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Experimental Therapeutics, Preclinical Pharmacology |
Cancer Research Campaign Centre for Cancer Therapeutics, E Block, Belmont [S. J. W., L. B., M. V., A. L. J.], and Cancer Research Campaign Centre for Cancer Therapeutics, Haddow Laboratories, Institute of Cancer Research, Sutton [J. T., P. M., G. W. A.], Surrey, SM2 5NG, United Kingdom
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ABSTRACT |
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Up-regulation of TS protein showed a time-, dose-, and cell-type-specific response to treatment with ZD9331. This response was observed in W1L2 cells treated for 24 h at equitoxic doses of raltitrexed (6-fold), ZD9331 (10-fold), fluorouracil (5-fold), LY231514 (7-fold), AG337 (7-fold), and BW1843U89 (3-fold). Up-regulation was observed over a range of doses. Elevation of TS protein only persisted up to 12 h after removal of drug. The extent of induction does not depend on basal TS levels. Nontransformed human fibroblasts showed significantly greater up-regulation of TS protein than tumor cells exposed to an equitoxic dose of ZD9331. In vivo experiments using the L5178Y thymidine kinase -/- mouse lymphoma implanted into DBA2 mice also showed greater up-regulation of TS protein in normal intestinal epithelial cells compared with tumor cells.
These results confirm that TS up-regulation is a common feature of TS inhibition in tumor cells and that it may occur to a greater extent in normal tissues, although the clinical implications of these findings remain to be determined.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The importance of TS as a chemotherapeutic agent is now well established. 5-FU, a fluoropyrimidine drug, is widely used in the treatment of breast, gastrointestinal, and head and neck cancers (3) . Several TS inhibitors have recently been clinically evaluated. These include the quinazoline antifolates, raltitrexed (Tomudex, ZD1694; Ref. 4 ) and ZD9331 (5 , 6) , MTA (7) , a multitargeted antifolate, BW1843U89 (8) , and the nonclassical lipophilic TS inhibitor, AG337 (9) .
Several acquired mechanisms of resistance to TS inhibitors have been reported: (a) overexpression of the target enzyme, resulting in elevated levels of TS protein (10 , 11 , 12) , (b) impaired drug uptake, e.g., because of down-regulation or mutation of reduced folate carrier (11 , 13) , and (c) diminished polyglutamation (11 , 14 , 15) .
However, another mechanism by which transient resistance to TS inhibitors can occur has been described and may have important consequences to therapeutic outcome (16 , 17) . In the G1 phase of the cell cycle, TS protein binds tightly to TS mRNA preventing translation of TS protein. On entering the S phase when thymidylate is required for DNA replication, TS undergoes a conformational change on binding its substrate dUMP and the folate cofactor. TS is no longer able to bind to TS mRNA, thereby allowing translation. A similar effect is seen when TS binds to an inhibitor with the result that TS can no longer bind to TS mRNA in any phase of the cell cycle leading to up-regulation of TS protein. Recently, an alternative model proposed that the up-regulation of TS protein after exposure to TS inhibitors is attributable to a stabilization of the TS protein rather than an increase in translation (18) . Regardless of the mechanism, the acute increase in TS observed after exposure to TS inhibitors may have important consequences to therapeutic outcome because induction of the target may result in decreased inhibition, although this may only be relevant if normal cells do not up-regulate TS to a similar or greater extent. Indeed, previous studies have suggested that induction of TS in normal cells is generally more responsive to the effects of 5-FU, particularly at higher doses (19) . In addition, normal mammary epithelial cells demonstrated a 40-fold up-regulation compared with only 10-fold in cancer mammary epithelial cells after treatment with raltitrexed (17) .
In the present study, a variety of human tumor cell lines and nontransformed fibroblasts were used to determine the extent of TS up-regulation in vitro after treatment with a range of TS inhibitors. In addition, in vivo experiments using a mouse lymphoma model were carried out. The results confirmed that TS up-regulation is a common feature of TS inhibition in tumor cells and that it may also occur to a greater extent in normal tissues.
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MATERIALS AND METHODS |
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Compounds.
All standard laboratory chemicals were AnalaR grade purchased
from either British Drug Houses (BDH, Poole, United Kingdom) or Sigma
(Poole, United Kingdom).
