Download Complementary DNA Cloning, Messenger RNA

(CANCER RESEARCH 52, 314-318, January 15, 1992]
Complementary DNA Cloning, Messenger RNA Expression, and Induction of
«-ClassGlutathione S-Transferases in Mouse Tissues1
Timo M. Buetler and David L. Eaton2
Department of Environmental Health and Institute for Environmental Studies, University of Washington, Seattle, Washington 98195
ABSTRACT
Glutathione 5-transferases (EC 2.5.1.18) are a multigene family of
related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important
determinant of susceptibility of that tissue to toxic injury or cancer.
Recent studies have suggested that «-class glutathione 5-transferase
isoforms may play an important role in the development of cancers.
Several a-class glutathione 5-transferase isozymes have been character
ized, purified, and cloned from a number of species, including rats, mice,
and humans. Here we report on the cloning, sequencing, and mRNA
expression of two a-class glutathione 5-transferases from mouse liver,
termed mYa and inYi. While mYa was shown to be identical to the
known a-class glutathione 5-transferase
complementary DNA clone
pGT41 (W. R. Pearson et al., J. Biol. Chem., 263:13324-13332,1988),
the other clone, mYc, was demonstrated to be a novel complementary
DNA clone encoding a glutathione 5-transferase homologous to rat Yc
(subunit 2). The mRNA for this novel complementary DNA is expressed
constitutively in mouse liver. It also is the major a-class glutathione 5transferase isoform expressed in lung. The levels of expression of the
butylated hydroxyanisole-inducible
form (mYa) are highest in kidney and
intestine. Treatment of mice with butylated hydroxyanisole had little
effect on the expression levels of mYc but strongly induced mYa expres
sion in liver. Butylated hydroxyanisole treatment increased expression
levels for both mYa and mYc to varying degrees in kidney, lung, and
intestine. The importance of the novel mouse liver a-class glutathione 5transferase isoform (mYc) in the metabolism of aflatoxin B, and other
carcinogens is discussed.
INTRODUCTION
GSTs3 are a multigene family of related proteins predomi
nantly involved in detoxification reactions (1,2). They are found
in all tissues and species, including bacteria (3), yeast (4), and
plants (2). In higher organisms, three classes of GST protein,
a, n, and 7T(1, 2), have been discriminated; a fourth class, 0,
has been tentatively identified, although relatively little is
known about this protein's structure and function (5, 6). Each
class consists of several closely related gene products which are
capable of forming dimeric proteins with other gene products
of the same class, but not with protein subunits from other
classes (1, 2). In the rat at least five members of the a class
have been described at either the DNA or protein level. Three
cDNA sequences and one gene (7) for rat a-class GSTs have
been published. The cDNAs code for subunits Ya4 (8-10)
(pGTB38; Ref. 11), Ya2 (pGTR261; Ref. 12), and Yc (pGTB42;
Ref. 13). Recently the amino acid sequence for two distinct rat
Received 8/5/91; accepted 10/31/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by NIH Grants ES05780, CA47561, and ES03933.
2To whom requests for reprints should be addressed, at Department of
Environmental Health, SC-34, and Institute for Environmental Studies, Univer
sity of Washington, Seattle, WA 98195.
3The abbreviations used are: GST, glutathione 5-transferase; AFB, aflatoxin
B,; BHA, 2(3)-fert-butyIhydroxyanisole;cDNA,
complementary DNA.
' Because of the disparity in numerical designations between mouse and rat
GST isoenzymes (8, 9), which may confuse the comparison of mouse GST
isoenzymes with rat GST isoenzymes, we adopted the "Y" designation of Hayes
(10).
a-class GST isozymes have been published (14, 15). These
include the complete amino acid sequence for rat liver GST Yk
(subunit 8) (14) and a partial amino acid sequence for rat liver
GST Yl (subunit 10) (15). In the mouse, one a-class GST
cDNA has been characterized (pGT41) (16). The sequence for
a very closely related mouse a-class GST (mus Ya) has been
characterized at the gene level (17). However, Southern blot
analysis of the murine genome suggested the presence of at
least four or five a-class GST genes (18). Indeed, analysis of
GST protein has revealed the presence of at least two different
a-class proteins in mouse liver (10, 19-21).
