(Received for publication, April 7, 1995)
From the
Buthionine sulfoximine (BSO) is a synthetic amino acid that
irreversibly inhibits an enzyme, -glutamylcysteine synthetase
(
-GCS), which is a critical step in glutathione biosynthesis. We
isolated three BSO-resistant sublines, KB/BSO1, KB/BSO2, and KB/BSO3,
from human epidermoid cancer KB cells. These cell lines showed 10-to
13-fold higher resistance to BSO, respectively, and had collateral
sensitivity to cisplatin, ethacrynic acid, and alkylating agents such
as melphalan and nitrosourea. Cellular levels of glutathione S-transferase
(GST-
) and its mRNA in BSO-resistant
cell lines were less than 10% of the parental cells. Nuclear run-on
assay showed that the transcriptional activity of GST-
was
decreased in BSO-resistant cells, and transient transfection of
GST-
promoter-chloramphenicol acetyltransferase constructs
revealed that the sequences between -130 and -80 base pairs
of the 5`-flanking region were at least partially responsible for the
decreased expression of the GST-
gene. By contrast,
-GCS mRNA
levels were 3-to 5-fold higher in resistant cell lines than in KB
cells, and the
-GCS gene was found to be amplified in the
BSO-resistant cell lines. GST-
mRNA levels appeared to be
inversely correlated with
-GCS mRNA levels in BSO-resistant cells.
We further established the transfectants, KB/BSO3-
1 and
KB/BSO3-
2, that overexpressed GST-
, from KB/BSO3, after
introducing a GST-
expression plasmid. These two transfectants had
similar levels in
-GCS mRNA, drug sensitivity to alkylating
agents, and glutathione content as those of KB cells. These findings
suggest that the cellular levels of GST-
and
-GCS might be
co-regulated in these novel BSO-resistant cells.
Intracellular non-protein sulfhydryl glutathione (GSH) has
multiple functions in catalysis, transport, and reductive
phenomena(1, 2) . Moreover it reacts with toxic
endogenous and exogenous substances, including free radicals and
anticancer agents. These functions are important in drug resistance
during cancer chemotherapy with agents such as nitrogen mustards,
nitrosourea, cisplatin, and anthracyclines (3, 4, 5, 6, 7) . GSH is a
tripeptide of glycine, glutamic acid, and cysteine, which is
synthesized intracellulary by the action of two enzymes: -GCS (
)and glutathione synthetase.
-GCS is a rate-limiting
enzyme in the synthesis of GSH and is feedback-inhibited by
GSH(8, 9) .
Glutathione metabolism is often
correlated with cellular sensitivity to anticancer agents. Indeed,
glutathione is protective against drug
cytotoxicity(1, 9, 10, 11) .
Acquired resistance to alkylating agents is frequently accompanied by
an elevation in the cellular non-protein sulfhydryl content.
Melphalan-resistant leukemia cells have a 2- to 4-fold higher level of
GSH than the sensitive parental cells(12, 13) . A
relevant study by Ozols and his colleagues (3, 14, 15) has demonstrated that ovarian
cancer cell lines resistant to adriamycin, cisplatin, and various
alkylating agents such as nitrosourea and cyclophosphamide have
increased GSH levels. Furthermore, lowering GSH levels by treatment
with the -GCS inhibitor, BSO, can potentiate the activity of
melphalan, cisplatin, and other anticancer agents in vitro as
well as in
vivo(6, 10, 16, 17, 18, 19) .
In our laboratory, we demonstrated that Chinese hamster ovary (CHO)
cell lines resistant to cisplatin have increasing levels of GST-,
suggesting the involvement of GST in the acquisition of the
cisplatin-resistant phenotype(20) . The introduction of
GST-
cDNA makes the recipient CHO cells 1.4- to 3.0-fold more
resistant to cisplatin(21) . The multidrug-resistant variant of
MCF-7 breast cancer cells, which is resistant to adriamycin and other
anticancer agents, overexpresses GSH-dependent enzymes GST and
glutathione peroxidase(22) . The GST-
cDNA-transfected
MCF-7 cells are more resistant to benzo(a)pyrene and
ethacrynic acid, but not to melphalan and cisplatin(23) . A
study by Nakagawa et al.(24) has demonstrated that
GST-
cDNA transfectants of mouse NIH3T3 cells are more resistant
to adriamycin and ethacrynic acid, but not to melphalan and cisplatin.
These findings suggest that increased GST levels play a role in
determining the sensitivity to some drugs, including alkylating agents,
but that the selectivity of drugs appears to differ according to the
origin of the cell line. Godwin et al.(25) have
reported that high cisplatin resistance in human ovarian cancer cells
is associated with the enhanced expression of mRNAs for
-GCS,
again suggesting the involvement of glutathione synthesis in the
acquisition of drug resistance.
