Yonago Acta medica 1996;39:151–154

Selective Enhancement of Expression of Class-Mu Glutathione STransferase Genes during Involution of Rat Ventral Prostate Inducedby Androgen Withdrawal
Yoshiko Hoshikawa and Takeshi Sairenji
Department of Biosignaling, Faculty of Medicine, Tottori University, Yonago 683, Japan
cDNA clones encoding class-Mu subunits (Yb1, Yb2 and Yb3) of glutathione Stransferases were isolated from a cDNA library of a rat ventral prostate. The mRNAlevels of Yb1, Yb2 and Yb3 were significantly elevated in the prostate of androgenablated rats. The enhancement of gene expression was characteristic for class-Mu
subunits, since the relative abundance of mRNAs for class-Alpha and class-Pi was notaffected by castration. The up-regulation of transcripts was repressed by immediateadministration of testosterone propionate after castration.
Key words: class-Mu subunits; glutathione S-transferase; mRNA; ventral prostate

Glutathione S-transferases (GSTs) are a familyof multifunctional enzymes involved in the detoxification of xenobiotic agents, drug biotransformation and protection of cells against peroxidative damage (Mannervik and Danielson,
1988; Pikett and Lu, 1989). Cytosolic GSTs
from rat tissues were designated as class-Alpha(subunits Ya1, Ya2 and Yc), class-Mu (subunitsYb1, Yb2 and Yb3), class-Pi (subunit Yp) andclass-Theta (subunit Yrs). Recently, we tried asubtraction hybridization cloning to isolate thegenes responding to the androgen withdrawal(Hoshikawa et al., 1991). Some of the isolated
clones contained mRNAs for class-Mu subunitsof GSTs. It has been reported that the mRNAlevel of Yb1 was elevated during the regressionof steroid-hormone dependent tissues (Chang etal., 1987; Flomerfelt et al., 1993). GSTs may
function in the cellular process of apoptosis
underlying tissue involution. In the present
study, we demonstrated that the expression ofclass-Mu GST genes was selectively elevatedduring the involution of a rat ventral prostatewith the specific probes for GST subunits
belonging to 3 distinct classes.

Materials and Methods

Isolation and sequencing of cDNA
clones for rat GST subunits
cDNA clones encoding class-Mu GSTs wereisolated from a cDNA library of a rat ventral
prostate as described previously (Hoshikawa etal., 1991). After the second cycle of differential
screening, we isolated 168 clones as candidatesof the mRNAs induced after castration. Basedon the results of Northern blot analyses, weselected 48 clones giving significantly different
hybridization signals with mRNAs from intact
and castrated rats. The 48 isolated clones wereshown to represent 10 distinct mRNA species.
Partial nucleotide sequences of inserted cDNA
fragments were determined with the representative clones by the dideoxy chain termination method (Sanger et al., 1977).

Generation of probes for Northern blotanalysis

Abbreviations: bp, base pair; DIG, digoxigenin;
GST(s), glutathione S-transferase(s)The inserted cDNA fragments of clones 74, 64and 3.5 were excised from plasmids with EcoR
I and used as the probes for subunits Yb1, Yb2
and Yb3, respectively. The probe used for subunit Ya1 was the DNA fragment that was 749

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Y. Hoshikawa and T. Sairenji

Table 1. cDNA clones for class-Mu GSTs
isolated from a rat ventral prostate
CloneNo. ofSize of mRNA†
No. related clones* (nucleotides)

Identification

7481062
Yb16411034
Yb2
3.591264
Yb3* Related clones were detected by cross hybridization among 168 clones selected in the second
cycle of screening as candidates of gene products
induced by castration.
† Sizes of the mRNAs were predicted by DNA sequence analyses of structural genes (Pickett andLu, 1989; Abramovitz et al., 1995).

base pairs (bp) in length and contained the complete coding sequence (Lai et al., 1984). Thiswas generated by reverse transcriptionpolymerase chain reaction using total RNA prepared from a rat liver and the primers 5'AGCTGAGTGGAGAAGAAGCC-3' and 5'AATTGGACAGTGCAGCTTCCG-3'. Theprobe for the rat Yp subunit was the 735-bp
DNA fragment that contained the complete
coding sequence. This was excised from theplasmid pGP5 (Suguoka et al., 1985) with EcoR
I and Sal I. pGP5 was provided from the Riken
Gene Bank. For the controls which showed thateach lane contained comparable amounts ofRNA, cDNA of human β-actin (Gunning et al.,1983) was used as the probe. Double-stranded
49696

ND

74 (Yb1)
328

661

887

64 (Yb2)-11/ 1675

1211

3.5 (Yb3)

Fig. 1. Schematic representation of cDNA clones 74(Yb1), 64 (Yb2) and 3.5 (Yb3) encoding class-Mu
GSTs isolated from a rat ventral prostate. Closed
and open boxes represent the protein coding andnon-coding sequences, respectively. Numerals
above the boxes indicate the positions of terminal
nucleotides of inserted cDNA fragments and protein
coding regions. Position 1 is the translational initiation site. The nucleotide sequence of 3'-terminal
region of clone 74 was not determined (ND).

DNAs were labeled with digoxigenin (DIG)11-dUTP using the DIG-high prime labeling kit(Boehringer Mannheim Inc., Mannheim,Germany).

Isolation of RNA and Northern blot
analysis
Ventral prostates of male Wistar rats (bodyweight 300–350 g) were used after castration at
24 or 48 h. As a replacement therapy, 5 mg of
testosterone propionate dissolved in 0.1 mL of
cotton seed oil was administrated immediately
after castration. Total cellular RNA was isolated from prostate tissues by the acid guanidinium thiocyanate-phenol-chloroform method(Chomczynski and Sacchi, 1987). Ten micrograms of RNAs were electrophoresed on 1.2%
agarose gels containing 2.2 M formaldehyde
and transferred to Biodyne Plus membrane (PallBiosupport Co., New York, NY). After hybridization DIG-labeled probes were detected byenzyme immunoassay using a DIG luminescentdetection kit (Boehringer Mannheim).

