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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Molecular Pharmacology [D. S. M., P. J. H., T. P. B.], Pharmaceutical Science [C. F. S., M. N. K.], and Biostatistics and Epidemiology [C. P.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, and Duke University Medical Center, Durham, North Carolina 27710 [H. S. F.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Temozolomide is believed to exert its toxic effects primarily by generating O6-methylguanine in DNA (4) . This adduct is subject to a single-step, error-free repair reaction that simply transfers the methyl group to a cysteine residue within the repair protein MGMT,3 thus restoring the DNA to its intact state. Hence, MGMT is a major determinant of temozolomide cytotoxicity (5 , 6) .
O6-Methylguanine in itself is not
deleterious to cells and does not inhibit DNA enzymatic processes such
as replication or transcription. However, the preferred base pairing
during DNA synthesis results in incorporation of thymine opposite
O6-methylguanine instead of cytosine
that results in a G:C to G:T transition mutation if not repaired. The
O6-methylG:T mismatch is recognized by
the MMR pathway of the cell (7)
, which proceeds to excise
the errant thymine residue in the daughter strand, however, unless the
O6-methylG is repaired before the
resynthesis step in MMR, thymine is likely to be reinserted opposite
the lesion. It is believed that the ensuing repetitive cycle of futile
MMR results in generation of a chronic strand break condition that
elicits an apoptotic response (Fig. 1)
.
Cells treated with methylating agents such as temozolomide have indeed
been observed to die via apoptosis (8)
.
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In the present study, we have tested these predictions by both determining the responses to temozolomide treatment of human solid tumor xenografts growing in mice and assessing the levels of MGMT and the MMR proteins MLH-1 and MSH-2 in these tumors. We have also related tumor sensitivity in vitro and in vivo to p53 genotype/phenotype. Our results indicate that MGMT and MMR repair functions do in fact correlate with in vivo response. However, the relationship between p53 status and temozolomide sensitivity is less clear.
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MATERIALS AND METHODS |
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Tumor Response and Tumor Failure Time.
For individual tumors, PR was defined as a volume regression >
50% but with measurable tumor (
0.10 cm3) at
all times. CR was defined as a disappearance of measurable tumor mass
(<0.10 cm3) at some point within 12 weeks after
initiation of therapy. MCR was defined as CR without
tumor regrowth within a 12-week study time frame. This time point was
chosen because most studies lasted 12 weeks. Because tumors were
implanted in both dorsal flanks of each mouse, a mouse is said to
achieve a CR only if tumors on both flanks have CRs and a PR only if
the tumor of at least one flank has a PR and the tumor response on the
other flank is not worse than a PR. If an initial tumor volume was less
than 0.20 cm3, data on that tumor were excluded.
Tumor failure time was defined as the time (in weeks) required by individual tumors to quadruple their volume from the initiation of therapy. Tumor failure times were censored if a mouse died prior to week 12 and before a tumor grew to four times its initial volume. Because tumors were implanted in both lateral flanks, the tumor failure times from each mouse are clustered observations. Evidence of high correlation between failure times has been reported previously (17) . Because the individual mouse is the unit of the experiment, the response of each mouse was taken to be the lesser of the two tumor failure times. This approach implicitly accounts for the clustering effect due to the mouse without explicitly specifying the correlation structure.
Statistical Methods.
For comparisons of time to tumor failure for different treatment
regimens, Kaplan-Meier estimates of failure distributions were
obtained, and survival distributions of each treatment group were
compared to the survival distribution of the control group using the
exact log-rank test. Experiment-wise, significance level was maintained
at 0.05 by using the Bonferroni procedure (18)
to adjust
for the multiplicity of tests of significance within each tumor line.
SAS 6.12 and StatXact-4 were used for statistical analysis.
Definition of Tumor Sensitivity.
