Biochimica et Biophysica Acta, 1048 (1990) 149-155
149
Elsevier BBAEXP 92028
Induction of a human carbonyl reductase gene located on chromosome 21 Gerald L. Forrest 1 Steven A k m a n 2, Siegfried Krutzik 3, R a y m o n d J. Paxton 4, Robert S. Sparkes 5, James Doroshow 2 Ronald L. Felsted 6 Constance J. Glover 6 Thomas M o h a n d a s 7 and Nicholas R. Bachur 8 Divisions of t Biology and 4 Immunology, Beckman Research Institute at the City of Hope and 2 Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA, 3 Disease Detection International, Irvine, CA, 5 Department of Medicine, UCLA Medical Center, Los Angeles, CA, 6 Laboratory of Biological Chemistry, National Cancer Institute, Bethesda, MD, 7 Department of Pediatrics, Harbor/UCLA Medical Center, Torrance, CA, and 8 University of Maryland Cancer Center, Baltimore, AID (U.S.A.)
(Received 2 June 1989)
Key words: cDNA; Daunorubicin reductase; Drug metabolism; MCF-7
Carbonyl reductase (EC 1.1.1.184) belongs to the group of enzymes called aldo-keto reductases. It is a NADPH-dependent cytosolic protein with specificity for many carbonyl compounds including the antitumor anthracycline antibiotics, daunoruhicin and doxorubicin. Human carbonyl reductase was cloned from a breast cancer cell line (MCF-7). The cDNA clone contained 1219 base paires with an open reading frame corresponding to 277 amino acids encoding a protein of M r 30375. Southern analysis of genomic DNA digested with several restriction enzymes and analyzed by hybridization with a labeled cDNA probe indicated that carbonyl reductase is probably ceded by a single gene and does not belong to a family of structurally similar enzymes. Southern analysis of 17 m o u s e / h u m a n somatic cell hybrids showed that carbonyl reductase is located on chromosome 21. Carbonyl reductase mRNA could be induced 3-4-fold in 24 h with 10/~M 2,(3)-t-butyl-4-hydroxyanisole (BHA), fl-naphthoflavone or Sudan 1.
Introduction Carbonyl reductase (secondary-alcohol : N A D P + oxidoreductase, EC 1.1.1.184) belongs to a group of N A D P H - d e p e n d e n t cytosolic enzymes called aldo-keto reductases [1,2]. These enzymes are ubiquitous in nature, metabolizing quinones and a variety of antitumor drugs containing carbonyl groups including the anthracycline antibiotics, daunorubicin and doxorubicin [1,2]. The role of aldo-keto reductases in drug metabolism is well documented. Carbonyl reductase, acting as an aldo-keto reductase, can reduce the methyl ketone in the carbon side-chain of the antitumor antibiotics, daunorubicin and doxorubucin, to its corresponding alcohol.
The sequence data in this paper have been submitted to the EMBL/ Genbank Data Libraries under accession number J04056.
Abbreviation: BHA, 2,(3)-t-butyl-4-hydroxyanisole. Correspondence: G. Forrest, Division of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, U.S.A.
Ahmed et al. [4] demonstrated differences in anthracycline drug metabolism in tissues from several animal species. H u m a n and rabbit tissue have an enhanced rate of daunorubicin metabolism which is accounted for by carbonyl reductase activity while the rat and mouse tissues are deficient in this activity. Although the mechanistic effects of drug metabolism in cells is not well understood, the reduction of daunorubicin and doxorubicin by carbonyl reductase affects the pharmacological properties of these drugs [5]. Wermuth et al. [3] demonstrated that carbonyl reductase can also act as a quinone reductase [3]. Carbonyl reductase acting as a quinone reductase may protect humans against quinone mediated carcinogenic, mutagenic and toxic effects similar to the protective role that another quinone reductase, DT-diaphorase, affords rats and mice [6-8]. Carbonyl reductase accounts for most of the N A D P H - r e d u c i n g activity in h u m a n liver, while DT-diaphorase accounts for less than 10% of the reducing activity [3]. In rat and mouse liver, carbonyl reductase appears to be low or absent while DT-diaphorase levels are higher and highly inducible [3,8]. Since several quinone substrates are metabolized by
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
150 both carbonyl reductase and DT-diaphorase, carbonyl reductase may supplant DT-diaphorase in the human liver [3,9]. We have cloned a human carbonyl reductase cDNA from liver and from a breast cancer cell line (MCF-7). Carbonyl reductase mRNA was induced in both breast and liver cells by compounds known to induce enzymes involved in protecting cells against the carcinogenic mutagenic and cytotoxic effects of quinones and other xenobiotics. The gene was localized to chromosome 21 which may have implications concerning genetic disease. Materials and Methods
Carbonyl reductase purification. Carbonyl reductases displaying daunorubicin reductase activity were isolated
and purified from human liver based on their low molecular weight and NADPH co-factor specificity [2,10]. The carbonyl reductases were purified as previously published, except that ion exchange chromatography was replaced by a second gel-filtration step using Sephacryl S-200 (3 x 80 cm) (Pharmacia, Piscataway, N J) and the isoenzymes were resolved by chromatofocusing (pH gradient 7.5-6.0) [11]. The enzymes were stored at - 7 0 ° C for 5 years. After thawing, a HPLC chromatography step was required to purify the protein for sequencing. Separation was obtained by reversephase HPLC on a /~Bondapak phenyl column (Waters, Milford, MA) through a linear gradient of 0-90% acetonitrile (vol/vol) in 0.1% trifluoroacetic acid (vol/vol) over 100 min with a flow rate of 1 ml/min. Peptide sequencing. Protein carboxymethylation [12], proteinase digestion with Lys-C [13] peptide sequencing
NUCLEOTIDE CAGACTCGATGTGCCTCTGGAACACGCTGCGGGGCTCCCGGGCCTGAGCCAGGTCTGTTCTCCACGCAGGTGTTCCGCGCGCCCCGT TCAGCC ATG TCG TCC GGC ATC CAT GTA GCG CTG GTG ACT GGA GGC AAC AAG GGC ATC GGC TTG M S S G I H V A L V T G G N K G I G L
GCC A
ATC I
GTG V
CGC R
GAC D
CTG L
TGC C
CGG R
CTG L
TTC F
TCG S
GGG G
GAC D
GTG V
GTG V
CTC L
ACG T
GCG A
CGG R
GAC D
GTG V
ACG T
CGG R
219
GGC G
CAG Q
GCG A
GCC A
GTA V
CAG Q
CAG Q
CTG L
CAG Q
GCG A
GAG E
GGC G
CTG L
AGC S
CCG P
CGC R
TTC F
CAC H
CAG Q
CTG L
GAC D
ATC I
285
GAC D
GAT D
CTG L
CAG AGC Q S
ATC I
CGC R
GCC A
CTG L
CGC R
GAC D
TTC F
CTG L
CGC AAG R K
GAG E
TAC Y
GGG G
GGC G
CTG L
GAC D
GTG V
351
CTG L
GTC V
AAC N
AAC N
GCG A
GGC ATC G I
GCC A
TTC F
AAG K
GTT V
GCT A
GAT D
CCC P
ACA T
CCC P
TTT F
CAT H
ATT I
CAA Q
GCT A
GAA E
417
GTG V
ACG T
ATG M
AAA K
ACA T
AAT N
TTC F
TTT F
GGT G
ACC T
CGA R
GAT D
GTG V
TGC C
ACA T
GAA E
TTA L
CTC L
CCT P
CTA ATA L I
AAA K
483
CCC P
CAA Q
GGG G
AGA R
GTG V
