Catalytic Cleavage of the Androgen Receptor Messenger RNA and Functional Inhibition of Androgen Receptor Activity by a Hammerhead Ribozyme
Shuo Chen,
Chung S. Song,
Yan Lavrovsky,
Baoyuan Bi*,
Robert Vellanoweth
,
Bandana Chatterjee and
Arun K. Roy
Department of Cellular and Structural Biology The University of
Texas Health Science Center at San Antonio San Antonio, Texas
78284
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ABSTRACT
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Androgen receptor (AR) plays a key role in cell
growth both in the normal prostate and in prostate cancer. Androgen
ablation and prolonged antiandrogen therapy can give rise to
AR-dependent prostate tumors, which nonetheless can grow in the
androgen-deprived milieu. Here we describe the ribozyme approach to
selectively degrading the AR mRNA and thereby inhibiting AR function. A
trans-acting hammerhead ribozyme was designed to cleave the
rat AR mRNA at the position +1827/1828, a region predicted to be
minimally involved in generating stable secondary structures. Using AR
mRNA fragments as substrates, it was established that this ribozyme can
specifically cleave the RNA target in a sequence-specific manner.
Kinetic experiments determined a Km for the
substrate of 77 nM and a
kcat/Km value of
1.8 x 107
M-1·min-1,
suggesting a catalytic efficiency similar to that of protein enzymes
such as the relatively nonspecific ribonuclease A and a
sequence-specific endonuclease EcoRI. Transient
cotransfections of prostate-derived PC3 cells with three plasmids, an
AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR
expression vector, and a ribozyme expression vector, showed that the
ribozyme was capable of reducing the functional activity of AR. At an
equimolar ratio of the AR expression plasmid to ribozyme expression
plasmid, androgen-inducible CAT activity was inhibited 70%. Similar
extents of inhibition were also observed at the cellular mRNA level
using ribonuclease protection assays, indicating that the ribozyme
functioned as an AR mRNA cleaving enzyme in cellulo.
Immunocytochemical examination revealed a decline of AR
immunoreactivity in ribozyme-transfected cells. In addition, no
morphologically detectable cellular abnormalities were associated with
ribozyme expression, indicating the absence of deleterious side
effects. These results offer a new avenue for the control of AR
function and cell growth, especially in the case of androgen-resistant,
but AR-dependent, prostate cancer cells.
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INTRODUCTION
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Androgen action in target cells is mediated by the androgen
receptor (AR), a member of the steroid-thyroid hormone-retinoid
superfamily of transcription factors (1). AR exhibits a prototypic
multidomain structure containing an N-terminal activation domain, a
C-terminal ligand-binding domain, and a centrally located DNA-binding
region with two zinc finger motifs (2, 3). Specific interaction with
androgenic ligands results in the activation of AR and its binding to
androgen response elements at target genes. Such DNA-receptor
association attracts additional regulatory factors at the gene
promoter, leading to an altered rate of transcription (4). Aberrant
androgen action results in various endocrine abnormalities, most
importantly neoplastic diseases of androgen targets such as the
prostate gland. Current approaches for clinical control of prostatic
diseases include depletion of the hormonal ligand by castration, use of
inhibitory ligands that compete with androgens for receptor occupancy,
and inhibition of the androgen-activating enzyme, steroid
5
-reductase. However, none of these regimens is applicable in the
control of the abnormal ligand-independent AR function that arises
during the recurrence of prostate cancer after prolonged androgen
deprivation (5). An unexplored and potentially exciting avenue for
inhibiting AR expression is the specific degradation of AR mRNAs in
target cells. This approach can also be modified for its future use in
gene therapy protocols. Because of these considerations, we have
examined the possible use of ribozyme technology for specific
inhibition of androgen action.