Tumors have been classified as demonstrating high, intermediate, or low
sensitivity based on the following response criteria. In the high
sensitivity group, all tumors regressed completely (CR) with no tumor
regrowths during the period of experimentation (mice were euthanized at
week 12 after start of treatment); intermediate sensitivity indicated
that all tumors demonstrated 50% regression (PR) but CRs were not
maintained at week 12; and low sensitivity indicated that treatment
groups had tumors that demonstrated <PR or progressive tumor growth
during treatment. To estimate the response nadir (i.e.,
maximum regression) and duration of regression (i.e., tumor
volume recovered to that at the start of treatment), mean relative
tumor volumes for groups of tumors within a treatment group were
calculated as relative tumor volume = volume at day x
after treatment/volume at initiation of treatment over the 12 week
period between starting treatment and terminating the experiments.
Drug Formulation and Administration.
Temozolomide, generously provided by Schering-Plow Research Institute,
was dissolved in sterile water and administered by oral gavage.
Efficacy was determined after administration for 5 consecutive days
repeated every 21 days for three cycles. The highest daily dose
tolerated for three cycles of therapy was 66 mg/kg on this schedule.
Pharmacokinetics.
Following a single oral dose of temozolomide (66 mg/kg), blood samples
were collected from mice (three animals per point) at 0, 0.25, 0.5, 1,
1.5, 2, 3, and 6 h. All samples were immediately centrifuged at
5.5 g for 2 min in a tabletop refrigerated centrifuge at 4°C.
Serum was then divided into aliquots for processing to assay either
temozolomide or MTIC. Temozolomide serum samples (800 µl) were
treated with 80 ml of 1.0 N HCl, and 100 µl of this
acidified plasma was further diluted with 400 ml of 0.1 N
HCl. Ethazolastone was added as an internal standard prior to
extraction with ethyl acetate. The organic phase was separated,
isolated and dried under a stream of nitrogen. The dried pellet was
resuspended in 800 ml of mobile phase, and 100 µl were injected onto
an isocratic high-performance liquid chromatograph using UV
detection (19
, 20)
. Plasma samples for MTIC (400 µl)
were treated with 800 ml of cold methanol, stored on ice for 5 min,
vortexed, and centrifuged at 5.5 x g for 2 min. An
aliquot (50 µl) was combined with 60 µl of mobile phase consisting
of methanol:50 mM ammonium phosphate, pH 6.5
[20:80 (v/v)], and analyzed by high-performance liquid chromatography
(21)
. These methods were determined to be precise
(intraday and interday CV, 3.1 and 4.9% for temozolomide and
5.1 and 7.6% for MTIC, respectively) at temozolomide concentrations
ranging from 50 to 1000 mg/ml and MTIC concentrations ranging from 0.2
mg/ml to 6 mg/ml
Temozolomide and MTIC plasma concentration-time data were analyzed using noncompartmental methods. The apparent time of maximum concentration (tmax) and maximum plasma concentration (Cmax) were noted. The AUC for temozolomide and MTIC was calculated using the logarithmic trapezoidal method (22) . The terminal elimination rate constant (ß) was determined by log-linear least-squares regression of the plasma concentration time points in the terminal phase of the plasma disposition curve. This value was used to extrapolate the area from the last measured concentration to infinity.
Cell Lines and Culture.
Human leukemic lymphoblasts (CCRF-CEM), a suspension line, were
a gift of A. Fridland (St. Jude Children’s Research Hospital,
Memphis TN). Human colon carcinoma (HCT 116) and human colon
adenocarcinoma (LoVo) cell lines were obtained from ATCC (Manassas,
VA). These cell lines served as controls for calibration of
immunosignals: CCRF-CEM for MGMT, HCT 116 for MSH-2 but not
for MLH-1, and LoVo for MLH-1 but not for MSH-2. CCRF-CEM cells
were grown in Eagle’s MEM containing 10% newborn calf serum. HCT 116
were grown in McCoy’s 5a medium containing 10% fetal bovine serum,
and LoVo cells were grown in Ham’s F-12 medium containing 20% fetal
bovine serum.
Extract Preparation.