Several investigators have demonstrated the presence of a
constitutively expressed a-class GST isoform in mouse liver as
well as a distinct isoform which is inducible by the antioxidant
BHA. For example, Benson et al. (19), using glutathione transferase activity toward l-chloro-2,4-dinitrobenzene
for detec
tion, demonstrated the presence of (a) a constitutively active aclass GST isoform (GST 10.6) in male and female CD-I mice
which was not significantly influenced by treatment with BHA
and (b) a BHA-inducible a-class GST isoform (GST 10.3).
McLellan and Hayes (20) showed that, in mice, BHA induced
an a-class GST isoform, termed Ya]Yai, that was essentially
absent from the livers of untreated mice. These investigators
also purified a second isoform, termed YasYa3, which was
constitutively expressed in mouse liver and was not affected by
BHA treatment. The mouse YaiYai isoform shared a high
structural identity with the rat YaYa (1-1) isoenzyme, while the
constitutively expressed form was less homologous to the rat
YaYa protein. Pearson et al. (16) isolated a mouse liver a-class
GST cDNA clone (pGT41) which was 95% homologous to the
rat Ya a-class GST sequence (pGTB38). Northern blot studies
showed that constitutive mRNA levels measured with this clone
were very low in liver but high in kidney. BHA treatment
induced this mRNA about 50-fold in liver but produced only a
slight induction in kidney. The RNA expression data from
untreated mice (16) correspond well with the protein data
published by Gupta et al. (22), who used antisera raised against
human a-class GST protein to detect a-class GST protein(s) in
mouse liver and kidney. The low levels of protein detected in
liver by Gupta et al., relative to that detected in kidney, would
suggest that the antibody used might specifically detect the
BHA-induced a-class GST protein, since Pearson et al. have
shown that the constitutive levels of expression of their a-class
GST clone, pGT41, are low in livers and high in kidneys of
untreated animals. Previous work from our laboratory (21) also
demonstrated the presence of a constitutively expressed a-class
GST isoform which was chromatographically distinct from a
BHA-inducible isoform.
Differences in the expression of specific a-class GSTs may
have significant physiological implications. For example, Mon
roe and Eaton (23) have demonstrated that GST activity toward
the carcinogenic epoxide metabolite of aflatoxin Br (AFB epoxide) differs by 50-100-fold between rats and mice. Such differ
ences appear to be related specifically to differences in the
activity of «-classGSTs (21). Recently, a-class GST proteins
have been implicated in the resistance of rat and mouse tissue
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GST EXPRESSION
IN MOUSE TISSUES
culture cells to chlorambucil (24, 25) and Adriamycin (26) and
in the metabolism of the nitrogen mustard melphalan (27).
Thus, while the existence of two a-class GST isoforms in mice
is well established, their contribution to the protection against
AFB-induced hepatocarcinogenicity and their role in the detox
ification of nitrogen mustards used in cancer therapy are not
known, and the relationship to their rat counterparts is unclear.
In this paper we report on the isolation of two cDNA clones
from mouse liver; one corresponds to the sequence published
by Pearson et al. (16), which we term mYa, and the other is a
novel sequence which shows the highest homology to the rat
Yc (subunit 2) sequence (pGTB42; Ref. 13) and which we term
mYc. Expression studies demonstrate that the mYc clone rep
resents the constitutively expressed mouse liver a-class GST
isoform, whereas the mYa clone represents the BHA-inducible
isoform.