A number of RNA enzymes, which include Group I and Group II introns,
hammerhead, hairpin, and delta virus ribozymes, and the RNA subunit of
ribonuclease P (RNase P), have been investigated extensively (6). Among
these, the most promising RNA enzyme for the purpose of gene therapy is
the small trans-acting hammerhead ribozyme that contains the
catalytic domain found in several plant viroids (7, 8). Hammerhead
ribozymes can associate with a larger target RNA by base-pairing to
cleave a specific phosphodiester bond. This trans-acting RNA
enzyme contains a tripartite structure consisting of a central
catalytic core that is flanked on both sides by two antisense side arms
that can form base pairs with the RNA substrate, thus providing the
sequence specificity of the endonuclease action. Cleavage of the RNA
substrate occurs via a transesterification reaction that generates
5'-hydroxyl and 2'- to 3'-cyclic phosphate termini from the
targeted phosphodiester bond. Catalytic cleavage of the RNA substrate
occurs at the 3'-end of a 5'HUX3' triplet
(where H can be any nucleotide and X is A, U, or C). However, G and C
are the preferred first and third bases of the triplet due to the base
preference for the transition states of the catalytic reaction and the
preferential ability of the cytidine phosphate to hold the ribose
moiety in south conformation during the cleavage reaction (9, 10). The
catalytic efficiency and biological effectiveness of specific ribozymes
also depend on a number of additional factors, including the secondary
structures of the RNA target, the base composition and length of the
specifier side arms, and the cellular stability of the ribozyme (9).
These conditions can be optimized for specific target substrates and
the hammerhead ribozyme has been successfully used to inhibit human
immunodeficiency virus (HIV) replication, inactivation of oncogenes,
and disruption of certain developmentally critical genes to generate
loss of function phenotypes (11, 12, 13, 14). However, no ribozyme-mediated
disruption of steroid hormone functions has yet been attempted. In this
report, we describe the development of a hammerhead ribozyme that can
function as a highly effective site-specific endonuclease for
inactivation of the AR mRNA both in vitro and in
cellulo.
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RESULTS
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Target Specificity and Reaction Kinetics of Ribozyme Action
Potential ribozyme target sites within the rat AR (rAR) mRNA were
initially selected by identification of the single-stranded regions
that do not contain any significant homology to other mRNA sequences.
Elimination of sites based on secondary structure (15) and homology
search reduced the number to seven putative targeting sites within the
rAR mRNA. Among these seven sites, two containing the highest A and U
contents were selected for subsequent analysis. The lengths of the two
flanking sequences and the extent of hydrogen bonding between the
ribozyme specifier side arms and the target RNA are the two most
important factors in determining the rate of turnover of the ribozyme
after the cleavage reaction. However, reduction of the base-pairing
region below 18 to 20 nucleotides (nts) can compromise substrate
specificity (16). We therefore decided on 18- and 19-bp recognition
sequences with an at least 50% or more AU-rich region. Cleavage
positions for the two potential ribozymes correspond to +1057/+1058
(R1) and +1827/+1828 (HR2). Numbering of the nt sequence of the rat AR
is based on the sequence data published by Chang et al.
(17). The 19-nt R1 ribozyme contains side arms with eight A and four U
and the 18-nt HR2 contains five A and four U residues, respectively.
These two hammerhead ribozymes were experimentally tested for specific
endonuclease activity in vitro. AR mRNA fragments containing
the expected cleavage sites for these two ribozymes were synthesized
and used as substrates for the in vitro reaction. The R1
substrate contains 144 nt residues and, when cleaved at the end of the
GUC triplet, is expected to yield 104- and 40-nt reaction products. The
234-nt HR2 substrate, if appropriately cleaved, would produce two
fragments of 182 nt and 52 nt in size. Initial analysis of the time
course of the enzymatic reaction revealed that the R1 ribozyme was less
than half as efficient as HR2. Furthermore, the targeted annealing site
of the HR2 ribozyme contains only a two-base mismatch for human AR
mRNA, and it was found to work equally well with both rat and human AR
mRNA substrates. Therefore, all subsequent experiments were conducted
with the ribozyme targeted to the HR2 attachment site.
Figure 1
shows the sequence structure of
the HR2 ribozyme and the corresponding complementary mRNA region of the
rAR. The figure also shows the predicted cleavage site of the 234-nt AR
mRNA fragment and the expected reaction products. A time course of the
in vitro endonuclease function of the HR2 ribozyme on this
234-nt mRNA fragment is presented in Fig. 2
. A substantial amount of cleavage
products can be observed even after 30 sec of incubation, and the
reaction is almost complete after 25 min. Swapping the RNA substrates
for R1 and HR2 and prolonged incubation (60 min) showed that the
endonuclease activity of HR2 is specific for its complementary
substrate only, and it cannot act on the substrate that corresponds to
the R1 ribozyme (Fig. 3
). Furthermore, a
two-base substitution within the catalytic core (G
U and A
C,
shown in Fig. 1
) caused more than 95% inactivation of the ribozyme
action (data not presented). These results, taken together, allow us to
conclude that HR2 functions as a highly effective and site-specific
endonuclease for the AR mRNA.

