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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, 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{alpha} knockout and not in ERß null mice (6, 7), ER{alpha} appears to provide the critical role in most of the estrogen-regulated processes. Thus, pharmacological inhibition of ER{alpha} 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{alpha} 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Design of the Human ER{alpha}-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{alpha} mRNA, we examined the potential secondary structure of the hER{alpha} mRNA sequence through the energy minimization approach (18). Figure 1AGo shows the potential stem-loop configuration of the hER{alpha} 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. 1AGo. 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 9–11 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. 1Go, B and C, respectively. GenBank search of the binding region of RZ-1 (hER{alpha} sequences from +947 to +966) and RZ-2 (hER{alpha} sequences from +880 to +900) indicated the absence of significant homology of these two sequence regions of the hER{alpha} 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. 1CGo) 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.



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Figure 1. Stem-Loop Secondary Structure of the hER{alpha} mRNA and the Nucleotide Sequence of Two hER{alpha}-Specific Hammerhead Ribozymes

A, Potential secondary structure of the hER{alpha} mRNA from 1- to 1300-nt residues encompassing the amino acid coding sequences for the N-terminal trans-activation domain, DNA-binding domain, hinge region, and part of the steroid-binding domain. Cleavage sites for the two ribozymes, RZ-1 and RZ-2, within open loop regions are indicated by arrows. B and C show the sequence structures of RZ-1 and RZ-2, the complementary mRNA sequences, and the expected endonuclease cleavage sites and reaction products. The circled P at the cleavage product specifies the 2',3'-cyclic terminal end. Base substitutions at A->C and G->U generate catalytically inactive mutant RZ-2. The expected points of cleavage at the mRNA sequence are indicated by arrowheads.

 
Catalytic Cleavage of the hER{alpha} 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{alpha}, 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. 2Go, 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-{alpha} mRNA substrate (Fig. 2AGo). 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. 2AGo) is about twice as effective in vitro as RZ-2 (Fig. 2BGo). 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. 2CGo). 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. 2DGo, lane 3). The results presented in Fig. 2DGo, 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. 1CGo) within the catalytic core of RZ-1 and RZ-2 almost completely abolished their enzymatic activities (Fig. 2DGo, 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. 2DGo, 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{alpha} 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 1–7). C, PhosphorImager quantification of the cleavage kinetics of the RZ-1 (filled circles) and RZ-2 (empty circles). D, Lanes 1–3, 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 4–7 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{alpha} substrate as in lanes 1–3. Both mutants contain two-point mutations at the catalytic core (as shown in Fig. 1Go), 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.

 
Inhibition of Estrogen Response Element (ERE)-Containing Promoter Function and Decrease in hER{alpha} Transcripts in COS-1 Cells Cotransfected with hER{alpha} 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{alpha} 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. 3AGo, 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{alpha} transcript. The results presented in Fig. 3BGo 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{alpha} mRNA level. This was indicated by the results of the ribonuclease (RNase) protection assay (Fig. 4Go). RZ-1, RZ-2, and RZ-1 plus RZ-2 all caused declines in the level of the hER{alpha} mRNA-protected radiolabeled antisense band to approximately 80%, and the mutant RZ-2 was only weakly effective (Fig. 4Go, 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. 4Go, 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{alpha} mRNA, thereby reducing the ER{alpha} 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{alpha} 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{alpha} expression vector) derived from cells transfected with the following expression vector combinations: 1, ERE-TK-Luc and hER{alpha}; 2, ERE-TK-Luc, hER{alpha}, and RZ-1; 3, ERE-TK-Luc and hER{alpha}+RZ-2; 4, ERE-TK-Luc, hER{alpha}, and a 50:50 mixture of RZ-1 plus RZ-2; 5, ERE-TK-Luc, hER{alpha}, 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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Figure 4. Quantitative Analysis of the hER{alpha} mRNA by RNase Protection Assay in COS-1 Cells Co-Transfected with hER{alpha} and Ribozyme Expression Vectors

RNA samples were derived from cells transfected with the hER{alpha} expression vector and pcDNA3.1 (control, lane 1) and hER{alpha} expression vector along with a 10-fold molar excess of ribozyme vectors as indicated on the top. The upper frame shows the autoradiogram of the hER{alpha} mRNA-protected antisense probe, the middle frame shows an autoradiogram of the ß-actin mRNA (invariant control)-protected antisense probe, and the bottom frame shows 5 µg total RNA samples from the corresponding cells, separated electrophoretically on a nondenaturing agarose gel and stained with ethidium bromide.

 
Effects of RZ-1 and RZ-2 on the Natural hER{alpha} 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{alpha} 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{alpha}, we used ER-positive MCF-7 cells. MCF-7 cells are highly estrogen sensitive for ERE-TK-Luc expression without any hER{alpha} cotransfection (Fig. 5Go, 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 2–5). 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.

 
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{alpha}-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. 6Go. 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{alpha} 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, ERß1, and ERß2 (2, 3, 4, 5). Among these receptor subtypes, ER{alpha} 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{alpha} 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{alpha} 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{alpha} 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{alpha} cDNA transcript, but also in the inhibition of the natural hER{alpha} 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).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Selection of Ribozyme Target Sites
Based on the primary and secondary structure analysis of the hER{alpha} 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{alpha} 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{alpha} 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{alpha}
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{alpha} 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{alpha} 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 [{alpha}-32P]UTP. An expression construct for the full-length human ER{alpha} 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 [{alpha}-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 manufacturer’s 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{alpha} mRNA was generated by SP6 polymerase-directed in vitro transcription of the NcoI-digested TEasy-ER plasmid construct in the presence of [{alpha}-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 manufacturer’s 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{alpha} 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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