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Experimental Therapeutics |
Departments of Oncology [C. L. N., A. F. C., J. G. C.], Pathology [C. L. N., A. F. C., J. G. C.], and Clinical Neurological Sciences [J. G. C.], University of Western Ontario, and London Regional Cancer Centre [C. L. N., A. F. C., J. G. C.], London, Ontario, Canada N6A 4L6, and Huntsman Cancer Institute, Salt Lake City, Utah 84112 [M. N.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, p53, MDR, and
MT. Oligodendrocytes were more sensitive to BCNU than
either astrocytes or O-2A progenitors. The increased resistance of
astrocytes in comparison to oligodendrocytes was modulated, at least in
part, by both O6-benzylguanine (BG) and
DL-buthionine-(S,R)-sulfoximine, suggesting
a role for both MGMT and glutathione in the resistance of astrocytes to
BCNU. The sensitivity of O-2A progenitors to BCNU following BG
pretreatment is virtually indistinguishable from that of
oligodendrocytes depleted of MGMT, suggesting that the down-regulation
of MGMT is sufficient to account for the increased sensitivity of
oligodendrocyte lineage cells to BCNU as they differentiate. These
experiments provide support for the hypothesis that properties of glial
cells retained in gliomas may contribute to the differential
chemosensitivity of glial neoplasms. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are two very different possible explanations for the expression of chemosensitivity or chemoresistance in tumors. One possible explanation, and the one that currently takes precedence in analysis of this problem, is that these differences are attributable to alterations in gene expression patterns associated with the process of neoplastic transformation. According to this hypothesis, it would be expected that certain tumors acquire a chemoresistant profile as a result of aberrant gene expression that protects against cell death. Expression of such genes as bcl-2, multidrug resistance genes, certain members of the glutathione-S-transferase family, and an increasing number of other genes are associated with the expression of a chemoresistant phenotype (8, 9, 10) . As yet, there is no direct evidence that genetic alterations causing glial tumors are also chemosensitizing alterations, but recently Cairncross et al. (11) reported that allelic loss of chromosome 1p, an early molecular event in the genesis of oligodendrogliomas, is significantly associated with sensitivity to procarbazine/CCNU/vincristine chemotherapy.
Another possible explanation for the expression of chemosensitivity or chemoresistance in tumors is that these differences are reflective of the biology of the specific cells of origin. Such a possibility is of particular interest in respect to the differential response of astrocytomas and oligodendrogliomas to chemotherapy because of the observations that, at least in rats, tumors of the oligodendroglial lineage are preferentially induced after transplacental exposure to ethylnitrosourea (12 , 13) . This result is consistent with the possibility that the normal cells that have been thought to give rise to oligodendrogliomas may be especially vulnerable to the effects of this particular nitrosourea.
Sensitivity to nitrosoureas (BCNU, CCNU, and methyl-CCNU being the most common) is of particular interest in respect to glioma therapy, because these lipid-soluble drugs are capable of penetrating into the central nervous system for treatment of intracranial tumors. Of these, BCNU has been the most frequently studied chemotherapy agent in the treatment of glial tumors (14, 15, 16) . Many mechanisms of resistance have been implicated for BCNU. One of the best-studied mechanisms is the DNA repair protein MGMT (17, 18, 19, 20) . MGMT repairs DNA damage by removing alkyl groups from the O6 position of guanine, a critical site of alkylation by the nitrosoureas. In some cells, resistance to BCNU and other alkylating agents can be mediated by MGMT, and depletion of MGMT activity by BG has been shown to reverse resistance (21, 22, 23, 24, 25) .
A second mechanism of resistance to the nitrosoureas that has received significant attention is the detoxification pathway of glutathione and its associated GSTs. The tripeptide glutathione plays a role in detoxification by nonenzymatic conjugation of alkylating agents and other electrophilic molecules with its thiolate moiety (26) . This conjugation of drug with glutathione results in a more water soluble and less toxic product (27) . GSTs are a family of enzymes that catalyze the conjugation of these drugs, thereby enhancing the rate of detoxification. GST isoenzymes are broadly grouped into three classes (28) , and each isoenzyme class exhibits different but potentially overlapping substrate specificities. Glutathione and GST levels have been studied in human glioma tissues and cell lines, and support for a role of these pathways in resistance to alkylating agents has been provided (29, 30, 31) . In addition, modulation of glutathione has been demonstrated to alter resistance to a number of alkylating agents, including BCNU (29 , 30 , 32, 33, 34, 35) .