MATERIALS
AND METHODS
Chemicals and Enzymes. The mouse cDNA library was purchased
from Clontech Laboratories, Inc. (Palo Alto, CA). The mRNA for the
library was derived from adult male BALB/c mouse liver, and the
cDNA was constructed by 5'-stretch, oligo-thymidine, and random
priming and cloned in Xgtll. Radioisotopes [35S]dATP, [«-"P]dCTP,
and [7-32P]ATP were purchased from ICN Biomedicals, Inc. (Costa
Mesa, CA). Restriction enzymes and other molecular biology-grade
reagents were purchased from Bethesda Research Laboratories (Gaithersburg, MD), Stratagene (La Jolla, CA), International Biotechnolo
gies, Inc. (New Haven, CT), United States Biochemical (Cleveland,
OH), Boehringer Mannheim (Indianapolis, IN), and Sigma (St. Louis,
MO). The Sequenase sequencing kit was purchased from United States
Biochemical Corp. Pre-made Luria broth was purchased from Gibco/
BRL (Gaithersburg, MD). Male Swiss-Webster mice were obtained
from Tyler Laboratories (Bellevue, WA).
Plasmids. The plasmids pGTB38 [containing the rat Ya (subunit 1)
cDNA] and pGTB42 [containing the rat Yc (subunit 2) cDNA] were a
generous gift from Dr. Cecil Picket! (Merck Frosst Canada, Inc.,
Québec,Canada).
Isolation of Mouse «-ClassGST cDNA Clones. The cDNA library in
Xgtl 1 was plated at 50,000 pfu/150-mm plate on Luria broth agarose
with Y1090 as indicator bacteria, following the protocol provided by
the manufacturer (Clontech Laboratories, Inc., Palo Alto, CA). Two
replica plaque lifts were made on Nytran filters (Schleicher & Schuell,
Keene, NH). A probe was made by digesting the rat Ya plasmid
pGTB38 with Pstl and isolating the 550-base pair insert fragment
comprising most of the protein coding region. Both filters were washed
at relatively low stringency (Ix standard saline citrate, composed of
150 HIMNaCl and 15 mivi Na citrate, pH 7.0, 0.1% sodium dodecyl
sulfate, at 63°C).cDNA inserts were subcloned into pUCIS plasmid
vector and transfected into Escherichia coli TG1 cells.
Sequencing. cDNA clones were sequenced using the universal primer
provided with the Sequenase kit (5'-GTAAAACGACGGCCAGT-3')
and the reverse primer. Nested deletions in the insert sequence were
created using the Exonuclease III/mung bean kit from Stratagene. The
vector was cut with Sphl to protect the vector from digestion with
£roIII in the 5' direction, and BamHl to allow ExolH action in the 3'
direction into the insert sequence. The remaining procedures were
conducted according to the protocol provided by the manufacturer.
Briefly, at room temperature £xoIIIactivity was determined to be about
100 nucleotides/min. Exolll incubations with DNA at room tempera
ture were stopped in 2.5-min intervals for five time points. Mung bean
nuclease was then added to digest the single-stranded DNA from the
5' end, leaving blunt-ended DNA. These DNAs were ligated and
transfected into E, coli TG1. Three to six colonies from each time point
were grown in 3 ml of Luria broth containing 25 Mgampicillin/ml, and
plasmid DNA was prepared using Plasmid Quick columns (Stratagene).
Two /¿g
of this preparation were used directly for double-stranded DNA
sequencing. Sequences were analyzed with GENEPRO software (Ver
sion 5.0; Riverside Scientific Enterprise, Bainbridge Island, WA) and
compared to the GenBank sequence data bank (Edition 64).
Oligonucleotide Synthesis. Oligonucleotides were synthesized on a
Cyclone Plus DNA synthesizer from MilliGen/Biosearch (Division of
Millipore, Novato, CA). The mouse Ya-specific oligo malO (5'CCA'TTA'GAG'GCC'AGT'ATC'TGC-3')
and Yc-specific oligo
mcl5 (5'-CTC'GTC'AGT'CAT'CAT'GTCTAC'CTG-3')
were cho
sen to have less than 17 of 21 nucleotides matching the other member
of the a class.
