Báo cáo khoa học: Inhibition of urokinase receptor gene expression and cell invasion by anti-uPAR DNAzymes in osteosarcoma cells

Inhibition of urokinase receptor gene expression and cell invasion by anti-uPAR DNAzymes in osteosarcoma cells Charles E. de Bock*, Zhen Lin*, Takashi Itoh, David Morris, George Murrell and Yao Wang

Orthopaedic Research Institute, Department of Medicine, St George Hospital, University of New South Wales, Sydney, NSW, Australia

Keywords DNAzyme; gene expression; osteosarcoma cell invasion; urokinase receptor

Correspondence Y. Wang, Orthopaedic Research Institute, St George Hospital, University of New South Wales, Sydney, NSW 2217, Australia Fax: +61 2 9350 3967 Tel: +61 2 9350 1422 E-mail: y.wang@unsw.edu.au

*These authors contributed equally to this work

(Received 11 October 2004, revised 28 February 2005, accepted 18 May 2005)

doi:10.1111/j.1742-4658.2005.04778.x

The urokinase-type plasminogen activator (uPA) receptor (uPAR) has been implicated in signal transduction and biological processes including cancer metastasis, angiogenesis, cell migration, and wound healing. It is a specific cell surface receptor for its ligand uPA, which catalyzes the formation of plasmin from plasminogen, thereby activating the proteolytic cascade that contributes to the breakdown of extracellular matrix, a key step in cancer metastasis. We have synthesized three different DNA enzymes (Dz372, Dz483 and Dz720) targeting uPAR mRNA at three separate purine (A or G)–pyrimidine (U or C) junctions. Two of these DNAzymes, Dz483 and Dz720, cleaved uPAR transcript in vitro with high efficacy and specificity at a molar ratio (uPAR to Dz) as low as 1 : 0.2. When analyzed over 2 h with a 200-fold molar excess of DNAzymes to uPAR transcript, Dz720 and Dz483 were able to decrease uPAR transcript in vitro by (cid:1) 93% and (cid:1) 84%, respectively. They also showed an ability to cleave uPAR mRNA in the human osteosarcoma cell line Saos-2 after transfection. The DNA- zyme Dz720 decreased uPAR mRNA within 4 h of transfection, and inhi- bited uPAR protein concentrations by 55% in Saos-2 cells. The decrease in uPAR mRNA and protein concentrations caused by Dz720 significantly suppressed Saos-2 cell invasion as assessed by an in vitro Matrigel assay. The use of DNAzyme methodology adds a new potential clinical agent for decreasing uPAR mRNA expression and inhibiting cancer invasion and metastasis.

receptor a potential target for anticancer drug development for inhibiting metastasis [3,4]. uPAR is a cellular

transforming growth factor-b,

Abbreviations ECM, extracellular matrix; DMEM, Dulbecco’s modified Eagle’s medium; PAI-1, plasminogen activator inhibitor-1; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor.

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The plasminogen activator system plays a central role in wound healing, inflammatory and tissue dissolution, and cell invasion and metastasis. Tumor cells need to penetrate the extracellular matrix (ECM) and the base- ment membrane in order to metastasize. The serine protease family, which includes plasmin and urokin- ase-type plasminogen activator (uPA), has been identi- fied as being involved in the metastatic process. In a clinical setting, members of the plasminogen activator system, including the uPA receptor (uPAR), have been found to be over-expressed in a large number of can- cers leading to a poor prognosis [1,2]. This over- expression of uPAR has resulted in its identification as for uPA, and is anchored to the extracellular side of the cell membrane via a glycosylphosphatidylinositol anchor. When uPA binds to its receptor, it has the ability to direct proteo- lytic activity toward the basement membrane and the ECM by catalyzing plasminogen activation to form plasmin. Plasmin then has the ability to either directly degrade the basement membrane and ECM or activate latent release basic fibroblast growth factor from its ECM-binding sites, and activate zymogens of matrix metalloproteinases,

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Inhibition of uPAR by DNAzymes

all of which contribute to focal proteolysis and regu- lation of wound healing, angiogenesis, and metastasis [5].

as ssDNA molecules composed of 31 deoxynucleotides, including 15 for the core catalytic motif and eight for each of the hybridizing arms [14]. These DNAzymes have the ability to bind specific mRNA targets and then catalyze a cleavage reaction in the presence of bivalent cations. This specific cleavage reaction is achieved by complementarity between the DNAzyme and the sequences flanking the target mRNA. This cleavage renders the mRNA incapable of undergoing successful translation. The DNAzymes have high enzymatic activity and cleave specific multiple target mRNA sequences in the absence of any energy source [15]. This report outlines the design of novel DNA- zymes that target uPAR mRNA as a method of inhib- iting its expression and thereby decreasing the invasive potential of tumor cells.