In the present study, we explored: (a) the expression of drug resistance genes in cultures of purified rat glial cells; (b) the sensitivity of rat astrocytes, O-2A progenitors, and oligodendrocytes to the bifunctional alkylating agent, BCNU; and (c) the effect of MGMT and glutathione modulation on resistance to BCNU in purified rat glial cells. We have found that there is an excellent correlation between the chemosensitivity of glial cells and the tumors to which they are thought to be related. In addition, the mechanisms that seem to be responsible for the differential response to BCNU also show an excellent correlation with mechanisms that are thought to play a role in the differing chemosensitivities of astrocytomas and oligodendrogliomas.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]dCTP and
[
-32P]dATP) were purchased from Amersham
Canada Ltd. (Oakville, Ontario, Canada). All molecular biology grade
chemicals were obtained from BDH, Inc. (Toronto, Ontario, Canada),
Fisher Scientific (Whitby, Ontario, Canada), Life Technologies, and
Sigma Chemical Co.
Isolation of Astrocyte Cultures.
Astrocyte cultures were isolated from newborn (1–3 days of age) rat
cerebrums using a method modified from McCarthy and De Vellis
(39)
. Cerebral hemispheres were isolated from
unanesthetized Sprague Dawley neonates and dissected free of meninges
and blood vessels. The tissue was minced and treated with 0.025%
trypsin for 30 min at 37°C. Trypsin inhibitor with 40 µg/ml DNase
was added and incubated for 5 min to stop the digestion of the tissue.
The tissue was centrifuged in a Beckman Model TJ-6 centrifuge (Beckman
Instruments, Inc., Fullerton, CA) for 
1 min at 1000 rpm, the pellet
was resuspended in 40 µg/ml DNase and triturated in a 10-ml pipette
to dissociate cells. The resultant single cell suspension was
centrifuged at 1000 rpm for 5 min, resuspended in DMEM supplemented
with 10% FBS and 50 units/ml penicillin/streptomycin, and seeded into
polylysine-coated, 80-cm2 flasks (2 x 107 cells/flask). To remove contaminating
cells, cultures were harvested after 48 h with 0.05% trypsin and
0.53 mM EDTA and replated. Cells were grown on coverslips
for various time intervals under the above-mentioned conditions before
being used for immunocytochemical analysis.
Isolation of Purified O-2A Progenitors and Oligodendrocytes.
Populations of purified O-2A progenitors and oligodendrocytes were
isolated from 7-day-old rat corpus callosa using an immunopanning
purification method modified from Mayer et al.
(40)
. Panning plates were prepared 1 day in advance by
incubating 10 ml of Tris buffer solution (50 mM,
pH 9.5) with 50 µg of goat antimouse IgG (Zymed Laboratories, Inc.,
San Francisco, CA) in a 10-cm Petri dish overnight at 4°C. The next
day, each dish was washed three times with PBS and incubated with
anti-RAN-2 hybridoma supernatant (1:4) in DMEM containing 0.8 mg/ml BSA
for 1 h at 37°C. The addition of BSA was required to block the
nonspecific adherence of cells to the panning plates. The dishes were
then washed three times with PBS, and PBS was left on the dishes until
they were required for panning. Twelve anti-RAN-2 plates were prepared
for each litter of 12–18 rats.
Corpus callosa were isolated from six to nine Sprague Dawley neonates, minced finely, and incubated in 750 µl of 667 units/ml solution of collagenase in L15 medium for 45 min at 37°C. The tissue was then centrifuged at 1000 rpm in a Beckman Model TJ-6 centrifuge for 5 min and resuspended in 3 ml of a 30 unit/ml papain solution in DMEM containing L-cysteine and 0.013% DNase. This suspension was incubated for 1 h at 37°C, centrifuged, and resuspended in 1 ml of papain inhibitor solution consisting of 2 mg/ml ovomucoid and 1 mg/ml BSA in L15 medium. The tissue was then triturated sequentially through 23- and 27-gauge needles to yield a single-cell suspension. Two of these preps were performed in parallel for each litter of 12–18 rats. The corpus callosa cell suspension was resuspended in B-S medium (41) as modified by Lillien et al. (42) . This defined medium consisted of 5.6 mg/ml glucose, 5 µg/ml insulin, 100 µg/ml transferrin, 100 µg/ml BSA, 0.06 ng/ml progesterone, 16 µg/ml putrescine, 40 ng/ml selenium, 40 ng/ml thyroxine, and 30 ng/ml tri-iodothyronine in DMEM.