Animal Treatment and RNA Isolation. Mice were fed Wayne Rodent
Blox ad libitum for 2 weeks prior to separation into two groups, one of
which was fed BHA (0.75% w/w) incorporated into ground chow while
the other was kept on control diet. After 5 days, the animals were killed
by cervical dislocation, and liver, kidney, lung, and intestine were
removed and placed in ice-cold phosphate-buffered saline. Intestinal
contents were removed by washing with ice-cold phosphate-buffered
saline. RNA was isolated from 0.3 g of tissue using the acid phenol
extraction protocol of Chomczynski and Sacchi (28). 20 Mg of total
RNA were separated on a 1% agarose/formaldehyde gel, blotted onto
Nytran membrane, and hybridized to either the "P-end-labeled oligonucleotides or random-primed 12P-labeled cDNA inserts from both
mouse a-class GST clones. An oligonucleotide of 20 nucleotides in
length corresponding to the 18S ribosomal RNA (from 943-962 of the
mouse 18S ribosomal RNA gene, 5'-CAC'CTC'TAG'AGG'
CGC'AAT'AC-3') was used to reprobe the Northern blots to normalize
the expression levels to variable amounts of RNA loaded.
RESULTS
A total of 300,000 plaques were screened with the rat Ya
probe, and 13 independent clones were isolated and purified.
Nine of these clones gave a strong signal with the rat Ya probe,
while the other four hybridized only weakly with the Ya probe.
Six of the strongly hybridizing clones were further analyzed.
DNA was isolated, and the £coRI inserts were cloned into
pUC18. Of these six cDNA inserts, only three were longer than
800 base pairs. From the cloning work of others in rats, humans,
and mice and based on the size of the GST protein, an 800base pair insert was considered the minimal length required to
contain a full-length cDNA insert. Restriction analysis of five
of the six clones revealed two different restriction patterns. The
pattern fordone 4.1 was similar to that of pGT41, the sequence
reported by Pearson et al. (16). The pattern of the four other
clones, two full-length (5.1 and 6.1) and two shorter (insert
sizes of 600-650 base pairs) clones were similar but distinct
from the pattern reported for any other mouse GST cDNA
sequence. Clone 5.1 seemed to be identical to clone 6.1 (both
about 1.0 kilobase in length) but was in inverted orientation in
the plasmid vector.
Partial sequencing from both ends using the universal primer
and the reverse primer for pUCIS suggested the sequence
identity of clone 4.1 with pGT41 and of clone 5.1 with clone
6.1. Clone 4.1 was completely sequenced in one direction, and
absolute sequence identity between this clone and the published
sequence for the mouse a-class GST clone pGT41 was found.
Clone 4.1 was 65 base pairs shorter at the 5' end but still
extended 29 base pairs 5' of the start codon and thus contained
the complete protein coding region. At the 3' end, this clone
was 22 base pairs longer and contained a polyadenylation signal.
Since clones 5.1 and 6.1 most probably encoded the same RNA
but were in inverted orientation in the cloning vector, each
clone was sequenced only in one direction in order to obtain
sequence confirmation of the opposite strand. Nested deletions
were created from the 5' end of each insert and sequenced using
the universal primer, which annealed in the pUCIS vector,
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GST EXPRESSION
IN MOUSE TISSUES
Table 1 Nucleolide and amino acid homology comparison ofa-class glutathione
S-transferases between different species
The sequences of «-classGSTs between different species are compared at the
nucleotide and amino acid levels. The sequences obtained from clones 4.1 (mYa)
and 5.1 6.1 (mYc) were compared to the GenBank databank (Edition no. 64)
using the Genepro 5.0 software on an IBM computer.
therefore bypassing the deletion. Complete sequencing of these
two clones revealed their identity; no mismatch was found
(Fig. 1).
