Ribozyme-Mediated Cleavage of the Estrogen Receptor Messenger RNA and Inhibition of Receptor Function in Target Cells
Yan Lavrovsky,
Rakesh K. Tyagi,
Shuo Chen,
Chung S. Song,
Bandana Chatterjee and
Arun K. Roy
Department of Cellular and Structural Biology (Y.L., R.K.T.,
S.C., C.S.S., B.C., A.K.R.) University of Texas Health
Science Center San Antonio, Texas 78284
Audie L. Murphy
Memorial Veterans Administration Hospital (B.C.) San Antonio, Texas
78284
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ABSTRACT
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Estrogen receptor (ER) functions as a
ligand-activated transcription factor for estrogen-regulated genes.
Because of the critical role of the ER in the proliferation of certain
estrogen-dependent cancer cell types such as the mammary tumor,
inhibitors of estrogen action at the level of receptor function are of
major clinical interest. Here we describe developments of two ribozymes
that can selectively degrade the human ER mRNA and inhibit
trans-activation of an artificial promoter containing the
estrogen response element. Two ribozymes, designated RZ-1 and RZ-2,
cleave the human ER
mRNA at nucleotide positions +956 and +889,
respectively. These cleavage sites lie within the coding sequence for
the DNA-binding domain of the receptor protein. Both RZ-1 and RZ-2 were
also effective in inhibiting the progression of quiescent MCF-7 breast
cancer cells to the S phase of the cell cycle after their exposure to
17ß-estradiol (10-9 M). These results
provide a new avenue for inhibition of estrogen action by selective
mRNA degradation with its potential therapeutic application through
targeted gene delivery vectors.
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INTRODUCTION
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Estrogenic hormones regulate target cell function and tissue
remodeling via signal transduction involving the estrogen receptor
(ER), which belongs to the steroid, thyroid, retinoid, and vitamin D
receptor superfamily of ligand-activated transcription factors (1). In
addition to one major form of the ER, i.e. ER
, two minor
subtypes, ERß1 and ERß2, have also been identified (2, 3, 4, 5).
Regulation of specific gene expression by the ligand-activated ER is
generally achieved in conjunction with certain coactivator proteins,
whereas ER-mediated tissue remodeling requires concerted action of the
receptor, other growth factors, cell cycle regulatory proteins, and
apoptotic signaling agents. As estrogen-dependent reproductive
abnormalities are only absent in ER
knockout and not in ERß null
mice (6, 7), ER
appears to provide the critical role in most of the
estrogen-regulated processes. Thus, pharmacological inhibition of ER
action has been of great endocrinological interest, especially for
therapeutic control of ER-positive breast cancer cells (8). Much of the
efforts in this regard have been limited to the design of estrogen
analogs, which when bound to the ER prevent its access to functional
estrogens. Such interactions also cause abnormal conformational change
in the receptor, thereby inhibiting its
trans-activational activity (9). Although this
strategy has helped generate a number of valuable antiestrogens, most
of these compounds possess a mixed agonist/antagonist activity, and
their inhibitory action may vary in a tissue- and gene-specific fashion
(10). An alternative strategy of gene-based inhibition of ER function
by dominant negative mutants of the ER is also being explored (11).
In a previous report, we showed that a hammerhead ribozyme can catalyze
site-specific endonuclease cleavage of the androgen receptor mRNA and
is highly effective in reducing the intracellular level of androgen
receptor mRNAs (12). We have used a similar strategy to design two
site-specific hammerhead ribozymes directed to the human (h) ER
mRNA, and here we describe their effectiveness in inhibiting ER
function in transfected ER-negative COS-1 cells (13) and ER-positive
MCF-7 cells (14). Expression vectors containing these ribozymes provide
an additional effective tool for selective inhibition of estrogen
action and ER-mediated tumor cell growth both in vivo and
in vitro.