Xenograft tissue was frozen in liquid nitrogen immediately after
excision and ground under liquid nitrogen with mortar and pestle. The
ground tissue was suspended in 3 volumes of extraction buffer (50
mM Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM EDTA, 1
mM DTT, containing protease inhibitors PMSF, aprotinin,
benzamidine, leupeptin, and pepstatin; Sigma Chemical Co., St. Louis,
MO) and homogenized three times for 10 s with a Brinkman Polytron
homogenizer. The homogenate was sonicated three times for 10 s and
centrifuged 30 min in a Beckman type 50 Ti rotor at 100,000 x
g.
CCRF-CEM, HCT 116, and LoVo cell lines were each harvested, resuspended in 2 volumes of extraction buffer, and disrupted by three cycles of freeze thawing in liquid N2. Cell lysates were then centrifuged 14,000 rpm for 10 min in an Eppendorf microcentrifuge, and the supernatant was assayed for protein. All protein concentrations were determined according to the method of Bradford (23) .
Immunoblot Analysis.
Protein extracts (50 µg) were separated by electrophoresis on Bio-Rad
SDS denaturing Ready gels, 12% gels for MGMT analysis, and 7.5% gels
for MLH-1 and MSH-2 analysis and then electroblotted to polyvinylidene
difluoride membranes (Millipore, Bedford, MA) using 140 mA for 90 min
in transfer buffer containing 25 mM Tris, 190
mM glycine, 15% methanol. Membranes were blocked with 5%
nonfat dry milk in TBS-T (0.8% NaCl, 20 mM Tris-HCl, pH
7.6, and 0.1% Tween 20) and then allowed to air dry. Monoclonal
antibody MT 3.1 (Chemicon, Temecula, CA) was biotinylated before use as
primary antibody to probe the 12% gel blots for MGMT, followed by
Streptavidin-biotinylated HRP complex (Amersham Pharmacia
Biotech, Buckinghamshire England). Polyclonal antibodies MLH-1
(Ab-2) and MSH-2 (Ab-3; Calbiochem, La Jolla, CA) were used as primary
antibodies to probe the 7.5% gel blots for MMR proteins, followed by
antirabbit HRP-linked secondary antibody (from donkey; Amersham
Pharmacia Biotech). ß-tubulin was probed as a loading control using
primary monoclonal antibody TUB 2.1 (Sigma) followed by antimouse HRP
linked antibody (from sheep; Amersham Pharmacia Biotech). The enhanced
chemiluminescence (ECL-Plus) system (Amersham Pharmacia Biotech) was
used to develop the HRP signals on the membrane followed by brief
exposure to X-ray film (Kodak X-Omat AR: Eastman Kodak Co., Rochester,
NY). ECL generated signals were quantitated following densitometric
scanning of fluorograms.
p53-Isogenic Cell Lines.
NB1643 cells were transfected using calcium phosphate precipitation
with either a control pcDNA3 plasmid or a pcDNA3 construct containing
p53TDN. p53TDN is mutated at positions 14, 19,
and 281 and exhibits a trans-dominant negative phenotype
without the "gain of function" phenotype associated with some p53
mutants (24)
. Cells were selected using G418, and colonies
were isolated and expanded. The colonies were then screened for
overexpression of p53 and failure of 
-radiation (10 Gy,
137Cs source) to induce a
p21cip1 response (see below). Two clones
(p53TDN-1 and p53TDN-6)
were used in the experiments reported. Clones were grown in RPMI 1640
containing 2 mM glutamine supplemented with 10%
fetal bovine serum (Life Technologies) and 50 u/ml penicillin/50
µg/ml streptomycin (Life Technologies) at 37°C with 5%
CO2. For growth assays, cells were trypsinized in
trypsin versene mixture (BioWhittaker) and plated at 2.5 x
105 cells per 35-mm well in six-well plates
(Costar, Corning NY). Triplicate wells of cells were treated with
various concentrations of temozolomide. After 7 days, cells were
harvested by trypsinization, and cell number was quantitated by nuclei
counting. Data were plotted using Prism (GraphPad). Data are presented
as mean ± SE.
p21cip1 Induction in NB1643 Cells.