When this sequence was compared with the 63 different GST
cDNA sequences in the GenBank (Edition No. 64) sequence
data bank, the highest homology (84%) was found with the rat
Yc sequence (13) (Fig. 1). Sequence homology to the mouse
and rat Ya sequences was only 70% and 67%, respectively
(Table 1). Thus, clone 5.1/6.1 apparently codes for a mouse
GST isoform which is orthologous to rat Yc (subunit 2) and is
referred to as mYc. The open reading frame of the mYc clone
is 663 base pairs long and encodes a protein of 221 amino acids
with a deduced M, of 25,360. Because rat Yl (subunit 10) and
rat Yc (subunit 2) are distinct but closely related, both had a
homology similar to that of the mouse Yc GST isozyme (85%)
(Table 1). The rat Yk (subunit 8) protein is apparently more
distantly related to other rat or mouse «-classGST isozymes,
since it has only a 57-59% amino acid sequence homology with
the other members of the a class.
Northern blots of 20 fig of total RNA from liver, kidney,
lung, and intestine of control and BHA-treated mice were
hybridized with cDNA inserts from either clone 4.1 (mYa) or
clone 5.1 (mYc). The mYa probe detected a specific signal at
0.8 kilobases, while the mYc-specific probe detected a signal at
1.0 kilobase (Fig. 2). The pattern of mYa expression was found
Mouse Ya
Mouse Yc
90
67
67
84
74
69
Nucleotide level
Rat Yal
Rat Ya2
Rat Yc
Human Hal
Human Ha2
87
69
73
72
Amino acid level
Rat Ya
Rat Yc
Rat Yk°
Rat Yl*
95
69
59
66
67
85
57
85
°Complete amino acid sequence of rat liver GST Yk (subunit 8) (14).
* Partial amino acid sequence of rat liver GST Y1 (subunit 10) ( 15).
to be different from that of mYc, and neither of the two probes
cross-hybridized with the mRNA encoding the other isoform.
Therefore, it can be concluded that each probe reacted only
with its homologous mRNA, resulting in a specific signal. The
specificity of the signal was confirmed by hybridizing Northern
blots with the isoform-specific oligonucleotides malO for mYa
mYc
rYc
-21 AACAAGA AAACCCAAGC
AGA GGGAGCAGCTT TTT
G
T
-43
AACTGCTGCC
--T
Met Ala Gly Lys Pro Val Leu His Tyr Phe Asp Gly Arg Gly Arg Met Glu Pro He Arg Trp Leu Leu Ala Ala
ATG GCG
c_- GGG AAG CCA GTC CTT CAT
__c TAC TTT
__c GAT GGC AGG GGA
__G AGA ATG GAG CCT
__c ATC CGG c__
TGG CTC TTG GCT GCA
__A
-
Pro
-------------
25
15
----------
Ala Gly Val Glu Phe Glu Glu Lys Phe Leu Lys Thr Arg Asp Asp Leu Ala Arg Leu Arg Ser Asp Gly Ser Leu 50
GCT GGT GTG GAG TTT GAA GAA AAA TTT CTG AAA ACT CGG GAT GAC CTG GCA AGG TTA CGA AGT GAT GGG AGT CTG 150
c__
-_c
c__ A_G _A_
T._
------Cln -----------ASn -
Met Phe Gin Gin Vil Pro Met Val Glu Ile Asp Gly Met Lys Leu Val Gin Thr Lys Ala Ile Leu Asn Tyr He
