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Kinetic characterization of directed against the cisplatin resistance±associated ABC transporter cMOAT/MRP2/ABCC2 Verena Materna, Per Sonne Holm, Manfred Dietel, and Hermann Lage

Institute of Pathology, Charite Campus Mitte, Humboldt University Berlin, Berlin D-10117, Germany.

The enhanced expression of the human ABC transporter, cMOAT (MRP2/ABCC2), is associated with resistance of tumor cells against platinum-containing compounds, such as cisplatin. Therefore, cMOAT represents an interesting candidate factor for modulation of antineoplastic drug resistance. Two different hammerhead ribozymes, which exhibit high catalytic cleavage activities towards specific RNA sequences encoding cMOAT, were designed. Cleavage sites of these ribozymes are the GUC sites in codons 704 and 708 of the open readingframe in the cMOAT-specific mRNA molecule. Hammerhead ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence. cMOAT-encodingsubstrate RNA molecules were created by a reverse transcription polymerase chain reaction usingRNA prepared from the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS overexpressingthe cMOAT-encoding transcript. In a cell-free system, both anti-cMOAT ribozymes cleaved their substrate in a highly efficient manner at a physiologic pH and temperature. The cleavage reaction was dependent on time and :substrate ratio for determining specific kinetic parameters. Cancer Therapy (2001) 8, 176±184

Key words: cMOAT; MRP2; ABCC2; ribozyme; drug resistance; cisplatin.

cquirement of resistance to antineoplastic agents, at of a broad range of endogenous and xenobiotic compounds Aconcentrations which were once effective for cancer across the apical canalicular membrane of the hepatocyte.4 chemotherapy, is a major obstacle in the clinical treatment Mutations in the cMOAT-encoding gene were shown to of human malignancies. The platinum-containing com- cause the Dubin-Johnson syndrome,5 a rare autosomal pound, cisplatin [cis-dichlorodiamin-platinum (II)], is recessive disorder characterized by chronic conjugated one of the most widely used anticancer agents active in the hyperbilirubinemia due to impaired hepatobiliary transport treatment of various cancers, including testicular, head and of nonbile salt organic anions. neck, esophageal, lung, ovarian, and bladder tumors. Methods for modulation of specific gene expression using Evidence was provided that cisplatin attacks guanine antisense oligonucleotides have been developed and have residues in DNA to form several types of adducts with been shown to be useful for suppressing activated DNA bases. The two major adducts are Pt-d(GpG)and Pt- including oncogenes6 and drug resistance±associated d(ApG)intrastrand cross-links, and the remaining include genes.7 Hammerhead ribozymes, catalytic RNA molecules Pt- (dG)monoadducts, Pt-d(GpNpG)intrastrand cross- exhibiting endoribonuclease activity directed against a 1 links, and Pt(dG) 2 interstrand cross-links. However, the specific mRNA target, have advantages over the use of rapid development of resistance to cisplatin represents an antisense oligonucleotides because of their side-specific important challenge to oncologists and laboratory investi- cleavage activities and catalytic potentials. They can cleave gators alike. any triplet of NUX (N=any ; X=A, C, or U)if The mechanisms conferring cisplatin resistance, among the flanking sequence is changed appropriately.8 It was others, include the enhanced expression of the ABC reported that ribozymes were effective against drug resis- ( binding cassette)transporter tance±mediating genes, including the P-glycoprotein± cMOAT (canalicular multispecific anion transporter), 2 also encoding mdr1 gene9,10 and the LRP (lung resistance designated MRP2 (multidrug resistance-associated protein protein)-encoding transcript. 11 However, a ribozyme 2),3 or with regard to the suggestions of the HUGO, Human directed against the cisplatin resistance±associated ABC Gene Nomenclature Committee as ABCC2. Physiologically, transporter cMOAT has not yet been applied. this ABC transporter mediates the ATP-dependent transport Accordingly, we designed two hammerhead ribozymes directed against the cMOAT-encoding transcript and characterized the kinetic parameters in a cell-free system. Address correspondence and reprint requests to Dr. Hermann Lage, The kinetic parameters revealed a high endoribonucleolytic InstituteofPathology,ChariteÂ,HumboldtUniversityBerlin,Schumannstr.20/ cleavage activity of both anti-cMOAT hammerhead ribo- 21, Berlin D-10117, Germany. E-mail address: [email protected] zymes.