To deplete astrocytes and meningeal cells, the suspension was divided evenly into six samples and incubated on anti-RAN-2 panning plates for 30 min at 37°C. This incubation also depletes microglia and macrophages that stick via their Fc receptors to the original IgG coating on the panning plate. The plates were agitated gently at 15 min to ensure access of the panning surface to all cells. The nonadherent cells were transferred to a second set of six anti-RAN-2 plates for an additional 30-min incubation. All cells remaining in the supernatant were seeded into polylysine-coated, 80-cm2 flasks in B-S medium containing 0.5% FBS and 10 ng/ml each of PDGF-AA and bFGF.
O-2A progenitor cells were grown in the presence of 10 ng/ml each of
PDGF-AA and bFGF to promote cell proliferation and prevent
differentiation. To obtain pure populations of oligodendrocytes, O-2A
progenitors were first grown to the required cell number. O-2A
progenitors were then allowed to undergo differentiation into
oligodendrocytes by removing bFGF from the medium. All medium was
removed from the flasks, and the progenitors were washed once with
citrate saline buffer (15 mM sodium citrate and 130
mM KCl). Fresh B-S medium containing 0.5% FBS and 10 ng/ml
PDGF-AA was added to the flasks, and the cells were allowed to
differentiate for 4 days. The state of differentiation was monitored
morphologically; oligodendrocytes displayed highly branched processes
in contrast to the bipolar morphology displayed by O-2A progenitors.
That such cultures consisted of >90% oligodendrocytes was confirmed
by staining with cell type-specific antibodies. Because the
oligodendrocytes began to undergo cell death within several days
after reaching terminal differentiation, all experiments were completed
within 1 week of the original change in morphology. In all cases, the
requisite growth factors were replenished every 24 h. Both cell
types were grown on coverslips for various time intervals under the
above-mentioned conditions before being used for immunocytochemical
analysis.
Immunocytochemistry.
Anti-GFAP antibody (rabbit IgG), A2B5 antibody (mouse IgM), and anti-GC
antibody (mouse IgG3) were used to ascertain the percentage of
astrocytes (GFAP-positive, A2B5-negative, and GC-negative), O-2A
progenitor cells (A2B5-positive, GC-negative, and GFAP-negative), and
oligodendrocytes (GC-positive, A2B5-negative, and GFAP-negative) in the
pure cultures using a triple staining protocol. All dilutions and
washes were carried out using Hank’s staining solution [HBSS without
sodium bicarbonate, 5% calf serum, 4.76 g/l HEPES
(Mr 238.3), 5.2 g/l HEPES
(Mr 260.3), and 0.05% sodium azide].
Briefly, coverslips containing cells were washed once by dipping the
coverslip in Hank’s staining solution, and cells were fixed with 4%
paraformaldehyde for 15–20 min. After all subsequent incubations, the
coverslips were washed by dipping in four changes of Hank’s staining
solution. A mixture of 1/2 GC-1/2 A2B5 hybridoma supernatants was added
for 20 min and washed off, and cells then were fixed in methanol for 15
min at -20°C to permeabilize the cell membranes. A mixture of
anti-GFAP (1:300), antimouse IgG3-fluorescein (1:100), and antimouse
IgM-rhodamine (1:100) was then added to the cells. Goat antirabbit
biotin (1:100) was added for 20 min, followed by
7-amino-4-methylcoumarin-3-acetic acid streptavidin (1:50) for 40 min.
After the final wash, coverslips were rinsed in distilled water,
mounted onto slides with a drop of antifade [2.5%
1,4-diazabicyclo-(2.2.2) octane in glycerol], and sealed with nail
polish. Cells were visualized with fluorescence optics on a Leitz
Diavert inverted microscope (Ernst Leitz Canada, Ltd., Midland,
Ontario, Canada), and culture purity was determined by counting the
percentage of positive cells.
Northern Blot Analysis.
Sequences for oligonucleotide probes are as follows: MGMT,
5'-tttcttacca gcaattagca gccctggcag gcaaccccaa agc-3'; GST µ,
5'-cggcacgaat ccgctcctcc tctgtctctc cacacaggtg-3'; and GST ,
5'-ggtaaccacc tcctccttcc agctctggcc ctggtcagcc-3'.