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RESULTS
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Design of the Human ER
-Specific Hammerhead Ribozymes and
Selection of Accessible and Optimum Cleavage Sites
Among various types of catalytic RNAs, the trans-acting
hammerhead ribozyme provides certain advantages as a tool for selective
degradation of the eukaryotic messenger RNA due to its high intrinsic
catalytic rate and small overall size (15, 16). The hammerhead ribozyme
consists of three distinct components: a central catalytic core and two
variable side arms that direct site-specific duplex formation with the
corresponding substrate mRNA. The length of the two side arms and their
A-U to G-C ratio determine the rate of turnover of the ribozyme after a
single round of catalytic reaction. Optimum catalytic cleavage occurs
at the end of a GUC triplet, producing a 2',3'-cyclic phosphate and a
5'-terminus (17). To provide the highest accessibility of the ribozyme
to the hER
mRNA, we examined the potential secondary structure of
the hER
mRNA sequence through the energy minimization approach (18).
Figure 1A
shows the potential stem-loop
configuration of the hER
mRNA from nucleotide positions +1 to +1300
encompassing coding sequences for the N-terminal
trans-activation domain, DNA-binding domain, hinge region,
and part of the steroid-binding pocket. Based on the computer search of
the looped regions with GUC triplets and at least 50% AU contents, we
selected two potential cleavage sites for the ribozyme at positions
+889 and +956. Cleavage sites of these ribozymes, ribozyme-1 (RZ-1) and
ribozyme-2 (RZ-2) are indicated with arrows in Fig. 1A
. Our
earlier experience with the design of ribozymes for the androgen
receptor mRNA (12) and the results of others (19, 20) suggest that an
optimum substrate specificity and turnover rate can be achieved with
two side arms of 911 residues each and with a close to 50% A-U base
pairs at the RNA-RNA duplex. The sequence structures of RZ-1 and RZ-2
are shown in Fig. 1
, B and C, respectively. GenBank search of the
binding region of RZ-1 (hER
sequences from +947 to +966) and RZ-2
(hER
sequences from +880 to +900) indicated the absence of
significant homology of these two sequence regions of the hER
mRNA
to any known human mRNAs, except for hERß and ER-related orphan
receptors hERR-1 and hERR-2 (21, 22). It has been established that
substitution of two bases at the catalytic core of the hammerhead
ribozyme (A
C and G
U, as indicated in Fig. 1C
) causes an almost
total loss of the catalytic activity without any significant change in
base pairing capacity (12). We, therefore, designed mutant ribozymes
with the two above-mentioned base substitutions to distinguish the
intracellular effects of antisense and catalytic functions. These
ribozymes were then tested for their catalytic activities in
vitro.
Catalytic Cleavage of the hER
mRNA Substrate by RZ-1 and
RZ-2
Ribozymes were synthesized by in vitro transcription of
the corresponding recombinant genes, and their specificities and
catalytic activities were examined on 32P-labeled RNA
substrates. For hER
, a 390-nucleotide (nt) long mRNA substrate
containing the expected cleavage sites for both ribozymes was used as
the substrate target. Electrophoretic autoradiograms of the cleavage
reaction catalyzed by RZ-1 and RZ-2 are displayed in Fig. 2
, A and B. The results show that both of
these ribozymes cleaved the mRNA substrate in a highly
sequence-specific fashion. RZ-1 produced two cleavage products of the
expected sizes (265 and 125 nt) from the 390-nt long hER-
mRNA
substrate (Fig. 2A
). Similarly, the same substrate was cleaved by RZ-2
to yield 201- and 189-nt long reaction products, as expected from the
site of excision at the end of the GUC triplet at the +889 position.
Based on the time course of the cleavage reaction, it appears that RZ-1
(Fig. 2A
) is about twice as effective in vitro as RZ-2 (Fig. 2B
). In the case of RZ-1, at an equimolar ratio of substrate to
ribozyme, 50% of the mRNA substrate was converted to reaction products
within 25 min of incubation. RZ-2 takes about 50 min to achieve 50%
cleavage of the mRNA substrate (Fig. 2C
). Additionally, when RZ-1 and
RZ-2 are allowed to function together in vitro on the same
substrate, they do not appear to affect each other either in a positive
or negative manner (Fig. 2D
, lane 3). The results presented in Fig. 2D
, lane 3, also show that one of the reaction products of RZ-1 (189 nt
long) containing the RZ-2 cleavage site was further cleaved into 125-
and 64-nt RNA fragments. Two base substitutions (as indicated in Fig. 1C
) within the catalytic core of RZ-1 and RZ-2 almost completely
abolished their enzymatic activities (Fig. 2D
, lanes 4 and 5).