Results

In vitro cleavage reaction and kinetic analysis

Of the components of the uPA system, uPAR is of particular interest as there is evidence that inhibition of uPAR expression in cancer cells prevents the proces- ses of invasion and metastasis. Our laboratory and others have focused on an antisense approach, in par- ticular aimed at decreasing the concentration of uPAR, as a potential strategy for therapeutic intervention. In early antisense strategies, a ribozyme approach with a 37-mer hammerhead ribozyme was utilized and was able to cleave uPAR mRNA in vitro to inhibit its translation in a concentration-dependent manner [6]. Similarly, an antisense oligonucleotide (18-mer) that covered the transcription start site of uPAR inhibited the invasive properties of transformed human fibro- blasts (VA-13) [7]. The down-regulation of uPAR in the human glioblastoma cell line SNB19 by an anti- sense construct of 300 bp to the 5¢ end of uPAR mRNA led to a significant decrease in uPAR expres- invasion sion and also markedly decreased Matrigel [8]. When human squamous carcinoma Hep3 cells were transfected with antisense uPAR, their ability to metastasize in a chorioallantoic membrane assay decreased [9]. We have previously cloned the human uPAR gene and characterized its promoter and tran- scription factors, including two AP-1 and one NF-jB motifs [10–12]. We have also found that colon cancer cell metastasis can be inhibited using antisense metho- dology. In a nude mouse model, HCT116 colon cancer cells with high invasive potential were transfected with a plasmid containing uPAR cDNA ((cid:1) 500 bp) in an antisense orientation. Pulmonary metastases were found in only 9% of mice injected with the clone intra- venously, but were present in 67% of mice injected with the parent HCT116 cells (P < 0.05). These data strongly support the importance of uPAR expression in colon cancer invasion and metastasis [13].

Table 1. Sequence of DNAzymes and site of cleavage of uPAR mRNA transcript. The catalytic core sequence is underlined. The correspond- ing mutant control DNAzyme for each was made by scrambling the sequence of the left and right arms.

DNAzyme

Sequence

Cleavage site

372 nt

483 nt

Dz372 (active) Dz372 (mutant) Dz483 (active) Dz483 (mutant) Dz720 (active) Dz720 (mutant)

720 nt

5¢-CTTCGGGAAGGCTAGCTACAACGAAGGTGACAG-3¢ 5¢-TTCGGGAACGGCTAGCTACAACGAAGTGGACGA-3¢ 5¢-GTCACCACAGGCTAGCTACAACGACCAGGCACT-3¢ 5¢-ACACCACTGGGCTAGCTACAACGATCACGGACC-3¢ 5¢-GAGCATCCAGGCTAGCTACAACGAGGGTGCTGT-3¢ 5¢-TAGAGCCACGGCTAGCTACAACGATTGGCGTGG-3¢

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To study DNAzyme-mediated suppression of uPAR expression, three DNAzymes carrying antisense uPAR arms (Dz372, Dz483 and Dz720) and their correspond- ing scrambled mutant controls were synthesized with the same phosphorothioate substitutions for the last three nucleotides at both ends (Table 1). The sites at which the DNAzymes cut uPAR mRNA are illustrated in Fig. 1. Dz372, Dz483 and Dz720 cut the uPAR mRNA at nucleotides 372, 483 and 720, respectively, according to the uPAR cDNA sequence published [16]. The uPAR Dzs can cleave purine–pyrimidine junctions. To select the target area, we scanned many purine–pyrimidine junctions and their flanking regions in the uPAR cDNA and compared their hybridization properties according to the primer design rules. The anti-uPAR Dzs contain 15 deoxynucleotides for the core catalytic domain and nine for each of the hybrid- izing arms. They have the ability to bind specific uPAR mRNA by complementary base-pairing between the Dz arms and the sequences flanking the uPAR mRNA and then catalyse a cleavage reaction specific- To develop a potential tool for uPAR gene therapy, we used a catalytic DNA enzyme (DNAzyme) approach. The DNAzymes were originally generated

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Fig. 1. DNAzyme targeting sites in uPAR cDNA. Schematic representation showing the exon regions of uPAR cDNA and the regions tar- geted by Dz372, Dz483 and Dz720. The hybridizing arm sequences are shown for each DNAzyme, and the catalytic core illustrated by a loop. The full sequence for each respective DNAzyme is shown in Table 1.

ally by Watson–Crick interactions. Using blast ana- lysis, we determined that there is no DNA sequence homology between our uPAR antisense (or Dz mutant) hybridizing arms and any known mRNA sequences.

ratios above 1, with DNAzyme in excess of uPAR sub- strate, Dz720 cleavage began with a 72% decrease at a molar ratio of 1 : 1, and a maximum reduction of 93% of uPAR substrate concentration at a molar ratio of 1 : 200 (Fig. 2C). A similar dose-dependent cleavage was observed for Dz483, with cleavage at a 1 : 1 ratio leading to a 65% decrease in uPAR substrate, and a maximum reduction of 84% of uPAR substrate concen- tration at a molar ratio of 1 : 200 (Fig. 2D). The speci- ficity of the DNAzymes was confirmed by the lack of cleavage by the untreated and mutant controls (Fig. 2A–D), with no cleavage at 100-fold excess DNA- zyme to uPAR substrate.