Glutathione function has been
studied extensively in relation to drug
metabolism(1, 9, 10) . However, it remains
unknown how GSH specifically detoxifies anticancer agents and how
cellular glutathione levels and GSH associated enzymes are selectively
modulated in cancer cells. In this study, we first isolated human
cancer cell lines resistant to the cytotoxic effects of BSO. GST-
gene expression was greatly abrogated, but that of
-GCS was
up-regulated in these BSO-resistant cell lines. The coordinate and
inverse co-regulation of GST-
and
-GCS genes is discussed in
relation to the acquisition of the novel BSO-resistant phenotype.
Figure 1:
Northern blot of -glutamylcysteine
synthetase (
-GCS) (A), glutathione peroxidase (GPX) (B),
-glutamyl transpeptidase (
-GTP) (C), MGMT (D) and GST-
(E) mRNAs, and immunoblot of GST-
(F). Total RNA
(15 µg) extracted from KB, KB/BSO1, KB/BSO2, and KB/BSO3 cells were
resolved by electrophoresis in a 1% agarose gel containing 2.2 M formaldehyde, transferred to Hybond N
, and
hybridized with various
P-labeled cDNA probes (B, C, D, and E). The equivalent loading of
total RNA is shown by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blot (A). For immunoblotting of GST-
,
100 µg of cytosolic fractions from each cell line were resolved on
12% SDS-polyacrylamide gel electrophoresis, transferred to a
nitrocellulose membrane, and then incubated with rabbit antibody
against human GST-
and biotinylated goat anti-rabbit IgG (F).
Figure 2:
Nuclear run-on analysis of GST-
and
-GCS. Transcription in the isolated nuclei was analyzed by
hybridization of
P-labeled transcript to 5 µg of
PUC-19 plasmid, GST-
,
-GCS, glutathione peroxidase (GPX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragments were immobilized on individual Hybond
N
membranes.
Figure 3:
Transcriptional activity of GST-
promoter-CAT fusion plasmid transiently transfected into KB and KB/BSO3
cells. GST-
promoter-CAT gene fusion plasmid, p-2203 GST-CAT,
p-130 GST-CAT, and p-80 GST-CAT were co-transfected with pSV2-
-gal
into KB and KB/BSO3 cells as described under ``Experimental
Procedures.'' CAT activities were corrected for differences in
transfection efficiency among the cell lines as estimated by
-galactosidase activity and then normalized to corrected activity
of RSV-CAT transfected cells. The plasmids were transfected in three
independent experiments; each bar represents the mean of three
experiments, and each error bar indicates S.D. from the mean.
*, significantly different (p < 0.05) between KB and
KB/BSO3 cells.
Figure 4:
Southern blot of the -GCS and
GST-
gene. DNA was extracted from KB and its BSO-resistant cell
lines and digested with EcoRI. The digest was then
electrophoresed, transferred to Hybond N
, and
hybridized with a
-GCS probe (A) and a GST-
probe (B). The molecular weight markers indicated by arrows are in kilobase pairs (kb).
Figure 5:
Northern blot (A), immunoblot (B) of GST-, Northern blot of
-glutamylcysteine
synthetase (C) from KB, KB/BSO3, KB/BSO3-
1, and
KB/BSO3-
2 cells. Total RNA (15 µg) extracted from KB, KB/BSO3,
KB/BSO3-
1, and KB/BSO3-
2 was separated on a
formaldehyde-agarose gel and transferred to a nitrocellulose membrane.
Preparation of the probes and hybridization proceeded as described in
the legend to Fig. 1(A and C). The
immunoblotting of GST-
proceeded as described in the legend to Fig. 1(F).
Table 2shows a comparison of the cellular
sensitivity of KB, KB/BSO3, KB/BSO3-1, and KB/BSO3-
2 to BSO,
cisplatin, ACNU, melphalan, and ethacrynic acid. Transfection of
GST-
cDNA into KB/BSO3 cells restored wild type KB cell BSO
sensitivity in the KB/BSO3-
1 and -
2 transfectants. These
results suggest that GST-
is involved in the modulation of
cellular sensitivity to BSO. In addition, increased expression of
GST-
in both KB/BSO3-
1 and KB/BSO3-
2 restored the
cellular resistance to melphalan, ACNU, and cisplatin to the levels
similar to those observed in KB cells (Table 2). Drug resistance
to ethacrynic acid in KB/BSO3 was also markedly increased in both
GST-
transfectants. By contrast, pSV2-neo transfectants without
GST-
overexpression showed almost similar sensitivities to various
drugs as their parent KB/BSO3 cells (data not shown).
