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Experimental Therapeutics |
Correlate with Etoposide Resistance in Multicell Spheroids and Xenograft Tumors1
British Columbia Cancer Research Centre, Vancouver, British Columbia, V5Z 1L3 Canada
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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are identical for monolayers or the
outer cells of spheroids, and the cell proliferation rate is the same,
our previous results indicated that phosphorylation of topo II is at
least 10 times higher in V79 monolayers than in spheroids. Because
phosphorylation of topo II has been associated with nuclear
translocation, we examined subcellular distribution of Topo II in
monolayers, spheroids, and xenograft tumors using immunohistochemistry.
Topo II was located predominantly in the nucleus of V79, human SiHa,
and rat C6 monolayers but was found mainly in the cytoplasm of the
proliferating outer cells of spheroids formed from these cell lines.
Conversely, the outer cells of WiDr human colon carcinoma spheroids
showed predominantly nuclear localization of topo II, and only WiDr
cells showed no increase in resistance to etoposide when grown as
spheroids. Cells sorted from xenografts resembled the spheroids in
terms of sensitivity to etoposide and location of topo II. When the
outer cells of V79 spheroids were returned to monolayer growth, the
rate of redistribution of topo II to the nucleus occurred with
similar kinetics as the increase in sensitivity to killing by
etoposide. Removal and return of individual outer V79 spheroid cells to
suspension culture resulted in the translocation of topo II to the
nucleus for the first 24 h, accompanied by an increase in
sensitivity to DNA damage by etoposide. Therefore, the cytoplasmic topo
II distribution in outer spheroid cells and tumors appears to
correlate not with morphological changes associated with growth in
suspension but rather with the presence of neighboring, noncycling
cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
or rate of uptake or efflux of [3
H]etoposide.
Moreover, the outer cells require 3 or more days of growth as spheroids
to develop maximum resistance, and, once returned to monolayer growth
conditions, cells require four or more cell divisions (about 48 h)
before they become as sensitive to etoposide as monolayers
(1)
.
Our previous results also showed that phosphorylation of topo II
was reduced at least 10-fold in the outer cells of V79 spheroids
relative to monolayers (2)
, and this observation could
explain the resistance of these spheroid cells to killing and DNA
damage by etoposide. In fact, several studies have implicated topo II
hypophosphorylation in resistance to topo II poisons (3)
.
However, the relationship of topo II phosphorylation to drug
sensitivity can be variable, and both stimulation and inhibition of
topo II activity have been reported (4)
. Phosphorylation
of the COOH terminus of topo II may play a role in nuclear
translocation of topo II (5
, 6)
. A significant decrease in
the amount of nuclear topo II would be expected to reduce the amount
of DNA damage by etoposide and could explain the 10-fold resistance of
outer spheroid cells to killing by etoposide (2)
.
Therefore, experiments were designed to compare the subcellular
distribution of topo II in monolayers with the outer cells of
spheroids. Results confirm that topo II was localized primarily
in the nucleus of the V79 monolayer cells and in the cytoplasm of the
external cells of spheroids. Three additional cell lines grown as
monolayers or spheroids were compared to determine the generality of
the observation. Because these cell lines could also be grown as
xenograft tumors, we were able to compare the spheroid results with the
response of the more relevant solid tumor models.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sequential Trypsinization.
Sequential trypsinization has been used for over 20 years as a method
to provide relatively pure populations of cells from various depths
within multicell spheroids (7
, 8)
. The outer cell layer of
spheroids approximately 0.6 mm in diameter was removed by agitating
spheroids for 5 min in cold 0.1% trypsin in PBS. Outer spheroid
cells were released by this process when spheroids were gently
transferred to cold medium and allowed to settle to the bottom of a
tube. The remaining cores were trypsinized to determine cell recovery
and to calculate the percentage of cells in the outer layer (generally
7–10% of the spheroid). The outer cells of spheroids were placed in
monolayer culture for exposure to etoposide and subsequently examined
for DNA damage and cell viability. Cell survival after etoposide
treatment was measured using a standard clonogenicity assay.