Additionally, neither of these ribozymes displayed any significant
degree of endonuclease activity on an mRNA fragment containing the
corresponding DNA-binding domain of the glucocorticoid receptor (GR;
Fig. 2D
, lanes 6 and 7). Collectively, these results demonstrate the
high degree of catalytic activity and substrate specificity of the two
ribozymes.

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Figure 2. Site-Specific Endonuclease Activity of RZ-1 and
RZ-2 on a 390-nt Long hER mRNA Fragment
A and B, Time course of the cleavage reaction of an equimolar mixture
of the RNA substrate and RZ-1 (A) and RZ-2 (B), respectively, after 0,
5, 15, 30, 60, 90, and 120 min of incubation (lanes 17). C,
PhosphorImager quantification of the cleavage kinetics of the RZ-1
(filled circles) and RZ-2 (empty
circles). D, Lanes 13, Cleavage pattern produced by RZ-1 or
RZ-2 singly or by a 50:50 mixture of the two ribozymes after 60 min of
incubation. Lane 1, RZ-1; lane 2, RZ-2; lane 3, RZ-1 plus RZ-2. The
cleavage products corresponded to expected cleavage sites as determined
with known size markers. Lengths of the ER mRNA substrate and reaction
products in nucleotide residues are indicated with
arrows. Lanes 47 show the results of control
experiments. Lanes 4 and 5 correspond to reactions with mutant RZ-1 and
mutant RZ-2, respectively, using the same 32P-labeled ER
substrate as in lanes 13. Both mutants contain two-point mutations at
the catalytic core (as shown in Fig. 1 ), and in neither case was
cleavage product generated. Lanes 6 and 7, The 32P-labeled
GR mRNA substrate incubated with RZ-1 (lane 6) and RZ-2 (lane 7). The
probe position corresponding to the GR mRNA substrate is shown by the
arrow at the right corner of the panel.
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Inhibition of Estrogen Response Element (ERE)-Containing Promoter
Function and Decrease in hER
Transcripts in COS-1 Cells
Cotransfected with hER
and Ribozyme Expression Constructs
COS-1 cells are ER negative, and they can be made estrogen
sensitive by transient transfection with an ER expression vector (13).
When COS-1 cells were transfected with the hER
expression plasmid
along with a promoter-reporter construct containing the ERE from the
vitellogenin gene promoter (23) and the luciferase-coding sequence, the
cells became estrogen sensitive (Fig. 3A
, bar 1). The addition of either RZ-1 (bar 2) or RZ-2 (bar 3) expression
vectors caused more than 80% reduction in luciferase activities. As
expected from the in vitro cleavage reaction, a 50:50
mixture of RZ-1 and RZ-2 did not show any significant difference in the
inhibition of the estradiol-ER-dependent increase in luciferase
activity (bar 4). The mutant form of RZ-2 (bar 5) caused only about
20% inhibition of the promoter function, possibly due to its antisense
effect on the hER
transcript. The results presented in Fig. 3B
show
that COS-1 cells, when transfected with a GR expression plasmid, showed
no significant inhibition of dexamethasone-induced chloramphenicol
acetyltransferase (CAT) expression from the mouse mammary tumor
virus (MMTV)-CAT promoter-reporter after cotransfection with
either RZ-1 or RZ-2. The ribozyme-mediated decrease in luciferase
activity in transiently transfected COS-1 cells is indeed due to a
concomitant decrease in the hER
mRNA level. This was indicated by
the results of the ribonuclease (RNase) protection assay (Fig. 4
). RZ-1, RZ-2, and RZ-1 plus RZ-2 all
caused declines in the level of the hER
mRNA-protected radiolabeled
antisense band to approximately 80%, and the mutant RZ-2 was only
weakly effective (Fig. 4
, upper panel). No significant
difference in the intensity of the protected bands resulting from the
ß-actin control antisense probe can be seen, and the overall quality
of the total cellular RNA remained unaltered after ribozyme
transfection (Fig. 4
, middle and bottom panels,
respectively). From these results, we conclude that expression of
either RZ-1 or RZ-2 transcripts can cause selective degradation of the
hER
mRNA, thereby reducing the ER
protein level in transfected
cells, which, in turn, is reflected in the decreased activity of the
ER-responsive promoter-reporter construct.