Results and Discussion

Cloning of class-Mu GST genes from acDNA library of a rat ventral prostate
In searching for the gene products involved in
the castration induced involution of the rat
ventral prostate, we isolated cDNA clones
containing mRNAs for class-Mu GSTs (Table1). The nucleotide sequences of clones 74, 64
and 3.5 were determined for about 200 bp inboth terminal regions of inserted cDNA fragments. By searching the sequence homology
with the registered sequences in the GenBank
database (Release 95.0), the nucleotide sequences of clones 74, 64 and 3.5 revealed 98%
identity to those of previously reported cDNAs
for Yb1 (Ding et al., 1985), Yb2 (Lai and Tu,1986) and Yb3 (Abramovitz and Listowsky,1987), respectively. The mRNA sequences ofclones 74, 64 and 3.5 were schematically represented in Fig. 1. Among rat class-Mu GSTs, theYb3 expression is restricted in some tissues.

152rat GST mRNA expression

The Yb3 gene was originally cloned from a rat
brain cDNA library and shown to be selectively
expressed in brain and testis (Abramovitz andListowsky, 1987; Abramovitz et al., 1995). In
addition we demonstrated that the Yb3 gene
was also expressed in the rat ventral prostate bycDNA cloning.

istration of testosterone propionate which has
androgenic hormonal activity (Izawa, 1990),
the levels of mRNA for class-Mu GSTs werereduced to the levels observed in the intact rats(lane 4). It was consistent with a conclusionthat the up-regulation of transcripts was dependent on the androgen withdrawal induced bycastration. The elevation of mRNA levels was
not observed in other male accessory sex organ,seminal vesicle (Hoshikawa et al., 1991).
These results suggest that the effects of androgen withdrawal on the expression of mRNAfor class-Mu subunits were variable among
androgen-dependent tissues. It is not clear atpresent whether the increase in the relative
abundance of mRNA for Yb1, Yb2 and Yb3 isdue to an increase in the rate of transcription orcaused by changes at the posttranscriptional
level.

The mRNA levels of Yb1, Yb2 and Yb3
were elevated during the involution ofprostate tissue
Northern blot analyses were performed for total
cellular RNA prepared from ventral prostates ofintact and castrated rats using the cDNA fragment of clones 74, 64 and 3.5 as probes (Figs.
2A–2C). The mRNAs for 3 class-Mu subunits
were detected in the prostate tissues from intact
rats (lane 1). The expressions of mRNAs forYb1, Yb2 and Yb3 were enhanced by castration(lanes 2 and 3). Chang and colleagues (1987)
have identified the mRNA for Yb1 to theandrogen-repressed 29-kDa protein by cDNA
cloning. The induction of the Yb1 gene wasalso observed early in the apoptotic pathway inT-lymphocytes treated with dexamethasone orcalcium ionophore A23817 (Flomerfelt et al.,1993). We demonstrated that the expressions of
not only Yb1 but also Yb2 and Yb3 were induced in the regressing prostate. By the admin-

Expressions of class-Mu genes, as opposed to -Alpha nor -Pi GST genes, wereselectively enhanced in the prostates of
androgen-ablated rats
To examine whether the expression of mRNAfor the other class-GSTs to class-Mu is alsoenhanced in the prostates of castrated rats,
Northern blot analysis was performed using
specific probes for Ya1 and Yp that are belonging to class-Alpha and -Pi, respectively. ThemRNA for Ya1 (about 900 nucleotides inlength) was not detected in the prostate (Fig.
2D), although the significant concentration ofmRNA was detected in the liver cells (data notshown). The mRNA for Yp (about 1,000
nucleotides in length) was expressed in theFig. 2. Effects of castration and testosterone propionate on theexpressions of GST genes in the rat ventral prostate. Ten micrograms of total cellular RNAs from the ventral prostates of intact(lane 1) and rats castrated for 24 h (lane 2) and 48 h (lane 3), andtreated immediately after castration with testosterone propionatefor 48 h (lane 4) was electrophoresed on 1.2% denaturing agarose
gels. RNAs were transferred onto nylon membranes and hybridized with DIG-labeled probes for rat GSTs; Yb1 (A), Yb2 (B),Yb3 (C), Ya1 (D) and Yp (E). cDNA for human β-actin (F) wasused as a probe for RNA amounts in each lane.

153Y. Hoshikawa and T. Sairenji

prostates of intact rats (Fig. 2E, lane 1).
However, the effect of castration on the expression of mRNA for Yp was insignificant (Fig.
2E, lanes 2 and 3). These results demonstrated
that the selective enhancement of expression ofclass-Mu GST genes was induced in the rat
ventral prostate following androgen withdrawal. The functions of GSTs were well characterized in the liver which is the major site forthe metabolism and detoxification of drugs andchemical carcinogens (Mannervik andDanielson, 1998; Pikett and Lu, 1989). However, very little is known about the roles ofGSTs in extrahepatic organs. GSTs are known
to function as as cellular detoxifying enzymes
by removing harmful metabolic byproducts that
may result from a variety of biochemicalreactions. As the cellular apoptotic pathway is
expected to increase such byproducts, the selective enhancement of mRNA expression ofclass-Mu GSTs may be associated with theearly signals of apoptosis in the regressing
prostates.

Acknowledgments: This work was supported by aGrant-in-Aid for the Second-Term Comprehensive
10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan.

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(Received September 3, Accepted September 18, 1996)

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