Cell Survival Measurement and Relative Resistance.
Single cells from monolayers, outer spheroid cells, or xenograft tumors
were placed in 100-mm tissue culture dishes containing 10 ml of
MEM + 10% FBS. After 10 days to 2 weeks, colonies (>50
cells) were stained with malachite green and counted. Experiments were
repeated at least three times to obtain the mean and SE. For V79 and
WiDr cells, survival as a function of etoposide concentration followed
a simple exponential curve, so relative resistance was constant at all
survival levels. Therefore, etoposide resistance for these cell lines
could be expressed as the ratio of the slopes obtained for the
monolayer and spheroid cell survival curves. For the SiHa and C6 cell
lines, survival was defined by a linear quadratic relationship, and the
lethal dose to 90% of the cells (LD90) was
calculated from these curves.
Xenograft Cell Sorting.
To obtain tumor cells closest to the blood vessels, mice bearing
approximately 0.5-gram tumors were injected i.v. with 4 mg/kg Hoechst
33342. Ten min later, tumors were excised, and a single cell suspension
was prepared by chopping the tumor finely and incubating the suspension
for 30 min with trypsin, collagenase, and DNase as described previously
(9)
. Filtered cells were centrifuged and resuspended in
complete medium for incubation with 0–15 µg/ml etoposide. After a
30-min incubation with etoposide, tumor cells were sorted on the basis
of the Hoechst 33342 diffusion gradient using a Becton Dickinson
FACS440 cell sorter with UV excitation (10)
. The 10% of
cells that contained the highest concentration of Hoechst 33342
(i.e., those closest to the functional tumor blood vessels)
were sorted and analyzed for survival using a colony formation assay.
DNA Damage Measured Using the Alkaline Comet Assay.
Cells from monolayers or the outer cells of spheroids were exposed for
30 min to etoposide (Bristol Meyers clinical formulation; 20 mg/ml).
After trypsin treatment, single cells in 0.5 ml of ice-cold PBS were
mixed with 1.5 ml of 1% low gelling temperature agarose (Sigma type
VII low gelling temperature agarose prepared in distilled water and
maintained at 40°C). After mixing, 1.5 ml of this
mixture was pipetted onto an agarose-precoated half-frosted microscope
slide and allowed to gel for about 1 min. Slides were then submersed
carefully in an alkaline lysis solution at room temperature containing
1.2 M NaCl, 0.03 M NaOH, and 0.1% sarkosyl for
1 h, followed by a 1-h wash in two rinses of 0.03 M
NaOH plus 2 mM EDTA to remove NaCl. Electrophoresis was
conducted at 0.6 V/cm for 25 min in a fresh solution of 0.03
M NaOH plus 2 mM EDTA. Slides were rinsed and
stained for 15–20 min in 2.5 µg/ml propidium iodide, and care was
taken in handling this mutagenic DNA stain. Individual cells or
"comets" were viewed using a Zeiss epifluorescence microscope with
an attached intensified solid state charged-coupled device camera as
described previously (11)
. Images were characterized using
"tail moment," which was defined as the product of the percentage
of DNA in the comet tail and the distance between the means of the head
and tail DNA distributions, and "DNA content," which was defined as
the total fluorescence associated with an image. Approximately 200
comets were analyzed from each slide, and experiments were repeated
three times.
Subcellular Fractionation and Immunoblotting.
Before preparation of nuclei, cells were incubated for 30 min with 10
µg/ml etoposide to stabilize DNA/topo II complexes. To prepare
nuclei, approximately 1.5 x 107
outer cells of spheroids or monolayers were rinsed in PBS and
resuspended in 100 µl of cold buffer A [10 mM HEPES (pH
7.9), 1.5 mM MgCl2, 10 mM
KCl, 300 mM sucrose, 1 mM EDTA (pH 8.0), 1
mM DTT, 0.1% NP40, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml protease inhibitor mixture (Sigma), and 0.1
mM Na3VO4].