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Figure 1. Sequence Structure of the HR2 Ribozyme and the
Corresponding Complementary Region of the rAR mRNA
Cleavage at the end of the GUC triplet of the 234-nt RNA substrate is
expected to produce two reaction products P1 (182 nt) and P2 (52 nt).
Substitution of two bases shown in this figure (A C and G U)
generates a mutant (disabled) ribozyme.
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Figure 2. Endonuclease Activity of the HR2 Ribozyme in
Vitro
The autoradiogram shows electrophoretically separated digestion
products (P1 and P2) derived from the 32P-labeled RNA
substrates (S, 234-nt fragment of rAR mRNA) after incubation at 37 C
for various time points ranging from 0 to 90 min.
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Figure 3. Substrate-Specific Endonuclease Activity of the
Ribozyme
AR mRNA fragments containing sequences complementary to the specifier
side arms of either the R1 (S1, lanes 13) or the HR2 (S2, lanes 46)
ribozymes were separately incubated for 60 min at 37 C with these two
ribozymes as indicated at the bottom. S1 and S2 are 144-nt and 234-nt
AR mRNA fragments containing the binding sites for the R1 and HR2
ribozymes, respectively. Appropriate cleavage products (P1 and P2) are
marked with arrows.
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To estimate the catalytic efficiency of the HR2 ribozyme, steady-state
cleavage velocities were measured with a constant amount (2
nM) of the ribozyme and varying amounts (865
nM) of the substrate (Fig. 4A
). As expected, the enzymatic reaction
displayed a reaction kinetics amenable to Michaelis-Menten analysis. An
Eadie-Hofstee plot of these results is shown in Fig. 4B
.
Michaelis-Menten parameters derived from these results were
Km = 77 nM, kcat = 1.39
min-1, yielding a relative catalytic efficiency
(kcat/Km) of 1.8 x 107
M-1·min -1. Considering that
Km values of RNA enzymes vary from the micro- to nanomolar
scale, the low Km of the HR2 ribozyme may be an important
contributing factor to its efficient catalytic function. It may be
noted that a nonspecific protein enzyme such as ribonuclease A has a
Km = 620 µM. However, high Km of
this protein enzyme is compensated by a higher kcat value
(8.4 x 104 min-1) (presumably due to
multiple target sites in contrast to the site-specific ribozyme),
resulting in a kcat/Km of 1.4 x
108 M-1·min-1 (18).
Thus, ribonuclease A is
10 times more efficient, albeit nonspecific,
compared with an RNA enzyme such as HR2. On the other hand,
EcoRI (19), which is a site-specific endonuclease that
cleaves a limited number of available sites on double-stranded DNA, has
a kcat/Km of 5.7 x 107
M-1·min -1, which is very close
to that of the HR2 ribozyme.

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Figure 4. Reaction Kinetics of the HR2 Ribozyme
A, Autoradiogram showing endonucleolytic cleavage of the AR mRNA
substrate by the HR2 ribozyme at a constant enzyme (2 nM)
and increasing concentrations of substrates: lane 1, 20 nM
substrate RNA without ribozyme; lanes 28, 2 nM ribozyme
plus 8, 14, 20, 30, 57, 60, and 65 nM substrate). B, An
Eadie-Hofstee plot of the data shown in panel A.
