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{dagger}, 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


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


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1Go 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. 2Go. 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. 3Go). Furthermore, a two-base substitution within the catalytic core (G ->U and A -> C, shown in Fig. 1Go) 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 1–3) or the HR2 (S2, lanes 4–6) 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.

 
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 (8–65 nM) of the substrate (Fig. 4AGo). 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. 4BGo. 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 2–8, 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.

 
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. 1Go, 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. 5Go). 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 (10–20%) 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 1Go). 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{alpha}-dihydrotestosterone (10-9 M); AR, pCMV-AR; HR2, pCMV-HR2; MT, pCMV-HR2 with two-base substitution as indicated in Fig. 1Go; 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

 
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. 6Go 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.

 
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. 7Go).



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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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 70–90% 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 10–20 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. 6Go). 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 12–18 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 HR2—all 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 [{alpha}-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 [{alpha}-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. {dagger}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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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