After 10 min of incubation on ice, cells were sheared by passing a few
times through a Gilson microtip and centrifuged for 10 min at 3000 rpm
at 4°C. The cytoplasmic supernatant was reserved for analysis by
Western blot, and the nuclear pellet was washed two times in buffer A
and examined by microscopy for the presence of cytoplasmic tags by
staining with nuclear and cytoplasmic fluorescent probes. After the
final wash, the nuclear pellet was resuspended in 75 µl of buffer B
[250 mM Tris-HCl (pH 7.9), 5 mM
MgSO4, 250 mM sucrose, 2
mM NaTT, 1% thiodiglycol, 1% NP40, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml protease inhibitor mixture
(Sigma), and 0.1 mM
Na3VO2]. Whole cell
lysates were also prepared by lysing 5.0 x 106 cells in buffer B. Equal amounts of protein
(Sigma Protein Assay) were denatured in sample loading buffer and
loaded into the wells of an 8% SDS-polyacrylamide gel. After
electrophoresis, gels were blotted onto polyvinylidene fluoride
membrane and analyzed for topo II using a monoclonal antibody
(Sigma-Genosys) diluted 1:250 in PBS containing 0.2% Tween 20.
Secondary goat antimouse antibody conjugated to horseradish peroxidase
(1:10,000 dilution) was incubated with blots for 1 h, and bands
were detected using the enhanced chemiluminescence detection system
(Amersham). Blots were also stained for lamin A (Santa Cruz
Biotechnology, Inc.), a nuclear protein, to confirm subcellular
fractionation.
Immunohistochemistry.
Cells suspended in ice-cold PBS were deposited by cytospinning
(CYTOSPIN II; Shandon, Pittsburgh, PA) at 800 rpm for 8 min onto
cleaned glass slides at a concentration of 2 x 104 cells in 100 µl of MEM plus 10%
FBS. Slides were air-dried for 10 min and then immersed in cold
1% paraformaldehyde (Sigma) in PBS for 30 min at 4°C. After rinsing
in PBS, slides were immersed in acetone for 30 s at room
temperature, and then they were rinsed two times in PBS and immersed in
PTN for 20 min at room temperature. Anti-topo II antibody
(Sigma-Genosys or TopoGEN, Columbus, OH) was diluted 1:100 in PTN, and
25 µl of diluted antibody was deposited on 1
cm2 parafilm strips. The strips were inverted
over slides that had been drained and placed in humidified chambers.
Chambers were placed at 4°C overnight. Slides were then removed from
the chambers and rinsed two times for 5 min in PBS and one time for 5
min in PTN. The secondary antibody, Alexa488 goat antimouse IgG (H + L) F(ab')2 fragment conjugate (Molecular
Probes, Eugene, OR), was diluted 1:150 in PTN, and 25 µl of
diluted secondary antibody were deposited on new parafilm strips
for incubation at room temperature for 2 h. Rinsed slides were
immersed in 0.05 µg/ml 4',6-diamidino-2-phenylindole dihydrochloride
hydrate (Sigma) in PBS for 5 min and then rinsed for 5 min in PBS,
drained, mounted in Fluorogard mounting medium (Bio-Rad, Mississauga,
Ontario, Canada), sealed, and viewed using a Zeiss Fluorescent
Microscope with an attached Sensicam Camera. The images were digitized
using Northern Eclipse 5.0 software (Empix, Toronto, Ontario, Canada).
For some images, NIH/Scion image software was used to analyze average
nuclear, cytoplasmic, and background fluorescence intensities from
several cells.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Etoposide resistance was also observed in cells recovered from the
outer layer of C6 rat glioma spheroids and SiHa human cervical cancer
spheroids. However, for WiDr human colon carcinoma cells, there was no
evidence of an increase in resistance to etoposide in the outer cells
of spheroids compared with monolayers. Relative resistance to
etoposide, based on comparison of the LD90, is given in
Table 1
for these four cell lines.
