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Vol. 3, 2459-2463, December 1997 Clinical Cancer Research 2459
06-Methylguanine.-DNA Methyltransferase Protein Levels in
Pediatric Brain Tumors1
Suradej Hongeng, Thomas P. Brent,
Robert A. Sanford, Hao Li, Larry E. Kun, and
Richard L. Heideman2Departments of Hematology-Oncology [S. H., R. L. H.], Molecular
Pharmacology [T. P. B.], Radiation Oncology [L. E. K.]. and
Biostatistics [H. L.], St. Jude Children’s Research Hospital, Memphis,
Tennessee 38105, and Neurosurgery Division, Department of Surgery.
LeBonheur Children’s Hospital, University of Tennessee, Memphis.Tennessee [R. A. S.]
ABSTRACT
Chloroethylnitrosoureas (CENUs) are commonly used
in the treatment of pediatric and adult central nervous
system (CNS) tumors. The antitumor activity of CENUs has
been hypothesized to be due to an alkylation occurring at the
06-position of guanine in DNA. The DNA repair protein
06-methylguanine-DNA methyltransferase (MGMT) is re-
sponsible for the repair of these potentially cytotoxic lesions
and may underlie tumor resistance to CENUs. The current
study is the largest report of MGMT levels among newly
diagnosed pediatric CNS tumors and the only study that has
quantitated MGMT by both biochemical and Western im-
munoblot assays. Our results show a good correlation be-
tween the two methods (r = 0.66). Medulloblastoma/primi-
tive neuroectodermal tumor and ependymoma had the
highest level of MGMT, followed by high-grade glioma and
low-grade glioma. These data may provide a guide to the use
of CENUs in the treatment of pediatric CNS tumors.
INTRODUCTIONCENUs,3 including lomustine (CCNU) and carmustine
[ 1 ,3-bis(2-chloroethyi)- 1 -nitrosourea], are bifunctional alkylat-
ing agents that are commonly used in the treatment of malignant
brain tumors ( 1). The often poor response of these tumors to
Received 4/30/97: revised 7/30/97: accepted 8/I 1/97.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
I Supported in part by Cancer Center Support CORE Grant PA3O; NIH
Grants CA 21765, CA 23099, and CA 14799; and the American Leb-anese Syrian Associated Charities.
2 To whom requests for reprints should be addressed. at Department ofHematology/Oncology, St. Jude Children’s Research Hospital, 332
North Lauderdale. Memphis, TN 38105. Phone: (901) 495-3604: Fax:(901) 495-3113.
3 The abbreviations used are: CENU, chloroethyinitrosourea: CCNU,
l-(2-chioroethyi)-3-cyclohexyl-l-nitrosourea; MGMT, 06-methylgua-
nine-DNA methyltransferase; CNS. central nervous system; LGG. low-
grade glioma; HGG, high-grade glioma; MBIPNET, medulloblastomai
primitive neuroectodermai tumor; EP, ependymoma: PFS, progression-
free survival; ECL, enhanced chemiiuminescence.
such therapy has been hypothesized to be the result of MGMT,
a DNA repair enzyme (2-4). MGMT can transfer mutagenic
and carcinogenic adducts from the 06 position of guanine, and
it incidentally repairs precursors of cytotoxic DNA cross-links
induced by the CENUs (5, 6). Although there have been several
studies outlining the presence and potential role of MGMT in
adult malignant gliomas (7, 8), there has been no systematic
investigation of MGMT among pediatric CNS tumors, except
for limited studies conducted in cell lines or as a small part of a
larger survey of adult and pediatric patients (9, 10). Unlike in
adults, malignant gliomas make up only a minority of pediatric
CNS tumors. More common are embryonal CNS tumors, such
as MBsIPNETs and ependymal CNS tumors, which together
constitute about 50% of childhood CNS tumors: these neo-
piasms are rare in adults ( 1 1 ). Here, we report the MGMT
content, as measured by both biochemical assay and Western
blot immunoassay, in a large group of newly diagnosed and
prospectively collected childhood CNS tumors. The data ob-
tamed not only provide a comparison of the standard biochem-
ical assay and Western blot immunoassay for MGMT but also
are potentially relevant to use of CENUs in the treatment of
pediatric brain tumors.