GTG V
AAC N
GTA V
TCT S
AGC S
ATC I
ATG M
AGC S
GTC V
AGA R
GCC A
CTT AAA L K
AGC S
TGC C
AGC S
CCA P
549
GAG E
CTG L
CAG Q
CAG Q
AAG K
TTC F
CGC R
AGT S
GAG E
ACC T
ATC I
ACT T
GAG E
GAG E
GAG E
CTG L
GTG V
GGG G
CTC ATG L M
AAC N
AAG K
615
TTT F
GTG V
GAG E
GAT D
ACA T
AAG K
AAG K
GGA G
GTG V
CAC H
CAG Q
AAG K
GAG E
GGC G
TGG W
CCC P
AGC S
AGC S
GCA A
TAC Y
GGG G
GTG V
681
ACG T
AAG K
ATT I
GGC G
GTC V
ACC T
GTT V
CTG L
TCC S
AGG R
ATC I
CAC H
GCC A
AGG R
AAA K
CTG L
AGT S
GAG E
CAG Q
AGG R
AAA K
GGG G
747
GAC D
AAG K
ATC [I
CTC L
CTG L
AAT N
GCC A
TGC C
TGC C
CCA P
GGG G
TGG W
GTG V
AGA R
ACT T
GAC D
ATG M
GCG A
GGA G
CCC P
AAG IK)
GCC A
813
ACC T
AAG K]
AGC S
CCA P
GAA E
GAA E
GGT G
GCA A
GAG E
ACC T
CCT P
GTG V
TAC Y
TTG L
GCC A
CTT L
TTG L
CCC P
CCA P
GAT D
GCT A
GAG E
879
GGT G
CCC P
CAT H
GGA G
CAA Q
TTT F
GTT V
TCA S
GAG E
AAG K
AGA R
GTT V
GAA E
CAG Q
TGG w
TGA *
GCTGGGCTCACAGCTCCATCCAT
GGGCCCCATTTTGTACCTTGTCCTGAGTTGGTCCAAAGGGCATTTACAATGTCATAAATATCCTTATATAAG~TGATCTC TTATCAATTAGCACTCACTAATGTACTACTAATTGAGCAACCTACGCACTCAGTTGACTACGTAAATCTGTCAGGTCTTTTGTGATT TCCTCTGATGCAGGAGAGGAAAAATTGTAATTGATGAAAATAATGAATGAAAATCAACAGATGAATAAATGGTTCTTTATAAGTGAA
87 153
950
1037 1124 1211 1219
Fig. 1. Nucleotide sequence of carbonyl reductase from a cDNA library. The clone is ]219 base pairs with an open reading-frame coding for 277 amino acids. Underlined amino acids were confirmed by amino acid sequencing of Lys-C peptides. Each peptide ends with a lysine residue conforming to the specificity of Lys-C. The peptide shown in brackets was the original peptide from which an oligonucleotide probe was generated and also contains a modified lysine residue shown in parentheses. The underlined polyadenylation signal AATAA,5_ is shown in the 3' noncoding region.
151 [14] and mass spectrometry [15] were performed as previously described. Cloning. A ?~gtll liver c D N A library (Clontech, Palo Alto, CA) and a human breast cancer cell (MCF-7) c D N A library constructed in ?~gtll with a cDNA synthesis kit (Bethesda Research L a b o r a t o r i e s , Gaithersburg, MD) was screened for carbonyl reductase. Oligonucleotide probes used for library screening were synthesized from amino acid sequence data obtained from carbonyl reductase peptides according to the rules described by Lathe [16]. Screening was performed according to the method of Berent et al. [17]. Sub-cloning into Bluescript vectors (Stratagene, San Diego, CA) was performed by standard procedures [18,19]. DNA sequencing. Double-stranded D N A sequencing was performed by a modified Sanger [20,21] procedure with a Sequenase kit (United States Biochemical Corporation, Cleveland, O H ) and 35S-labeled-dATP (New England Nuclear, Boston, MA). Both strands of c D N A were sequenced. Southern analysis. Genomic D N A was digested with different restriction enzymes (10 U / ~ g ) overnight at 37 ° C. In gel hybridizations were performed according to the method of Miyada and Wallace [22].
Results
Messenger RNA induction
Carbonyl reductase cDNA isolation
MCF-7 cells were grown in Eagle's minimum essential medium with Eagle's salts supplemented with tglutamine, sodium pyruvate and 5% heat inactivated fetal calf serum (Gibco Laboratories, G r a n d Island, NY). Cells were inoculated into 150 m m tissue culture
Amino acid sequencing of Lys-C peptides yielded a peptide with the sequence I L L N A C C P G W V R T D M A G P . A synthetic oligonucleotide probe (45mer) was synthesized with the corresponding sequence G G G G C CAGCCATGTCTGTCCGCACCCAGCCAGGGCAG C A G G C A T T . The probe was labeled with [32p]ATP and used to screen a ?~gtll human liver library under conditions corresponding to 80% homology [16]. Several positive c D N A clones were purified and sequenced. The longest clone was 1040 base pairs. The c D N A was labeled with [32p]dCTP by random priming and used as a probe to detect m R N A isolated from MCF-7 cells by northern analysis. A single band corresponding to a molecular size of approx. 1.2 kilobases was determined (Fig. 2A1). Since MCF-7 cells displayed a high abundance of carbonyl reductase m R N A , a c D N A library was constructed from MCF-7 poly (A) R N A and screened for a full-length c D N A with the previous clone. Several clones were isolated, one of which contained a full length sequence. The c D N A contained 1219 base pairs and is shown in Fig. 1. An open reading frame of 831 base pairs corresponding to 277 amino acids extends from the A T G start codon at position 94 to the T G A stop codon at position 924. The 3' noncoding region contains 295 base pairs and includes the polyadenylation signal AATAAA. The underlined sequences represent sequences of carbonyl reductase confirmed by amino acid sequence data from peptides produced by proteolytic digestion. The isolation of the C-terminal
A
MCF-7 1 234
B Hep-G2 1234
Fig. 2. Induction of carbonyl reductase mRNA in human cells. Northern blot analyses of a human breast cancer cell line (MCF-7) and of a human hepatoma cell line (HepG2). Cells were induced with 10 /~M final concentration of inducer for 24 h. Total cytoplasmic RNA was isolated from MCF-7 cells and 25 pg perlane was analyzed. Poly(A) RNA was isolated from HepG2 cells by binding to oligo dT cellulose. 10 #g of poly(A) RNA was analyzed. A1, control; A2, BHA; A3, fl-naphthoflavone; A4, sudan I. B1, control; B2, fl-naphthoflavone; B3, sudan I; B4, 20 /xg of ribosomal and nonpoly(A) mRNA from oligo-dT isolation.
dishes and allowed to grow to 70% confluency. Inducer was dissolved in D M S O to give a 10 m M solution. The inducer w,a,g added to the cells at a final concentration of 10 /L'm. Total cytoplasmic R N A was isolated 24 h after induction by a standard procedure [23], except that 1 m M dithiothreitol and 1000 U / m l of Rnasin (Promega, Madison, WI) were added to the lysis buffer. Northern analysis. Cytoplasmic R N A was denatured in f o r m a l d e h y d e / f o r m a m i d e and separated in a 1.3% agarose formaldehyde gel [24]. The R N A was blotted to Genetrans nylon membranes (Plasco, Woburn, MA), prehybridized for 4 h and hybridized at 50 ° C overnight according to instructions supplied with zetaprobe nylon membranes (Bio-Rad, Richmond, CA) Band intensity was quantified with a Bio-Rad model 620 video densitometer. Somatic cell hybrids. A panel of 17 m o u s e / h u m a n somatic cell hybrids were derived from the fusion of thymidine kinase deficient mouse cells (B82, G M 0347A) and normal human male fibroblasts ( I M R 91) as described previously [25]. Somatic cell hybrid D N A isolation and filter hybridization was accomplished as previously described [26-28].
