resuspension/medium change
at 24 h. Thus
is important to compare VP-16-treated
samples with the parallel control included in each treatment
group (Table I).
The data show that, although VP-16 alone results in the
accumulation of cells in G2 as a function of dose, there is no
evidence of trapping of cells in mitosis. The percentage of
cells that have attempted mitosis, in the presence of colcemid,
decreases as a function of dose and is approximately 1% at
2 >IM VP-16. There is no evidence of significant trapping of
percentage of cells in the combined GI and S-phase compart-
ment r[.e. 100-(G2 + M)] for the low dose range (0.0625-
0.25gMVP-16), whereas a high dose (21.M)of VP-16 induces
a significnt delay in the delivery of cells to G2 and a
complete block to G2 exit (condition a in Table I). Parallel
VP-16 concentration (pM)
number, relative to untreated controls, for different exposure
periods. Symbols: 0, 24 h; A, 48 h; 0, 5 days. Data are mean
values (± s.d.) for three experiments.
VP-16 dose-dependent changes in H69 viable cell
VP-16 dose (pm x 24 h)
VP-16 dose (pM x 48 h)
induced by continuous VP-16 treatment for 24 h (a) or 48 h (b).
Control samples gave mean values (± s.e. for 13 determinations)
of 40.5 ± 1.9, 38.4 ± 1.7 and 21.1 ± 0.8% cells in G,, S phase and
experiments. Symbols: 0. G,; A. S-phase; 0. G,.
Dose dependency of the H69 cell cycle perturbations
af .. ..
EFFECTS OF ETOPOSIDE IN SCLC 917
Total cells in cell cycle compartment for different
treatment and recovery periods (%)
Data derived from a single representative experiment. 124 h continuous exposure
to VP-16 (colcemid added at
resuspended in their own media at 24 h (cobemid added at
continuous VP-16 exposure; cells resuspended in fresh media at 24 h (colcemid
added at t=24h).
t =Oh). b48 h continuous VP-16 exposure; cells
t = 24 h). C48 h
Flow cytometric analysis of proportions of cells actively engaged in DNA synthesis
following 24 h exposure to VP-16
Mean data (± range) derived from two determinations.
Cells in region (per cent of total) at end of 24 h VP-16 treatment
37.3 ± 4.7
32.9 ± 0.9
10.2 ± 2.0
19.2 ± 3.6
studies (data not shown), which included a wild-type p53-
expressing human cell line (Smith et al., 1994), confirm that
the H69 cell line also shows a defect in arrest at GI/S
following acute X-irradiation (S. Soues & PJ. Smith manu-
script in preparation). Collectively, the results are consistent
with the evasion of the G1/S checkpoint by VP-16-treated
During the incubation period of 24-48 h for a continuous
(48 h; condition b in Table I) exposure to doses of <0.25gAM
VP-16 there is evidence of cells attempting mitosis, whereas
at the 2 FLM concentration the majority of cells become trap-
ped in G2. Thus, it appears that at low doses, over the first
48 h, cells are progressing through the cell cycle relatively
normally except that there is an increasing probability, with
time, of a given cell being trapped in G2. In contrast, at the
higher concentration, over the first 24 h period, a large pro-
portion of cells are slowed down or blocked in S-phase with
a very high probability that any cell delivered to G2 will
remain trapped in that cell cycle compartment.
Cells released from a 24 h exposure to low doses of VP-16
(condition c in Table I) re-enter cycle during the subsequent
24 h incubation in fresh medium. This is in contrast to
high-dose VP-16-treated cells, which when released at 24h
continue to accumulate in G2 over the following 24 h period
and remain blocked in G2 with very few cells attempting
S-phase delay analysed by BrdUrd incorporation
Flow cytometric measurements of BrdUrd incorporation
were used to analyse S-phase delay in order to determine the
proportion of cells synthesising DNA in treated and un-
treated cultures. Gates were set around two regions on con-
tour plots of DNA content and BrdUrd incorporation.
and this region was analysed by the standard cell cycle
phase-fitting programme and the calculated GI, 'inactive' S
1 contained cells that did not incorporate BrdUrd,
and G2 percentages converted into percentages of total cells
within regions. Region 2 was designated 'active S-phase', as
this contained cells that incorporated BrdUrd above back-
ground levels. Typical results, shown in Table II, indicate
that with increasing VP-16 dose after 24 h there is an increase
in the percentage of cells engaged in DNA synthesis. The
reduced G2 arrest observed upon VP-16 treatment may arise
from a direct effect of the thymidine analogue on cell cycle
traverse. The contour plots show that the extent of BrdUrd
incorporation relative to position in S-phase is similar for
control and VP-16-treated cells (data not shown). Parallel
studies using thymidine incorporation also show that such
DNA synthesis detected in VP-16-treated cells is resistant to
the inhibitory effects of acute
VP-16 despite continued sensitivity to the DNA-protein
cross-link-ing action of such high doses of drug (data not
shown). The results consolidate the stathmokinetic results in
that at the 2gM VP-16 dose level initially delayed cells
continue to traverse S-phase after a 24 h drug exposure in an
apparently normal manner. Importantly, the percentage of
cells in 'inactive' S-phase did not increase above the numbers
gated in control cultures in response to VP-16 treatment,
suggesting that no cells are actively blocked at 24 h in S-
phase for any of the doses studied.