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Figure 3. Ribozyme-Mediated Inhibition of the
Xenopus Vitellogenin Promoter-Derived ERE (A) and MMTV
Long Terminal Repeat (B) Activation in Transfected COS-1 Cells
Luciferase activities are expressed as arbitrary light units per µg
protein, and CAT values as optical densities x 103
per µg protein. A, The cells were cotransfected with the hER
expression vector (1 µg), ERE-TK-Luc promoter-reporter vector (1
µg), and either the ribozyme expression vector (1 µg) or the same
amount of the empty expression vector (pcDNA3.1). Luciferase activity
was determined at 48 h after transfection. All culture media
except the negative control contained 10-9 M
17ß-estradiol. The numbers on histograms represent reporter activity
(minus the background activity of the estrogen-free negative control
containing the hER expression vector) derived from cells transfected
with the following expression vector combinations: 1, ERE-TK-Luc and
hER ; 2, ERE-TK-Luc, hER , and RZ-1; 3, ERE-TK-Luc and hER +RZ-2;
4, ERE-TK-Luc, hER , and a 50:50 mixture of RZ-1 plus RZ-2; 5,
ERE-TK-Luc, hER , and mutant RZ-2. Each histogram is a mean of four
determinations ± SD. B, Cells were transfected with
the GR expression vector (1 µg) and the reporter MMTV-CAT (1 µg)
together with 1 µg of the empty vector, pcDNA3.1 (lane 1), or the
expression vector encoding RZ-1 (lane 2) or RZ-2 (lane 3). All culture
media contained 10-8 M dexamethasone. The
numbers represent values minus the background activity of the negative
control containing GR expression vector but not dexamethasone. The
points in each histogram indicate the results from three
independent transfections.
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Effects of RZ-1 and RZ-2 on the Natural hER
Transcript in
ER-Positive MCF-7 Cells and on the Estrogen-Dependent Cell Cycle
Progression into the S Phase
The inhibitory effects of the two ribozymes on
promoter-reporter function in COS-1 cells transfected with the
hER
expression vector only indicate the efficacy of these ribozymes
on the transcripts derived from processed complementary DNAs (cDNAs).
To examine the ribozyme effects on the natural transcript of hER
, we
used ER-positive MCF-7 cells. MCF-7 cells are highly estrogen sensitive
for ERE-TK-Luc expression without any hER
cotransfection (Fig. 5
, bars 1 and 2). Cotransfection with the
empty vector (pcDNA3.1) did not significantly alter luciferase activity
(bar 3). However, both RZ-1 and RZ-2 caused more than 60% inhibition
of luciferase activity.

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Figure 5. Inhibition of the Endogenous ER-Mediated
Trans-Activation of the ERE-TK-Luc in Transfected MCF-7
Cells
Estrogen-depleted cells were transfected with ERE-TK-Luc and cultured
either in estrogen-free medium (bar 1) or in the presence of
10-9 M 17ß-estradiol (bars 25). Histograms
1 and 2 were derived from cells transfected only with ERE-TK-Luc.
Histograms 4 and 5 indicate transfections with ERE-TK-Luc plus RZ-1 and
RZ-2 expression constructs, respectively. Histogram 3 indicates
transfection with the control vector (pcDNA3.1). Results are average of
duplicate experiments, with individual values presented as
dots.
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MCF-7 cells become quiescent when deprived of estrogens, but subsequent
estrogen supplementation propels them into the synthetic phase of the
cell cycle, leading to mitosis (24). Earlier studies have shown about
50% reduction of S phase cells 24 h after inhibition of estrogen
action by the antiestrogenic ligand tamoxifen (25). Despite the
limitation of transient transfections, where only a certain percentage
of the cell population picks up the transfected DNA, we examined the
effects of the hER
-specific ribozymes on the percentage of S phase
population 26 h after 17ß-estradiol was reintroduced into the
ribozyme-expressed quiescent MCF-7 cells. The percentages of S phase
populations as determined by fluorescence-activated cell sorting (FACS)
are shown in Fig. 6
. The results suggest
that both RZ-1 and RZ-2 can cause a reduction in the number of cells
that enter into the S phase, a prelude to mitosis. All of these results
taken together indicate that both RZ-1 and RZ-2 can serve as effective
inhibitors of ER
expression, and they can also inhibit
estrogen-dependent transcriptional activation and cell
proliferation.