152 TABLE I
Segregation of carbonyl reductase gene with human chromosomes in mouse-human somatic cell hybrids Hybrid
CR *
Human chromosomes * *
clone 84- 5 84-13 84-20 84-21 84-25 84-26 84-27 84-30 84-37 84- 2 84- 3 84- 4 84- 7 84-34 84-35 84-38 84-39
+ + + + + + + + + . -
.
N o . o f discordant hybrids
1
2
3
4
5
6
7
8
9
10
11
. + + + + . + -
+ (+ )
+
+ +
+ + -
+ + + + -
+ + + . + + + + +
-
+ + +
+ + + + + + + + + + + -
+ + + (+ ) + + (+ ) -
+ + + + + + + + + + + + -
+ + + + + + + + + + + +
+ + + + + (+ ) + + + + + + + +
-
+ + + + + + + + + -
(+ ) + + + + + + -
9
10
11
4
9
13
11
9
8
8
.
.
.
.
.
12
* + , i n d i c a t e s p r e s e n c e o f the C R s e q u e n c e s in the h y b r i d c l o n e d e t e r m i n e d b y the p r e s e n c e o f the h u m a n b a n d ; - ,
i n d i c a t e s a b s e n c e o f the
gene. * * + , i n d i c a t e s p r e s e n c e o f the h u m a n c h r o m o s o m e in g r e a t e r t h a n 30% o f m e t a p h a s e s a n a l y z e d ; ( + ), i n d i c a t e s p r e s e n c e o f the c h r o m o s o m e in 1 0 - 3 0 % of the m e t a p h a s e s a n a l y z e d ; - , i n d i c a t e s a b s e n c e o f the h u m a n c h r o m o s o m e .
peptide
RVEQW
a tryptophan correct. lysine
The
peptide at
at
coded
analysis this for
a
indicated mass
ified
lysine
residues
later
did
lysine
was
ended
with
termination
codon
was
in brackets
contained
not
that
did
giving
position show
residue.
A
the mass
Lys-C cleave
did
mass
sequence
spectrum
of
of the modifying
the
rise to a peptide
minor
peptide
unmodified
19
was
amino
acids
also isolated
containing
and
the
sequenced.
the
the
group
Induction o f carbonyl reductase m R N A A human
hepatoma
breast
cancer
final
concentration
(BHA),
lysine
three
MCF-7
total
of 22 amino
acids.
poly(A)
RNA
of
B-naphthoflavone, RNA (Fig.
electrophoresis,
A ~,..,~,..,~ B ,..,~
cell line
cell line (MCF-7)
mod-
next
of
lysine
acid amino
eDNA
recognize
A
a modified Amino
recognizable
the
not at
821.
any
However,
that
units. but
the protein
the TGA
nucleotide
position.
peptide 72
that
residue
sequence acid
confirmed
and
hybridized
or Sudan 2A) were
transferred
to
the
and
exposed
a human to 10/~M
2,(3)-t-butyl-4-hydroxanisole
(Fig. 2B)
(HepG2) were
labeled
A BCD
and
I for 24 h. 25/tg 10
analyzed to
by
nylon
carbonyl
/~g of
agarose
membranes reductase
E FGH
of
HepG2 gel and cDNA
I
1 3.06.1_
iiii!ii!~ilil
Fig. 3. In-gel S o u t h e r n a n a l y s i s o f r e s t r i c t i o n e n z y m e d i g e s t e d h u m a n D N A . A, h u m a n l y m p h o c y t e D N A ; B, M C F - 7 D N A . D N A w a s d i g e s t e d o v e r n i g h t at 3 7 ° C w i t h 10 U / / ~ g o f e n z y m e . T h e D N A w a s s e p a r a t e d o n a 0.8% a g a r o s e gel a n d h y b r i d i z e d w i t h l a b e l e d c a r b o n y l r e d u c t a s e cDNA.
Fig. 4. S o u t h e r n b l o t o f D N A f r o m m o u s e / h u m a n h y b r i d s h y b r i d i z e d w i t h c a r b o n y l r e d u c t a s e c D N A . L a n e s : A - G s o m a t i c cell h y b r i d s (respectively, 84-27, 84-30, 84-34, 84-35, 84-37, 84-38, 84-39); H , m o u s e p a r e n t ; a n d I, h u m a n p a r e n t . T h e p r e s e n c e o f the 6.1 k i l o b a s e h u m a n b a n d w a s u s e d to s c o r e t h e s o m a t i c cell h y b r i d s . L a n e s A , B a n d E c o n t a i n the h u m a n gene.
153
12
13
14
15
16
17
18
19
20
21
22
X
-
(+)
+
+
-
+
-
+
-
+
+
+
+
+
+
--
+
--
+
+
--
+
+
--
_
_
(+) + (+)
+ .
+ + .
-
_ _
+ + +
+ -
_ _
+ + _
+ + +
+ -
+
+
+
+
+
+
+
+
+
+
+
--
+
--
_
+
--
+
+
--
+
+
--
_
_
+
+
-
_
+
+
-
+
+
-
_
+
--
+
--
_
+
--
_
+
+
+
--
_
+
--
+
-t-
+
+
+
+
+
--
+
--
--
--
+
+
+
--
+
+
+
+
.
.
.
.
--
+
+
+
--
+
+
+
+
.
.
.
.
+
--
+
--
+
+
--
_
+
.
.
.
.
_
_
+
-
_
+
-
(+)
+
-
-
_
+
--
+
+
--
q-
+
.
+
+
+
+
-
+
+
-
_
_
_
_
+
--
+
.
10
11
10
(+)
5
.
7
.
8
9
p r o b e . A single b a n d c o r r e s p o n d i n g to a 1.2 k i l o b a s e m e s s a g e was i n d u c e d in b o t h cell lines. Fig. 2 A shows t h a t c a r b o n y l r e d u c t a s e in M C F - 7 cells is i n d u c e d 3-fold, 4.6-fold a n d 4.1-fold b y B H A (lane 2), f l - n a p h t h o f l a vone (lane 3), a n d S u d a n I (lane 4), respectively. L a n e 1 is the u n i n d u c e d control. Fig. 2B shows i n d u c t i o n in H e p G 2 cells of 2.5-fold a n d 4.5-fold b y f l - n a p h t h o f l a v o n e (lane 2) a n d S u d a n I (lane 3), respectively. L a n e 1 is the u n i n d u c e d control. L a n e 4 c o n t a i n s 20 # g of ribos o m a l a n d p o l y ( A ) m i n u s R N A o b t a i n e d f r o m chrom a t o g r a p h y of total R N A on an o l i g o - d T cellulose column.
Southern analysis H u m a n l y m p h o c y t e D N A (Fig. 3A) a n d M C F - 7 D N A (Fig. 3B) were digested with several restriction e n z y m e s a n d a n a l y z e d on an 0.8% agarose gel. T h e c D N A p r o b e was l a b e l e d a n d h y b r i d i z e d to the D N A in the gel (Fig. 3). T h e results show a single m a j o r b a n d a n d s o m e t i m e s a m i n o r b a n d h y b r i d i z i n g to the probe. T h e r e is an i n t e r n a l BamHI site in the c D N A which s h o u l d result in two bands. T h e d a t a are consistent with c a r b o n y l r e d u c t a s e being c o d e d for b y a single gene.
Chromosomal location T h e c h r o m o s o m a l l o c a t i o n o f the h u m a n c a r b o n y l r e d u c t a s e gene was d e t e r m i n e d b y e x a m i n i n g the p a n e l of h u m a n / m o u s e s o m a t i c cell h y b r i d s with the c D N A
(+)
.