1 h high-dose exposures to
Cell cycle delay and recovery in synchronised cultures
Figure 3a and b shows the effects of VP-16 on the traverse of
S-phase as monitored by G2 accumulation. Asynchronous
cultures were compared with those released from partial
synchrony in early S-phase achieved by aphidicolin treat-
ment. Control cells, released from G,/S synchrony, traverse
S-phase during the first 6 h of release, pass as a cohort
through G2 and re-enter GI by 24 h. Following release into
0.25 or 2pAMVP-16 (Figure 3b), cells are delayed in S-phase
and accumulate in G2. After a 24 or 30 h VP-16 exposure
only cells exposed to the low dose (0.25gM)can exit G2 and
P.J. SMITH et al.
accumulation, for asynchronous H69 cells (a) and cells released
from synchrony G, S (b). Mean data derived from three indepen-
dent experiments (s.e. <10%). Cultures were treated for 6, 24 or
30h with VP-16 at OsM (=E), 0.25Mm (
Following 24 or 30 h treatments cells were released for the
indicated recovery (R) period.
Effects of VP-16 on S-phase traverse, monitored by G,
) or 2 .tM (_).
(total fluorescence intensity, arbitrary units)
re-enter GI. Synchronisation increases the number of cells
S-phase during VP-16
reduces the number of cells capable of recovery from G2
arrest compared with asynchronous cultures. Asynchronous
cultures contain fewer cells in early S-phase and show a
greater recovery from 0.25glM VP-16 compared with syn-
chronised cultures. Cells exposed to 2AM VP-16, under either
culture condition remain trapped in G2, indicating the essen-
tially irreversible nature of the arrest for the lowest concen-
tration capable of inducing an overall S-phase delay.
DNA damage as afunction ofcell cycle position
We have used an adaptation of the 'comet' assay (Singh et
al., 1988; Smith & Sykes, 1992) to detect DNA breakage
resulting from cleavage of trapped topoisomerase complexes
or secondary fragmentation events
nucleoid volume measurements. Figure 4 a-c shows the
essentially linear relationship between nucleoid volume and
DNA content for control cultures and the increase in the
proportion of cells with high DNA contents in VP-16-treated
cultures. The results reveal heterogeneity in the responses of
cells to VP-16, with only a few cells (<15%) showing low
levels of damage at 0.25
cells (>80%) show substantial damage at2glM VP-16. The
GI fraction in cultures exposed to 2gM VP-16 was too
infrequent for detailed analysis. However, some cells with
significnt levels of DNA damage and may represent a sub-
population not undergoing either S-phase delay or elevation
of damage levels.
as monitored by the
iM VP-16, whereas the majority of
24 h exposure to VP-16 as a function of total cellular DNA
content determined by nucleoid volume (halo assay). a, Control
R2=0.718; reproduced in b and c). b and c, 0.25gM or 29LM
VP-16 x 24 h respectively.
Induction of DNA damage in individual H69 cells by
(y = 0.934 + 0.388x,
VP-16-induced changes in topoisomerase II availability
Whole population studies
parations of H69 cultures exposed to VP-16 for 24 h showed
a major immunoreactive band with a mean molecular weight
(± s.d. for ten determinations) of 171.3 ± 2.9 kDa correspon-
ding to that expected for the p170 form of human DNA
topoisomerase IIa. An increase in the intensity of the p170
band above control was observed for all VP-16-treated sam-
ples, and densitometry (Figure 5) was used to quantify the
changes with respect to control samples. The p170 band
intensity increased with drug dose and exposure period,
reaching a maximum of 3.7-fold at doses of l-2 M VP-16
with a reduction in band intensity evident at 8 LM VP-16.
