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Experimental Therapeutics |
Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The response of cells to cytotoxic stress and DNA damage may depend on the cell type, together with the type and extent of DNA damage. Some reports have indicated that certain DNA-damaging agents may induce apoptosis via Fas signaling (6, 7, 8, 9) , whereas others suggest that stress-induced apoptosis caused by drug treatment may be independent of Fas (10, 11, 12) . The objective of this study was to elucidate whether, in addition to DNA damage induced by dThd starvation, other modes of DNA damage induced in TS- cells by chemotherapeutic agents could also induce cell death by signaling via the Fas death receptor. Data demonstrated a close correlation between loss in clonogenic survival, decrease in numbers of cells, the acute induction of apoptosis, and up-regulated expression of FasL in TS- cells in response to thymineless stress and that blocking of Fas/FasL interactions by the NOK-1 inhibitory MoAb completely protected cells from loss in clonogenic survival over a period of 5 days. In contrast, other types of DNA damage induced by doxorubicin (intercalator and dual topoisomerase I and II inhibitor), VP-16 (inhibitor of topoisomerase II), and topotecan (inhibitor of topoisomerase I) induced loss in clonogenic survival at considerably lower drug concentrations (50–100-fold) than those that initiated acute apoptosis. Under these conditions, no protection from loss in clonogenic potential or the induction of apoptosis was obtained by blocking Fas/FasL interactions. The loss in ability of cells to clone at low drug concentrations was due to the induction of a prolonged G2-M-phase arrest that ultimately led to cell death and apoptosis after 6 days. This delayed form of apoptosis could be inhibited by caspase inhibitors, but loss in clonogenic survival could not be prevented by inhibition of caspase activation. At high drug concentrations, an acute form of cell death was apparent at 24 h, where a close correlation existed between the induction of apoptosis and loss in numbers of cells. However, despite drug-induced expression of FasL, this also appeared to be independent of Fas signaling because blocking Fas/FasL interactions did not prevent the drug-induced acute apoptotic response. Data indicated that at low concentrations of DNA-damaging agents, cells accumulated in the G2-M phase of the cell cycle in the absence of acute apoptosis, which resulted in delayed apoptosis and loss in clonogenic survival after drug removal, independent of signaling via Fas but dependent on caspase activation. In contrast, the regulation of thymineless death in TS- cells was dependent on signaling via the Fas death receptor, indicating that that not all forms of DNA damage induce subsequent apoptosis via the Fas signaling pathway in colon carcinoma cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clonogenic Assays.
TS- cells were plated at a density of 3000
cells/well in 6-well plates (Falcon). Cells were also treated with the
NOK-1 MoAb (PharMingen; 100 ng/ml) or a mouse IgG1 isotype-matched
control MoAb (PharMingen; 100 ng/ml) at the time of plating. After
overnight attachment, cells were washed with 2 ml of HBSS (37°C) and
subsequently deprived of dThd by refeeding with dThd-free medium
containing the respective Abs. At various times for up to 7 days, cells
were rescued by adding dThd (20 µM) to individual wells,
and clonogenic survival was determined 11 days after dThd restoration
(1)
. Alternatively, cells were plated in the presence of
NOK-1 and subsequently treated for 72 h with doxorubicin
(0.01–100 nM), topotecan (0.01–100 nM), or
VP-16 (0.1–1 µM) in the presence of dThd and in either
the absence or presence of Abs. Clonogenic survival was determined 11
days after removal of the drugs (1)
. To determine the
influence of caspase activation on loss in clonogenic survival after
the induction of thymineless stress or treatment with anti-Fas
(CH-11; 3–30 ng/ml; MBL International Corp.) or VP-16
(0.1–1 µM; Bristol-Myers-Squibb), additional clonogenic
survival assays were conducted in the presence of the general caspase
inhibitor Z-VAD-FMK (100 µM; Enzyme Systems
Products) during 72-h exposures.
Analysis of Cell Numbers.