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Figure 6. Flow Cytometric Analysis of the S Phase Cell
Population in MCF-7 Cells Transfected with the Ribozyme Expression
Vector
Estrogen-depleted quiescent cells were transfected with the following
plasmids: pcDNA3.1 empty vector (panel 1), RZ-1 (panel 2), RZ-2 (panel
3), and mutant RZ-2 (panel 4). Transfected cells were cultured for
26 h in the presence of 10-9 M
17ß-estradiol before harvesting for FACS analysis. The shaded
area within the distribution profile shows S phase cell cycle
populations. The FACS analysis was repeated three different times, and
the figure represents the results of one of the experiments. Two other
experiments produced similar distribution patterns, with RZ-1 causing
19% and 38% inhibition and RZ-2 causing 34% and 25% inhibition of
the S phase population over the vector-treated control.
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DISCUSSION
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Estrogens are essential not only for the regulation of female
reproductive functions, but also play critical roles in the propagation
of a number of tumor phenotypes of estrogen target organs, such as the
mammary gland. All of these hormonal functions are mediated through
ERs, i.e. ER
, ERß1, and ERß2 (2, 3, 4, 5). Among these
receptor subtypes, ER
provides the dominant regulatory role in most
target tissues (6). Primarily because of the important clinical use in
the management of estrogen-dependent cancers, the search for an
improved inhibitor of estrogen action has always been of significant
endocrinological interest. Historically, estrogen analogs that bind to
the receptor, but do not promote coactivator association and block
trans-activation function, received major attention (8, 9, 10).
These efforts have led to the development of several important
antiestrogens, such as tamoxifen, raloxifene, and ICI 182780 (26, 27, 28, 29),
all of which are extensively used as pharmacological inhibitors of
estrogen action. However, some of these antagonists also act as partial
agonists, and most of them when used for a prolonged period give rise
to drug resistance, possibly due to progressively increased metabolic
inactivation. Additionally, the potential role of the
ligand-independent constitutively active mutant forms of ER in the
proliferation of certain types of cancer cells has been reported (30, 31). The above considerations, and recent advances in the development
of gene-based therapeutic agents, have provided the impetus for the
development of alternate strategies for inhibition of ER function. One
such nonconventional molecular approach involves targeted
overexpression of a dominant negative form of ER (11). This approach is
based on the principle that a defective form of ER that can dimerize
with the wild-type natural subunit will, upon overexpression in
sufficient amounts, disable enough normal subunits and thereby inhibit
the estrogen signaling cascade. The success of this approach for its
therapeutic application may be dependent on massive overexpression of
the defective subunit sufficient for inactivation of the wild-type
receptor below a critical threshold level. A third approach with its
immediate potential for incorporation into gene-based therapy is the
selective intracellular destruction of ER mRNAs in target cells.
Antisense ER transcripts can potentially function in this manner, and a
major improvement in the antisense approach is achieved when the
antisense specificity is combined with catalytic cleavage of the
phosphodiester bond of the RNA target (12, 15, 16, 17, 18, 19, 20). Earlier studies
with estrogen antagonists such as tamoxifen and ICI 182780 have shown
that even at concentrations of 10- to 100-fold molar excesses over
estradiol, these compounds can cause more than 50% inhibition of
ERE-TK-Luc trans-activation and MCF-7 cell cycling (25, 32).
In the transient transfection assay, the ER-specific ribozymes and the
hER
expression vector, only at an equimolar ratio, resulted in about
80% inhibition of ERE-TK-Luc trans-activation.
Additionally, both of these potent antiestrogens have unique
disadvantages, such as differential effects on target genes, the need
for systemic administration, and the development of drug resistance
after prolonged use. Most of these problems can be potentially avoided
by targeted tissue-specific delivery of the ribozyme expression vector.
Thus, a combination therapy based on antiestrogens, overexpression of
the dominant negative mutants, and selective degradation of ER mRNAs
can potentially provide the most effective means to achieve almost a
total blockage of estrogen action.