In vitro cleavage reactions of a-32P-labeled uPAR transcript were used to determine the kinetic and sequence specificity of cleavage by the DNAzymes and their controls. The amount of cleavage was examined using a truncated a-32P-labeled 1113-nucleo- tide uPAR transcript. The resulting two fragments were resolved on a denaturing acrylamide gel. In vitro cleavage results showed the expected two cleavage products for all three DNAzymes. However, Dz372 had weak activity compared with Dz483 and Dz720 and was therefore omitted from further analysis (data not shown).

To determine the time-dependent reaction, experi- ments were carried out with a 100-fold excess of DNA- zyme to uPAR transcript substrate, and the cleavage products analyzed over a 2 h period. For both Dz720 and Dz483, there was a rapid decrease of (cid:1) 50% of uPAR transcript substrate within 10 min (Fig. 3). After 2 h incubation, the final uPAR transcript con- centrations were decreased (cid:1) 90% and (cid:1) 75% for Dz720 and Dz483 cleavages, respectively (Fig. 3C,D).

ratios

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To further confirm that the wild-type DNAzymes and their mutants have no effect on other transcripts, the DNAzymes were tested on labeled uPA and its inhibitor PAI-1 mRNAs by in vitro transcription and cleavage assays. To perform the assays, pGEM3Z vec- tor containing a uPA cDNA fragment and pGEM3 vector carrying a PAI-1 cDNA fragment were restric- tion digested. The linearized cDNAs were used as tem- plates for in vitro RNA transcription using T7 or SP6 RNA polymerase. The RNA transcripts were labeled To determine dose-dependent cleavage by the anti- uPAR DNAzymes, experiments were carried out using a range of molar ratios of substrate to DNAzyme con- centrations (above and below a ratio of 1 : 1). As shown in Fig. 2, over a 2 h incubation, uPAR transcript was cleaved by Dz720 in a dose-dependent manner at increased molar (Fig. 2A, uPAR ⁄ Dz720 ¼ 1 : 0.2, 0.5 or 1; Fig. 2C, uPAR ⁄ Dz720 ¼ 1 : 1, 10, 50, 100 or 200). A similar effect of Dz483 on uPAR tran- script cleavage is shown in Fig. 2B,D. Initially Dz720 and Dz483 were evaluated at a molar ratio below 1 over a 2 h period. For these low DNAzyme molarities, Dz720 and Dz483 cleavage products were evident when there was a fivefold excess of substrate to DNAzyme (uPAR ⁄ Dz ¼ 1 : 0.2; Fig. 2A,B). At the higher molar

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A

B

C

D

Fig. 2. Dose-dependent in vitro cleavage of uPAR mRNA substrate by Dz720 and Dz483. Different molar ratios of uPAR tran- script to DNAzyme were incubated for 120 min at 37 (cid:1)C. (A) Ratio of uPAR tran- script ⁄ Dz720 from 1 : 0.2 to 1 : 1. (B) Ratio of uPAR transcript ⁄ Dz483 from 1 : 0.2 to 1 : 1. (C) Ratio of uPAR transcript ⁄ Dz720 from 1 : 1 to 1 : 200. (D) Ratio of uPAR tran- script ⁄ Dz483 from 1 : 1 to 1 : 200. The posi- tions of the unreacted substrate (1113 nucleotides) and products are indicated. Each experiment was repeated at least three times.

with [32P]UTP[aP] and processed as described in Experimental procedures. These experiments were car- ried out with about a 200-fold excess of DNAzyme to the transcript substrates and the cleavage products analyzed. After 2 h incubation, the labeled uPAR mRNAs were cleaved significantly by Dz720 or Dz483. However, the labeled uPA and PAI-1 mRNA sub- strates were not cleaved by a 200-fold excess of Dz720, Dz483, and their mutants (data not shown).