NB1643pcDNA, p53TDN-1, or
p53TDN-6 were plated at either 2 or 4 x
106 cells/10-cm dish (Costar) and cultured for
either 2 or 1 days, respectively, or at 1 x
106/6-cm dish and cultured for 2 days. Cells were
then treated with radiation (137Cs source),
temozolomide, or doxorubicin at the doses indicated in the figures.
Cells were then lysed 24 h later in 400 µl (10-cm dishes) or 100
µl (6-cm dishes) of SDS-PAGE sample buffer preheated to 100°C.
Lysates were repipetted four times through a 25 G needle. Forty µl of
each sample were loaded for resolution using SDS-PAGE. The gels were
transferred to Immobilon-P (Millipore) using the Trans-Blot
electrophoretic transfer cell (Bio-Rad) containing
Tris/glycine/methanol transfer buffer [25 mM
Tris, 192 mM glycine, pH 8.3, and 20% (v/v)
methanol] under a constant voltage of 100 V for 1 h. Membranes
were blocked for 1 h in 5% milk in TBST (10
mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.05% Tween-20) at 22°C. Blots were washed once for 15 min and
twice for 5 min with TBST and then incubated with
anti-p21cip1 antibodies (Santa Cruz) in 1%
milk/1% BSA in TBST for 1 h. The membranes were then washed as
above and incubated with antirabbit or antimouse horseradish peroxidase
in 1% milk/1% BSA in TBST for 1 h. The membranes were then
washed for 15 min and then four times more for 5 min in TBST. Protein
bands were then visualized using chemiluminescent detection with the
ECL (Amersham Pharmacia Biotech) and XAR 5 film (Kodak).
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RESULTS |
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2
3
show the results of the exact log-rank tests used to compare
differences in the distributions of tumor failure times among
temozolomide treatment groups compared to control groups for
neuroblastoma, rhabdomyosarcoma, and brain tumors, respectively.
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2
3
, temozolomide induced CR that was maintained at
week 12 in all mice bearing NB-1382, NB-1771 neuroblastomas, Rh30 and
Rh30c rhabdomyosarcoma, and SJ-BT27 medulloblastoma. These tumors were
classified as having high sensitivity to treatment with this agent. At
least PRs were obtained against Rh28 and neuroblastomas NB-EB and
NB-1643. In one experiment, all D283 medulloblastomas demonstrated
PR, but in other experiments, although temozolomide significantly
inhibited growth, few PRs were obtained (Table 3)
. Approximately half
of all of the tumors examined were classified as having low sensitivity
to temozolomide. In these tumor lines there were relatively few PRs,
although significant tumor growth inhibition was observed for some
lines (notably NB-SD, Rh12, and GBM2; Tables 1
2
3
). The time for tumors
in a treatment group to regress to the group nadir, and duration of
regression is also presented for each tumor (Tables 1
2
3)
. Overall,
temozolomide induced objective regressions (>50% decrease in volume)
in 8 of 17 tumor lines examined at the MTD on this daily times 5
schedule.
Pharmacokinetics.
To determine whether temozolomide plasma systemic exposure was relevant
to responses of human cancer at the dose level administered, we
determined the plasma levels of temozolomide and MTIC. Temozolomide and
MTIC concentrations exceeded the limit of assay sensitivity for the
duration of the study. Following oral administration (66 mg/kg), the
apparent tmax was 15 min for
temozolomide and 30 min for MTIC. The
Cmax values for temozolomide and MTIC
were 20 and 0.8 mg/ml respectively. The plasma
AUC0
for temozolomide and MTIC
were 40 and 1.9 mg/l-h, respectively.
DNA Repair Phenotypes.