The two hammerhead ribozymes, RZ-1 and RZ-2, which we describe in this
article, selectively inhibit estrogen action by cleaving the hER
mRNA within its DNA-binding domain. The specifier side arms of both
RZ-1 and RZ-2 do not show any significant homology to any known human
mRNA species, except three related receptors, hERR-1, hERR-2, and
hERß. RZ-2 possesses a slightly greater homology with hERß (90%
sequence homology with respect to both side arms) than RZ-1 (one side
arm, 90%; the other, 70%). It is possible that because of such
sequence homology to the ERß mRNA, RZ-2 provides a slightly better
inhibitory function on the activity of the ERE-TK-Luc plasmid in
transfected MCF-7 cells. However, we have not experimentally tested
this possibility. Both RZ-1 and RZ-2 were designed on the basis of
predicted sequence specificity with an optimum cleavage site (GUC
triplet) within a region that is free of any secondary structure.
Analysis of the mRNA sequence by the MFOLD computer program provides
suboptimal stem-loop structures on the basis of energy minimization.
However, the predicted secondary structure is only an approximation,
and in the cellular context the structure may exist in a thermodynamic
equilibrium of more than one conformational variation. The local
subcellular environment and protein-RNA interactions can also
significantly distort the RNA secondary structure over its minimum free
energy content. Thus, ribozymes that are optimized from theoretical
considerations and are effective in sequence-specific cleavage in
vitro may not necessarily function with similar effectiveness
within the target cell. Additionally, the stability of the ribozyme
transcript is considered to be a significant complicating factor. RZ-2,
which is only about 50% as efficient as RZ-1 in the in
vitro cleavage of the hER
mRNA substrate, was found to function
with equal or better efficiency in inhibiting ERE-TK-Luc expression in
transfected cells. The intracellular efficacy of these two ribozymes is
evident not only in the inactivation of the expressed hER
cDNA
transcript, but also in the inhibition of the natural hER
gene
transcript in MCF-7 breast cancer cells, where the receptor mRNA
undergoes normal processing steps in subcellular compartments. It
should also be noted that the ribozyme expression vector that we used
for this study provides 5' capping and polyadenylation of the
transcribed RNA, both of which are expected to enhance the
intracellular stability of the ribozyme transcript (33). Finally, from
the standpoint of its therapeutic application, it is noteworthy that
RZ-1 and RZ-2 not only block intracellular trans-activation
of the model ER target, i.e. ERE-TK-Luc, but are also
effective in inhibiting a complex regulatory function such as cell
cycling. The ribozyme-mediated decrease in the population of MCF-7
cells that enters into the S phase after estrogen supplementation of
the quiescent cells attests to the therapeutic potential of this new
class of inhibitors of estrogen action. Recent improvements of the
adenovirus-mediated regulable gene delivery system are expected to
facilitate utilization of the ribozyme approach for its eventual
therapeutic applications (34).
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MATERIALS AND METHODS
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Selection of Ribozyme Target Sites
Based on the primary and secondary structure analysis of the
hER
mRNA sequence, an optimum target site for each hammerhead
ribozyme was chosen. The sites free of any potential secondary
structure were identified by analysis of the ER
mRNA sequence using
the MFOLD program (Genetics Computer Group, version
8.1, Madison, WI), which predicts optimal and suboptimal RNA secondary
structures based on the energy minimization method (18). The linear
structure of the mRNA was converted into a two-dimensional stem-loop
format by processing the results of the MFOLD analysis in a VAX
computer using the SQUIGGLES graphic program. The structure containing
the least free energy change of formation was used for further
consideration. To eliminate non-ER
mRNA targets, we selected
approximately 20-nucleotide sequence stretches surrounding GUC triplets
within the single-stranded, looped regions of the two-dimensional
structure, and those sequences that lack any substantial homology with
non-ER mRNA sequence entries in the GenBank database were selected.
Expression Constructs for Ribozymes and ER
Oligodeoxynucleotides corresponding to the wild-type RZ-1 and
RZ-2 as well as the corresponding mutant ribozymes were synthesized at
the institutional DNA synthesis core facility, and the oligos were
purified on a 16% polyacrylamide/8 M urea gel. The
Bluescript SK plasmid (Stratagene, La Jolla, CA) was
digested with SacI and EcoRI restriction enzymes,
and each oligonucleotide was subcloned into the plasmid vector via the
SacI/EcoRI cloning sites. The human ER
mRNA
fragment from 748-1047 nt positions (GenBank accession no. M12674) was
used as the substrate for the ribozyme-catalyzed cleavage reaction.