Effect of DNAzymes on uPAR mRNA reduction in Saos-2 cells

[17] After transfection of 1.6 lgÆmL)1 either Dz720 or Dz483 into the Saos-2 cells, total RNA was isolated after various time points. As shown in Fig. 4, reduc- tion of uPAR mRNA was noted within 4 h of trans- fection for Dz720, leading to about 37% inhibition of uPAR expression after 24 h compared with its scrambled mutant control. However, with Dz483, the first 24 h showed (cid:1) 24% decrease in uPAR mRNA concentrations. The concentration of uPAR mRNA was further decreased after 48 h, resulting in a 46% reduction compared with its scrambled mutant con- trol (Fig. 5B). To determine whether there was an additive or synergistic effect by using a combination of both active DNAzymes, northern blot analysis was performed as above. We did not find any additive or synergistic effect using both Dz720 and Dz483 simul- taneously (data not shown).

Effect of DNAzymes on uPAR protein reduction in Saos-2 cells

range

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To investigate the effects of Dz720 and Dz483 on uPAR mRNA changes, the DNAzymes were trans- fected into Saos-2 cells. Previous studies by Zhang et al. investigating the stability of DNAzymes, found detectable levels of DNAzymes within 2 h of transfection. They were maintained in both cell cyto- plasm and nucleus for the first 24 h before progres- sively decreasing, and were still detectable after 48 h. Our pilot dose-dependent analysis of DNAzyme con- transfection ranged from 1.6 to centration for cell 10 lgÆ(mL cell medium))1 and showed optimum doses in the range 1.6–3.2 lgÆmL)1 for minimum cell toxic- ity and maximum decrease in uPAR mRNA. We then to analyse uPAR mRNA used this dose concentrations using northern blot analysis over a 24–48 h period. To determine whether this decrease in uPAR mRNA was concomitant with a decrease in its protein, uPAR protein was analysed using western blotting. Single and double dose transfections did not result in any significant decrease in uPAR protein, and therefore a triple transfection protocol was under- taken. The triple transfection protocol began with a single dose using 3.2 lgÆmL)1 Dz720, followed by

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A

B

C

D

Fig. 3. In vitro cleavage of uPAR mRNA substrate over time by Dz720 and Dz483. The reactions were performed at 37 (cid:1)C at a ratio of uPAR substrate to DNAzyme of 1 : 100. The cleavage reaction was analyzed at different time points. The resulting frag- ments were separated on denaturing 5% acrylamide gel. (A) Dz720 cleavage of the uPAR transcript over 120 min. The reaction time and positions of the unreacted sub- strate (1113 nucleotides) and products (720 nucleotides and 393 nucleotides) are indica- ted. (B) Dz483 cleavage of the uPAR tran- script over 120 min. The reaction time and positions of the unreacted substrate (1113 nucleotides) and products (630 nucleotides and 483 nucleotides) are indicated. (C) The corresponding densitometry results of (A). (D) The corresponding densitometry results of (B). Each experiment was repeated at least three times.

in cell uPAR protein concentrations between

two consecutive transfections of 1.6 lgÆmL)1 each at 24 h intervals. The corresponding level of uPAR pro- tein expression was also determined 24 h after the third transfection. The Dz720 transfection resulted in decreases of (cid:1) 72% and (cid:1) 57% in uPAR protein after treatment with active DNAzyme compared with untreated control and mutant control, respectively (P < 0.01). There was no significant difference in the the untreated control and mutant DNAzyme-treated cells (Fig. 5). compared with untreated control

Inhibition of Saos-2 cell invasion by Dz720

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to ameliorate The plasminogen activator system can promote the breakdown of ECM and facilitate cell invasion and metastasis. Increased expression of uPAR is associ- ated with invasive cancer cell phenotypes and a poor prognosis. The ability to decrease uPAR expression levels would be advantageous this effect. We therefore evaluated the capacity of Dz720 to inhibit cellular invasion of the Saos-2 cells through basement membrane matrices using an in vitro Matri- gel invasion assay. The cells were transfected three times as described in western blot analysis, collected 24 h after the final transfection, and seeded into wells inserts and a filter pore size of containing Matrigel 8 lm. After treatment with Dz720, there was a signi- ficant decrease invasion compared with untreated and scrambled mutant controls (P < 0.01) (Fig. 6). Saos-2 cells treated with the active Dz720 had 42% and 17% fewer cells on the lower side of the filter cells and cells treated with Dz720 mutant, respectively. Although there was also a significant difference between the untreated control and scrambled mutant Dz720-treated cells, this may be caused by toxicity of the DNAzyme molecules or the transfection reagent Lipofectamine or their complex. This result suggests that the targeting of uPAR by Dz720 can decrease the potentially invasive phenotype of cells over- expressing uPAR.