All cDNA inserts used for Northern hybridization were generously donated by other investigators. A 1.35-kb mouse p53 partial cDNA in pUC-18, designated pMO53, was a gift from Dr. Sam Benchimol (Ontario Cancer Institute, Toronto, Ontario, Canada). A derivative of pMO53, pECM53, has been described by Johnson et al. (43) . The hamster MDR cDNA in pUC-9, designated pEX/172 (44) , and the primers (A100 and B100) required to label the probe using a PCR protocol were kindly provided by Dr. Victor Ling (British Columbia Cancer Research Center, Vancouver, British Columbia, Canada). The MT cDNA was generously provided by Dr. Jim Koropatnick (London Regional Cancer Center, London, Ontario, Canada).
Total RNA was isolated from cultured cells using TRIzol reagent (Life Technologies, Inc., Burlington, Ontario, Canada), following the manufacturer’s instructions. Selection of mRNA was performed using Oligo(dT) Cellulose Columns (Life Technologies). As described (45) , samples and molecular weight markers (0.24–9.5 kb; Life Technologies) were electrophoresed at 50 mA for 3–4 h in 1.1% agarose gels containing formaldehyde; mRNA then was transferred to Gene Screen Plus membranes (DuPont Canada, Mississauga, Ontario, Canada) in 10x SSC buffer (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) by capillary action.
To block nonspecific binding, membranes were prehybridized in a VWR Scientific Model 2710 hybridization oven (Toronto, Ontario, Canada) at 42°C in a buffer containing 50% formamide, 10% dextran sulfate, 1% SDS, 1 M NaCl and 100 µg/ml herring sperm DNA. After 1–4 h, radiolabeled probe (106 cpm/ml) was added to the prehybridization solution and incubated with constant rotation for 16–20 h at 42°C. 5'-Labeling of oligonucleotide probes was carried out with T4 polynucleotide kinase, and labeling of cDNA probes was performed using the Life Technologies Random Primers DNA Labeling System, following the manufacturer’s instructions (Life Technologies). A PCR-based method was used to label the pEX/172 (MDR) cDNA probe (44) . Subsequently, probe-specific washing procedures were followed.
Membranes were exposed to a Storage Phosphor Screen (Molecular Dynamics, Inc., Sunnyvale, CA), screens were scanned by a Personal Densitometer SI Model 375A (Molecular Dynamics, Inc.), and images were quantitated using ImageQuant software (Molecular Dynamics, Inc.). All images were below saturation and corrected for background. Expression of standard control probes, ß-actin and glyceraldehyde-3-phosphate dehydrogenase, varied slightly with the degree of differentiation of oligodendrocyte lineage cells (data not shown); as such, quantified signal intensities for each probe were normalized to a poly-dT probe. Images were assembled into composites using CorelDraw software (Corel Corp., Ottawa, Ontario, Canada), and signal intensity data were analyzed using Microsoft Excel software (Microsoft Canada, Inc., Mississauga, Ontario, Canada).
Cytotoxicity Analysis.
BCNU was dissolved in absolute alcohol and diluted to 3.3 mg/ml with
sterile water. The stock solution was stored at -80°C, and
subsequent dilutions were made with culture medium at the time of cell
treatment. BG stock solution (100 mM) was made in
filter-sterilized DMSO and stored at -80°C. At the time of use, BG
was diluted to 1 mM in PBS, and subsequent dilutions were
made with culture medium. Twenty-five µM BSO was prepared
in medium at the time of treatment.
To test for cytotoxicity, 96-well plates were seeded at 104 cells/well and incubated at 37°C. Forty-eight h later, cells were treated and incubated for an additional 96 h, at which time cytotoxicity was measured using the MTT assay. BCNU treatments were performed for 2 h. On the basis of previous reports, pretreatment with modulators consisted of a 2-h incubation for BG (46) and a 24-h incubation for BSO (47 , 48) to modulate MGMT and glutathione, respectively.