+ .
.
.
(+) .
12
.
8
(+) _ _
+
Y
(+) _ +
.
.
+ .
0
.
7
7
6
p r o b e (Fig. 4). T h e results of these studies are s h o w n in T a b l e I. H u m a n c a r b o n y l r e d u c t a s e is assigned to chrom o s o m e 21. Discussion
W e have d e t e r m i n e d the sequence of a c D N A c o d i n g for a h u m a n c a r b o n y l r e d u c t a s e gene i s o l a t e d f r o m the b r e a s t cancer cell line, M C F - 7 . P a r t i a l c D N A clones f r o m a h u m a n liver l i b r a r y were also isolated a n d were identical to the M C F - 7 c D N A clone. T h e c a r b o n y l r e d u c t a s e clone was i d e n t i c a l to a r e c e n t l y p u b l i s h e d c D N A clone f r o m h u m a n p l a c e n t a [29]. Thus, c a r b o n y l r e d u c t a s e m R N A has n o w b e e n i d e n t i f i e d in liver, b r e a s t a n d p l a c e n t a l tissue. C a r b o n y l r e d u c t a s e s are u b i q u i t o u s in n a t u r e a n d m e t a b o l i z e a variety of a l d e h y d e a n d k e t o n e c o m p o u n d s i n c l u d i n g m a n y d r u g s [1,2]. Based on f u n c t i o n a l studies, one m i g h t have e x p e c t e d to find a large class of enzymes s t r u c t u r a l l y r e l a t e d to c a r b o n y l reductase. However, h o m o l o g y searches o f the P I R a n d Swiss-Protein d a t a b a n k s t h r o u g h Bionet d i d n o t d e t e c t a n y significant h o m o l o g y with o t h e r k n o w n proteins. Therefore, c a r b o n y l r e d u c t a s e s m u s t b e f u n c t i o n a l l y related r a t h e r t h a n s t r u c t u r a l l y related. I d e n t i f i c a t i o n of a m o d i f i e d lysine r e s i d u e at a m i n o acid 239 was observed. T h e m a s s of the m o d i f y i n g g r o u p was 72 a n d d i d n o t c o r r e s p o n d to a n y c o m m o n lysine m o d i f y i n g group. T h e significance of this m o d i f i -
154 cation is not known. The modification of lysine residues may explain the multiple bands of activity observed in reports on purification of the enzyme by isoelectrofocusing or DEAE chromatography. Differences in molecular weight have also been observed [10,11], indicating the possible presence of other proteins or additional protein modifications. A potential glycosylation site occurs at amino acid 137. Glycosylation could generate proteins with molecular mass differences of 1400-2000 Da giving rise to observed differences reported in the literature. Induction of carbonyl reductase mRNA by compounds that induce enzymes involved in xenobiotic detoxification imply that carbonyl reductase is also important in cell protection. The mechanism of induction of carbonyl reductase mRNA is presently unkown. However, one possible gene regulatory mechanism which may be involved in the induction of carbonyl reductase involves the Ah locus. The Ah locus controls induction of the aryl hydrocarbon hydroxylase system which is a major metabolic system for xenobiotic detoxification [30]. This locus is involved in the induction of many drug metabolizing enzymes and in the induction of DT-diaphorase [31-33]. DT-diaphorase is induced by many structurally unrelated compounds such as phenolic antioxidants, azo-dyes and polycyclic aromatic hydrocarbons [7,8,34]. It has been suggested that regulatory control by the Ah locus acts indirectly by controlling induction of the aryl hydrocarbon hydroxylase system [35]. The aryl hydrocarbon hydroxylase enzymes oxidize the inducer producing a product(s) which activates transcription of the DT-diaphorase gene. Azo-dyes are activated by direct oxygenation from the aryl hydrocarbon hydroxylase system and are also inducers of DT-diaphorase [5] and carbonyl reductase (Fig. 2). The similarities in induction of DT-diaphorase and carbonyl reductase with azo-dyes may indicate that the Ah locus controls both enzymes. Identifying regulatory sequences in genomic clones of carbonyl reductase and DT-diaphorase will determine whether common regulatory sequences exist. Localization of carbonyl reductase to chromosome 21 may have implications to certain genetic diseases such as Down's syndrome. Some aspects of Down's syndrome such as premature aging, increased incidence of leukemia, brain damage similar to Alzheimer's disease, and lipofusin accumulation are symptoms that have been associated with oxidative damage [36]. Superoxide dismutase, an enzyme coded for on the distal arm of chromosome 21, shows a gene dosage effect in trisomy 21 patients and is involved in oxygen free radical metabolism. It is thought to be involved in the pathology of Down's syndrome. Carbonyl reductase, acting as a quinone reductase, could influence both oxygen radical generation and cellular antioxidant defenses by modulating the redox state of important cellular
metabolites [3,37,38]. Presently this is highly speculative and one must wait for more detailed chromosome mapping, gene dosage and metabolite data. The role of anthracycline metabolism in human leukemic cells is well documented and has profound effects upon the pharmacological properties of these drugs [5,39,40]. However, the mechanisms by which the pharmacological changes occur are not understood and are somewhat controversial. In addition, the metabolism of other quinone and carbonyl substrates and the entire process of xenobiotic protection is currently being investigated. The cloning of carbonyl reductase will allow us to assess its importance in xenobiotic cell protection and drug metabolism.
Acknowledgements We thank William Kaplan, Leonar Directo and Camilla Heinzmann for expert technical assistance. This work was supported by the City of Hope Cancer Center Support Grant CA 33572.
References 1 Bachur, N.R. (1976) Science 193, 595-597. 2 Felsted, R.L. and Bachur, N.R. (1980) in Drug Metabolism Reviews (DiCarol, F.J., eds.), Vol. 11, pp. 1-70, Marcel Dekker, New York. 3 Wermuth, B., Platts, K.L., Seidel, A. and Oesch, F. (1986) Biochem. Pharmacol. 35, 1277-1282. 4 Ahmed, N.K., Felsted, R.L. and Bachur, N.R. (1978) Biochem. Pharmacol. 27, 2713-2719. 5 Bachur, N.R., Steele, M., Meriwhether, W.D. and Hildebrand, R.C. (1976) J. Med. Chem. 19, 651-654. 6 Huggins, C.B., Ueda, N. and Russo, A. (1978) Proc. Natl. Acad. Sci. USA 75, 4524-4527. 7 Wattenberg, L.W. and Lam, L.K.T. (1981) in lnhibitors of Tumor Induction and Development (Zedeck, M.S. and Lipkin, M., eds.), pp. 1-22, Plenum Press, New York. 8 Ernster, L. (1986) Chem. Scripta 27A, 1-13. 9 Wermuth, B. (1981) J. Biol. Chem. 256, 1206-1213. 10 Felsted, R.L. and Bachur, N.R. (1980) in Enzymatic Basis of Detoxification (Jacoby, W.B., eds.), Vol. 1, pp. 281-293, Academic Press, New York. 11 Ahmed, N.K., Felsted, R.L. and Bachur, N.R. (1979) J. Pharmacol. Exper. Ther. 209, 12-19. 12 Waxdal, M.J., Konigsberg, W.H., Itenley, W.L. and Edelman, G.M. (1968) Biochem. 9, 1959-1966. 13 Jekel, P.A., Weijer, W.J. and Geintema, J.J. (1983) Anal. Biochem. 134, 347-354. 14 Paxton, R.J., Mooser, G., Pande, H., Lee, T. and Shively, J.E. (1987) Proc. Natl. Acad. Sci. USA 84, 920-924. 15 Haniu, M., Iyanagi, T., Miller, P., Lee, T. and Shively, J.E. (1986) Biochem. 25, 7906-7911. 16 Lathe, R. (1985) J. Mol. Biol. 183, 1-12. 17 Berent, S.L., Mahmoudi, M., Torcyznski, R.M., Bragg, P.W. and Bollen, A.P. (1985) Biotechniques 3, 208-220. 18 Tautz, D. and Renz, M. (1983) Anal. Biochem. 132, 14-19. 19 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor. 20 Hattori, M. and Sakaki, Y. (1986) Anal. Biochem. 152, 232-238.