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A

C

B

D

Fig. 4. Effect of Dz720 and Dz483 on uPAR mRNA concentrations in cultured Saos-2 cells. (A) The cells were transfected with Dz720 (1.6 lgÆmL)1) for 4, 8, 12 and 24 h. 20 lg total RNA was used for northern blot, and the blot was hybridized with [32P]dCTP[aP]-labeled uPAR cDNA and 18S rDNA probes as a control. Autoradiographic exposure time was 24 h (for uPAR as probe) and 2 h (for 18S rDNA as probe). (B) The corresponding densitometry results are shown. After normalization for loading with 18S rRNA, the decrease in uPAR is calcu- lated as the fraction compared with the mutant DNAzyme control. (C) The cells were transfected with Dz483 (1.6 lgÆmL)1) for 24, 48 and 72 h. Total RNA (20 lg) was used for northern blot analysis and processed as described above. Autoradiographic exposure time was 24 h (for uPAR as probe) and 2 h (for 18S rDNA as probe). (D) The corresponding densitometry results are shown after normalization for loading with 18S rRNA. The decrease in uPAR was calculated as the fraction compared with the mutant DNAzyme control. Each experiment was repeated at least three times and a representative data set is shown in the figure.

Discussion

in its

In this paper we have shown that anti-uPAR DNA- zymes were able to cleave uPAR mRNA in an in vitro cleavage assay with high efficiency. When the DNA- zymes were transfected into the osteosarcoma cell line, Saos-2, it resulted in a significant loss of uPAR mRNA expression and decreased concentrations of uPAR pro- tein. This observed decrease in uPAR had the ability to inhibit the invasive potential of human osteosarcoma Saos-2 cells. This DNAzyme methodology offers an alternative approach to decreasing uPAR concentra- tions, which may be useful in a clinical setting. The ulti- mate goal of the project is to develop an anti-uPAR DNAzyme for anticancer therapy. This paper describes the first step in the development of a potential thera- peutic drug. It would be interesting to perform further studies in other cancer cell lines and in an animal model to show the efficacy of anti-uPAR DNAzymes in preventing cancer cell growth and metastasis. substrate. As

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A number of approaches have been used to disrupt the interaction between uPA and uPAR, in an attempt to decrease cancer invasion and metastasis. These methods include the use of antibodies, small molecule antagonists, and antisense strategies and have been reviewed elsewhere [3]. Although antisense oligonucleo- tides are one of the most well established methods for suppressing gene expression, they have a number of limitations; a useful review can be found elsewhere [18]. The DNAzyme technique offers a new way of suppressing gene expression. In comparison with other antisense technologies, including antisense oligonucleo- tides, ribozymes, and RNAi, the strength of the DNA- inexpensive production, zyme approach lies excellent catalytic activity, and the ability to modify its backbone for systemic delivery in the absence of a vec- tor [19]. We have therefore designed DNAzymes that target uPAR mRNA. Using an in vitro cleavage assay, we found dose-dependent cleavage of uPAR mRNA at molar ratios (uPAR ⁄ Dz) from 1 : 1 to 1 : 200 resulting in 65% to 93% decreases, respectively, in uPAR tran- shown in Fig. 2, DNAzymes script Dz720 and Dz483 can cleave their target uPAR tran- script at a molar ratio as low as 1 : 0.2.

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A

B

Fig. 5. Expression of uPAR protein concentrations in Saos-2 cells after Dz720 treatment. The cells were transfected three times at 24 h intervals with 3.2 lgÆmL)1, 1.6 lgÆmL)1, and 1.6 lgÆmL)1, respectively. They were then lysed 24 h after the third transfection. (A) Representative levels of uPAR protein expression are shown. Each sample (untreated control, Dz720 mutant and Dz720 active) was electrophoresed in duplicate. (B) The corresponding densito- metry of three replicate experiments is shown. Columns, means of three separate experiments normalized to b-actin expression and as a fraction of untreated control. Statistical evaluation showed that the effect of Dz720 in reduction of uPAR protein concentrations in the Saos-2 cells was significant as shown by ***P < 0.01.

thern blot analysis showed that the concentration of uPAR mRNA was (cid:1) 51% less than that of the cor- responding mutant control after the third Dz720 transfection for 24 h using the same transfection method as in Fig. 4 (data not shown). This result shows that two more transfections further decreased uPAR mRNA as compared with data in Fig. 4. The requirement for the triple transfection for uPAR pro- tein reduction may be due to differences in half-life between uPAR protein and mRNA. The plasminogen system is complicated because of the presence of uPAR not only on the cell surface, but also in an internalized compartment after binding of uPA and its inhibitor PAI-1. Western blot was used to analyze total uPAR protein of both intracellular and extra- cellular compartments. The triple transfection used to observe a decrease in uPAR protein is speculated to be a combination of both DNAzyme half-life and the multicompartmental state of uPAR, such that the concentration of uPAR mRNA must be maintained at low concentrations to ensure that all compartments are depleted of uPAR protein. The long half-life of the uPAR protein may be due to the discrepancy between a single and triple transfection approach. A future useful experiment would be to treat cells with cycloheximide to inhibit translation, and then treat them with Dz720 to determine whether uPAR protein decreases at a faster rate as the result of existing uPAR mRNA cleavage.