Tumor sensitivity to temozolomide administered at the MTD on the daily
times 5 schedule is related to the tumor phenotype in Table 4
. The primary repair mechanism for
resistance to temozolomide is MGMT. We therefore determined the MGMT
levels in (untreated) tumor extracts from the xenograft lines that had
been characterized for temozolomide response. Immunoblots shown in Fig. 2
indicate that MGMT varies from
relatively high levels to complete suppression. The amounts of MGMT
protein were quantitated by densitometry relative to CEM cells and
ranked as described in Table 4
. How MGMT protein levels relate to the
tumor responses to temozolomide is shown in Table 4
. Generally, the
most responsive tumors (four of five) are totally deficient or have
very low MGMT. In contrast, among the most resistant tumors, five of
nine had high levels of MGMT. Responses of two rhabdomyosarcoma lines
that differ primarily in their MGMT levels (their MMR proteins being
similar) are shown in Fig. 3, A, B, C, and D
.
Temozolomide, even at the highest tolerated dose level (66 mg/kg),
failed to cause appreciable regressions in Rh12 tumors with high level
MGMT, whereas MGMT-deficient Rh30c tumors treated with only 28 mg/kg
temozolomide regressed completely with no regrowth.
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, and the
values relative to control cells LoVo (MLH-1) and HCT116 (MSH-2) are
indicated in Table 4
. Except for SJ-BT27 and Rh30, which have low MLH-1
levels, the MMR proteins appear to be present in significant amounts in
all of the sensitive tumors, as would be expected.
Among nine resistant (and three intermediate sensitivity) tumors, four
are completely deficient and two have only marginal levels of at least
one of the MMR proteins. Most significantly, those tumors in this group
that are deficient in MGMT (Rh28/Rh28c and GBM2) and that, based on
this criterion alone, we would predict to be drug sensitive, are also
compromised by impaired MMR. Responses of rhabdomyosarcomas Rh30c and
Rh28c, which differ notably in their levels of MLH-1, are shown in Fig. 3, A, B, E and F
. Both lack
MGMT; however, MLH-1-competent Rh30c tumors were highly sensitive to
temozolomide at a dose of 28 mg/kg, whereas MLH-1 deficient Rh28c
xenografts were highly refractory to treatment at the MTD of 66 mg/kg.
Role of p53 in Temozolomide Sensitivity.
Temozolomide-induced apoptosis may be dependent or independent of p53
function (12)
, although p53 may sensitize cells to this
agent (8)
. Because there is a report that temozolomide may
induce p53 protein (13)
, we tested the role that p53 has
in temozolomide sensitivity using paired neuroblastoma cell lines
differing in functional p53 status. NB1643 cells were engineered to
overexpress a TDN p53 protein. This TDN p53 protein, which has
mutations at positions 14, 19, and 281, does not exhibit the gain of
function phenotype that is associated with some mutations in p53.
-Radiation induces p21cip1 expression in
NB1643pcDNA3, whereas -radiation does not induce
p21cip1 expression in either NB1643p53
TDN-1 or p53 TDN-6 (Fig. 4A
). We conclude that
NB1643pcDNA retains wild type functional p53, whereas the resulting
cell clones, NB1643p53TDN-1 and
NB1643p53TDN-6, do not have functional p53
status. Growth inhibition assays were performed with temozolomide using
these paired cell lines; temozolomide has similar activity against both
the parental line with functional p53 and
NB1643p53TDN-1 (Fig. 4B
).
Unexpectedly, one of the nonfunctional p53 lines,
NB1643p53TDN-6, is more sensitive to
temozolomide. Further characterization showed that MGMT was
undetectable in NB1643p53TDN-6, whereas both the
NB1643p53pcDNA3 and NB1643p53TDN-1 clones
expressed MGMT, albeit at very low levels (Table 5)
. Both of the MMR proteins were present
at appreciable levels in all three clones. Therefore, it seems likely
that the differing drug sensitivities of these lines do not involve
functional p53 but rather other proteins involved in determining drug
sensitivity to temozolomide, such as MGMT. We next tested whether
temozolomide induces p21cip1 in these paired
neuroblastoma clones. In contrast to -radiation (5 Gy), temozolomide
did not induce p21cip1 in either parental or
p53TDN-1 cells 4 h after exposure (Fig. 4C
). To determine whether there was a delayed response to
temozolomide (that required at least one cell cycle time), NB1643pcDNA3
cells were exposed to temozolomide or the topoisomerase II poison
doxorubicin for up to 72 h (Fig. 4D
). Doxorubicin
induces p21cip1 in the vector control cells.