Cells were plated in 6-well plates at a density of 400,000 cells/well.
After overnight attachment, cells were treated with doxorubicin (0.3–3
µM), topotecan (0.3–3 µM), and VP-16
(10–30 µM) for periods of up to 96 h. Cells were
subsequently enumerated using a Coulter Particle Counter
(5)
.
Apoptosis and Cell Cycle Analyses.
TS- cells were plated at a density of 400,000
cells/well in 6-well plates. After overnight attachment, cells were
deprived of dThd or treated with varied concentrations of doxorubicin,
topotecan, or VP-16 for periods of up to 96 h before harvest. Both
the floating cells and attached cells were pooled after trypsinization,
fixed in 70% ethanol, and stored at -20°C before analysis.
Apoptotic cells were detected as a sub-G1
fraction after propidium iodide staining and analysis using a Becton
Dickinson FACScan (13
, 14) . The influence of cotreatment
with Z-VAD-FMK (100 µM) for 72 h on the induction of
acute apoptosis (72-h exposure to 10–100 µM
VP-16) or delayed apoptosis (72 h after a 72-h coincubation with
0.1–1 µM VP-16) was also examined. Furthermore,
the effect of NOK-1 (50 µg/ml) or Fas-Fc (50 µg/ml; a chimeric
fusion protein generously provided by Dr. Carl Ware; the La Jolla
Institute for Allergy and Immunology, San Diego, CA), which prevent
Fas/FasL interactions, was examined on the induction of rapid apoptosis
(72-h exposure to 30 µM VP-16) or delayed apoptosis (72 h
after a 72-h exposure to 1 µM VP-16). For evaluation of
the cell cycle distribution, samples were collected as described and
immediately centrifuged and resuspended in 0.005% propidium iodide
containing 0.1% sodium citrate and 0.1% Triton X-100, filtered, and
analyzed by FACScan.
Expression of FasL.
FasL expression was determined by semiquantitative RT-PCR in
TS- cells at various times during dThd
deprivation or during treatment with doxorubicin (0.3–3
µM), topotecan (0.3–3 µM), or VP-16
(10–90 µM) for up to 96 h, as described previously
(Ref. 5
; acute apoptosis). FasL expression was also
determined at 72 h after exposure to 1 µM VP-16 and
at 72 h after the end of drug exposure (delayed apoptosis).
ß-Actin was used as a control to monitor RT-PCR amplification
efficiency and the quality of the cDNA from 2 µl of the template at
25 cycles, as reported previously (5)
. PCR products were
separated by electrophoresis in a 2% agarose gel and visualized by
ethidium bromide staining and UV light illumination. Quantitation was
by optical densitometry of the reverse gel image using a Hewlett
Packard Scan Jet IIC. Intensity of the signal was determined by
comparison with a calibrated photographic gray scale (Kodak) in the
linear range of detection.
Selection of CH-11-resistant Clones.
TS- cells were selected for resistance to
Fas-mediated apoptosis by treatment with the cytolytic anti-Fas MoAb
CH-11 (MBL International Corp.). Cells (100 x 106) were mutagenized with EMS (300
µg/ml) in T-175 flasks for 22 h. Cells were cultured for an
additional 48 h in the absence of EMS and subsequently selected
for 72 h in 200 ng/ml CH-11. After growth for an additional 7 days
in the absence of CH-11, surviving cells were mutagenized with EMS and
selected in CH-11 (200 ng/ml) for a second time. Surviving colonies
were allowed to grow for 2 weeks and were subsequently ring-cloned and
placed in T-12.5 flasks in 2 ml of dFBS-containing medium with
dThd (20 µM), and the colonies were expanded. Three
colonies were selected for further evaluation.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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demonstrates that after dThd withdrawal from asynchronously
growing TS- cells, complete protection from
thymineless death and loss in clonogenic survival occurred when cells
were incubated simultaneously with NOK-1. Cells incubated with the IgG1
isotype-matched control in the absence of dThd demonstrated the same
kinetics of cell kill as cells exposed to dThd deprivation alone: cells
lost 50% of clonogenic potential in 72 h; and only 5% of cells
formed colonies at 5 days after dThd deprivation. In contrast,
cells coincubated with NOK-1 in the absence of dThd were completely
protected from thymineless death at this time.