MATERIALS AND METHODS
Patient Population and Specimen Procurement
Between 1990 and 1995, tumor samples were obtained
from 60 patients with newly diagnosed CNS neoplasms at St.
Jude Children’s Research Hospital. Patients were between 0.1
and 19.3 years old (median, 5 years) at diagnosis; 33 were male,
and 27 were female. Samples were obtained at the time of each
patient’s initial definitive surgery and were kept on wet ice in
saline-soaked gauze before being snap frozen at -70#{176}C. gener-
ally within 2 h of collection. Patients were selected for tissue
procurement only by the availability of fresh tissue in sufficient
quantity to ensure both MGMT quantification and histological
interpretation. Samples were chosen as being representative of
the tumor and essentially free of normal brain or necrotic debris.
Cells and Tissues
CEM cells (CEM-CCRF line, originally obtained from A.
Fridland, St. Jude Children’s Research Hospital) were used as a
control; cells were grown in Eagle’s MEM supplemented with
10% newborn calf serum. Prior to preparation of extracts, cells
were centrifuged at low speed and washed twice with PBS, and
the pellet was quick-frozen in liquid nitrogen and stored at
-70#{176}C.
Extract Preparation
Frozen tumor samples were weighed before being ground
under liquid nitrogen with a mortar and pestle. The powdered
tissue was thawed by addition of 2-4 volumes of TEDN buffer
[I 0 msi Tris-HC1 (pH 7.5), with 0. 1 M NaCl, 2 mtvt EDTA. 1 mM
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2460 MGMT in Pediatric Brain Tumors
DTF, 0.02% sodium azide, 0.2 mrvi phenylmethylsulfonyl fluo-
ride, aprotinin (20 trypsin-inhibitor units/liter), and leupeptin
(20 jig/mi); Chemicon, Temecula, CA] and disrupted with three
1 5-s bursts of sonication followed by centrifugation at 98,000 X
g (30,000 rpm) in a Beckman Ty 65 rotor for 30 mm. The
supernatant was removed, quick-frozen and stored at -70#{176}C.
CEM cell pellets were thawed in 2 volumes of TEDN disrupted
by freeze-thawing three times. After high-speed centrifugation,
the supernatant was quick-frozen in liquid nitrogen and stored at
-70#{176}C.
Methods of Quantitation
Chemicals. Unless otherwise specified, chemicals were
of molecular biology grade and were purchased from Sigma
Chemical Co. (St. Louis, MO).
Total Protein Assay. Protein concentration in cell and
tissue extracts was determined by the Bradford method (12)
using Bio-Rad Protein Assay reagent (Bio-Rad Laboratories,
Richmond, CA). BSA was used as the standard.
MGMT Biochemical Assay. MGMT activity in cell and
tissue extracts was assayed using [3H]methylnitrosourea-treated
calf thymus DNA (13) as a substrate, as described previously
(14). Briefly, substrate DNA containing 0.1 pmoi of [3H]06-
methylguanine was incubated at 37#{176}Cfor 30 mm with the
sample in a final volume of 210 p.1 of TEDG buffer [50 mrvt
Tris-HC1 (pH 7.5), 2 mM EDTA, 1 m�i DTF, and 10% glycerol].
The reaction was stopped by addition of 3 ml of 5% trichioro-
acetic acid, and excess DNA was hydrolyzed by heating it at
80#{176}Cfor 30 mm. Radiolabeled protein was recovered by filtra-
tion through Whatman GF/F filters. Following two washes with
10 ml of 5% trichloroacetic acid and one with 5 ml of 95%
ethanol, filters were transferred to scintillation-counting vials,
before addition of NCS tissue solubilizer (Amersham Corp.) and
nonaqueous scintillation fluid for scintillation spectrometry.
MGMT activity was calculated as fmol of [3Hlmethyl trans-
ferred from DNA to protein. The levels in brain tumor samples
were calculated relative to MGMT in an equivalent amount of
extract protein from CEM cells, and after normalizing them to
MGMT level in CEM cells (- 1 fmoi/pg) levels are expressed
as fmoi MGMT/p.g extract protein.