176 Cancer Gene Therapy, Vol 8, No 3, 2001: pp 176±184 MATERNA, HOLM, DIETEL, ET AL: CMOAT RIBOZYME 177

MATERIALS AND METHODS specific RNA fragments (substrates-1 and -2)were obtained by performing an in vitro transcription as described Truncated target transcripts previously.12 In each case, the sequences and nt positions were The complete sequence of the cMOAT-encoding mRNAwas derived from human cMOATcDNA sequence (accession no. analyzed concerning putative secondary structures using the X96395). 13 Nucleotide position +1 is assigned to the A DNASIS 3.0 (Hitachi, Yokohama, Japan)and the mfold 3.0 residue of the ATG translation start codon. (Washington University, St. Louis, MO; E-mail address: http:/ /mfold2.wustl.edu/mfold/rna/form1.cgi)software. Construction and synthesis of hammerhead ribozymes Sequence regions were estimated to be particularly suitable According to the secondary structure analysis used for for ribozyme cleavage, if these regions contained potential cMOAT substrate selection, the potential hammerhead ribozyme cleavage sites in nonbase-paired structures, such ribozyme cleavage sites were determined at nt 99 and nt as hairpin loops. Two different truncated cMOAT-encoding 110 of both substrate. These target sequences are not blocked DNA molecules were generated by polymerase chain by any base pairing. Additionally, the different sizes of the reaction (PCR), which appeared to contain such suitable cleavage products of the truncated RNA transcripts should secondary structures and should allow an easy detection of allow an easy identification of each fragment. Hammerhead the cleavage products. Total cellular RNA, prepared from the ribozymes were named RzM1 and RzM2 and are shown in cMOAT-overexpressing, cisplatin-resistant human ovarian Figure 1. The cleavage reactions with RzM1 should yield a carcinoma cell line A2780RCIS (not yet published), acted 99-nt 50 -product and a 101-nt 30 -product using substrate-1, as a template for arbitrarily primed synthesis of cMOAT- and a 99-nt 50 -product and a 134-nt 30 -product using encoding cDNA used in PCR. The unique forward sense substrate-2. In contrast, the endoribonucleolytic digestion primer cMOAT/sub-fw (nucleotide [nt] +2014 to nt with RzM2 should yield a 110-nt 50 -product and a 90-nt 30 - +2036)was 5 0 -linked with a T7 RNA polymerase promoter product using substrate-1, and a 110-nt 50 -product and a sequence: 50 -TAATACGACTCACTATAGCCTGTCGGCTC- 123-nt 30 -product using substrate-2. For each hammerhead TGGGAAATCCTC-30 (T7 promoter±encoding sequence ribozyme, an encoding sense ssDNA 50 -linked with a T7 italicized). Oligonucleotide primers used for the am- RNA polymerase promoter sequence, as well as the plification of cMOAT substrate-1±encoding sequences homologous antisense ssDNA, was synthesized on an were cMOAT/sub-fw and cMOAT/sub1-rev (nt +2194 oligonucleotide synthesizer. Sense sequence for RzM1 is to nt +2213), 5 0 -GCACAGGCCTCCAGTACTTG-30, 50 -TAATACGACTCACTATAGTGCTGTGGCTGATGAG- yielding an expected product of 200 nt; for amplification TCCGTGAGGACGAAACATAGGC-30 and for RzM2 50 - of substrate-2±encoding sequences were cMOAT/sub-fw TAATACGACTCACTATAGGAATCCAGCTGATGAGTC- and cMOAT/sub2-rev (nt +2223 to nt +2246), 5 0 - CGTGAGGACGAAACTGCTGT-30, whereby T7 promo- CCTCCAGGCAGCATTTCCAAGTCT-30, yielding an ter±encoding sequences are underlined, respectively. Using expected product of 233 nt. To generate RNA-encoding these template molecules, the hammerhead ribozymes were DNA templates, the reaction mixture contained 100 ng of obtained by in vitro transcription as described previously.12 cDNA prepared from A2780RCIS cells, 0.2 M of each oligonucleotide primer, 0.2 mM of each dNTP, 50 mM KCl, Cleavage of substrate 10 mM Tris±HCl, pH 8.3, 1.5 mM MgCl2, 0.001% gelatin, and 2 units of AmpliTaq Gold (Perkin Elmer)in a final The ability of the both anti-cMOAT hammerhead ribozymes volume of 50 L. Cycling conditions were 12 minutes at to cleave their target RNA was assayed as described earlier 12 948C followed by 36 cycles consisting of 60 seconds at with some modifications. In brief, cleavage reactions were 948C, 90 seconds at 628C, and 90 seconds at 728C. The last carried out in a final volume of 10 L containing 200 nM cycle contained a 5-minute extension step at 728C. hammerhead ribozyme, 200 nM of the target molecule, 40 Subsequently, the PCR products were purified with mM Tris±HCl, pH 7.5, and 12 mM MgCl2 at 378C. Samples Sephadex G-50 columns. The two different cMOAT- were removed after various time intervals and quenched by

Figure 1. Sequences of the anti-cMOAT hammerhead ribozymes, RzM1 and RzM2, indicating the base-pairing interactions between the ribozymes and the cMOAT-encoding target RNA (accession no. X96395)13 shown in the prototypical hammerhead confirmation. Arrows indicate the predicted sites of cleavage.