The toxic effects of the above treatments were compared using the MTT assay as modified for chemosensitivity testing as described by Cole (49) . Ninety-six h after treatment, 100 µl of medium were removed, 25 µl of MTT solution (2 mg/ml in PBS) were added, and the plates were incubated for 2 h at 37°C. To solubilize formazan crystals, 0.04 M hydrochloric acid in isopropanol was added to each well and mixed thoroughly. Plates were kept at 37°C for 1 h, and cell viability was quantified by measuring light absorbance (570 nm) in a Bio-Rad Model 3550 automated microplate reader (Bio-Rad Laboratories, Richmond, CA). To control for differential growth rates, dose-response curves were normalized by expressing absorbance values relative to nontreated control values. Cytotoxicity assay results were analyzed using the two-tailed t test for independent means.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genes Associated with Chemoresistance Are Expressed at Different
Levels in Different Glial Cell Populations.
To explore the expression of drug resistance genes in cultures of
purified glia, RNA was isolated from astrocytes, O-2A progenitor cells,
and oligodendrocytes, poly(A) selected, and subjected to Northern blot
analysis. The Northern blots were probed for MGMT, GST
µ, GST , p53, MDR, and
MT transcripts (Fig. 1)
. Hybridization signals were normalized to a poly-dT probe (data not
shown). A differential pattern of mRNA expression was observed for each
of the six probes. Among differentiated glial cells (i.e.,
astrocytes and oligodendrocytes), expression levels of MGMT,
p53, and MDR mRNA were higher in astrocytes;
astrocytes displayed a 5-fold increase in both MGMT and
MDR expression over oligodendrocytes; and an approximate
2-fold increase was exhibited in p53 expression. In
comparison, expression levels of GST µ and
MT mRNAs were slightly higher in oligodendrocytes than in
astrocytes.
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, p53, and
MDR expression as compared with oligodendrocytes (Fig. 1)
.
Progenitors displayed an 5-fold increase in MGMT
expression over oligodendrocytes, GST and
p53 expression in progenitors was approximately twice that
of oligodendrocytes, and an 3.5-fold higher level of MDR
expression was displayed over oligodendrocytes. In contrast,
oligodendrocytes displayed a higher level of expression of GST
µ and MT mRNAs; the expression of GST
µ was approximately twice that seen in O-2A progenitor
cells, and MT mRNA levels were slightly higher in
oligodendrocytes than in the progenitor cells from which they were
derived. Two mRNA transcripts were detected for each of p53 and MDR. Although two transcripts have not been demonstrated previously in rat tissue, alternatively spliced forms of p53 have been documented in mouse tissues (50 , 51) . Two transcripts have been detected previously for MDR in rat and are known to correspond to the MDR1a and MDR1b genes (larger and smaller transcripts respectively; Ref. 52 ).
Oligodendrocytes Are More Sensitive to Killing by BCNU Than Are
O-2A Progenitor Cells or Astrocytes.
To explore the potential functional consequences of differential
expression of drug resistance genes in glial cells, we next examined
the effect of BCNU treatment on the viability of purified rat glial
cells. It had been observed previously that mixed cultures of rat cells
from the oligodendrocyte lineage were more sensitive to the
bifunctional alkylating agent BCNU than astrocytes (53)
.
To expand upon this finding, we determined the sensitivity of purified
rat astrocytes, O-2A progenitor cells, and oligodendrocytes to
treatment with BCNU. Cells were isolated from newborn rats and treated
with increasing concentrations of BCNU, and cell viability was
determined using the MTT assay. Oligodendrocytes were more sensitive to
BCNU than either astrocytes or O-2A progenitor cells (Fig. 2)
. Astrocytes appeared somewhat more sensitive to BCNU than O-2A
progenitors, but this difference did not reach statistical
significance.
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Sensitivity of Astrocytes to BCNU Can Be Induced by Depletion of
MGMT Activity or by Inhibition of Glutathione Synthesis.
There may be many reasons for resistance of astrocytes to BCNU. On the
basis of previous studies, however, levels of MGMT activity and levels
of glutathione were of particular interest. Rat astrocyte cultures have
been found previously to have 3-fold higher levels of MGMT activity
than mixed cultures of oligodendrocyte lineage cells (53)
.
Moreover, modulation of glutathione levels has been demonstrated to
alter resistance to a number of alkylating agents, including BCNU
(29
, 30
, 32, 33, 34, 35)
. In this regard, it is potentially
relevant that Thorburne and Juurlink (54)
demonstrated
that astrocytes had 3-fold higher glutathione concentrations than O-2A
progenitor cells.