In vitro transcription by T7 RNA polymerase was employed to
synthesize the RNA substrate from the cDNA template spanning positions
748-1047 of the human ER
mRNA. This cDNA was generated by RT-PCR of
the total mRNAs of MCF-7 cells in the presence of appropriate sense and
antisense primers (each 20 nt long) and cloned into the plasmid vector
pGEM-TEasy (Promega Corp., Madison, WI) to generate
TEasy-ER plasmid. To generate the mRNA substrate corresponding to the
GR DNA-binding domain (+1387 to +1642, GenBank accession no. M14053),
the cDNA was amplified using GR-specific primers
(5'-GCCTGGTGTGCTCCGATGAAGC and 5'-CCTGCAGTGGCTTGCTGAATCC), and the
256-bp PCR product was subcloned into the pCRII cloning plasmid
(Invitrogen). The radiolabeled RNA substrate was
synthesized by T7 RNA polymerase-directed in vitro
transcription of BamHI-digested plasmid construct in the
presence of [
-32P]UTP. An expression construct for the
full-length human ER
cDNA driven by the Rous sarcoma virus long
terminal repeat (RSV-ER, a gift from Dr. Sophia Tsai, Baylor College of
Medicine, Houston, TX) was used to assess ribozyme function in
transfected COS-1 cells. The GR expression plasmid was driven by the
cytomegalovirus (CMV) promoter. The original full-length GR cDNA clone
was obtained from American Type Culture Collection
(Manassas, VA) and was subsequently cloned into the plasmid pCMV5.
Luciferase expression from the plasmid ERE-TK-Luc containing the
vitellogenin ERE ligated to the luciferase reporter gene provided
measures of ER functionality in transfected cells. Ribozyme expression
in transfected cells was driven by the CMV promoter of the ribozyme
expression plasmids, which were produced by cloning the ribozyme
sequences (wild-type and mutant) into pcDNA3.1
(Invitrogen, San Diego, CA). All constructs were
authenticated by DNA sequencing.
Substrate Cleavage in Vitro by Ribozymes
The radiolabeled RNA substrate was prepared by T7 RNA
polymerase-directed in vitro transcription of the
SalI-digested TEasy-ER plasmid in the presence of
[
-32P]UTP and the other three unlabeled ribo-NTPs
using standard conditions as previously described (12). The hammerhead
ribozyme transcripts were synthesized from the
EcoRI-digested ribozyme expression constructs by in
vitro transcription directed by T7 RNA polymerase. The in
vitro cleavage reactions were performed as previously described
(12) with some modifications. Briefly, the radiolabeled mRNA substrate
and the ribozyme(s) were separately preincubated at 37 C for 3 min in
50 mM Tris-HCl (pH 7.5), 2 mM spermine, and 1
mM EDTA. Each preincubated mixture was then brought to 10
mM MgCl2 and mixed together to initiate the
cleavage reaction by incubation at 37 C. At the end of the incubation,
the reaction products along with 5 µg yeast transfer RNA (carrier)
were precipitated at -70°C (30 min) in the presence of 2.5
M ammonium acetate and 70% ethanol. The precipitates were
pelleted, washed once with 70% ethanol and once with absolute ethanol,
and then air-dried. The dry pellets were suspended in a RNA sample
buffer containing 10 mM EDTA, 90% formamide, 0.1%
bromophenol blue, and 0.1% xylene cyanol and heated for 5 min at 95 C.
Afterward, the cleavage products were resolved electrophoretically on a
5% polyacrylamide/8 M urea gel. The products along with
the uncleaved substrate were visualized by autoradiography. To monitor
the kinetics of the enzymatic cleavage, 100 µl of a mixture
containing the unlabeled ribozyme and the 32P-labeled ER
mRNA substrate (1:1 molar ratio) were incubated at 37 C; reaction
mixtures were removed in aliquots at different time points,
precipitated, and processed as described above. The percentage of
cleavage was quantified by PhosphorImager (GS-363, Bio-Rad Laboratories, Inc., Richmond, CA) analysis of the radiolabeled
bands after subtracting background values.