We then assessed the ability of Dz720 and Dz483 to suppress uPAR mRNA expression in the Saos-2 cells. A reduction of 37% was seen within 24 h for Dz720, but a slower decrease was evident with Dz483, with only (cid:1) 24% reduction seen 24 h after the initial trans- fection. The significant kinetic differences and ability to cleave uPAR mRNA between Dz720 and Dz483 may be related to decreased accessibility to the partic- ular region being targeted. This has been hypothesized to be due to tertiary RNA structures inhibiting access to some sites. It has been postulated that up to 90% of putative targets on long RNAs are resistant to clea- vage by DNAzymes, with different constraints on the ability of DNAzymes to hybridize (in comparison with antisense oligonucleotides) because of the bulky nature of the activity centre acting as a barrier to accessible sites [20,21]. One method used to increase the efficacy of DNAzymes is to introduce 2¢-O-methyl RNA or locked nucleic acid monomers into the binding arms of the DNAzyme. This can result in cleavage of sites that were previously unsuitable [20]. Regardless of the efficiency of cleavage, one of the major challenges facing the future application of DNAzymes in a clinical setting is cellular uptake and its half-life within a cell for long-term gene suppres- sion. One attempt to increase the half-life has been made by designing and constructing a novel endogen- ously replicating circular DNAzyme. This was devel- oped by cloning a specific DNAzyme into the vector M13mp18. The circular DNAzyme was able to cleave b-lactamase mRNA both in vitro and in bacteria. However, it should be noted that, although the half- life may have increased, the overall efficacy of the cir- cular DNAzyme was less than its linear counterpart. This was postulated to be due to the super-coiled nature of a proportion of the plasmid, trapping the active center [22]. To overcome the obstacle of tumor- specific delivery in vivo, the use of transferrin-modified, cyclodextrin polymer-based polycations has resulted in the intracellular delivery of DNAzymes after intraven- ous administration to mice. The system showed longer tumor retention and efficient cell targeting compared with unformulated and targeted DNAzymes [23].

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As shown in Fig. 5, a triple transfection procedure was used to detect changes in uPAR protein. Nor- This study has shown that the anti-uPAR DNA- invasion. The zyme Dz720 can inhibit cancer cell

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A

Fig. 6. Invasion of Saos-2 cells measured in Matrigel-coated tran- swell chambers. Invading cells were stained with 1% toluidine blue and visualized by microscopy. (A) Untreated cells. (B) Cells trans- fected with Dz720 mutant. (C) Cells transfected with Dz720 active. (D) Graphical representation of toluidine blue-stained cells after invasion, calculated by AREA PERCENT software (Carl Zeiss). Each experiment was repeated at least three times. Columns, means of three separate experiments as a fraction of untreated control. Sta- tistical evaluation showed that the effect of Dz720 in inhibition of Saos-2 cell invasion was significant as shown by ***P < 0.01.

B

reduction in in vitro invasion of the Saos-2 cells was modest. This could be for a variety of reasons. For example, in vitro invasion is a complex process, and redundant proteolytic systems might compensate for the repression of uPAR expression. It may be specula- ted that the observed decrease can be attributed to uPAR, with other compensatory mechanisms allowing the cell invasion process to continue.

C

uPA binding to uPAR has been shown to induce the proliferation of Saos-2 cells, therefore down-regulation of uPAR expression in these cells may have antiproli- ferative effects. To test whether the decreased number of cells on the Matrigel filters in the Dz720-treated wells is due to an anti-invasive effect rather than an antiproliferative effect, a cell proliferation assay was carried out. Only an 8% decrease in cell proliferation was found in Dz720 mutant controls compared with active Dz720 (data not shown), whereas our cell inva- sion assay (Fig. 6) showed a 17% difference between active Dz720 and Dz720 mutant controls. Therefore, the inhibition of invasion seen in this study is due mainly to a decrease in invasion and partly to a decrease in cell proliferation. These results are consis- tent with the known roles of uPAR in both cell proli- feration and cell invasion.

D

Previously we found that an antisense uPAR frag- ment stably integrated into the colon cancer cell line HCT116 led to the suppression of the Erk-MAP kinase pathway and reductions in uPA secretion, cell adhe- sion, and plasminogen-dependent matrix degradation. Importantly, the uPAR–b1 integrin complex was also disrupted in the antisense cell clone, with this inter- action important in maintaining the invasive pheno- type of colon cancer cells [24]. It may be speculated that the decreased uPAR expression caused by Dz720 leads to a decrease in activation of downstream mole- cules involved in the uPAR signaling pathway such as the Erk-MAP kinase pathway. It will be interesting to investigate further the exact nature of the inhibition of cell invasion by the anti-uPAR DNAzyme Dz720.