Cancer Gene Therapy, Vol 8, No 3, 2001 178 MATERNA, HOLM, DIETEL, ET AL: CMOAT RIBOZYME adding an equal volume of 8 M urea, 8.7% glycerin, 0.02% the percentage of cleavage products at t=1 and t=0. This xylene cyanole, 0.02% bromephenole blue, 0.02% orange G. calculation is based on the supposition that the cleavage of Each sample was size-fractionated on an 8% polyacrylamide the first 30% of the substrate follows a linear curve as well, gel containing 7 M urea. Substrates and cleavage products but, in contrast to the upper calculation, regards only that were identified by autoradiography, scanned using the amount of substrate cleavable, which is actually cleaved after BioRad molecular analyst software package (BioRad, a considerable amount of time (here 5 hours; described as Hercules, CA), and counted to determine the extent of t=1). For these k cat /k m values corresponding to the ascent cleavage. This way, both anti-cMOAT hammerhead ribo- of the linear beginning of the graphs, it is obvious that the zymes, RzM1 and RzM2, were estimated to have a high results gained by these two procedures must differ. The linear catalytic cleavage activity and were characterized in detail. range of the curve was determined as described above.

Turnover kinetics

Determinations of the values for the initial velocities (v ini ) and the reaction parameters k cat /k m of the hammerhead ribozymes were performed as described earlier 12 with some modifications. Additionally, the values for the observed cleavage constants k obs were determined. The value of the initial velocity v ini of each cleavage reaction was estimated by measuring the amount of 32P-labeled substrate and formed cleavage products appearing as time-dependent using 40 nM substrate and 200 nM hammerhead ribozyme. The reaction was started by adding substrate RNA to the reaction mix containing the ribozyme RNA, 40 mM Tris± HCl, pH 7.5, and 12 mM MgCl2 at 378C. All experiments were performed at least in triplicate. Substrate:product ratios were estimated following polyacrylamide gel electro- phoresis of the reaction mixtures and autoradiography by densitometric measurement using the BioRad molecular analyst software. The values for the initial velocities were calculated from the ascent of the graph in the product±time diagrams. The linear range of the graphs was assessed by selection of the highest correlation coefficients obtained by the calculation of different linear regression curves using various breakpoints. For better comparability with kinetic values from the literature determined by different methods, the reaction parameter k cat /k m was calculated using two different commonly used mathematical approaches. (i)The value of k cat /k m was assessed following the equation given by Heidenreich and Eckstein:14

Àkcatt‰ribozymeŠ Su ˆ 1 À Pt ˆ e km where the percentage of uncleaved substrate (S u)per reaction time (t)was plotted against the ribozyme concentration ( [ribozyme] ). P t is the amount of cleavage products at time (t). This calculation is based on the accepted supposition that the cleavage of the first 30% of the substrate follows a linear curve and, upon that, the whole amount of given substrate is cleavable. The linear range of Figure 2. In vitro cleavage of substrate-1 (200 nt) and substrate-2 (233 nt) by the anti-cMOAT hammerhead ribozymes, RzM1 and the graphs was assessed as described above. Under these RzM2. In each case, hammerhead ribozyme RNA (200 nM) was circumstances, the k cat /k m value corresponds to the ascent of incubated with substrate RNA (200 nM) at 378C for 2 hours. 0 0 the linear beginning of the graph. (ii)The value for k cat /k m Substrate RNA, 5 - and 3 -cleavage products, and ribozymes were was determined by the equation of Hendry et al:15 separated on an 8% polyacrylamide gel containing 7 M urea. Lane 1, RzM1 plus substrate-1, cleavage products are 99 nt (50 -product) Àkcatt‰ribozymeŠ Pt ˆ P1 À PÁe km and 101 nt (30 -product); lane 2, RzM1 plus substrate-2, cleavage products are 99 (50 -product) and 134 nt (30 -product); lane 3, RzM2 P t and t are the same as above, P1 is the amount of plus substrate-1, cleavage products are 110 nt (50 -product) and 90 cleavage products at that point of time t=1 when the nt (30 -product); lane 4, RzM2 plus substrate-2, cleavage products 0 0 reaction rate was constant, and P Á is the difference between are 110 (5 -product) and 123 nt (3 -product).