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In Cellulo Efficacy of the HR2 Ribozyme
Three cytomegalovirus (CMV) promoter-based mammalian expression
plasmids were used to examine the efficacy of the HR2 ribozyme in
transiently transfected cells. These expression plasmids contain cDNA
inserts corresponding to either the HR-2 ribozyme, or a disabled HR2
ribozyme which has two point mutations within the catalytic core as
shown in Fig. 1
, or an antisense sequence alone, covering the specifier
side arms of HR2 without the catalytic core. Relative inhibitory
effects of these expression plasmids on AR action were tested in the
prostate cancer-derived PC3 cells cotransfected with the rAR expression
plasmid and the mouse mammary tumor virus (MMTV)-chloramphenicol
acetyltransferase (CAT) reporter construct. It should be noted that PC3
cells are AR-negative (20) and show an androgen-dependent response only
after transfection of the AR expression plasmid. Furthermore, CAT
expression in this system is strictly dependent on the presence of
added androgen [10-9 M dihydrotestosterone
(DHT)] (Fig. 5
). Androgen-dependent
transactivation of the MMTV promoter was inhibited by the HR2 ribozyme
expression plasmid in a dose-dependent manner, with
70% inhibition
at an equimolar ratio of the ribozyme to AR expression plasmid. The
same ratio of the corresponding antisense plasmid or the disabled
(mutant) ribozyme caused only minor (1020%) inhibition of the
AR-mediated transactivation function. We also tested the efficacy of
the R1 ribozyme in cell transfection and found that, compared with HR2,
R1 is approximately less than half as efficient in reducing MMTV
promoter transactivation (data not presented). Intracellular
specificity of the ribozyme targeting was further substantiated by
control experiments using glucocorticoid receptor (GR) instead of the
AR expression plasmid. We inserted the rat glucocorticoid cDNA into the
same CMV promoter-based expression vector that was used for the AR
expression. The prostate cancer-derived PC3 cells transfected with
pMMTV-CAT and pCMV-GR showed about a 200-fold increase of CAT
expression in the presence of 10-8 M
dexamethasome. No major difference in CAT expression was observed when
these cells were cotransfected with either the empty vector (pCMV) or
HR2 expression plasmid (pCMV-HR2) at an equimolar ratio of the ribozyme
to pCMV-GR plasmid (Table 1
). From all of
these results, we conclude that the specific inhibitory effect of HR2
on AR function is primarily due to the catalytic function of the
ribozyme and not to its antisense effect.

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Figure 5. Dose-Dependent Inhibition of the AR-Mediated
Transactivation of the MMTV-CAT Promoter-Reporter System in Transfected
PC3 Cells
A, Structures of the HR2 ribozyme, mutant HR2 ribozyme, and the
antisense expression plasmids. Abbreviations used are: CMV,
cytomegalovirus; HR2, ribozyme; MT, mutant HR2 ribozyme; AS, antisense
corresponding to the mRNA sequences used for the HR2 ribozyme; bGH,
bovine GH. B, Inhibition of CAT expression after transfection with
different HR2:AR expression plasmid ratios as indicated on the
top of the bar graphs. DHT, 5 -dihydrotestosterone
(10-9 M); AR, pCMV-AR; HR2, pCMV-HR2; MT,
pCMV-HR2 with two-base substitution as indicated in Fig. 1 ; AS, pCMV
expression plasmid containing only the specifier side arms of the HR2
ribozyme.
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Table 1. Effect of the HR-2 Ribozyme on a Heterologous
Steroid Hormone Receptor (GR) Dependent Transactivation of MMTV-CAT
Promoter-Reporter in Transfected PC-3
Cells
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The dose-dependent inhibition of MMTV-CAT expression by the HR2
expression plasmid is indeed due to the degradation of AR mRNA, and
this was established by RNase protection analysis. Results presented in
Fig. 6
show that, in contrast to the RNA
samples from cells transfected with the antisense expression construct,
the plasmid encoding HR2 caused a substantial reduction in the
steady-state levels of AR mRNA with about
90% reduction at a
ribozyme-AR ratio of 50.

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Figure 6. RNase Protection Assay of the AR mRNA in PC3 Cells
Cotransfected with the Ribozyme or the Antisense Expression Vectors
The first and the last lanes show the protected RNA fragment derived
from cells transfected with the pCMV-AR without the ribozyme expression
plasmid and the undigested AR mRNA probe, respectively. Lanes marked
HR2 and AS represent RNA samples derived from cells cotransfected with
either the ribozyme or the antisense expression plasmid along with
pCMV-AR expression vector. Fold molar excesses of the ribozyme and
antisense expression plasmids to AR expression plasmid are indicated on
the top of each lane. Levels of the ß-actin mRNA
(bottom) from corresponding samples are used as
invariant controls.