ZD9331, raltitrexed, and AG337 were synthesized at Zeneca Pharmaceuticals (Macclesfield, United Kingdom). MTA and BW1843U89 were generously supplied by Eli Lilly and Company (Indianapolis, IN) and Glaxo-Welcome Pharmaceuticals (Stevenage, Hertfordshire, United Kingdom), respectively. 5-FU was purchased from Sigma. All compounds except AG337 were dissolved at 10 mM in 0.1 M NaHCO3 (pH 8.3). AG337 was dissolved at 10 mM in 100% DMSO. The dissolved compounds were then passed through a 0.22-µm filter and stored at -20°C for a maximum of 3 months.
Drug Exposure.
To measure changes in TS levels, cell lines were exposed to TS
inhibitors for 16 or 24 h at doses standardized to
IC50 values. IC50 values
were determined using 72-h (all cell lines except A549) or 120-h (A549)
3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assays as described previously (Ref. 22
; adherent cell
lines) or by cell counting using a Coulter counter (suspension cell
lines). At the end of drug exposure, cells were harvested and washed
with PBS, and protein concentrations for Western blotting were measured
using the bicinchoninic acid protein assay according to the
manufacturers instructions (Pierce, United Kingdom).
Western Blotting.
Analysis of TS protein levels was performed as described previously
(23)
. Briefly, total cellular protein was isolated from
cells in lysis buffer containing proteinase inhibitors, and equal
amounts (50 µg) of protein were separated by SDS-PAGE (8–16%
tris/glycine gels purchased from NOVEX, Germany) and electroblotted to
a nitrocellulose membrane. Recombinant human TS (generously supplied by
Agouron, San Diego, CA) was used as a positive control. Immunoblotting
was performed using a rabbit polyclonal antibody to recombinant human
TS (23)
, followed by horse radish peroxidase-conjugated
antirabbit secondary antibody. Proteins were detected using enhanced
chemiluminescence according to the manufacturer’s instructions
(Amersham Life Sciences, Bucks, United Kingdom).
Confocal Microscopy.
A549 cells were grown to 60% confluency on coverslips. The cells were
then treated for 24 h with varying doses of ZD9331. After
treatment, the cells were fixed (4% paraformaldehyde in PBS, room
temperature, 30 min), washed three times with PBS, and permeabilized
(0.1% Triton X100, 10 min). The cells were washed three times with
PBS, nonspecific binding was blocked with PBS/0.5% BSA for 30 min, and
the cells were incubated for 1 h in primary antibody [anti-TS
(23)
] diluted 1:150 in PBS/0.5% BSA. After a further
three washes in PBS, the cells were incubated for 1 h with
secondary antibody (Alexa 488 goat antirabbit IgG conjugate, Molecular
Probes, Cambridge, United Kingdom) diluted 1:200 in PBS/0.5%
BSA, and nuclei were labeled with propidium iodide after treatment with
RNase. Slides were then mounted using Vectashield (Vector Laboratories,
Peterborough) and examined at 488 nm on a TCS-SP confocal microscope
(Leica, Milton Keynes, United Kingdom).
ELISA.
TS protein concentrations in W1L2 cells (wild-type and acquired
resistant variants; Refs. 5
, 11
,
20
, and 21
) were determined using ELISA
(23)
. Information generated from this method was used to
validate and quantify the flow cytometry method described below.
Flow Cytometry to Determine TS Protein Concentrations.
A flow cytometry method was evaluated to measure TS protein in cells.
Appropriately drug-treated cells (1 x 106)
were washed with PBS + 1% FCS + 0.01% NaN3
(sodium azide; 5 min, 1500 rpm, 25°C), fixed (1 ml 2%
paraformaldehyde, 10 min), and permeabilized (50 µl 0.1% Triton
X-100, 10 min). The cells were split into two aliquots and washed twice
(as above). One aliquot of cells was then treated with 100 µl of TS
antibody (Ref. 23
; 1/60 in PBS, 1 h), the other was
treated with 100 µl of normal rabbit serum (1/60 in PBS, 1 h).
The optimal dilutions were found by titration of the antibodies. One
hundred µl of second antibody [goat antirabbit FITC (Amersham,
Buckinghamshire, United Kingdom) 1/100 in PBS] were added to each
aliquot after washing (as above), and samples were analyzed within
24 h by flow cytometry after two washes (as above). To quantify
the difference in TS between samples, the following ratio was
calculated:
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In Vivo Studies.