Exposure for up to 72 h to temozolomide (100–200
µM) did not induce
p21cip1 in NB1643pcDNA3 cells.
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DISCUSSION |
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The results of our pharmacokinetic studies show that the temozolomide AUC was similar to that reported by other investigators in both rodents and patients. Gallo and colleagues (25) administered temozolomide intraarterially at a dose of 40 mg/kg in the glioma-bearing rat model and reported a temozolomide AUC of 17.3 mg/l-h. In addition, Stevens et al. (26) administered temozolomide p.o. to mice (20 mg/kg), and based upon interpolation of data presented in a figure, the temozolomide AUC was approximately 38 mg/l-h. Data from patients receiving temozolomide 100 mg/m2 showed an AUC of 16 mg/l-h (27) , which is consistent with the results presented in the present study. Moreover, Reid et al. (21) administered a dose of 200 mg/m2 and reported a temozolomide AUC of approximately 30 mg/l-h. However, the MTIC values reported by Baker et al. (27) and Reid et al. (Ref. 21 ; 0.628 and 0.636 mg/l-h) are somewhat lower than levels observed in our xenograft model.
Sensitivity of each tumor to temozolomide was next compared to the
expression of MGMT and MMR proteins MSH-2 and MLH-1. Each tumor
classified as highly sensitive or of intermediate sensitivity has low
levels of MGMT. Of note is that MGMT could not be detected by
immunoblot analysis in one neuroblastomas (NB-1643), two
rhabdomyosarcomas and their sublines (Rh28, Rh28c, and Rh30, Rh30c),
one medulloblastoma (SJ-BT27), and the glioma (SJ-GBM2). Each of the
tumors having high or intermediate sensitivity demonstrated detectable
MSH-2 and MLH-1 proteins. Tumors classified as having low sensitivity
appear to fall into two groups: those with high MGMT and detectable
expression of MSH-2 and MLH-1 and those with a wide range of MGMT
(Rh12, Rh66, D283, and SJ-BT29) but with undetectable or
marginal MLH-1 (Rh18, Rh28c, NB-SD, NB-1691, and SJ-GBM2). The
contribution of MGMT to intrinsic resistance is best judged from
comparison of responses in sensitive tumors, such as Rh30c (Fig. 3, A and B
), and NB-1643 versus
insensitive tumors, such as Rh12 (Fig. 3, C
and
D) and Rh66, that all have similar expression of MLH-1 and
MSH-2 but differ only in expression of MGMT. Similarly, MGMT status
appears to be the major difference between two medulloblastomas,
SJ-BT27 and SJ-BT29. The importance of MMR can be estimated by
comparison of sensitive tumors that are MGMT negative but MMR competent
(Rh30c, NB-1643, and NB-EB) with tumors that are MGMT negative but also
MLH-1 negative [Rh28c (Fig. 3, E and F
) and
SJ-GBM2]. These tumors responded poorly to temozolomide, although
treatment caused significant growth inhibition. Our results
suggest that high levels of MGMT predict intrinsic resistance to
temozolomide in agreement with other reports. However, for tumors with
low or undetectable MGMT, MMR-status is an important determinant of
response. These results are in general consistent with the mechanisms
shown in Fig. 1
. However it should be pointed out that other factors,
such as methylation adducts other than
O6-methylguanine in DNA, and other
cellular resistance mechanisms or extracellular factors, such as drug
distribution, may also play a role in the responses to temozolomide
therapy.