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shows the surviving fraction of cells obtained from xenograft
tumors (
). To minimize the presence of nonproliferating tumor cells,
a cell-sorting method was used to select tumor cells that were close to
the functional vasculature. Exposure to etoposide, like exposure for
monolayers and spheroids, was performed in vitro for 30 min.
Results from the xenograft tumors were not significantly different from
those obtained using the outer cells of spheroids.
The intracellular distribution of topo II was examined in
monolayers and cells from the outer layer of spheroids. Representative
results in Fig. 2a
indicate the predominantly nuclear location of topo II
antibody in V79 monolayers but show cytoplasmic localization in the
outer cells from V79 spheroids (Fig. 2b)
, as observed in
more than 10 separate experiments. Secondary antibody alone, under the
same conditions of illumination, showed no significant fluorescence.
Similarly, C6 monolayers and SiHa monolayers (Fig. 2, c and g)
showed more nuclear staining than outer spheroid cells
(Fig. 2, d and h)
. However, WiDr spheroids and
monolayers showed a similar distribution, with topo II localized
primarily in the nucleus (Fig. 2, e and f)
. Some
intercellular variability in pattern of fluorescence was also observed,
especially within the human tumor cell populations, presumably as a
result of differences in topo II levels through the cell cycle or
the occasional noncycling cell (12)
.
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(Fig. 3)
. Cells closest to tumor blood vessels were again sorted for this
analysis. Results are in agreement with the spheroid
immunohistochemistry results and show a predominantly cytoplasmic
localization of topo II in C6 and SiHa tumor cells but a
predominantly nuclear localization in cells from WiDr xenografts.
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in monolayer but much
less nuclear topo II in nuclei from outer spheroid cells was
confirmed by Western blotting (Fig. 4)
. Conversely, cytoplasmic topo II was predominant in spheroid cells
but not in monolayer cells. Lamin A, a nuclear protein, was absent from
cytoplasmic samples.
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antibody staining (Fig. 5, b and c)
. The pattern of antibody binding 24–48
h after return to monolayer growth was indistinguishable from the
monolayer control (Fig. 5, f–h)
.
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) was used as a measure of the degree of etoposide
resistance. With time after removal of outer cells and their return to
monolayer growth, there was a loss of resistance of these cells to
killing by etoposide (Fig. 6b)
. The rate of recovery of nuclear fluorescence shown in
Fig. 6a
appeared to correlate with the rate of recovery of
sensitivity to killing by etoposide.
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. After 12 h, outer spheroid cells cultured as
monolayers or in suspension showed predominantly cytoplasmic topo II,
but by 24 h, much of the topo II was localized to the nucleus
(Fig. 7)
. Therefore, despite the fact that the outer spheroid cells were
returned to suspension culture, they failed to maintain cytoplasmic
fluorescence, indicating that cell-cell associations that occur in
larger spheroids must be necessary to maintain cytoplasmic topo II.
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show a gradual increase in DNA damage by etoposide for both
monolayer and suspension culture cells over the first 24 h after
outer cells were removed from spheroids and grown either on plastic
dishes or in suspension culture. These results are consistent with
images shown in Fig. 7
. However, as suspension-cultured cells began to
reform and grow as spheroids, there was a return to the expected
resistant phenotype (Fig. 8e)
.
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, demonstrating the
decrease in growth fraction that begins to occur in suspension cultured
cells after 24 h. This change corresponds with the time when
spheroids begin to develop greater resistance to DNA damage by
etoposide. However, only a part of this change can be attributed to the
presence of a population of noncycling cells. This is indicated by the
distribution of DNA damage in individual cells (Fig. 8, a–d)
. The distribution is similar whether cells are grown
in suspension or as monolayers when examined 24 h after
trypsinization (Fig. 8, a and b)
. However, by
48 h, there is a substantial proportion of noncycling cells in the
population grown in suspension (tail moment 
3), but
not in those grown as monolayers (Fig. 7, c and d)
. More importantly, for those suspension culture cells
that did respond to etoposide, the average amount of DNA damage was
lower for cells grown in suspension than for those grown as monolayers.