Six-week-old female DBA2 mice were obtained from Harlan UK Ltd
(Bicester, Oxon) and randomly allotted to one of three treatment groups
and allowed free access to a standard diet (RM1). The experiment was
approved by the local ethical committee. Animals were treated when 8
weeks old. L5178Y TK -/- cells were grown to 60% confluency in RPMI
medium. The cells were then harvested (1500 rpm, 5 min), counted using
a hemocytometer, and diluted to 1 x 108
cells/ml in sterile saline. Cells (5 x
106; 50 µl) were implanted into the
gastrocnemius muscle of 17 mice. The tumor was allowed to reach 8–9 mm
in diameter after which 10 mice were treated with ZD9331 (50 mg/kg i.p.
bolus). Five drug-treated mice and three (7 h) or four (16 h) untreated
mice were sacrificed by cervical dislocation either 7 or 16 h
after injection. Tumors and gut scrapes were collected immediately.
Fifteen cm of gut starting 5 cm from the stomach were removed, flushed
with ice-cold PBS, slit along its longitudinal axis, and scraped using
the back of a scalpel blade. All tissue harvested was immediately
homogenized in 0.3 ml of lysis buffer (23)
and snap-frozen
in liquid nitrogen. Samples were stored at -70°C before analysis for
TS protein using Western blotting.
Statistical Analysis.
An ANOVA two-way statistical analysis was performed using Graphpad
Prism.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). When flow cytometry and ELISA data
for four other cell lines were included in the correlation, the slope,
Y intercept, and r2 value
did not change significantly [data points for A2780, SW480, HX62, and
HT29 are superimposed on the standard curve (Fig. 1)
]. This procedure
enabled quantitation of TS levels for the remainder of this study.
Although the flow cytometry ratio was low (for example, in W1L2 cells
after a 24-h exposure of 1 x IC50 ZD9331,
the ratio was 1.07 ± 0.02 compared with 1.04 ± 0.0006 for
untreated cells), results were highly reproducible, and the coefficient
of variation percentage of the measurements were within
acceptable levels.
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). Confocal microscopy confirmed
the up-regulation observed over 24 h of treatment in A549 cells
with ZD9331 (Fig. 2)
. A dose response was
also observed using this method. Treated cells were enlarged compared
with untreated controls—a characteristic of thymineless death
(26)
.
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shows results on W1L2 and A549
cells using flow cytometry. Greater induction of TS protein can be seen
using flow cytometry than using Western blotting (9-fold compared with
3-fold up-regulation in A549 cells under similar conditions of ZD9331
exposure). A dose- and time-dependent response can be observed in both
A549 and W1L2 cells after treatment for either 16 or 24 h with
increasing concentrations of ZD9331. No statistically significant
up-regulation is observed in either cell line below 7 x
IC50 values after 16-h treatment with ZD9331, but
statistically significant up-regulation is observed with 1 x
IC50 values after 24-h treatment with ZD9331
(P < 0.001). Over a 24-h time course, W1L2 cells show
significantly greater up-regulation of TS protein than A549 cells
(5-fold up-regulation in A549 cells compared with 10-fold in W1L2 cells
at 100 x IC50 values of drug;
P < 0.001).
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. Increased levels
of TS protein were measured after 24-h treatment of W1L2 cells with all
of the TS inhibitors tested. The specific TS inhibitors, ZD9331, AG337,
and raltitrexed, caused greatest up-regulation of TS protein
(
9.8-fold, 7.3-fold, and 6.0-fold up-regulation with 100 x
IC50 doses, respectively). BW1843U89, a specific
polyglutamatable benzoquinazoline TS inhibitor, gave the least
up-regulation (3.1-fold using 100 x IC50
dose).
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). TS protein was up-regulated to a
similar extent as in previous experiments, but a significant decrease
in TS levels was observed within 4 h of resuspension of the cells
in DFM (P < 0.001). After 12 h, the amount of
protein was less than in untreated controls and remained at this level
for up to 24 h after resuspension in DFM.