Cancer Gene Therapy, Vol 8, No 3, 2001 MATERNA, HOLM, DIETEL, ET AL: CMOAT RIBOZYME 179

Experiments were performed for 15 minutes using 40 nM the percentage of product at t=1 and t=0. This approach substrate and increasing concentration of hammerhead regards that the substrate is not cleaved completely in the ribozymes (0±400 nM). The reaction was started by adding ribozyme-catalyzed reaction. the substrate RNA to the reaction mix containing a ribozyme, 40 mM Tris±HCl, pH 7.5, and 12 mM MgCl2 at 378C. All experiments were performed at least in triplicate and RESULTS analyzed as described above. The 50 -linked T7 promoter carrying oligonucleotide primers According to the comparability with alternative published used for generation of truncated cMOAT-specific RNA kinetic values, the observed cleavage rates k were obs substrates was successfully applied for PCR-based DNA determined by two methods. (i)The k value was obs template synthesis. These truncated cMOAT-encoding DNA calculated according to Heidenreich et al:16 templates were suitable for direct in vitro transcription ln S 0 k ˆÀ u without any cloning procedure. The same was true for 5 - obs t linked T7 promoter sequence containing synthetic DNA molecules, which encoded the two anti-cMOAT hammer- whereby the reaction constant k obs equals the negative head ribozymes. natural logarithm of the uncleaved substrate (Su )divided by The two anti-cMOAT hammerhead ribozymes, RzM1 and the reaction time t. This equation does not take in RzM2, with symmetric 8-nt 50 -antisense and 8-nt 30 - consideration how much substrate is effectively cleaved. antisense flanks were designed according to the method of (ii)The value of k obs was assessed by the improved method 17 15 Haseloff and Gerlach. Both catalytic RNA molecules were of Hendry et al: tested in a cell-free assay for their ability to cleave in vitro Àkobst transcribed truncated fragments of the cMOAT-encoding Pt ˆ P1 À e PÁ mRNA. The substrate fragment size ranged from 200 to 233 where P t is the amount of formed product at time t, P 1 is the nt. Anti-cMOAT hammerhead ribozymes, RzM1 and RzM2, amount of product at t=1, and P is the difference between targeting the GUC motifs 704 and 708, respectively, of the

Figure 3. In vitro cleavage of truncated cMOAT-encoding RNA substrates by anti-cMOAT hammerhead ribozymes. For cleavage, 20 nM substrate RNA was incubated with 100 nM ribozyme at 378C. Substrate molecules, 50 -products, 30 -products, and ribozymes were size- fractionated on an 8% polyacrylamide gel containing 7 M urea. Lane 1, ribozyme; lane 2, substrate; lane 3, substrate plus ribozyme (t=0 minute); lane 4, substrate plus ribozyme (t=0.5 minute); lane 5, substrate plus ribozyme (t=1 minute); lane 6, substrate plus ribozyme (t=2 minutes); lane 7, substrate plus ribozyme (t=3 minutes); lane 8, substrate plus ribozyme (t=5 minutes); lane 9, substrate plus ribozyme (t=10 minutes); lane 10, substrate plus ribozyme (t=20 minutes); lane 11, substrate plus ribozyme (t=30 minutes); lane 12, substrate plus ribozyme (t=60 minutes); lane 13, substrate plus ribozyme (t=120 minutes); lane 14, substrate plus ribozyme (t=180 minutes); lane 15, substrate plus ribozyme (t=300 minutes). A: Digestion of substrate-2 (233 nt) by RzM1 (38 nt) and visualization of the 99 nt 50 -product and the 134 nt 30 - product. B: Cleavage of substrate-1 (200 nt) by RzM2 (38 nt) and demonstration of the 110 nt 50 -product and the 90 nt 30 -product.