Cell Transfection
MCF-7 (ER-positive) and COS-1 (ER-negative) cells were obtained
from American Type Culture Collection and cultured in
serum-containing medium as recommended by the supplier. The COS-1 cells
were plated in six-well culture flasks at 2 x 105
cells/well, grown overnight, and then, using the LipoTaxi
(Stratagene) reagent, cotransfected with the ERE-TK-Luc
reporter construct (35), pRSV-ER target vector, and the ribozyme
expression construct. After 4 h, the cells were placed in the
growth medium (MEM and phenol red-free, 5% charcoal-stripped FBS) with
or without 10-9 M 17ß-estradiol. At the end
of 48 h, the cells were harvested, and cell extracts were assayed
for luciferase activity (assay kit, Promega Corp.), and
protein concentrations were determined by the Bradford procedure (36).
The MMTV-CAT plasmid was used as the reporter construct to examine the
effect of the ribozyme on GR-activated reporter expression in COS-1
cells, using transfection conditions similar to those described above
in the presence or absence of dexamethasone (10-8
M). The cell extracts were assayed for CAT expression by
enzyme-linked immunosorbent assay using 50 µg protein extracts
according to the manufacturers protocol (Boehringer Mannheim, Indianapolis, IN). Results were expressed as optical
densities (x1000) per µg protein. For RNase protection, COS-1 cells
were seeded in T75 flasks (
1 x 106/flask),
cultured overnight, and transfected with the ER expression plasmid and
appropriate ribozyme expression constructs. To achieve the highest
possible transfection efficiency for the flow cytometric analysis of
MCF-7 cells, the high efficiency FuGENE6 (Boehringer Mannheim) transfection reagent was used.
RNase Protection
Total RNA from transfected cells was isolated using the RNeasy
Kit (QIAGEN, Chatsworth, CA). The antisense probe for
hER
mRNA was generated by SP6 polymerase-directed in
vitro transcription of the NcoI-digested TEasy-ER
plasmid construct in the presence of [
-32P]UTP and
three other ribo-NTPs, and RNase protection was performed using an
RPAII assay kit (Ambion, Inc., Houston, TX). The ß-actin
antisense RNA probe was used as an internal control. Radiolabeled bands
were quantitated by PhosphorImager analysis.
Flow Cytometry
Cell cycle distributions of the ribozyme-expressed and control
vector-expressed MCF-7 cells were examined by FACS analysis of the
propidium iodide-stained cells. MCF-7 cells were seeded in T75 flasks
at about 0.5 x 106 cells, cultured overnight, and
then transfected with 10 µg plasmid using 20 µl FuGENE6
transfection reagent (Boehringer Mannheim) according to
the manufacturers recommended protocol. At the end of 12 h of
transfection, the cells were washed and placed in the fresh culture
medium containing 1 nM 17ß-estradiol. The cells were
cultured for additional 26 h and harvested for analysis by flow
cytometry. Briefly, the harvested cells were pelleted, washed with PBS
(pH 7.5), and incubated with 500 µl 70% ethanol at -20 C for
2.5 h. After washing with PBS containing 0.5% BSA, the pelleted
cells were resuspended in 150 µl fresh PBS. To the cell suspension
were added 1 vol propidium iodide (100 µg/ml) and 0.5 vol RNase A
solution (1 mg/ml), and the stained cells were filtered through nylon
mesh. The cells were then analyzed in a FACS (FACStar Plus,
Becton Dickinson and Co.). Data were analyzed using the
ModFit LT program (Verity House Software).
 |
ACKNOWLEDGMENTS
|
---|
We thank Nadarajan Velu, Paramita Ghosh, Charles Thomas, Gilbert
Torralva, and Annalise Castro for their assistance during different
phases of this investigation. We thank Sophia Tsai and Douglas Yee for
providing the hER
expression vector and ERE-TK-Luc construct,
respectively.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Arun K. Roy, Ph.D., Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284. E-mail:
roy{at}uthscsa.edu
This work was supported by the NIH Grants R37-AG-10486 and
RO1-DK-14744. S.C. was partly supported by the NIH Training Grant T32
AG-00165. B.C. is a career scientist of Veterans Affairs.
Received for publication February 11, 1999.
Revision received March 22, 1999.
Accepted for publication March 22, 1999.
 |
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