75
ATG TTC CAG CAA GTG CCC ATG GTA GAG ATC GAC GGG ATG AAA CTG GTG CAG ACC AAA GCC ATT CTC AAC TAC ATT 225
--G
--T _-T
-
—¿G
-
-
-
-
_G_
Arg
-----
-
Ala Ser Lys Tyr Asn Leu Tyr Gly Lys Asp Met Lys Glu Arg Ala Ile Ile Asp Met Tyr Thr Glu Gly Val Ala 100
GCC A-TCC AAA TAC AAC CTC TAT GGG AAG GAC ATG AAG GAG AGA GCC ATC
C— ATT
--C GAC ATG TAC
--T G-ACÕ GAA GGA GTG GCG 300
-
Thr -------------
Leu -
-
-
- Ala -
-
-
-
Fig 1 Sequence alignment of clone 5 I/
6.1 with the rat Yc sequenceof clone PGTB42
Asp Leu Glu Ile Met Ile Leu Tyr Tyr Pro His Met Pro Pro Glu Glu Lys Glu Ala Ser Leu Ala Lys Ile Lys 125
GATCTG GAGATA ATG ATT CTC TAT TAC ccc CACATG ccc CCT GAGGAGAAAGAGGCAAGCCTT Gcc AAGATC AAG375
T T
"£ GA' A*¡G" . £ .' "T £ '£ T
T
cTy "-' "-' "-' "-' "* ~
'" "-* ~
"-'
(13). The translated ammo acid sequencesare
Shown above and below the respective nucleotide sequences. -, identical nucleotide or
amino acid at that position; *, a gap for best
alignment. Nucleotide 1 is the A of the ATG
Glu Gin Thr Arg Asn Arg Tyr Phe Pro Ala Phe Glu Lys Val Leu Lys Ser His Gly Sin Asp Tyr Leu Val Gly 150
GAACAAACCAGGAACCGT TAC TTC CCT GCCTTT GAAAAGGTGTTG AAGAGCCAT GGACAAGAT TAT CTC GTT GGC450
—¿-e
A-- G-A —¿â€”¿â€”¿â€”¿â€”¿T
—¿â€”¿â€”¿â€”¿â€”¿â€”¿â€”¿â€”¿â€”¿â€”¿
—¿â€”¿â€”¿â€”¿â€”¿
Asf L** *la ~
''
°'
Asn Arg Leu Ser Arg Ala Asp Ile Ala Leu Val Glu Leu Leu Tyr His Val Glu Glu Leu Asp Pro Gly Val Val 175
AAC AGG CTG AGC AGG GCT GAT ATT GCC CTG GTT GAA CTC CTC TAC CAT GTG GAA GAG CTG GAC CCC GGC GTT GTG 525
—¿T
G— TA
A --- c— G-T
A
c. T—
-
Val Tyr -
- Gin Val -
-
-
-
-
-
-
-
-
Ser Ala Leu
Asp Asn Phe Pro Leu Leu Lys Ala Leu Arg Ser Arg Val Ser Asn Leu Pro Thr Val Lys Lys Phe Leu Gin Pro 200
GAC AAC TTC CCT CTC CTG AAA GCG CTG AGA AGC AGA GTC AGC AAC CTC CCC ACA GTG AAG AAG TTT CTT CAA CCT 600
_c_
-_G -_c
-c__G
Aja --------r/]r -------------Gly Ser Gin Arg Lys Pro Phe Asp Asp Ala Lys Cys Val Glu Ser Ala Lys Lys Ile Phe Ser
GGC AGC CAG AGG AAG CCT
--A TTT
—¿A
-_G
GAT _-T
GAC -AG
GCA AAA TGT GTT
-_A GAG
-_A TCA
_-T GCA GTT
AAG AAG ATT
--c TTC AGT TAATTCAGGC
------
Leu Glu
-
Glu
-
-
-
ATAAGT ACATAGCCCC
—¿GGA- CT-TA--
CACAAAGCCA
ACCTTCTAAA
G
G
GCTAAC AAGTTTTCTA
--C
-c
AGGCGTCTGT
-T--T
GTCAATTCAG GTAGACATGA
--T
T A
GAAACT CATGATCACT
TG--A-
TCCTCGGATA
A
TTTTCTTCTG
--ACT--GAA
-
-
ATTTTGCATC ACATTGAAGT
GC
A- ----C-C-"-
-
Val
221
AACT
_T._ 615
-
GTTTTGACTA
A
AGTGTTGACC
-A
CTACTTA«** GAAA 141
TTG -G-G 148
CTGACGAGGA
T
ACGGCCGGGA
-TT--T
TGCTCTCTAG TTGTAGTTAA
A-T-G-
T'TCAAT'AA AACAAAACAA
-C
A—
TTCG
GCTTCTTAGA
CTCTGG
AATT 823
824
316
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923
915
GST EXPRESSION
li ki
.+.+.+.