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Potential cellular toxicity and morphological changes due to ribozyme
expression were examined in transfected cells, which were immunostained
in situ with the anti-AR antibody and lightly counterstained
with hematoxylin. Both at 24 and 48 h, posttransfection culture
dishes without the ribozyme expression plasmid showed
10% of
the cells with strongly immunostained cell nuclei. However, the extent
of immunostaining of the ribozyme-transfected cells was greatly muted.
No apparent morphological differences between cells with and without
ribozyme transfection were observed (Fig. 7
).

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Figure 7. Immunostaining of PC3 Cells Cotransfected with the
AR Expression Plasmid and the HR2 Ribozyme Expression Plasmid
The two upper frames show control cells transfected with
the AR expression plasmid (pCMV-AR) at 24 and 48 h
posttransfection. The two lower frames are corresponding
cells cotransfected with pCMV-AR and the ribozyme expression plasmid
(pCMV-HR2). Control panels show strong nuclear immunoreactivity in
several AR-positive cells. Each of the two lower frames
shows barely detectable immunoreactive cells that are marked with
arrows.
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DISCUSSION
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Ribozymes are potentially powerful tools for the degradation of
selective mRNAs (21). Carefully designed and appropriately selected
ribozymes can be much more effective inhibitors of mRNA function than
are the corresponding antisense oligonucleotides. Sequence-specific
hammerhead ribozymes have been successfully used to inhibit HIV
replication (11) and to selectively degrade mRNAs encoding a number of
oncoproteins such as c-Ha-ras, c-fos, bcr-abl, bcl-2, and Her-2/neu
(12, 22, 23, 24, 25), thereby causing reversion of the transformed phenotype.
Additionally, conditional expression of targeted ribozyme transgenes in
Drosophila (13), Zebrafish (14), and mouse (26) has resulted
in a 7090% tissue-selective reduction of targeted mRNAs, causing
loss of specific gene function. Our results show that this novel
approach can be successfully adapted to selectively degrade the AR mRNA
both in vitro and in cellulo.
Although a number of guidelines can be used to optimize the parameters
for designing the most effective ribozyme specific for any mRNA, a
large number of uncertain variables make it impossible to predict
enzymatic efficiency without experimental verification. Among many
factors, two that critically affect the in vitro function of
a trans-acting hammerhead ribozyme are the secondary
structure and length of the substrate mRNA, and both the length and
base composition of the specifier side arms. Several studies have shown
that a ribozyme targeted to an open stem-loop structure within a large
RNA substrate is more effective in catalyzing cleavage reaction than
when it is targeted to a base-paired region (9, 27, 28). Although the
currently available computer program (MFOLD) provides a theoretical
prediction of the secondary structure of any particular mRNA on the
basis of the energy minimization method (15), it is by no means an
accurate representation of the secondary structures of various segments
of a large RNA molecule, which exist naturally in solution. Despite
this limitation, targeting of the ribozyme cleavage site to
theoretically predicted open-loop areas appears to be the most
judicious approach (9). The length and base composition of the
specifier side arms greatly contribute to the turnover rate of the
ribozyme, thereby influencing its catalytic function (9, 29, 30). The
effective length of specifier side arms for any particular RNA
substrate can vary within a certain limit, and maximum catalytic
efficiency has been observed with relatively short sequences containing
1020 base residues (16). Although longer flanking sequences increase
specificity, they also decrease the efficiency of the ribozyme due to a
slower turnover rate. Our selection of 18-residue flanking sequences
with a 50% AU content for the HR2 ribozyme has generated an AR
mRNA-specific ribozyme of high catalytic efficiency with a turnover
number of (kcat/Km) 1.8 x 107
M-1·min-1. This value is
comparable to the catalytic efficiency of protein enzymes such as
ribonuclease A and EcoRI (18, 19).
Based on in vitro activity alone, it is not possible to
predict the efficacy of a ribozyme in degrading the targeted mRNA in
intact cells. This is because of the uncertainty concerning the access
of the ribozyme to the target site due to the formation of
ribonucleoprotein complexes, altered RNA conformation, and differential
compartmentalization of the RNA target and the ribozyme. The
intracellular stability of the ribozyme is of additional concern (31).