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sequences to both uPA and uPAR in a single adeno- viral vector. The bicistronic construct had the ability to significantly reduce the concentrations of uPA and uPAR in a glioma cell line and thereby diminish inva- siveness and tumorigenicity [25]. This research shows Recently, down-regulation of the uPA–uPAR inter- action was attained by the delivery of antisense

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complex, the medium was replaced with DMEM contain- ing 10% fetal bovine serum. Cells were collected and assayed by northern and western blot analyses at variable time points after transfections. For multiple transfections, the same method was used with 24 h intervals between successive transfections.

that the simultaneous targeting of two components in a single system can have a synergistic effect rather than only an additive effect. Our study provides opportunit- ies for a similar approach using DNAzymes that target both uPA and uPAR to further inhibit cancer cell invasion and metastasis.

Northern blot analysis

Experimental procedures

The DNAzymes were synthesized by Geneworks Pty Ltd (Hindmarsh, SA, Australia). The TRIzol reagent was pur- chased from Invitrogen Corporation (Carlsbad, CA, USA). Murine IgG anti-human uPAR (No. 3931) monoclonal antibody was purchased from American Diagnostica Inc (Greenwich, CT, USA). The enhanced chemiluminescence was purchased from Pierce (Rockford, IL, USA).

Materials

in cleavage buffer

Total RNA was isolated from Saos-2 cells with TRIzol Reagents using standard protocols of Invitrogen. Aliquots of 20 lg total RNA were size separated on a 1.2% agarose gel containing 2.2 m formaldehyde and 1· Mops buffer. The RNA was transferred to a nitrocellulose membrane and immobilized by baking at 80 (cid:1)C for 2 h and UV cross- linking 30 000 lJÆcm)2 (UVC 508 ultraviolet cross linker; Ultra-Lum Inc, Claremont, CA, USA). The cDNA probes radiolabeled with for uPAR and 18S rRNA were [32P]dCTP[aP] using the Prime-a-Gene Labeling system (Promega) and purified using ProbeQuant G-50 micro columns (Amersham Biosciences, Piscataway, NJ, USA). The membranes were then processed in hybridization buffer [5· NaCl ⁄ Cit, 5· Denhardt’s, 50% formamide, 50 mm sodium phosphate buffer (pH 6.7), 0.1% (w ⁄ v) SDS, 100 lgÆmL)1 heat-denatured herring sperm DNA] contain- ing labeled probe at 1 · 106 c.p.m.ÆmL)1 at 42 (cid:1)C for 48–72 h. After hybridization, the membranes were washed and exposed to Kodak Biomax MR film. Multiple film exposures were used to ensure linearity of band intensities. The intensities of mRNA bands in the autoradiographs were scanned and quantified by an Imaging Densitometer (model GS-700 ⁄ 690; Bio-Rad, Hercules, CA, USA). Intensi- ties of uPAR mRNA were calculated relative to the inten- sity of the 18S rRNA internal control.

In vitro translation and cleavage assay

The 1.4-kb cDNA fragment of uPAR contained in pBlue- script was restriction-digested, separated on a 1% agarose gel, and purified. The product was then subcloned into the pGEM vector of the Riboprobe Combination system (Promega, Madison, WI, USA) and used as a template for in vitro RNA transcription using T7 RNA polymerase. The RNA transcript was labeled with [32P]UTP[aP] for 90 min. DNA template was digested by DNase I, and the RNA purified. The RNA pellet was collected by centrifu- gation and dissolved in diethyl pyrocarbonate-treated dH2O. The quantified transcript was subjected to cleavage (50 mm Tris ⁄ HCl, by DNAzymes pH 7.5, 10 mm MgCl2). Cleavage products were separated (1 · Tris ⁄ borate ⁄ EDTA buffer ⁄ 7 m on a denaturing urea ⁄ 5% acrylamide) gel. The gel was then exposed to Kodak Biomax MR film.

Western blot analysis

Invitrogen)

Cells were washed with NaCl ⁄ Pi, trypsinized, and collected. For protein extraction, the cells were lysed in 1· cell lysis buffer (20 mm Tris ⁄ HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm b-glycerophosphate, 1 mm sodium orthovanadate, 1 lgÆmL)1 leupeptin, 10 lgÆmL)1 aprotinin, 1 mm phenylmethanesulfonyl fluoride, 1 lgÆmL)1 pepstatin A, 100 lgÆmL)1 aminoethylbenzenesulfonyl fluoride). Sup- ernatants containing equal amounts of protein (40 lg) in sample buffer [62.5 mm Tris ⁄ HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mm dithiothreitol] were separated on a SDS ⁄ 12% (v ⁄ v) polyacrylamide gel. Fractionated proteins were then transferred to poly(vinylidene difluoride) mem- brane that was then probed with uPAR antibody (No. 3931). Immunoreactive bands were visualized by Super- signal West Pico Chemiluminescence (Pierce) and quantified by densitometry (model GS-700 ⁄ 690; Bio-Rad). To ensure