155 21 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 22 Miyada, C.G. and Wallace, R.B. (1986) Methods Enzymol. 154, 94-107. 23 Gilman, M. (1989) in Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kinston, R.E., Moore, D.D., Smith, J.A., Seidman, J.G. and Struhl, K., eds.), Vol. 1, pp. 4.1.2-4.1.6, John Wiley & Sons, New York. 24 Fourney, R.M., Miyakoshi, J., Day, R.S., III and Paterson, M.C. (1988) Focus 10, 5-7. 25 Mohandas, T., Heinzmann, C., Sparkes, R.S., Wasmuth, J., Edwards, P. and Lusis, A.J. (1986) Somat. Cell and Mol. Genet. 12, 89-94. 26 Fodor, E.J.B. and Doty, P. (1979) Biochem. Biophys. Res. Commun. 77, 1478-1485. 27 Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. 28 Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 29 Wermuth, B., Bohren, K.M., Heinemann, G., Von Warthburg, J.P. and Gabbay, K.H. (1988) J. Biol. Chem. 263, 16185-16188. 30 Gonzalez, F.J., Tukey, R.H. and Nebert, D.W. (1984) Mol. Pharmacol. 26, 117-121.
31 Kumaki, K., Jensen, N.M., Shire, J.G.M. and Nebert, D. (1977) J. Biol. Chem. 252, 157-165. 32 Robertson, J.A., Chen, H.C. and Nebert, D.W. (1986) J. Biol. Chem. 261, 15794-15799. 33 Jaiswal, A.K., McBride, O.W., Adesnik, M. and Nebert, D. (1988) J. Biol. Chem. 263, 13572-13578. 34 Benson, A.M., Hunkeler, M.J. and Talalay, P. (1980) Proc. Natl. Acad. Sci. USA 77, 5216-5220. 35 Talalay, P. and Prochaska, H.J. (1987) Chem. Scr. 27A, 61-66. 36 Sinet, P.M. (19820 Ann. NY Acad. Sci. 396, 83-94. 37 Akman, S., Dietrich, M., Chlebowski, R., Limberg, P. and Block, J.B. (1985) Cancer Res. 45, 5257-5262. 38 Akman, S., Doroshow, J.H., Dietrich, M.F., Chlebowski, R.T. and Block, J.S. (1987) J. Pharmacol. Exper. Ther. 240, 486-491. 39 Speth, P.A.J., Van Hoesel, Q.G.C.M. and Haanen, C. (1988) Clin. Pharmacol. 15, 15-31. 40 Olson, R.D., Mushlin, P.S., Brenner, D.E., Fleischer, S., Cusack, B.J., Chang, B.K. and Boucher, R.J., Jr. (1988) Proc. Natl. Acad. Sci. USA 85, 3585-3589.
149
Elsevier BBAEXP 92028
Induction of a human carbonyl reductase gene located on chromosome 21 Gerald L. Forrest 1 Steven A k m a n 2, Siegfried Krutzik 3, R a y m o n d J. Paxton 4, Robert S. Sparkes 5, James Doroshow 2 Ronald L. Felsted 6 Constance J. Glover 6 Thomas M o h a n d a s 7 and Nicholas R. Bachur 8 Divisions of t Biology and 4 Immunology, Beckman Research Institute at the City of Hope and 2 Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA, 3 Disease Detection International, Irvine, CA, 5 Department of Medicine, UCLA Medical Center, Los Angeles, CA, 6 Laboratory of Biological Chemistry, National Cancer Institute, Bethesda, MD, 7 Department of Pediatrics, Harbor/UCLA Medical Center, Torrance, CA, and 8 University of Maryland Cancer Center, Baltimore, AID (U.S.A.)
(Received 2 June 1989)
Key words: cDNA; Daunorubicin reductase; Drug metabolism; MCF-7
Carbonyl reductase (EC 1.1.1.184) belongs to the group of enzymes called aldo-keto reductases. It is a NADPH-dependent cytosolic protein with specificity for many carbonyl compounds including the antitumor anthracycline antibiotics, daunoruhicin and doxorubicin. Human carbonyl reductase was cloned from a breast cancer cell line (MCF-7). The cDNA clone contained 1219 base paires with an open reading frame corresponding to 277 amino acids encoding a protein of M r 30375. Southern analysis of genomic DNA digested with several restriction enzymes and analyzed by hybridization with a labeled cDNA probe indicated that carbonyl reductase is probably ceded by a single gene and does not belong to a family of structurally similar enzymes. Southern analysis of 17 m o u s e / h u m a n somatic cell hybrids showed that carbonyl reductase is located on chromosome 21. Carbonyl reductase mRNA could be induced 3-4-fold in 24 h with 10/~M 2,(3)-t-butyl-4-hydroxyanisole (BHA), fl-naphthoflavone or Sudan 1.
Introduction Carbonyl reductase (secondary-alcohol : N A D P + oxidoreductase, EC 1.1.1.184) belongs to a group of N A D P H - d e p e n d e n t cytosolic enzymes called aldo-keto reductases [1,2]. These enzymes are ubiquitous in nature, metabolizing quinones and a variety of antitumor drugs containing carbonyl groups including the anthracycline antibiotics, daunorubicin and doxorubicin [1,2]. The role of aldo-keto reductases in drug metabolism is well documented. Carbonyl reductase, acting as an aldo-keto reductase, can reduce the methyl ketone in the carbon side-chain of the antitumor antibiotics, daunorubicin and doxorubucin, to its corresponding alcohol.
The sequence data in this paper have been submitted to the EMBL/ Genbank Data Libraries under accession number J04056.
Abbreviation: BHA, 2,(3)-t-butyl-4-hydroxyanisole. Correspondence: G. Forrest, Division of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, U.S.A.