lu
int
*
IN MOUSE TISSUES
DISCUSSION
BHA
+•1.0
kb
Ya
«-0.8 kb
+ 1.0 kb
Ye
«-0.8 kb
18S rRNA
Fig. 2. Expression of mYa and mV'c GST mRNA in mouse tissues. RNA was
prepared from liver (//), kidney (ki), lung (lu), and intestine (ini) of mice fed
either a control diet (—)or a diet containing 0.75% BHA (+) for 5 days prior to
sacrifice. Twenty ng of total RNA were separated on agarose gels and blotted
onto Nytran membranes. The blots were hybridized to one of the following
probes: thecDNA insert of clone 6.1 (top), thecDNA insert of clone 4.1 (middle),
or an oligonucleotide specific for 18S rRNA (bottom).
and mclS for mYc. The resulting expression pattern was found
to be identical to that using the cDNA probes, with no observ
able cross-hybridization (data not shown). Thus, these probes
detected a specific mRNA for mYa and mYc at 0.8 and 1.0
kilobase, respectively. These sizes correspond well with the
sizes of the cDNA clones, 849 base pairs for mYa (pGT41) and
950 base pairs for mYc (5.1), with the mYc clone longer in its
3' noncoding region.
Two cDNA clones were isolated from a commercially avail
able mouse liver cDNA library using the insert of a rat Ya
cDNA clone as a probe. One clone (4.1) was found to be
identical to the published cDNA sequence of pGT41 (16), a
mouse a-class GST, and was therefore termed mYa. The other
clone encoded a novel sequence which showed the highest
homology to the rat Yc (subunit 2) sequence (84% nucleotide
homology) and was termed mYc. The homology between the
rat and the mouse Yc isoforms proved to be significantly lower
than for mouse and rat Ya isoforms (85% versus 95% homol
ogy). At high stringency of hybridization we found that the
mYa probe did not cross-hybridize with the mYc mRNA and
vice versa. This may explain why Pearson et al. did not detect
the constitutively expressed liver a-class GST isoform (mYc)
with their pGT41 cDNA probe on a Northern blot (16). The
specificity of the hybridization using the cDNA inserts was
confirmed by hybridization with mYa- and mYc-specific oligonucleotides (malO and mclS, respectively). Although hybrid
ization with the mYa-specific oligonucleotide resulted in a very
weak signal, it was adequate to determine the expression pattern
for mYa. Probing a Northern blot with the mYc-specific oli
gonucleotide yielded results identical to those obtained with the
mYc cDNA insert. Based on the protein expression pattern
known from the literature, the two «-classGST clones described
here represent the two major «-classGST isoenzyme forms
found in liver and probably in other tissues of the mouse. In
liver, mYc represents the constitutively expressed, and mYa the
BHA-inducible, isoform.
Previous studies from our laboratory have demonstrated that
mice are resistant to the hepatocarcinogenic effects of aflatoxin
B, and have 100-fold lower AFB-DNA adduci formation than
rats (23). This relatively low level of DNA binding was associ
ated with a 50-100-fold higher level of cytosolic GST activity
toward AFB epoxide in mice relative to rats, even though
specific activity toward l-chloro-2,4-dinitrobenzene
was com
parable between the two species (29). The efficient detoxifica
tion of AFB epoxide by mice appears to be a common charac
teristic of the mus species, inasmuch as nine different strains
Quantitation of expression by densitometric scanning re
vealed that mouse liver contained a low basal level of mYa
message, which was induced about 15-fold by BHA (Fig. 3).