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. All of these cell lines
express higher levels of TS protein than W1L2 (lowest levels) or A549
cells. The extent of up-regulation of TS protein in the ovarian and
colon cell lines was then determined after 24-h treatment with ZD9331
(Fig. 4B)
and compared with basal TS levels. No correlation
was found between basal TS protein levels and extent of up-regulation
or with doubling times of the cell lines, which ranged from 18 to
24 h (data not shown).
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. Up-regulation of TS protein was
generally higher at both time points with all concentrations of ZD9331
compared with tumor cells. Up-regulation showed both a time- and
dose-dependent response reaching 20-fold greater quantities of TS
protein than in untreated control cells compared with 8-fold under
the same conditions in W1L2 cells.
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. Up-regulation of TS in
L5178Y TK -/- cells was then measured in vivo after
implantation into DBA2 mice and administration of ZD9331 at a dose
resulting in antitumor activity (5)
. TS protein levels in
the lymphoma and in normal gut cells were then compared. Gut cells
showed a significantly greater up-regulation of TS protein than tumor
cells (P < 0.05; Fig. 6B
). There was no
difference between the up-regulation observed within the same tissues,
i.e., gut or tumor after 7 h or 16 h of treatment.
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DISCUSSION |
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This study has demonstrated that "short-term" induction of TS
occurs to a significant extent (up to 10-fold after treatment with
ZD9331 compared with untreated control samples in the human tumor cell
lines W1L2 and A549; Fig. 2
and Table 2
). Statistically significant
up-regulation occurred by 16 h at doses of ZD9331 >0.07
µM and 0.7 µM (7 x
IC50) in W1L2 and A549 cells, respectively
(P < 0.001). However, statistically significant
up-regulation occurred at doses as low as 0.01
µM and 0.1 µM in the
same cell lines (1 x IC50) after 24-h
exposure to ZD9331 (P < 0.001). ZD9331 has been shown
to be present in the plasma of mice bearing the L5178Y TK -/- tumor
at concentrations exceeding 0.5 µM for 12 h after bolus administration of ZD9331 (19 h following s.c. infusion;
Refs. 41
and 42
). Tumor drug levels were
shown to be 2-fold higher than plasma levels (42)
. The
concentration of free drug in human plasma has been estimated at 20
µM in humans, and pharmacokinetic data from
Phase I trials have demonstrated a long terminal elimination half-life
(83 h; Ref. 43
). These results suggest that up-regulation
could occur at drug concentrations relevant to pharmacologically active
doses in both mice and man, although protein binding of the drug also
needs to be taken into account.
The up-regulation was not retained in W1L2 cells after removal of drug
(Fig. 3)
, and TS protein levels fell to below pretreatment levels
within 12 h of resuspension in DFM, demonstrating the reversible
nature of this response. The levels remained up to 4-fold less than
pretreatment values 24 h later. This drop below control levels of
TS protein could be explained in three ways. Firstly, it could be
attributable to overcompensation of the regulatory mechanism when the
cell is no longer exposed to a TS inhibitor. Alternatively, this may
simply represent the lack of cell replication immediately after removal
of the TS inhibitor when little TS activity is required pending
downstream DNA repair or cell death. Lastly, TS levels and activity may
vary 14- to 24-fold between the peak exponential and confluent growth
phase (44)
, and this may account for the down-regulation
observed because a doubling time has passed over the course of
the experiment.
Up-regulation of TS protein was observed using a range of TS inhibitors
of varying structure (Table 3)
. Up-regulation ranged between 3- and
10-fold in W1L2 cells treated for 24 h. Previous studies have
demonstrated similar increases in TS activity after exposure to 5FU and
raltitrexed (17
, 25
, 27)
. No correlation was observed
between the extent of up-regulation and (a) whether drugs
are specific TS inhibitors; (b) whether the drugs undergo
polyglutamation within cells; or (c) the mechanism of uptake
into the cell. Consistent with previous studies, no elevation in TS
mRNA levels were measured using Northern blotting after treatment for
up to 72 h (100 x IC50) with TS
inhibitors (data not shown; Refs. 17
and 18
).
Several researchers have reported a direct correlation between TS
protein expression and response to 5FU (25
, 30)
, with
lower TS protein expression predicting for better response to TS
inhibitors, although other studies failed to show any correlation
(45)
. Considerable variation in TS expression has also
been reported between sensitive and resistant cell lines in
vitro (25)
. The extent of up-regulation of TS protein
was shown to vary between cell lines in this study (Table 2)
. It was
therefore proposed that the extent of up-regulation might depend upon
basal TS protein levels. Four cell lines were selected based upon their
basal TS protein levels. However, no correlation was observed between
predrug exposure TS protein levels and extent of up-regulation of TS.