Ahmed et al. [4] demonstrated differences in anthracycline drug metabolism in tissues from several animal species. H u m a n and rabbit tissue have an enhanced rate of daunorubicin metabolism which is accounted for by carbonyl reductase activity while the rat and mouse tissues are deficient in this activity. Although the mechanistic effects of drug metabolism in cells is not well understood, the reduction of daunorubicin and doxorubicin by carbonyl reductase affects the pharmacological properties of these drugs [5]. Wermuth et al. [3] demonstrated that carbonyl reductase can also act as a quinone reductase [3]. Carbonyl reductase acting as a quinone reductase may protect humans against quinone mediated carcinogenic, mutagenic and toxic effects similar to the protective role that another quinone reductase, DT-diaphorase, affords rats and mice [6-8]. Carbonyl reductase accounts for most of the N A D P H - r e d u c i n g activity in h u m a n liver, while DT-diaphorase accounts for less than 10% of the reducing activity [3]. In rat and mouse liver, carbonyl reductase appears to be low or absent while DT-diaphorase levels are higher and highly inducible [3,8]. Since several quinone substrates are metabolized by
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
150 both carbonyl reductase and DT-diaphorase, carbonyl reductase may supplant DT-diaphorase in the human liver [3,9]. We have cloned a human carbonyl reductase cDNA from liver and from a breast cancer cell line (MCF-7). Carbonyl reductase mRNA was induced in both breast and liver cells by compounds known to induce enzymes involved in protecting cells against the carcinogenic mutagenic and cytotoxic effects of quinones and other xenobiotics. The gene was localized to chromosome 21 which may have implications concerning genetic disease. Materials and Methods
Carbonyl reductase purification. Carbonyl reductases displaying daunorubicin reductase activity were isolated
and purified from human liver based on their low molecular weight and NADPH co-factor specificity [2,10]. The carbonyl reductases were purified as previously published, except that ion exchange chromatography was replaced by a second gel-filtration step using Sephacryl S-200 (3 x 80 cm) (Pharmacia, Piscataway, N J) and the isoenzymes were resolved by chromatofocusing (pH gradient 7.5-6.0) [11]. The enzymes were stored at - 7 0 ° C for 5 years. After thawing, a HPLC chromatography step was required to purify the protein for sequencing. Separation was obtained by reversephase HPLC on a /~Bondapak phenyl column (Waters, Milford, MA) through a linear gradient of 0-90% acetonitrile (vol/vol) in 0.1% trifluoroacetic acid (vol/vol) over 100 min with a flow rate of 1 ml/min. Peptide sequencing. Protein carboxymethylation [12], proteinase digestion with Lys-C [13] peptide sequencing
NUCLEOTIDE CAGACTCGATGTGCCTCTGGAACACGCTGCGGGGCTCCCGGGCCTGAGCCAGGTCTGTTCTCCACGCAGGTGTTCCGCGCGCCCCGT TCAGCC ATG TCG TCC GGC ATC CAT GTA GCG CTG GTG ACT GGA GGC AAC AAG GGC ATC GGC TTG M S S G I H V A L V T G G N K G I G L
GCC A
ATC I
GTG V
CGC R
GAC D
CTG L
TGC C
CGG R
CTG L
TTC F
TCG S
GGG G
GAC D
GTG V
GTG V
CTC L
ACG T
GCG A
CGG R
GAC D
GTG V
ACG T
CGG R
219
GGC G
CAG Q
GCG A
GCC A
GTA V
CAG Q
CAG Q
CTG L
CAG Q
GCG A
GAG E
GGC G
CTG L
AGC S
CCG P
CGC R
TTC F
CAC H
CAG Q
CTG L
GAC D
ATC I
285
GAC D
GAT D
CTG L
CAG AGC Q S
ATC I
CGC R
GCC A
CTG L
CGC R
GAC D
TTC F
CTG L
CGC AAG R K
GAG E
TAC Y
GGG G
GGC G
CTG L
GAC D
GTG V
351
CTG L
GTC V
AAC N
AAC N
GCG A
GGC ATC G I
GCC A
TTC F
AAG K
GTT V
GCT A
GAT D
CCC P
ACA T
CCC P
TTT F
CAT H
ATT I
CAA Q
GCT A
GAA E
417
GTG V
ACG T
ATG M
AAA K
ACA T
AAT N
TTC F
TTT F
GGT G
ACC T
CGA R
GAT D
GTG V
TGC C
ACA T
GAA E
TTA L
CTC L
CCT P
CTA ATA L I
AAA K
483
CCC P
CAA Q
GGG G
AGA R
GTG V
GTG V
AAC N
GTA V
TCT S
AGC S
ATC I
ATG M
AGC S
GTC V
AGA R
GCC A
CTT AAA L K
AGC S
TGC C
AGC S
CCA P
549
GAG E
CTG L
CAG Q
CAG Q
AAG K
TTC F
CGC R
AGT S
GAG E
ACC T
ATC I
ACT T
GAG E
GAG E
GAG E
CTG L
GTG V
GGG G
CTC ATG L M
AAC N
AAG K
615
TTT F
GTG V
GAG E
GAT D
ACA T
AAG K
AAG K
GGA G
GTG V
CAC H
CAG Q
AAG K
GAG E
GGC G
TGG W
CCC P
AGC S
AGC S
GCA A
TAC Y
GGG G
GTG V
681
ACG T
AAG K
ATT I
GGC G
GTC V
ACC T
GTT V
CTG L
TCC S
AGG R
ATC I
CAC H
GCC A
AGG R
AAA K
CTG L
AGT S
GAG E
CAG Q
AGG R
AAA K
GGG G
747
GAC D
AAG K
ATC [I
CTC L
CTG L
AAT N
GCC A
TGC C
TGC C
CCA P
GGG G
TGG W
GTG V
AGA R
ACT T
GAC D
ATG M
GCG A
GGA G
CCC P
AAG IK)
GCC A
813
ACC T
AAG K]
AGC S
CCA P
GAA E
GAA E
GGT G
GCA A
GAG E
ACC T
CCT P
GTG V
TAC Y
TTG L
GCC A
CTT L
TTG L
CCC P
CCA P
GAT D
GCT A
GAG E
879
GGT G
CCC P
CAT H
GGA G
CAA Q
TTT F
GTT V
TCA S
GAG E
AAG K
AGA R
GTT V
GAA E
CAG Q
TGG w
TGA *
GCTGGGCTCACAGCTCCATCCAT
GGGCCCCATTTTGTACCTTGTCCTGAGTTGGTCCAAAGGGCATTTACAATGTCATAAATATCCTTATATAAG~TGATCTC TTATCAATTAGCACTCACTAATGTACTACTAATTGAGCAACCTACGCACTCAGTTGACTACGTAAATCTGTCAGGTCTTTTGTGATT TCCTCTGATGCAGGAGAGGAAAAATTGTAATTGATGAAAATAATGAATGAAAATCAACAGATGAATAAATGGTTCTTTATAAGTGAA
87 153
950
1037 1124 1211 1219
Fig. 1. Nucleotide sequence of carbonyl reductase from a cDNA library. The clone is ]219 base pairs with an open reading-frame coding for 277 amino acids. Underlined amino acids were confirmed by amino acid sequencing of Lys-C peptides. Each peptide ends with a lysine residue conforming to the specificity of Lys-C. The peptide shown in brackets was the original peptide from which an oligonucleotide probe was generated and also contains a modified lysine residue shown in parentheses. The underlined polyadenylation signal AATAA,5_ is shown in the 3' noncoding region.
151 [14] and mass spectrometry [15] were performed as previously described. Cloning. A ?~gtll liver c D N A library (Clontech, Palo Alto, CA) and a human breast cancer cell (MCF-7) c D N A library constructed in ?~gtll with a cDNA synthesis kit (Bethesda Research L a b o r a t o r i e s , Gaithersburg, MD) was screened for carbonyl reductase. Oligonucleotide probes used for library screening were synthesized from amino acid sequence data obtained from carbonyl reductase peptides according to the rules described by Lathe [16]. Screening was performed according to the method of Berent et al. [17]. Sub-cloning into Bluescript vectors (Stratagene, San Diego, CA) was performed by standard procedures [18,19]. DNA sequencing. Double-stranded D N A sequencing was performed by a modified Sanger [20,21] procedure with a Sequenase kit (United States Biochemical Corporation, Cleveland, O H ) and 35S-labeled-dATP (New England Nuclear, Boston, MA). Both strands of c D N A were sequenced. Southern analysis. Genomic D N A was digested with different restriction enzymes (10 U / ~ g ) overnight at 37 ° C. In gel hybridizations were performed according to the method of Miyada and Wallace [22].