The basal levels of expression of mYa in intestine and kidney
were 3- and 4-fold greater than in liver, respectively, and undetectable in lung. BHA treatment resulted in a 7- to 15-fold
induction of mYa message levels in intestine and a 3-fold
induction in kidney. No expression or induction was observed
in lung. These data are in good agreement with the data from
Pearson et al. (16), although the extent of induction by BHA
was not as large as that found by these investigators.
In liver, mYc was found to be constitutively expressed at a
level approximately 25-fold greater than mYa (Fig. 3). BHA
had no significant effect on mYc expression levels in liver.
Constitutive expression of mYc was substantial in lung (about
10% of the constitutive level in liver). BHA increased mYc
expression in lung to 40% of the level found in liver. In contrast
to mYa, mYc expression levels were very low to undetectable
in normal kidney and intestine. BHA treatment induced mYc
expression in kidney, lung, and intestine 10-, 3-, and 6-fold,
respectively (Fig. 3).
liver
kidney
lung
intestine
kidney
lung
intestine
Fig. 3. Relative expression levels of mYa
tissues. The amounts of RNA were determined
GS-300; Hoefer Scientific Instruments, San
expression levels, relative to 18S rRNA, from
and mYc GST mRNA in mouse
by densitometric scanning (Hoefer
Francisco, CA). Columns, mean
at least 5 independent scannings.
317
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had similarly high specific activities (30). We have also dem
onstrated that the high specific activity of GST toward AFB
epoxide in mice is largely attributable to an «-classGST (21).
Thus, because normal (untreated) mice have high AFB epoxideconjugating activity and do not constitutively express GST
mYaYa, it is tempting to speculate that the mYcYc isoform is
responsible for the high AFB epoxide-detoxifying ability of
mouse liver cytosol. In fact, preliminary results of bacterially
expressed mouse and rat isozymes (Ya and Yc) suggest that
indeed only the mouse YcYc isozyme has substantial AFB
epoxide-conjugating activity.5 The mouse Yc subunit may also
prove to play an important role in the acquired resistance of
tumor cells to nitrogen mustards like melphalan (27), chlorambucil (24, 25), and/or Adriamycin (26). Bolton et al. (27)
have demonstrated that only «-classGST protein(s) from un
treated mice are capable of forming the melphalan-glutathione
conjugate. Since mYc is the predominant «-classGST isoform
in untreated mouse liver, it may well be involved in this meta
bolic reaction. Yang et al. (25) have observed an increased
expression of «-classGST mRNA in chlorambucil-resistant
mouse tissue culture cells. Schisselbauer et al. (26) have ob
served an increase in «-classGST protein in two Adriamycinresistant Friend erythroleukemia cell lines.
Further experiments utilizing site-directed mutagenesis and
chimeric cDNA constructs should provide a means of identify
ing the importance of specific sequences within «-classGST
proteins which may account for the differences in GST activity
among various species and target tissues, thus enhancing the
ability to predict potential sensitivity to epoxide carcinogens or
antineoplastic drugs across species and target tissues.
ACKNOWLEDGMENTS
We are grateful to Drs. Curtis Omiecinski and Christopher Masseti
for helpful discussions and review of the manuscript and to Dr. Cecil
Pickett (Merck Frosst Center for Therapeutic Research, Québec,Can
ada) for his generous gift of the rat cDNA clones pGTB38 and pGTB42.
Note Added in Proof
J.D. Hayes et al. (Biochem.J., 279:385-398,1991) recently reported
the isolation of a new, nonconstitutively expressed rat «-classGST
subunit from ethoxyquin-induced rat liver, termed Yc2. The partial
(-70%) amino acid sequence for rat Yc2 was 91% homologous to the
mouse Yc clone reported here and had high activity toward aflatoxin8,9-epoxide. Thus, it appears that this new rat Yc2 subunit may be the
orthologous form to the mouse Yc subunit reported here.
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Complementary DNA Cloning, Messenger RNA Expression, and
Induction of α-Class Glutathione S-Transferases in Mouse
Tissues
Timo M. Buetler and David L. Eaton
Cancer Res 1992;52:314-318.
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