Figure 6:
Total intracellular GSH levels in KB,
KB/BSO3, KB/BSO3-1, and KB/BSO3-
2 cells. Exponentially
growing cells were exposed to various concentrations of BSO for 18 h,
then the GSH content was determined.
, KB;
, KB/BSO3;
▪, KB/BSO3-
1;
, KB/BSO3-
2. Each point is the
average of triplicate dishes. Bars,
±S.D.
We first selected three human cancer KB cell lines resistant
to BSO, a synthetic amino acid inhibitor of
-GCS(18, 19) . The expression of GST-
was
dramatically decreased with a concomitant increase of
-GCS levels
in the BSO-resistant cell lines. Three BSO-resistant cell lines,
KB/BSO1, KB/BSO2, and KB/BSO3, were 10- to 13-fold more resistant to
BSO than were parental KB cells. Cellular GSH levels are balanced
through glutathione metabolism, and the critical reaction for GSH
synthesis is catalyzed by
-GCS and
-glutamyl
transpeptidase(1, 9) . These three BSO-resistant cell
lines had similar levels of GSH (
)and
-GCS mRNA (Fig. 1). These findings suggest that the increased GSH and
-GCS levels are associated with their BSO-resistant phenotypes.
Nuclear run-on assay also showed that the
-GCS mRNA transcribed in
KB/BSO3 was higher than that of parent KB cells (Fig. 2). In
addition, Southern blot analyses revealed that increased
-GCS
levels were due to gene amplifications in BSO-resistant cells. Thus, it
is likely that the increased mRNA levels of
-GCS are mainly
attributable to gene amplification rather than activation of its
promoter activity. Richman and Meister (8) have reported that
both the synthesis and activity of
-GCS are inhibited by GSH
through a feedback mechanism.
In GSH metabolism, the cellular level
of GSH is also controlled by glutathione peroxidase and
GST(9) . The expression of GST-, but not of glutathione
peroxidase, was markedly reduced specifically in all the BSO-resistant
cell lines (Fig. 1). Nuclear run-on assay (Fig. 2) and
GST-
promoter-CAT experiment (Fig. 3) revealed that the
decreased expression of GST-
gene in BSO-resistant cells was due
to reduced transcriptional activity. Furthermore, transient
transfection assays of GST-
promoter-CAT (Fig. 3) suggest
that the cis-regulatory elements between -130 bp and
-80 bp from the initiation site might be at least partially
responsible for decreased GST-
expression in BSO-resistant cells.
As Southern blots revealed no deletion and rearrangement of the
GST-
gene in BSO-resistant cells (Fig. 4B), we
conclude that decreased expression of GST-
gene is mainly due to
altered transcriptional regulation. Human GST-
promoter sequences
contain no binding sites of known regulatory factors between -130
and -80 from the initiation site(38) . In mouse, rat, and
human GSTs gene, a TPA-responsive element or AP-1 binding site is the
common motif of their promoter
regions(23, 46, 47, 48, 49, 50) .
The mouse GST-Ya subunit gene is controlled by an EpRE enhancer which
contains two AP-1-like binding sites(46, 47) .
Bergelson et al.(48) have recently reported that
Fos-Jun heterodimeric complex (AP-1)-mediated transcription activation
of the rat GST-Ya gene acts through a common mechanism involving the
production of reactive oxygen species and the depletion of GSH. Rat
GST-P, which is a homologue of human GST-
, contains two
TPA-responsive elements in its promoter region: one is in the enhancer
element GPE1 (GST-P enhancer I) at the position of -2.5
kilobases, and the other is at -61 bp upstream of the GST-P
transcriptional start point(49) . TPA treatment stimulated
endogenous GST-P transcription (49) , but Morimura et al.(50) have reported that involvement of Fos-Jun in rat
GST-P gene expression is less likely in rat hepatoma cells. Morrow et al.(38) revealed that neither TPA treatment nor
co-transfection of Fos-Jun expression vectors induced human GST-
promoter activity. There appears to be no consistent regulatory
mechanism for GST gene expression, in mammalian cells. Further study is
required to understand the molecular mechanism underlying defective
GST-
gene expression in our BSO-resistant cell lines.
Furthermore, the introduction of GST- cDNA into KB/BSO3 cells
resulted in a decrease of the GSH levels (Fig. 6). GSTs play an
important role in detoxification by conjugating many xenobiotics and
other hydrophobic/electrophilic compounds with a concomitant decrease
in GSH levels(51) . Consistent with this notion, GST-
may
play a critical role in the balance of cellular GSH levels and also in
the acquisition of BSO-resistant phenotypes in KB cells. On the other
hand, cellular GSH levels were efficiently depleted when incubated with
various doses of BSO up to 5 µM (Fig. 6). KB and
KB/BSO3 showed similar percent depletion of GSH levels in response to
BSO treatment, suggesting that the membrane permeability of BSO is not
blocked in the resistant cell line.