The importance of p53 function is less readily defined from these studies. Temozolomide did not induce p53 or p21cip1 expression in NB1643 cells at a concentration of drug that inhibited growth by >90%. In vitro, suppression of p53 function in NB-1643 clones, engineered to express a trans-dominant p53, did not lead to temozolomide resistance. Rather, one clone was more sensitive possibly due to decreased MGMT, and one clone that was slightly more resistant, expressed slightly higher MGMT than the vector control clone. It should be noted that MGMT levels in all of these clones were extremely low in comparison to CCRF-CEM leukemic cells used as the standard for MGMT expression. Although these NB-1643 clones are derived from a common parental cell line, a loss of p53 may facilitate rapid accumulation of other changes. Hence, it is quite possible that the differences in temozolomide sensitivity of these clones are due to damage or resistance factors unrelated to MGMT or MMR.
The notion that p53 is required for MMR-induced apoptosis is not supported by data from the NB1643 model. Although p53 may be induced by MMR processing of temozolomide damage, there probably are other redundant pathways for apoptosis.
In summary, at therapeutically relevant systemic exposures, temozolomide demonstrates significant activity against a panel of childhood solid tumors heterografted in immune-deprived mice. The dose level used achieves plasma levels of MTIC consistent with plasma systemic exposures that are reported in children using 200 mg/m2. These results suggest temozolomide may have activity in childhood neoplasms other than brain tumors.
The major mechanism for temozolomide resistance in vivo appears to be MGMT; however, potentially responsive MGMT-deficient tumors may escape by virtue of defective MMR. Consequently, based on the data presented, it should be possible to predict therapeutic outcome and guide the clinical use of this drug in combination with an MGMT inhibitor such as O6-benzylguanine. However, further studies to determine MMR and MGMT status of patient tumors will be necessary to validate the predictions based on the xenografts used in this study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by United States Public
Health Service awards CA71628, CA23099, CA14799, and CA21765 (Cancer
Center Support Grant) from the National Cancer Institute and by
American, Lebanese, Syrian Associated Charities. 
2 To whom requests for reprints should be
addressed, at Molecular Pharmacology, St. Jude Children’s Research
Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794. Phone:
(901) 495-3440; Fax: (901) 521-1668; E-mail: david.middlemas{at}stjude.org
3 The abbreviations used are: MGMT,
O6-methylguanine-DNA methyltransferase; CR,
complete response; PR, partial response; MMR mismatch repair; TDN,
trans-dominant negative; MCR, maintained complete
response; MTD, maximum tolerated dose; AUC, area under the
concentration-time curve; HRP, horseradish peroxidase.
Received 10/ 7/99; revised 12/13/99; accepted 12/13/99.
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L. M. Wagner, R. E. McLendon, K. J. Yoon, B. D. Weiss, C. A. Billups, and M. K. Danks Targeting Methylguanine-DNA Methyltransferase in the Treatment of Neuroblastoma Clin. Cancer Res., September 15, 2007; 13(18): 5418 - 5425. [Abstract] [Full Text] [PDF]
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I. F. Pollack, R. L. Hamilton, R. W. Sobol, J. Burnham, A. J. Yates, E. J. Holmes, T. Zhou, and J. L. Finlay O6-Methylguanine-DNA Methyltransferase Expression Strongly Correlates With Outcome in Childhood Malignant Gliomas: Results From the CCG-945 Cohort J. Clin. Oncol., July 20, 2006; 24(21): 3431 - 3437. [Abstract] [Full Text] [PDF]