MGMT Western Blot Immunoassays. Proteins were
separated by electrophoresis in a Bio-Rad (Richmond, CA)
minigel apparatus at 200 V for 45 mm on 0.75 mm 9% SDS-
PAGE slab gels according to the method of Laemmli (15) as
described previously (16). Gels were calibrated with Bio-Rad
low molecular weight standards. Proteins were transferred onto
polyvinylidene difluoride membranes (Immobiion-P; Millipore,
Bedford, MA) according to the method of Matsudaira (17) using
a Bio-Rad Mini-Trans-Blot cell for 2 h at 140 mA. Blots were
blocked with 5% BSA (Immunogoid quality; Amersham) and
probed with the anti-MGMT mouse monoclonal antibody MT
3.1 (16).
Primary antibody binding was visualized either with gold-
labeled secondary antimouse IgG antibody using Auroprobe and
IntenSE reagents (Amersham) or by enhanced luminescence
using horseradish peroxidase-labeled secondary antibody and
ECL reagents (Amersham), in each case following the manu-
facturer’s instructions. Protein bands in ECL-processed mem-
branes were visualized by exposure to X-ray film (Kodak X-
Omat AR, Eastman Kodak Co., Rochester, NY) for time
intervals ranging from a few seconds to 30 mm.The intensities of the silver-enhanced gold-stained bands
were quantitated by densitometry of bands in transparencies
prepared by direct positive photography, and by whole-band
analysis on a Bio-Image Visage I 10 analytical imaging instru-
ment (Millipore). Bands in ECL-generated X-ray film were
quantitated in the same way. Results obtained by the two West-
ern blot immunoassay procedures were essentially identical.
Each set of analyses for brain tumor samples was accom-
panied by a set ofCEM extracts (2-10 p.g ofprotein) containing
4-20 fmol of MGMT as calibration standards. Intensities of
brain tumor MGMT bands were calculated relative to the band
intensity of equivalent amounts of CEM extract protein, and
after normalizing to CEM cell MGMT levels (-1 fmol/p.g),
tumor MGMT levels were expressed as fmol MGMT/p.g extract
protein.
Statistical Methods
The association between the MGMT levels obtained with
each of the two assay methods was evaluated using the Spear-
man rank-order correlation coefficient.
The Kruskai-Wallis test was used to compare the distribu-
tion of MGMT levels obtained using the two assay methods
among the five tumor groups. Later, the pairwise Wilcoxon
rank-sum test was used to look for differences between the
tumor groups.
To compare the biochemical and Western immunobiot
assay levels of MGMT versus patient age, the Spearman rank-
order correlation coefficient was again used. A comparison of
MGMT levels versus overall and progression-free survival was
examined using univariate Cox regression models.
RESULTS
The histological distribution of brain tumor samples eval-
uated was nearly identical to that observed in the pediatric CNS
tumor population at large. LGGs (n = 25) accounted for 42%
of tumor samples, MBsIPNETs (n 14), for 23%, and HOGs
(n = 8) for 13%. EPs (n = 6) and anaplastic EPs (n = 2) were
combined into the single category of EP, which collectively
accounted for 13% of patient samples. The miscellaneous other
tumors included choroid plexus carcinoma (n = 2), craniopha-
ryngioma (n = 1), mixed germ cell tumor (n = 1), and menin-
gioma (n = 1 ). Among the medulioblastoma samples. six came
from patients with metastatic disease at diagnosis and eight from
patients with local tumor only.
The MGMT levels of essentially all samples were deter-
mined by both the biochemical assay (n = 58) and the Western
blot immunoassay (n = 60). The values obtained by the
two assay techniques were well correlated, as outlined in Fig. I
(r 0.66, and P 0.0001).
The rank order of median MGMT levels among the mdi-
vidual tumor groups was similar for both methods of quantita-
tion, and in both the range was large; the difference between the
lowest and highest value was 100-fold. The highest levels were
observed in MBs/PNETs and EPs, followed by the glial and
other tumors. LOGs had the lowest level (Table 1 , Fig. 2).
Among those patients in the MBIPNET group, there was no
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1.0
0.8
0.6
0.4
>‘
CO.-.
U
“ EpendJAE, ependymoma/anaplastic ependymoma.