BSO-resistant cell lines were
collaterally sensitive to alkylating agents such as nitrosourea (ACNU)
or melphalan as well as ethacrynic acid (Table 1). The expression
of the MGMT gene often confers resistance to the toxic effects of
alkylating agents in mammalian cells (31, 45, 52) . However, all BSO-resistant
cell lines had similar levels of MGMT, suggesting that this enzyme is
probably not involved in the collateral sensitivity to alkylating
agents. The transfection of KB/BSO3 cells with GST- cDNA, however,
made them more than 3-fold resistant to ethacrynic acid than the wild
type KB cells (Table 2). Transfection with the GST-
gene
confers upon human breast cancer MCF-7 cells drug resistance to
ethacrynic acid (23) and mouse NIH3T3 cells(24) . These
findings consistently support the notion that cellular sensitivity to
ethacrynic acid, a diuretic substrate which is a substrate for
GST(53) , is closely correlated with GST-
levels.
Cisplatin is an effective anticancer agent, which functions by
interacting with its target DNA through interstrand or intrastrand
cross-links. Drug resistance to cisplatin is mediated through
pleiotropic mechanisms, including protection by GSH compounds, drug
transport, DNA repair, and
metallothionein(54, 55, 56) . We also
demonstrated that the acquisition of cisplatin resistance is due to
elevated GST- levels(20, 21) , decreased
intracellular accumulation of the drug(57, 58) ,
decreased levels of DNA topoisomerase I(44) , and DNA
repair(59) . The cellular GSH levels often influence drug
resistance to cisplatin as well as to alkylating agents(10) .
Increased cellular GST-
levels are closely associated with
resistance to cisplatin in Chinese hamster ovary cells (20, 21) or in patients with gastric
cancer(60) , but not with resistance to cisplatin in human
breast cancer cells or mouse fibroblasts(23, 24) . In
this study, KB/BSO3 cells showed a slight collateral sensitivity,
whereas relative resistance was restored to 1.2 by introducing the
GST-
gene (Table 2). Drug sensitivity to cisplatin in human
cancer KB cells is thus only slightly if at all modulated by the
GST-
levels. On the other hand, Godwin et al.(25) have reported that cisplatin resistance in human
ovarian cancer cells is closely associated with increased cellular
-glutamyl transpeptidase and
-GCS levels. Their
cisplatin-resistant ovarian cancer cell lines show very high degrees of
drug resistance, being 30- to 1000-fold more resistant than their
parental cells. However, all cell lines had similar GST
activities(25) . The
-GCS levels were 3- to 5-fold higher
in BSO-resistant cell lines than in KB cells, whereas the levels of
-glutamyl transpeptidase were similar among our resistant and
sensitive cell lines (Fig. 1). BSO-resistant cell lines were not
cross-resistant, but rather collaterally sensitive, to cisplatin,
suggesting that increased
-GCS levels do not directly influence
the sensitivity to cisplatin.
Among the BSO-resistant cell lines
with increased levels of -GCS mRNA, cellular GST-
mRNA levels
were decreased. Transfection of GST-
into BSO-resistant KB/BSO3
cells restored
-GCS mRNA, GSH contents, and sensitivity to BSO and
other drugs to levels similar to those of wild type KB cells (see Table 3). Our present findings suggest that GST-
levels
might be obligatorily coupled with
-GCS levels in human cancer KB
cells. However, this hypothesis comes from our experiments with
GST-
transfectants, which were established during incubation for
about 1 month in the absence of BSO. One could argue that
-GCS
gene amplification in KB/BSO lines is unstable, resulting in rapid loss
during selection of the transfectants in the absence of BSO. However,
both BSO-resistant phenotype and
-GCS amplification had been
stably maintained for 1 month in KB/BSO3 cells in the absence of BSO,
and pSV2-neo transfectants of KB/BSO3 without overexpression of
GST-
gene showed similar levels of drug sensitivities and
-GCS gene amplification as those of KB/BSO3 cells.
In
our present study,
-GCS gene expression was not determined in
KB/BSO3 cells immediately after the exogenous GST-
expression
plasmid was introduced. Furthermore, it remains unknown whether
overexpression of
-GCS gene could also affect GST-
gene
expression. The hypothesis whether cellular GST-
levels could
directly modulate expression of
-GCS gene is speculative pending
further study. To examine whether this selective reduction of GST-
mRNA levels in BSO-resistant cells is a common phenomenon, further
isolation and characterization of BSO-resistant variants from other
cell types will be required.