Immunoblots of whole-cell pre-
Single-cell analysis and cell cycle distribution
cell cycle perturbations to the changes in topoisomerase lIIK
we have utilised a flow cytometric technique (Smith &
Makinson, 1989), which provides a simultaneous analysis of
DNA content and nuclear DNA topoisomerase II. Initial
studies established the cell cycle distribution of topoisomerase
II. Bivariate plots of DNA versus topoisomerase II content
revealed the expected distribution of the p170 form of DNA
topoisomerase II throughout the cell cycle: low levels inGI,
To relate the
EFFECTS OF ETOPOSIDE IN SCLC
VP-16 dose (pM)
band for Western blots for H69 whole-cell preparations. Cultures
were exposed to VP-16 for 24 h (0) or 48 h (0). Data are mean
values (± s.d.) derived for four independent blots.
Densitometric analysis of the p170 topoisomerase
levels increasing through S-phase, high levels in G2 with a
subset in G2 showing the highest levels before recycling into
G,. Setting the anti-topoisomerase II antibody staining ofGI
3.47 ± 0.61-fold
for S-phase and GJM
(± s.d.; three experiments). Extensive redistribution of cells
in the cell cycle by exposure to colcemid (see above) for 24 h
gave corresponding values of 1.85 ± 0.28 and 3.47 ± 0.19 for
S-phase and GJM
analysis for immunoreactivity is independent of the propor-
tions of cells within the gated regions. The control data (not
enzyme content through the cell cycle and support the interp-
retation that the changes seen above by Western blotting
represent, at least in part, cell cycle redistribution. DNA
content analysis for drug-treated cells (2gMVP-16 for 24 h)
clearly showed the expected VP-16-induced accumulation of
cells in G, and S phase. DNA distributions were divided into
six compartments of increasing DNA content representing:
GI, early S, early to mid S, mid to late S, late S and G2/M
fractions respectively (Figure 6). Median 530 nm fluorescence
intensities were calculated for each gated compartment, cor-
rected for background fluorescence and expressed as a value
relative to control samples (Figure 6). No increase above
control values was observed for cells exposed to 0.255kM
VP-16 (data not shown). At 2gM VP-16 there is an uns-
S-phase and in GJ/M.
1.7 ± 0.18-fold and
in nuclear topoisomerase
II content in
This study has demonstrated the strict dose dependency of
the cell cycle perturbations underlying the cytostatic action of
VP-16 on a human SCLC cell line. The disruption of the
traverse of S-phase, in particular a delay in early S-phase
progression appears to be a key component in the irreversible
cytostatic action of VP-16. Continuous exposure to low doses
of VP-16 ( .0.25luM) results in significant enrichment of cells
within cell cycle compartments which normally express high
levels of topoisomerase II. VP-16 induced a cell cycle block
in G2 with a significant delay of cells in S-phase being
apparent at high VP-16 concentrations. Similar observations
have been reported for other human cells including trans-
formed fibroblasts (Smith et al., 1986), lymphoblasts (Kal-
winsky et al., 1983) and breast tumour cells (Epstein et al.,
Dysfunction of the p53 proto-oncogene would be expected
to contribute to a loss of the ability of cells to arrest atGI/S
to DNA damage. X-irradiation of the p53
mutant cell line H69 resulted in no detectable arrest of cells
cell cycle compartments measured 24 h after continuous exposure
to 2 kM VP-16 for 24h. Data are means (± s.e.) of the corrected
Median 530 nm RF values were background corrected and ex-
pressed as a ratio of the value obtained from unperturbed control
cells such that a value of zero indicates no change
isomerase Il-associated immunofluorescence compared with the
control. S phase has been divided into four equal compartments
of increasing DNA content.
II. relative to control levels, in cells within gated
VP- I 6-induced
atGI/Sbut progressive accumulation at the GJM boundary
(S. Soues &
P.J. Smith, unpublished data). The results
obtained with protracted VP-16 exposure also reveal
inability to arrest at GI/S. In viewing the relevance of the
concentration dependency of VP-16 effects, it is important to
relate the drug doses used in this model study with those
found in clinical practice. In a study by Slevin et al. (1989ab),
patients with SCLC demonstrated that a 5 day oral regimen
could maintain plasma VP-16 levels above 1.75kM. Thus the
present study describes cellular effects at clinically relevant
doses of VP-16. The relationship between S-phase delay and
subsequent cellular recovery from G2 arrest was studied in
synchronised cells exposed to VP-16 upon release from the
GI/S transition point. Cells not experiencing a VP-16-induced
S-phase delay entered a long-term G2 arrest dependent upon
the continued presence of VP-16. Removal of VP-16 resulted
in the progression of the majority of these cells through
mitosis during the 6-24h period after drug removal. Cells
remained in long-term G, arrest with no evidence of progres-
sion from G2.
Continuous exposure to an irreversibly cytostatic concen-
tration of VP-16 also results in an increased immunoreac-
tivity of nuclei to an anti-topoisomerase
suggest that two effects can account for this observation.