Analysis of the effects of BG and BSO on BCNU sensitivity in astrocytes
indicates that depleting MGMT activity or inhibiting glutathione
production both increase sensitivity to this alkylating agent. Cells
were pretreated with 25 µM BG (to deplete MGMT activity)
for 2 h and exposed to increasing concentrations of BCNU, and cell
viability was determined using the MTT assay. As shown in Fig. 3A
, such treatment increased significantly the sensitivity of
astrocytes to BCNU. Similar results were obtained by treating
astrocytes with BSO to inhibit glutathione synthesis. As shown in Fig. 3B
, a 24-h pretreatment with 25 µM
BSO was also associated with a significant increase in sensitivity of
these cells to treatment with BCNU.
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. BG pretreatment also somewhat increased the sensitivity of
oligodendrocytes to BCNU, although to a lesser extent than was seen
with O-2A progenitor cells (Fig. 4A
, and 4B)
.
Interestingly, after negating the effects of MGMT on drug resistance by
pretreatment with BG, O-2A progenitors and oligodendrocytes had similar
sensitivities to BCNU (Fig. 4C)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The lineage origin of oligodendrogliomas has been a topic of interest
for many years and remains uncertain. Previous studies have suggested
that oligodendrogliomas may express some properties of O-2A lineage
cells (55)
. More recent evidence that tumors of this
classification express NG2 proteoglycan and the PDGF- receptor
(which are expressed by O-2A progenitor cells) lends support to the
view that, as implied by their name, oligodendrogliomas may indeed be
related to the oligodendrocyte lineage (56
, 57)
. Thus, it
is becoming clearer that consideration of the properties of cells of
the O-2A lineage may provide information of relevance to understanding
the properties of oligodendrogliomas.
A differential pattern of mRNA expression was observed for each of the
six drug resistance genes that we examined: MGMT, GST
µ, GST , p53,
MDR, and MT. Rat astrocytes expressed higher
levels of MGMT, p53, and MDR than
oligodendrocytes. The results for MGMT are consistent with
our previous observations of increased MGMT activity in rat astrocytes
relative to mixed cultures of oligodendrocyte lineage cells
(53)
.
One of the best-studied mechanisms of resistance to BCNU is MGMT (17, 18, 19, 20) . MGMT levels show a striking correlation with responsiveness to BCNU in patient populations (58) . In addition, it has been found that levels of MGMT in oligodendrogliomas and mixed oligo-astrocytomas are significantly lower than in astroglial tumors (59) . Because MGMT protein activity has been found to correlate with resistance to BCNU in cultures of mixed glial cells (53) , the effect of pretreatment with the modulator, BG, on the sensitivity of astrocytes to BCNU was determined. BG pretreatment significantly increased the sensitivity of astrocytes to BCNU, consistent with the hypothesis that this protein is important in conferring resistance to these cells. It was interesting to see, however, that astrocytes treated with BG nevertheless remained more resistant to BCNU than untreated oligodendrocytes. From these experiments, we conclude that MGMT contributes to the resistance of rat astrocytes to BCNU, but that other mechanisms of resistance also must be operative in these cells.
We suggest that a second component of BCNU resistance in astrocytes is
related to levels of glutathione in these cells, which have elsewhere
been shown to be higher than in O-2A progenitor cells
(54)
. Modulation of glutathione has been demonstrated to
alter resistance to a number of alkylating agents, including BCNU
(29
, 30
, 32, 33, 34, 35)
. For this reason, the effect of BSO
pretreatment on the sensitivity of astrocytes was examined. BSO, an
inhibitor of -glutamyl cysteine synthetase (the rate-limiting enzyme
in glutathione biosynthesis), has been shown to cause depletion of
cellular glutathione levels (30
, 32, 33, 34
, 48)
. BSO
pretreatment increased the sensitivity of astrocytes to BCNU,
suggesting that the glutathione-GST detoxification system, or some
other aspect of glutathione physiology, may also play a role in the
resistance of astrocytes to BCNU.