Clinical Cancer Research 2461
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Biochemical Assay
(fmoV�sg protein)
Fig. I Regression plot showing the correlation between Western im-
munoblot assays and biochemical assays for MGMT (r = 0.66;
P = 0.0001).
significant relationship between MGMT level and the presence
or absence of metastatic disease (P = 0.35). There was no
significant relationship between age and MGMT levels in all
groups combined, whether the cutoff was 2 or 4 years of age at
diagnosis (P > 0.5).
Because of small numbers of patients in most tumor
groups, the correlation between the MGMT levels and PFS and
overall survival was examined only among all patients com-
bined and in the MB/PNET and LGG groups; no statistically
significant association with either PFS and overall survival was
present for any of these groups (P > 0.5). A potential limitation
to such an analysis is that none of these patients were treated
with CENUs or 06-guanine alkylators, such as procarbazine.
DISCUSSIONMGMT is ubiquitously present in both prokaryotic and
eukaryotic organisms, suggesting an important evolutionary role
related to protection against DNA damage from environmen-
tally and metabolically derived aikylating agents (5). Elevated
levels of this protein have been found in several human tumor
cell lines, such as colon cancer (1 8), lung cancer ( 1 8, 19),
ovarian cancer ( 19), leukemia (20), and brain tumors (9, 19, 21).
These findings suggest that MGMT may be potentially impor-
tant in determining resistance to CENUs and monofunctional
methylating agents, such as procarbazine or temozolamide,
which produce 06-guanine DNA adducts. Studies in CNS tumor
cell lines and xenografts, and clinical correlations in adult CNS
tumors, have suggested an inverse relationship between MGMT
levels and survival or response to CENUs (4, 7, 8, 22). Although
two previous reports have evaluated MGMT in small numbers
of pediatric CNS tumors, one measured activity only in tumor-
derived cell lines (9), and the other quantitated MGMT (fmol/
cell) in a fashion that does not allow direct comparison of their
results with this or other published studies (10). The current
report is the largest study of MGMT in pediatric CNS tumors
Table I MOM T (fmol4i.g protein) among different histological
types of pediatric brain tumors
Method Group N Median Range
Biochemical assay MBIPNET 14 0.333 0.095-0.524
Epend/AE” 7 0.237 0.044-0.512
HOG 8 0.181 0.035-0.387
LOG 24 0.117 0.011-0.321
Other S 0. 144 0.075-1.20
Total 58Western blot immunoassay MBIPNET
Epend/AE”HOG
LOG
OtherTotal
14
8
8
25
560
0.239
0. 1640.127
0.170
0.140
0.084-1.05
0.052-1.09
0.006-0.301
0.072-0.339
0.072-1.12
and the first to quantitate levels using separate and confirmatory
methods.
The quantitation of MGMT has relied typically on a bio-
chemical assay based on the transfer of the radiolabel from
methylated (methy!-3H) DNA to cellular extracts of the protein.
as described here in “Materials and Methods.” The recent avail-
ability of monoclonal antibodies to MGMT has allowed an
assay based on antibody probing and identification of MGMT in
Western blots of cellular protein (21. 23). Both methods of
quantitation were used in this study, thus allowing confirmation
of the results of one method with a separate determination by the
other, as well as providing a comparison of the fidelity and
facility of these two methods for measuring MGMT. As shown
in Fig. 1 and Table 1, the correlation between these methods is
high: there was no instance of a tumor negative for MGMT, and
both methods produced similar results. Our experience with
these methods suggests that the Western blot immunoassay is
more sensitive and requires less patient material: only 1/10 of
the tumor material needed for reliable quantitation by the bio-
chemical assay is needed for the Western blot immunoassay.
The Western blot immunoassay may thus be the method of
choice.
The level of MGMT has been reported to correlate in-
versely with response to CENU therapy. Hotta et a!. (7) showed
that in tumors with MGMT levels >0.2 fmol/pg protein. re-
sponses to CENU treatment were significantly fewer than
among patients whose tumors had lower levels of the protein.
Similar results have been reported by Belanich et a!. (8). Addi-
tionally, a significant relationship between MGMT levels and
survival after treatment with procarbazine has been noted in
human xenografts (24). Investigations by others have under-
scored the relationship between MGMT and tumor response by
showing that the depletion of the MGMT by pretreatment with
06-benzylguanine or streptozotocin markedly increases the cy-
totoxicity of CENUs in vitro as well as in xenografts (25-27).