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), doxorubicin (Fig. 1C
), and VP-16 (Fig. 1D
) was determined in the absence or presence of NOK-1.
Thus, loss in clonogenic survival was induced by all three agents, with
less than 10% clonogenicity remaining at topotecan, doxorubicin, and
VP-16 concentrations of 30 nM, 30 nM, and 1
µM, respectively, and
IC50 values of 10, 5, and 150
nM, respectively. In contrast to the protection
of TS- cells from thymineless death by
simultaneous incubation with the NOK-1 MoAb, no protection from the
cytotoxic effects of these three agents was provided during
coincubation with the inhibitory Ab for 72 h. Furthermore, no
protection was provided by NOK-1 when cells were coincubated with the
MoAb additionally after drug treatment (data not shown).
Sensitivity of Fas-resistant TS- Clones to
DNA-damaging Agents.
Characterization of the Fas resistance phenotype demonstrated wild-type
sequence for Fas in all clones after DNA sequencing of the death domain
(and extracellular binding domain). However, Fas-resistant clones were
found to express lower levels of Fas (reduced by 40%) as determined by
RNase protection and FACS analyses (data not shown). To confirm the
lack of involvement of the Fas death receptor in regulation of the
cellular sensitivity of TS- cells to
doxorubicin, topotecan, and VP-16, studies were conducted in five
clones. When asynchronous populations of Fas-resistant clones were
exposed to thymineless stress, >80% survival was obtained at 5 days
after dThd withdrawal, in contrast to only 5% in the case of
TS- cells (Fig. 2A
), demonstrating that TS- cells
selected for resistance to CH-11 and hence for resistance to signaling
via Fas were also cross-resistant to the induction of thymineless death
after dThd deprivation.
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,
B–D, in comparison to the sensitivity of
TS- cells to the three agents. No change in the
sensitivities to the three DNA-damaging agents was obtained in
CH-11-resistant clones, confirming the lack of influence of signaling
via Fas on the sensitivity of TS- cells to these
agents.
Induction of Apoptosis and Expression of FasL.
The influence of thymineless stress or drug exposure on the induction
of apoptosis in TS- cells, on the numbers of
cells surviving after 72 h, and on the expression of FasL was
determined. After dThd withdrawal (Fig. 3
) apoptosis determined by FACS analysis was detected by 24 h and
increased to almost 45% at 5 days (Fig. 3
). This correlated temporally
with the loss in numbers of surviving cells, which decreased to <50%
at 5 days, and to the progressive induction of the expression of FasL
(Fig. 3
).
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), but not at lower drug concentrations. Analysis of the
drug concentration-dependent loss in the number of surviving cells
after treatment for 72 h correlated with the percentage of cells
undergoing apoptosis at this time (Fig. 4
). FasL expression, as
determined by RT-PCR, was induced at the same concentrations of
doxorubicin, topotecan, and VP-16 after 72-h exposures that induced
apoptosis and loss of cells after drug exposure. Furthermore,
expression of FasL also increased with increasing drug concentrations
(Fig. 4C
). There was also a close correlation between the
three parameters during drug treatment and incubation time at 1–30
µM drug concentrations (Fig. 5
). However, the concentrations of drugs that induced apoptosis and
up-regulated expression of FasL were considerably higher (50-fold) than
those that effectively reduced clonogenic survival. Apoptosis was thus
determined to be acute under these conditions.