The human osteosarcoma cell line Saos-2 was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s supplemented with 10% medium (DMEM; fetal bovine serum (Hyclone, Tauranga, New Zealand) and 1% antibiotics at 37 (cid:1)C and 5% CO2. Before cell har- vesting, cell viability was consistently found to be > 90%. For transfections, the Saos-2 cells were plated in 100-mm culture dishes containing DMEM with 10% fetal bovine serum without antibiotics and then treated with Lipofecta- mine 2000 (Invitrogen) with or without active DNAzyme in the presence of opti-MEM I or scrambled mutant reduced serum medium (Invitrogen). After 4 h of serum starvation in the presence of the DNAzyme–Lipofectamine

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equal protein loading, b-actin was used as an internal control.

4 Sidenius N & Blasi F (2003) The urokinase plasminogen activator system in cancer: recent advances and implica- tion for prognosis and therapy. Cancer Metastasis Rev 22, 205–222.

5 Andreasen PA, Egelund R & Petersen HH (2000) The plasminogen activation system in tumor growth, inva- sion, and metastasis. Cell Mol Life Sci 57, 25–40.

6 Kariko K, Megyeri K, Xiao Q & Barnathan ES (1994) Lipofectin-aided cell delivery of ribozyme targeted to human urokinase receptor mRNA. FEBS Lett 352, 41–44.

7 Quattrone A, Fibbi G, Anichini E, Pucci M, Zamperini A, Capaccioli S & Del Rosso M (1995) Reversion of the invasive phenotype of transformed human fibroblasts by anti-messenger oligonucleotide inhibition of urokinase receptor gene expression. Cancer Res 55, 90–95. 8 Mohanam S, Chintala SK, Go Y, Bhattacharya A,

Venkaiah B, Boyd D, Gokaslan ZL, Sawaya R & Rao JS (1997) In vitro inhibition of human glioblastoma cell line invasiveness by antisense uPA receptor. Oncogene 14, 1351–1359.

The migratory and invasive responses of Saos-2 cells were determined using the BD Biocoat Matrigel Invasion Cham- ber (24-well, 8 lm pore size) according to the manufacturer’s instructions (Becton and Dickinson, Franklin Lakes, NJ, USA). Briefly, Saos-2 cells were transfected three times with Dz720 at 24 h intervals and then collected 24 h after the final transfection. Approximately 5 · 105 cells were seeded into the 24-well chambers in DMEM containing 1% fetal bovine serum. The mixture 10% fetal bovine serum ⁄ 0.5 mgÆmL)1 collagen I (Sigma) was used as a chemoattractant. After 24 h, the Matrigel inserts were removed, and the noninvad- ing cells removed from the upper surface of the membrane by gentle scrubbing using a cotton tipped swab. The cells on the lower surface were fixed and stained with 100% meth- anol and 1% toluidine blue, respectively. The membranes were then mounted and analyzed by microscopy. Four fields per filter were used to quantify cell invasion using area per- cent 1.0 software (Carl Zeiss, Thornwood, NY, USA).

9 Kook YH, Adamski J, Zelent A & Ossowski L (1994) The effect of antisense inhibition of urokinase receptor in human squamous cell carcinoma on malignancy. EMBO J 13, 3983–3991.

Cell invasion assay

10 Wang Y, Dang J, Johnson LK, Selhamer JJ & Doe WF (1995) Structure of the human urokinase receptor gene and its similarity to CD59 and the Ly-6 family. Eur J Biochem 227, 116–122.

All values are expressed as the mean ± SD. Statistical sig- t-test where nificance was calculated using Student’s appropriate.

11 Dang J, Boyd D, Wang H, Allgayer H, Doe WF &

Data analysis

Acknowledgements

Wang Y (1999) A region between )141 and )61 bp con- taining a proximal AP-1 is essential for constitutive expression of urokinase-type plasminogen activator receptor. Eur J Biochem 264, 92–99.

12 Wang Y, Dang J, Wang H, Allgayer H, Murrell GA & Boyd D (2000) Identification of a novel nuclear factor- kappaB sequence involved in expression of urokinase- type plasminogen activator receptor. Eur J Biochem 267, 3248–3254.

We thank Dr Lunquan Sun for technical advice, and A. Q. Wei for her help. This work was supported by Foun- dation for Research Science and Technology, New Zealand; St George Hospital ⁄ South-eastern Sydney Area Health Service, St George Private Hospital ⁄ Health Care of Australia, the Australian Kidney Foundation (S1002) and Arthritis Foundation of Australia.

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