Therefore, for suspension cultured cells after 48 h, not only is
the growth fraction lower, but the sensitivity of the growing cells to
etoposide is also decreased. Fig. 7f
indicates that the
cells grown as monolayers continue to proliferate for 96 h, with a
doubling time of 11.1 h. However, despite the fact that the
majority of these cells are still in cycle at 80 h, they are
crowded on dishes and show a dramatic decrease in sensitivity to
etoposide. When examined for topo II, these confluent monolayer
cells also exhibited a significantly reduced intensity of fluorescence,
making it difficult to observe any cytoplasmic topo II (data not
shown). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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has been recognized as a useful cell proliferation
marker for solid tumors (14
, 15)
. This enzyme is generally
found in the interphase nucleus of proliferating cells; however,
cytoplasmic topo II has also been observed in some cell types
(16)
, and it may be more prevalent in late log phase and
early plateau phase cultures (17)
. Induction of cell
differentiation has been shown to be associated with a decrease in topo
II phosphorylation that precedes reduction in catalytic activity
(18)
. Whereas the outer cells of spheroids divide at the
same rate as monolayers and are distributed through the cell cycle in
the same proportions (2)
, many of these cells will be
exiting the cycle within a day or two as they pass into the interior of
the spheroid. Noncycling cells in the center of spheroids lack topo
II and show relatively little or no DNA damage when treated with
etoposide (1)
. A possible explanation for the cytoplasmic
localization of topo II in the external cells of spheroids is that
these cells are preparing for the approaching exit from the cell cycle,
although they continue to progress through the cycle at a normal rate
at that point in time. In 1981, Wilson et al.
(19)
examined the response of the outer cells of V79
spheroids to another topo II inhibitor, amsacrine. These authors also
concluded that resistance to this drug occurred "well before
departure from exponential growth can be detected, and may thus be a
consequence of metabolic changes more subtle than the transition from a
cycling to a non-cycling state." It should be noted that even a
drastic reduction in total cellular topo II in V79 mutant cells
caused only minor perturbations of cell growth (20)
,
indicating that only a small portion of the nuclear topo II is
involved in functions essential for replication and chromosome
segregation.
The dramatic decrease in topo II phosphorylation that we reported
previously is most likely responsible for the change in intracellular
distribution of topo II in outer spheroid cells. Phosphorylation of
the noncatalytic domain apparently does not affect catalytic activity
(21)
but can influence intracellular distribution of topo
II because it results in nuclear translocation (22)
.
The fact that topo II phosphorylation has little effect on the
catalytic activity is consistent with our previous results that showed
that topo II decatenation activity was similar for extracts from
monolayers and outer cells of spheroids (1)
. The long
half-life of phosphorylated topo II (12–27 h; Ref. 4
) may
explain in part the requirement for at least 3 days in suspension to
achieve maximum etoposide resistance when V79 cells are grown as
spheroids. When spheroids were returned to monolayer growth conditions,
topo II increased in the nucleus at the same time as cells became
more sensitive to etoposide. By 24 h after return to monolayer
growth, nuclear localization is apparent, and cells are only 1.6 times
less sensitive to etoposide than monolayers (Fig. 6)
. Therefore
phosphorylation of topo II may also occur relatively slowly, which
would be consistent with the possibility that loss of a topo II
phosphatase activity in cells grown as spheroids occurs mainly by
dilution during cell division.
Is it growth of cells in suspension or cell-cell contact that is
responsible for the change in topo II phosphorylation in spheroids?