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). At the end of drug exposure (72 h), only a small fraction of cells
were undergoing apoptosis (15%). However, after removal of drugs and
further incubation in drug-free medium, 40–50% of treated cells
demonstrated evidence of apoptosis after an additional 3 days, and
apoptosis continued to increase with increasing time of incubation. On
examination of the expression of FasL, a low level of FasL expression
was detected after a 72-h exposure to VP-16 (1 µM), and
FasL expression was further increased after an additional 3 days in the
absence of drug (data not shown), reaching levels comparable to those
induced by high concentrations at 72 h. Thus, at low
concentrations of drugs that reduced clonogenic survival, apoptosis
was delayed after a 72-h drug exposure.
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. In response to doxorubicin and topotecan, there was an
increase in G2-M-phase cells from 20% to between
60% and 75% after drug treatment, and in response to VP-16, there was
an increase in G2-M-phase cells up to 44%. As
the concentrations of drugs were increased in cells undergoing acute
apoptosis, the percentage of cells in G2-M phase
decreased, correlating with the increase in percentage of apoptotic
cells.
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). Furthermore, there was a decline
in the fraction of TS- cells in
G2-M phase with increasing time of incubation.
Data suggested that TS- cells arrested in
G2-M phase at low concentrations of DNA-damaging
agents that reduce clonogenic survival enter a prolonged and
irreversible G2-M-phase arrest that ultimately
leads to apoptosis.
Effect of Inhibition of Caspase Activation on Apoptosis and
Clonogenic Survival.
The effect of the general caspase inhibitor Z-VAD-FMK (100
µM) on the induction of both acute and delayed apoptosis
during a 72-h exposure to VP-16 was determined and compared with the
effect on apoptosis induced by thymineless stress and after treatment
with anti-Fas (CH-11, 3–50 ng/ml). During incubation with VP-16
(10–30 µM) or CH-11 (50 ng/ml) and under dThd-free
conditions, Z-VAD-FMK inhibited the induction of acute apoptosis (Fig. 8A
). In addition, 72-h exposure to low concentrations of VP-16
(0.1–1 µM) in the presence of the caspase
inhibitor also resulted in a reduction in the degree of apoptosis
induced (Fig. 8B
). Although Z-VAD-FMK protected
TS- cells from loss in clonogenic survival after
the induction of thymineless stress and ligation of Fas after treatment
with CH-11, no protection was demonstrated after treatment with 0.1
µM VP-16 (Fig. 9
).
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). Blocking ligation of Fas under both conditions did not prevent
either acute or delayed apoptosis induced by VP-16.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cytotoxic activity of anticancer agents has been described to be both dependent (6, 7, 8, 9) and independent (10, 11, 12) of signaling via Fas. In Jurkat T leukemia cells, apoptosis induced by doxorubicin appeared to proceed via activation of FasL, analogous to the dependence of activation-induced apoptosis in T lymphocytes on transcription and expression of FasL (6) . Furthermore, competitive binding of either FasL (using a Fas-Fc chimeric protein; Ref. 6 ) or Fas (using a neutralizing anti-Fas Ab) effectively inhibited death induced by VP-16 and VM-26 (7) , suggesting that apoptosis induced by these agents was mediated via Fas signaling. Of interest was that interference with Fas/FasL interactions inhibited anisomycin- but not radiation-induced apoptosis in Jurkat cells, suggesting that not all stress stimuli involving DNA damage use the same cell death pathway (10) .
In this study, we demonstrated that Fas-dependent loss in clonogenic
survival of TS- cells after dThd withdrawal
correlated closely with the loss in cell numbers over a period of 5
days, the onset and induction of apoptosis determined by FACS analysis,
and the up-regulated expression of FasL. The importance of thymineless
death in the mechanism of cell killing by FUra/LV combinations in human
colon carcinoma cell lines (16)
, xenografted tumors in
mice (20
, 21)
, and colon carcinomas in humans (22
, 23)
has been well established. Furthermore, up-regulation of the
expression of Fas by IFN-
in colon carcinoma cell lines has been
shown to be important in increasing the cytotoxic activity of FUra/LV,
which is dependent on thymineless stress-induced DNA damage
(16)
. Therefore, taken together, data suggest that Fas is
an important determinant of the extent to which colon carcinomas are
sensitive to the induction of thymineless death.