First, the principal effect
topoisomerase II levels. This increase arises from the delay of
cells in S-phase during a period in which there is a scheduled
increase in topoisomerase IIa. Accordingly, there may be no
cellular feedback to
merase levels once a cell is committed to active DNA syn-
topoisomerase poisons and for the modulation of other cell
cycle-regulated proteins. Second, a minor component of the
increase may represent enhanced stabilisation of cleavable
complexes. In support of this latter possibility is the observa-
tion that the sensitive nucleoid expansion method, performed
under conditions which cleave DNA at trapped complexes,
reveals significant levels of DNA fragmentation in delayed
cells. However, the levels of cleavage detected are commen-
II antibody. We
link cell cycle progression to topoiso-
_Z E *4D
P.J. SMITH et al.
surate with only low levels of complex trapping detectable by
the conventional K+SDS precipitation method (P.J. Smith &
S.J. Falk. unpublished data).
It does not appear that the fragmentation revealed by
nucleoid scanning represents typical apoptosis given the
results of the viability measurements and our observation
that acridine orange-stained preparations showed that <1%
of cells displayed typical apoptotic nuclei. Furthermore, ex-
tractions of low molecular weight DNA fractions from VP-
16-treated cell populations showed no detectable levels of
nucleosome laddering as assessed by conventional agarose gel
electrophoresis (P.J. Smith, unpublished data). The lack of
early induction of apoptosis is not surprising since it has
been reported that SCLC cell lines may differ in their ability
to express VP-16-induced apoptosis (Okamoto-Kubo et al.,
1994) and the potential requirement of functional p53 for
efficient induction (Clarke et al., 1993; Lowe et al., 1993).
Additional studies are required to determine whether the
damage visualised by nucleoid scanning represents target
enzyme trapping or a secondary process of preapoptotic,
perhaps DNA domain-limited, fragmentation.
We suggest that the minimal cytotoxic dose threshold for
low-dose, protracted VP-16 exposures is defined by the dose
intensity required to impose an early S-phase delay rather
than effect G2 arrest per se. In the in vivo situation, the
maintenance of a low but bioactive drug concentration would
allow the continued recruitment of tumour cells into S-phase
delay as they opt to enter the cell cycle since it is unlikely
that the G,,S checkpoint
evident that SCLC cells can overcome the initial restriction
to early S-phase traverse even in the continued presence of
VP-16 and cells eventually enter a G, delayed state with
enhanced availability of topoisomerase II.
Clinical studies have supported the concept that prolonged
low-dose VP-16 treatment offers the combined benefits of
efficacy and low toxicity. On the other hand, it is clear that
such treatment will not break new ground in terms of in-
creasing response duration or survival either when used alone
is functional in SCLC and GI
is not affected even by high drug doses.
designed regimen (Murphy et al., 1992). However, prolonged
schedules of VP-16 treatment may be a foundation for novel
combination regimens which capitalise on the consequences
of continuous topoisomerase
synchronisation or modulation of topoisomerase II levels.
This hypothesis was evaluated by exposing H69 cells to
VP-16 for 24 h prior to treating washed cultures with selected
agents and assaying growth potential using the conventional
MTT assay (data not shown). The results indicated that
VP-16 pretreatment results in a greater than 2-fold enhance-
ment of growth inhibition potential for cisplatin, Amsacrine
(mAMSA) and VP-16, while the other agents (camptothecin,
mitoxantrone and doxorubicin) gave values of less than 1.3-
The absence of an effect on sensitivity to the topoiso-
merase I poison camptothecin is consistent with the non-cell
cycle-regulated nature of the target enzyme (Heck et al.,
1988). VP-16 pretreatment did not interfere with the cyto-
toxic potential of the topoisomerase poisons doxorubicin and
mitoxantrone, suggesting that the initial capacity to induce
topoisomerase II cross-linking not being an important factor
in the cytotoxic action of anthracyclines and related drugs
(Fox & Smith,
1990; Smith et
enhancement of cytotoxicity observed for the topoisomerase
II poisons mAMSA and VP-16 is itself consistent with the
effects of topoisomerase II modulation. The observations of
enhanced cisplatin sensitivity is interesting given the capacity
of this agent to induce DNA-DNA and DNA-protein
cross-linking in what may be topologically compromised
DNA molecules in VP-16-pretreated cells.
dynamic changes in cell cycle distribution and target enzyme
presentation in tumour cells exposed for protracted periods
to low doses of VP-16 may offer novel opportunities for the
introduction of other agents in combined regimens. The
single-cell analytical approach described here offers a method
of monitoring the effects of chronic VP-16 exposure in vivo
within defined tumour target populations.
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