Differences in doubling times of cell lines used during this study also
did not correlate with the extent of up-regulation of TS (data not
shown).
TS protein expression was also shown to be increased in normal,
nontransformed human fibroblasts (Fig. 5)
. Up-regulation was
significantly greater in these cells than in any of the tumor cell
lines tested (up to 20-fold compared with 9-fold in W1L2 cells after
24-h treatment with equitoxic doses of ZD9331). Up-regulation was also
significantly greater (P < 0.05) in fibroblasts than
tumor cells when equimolar doses of ZD9331 are compared (10-fold
up-regulation in W1L2 cells compared with 15-fold in fibroblasts after
treatment with 1 µM ZD9331 for 24 h). This
is in agreement with previous studies that have shown a 40-fold
increase in TS activity in human normal mammary epithelial cells
compared with a 10-fold increase in tumor cells after treatment with
equimolar concentrations of raltitrexed for 36 h
(17)
. Induction of TS in normal cells has also been
reported to be generally more responsive to treatment with 5-FU
compared with malignant cells (19)
.
Significantly greater up-regulation was observed in vivo in gut compared with lymphoma cells (2 and 1.2, respectively; P < 0.05) after treatment for either 7 or 16 h. The extent of up-regulation observed both in vitro and in vivo in the tumor tissue was small compared with that observed in other tumor cell lines studied. Up-regulation of TS protein was identical at both time points, suggesting that a threshold level of TS protein up-regulation had been reached for this concentration of ZD9331. These results indicate that normal proliferating cells may up-regulate TS protein to a greater extent than tumor cells upon treatment with a TS inhibitor, which could in turn lead to an increase in therapeutic index. Indeed, this is indicated by data showing levels of TS inhibition in solid human tumor and normal liver biopsies treated with 5-FU. Seventy to 80% inhibition of TS was observed in tumor tissues compared with 50% inhibition in histologically normal tissue. Also, patients whose tumors were responsive to 5-FU had greater inhibition of TS (46) . This is consistent with our data.
Several studies have hypothesized that antisense oligonucleotide down-regulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS. Indeed, these studies have demonstrated that TS mRNA levels are reduced, and growth of human colon and cervical cancer RKO, HT29, and HeLa cells may be inhibited after treatment with antisense oligoribonucleotides or ODNs (47, 48, 49) . ODN 85 also enhanced the cytotoxicity of raltitrexed, 5-fluoro-2'-deoxyuridine, 5-FU, and methotrexate (49) . These results suggest that the enhanced therapeutic efficacy suggested by the present study may be further exploited by the use of tumor-directed oligoribonucleotides or ODNs to enhance the selectivity of TS inhibitors by further increasing the difference in TS levels between tumor and normal cells after treatment with TS inhibitors.
In summary, these results confirm that up-regulation of TS protein occurs to a significant extent both in vitro and in vivo at pharmacologically active doses of ZD9331, which may be detrimental to response after treatment with TS inhibitors. Up-regulation occurs within 16 h of treatment with a range of TS inhibitors. However, significant decreases in protein levels are observed within 4 h of resuspension of W1L2 cells in DFM. Significantly greater up-regulation was apparent in normal compared with tumor cells both in vitro and in vivo, suggesting that up-regulation of TS protein may be beneficial in normal tissues by providing protection from the cytotoxic effects of TS inhibitors, leading to increased therapeutic efficacy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a studentship from the Institute of
Cancer Research and a grant from the Cancer Research Campaign (Program
Grant SP2330/0201). Tomudex is a trademark, property of Zeneca Limited
(part of AstraZeneca). 
2 To whom requests for reprints should be
addressed, at CRC Center for Cancer Therapeutics, Haddow Laboratories,
15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom. E-mail: wynne{at}icr.ac.uk
3 The abbreviations used are: TS,
thymidylate synthase; 5-FU, fluorouracil; MTA, LY231514; TK, thymidine
kinase; DFM, drug-free medium; ODN, oligodeoxynucleotide.
Received 12/17/99; revised 3/ 6/00; accepted 3/ 7/00.
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