Furthermore, retroviraily mediated transfer of either human or
bacterial MGMT genes into mammalian hematopoietic stem
cells has been shown to confer CENU resistance (28. 29).
Among the pediatric tumors evaluated in the current study,
median MGMT levels were highest in MBs/PNETs and EPs,
followed by the HGG and LOG groups. This relationship was
the same for both methods of MGMT quantitation. Statistically,
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1.0�
0.8�
C00
�. 0.6at
0
E 0.4�
0.2�
0.0�
MB/PNET EP LGG HOG
Tumor Group
2462 MGMT in Pediatric Brain Tumors
Fig. 2 MGMT levels in individual tumorgroups (Western immunoblot assay). Bracketsshow the range, boxes show the 25th and 75thpercentiles, and the thick horizonta! lines aremedians. MGMT levels outside these ranges
(one each in the MG/PNET and EP groups) areshown separately as thin horizontal lines nearthe top of the plot.
the range of levels within each of the histological groups over-
lapped broadly, and there was no significant difference among
any of the groups, with the exception of the comparison of
MB/PNET versus LGG (P 0.004) using the biochemical
method. A similar rank ordering of MGMT among a variety of
CNS tumors from both adults and pediatric patients has been
reported by Silber et a!. (10), using a different method of
quantitation.
Most prior information regarding the levels of MGMT in
brain tumors has been obtained in adult HGGs, in which levels
have generally been measured to be >0.2 fmol/�i.g protein (7,
21). The median level of MGMT among the pediatric HOGs in
the current study, 0.18 fmol/p.g protein, does not differ mark-
edly from that in adults. However, the median MGMT levels in
MBs/PNETs and EPs, 0.33 and 0.24 fmol/p�g protein by the
biochemical and Western immunoblot assays, respectively, ap-
pear to be higher than those reported in most adult HGGs; these
levels are within the range associated with CENU resistance
(>0.2 fmol/p�g protein). Relevant to MGMT elevation in these
tumors are clinical studies of CENU-containing combination
chemotherapy regimens in childhood MBsIPNETs (30) and EPs
(31), which showed no significant impact of such treatment on
survival. Likewise, the use of these regimens in HGG has
generally been associated with only marginal improvement in
outcome in pediatric patients (7, 8, 32, 33).
In a report on MGMT in five MB/PNET cell lines, He et a!.
(9) showed levels of 0.05-1.68 fmol/p.g protein using the bio-
chemical assay; three of the five had levels greater than 1.36
fmolfli.g protein. These levels are in contrast to the range of
0.1-0.5 fmol/p�g protein noted in the 14 MB/PNET samples
reported here. This suggests the possibility that the selective
pressure associated with cell line development may lead to
MGMT levels greater than would otherwise be seen in vitro and
affirms the necessity of evaluating the levels of this protein in
freshly obtained tissues rather than in cell lines.
The levels of MGMT we have observed in MBsIPNETs
question the recent enthusiasm for CENUs in the therapy of
these diseases. MGMT levels similar to those noted in MBs/
PNETs have been associated with clinical resistance to CENUs
in other CNS tumors. Although a recently reported adjuvant
chemotherapy regimen of CCNU, vincristine, and cisplatin has
been associated with a significant increase in PFS among pa-
tients with MBIPNET (34), the limited efficacy of single-agent
CCNU in this and other CNS tumors and the well-documented
efficacy for platinating agents raise a question regarding the
contribution of CCNU to the activity of this regimen. The
risk:benefit ratio of the CENUs must be weighed carefully; the
prominent hematological suppression and hematological stem
cell toxicity, late-onset pulmonary fibrosis, and second malig-
nant tumors associated with these agents suggest they should be
used with caution (35).The newly activated North American pediatric cooperative
group study in standard-risk MBIPNET may help to document
the clinical activity of CCNU, by comparing similar adjuvant
chemotherapy regimens differing only by the use of CCNU
versus cyclophosphamide. In the interim, studies correlating
MGMT levels with outcome in CENU-based trials or explora-
tion of the MGMT-modulating effects of agents such as
benzylguanine in MBsIPNETs and other tumors may be of
interest.
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