In contrast, the cytotoxic response to both nanomolar and micromolar concentrations of doxorubicin, topotecan, and VP-16 was independent of regulation via Fas. At low drug concentrations, drug-induced loss in clonogenic survival was not inhibited by the anti-Fas inhibitory MoAb NOK-1. Of interest was that FasL expression was induced under these conditions. However, this appeared to be an associated stress response and was not causative of the induction of apoptosis because the delayed apoptotic response after VP-16-induced G2-M-phase arrest at a low drug concentration could not be inhibited by blocking Fas/FasL interactions with either NOK-1 or Fas-Fc. A close correlation between the loss in numbers of surviving cells, induction of apoptosis, and up-regulated expression of FasL was obtained in response to treatment with doxorubicin, topotecan, and VP-16, but at considerably higher drug concentrations (50-fold) than required to induce loss in clonogenic survival. Whereas evidence suggests that TS- cells undergo apoptosis and cell loss after dThd deprivation as cells enter S phase, resulting in no apparent accumulation of cells in S phase during this time, the cellular response to DNA-damaging agents was different. It was apparent that at all drug concentrations, TS- cells were arrested in the G2-M phase of the cell cycle and that at the higher concentrations (micromolar concentrations), this was associated with acute induction of apoptosis and up-regulated expression of FasL. At lower drug concentrations (nanomolar concentrations), the initial arrest of cells in G2-M occurred in the absence of apoptosis. However, it is evident that these cells were already committed to cell death because apoptosis ultimately took place after the equivalent of several cell doublings. Furthermore, the caspase inhibitor Z-VAD-FMK did prevent the induction of delayed apoptosis induced by VP-16, indicating that this late form of cell death was a caspase-dependent apoptosis, after commitment of TS- cells to cell death in the G2-M phase of the cell cycle. Thus, studies conducted at drug concentrations that induce apoptosis in the acute phase may not necessarily be indicative of the actual mechanism by which cells ultimately commit to cell death. It is also evident that the induction of acute apoptosis was not dependent on Fas/FasL interactions because simultaneous incubation of VP-16 with NOK-1 or Fas-Fc failed to prevent induction of apoptosis. This suggested that, like the delayed apoptotic response, the induction of acute apoptosis was independent of Fas. Both acute and delayed apoptosis required caspase activation, as demonstrated by an inhibitory response in the presence of Z-VAD-FMK.
There have been several reports of dual mechanisms of action demonstrated by DNA-damaging agents including doxorubicin (24) , daunomycin (24) , VP-16 (25 , 26) , and bleomycin (27) , dependent on the concentrations of the agents used. For doxorubicin and daunomycin, EC90 concentrations led to G2-M-phase arrest and cytostasis in HeLa cells in the absence of inhibition of DNA synthesis, ultimately resulting in delayed apoptosis and cell death. In contrast, at 10x EC90 concentrations, apoptosis was detected as early as 3 h after drug treatment (24) . Different cellular responses to VP-16 have been reported in thymocytes at different drug concentrations (25) . In addition, Lock and Stribinskiene (26) reported dual modes of death in HeLa cells induced by VP-16. One mode was prevented by Bcl-2, which inhibited loss in cell viability and induction of apoptosis, whereas the second, which induced loss in clonogenic survival and comprised the formation of giant, multinucleated cells characteristic of mitotic catastrophe, was not affected by Bcl-2. Similarly, at low concentrations of bleomycin exposure, DC-3F Chinese hamster lung fibroblasts arrested in G2-M phase and became enlarged and polynucleated before dying. This response paralleled the mitotic death observed after ionizing radiation (27) and also after cisplatin-induced cellular cytotoxicity (28 , 29) . In contrast, apoptosis and rapid DNA fragmentation were observed at high bleomycin concentrations.