In the case of HR2, results of the transfection experiments show that
it can function efficiently in cellulo to degrade the AR
mRNA and inhibit the AR-dependent transactivation of the MMTV-CAT
promoter-reporter plasmid in a dose-dependent manner. Compared with
other published reports on ribozyme function (32, 33, 34), our results
showing an approximately 70% inhibition of the AR transactivation
function at a 1:1 ribozyme-AR ratio indicate that HR2 is a highly
efficient enzyme. Although HR2, at an equimolar ratio, could cause
approximately 70% inhibition of transactivational function, reduction
in the steady state level of AR to a similar extent required higher
concentrations of the ribozyme (Fig. 6
). The reasons for this
difference are presently unclear and may be partly due to the fact that
inhibition of transactivational function reflects both mRNA
sequestration interfering with its translation, and its cleavage by the
ribozyme molecule. It should also be noted that the transfectional
analysis described in the present study does not address the effect of
the ribozyme on pre-mRNA processing steps within the nuclear
compartment. However, because of the transient nature of the nuclear
processing step, most of the ribozyme action is expected to occur at
the level of the cytoplasmic AR mRNA.
Polyadenylation at the 3'-end and 5'-capping are two important
determinants of RNA stability (35), and the expression vector that we
have used is expected to facilitate both of these posttranscriptional
modifications to stabilize the ribozyme RNA. Furthermore, in
situ staining of the transfected cells shows that the HR2 ribozyme
can cause a marked reduction of the immunoreactive AR without imparting
any deleterious changes on the cell morphology, a problem often
encountered with the use of antisense oligonucleotides in the
inhibition of gene expression (36).
AR is a critical factor for cell growth in prostatic carcinoma. At an
early stage of the disease, about 70%80% of patients respond
favorably to endocrine therapy involving androgen deprivation
(castration or treatment with LHRH agonist and antiandrogens). However,
after 1218 months, the prostate cancer generally progresses to an
androgen-independent state in which even the alternative approach
utilizing cytotoxic chemotherapy is mostly ineffective (37). In
approximately 30% of cases in which tumors reemerge after androgen
ablation, cancer cells show high levels of AR gene amplification with a
concomitant elevation in the level of functional AR (38). Irrespective
of AR gene amplification, increased AR expression appears to be the
selective driving force for progression to the androgen-refractory
phase of prostate cancer (39). A similar phenomenon in the prostate
cancer-derived LNCaP cell culture model has also been reported. After
prolonged passage in an androgen-free medium, these cells enter into an
accelerated growth phase with more than a 5-fold increase in AR (40).
Thus, prostate cancer that recurs after androgen deprivation therapy is
clearly AR-dependent. However, the mechanism that activates such a high
level of AR in these recurrent tumor cells is still unknown and may be
due to the presence of a very low level of androgenic steroids or to
ligand-independent activation pathways involving protein kinase A
and/or peptide growth factors (41, 42). Irrespective of the activation
mechanism, specific reduction of AR mRNAs, and thus functional AR, is
expected to deprive these cells of a critical growth advantage.
Efficacy of specific ribozymes in inhibiting HIV replication and
oncogene function has already provided the impetus for using these
sequence-specific RNA enzymes in clinical trials (24). Results
presented in this paper furnish the initial step toward application of
this novel approach to the management of AR-dependent prostate cancer,
and potentially to targeted gene therapy.
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MATERIALS AND METHODS
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Selection of Ribozyme-Targeting Sequence
To select the best possible site for targeting the hammerhead
ribozyme, we analyzed the primary and secondary structures of the
entire length of the rAR mRNA. The first phase of this analysis
involved selection of target sites free of any potential secondary
structure. This was achieved by processing the mRNA sequence through
the MFOLD computer program of the University of Wisconsin Genetics
Computer Group (GCG), version 8.1. The MFOLD program predicts optimal
and suboptimal RNA secondary structures based on the energy
minimization method (15). Results were processed through a VAX computer
to convert the linear structure into a two-dimensional stem-loop format
by using the SQUIGGLES graphic program. The structure containing the
least free energy change of formation was used for further
consideration. We selected sequence stretches containing 20-nt domains
surrounding GUC triplets within the single-stranded, looped regions of
the two-dimensional structure and scanned them through the GenBank
sequence database to eliminate potential targets with substantial
homology to other non-AR mRNAs.