We also were surprised to find that sensitivity to BCNU treatment increases as O-2A progenitors differentiate into oligodendrocytes. Because MGMT levels were 5-fold higher in O-2A progenitor cells than in oligodendrocytes, we examined the effect of MGMT modulation on sensitivity to BCNU in purified cultures of O-2A progenitor cells and oligodendrocytes. BG pretreatment appeared to increase the sensitivity of both cell types to BCNU, although a greater increase in sensitivity was observed in the cultures of O-2A progenitors. These results suggested that MGMT contributes to BCNU resistance in cells of the oligodendrocyte lineage. Interestingly, the sensitivity of O-2A progenitors to BCNU after BG pretreatment is virtually indistinguishable from that of oligodendrocytes depleted of MGMT, suggesting that the down-regulation of MGMT is sufficient to account for the increased sensitivity of oligodendrocyte lineage cells to BCNU as they differentiate. If our general hypothesis that cellular lineage contributes to chemoresponsiveness is correct, then it will be of interest in future studies to determine whether the levels of MGMT in oligodendrogliomas or oligo-astrocytomas correlate with the extent to which maturation is precluded in these tumors.
In summary, we have demonstrated that rat glial cell subtypes exhibit differential expression of a number of drug resistance genes. In addition, it was determined that rat glial cell subtypes display a differential sensitivity to the alkylating agent BCNU. The increased resistance of astrocytes in comparison to oligodendrocytes was modulated, at least in part, by both BG and BSO, suggesting a role for both MGMT and glutathione in the resistance of astrocytes to BCNU. Thus, the sensitivity of normal rat glial cells to BCNU appeared to be dependent on more than one mechanism of drug resistance. Our experiments provide support for the hypothesis that the fundamental properties of glial cells may be retained in gliomas and may contribute to the differential chemosensitivity of glial neoplasms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant A178 from the London Regional
Cancer Centre Endowment Fund and Grant MOP37849 from the Medical
Research Council of Canada. C. L. N. received an Ontario Graduate
Scholarship for this work and is currently a Research Fellow of the
National Cancer Institute of Canada supported with funds provided by
the Terry Fox Run. 
2 Present address: Section of Neurobiology, Yale
University School of Medicine, New Haven, CT 06510.
3 To whom requests for reprints should be
addressed, at London Regional Cancer Centre, 790 Commissioners Road
East, London, Ontario, N6A 4L6 Canada.
4 The abbreviations used are: CCNU,
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; anti-RAN-2, anti-rat
neural antigen-2; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; bFGF,
basic fibroblast growth factor; BG,
O6-benzylguanine; B-S, Bottenstein-Sato;
BSO, DL-buthionine-(S,R)-sulfoximine; FBS,
fetal bovine serum; GC, galactocerebroside; GFAP, glial fibrillary
acidic protein; GST, glutathione-S-transferase; MDR,
multidrug resistance; MGMT,
O6-methylguanine-DNA methyltransferase; MT,
metallothionein; MTT,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; O-2A,
oligodendrocyte-type-2 astrocyte; PDGF, platelet-derived growth
factor.
Received 2/24/00. Accepted 7/ 5/00.
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REFERENCES |
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expression and subcellular localization in human gliomas. Clin. Cancer Res., 3: 2253-2261, 1997.This article has been cited by other articles:
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  J. P. Fruehauf, H. Brem, S. Brem, A. Sloan, G. Barger, W. Huang, and R. Parker In vitro Drug Response and Molecular Markers Associated with Drug Resistance in Malignant Gliomas Clin. Cancer Res., August 1, 2006; 12(15): 4523 - 4532. [Abstract] [Full Text] [PDF]
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N. D. Doolittle, L. E. Abrey, N. Ferrari, W. A. Hall, E. R. Laws, R. E. McLendon, L. L. Muldoon, D. Peereboom, D. R. Peterson, C. P. Reynolds, et al. Targeted Delivery in Primary and Metastatic Brain Tumors: Summary Report of the Seventh Annual Meeting of the Blood-Brain Barrier Disruption Consortium Clin. Cancer Res., June 1, 2002; 8(6): 1702 - 1709. [Abstract] [Full Text] [PDF]
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 C. Geroni, S. Marchini, P. Cozzi, E. Galliera, E. Ragg, T. Colombo, R. Battaglia, M. Howard, M. D'Incalci, and M. Broggini Brostallicin, a Novel Anticancer Agent Whose Activity Is Enhanced upon Binding to Glutathione Cancer Res., April 1, 2002; 62(8): 2332 - 2336. [Abstract] [Full Text] [PDF]
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, O-2A progenitors; 
,
oligodendrocytes. Bars, SE; asterisks,
level of statistical significance (* *, P < 0.005).