Results
Messenger RNA induction
Carbonyl reductase cDNA isolation
MCF-7 cells were grown in Eagle's minimum essential medium with Eagle's salts supplemented with tglutamine, sodium pyruvate and 5% heat inactivated fetal calf serum (Gibco Laboratories, G r a n d Island, NY). Cells were inoculated into 150 m m tissue culture
Amino acid sequencing of Lys-C peptides yielded a peptide with the sequence I L L N A C C P G W V R T D M A G P . A synthetic oligonucleotide probe (45mer) was synthesized with the corresponding sequence G G G G C CAGCCATGTCTGTCCGCACCCAGCCAGGGCAG C A G G C A T T . The probe was labeled with [32p]ATP and used to screen a ?~gtll human liver library under conditions corresponding to 80% homology [16]. Several positive c D N A clones were purified and sequenced. The longest clone was 1040 base pairs. The c D N A was labeled with [32p]dCTP by random priming and used as a probe to detect m R N A isolated from MCF-7 cells by northern analysis. A single band corresponding to a molecular size of approx. 1.2 kilobases was determined (Fig. 2A1). Since MCF-7 cells displayed a high abundance of carbonyl reductase m R N A , a c D N A library was constructed from MCF-7 poly (A) R N A and screened for a full-length c D N A with the previous clone. Several clones were isolated, one of which contained a full length sequence. The c D N A contained 1219 base pairs and is shown in Fig. 1. An open reading frame of 831 base pairs corresponding to 277 amino acids extends from the A T G start codon at position 94 to the T G A stop codon at position 924. The 3' noncoding region contains 295 base pairs and includes the polyadenylation signal AATAAA. The underlined sequences represent sequences of carbonyl reductase confirmed by amino acid sequence data from peptides produced by proteolytic digestion. The isolation of the C-terminal
A
MCF-7 1 234
B Hep-G2 1234
Fig. 2. Induction of carbonyl reductase mRNA in human cells. Northern blot analyses of a human breast cancer cell line (MCF-7) and of a human hepatoma cell line (HepG2). Cells were induced with 10 /~M final concentration of inducer for 24 h. Total cytoplasmic RNA was isolated from MCF-7 cells and 25 pg perlane was analyzed. Poly(A) RNA was isolated from HepG2 cells by binding to oligo dT cellulose. 10 #g of poly(A) RNA was analyzed. A1, control; A2, BHA; A3, fl-naphthoflavone; A4, sudan I. B1, control; B2, fl-naphthoflavone; B3, sudan I; B4, 20 /xg of ribosomal and nonpoly(A) mRNA from oligo-dT isolation.
dishes and allowed to grow to 70% confluency. Inducer was dissolved in D M S O to give a 10 m M solution. The inducer w,a,g added to the cells at a final concentration of 10 /L'm. Total cytoplasmic R N A was isolated 24 h after induction by a standard procedure [23], except that 1 m M dithiothreitol and 1000 U / m l of Rnasin (Promega, Madison, WI) were added to the lysis buffer. Northern analysis. Cytoplasmic R N A was denatured in f o r m a l d e h y d e / f o r m a m i d e and separated in a 1.3% agarose formaldehyde gel [24]. The R N A was blotted to Genetrans nylon membranes (Plasco, Woburn, MA), prehybridized for 4 h and hybridized at 50 ° C overnight according to instructions supplied with zetaprobe nylon membranes (Bio-Rad, Richmond, CA) Band intensity was quantified with a Bio-Rad model 620 video densitometer. Somatic cell hybrids. A panel of 17 m o u s e / h u m a n somatic cell hybrids were derived from the fusion of thymidine kinase deficient mouse cells (B82, G M 0347A) and normal human male fibroblasts ( I M R 91) as described previously [25]. Somatic cell hybrid D N A isolation and filter hybridization was accomplished as previously described [26-28].
152 TABLE I
Segregation of carbonyl reductase gene with human chromosomes in mouse-human somatic cell hybrids Hybrid
CR *
Human chromosomes * *
clone 84- 5 84-13 84-20 84-21 84-25 84-26 84-27 84-30 84-37 84- 2 84- 3 84- 4 84- 7 84-34 84-35 84-38 84-39
+ + + + + + + + + . -
.
N o . o f discordant hybrids
1
2
3
4
5
6
7
8
9
10
11
. + + + + . + -
+ (+ )
+
+ +
+ + -
+ + + + -
+ + + . + + + + +
-
+ + +
+ + + + + + + + + + + -
+ + + (+ ) + + (+ ) -
+ + + + + + + + + + + + -
+ + + + + + + + + + + +
+ + + + + (+ ) + + + + + + + +
-
+ + + + + + + + + -
(+ ) + + + + + + -
9
10
11
4
9
13
11
9
8
8
.
.
.
.
.
12
* + , i n d i c a t e s p r e s e n c e o f the C R s e q u e n c e s in the h y b r i d c l o n e d e t e r m i n e d b y the p r e s e n c e o f the h u m a n b a n d ; - ,
i n d i c a t e s a b s e n c e o f the
gene. * * + , i n d i c a t e s p r e s e n c e o f the h u m a n c h r o m o s o m e in g r e a t e r t h a n 30% o f m e t a p h a s e s a n a l y z e d ; ( + ), i n d i c a t e s p r e s e n c e o f the c h r o m o s o m e in 1 0 - 3 0 % of the m e t a p h a s e s a n a l y z e d ; - , i n d i c a t e s a b s e n c e o f the h u m a n c h r o m o s o m e .
peptide
RVEQW
a tryptophan correct. lysine
The
peptide at
at
coded
analysis this for
a
indicated mass
ified
lysine
residues
later
did
lysine
was
ended
with
termination
codon
was
in brackets
contained
not
that
did
giving
position show
residue.
A
the mass
Lys-C cleave
did
mass
sequence
spectrum
of
of the modifying
the
rise to a peptide
minor
peptide
unmodified
19
was
amino
acids
also isolated
containing
and
the
sequenced.
the
the
group
Induction o f carbonyl reductase m R N A A human
hepatoma
breast
cancer
final
concentration
(BHA),
lysine
three
MCF-7
total
of 22 amino
acids.
poly(A)
RNA
of
B-naphthoflavone, RNA (Fig.
electrophoresis,
A ~,..,~,..,~ B ,..,~
cell line
cell line (MCF-7)
mod-
next
of
lysine
acid amino
eDNA
recognize
A
a modified Amino
recognizable
the
not at
821.
any
However,
that
units. but
the protein
the TGA
nucleotide
position.
peptide 72
that
residue
sequence acid
confirmed
and
hybridized
or Sudan 2A) were
transferred
to
the
and
exposed
a human to 10/~M
2,(3)-t-butyl-4-hydroxanisole
(Fig. 2B)
(HepG2) were
labeled
A BCD
and
I for 24 h. 25/tg 10
analyzed to
by
nylon
carbonyl
/~g of
agarose
membranes reductase
E FGH
of
HepG2 gel and cDNA
I
1 3.06.1_
iiii!ii!~ilil
Fig. 3. In-gel S o u t h e r n a n a l y s i s o f r e s t r i c t i o n e n z y m e d i g e s t e d h u m a n D N A . A, h u m a n l y m p h o c y t e D N A ; B, M C F - 7 D N A . D N A w a s d i g e s t e d o v e r n i g h t at 3 7 ° C w i t h 10 U / / ~ g o f e n z y m e . T h e D N A w a s s e p a r a t e d o n a 0.8% a g a r o s e gel a n d h y b r i d i z e d w i t h l a b e l e d c a r b o n y l r e d u c t a s e cDNA.
Fig. 4. S o u t h e r n b l o t o f D N A f r o m m o u s e / h u m a n h y b r i d s h y b r i d i z e d w i t h c a r b o n y l r e d u c t a s e c D N A . L a n e s : A - G s o m a t i c cell h y b r i d s (respectively, 84-27, 84-30, 84-34, 84-35, 84-37, 84-38, 84-39); H , m o u s e p a r e n t ; a n d I, h u m a n p a r e n t . T h e p r e s e n c e o f the 6.1 k i l o b a s e h u m a n b a n d w a s u s e d to s c o r e t h e s o m a t i c cell h y b r i d s . L a n e s A , B a n d E c o n t a i n the h u m a n gene.