Results shown in Fig. 8
indicate that removing the outer cells of
spheroids and returning them to spinner culture produces an initial
increase in etoposide sensitivity that is identical to that
observed when cells are returned to monolayer growth conditions. We
therefore conclude that growth in suspension, with attendant
morphological changes, is not responsible for the change in topo II
distribution. However, cell-cell contact or sequelae arising from
contact appear to be a requirement. Because C6 glioma cells lack
significant gap junctional communication (23)
, it is
unlikely that gap junctions are required for this effect. Similarly,
development of areas lacking oxygen or glucose is generally associated
with spheroids much older than 2 days (24)
. However, the
kinetics shown in Fig. 8
is consistent with a developing population of
noncycling cells in the spheroids, which begin to emerge after 1–2
days in culture. Recent results indicate that the cyclin-dependent
kinase inhibitor p27Kip-1 is highly expressed in
mammary tumor cell spheroids (25
, 26)
. Our preliminary
results indicate that p27 is also elevated in the outer cells of V79
and SiHa but not WiDr spheroids, and we are currently examining the
possibility that expression of p27 or cell cycle-regulatory molecules
is related in some way to the cytoplasmic localization of topo II.
Previously, we explained the resistance of spheroids to etoposide as a
result of a change in DNA conformation that occurs when V79 cells are
grown in spinner culture (1)
. Our results do not eliminate
the possibility that chromatin organization contributes to etoposide
resistance or that changes in topo II localization contribute to
changes in chromatin structure. In fact, it has been suggested that
alterations in topo II phosphorylation may affect nuclear
architecture (18)
, and DNA topoisomerases have been
suggested to play a role as repair enzymes through both damage
recognition and recombinase activities (27)
. Exciting work
from Bojanowski et al. (28)
indicates that topo
II can change chromatin architecture through direct binding
interactions, apparently independently of catalytic activity.
Interestingly, the only cell line that failed to show a change in
intracellular distribution of topo II on growth as spheroids was the
WiDr colon carcinoma cell line (Fig. 2f)
. Cells from the
outer layer of WiDr spheroids or xenografts also failed to develop
greater resistance to killing by etoposide (Fig. 1)
. In previous
studies, we found that WiDr cells grown as monolayers showed resistance
to radiation-induced DNA unwinding when cells were lysed in alkali and
high salt, indicating that chromatin organization may differ for this
cell line (29)
. It is possible that cell contact-induced
changes in signaling may be a more general phenomenon that may also
influence chromatin organization and may help to explain the resistance
of spheroids to other agents, including ionizing radiation
(30)
.
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FOOTNOTES |
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1 Supported by NIH Grant CA37879 and by the
National Cancer Institute of Canada with funds provided by the Canadian
Cancer Society. 
2 To whom requests for reprints should be
addressed, at the Medical Biophysics Department, British Columbia
Cancer Research Centre, 601 West 10th Avenue, Vancouver, British
Columbia, V5Z 1L3 Canada. Phone: (604) 877-6000, ext. 3024; Fax:
(604) 877-6002; E-mail: polive{at}bccancer.bc.ca
3 The abbreviations used are: topo, topoisomerase;
FBS, fetal bovine serum; PTN, PBS with 1% BSA plus 0.1% Tween 20.
Received 1/26/00. Accepted 8/11/00.
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phosphorylation. Cancer Res., 56: 4192-4199, 1996. associated with major acquired resistance to topoisomerase II active agents but minor perturbations of cell growth. Oncol. Res., 7: 407-416, 1995.[Medline]
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) and outer spheroid cells
(•) to etoposide. Monolayers and the outer cell layer of spheroids
were removed by a mild trypsin exposure and immediately exposed to
etoposide for 30 min. Cells were then examined for survival using a
standard colony formation assay. The means and SEs for three
independent experiments are shown. Curves are fit using an exponential
fit (V79 and WiDr) or a linear quadratic fit (SiHa and C6). For the
xenograft tumors (
indicates the monolayer value.
b shows the response of outer V79 spheroid cells to
etoposide after return to monolayer growth conditions. The degree of
resistance to etoposide was determined using a clonogenic assay,
and calculating the ratio of the slopes such as those shown for V79
cells in Fig. 1
). Results from three independent experiments
(dotted line indicates 95% confidence limits) are
shown. f indicates the total cell numbers in the two
environments showing a decrease in growth rate (growth fraction) of the
suspension cultured cells after 24 h.