Plasmid Constructs
Oligodeoxynucleotides corresponding to ribozymes and antisense
RNA sequences were synthesized by the phosphoramidite method and
purified on a 16% polyacrylamide/8 M urea gel. Each
oligonucleotide was ligated to a Bluescript SK plasmid (Stratagene, La
Jolla, CA) that had been digested with SacI and
EcoRI, to allow expression of the hammerhead ribozymes or
antisense oligonucleotides under the control of a T3 RNA polymerase
promoter. Two nonoverlapping segments of the rAR cDNA containing the
target sites of R1 and HR2 hammerhead ribozymes (144 and 234 bp,
corresponding to +954/+1097 and +1646/+1879 positions, respectively)
were cloned into the Bluescript plasmid to generate the AR mRNA
fragments that were used as ribozyme substrates. For in
cellulo studies, the full-length AR cDNA was cloned into a
mammalian expression vector containing the human CMV promoter to create
the recombinant expression vector. The MMTV long-terminal repeat
containing the AR/GR response elements was ligated to the CAT gene to
create a pMMTV-CAT plasmid as a promoter-reporter construct. Ribozymes
R1 and HR2, the mutant HR2, and the antisense HR2 oligo all contain a
HindIII site at the 5'-end and an Xbal site at
the 3'-end. This allowed convenient cloning of these DNAs into the
HindIII/Xbal sites of the mammalian expression
vector, pcDNA3 (Invitrogen, San Diego, CA). After these steps, we
generated pCMV-HR2, pCMV-mut-HR2, and pCMV-AS expression vectors that
contained ribozymes HR2, mutant HR2, and the antisense sequence
corresponding to HR2all under the control of the CMV promoter.
Sequences of all constructs were confirmed by DNA sequencing.
In Vitro Assays of Ribozyme Activity
Bluescript plasmids containing the AR cDNA and different
hammerhead ribozyme DNA constructs were linearized with appropriate
restriction enzyme digestion. The transcription reactions were carried
out with T3 or T7 RNA polymerase (Promega, Madison, WI). The AR gene
transcripts were either synthesized with unlabeled nucleoside
triphosphates, or labeled with
[
-32P]UTP. The products were purified by
electrophoresis in a 10% polyacrylamide/8 M urea gel. The
AR mRNA substrate and the hammerhead ribozyme RNA were separately
preincubated at 37 C for 3 min in 50 mM Tris-HCl, pH 7.5, 2
mM spermine, and 1 mM EDTA. This was followed
by addition of 100 mM MgCl2 to a final
concentration of 10 mM and mixing of the ribozyme and the
substrate. The enzymatic activity was terminated with an equal volume
of stop buffer (10 mM EDTA, 90% formamide, 0.1%
bromophenol blue, and 0.1% xylene cyanol). After heating at 95 C for 2
min, the cleavage products were resolved by electrophoresis in a 10%
polyacrylamide/8 M urea gel. The products were detected by
autoradiography or by ethidium bromide staining. For time-course
experiments, 100 µl of a mixture containing the unlabeled hammerhead
ribozyme and the 32P-labeled AR mRNA substrate (at a 1:1
molar ratio) were incubated at 37 C under the conditions described
above. The cleavage reaction was followed by removing 10 µl of the
mixture at different times, and reaction was stopped by adding 5 µl
of stop buffer and quickly freezing the mixture at -20 C until further
analysis. The labeled cleavage products were separated on a 10%
polyacrylamide/8 M urea gel. The percentage of cleavage was
quantified by a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale,
CA), using the equation: % cleavage = (P1+P2)/(P1+P2+S) x 100%,
where P1 and P2 are 5' and 3' cleaved fragment band intensities and S
is the band intensity of the uncleaved substrate.
Analysis of Enzyme Kinetics
Kinetics constants Km and
kcat were determined from an Eadie-Hofstee plot
of initial velocities under multiple turnover conditions (29, 30).
Mixtures of the HR2 ribozyme (2 nM) and substrate ranging
from 8 nM to 65 nM were incubated under the
conditions described above. The reactions were linear for the first 40
min, during which time the reaction products were analyzed on a 10%
polyacrylamide/8 M urea gel and quantified with
phosphorimaging. All experiments were performed in duplicate.