153
12
13
14
15
16
17
18
19
20
21
22
X
-
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+
+
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p r o b e . A single b a n d c o r r e s p o n d i n g to a 1.2 k i l o b a s e m e s s a g e was i n d u c e d in b o t h cell lines. Fig. 2 A shows t h a t c a r b o n y l r e d u c t a s e in M C F - 7 cells is i n d u c e d 3-fold, 4.6-fold a n d 4.1-fold b y B H A (lane 2), f l - n a p h t h o f l a vone (lane 3), a n d S u d a n I (lane 4), respectively. L a n e 1 is the u n i n d u c e d control. Fig. 2B shows i n d u c t i o n in H e p G 2 cells of 2.5-fold a n d 4.5-fold b y f l - n a p h t h o f l a v o n e (lane 2) a n d S u d a n I (lane 3), respectively. L a n e 1 is the u n i n d u c e d control. L a n e 4 c o n t a i n s 20 # g of ribos o m a l a n d p o l y ( A ) m i n u s R N A o b t a i n e d f r o m chrom a t o g r a p h y of total R N A on an o l i g o - d T cellulose column.
Southern analysis H u m a n l y m p h o c y t e D N A (Fig. 3A) a n d M C F - 7 D N A (Fig. 3B) were digested with several restriction e n z y m e s a n d a n a l y z e d on an 0.8% agarose gel. T h e c D N A p r o b e was l a b e l e d a n d h y b r i d i z e d to the D N A in the gel (Fig. 3). T h e results show a single m a j o r b a n d a n d s o m e t i m e s a m i n o r b a n d h y b r i d i z i n g to the probe. T h e r e is an i n t e r n a l BamHI site in the c D N A which s h o u l d result in two bands. T h e d a t a are consistent with c a r b o n y l r e d u c t a s e being c o d e d for b y a single gene.
Chromosomal location T h e c h r o m o s o m a l l o c a t i o n o f the h u m a n c a r b o n y l r e d u c t a s e gene was d e t e r m i n e d b y e x a m i n i n g the p a n e l of h u m a n / m o u s e s o m a t i c cell h y b r i d s with the c D N A
(+)
.
+ .
.
.
(+) .
12
.
8
(+) _ _
+
Y
(+) _ +
.
.
+ .
0
.
7
7
6
p r o b e (Fig. 4). T h e results of these studies are s h o w n in T a b l e I. H u m a n c a r b o n y l r e d u c t a s e is assigned to chrom o s o m e 21. Discussion
W e have d e t e r m i n e d the sequence of a c D N A c o d i n g for a h u m a n c a r b o n y l r e d u c t a s e gene i s o l a t e d f r o m the b r e a s t cancer cell line, M C F - 7 . P a r t i a l c D N A clones f r o m a h u m a n liver l i b r a r y were also isolated a n d were identical to the M C F - 7 c D N A clone. T h e c a r b o n y l r e d u c t a s e clone was i d e n t i c a l to a r e c e n t l y p u b l i s h e d c D N A clone f r o m h u m a n p l a c e n t a [29]. Thus, c a r b o n y l r e d u c t a s e m R N A has n o w b e e n i d e n t i f i e d in liver, b r e a s t a n d p l a c e n t a l tissue. C a r b o n y l r e d u c t a s e s are u b i q u i t o u s in n a t u r e a n d m e t a b o l i z e a variety of a l d e h y d e a n d k e t o n e c o m p o u n d s i n c l u d i n g m a n y d r u g s [1,2]. Based on f u n c t i o n a l studies, one m i g h t have e x p e c t e d to find a large class of enzymes s t r u c t u r a l l y r e l a t e d to c a r b o n y l reductase. However, h o m o l o g y searches o f the P I R a n d Swiss-Protein d a t a b a n k s t h r o u g h Bionet d i d n o t d e t e c t a n y significant h o m o l o g y with o t h e r k n o w n proteins. Therefore, c a r b o n y l r e d u c t a s e s m u s t b e f u n c t i o n a l l y related r a t h e r t h a n s t r u c t u r a l l y related. I d e n t i f i c a t i o n of a m o d i f i e d lysine r e s i d u e at a m i n o acid 239 was observed. T h e m a s s of the m o d i f y i n g g r o u p was 72 a n d d i d n o t c o r r e s p o n d to a n y c o m m o n lysine m o d i f y i n g group. T h e significance of this m o d i f i -
154 cation is not known. The modification of lysine residues may explain the multiple bands of activity observed in reports on purification of the enzyme by isoelectrofocusing or DEAE chromatography. Differences in molecular weight have also been observed [10,11], indicating the possible presence of other proteins or additional protein modifications. A potential glycosylation site occurs at amino acid 137. Glycosylation could generate proteins with molecular mass differences of 1400-2000 Da giving rise to observed differences reported in the literature. Induction of carbonyl reductase mRNA by compounds that induce enzymes involved in xenobiotic detoxification imply that carbonyl reductase is also important in cell protection. The mechanism of induction of carbonyl reductase mRNA is presently unkown. However, one possible gene regulatory mechanism which may be involved in the induction of carbonyl reductase involves the Ah locus. The Ah locus controls induction of the aryl hydrocarbon hydroxylase system which is a major metabolic system for xenobiotic detoxification [30]. This locus is involved in the induction of many drug metabolizing enzymes and in the induction of DT-diaphorase [31-33]. DT-diaphorase is induced by many structurally unrelated compounds such as phenolic antioxidants, azo-dyes and polycyclic aromatic hydrocarbons [7,8,34]. It has been suggested that regulatory control by the Ah locus acts indirectly by controlling induction of the aryl hydrocarbon hydroxylase system [35]. The aryl hydrocarbon hydroxylase enzymes oxidize the inducer producing a product(s) which activates transcription of the DT-diaphorase gene. Azo-dyes are activated by direct oxygenation from the aryl hydrocarbon hydroxylase system and are also inducers of DT-diaphorase [5] and carbonyl reductase (Fig. 2). The similarities in induction of DT-diaphorase and carbonyl reductase with azo-dyes may indicate that the Ah locus controls both enzymes. Identifying regulatory sequences in genomic clones of carbonyl reductase and DT-diaphorase will determine whether common regulatory sequences exist. Localization of carbonyl reductase to chromosome 21 may have implications to certain genetic diseases such as Down's syndrome. Some aspects of Down's syndrome such as premature aging, increased incidence of leukemia, brain damage similar to Alzheimer's disease, and lipofusin accumulation are symptoms that have been associated with oxidative damage [36]. Superoxide dismutase, an enzyme coded for on the distal arm of chromosome 21, shows a gene dosage effect in trisomy 21 patients and is involved in oxygen free radical metabolism. It is thought to be involved in the pathology of Down's syndrome. Carbonyl reductase, acting as a quinone reductase, could influence both oxygen radical generation and cellular antioxidant defenses by modulating the redox state of important cellular
metabolites [3,37,38]. Presently this is highly speculative and one must wait for more detailed chromosome mapping, gene dosage and metabolite data. The role of anthracycline metabolism in human leukemic cells is well documented and has profound effects upon the pharmacological properties of these drugs [5,39,40]. However, the mechanisms by which the pharmacological changes occur are not understood and are somewhat controversial. In addition, the metabolism of other quinone and carbonyl substrates and the entire process of xenobiotic protection is currently being investigated. The cloning of carbonyl reductase will allow us to assess its importance in xenobiotic cell protection and drug metabolism.
Acknowledgements We thank William Kaplan, Leonar Directo and Camilla Heinzmann for expert technical assistance. This work was supported by the City of Hope Cancer Center Support Grant CA 33572.
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