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Inhibitionofurokinasereceptorgeneexpressionandcellinvasionbyanti-uPARDNAzymesinosteosarcomacells

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CharlesE.DeBock

UniversityofLeuven

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ZhenLin

KollingInstituteofMedicalResearch

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GeorgeACMurrell

UniversityofNewSouthWales

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Inhibition of urokinase receptor gene expression and cellinvasion by anti-uPAR DNAzymes in osteosarcoma cellsCharles 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

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

a potential target for anticancer drug development for

inhibiting metastasis [3,4].

uPAR is a cellular receptor 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 transforming growth factor-b, release basic

fibroblast growth factor from its ECM-binding sites,

and activate zymogens of matrix metalloproteinases,

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 � 93% and

� 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.

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.

3572 FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS

all of which contribute to focal proteolysis and regu-

lation of wound healing, angiogenesis, and metastasis

[5].

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-

sion and also markedly decreased Matrigel invasion

[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-jBmotifs [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 (� 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].

To develop a potential tool for uPAR gene therapy,

we used a catalytic DNA enzyme (DNAzyme)

approach. The DNAzymes were originally generated

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

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-

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

Dz372 (active) 5¢-CTTCGGGAAGGCTAGCTACAACGAAGGTGACAG-3¢Dz372 (mutant) 5¢-TTCGGGAACGGCTAGCTACAACGAAGTGGACGA-3¢ 372 nt

Dz483 (active) 5¢-GTCACCACAGGCTAGCTACAACGACCAGGCACT-3¢Dz483 (mutant) 5¢-ACACCACTGGGCTAGCTACAACGATCACGGACC-3¢ 483 nt

Dz720 (active) 5¢-GAGCATCCAGGCTAGCTACAACGAGGGTGCTGT-3¢Dz720 (mutant) 5¢-TAGAGCCACGGCTAGCTACAACGATTGGCGTGG-3¢ 720 nt

C. E. de Bock et al. Inhibition of uPAR by DNAzymes

FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS 3573

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.

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 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 ratios (Fig. 2A, uPAR ⁄Dz720 ¼1 : 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

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.

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 � 50% of

uPAR transcript substrate within 10 min (Fig. 3).

After 2 h incubation, the final uPAR transcript con-

centrations were decreased � 90% and � 75% for

Dz720 and Dz483 cleavages, respectively (Fig. 3C,D).

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

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.

Inhibition of uPAR by DNAzymes C. E. de Bock et al.

3574 FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS

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

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. [17] 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-

centration for cell transfection ranged from 1.6 to

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

used this dose range to analyse uPAR mRNA

concentrations using northern blot analysis over a

24–48 h period.

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 � 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

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

A

C

B

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 �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.

C. E. de Bock et al. Inhibition of uPAR by DNAzymes

FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS 3575

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 � 72% and � 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 uPAR protein concentrations between the

untreated control and mutant DNAzyme-treated cells

(Fig. 5).

Inhibition of Saos-2 cell invasion by Dz720

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 to ameliorate 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

containing Matrigel inserts and a filter pore size of

8 lm. After treatment with Dz720, there was a signi-

ficant decrease in cell 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 compared with untreated control 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.

A B

C DFig. 3. In vitro cleavage of uPAR mRNA

substrate over time by Dz720 and Dz483.

The reactions were performed at 37 �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.

Inhibition of uPAR by DNAzymes C. E. de Bock et al.

3576 FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS

Discussion

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.

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-

zyme approach lies in its inexpensive production,

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-

script substrate. As shown in Fig. 2, DNAzymes

Dz720 and Dz483 can cleave their target uPAR tran-

script at a molar ratio as low as 1 : 0.2.

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.

C. E. de Bock et al. Inhibition of uPAR by DNAzymes

FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS 3577

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 � 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].

As shown in Fig. 5, a triple transfection procedure

was used to detect changes in uPAR protein. Nor-

thern blot analysis showed that the concentration of

uPAR mRNA was � 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.

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].

This study has shown that the anti-uPAR DNA-

zyme Dz720 can inhibit cancer cell invasion. The

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.

Inhibition of uPAR by DNAzymes C. E. de Bock et al.

3578 FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS

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.

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.

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.

Recently, down-regulation of the uPA–uPAR inter-

action was attained by the delivery of antisense

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

A

B

C

D

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.

C. E. de Bock et al. Inhibition of uPAR by DNAzymes

FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS 3579

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.

Experimental procedures

Materials

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).

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

by DNAzymes in cleavage buffer (50 mm Tris ⁄HCl,

pH 7.5, 10 mm MgCl2). Cleavage products were separated

on a denaturing (1 · Tris ⁄ borate ⁄EDTA buffer ⁄ 7 m

urea ⁄ 5% acrylamide) gel. The gel was then exposed to

Kodak Biomax MR film.

Cell culture and transfection

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

medium (DMEM; Invitrogen) supplemented with 10%

fetal bovine serum (Hyclone, Tauranga, New Zealand)

and 1% antibiotics at 37 �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

or scrambled mutant in the presence of opti-MEM I

reduced serum medium (Invitrogen). After 4 h of serum

starvation in the presence of the DNAzyme–Lipofectamine

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.

Northern blot analysis

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 �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

for uPAR and 18S rRNA were radiolabeled with

[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 �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.

Western blot analysis

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

Inhibition of uPAR by DNAzymes C. E. de Bock et al.

3580 FEBS Journal 272 (2005) 3572–3582 ª 2005 FEBS

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

control.

Cell invasion assay

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).

Data analysis

All values are expressed as the mean ± SD. Statistical sig-

nificance was calculated using Student’s t-test where

appropriate.

Acknowledgements

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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