It is therefore evident that the acute induction of apoptosis after
treatment of cells with DNA-damaging agents is not a prerequisite for
the induction of cellular sensitivity to these agents. The data of
Smith et al. (30)
in a small cell lung
carcinoma cell line suggested that traverse of
G1-S phase and early S phase in the presence of
VP-16 led to trapping and enhanced availability of topoisomerase II
and subsequent irreversible arrest of cells in
G2-M phase in the presence of limited DNA
fragmentation. After G2-M phase arrest of DC-3F
cells at low bleomycin concentrations, cells died after a time period
equivalent to three doubling times (27)
. In the current
study, we demonstrated a similar G2-M-phase
arrest in the TS- human colon carcinoma cell
line at low, nanomolar concentrations of doxorubicin, topotecan, and
VP-16, resulting in loss of clonogenic survival. In contrast to the
induction of thymineless death, this was independent of regulation via
Fas. However, similar to the studies described above, the induction of
G2-M-phase arrest in TS-
cells at nanomolar drug concentrations ultimately led to the induction
of apoptosis. Both acute and delayed forms of apoptosis are caspase
dependent; however, inhibition of apoptosis by caspase inhibitors did
not restore clonogenic survival.
It is not clear which form of cell death may be responsible for the antitumor activity of these drugs in vivo. However, inhibitors of both topoisomerases I and II have demonstrated marked schedule dependence. Against human tumor xenograft models, topotecan and irinotecan have demonstrated superior therapeutic efficacy when administered at low doses in protracted schedules in comparison to more intense shorter-term schedules of administration (31 , 32) . In clinical trials, prolonged administration of VP-16 for 3–5 days was more effective than a single high-dose bolus administration for 24 h in small cell lung cancers (33) and in non-Hodgkin’s lymphomas (34) . The plasma levels of VP-16 during high-dose therapy reached 10–50 µM (35) , whereas continuous infusion or oral administration maintained plasma levels of VP-16 from 0.5–1 µM for prolonged periods (33 , 36) . These findings are consistent with the marked efficacy of low concentrations of these agents in TS- cells determined in the current study.
In summary, we have demonstrated that thymineless stress induces cytotoxicity and apoptosis in TS- colon carcinoma cells by signaling via the Fas death receptor, in contrast to DNA-damaging agents that induce cytotoxicity independent of Fas in these cells. After DNA damage, topotecan, VP-16, and doxorubicin reduce clonogenicity via a prolonged G2-M-phase arrest, which ultimately results in the commitment of TS- cells to undergo apoptosis, and this mechanism is independent of Fas. In contrast, acute apoptosis is induced only at considerably higher drug concentrations, which may be less relevant therapeutically than those concentrations that initiate a delayed apoptotic response and are also independent of Fas in TS- colon carcinoma cells.
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FOOTNOTES |
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1 Supported by NIH Award R37 CA 32613, Cancer
Center Support (CORE) Grant CA 21765, and the American Lebanese Syrian
Associated Charities (ALSAC). I. P. was supported in part by a
scholarship from the Fulbright Program and by the First Institute of
Pathology and Experimental Cancer Research, Semmelweiss University
(Budapest, Hungary), and R. M. by a Rolyai Research \E
Fellowship and Grants OTKA T029611 and FK FP0091. 
2 To whom requests for reprints should be
addressed, at Department of Molecular Pharmacology, St. Jude
Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN
38105. Phone: (901) 495-3440; Fax: (901) 521-1668.
3 The abbreviations used are: TS-,
thymidylate synthase-deficient; FasL, Fas ligand; dThd, thymidine;
MoAb, monoclonal antibody; VP-16, etoposide; FUra/LV,
5-fluorouracil/leucovorin; Ab, antibody; RT-PCR, reverse
transcription-PCR; FACS, fluorescence-activated cell-sorting.
Received 5/19/99. Accepted 3/17/00.
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