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K. E. Warren, A. A. Aikin, M. Libucha, B. C. Widemann, E. Fox, R. J. Packer, and F. M. Balis Phase I Study of O6-Benzylguanine and Temozolomide Administered Daily for 5 Days to Pediatric Patients With Solid Tumors J. Clin. Oncol., October 20, 2005; 23(30): 7646 - 7653. [Abstract] [Full Text] [PDF]
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R. C.A.M. van Waardenburg, L. A. de Jong, F. van Delft, M. A.J. van Eijndhoven, M. Bohlander, M.-A. Bjornsti, J. Brouwer, and J. H.M. Schellens Homologous recombination is a highly conserved determinant of the synergistic cytotoxicity between cisplatin and DNA topoisomerase I poisons Mol. Cancer Ther., April 1, 2004; 3(4): 393 - 402. [Abstract] [Full Text] [PDF]
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L. A. Hammond, J. R. Eckardt, J. G. Kuhn, S. L. Gerson, T. Johnson, L. Smith, R. L. Drengler, E. Campbell, G. R. Weiss, D. D. Von Hoff, et al. A Randomized Phase I and Pharmacological Trial of Sequences of 1,3-bis(2-Chloroethyl)-1-Nitrosourea and Temozolomide in Patients with Advanced Solid Neoplasms Clin. Cancer Res., March 1, 2004; 10(5): 1645 - 1656. [Abstract] [Full Text] [PDF]
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L. M. Wagner, K. R. Crews, L. C. Iacono, P. J. Houghton, C. E. Fuller, M. B. McCarville, R. E. Goldsby, K. Albritton, C. F. Stewart, and V. M. Santana Phase I Trial of Temozolomide and Protracted Irinotecan in Pediatric Patients with Refractory Solid Tumors Clin. Cancer Res., February 1, 2004; 10(3): 840 - 848. [Abstract] [Full Text] [PDF]
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L. Tentori, C. Leonetti, M. Scarsella, G. d'Amati, M. Vergati, I. Portarena, W. Xu, V. Kalish, G. Zupi, J. Zhang, et al. Systemic Administration of GPI 15427, a Novel Poly(ADP-Ribose) Polymerase-1 Inhibitor, Increases the Antitumor Activity of Temozolomide against Intracranial Melanoma, Glioma, Lymphoma Clin. Cancer Res., November 1, 2003; 9(14): 5370 - 5379. [Abstract] [Full Text] [PDF]
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L. S. Lashford, P. Thiesse, A. Jouvet, T. Jaspan, D. Couanet, P. D. Griffiths, F. Doz, J. Ironside, K. Robson, R. Hobson, et al. Temozolomide in Malignant Gliomas of Childhood: A United Kingdom Children's Cancer Study Group and French Society for Pediatric Oncology Intergroup Study J. Clin. Oncol., December 15, 2002; 20(24): 4684 - 4691. [Abstract] [Full Text] [PDF]
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P. J. Houghton, P. C. Adamson, S. Blaney, H. A. Fine, R. Gorlick, M. Haber, L. Helman, S. Hirschfeld, M. G. Hollingshead, M. A. Israel, et al. Testing of New Agents in Childhood Cancer Preclinical Models: Meeting Summary Clin. Cancer Res., December 1, 2002; 8(12): 3646 - 3657. [Abstract] [Full Text] [PDF]
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D. B. Bocangel, S. Finkelstein, S. C. Schold, K. K. Bhakat, S. Mitra, and D. M. Kokkinakis Multifaceted Resistance of Gliomas to Temozolomide Clin. Cancer Res., August 1, 2002; 8(8): 2725 - 2734. [Abstract] [Full Text] [PDF]
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M. S. Bobola, M. S. Berger, R. G. Ellenbogen, T. S. Roberts, J. R. Geyer, and J. R. Silber O6-Methylguanine-DNA Methyltransferase in Pediatric Primary Brain Tumors: Relation to Patient and Tumor Characteristics Clin. Cancer Res., March 1, 2001; 7(3): 613 - 619. [Abstract] [Full Text]
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B. A. Teicher, K. Menon, E. Alvarez, E. Galbreath, C. Shih, and M. Faul Antiangiogenic and Antitumor Effects of a Protein Kinase C{beta} Inhibitor in Human T98G Glioblastoma Multiforme Xenografts Clin. Cancer Res., March 1, 2001; 7(3): 634 - 640. [Abstract] [Full Text]
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