Cell Culture and Transfectional Assay
PC-3 cells, which are AR negative and derived from human
prostate adenocarcinoma, were transfected using carrier liposomes
(LipoTaxi, Stratagene, San Diego/La Jolla, CA). Briefly, the PC-3 cells
were plated, grown, and cotransfected with a reporter construct
(pMMTV-CAT), a target vector (pCMV-AR), and pCMV control vector
together with hammerhead ribozyme/antisense expression vectors
pCMV-HR2, pCMV-mut-HR2, and pCMV-AS. After 4 h, the transfection
medium was replaced with normal growth medium (MEM +7%
charcoal-stripped FBS) with or without 10-9 M
DHT. Cells were harvested 36 h later, and CAT activity was
determined by ELISA, and protein concentration by the Bradford
procedure (43). The CAT ELISA was perfomed with 50 µg protein extract
according to the manufacturer protocol (Boehringer Mannheim,
Indianapolis, IN). Separate standard curves and CAT activity for the
vector control were established for each set of experiments. Results
were expressed as the percent CAT activity relative to the vector
control.
RNase Protection Analysis
Total RNA from the transfected cells was isolated according to
the protocol provided with the RNeasy Kit (Qiagen, Chatsworth, CA).
Briefly, 106 cells were washed three times with ice-cold
PBS without Ca++ and Mg++ and lysed with a
solution containing guanidinium isothiocyanate. The lysate was mixed
with an equal volume of 70% ethanol and centrifuged through the RNeasy
spin column. Column-purified products were treated with RNase-free
pancreatic DNaseI (Promega) in 10 mM MgCl2/0.1
mM dithiothreitol/10 mM RNase inhibitor for 30
min at 37 C (44). The samples were further purified through the RNeasy
spin column. To generate an antisense AR RNA probe, rAR cDNA was
digested with SstI to release a 169-bp fragment spanning
+1697- to +1865-bp positions. The fragment was cloned into the
SstI site of the Bluescript vector. The vector containing
the 169-bp fragment of the AR cDNA was linearized with XbaI,
and an antisense AR RNA probe was synthesized with
[
-32P]UTP, CTP, ATP, and GTP and T7 RNA polymerase.
The probe was purified by electrophoresis through a 5%
polyacrylamide/8 M urea gel. The ß-actin antisense RNA
probe was used as an internal control. RNase protection assays were
performed using a RNase protection assay kit RPAII (Ambion, Inc.,
Austin, TX). Briefly, 1 and 8 µg of total RNA were hybridized with
5 x 105 cpm of radiolabeled antisense ß-actin RNA
probe and 5 x 105 cpm of radiolabeled antisense AR
RNA probe, respectively. The products were digested with an RNaseA/T1
mixture and precipitated with ethanol. The protected AR mRNA and the
ß-actin mRNA products were separated on 5% polyacrylamide/8
M urea gels. The gels were dried, and AR mRNA was
quantified by phosphorimaging. The level of the AR mRNA was normalized
to the ß-actin mRNA in each sample.
Immunostaining
PC-3 cells transfected with or without the hammerhead ribozyme
were immunostained using the experimental conditions previously
described (45). Cells were washed with PBS three times and fixed in PBS
containing 2% paraformaldehyde and 10% sucrose, pH 7.2, for 20 min,
and then permeabilized with PBS containing 0.3% Triton X-100 for 30
min. After initial blocking of nonspecific binding sites by treatment
with PBS containing 10% mouse serum for 30 min, cells were incubated
with primary AR antibody (affinity-purified rabbit antibody directed to
the 19 N-terminal residue of rAR) in the blocking reagent overnight at
room temperature. Cells were then washed three times with PBS and
exposed to biotinylated goat anti-rabbit IgG (1:100) as secondary
antibody in vectastain elite ABC reagent (Vector Laboratories,
Burlingame, CA) for 30 min at room temperature. After three washes in
PBS, AR-positive immunoreactivity was identified with the chromogenic
reagent, 3,3'-diaminobenzidine containing
H2O2.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Jacques R. Fresco and Nadarajan K. Velu for their
interest and criticism, and Ms. Nyra White for secretarial
assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Arun K. Roy, Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7762.
This work was supported by NIH Grant DK-14744. S.C. was supported in
part by NIH Training Grant T32 AG-00165. *Present Address: Section of
Immunobiology, Yale University School of Medicine, New Haven,
Connecticut 06510.
Present Address: Department of Chemistry &
Biochemistry, California State University, Los Angeles, California
90032.
Received for publication May 20, 1998.
Revision received July 7, 1998.
Accepted for publication July 11, 1998.
 |
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