Cancer Gene Therapy, Vol 8, No 3, 2001 180 MATERNA, HOLM, DIETEL, ET AL: CMOAT RIBOZYME cMOAT-encoding open reading frame were very efficiently cleavage products were assessed when the turnover rate in cleaving their RNA substrates at physiologic pH and became constant. For both hammerhead ribozymes, these temperature at equimolar concentrations of hammerhead values (P 1 )were typically in the region of 0.8, i.e., 80% ribozyme and substrate RNA in the cleavage reaction (Fig of the substrate molecules was cleaved at the end of the 2). For better separation and visualization of the endor- reaction. The initial velocities (v ini )of the ribozyme- ibonucleolytic cleavage products after electrophoresis, the catalyzed reactions were calculated as follows: (i)for À 1 kinetic characterization of RzM1 was performed using ribozyme RzM1, v ini =0.127‹0.006 fmol s (Fig 5A)and À 1 substrate-2 (Fig 2 demonstrates the excellent possibility to (ii)for ribozyme RzM2, v ini =0.086‹0.005 fmol s (Fig distinguish both products). In contrast, for the characteriza- 5B). The values of the reaction parameter k cat /k m were À 1 tion of hammerhead ribozyme RzM2, substrate-1 was calculated for ribozyme RzM1 as k cat /k m =9307‹312 M applied in the experimental procedures. s À 1 (Fig 6A)according to the equation of Hendry et al 15 À 1 À 1 The capacity of both anti-cMOAT hammerhead ribo- and as k cat /k m =7719‹284 M s (Fig 7A)using the zymes, RzM1 and RzM2, to cleave the target molecules was equation given by Heidenreich and Eckstein.14 The ham- determined on time (Fig 3A and B)and substrate:ribozyme merhead ribozyme±specific values of the reaction rate k obs À 4 ratio (Fig 4A and B). The amounts of endoribonucleolytic were determined for ribozyme RzM1 as k obs =8.73Â10

Figure 4. Endoribonucleolytic cleavage of truncated cMOAT encoding RNA substrates with increasing concentrations of anti-cMOAT hammerhead ribozymes. A total of 40 nM substrate RNA was cleaved at 378C for 15 minutes with increasing amounts of ribozymes. Substrates, cleavage products, and ribozymes were separated on an 8% polyacrylamide gel containing 7% urea. Lane 1, ribozyme; lane 2, substrate; lane 3, substrate plus 80 nM ribozyme (t=0 minute); lane 4, substrate plus 20 nM ribozyme (t=15 minutes); lane 5, substrate plus 40 nM ribozyme (t=15 minutes); lane 6, substrate plus 80 nM ribozyme ( t=15 minutes); lane 7, substrate plus 120 nM ribozyme (t=15 minutes); lane 8, substrate plus 200 nM ribozyme (t=15 minutes); lane 9, substrate plus 300 nM ribozyme (t=15 minutes); lane 10, substrate plus 400 nM ribozyme (t=15 minutes). A: Cleavage of substrate-2 (233 nt) by RzM1 (38 nt) and visualization of the 99 nt 50 -product and the 134 nt 30 - product. B: Digestion of substrate-1 (200 nt) by RzM2 (38 nt) and demonstration of the 110 nt 50 -product and the 90 nt 30 -product.

Cancer Gene Therapy, Vol 8, No 3, 2001 MATERNA, HOLM, DIETEL, ET AL: CMOAT RIBOZYME 181

DISCUSSION

Two hammerhead ribozymes, RzM1 and RzM2, were constructed to cleave the cisplatin resistance±associated cMOAT-encoding mRNA transcripts, and the efficiency of cleavage by these ribozymes was examined in a cell-free system. Although hammerhead ribozymes have been applied successfully to cleave the mRNA transcripts of drug resistance±mediating factors, such as the ABC transporter P-glycoprotein,9,10 dihydrofolate reductase,18 or the vault component LRP,11 no hammerhead ribozymes have been designed to inactivate RNA molecules encoding the cisplatin resistance±mediating ABC transporter, cMOAT. Therefore, the two anti-cMOAT ribozymes, RzM1 and RzM2, appear as excellent new candidate molecules for modulating cytostatic drug resistance, both as laboratory tools for the elucidation of the relationship between mRNA expression

Figure 5. Product±time diagrams for experimental determination of the values of v ini between the anti-cMOAT ribozymes, RzM1 and RzM2, and their substrates. To estimate v ini, the concentrations of the formed cleavage products (P t ) after various reaction times were plotted against the time. The values of v ini correspond to the apparent linear first part of the curve (small parts of the figures). All experiments were performed at least in triplicate. A: Product±time diagram using RzM1 and substrate -2. The value of v ini was À 1 calculated to be v ini =0.127‹0.006 fmol s . B: Product± time diagram using RzM2 and substrate -1. The value of v ini was À 1 determined to be v ini =0.086‹0.005 fmol s .

‹0.33Â10 À 4 s À 1 (Fig 8A)using the method of Hendry et 15 À 4 À 4 À 1 al and as k obs =7.66Â10 ‹0.29Â10 s (Fig 9A) 16 according to Heidenreich et al. Assessment of the k cat /k m values for anti-cMOAT ribozyme RzM2 revealed a k cat / À 1 À 1 k m =4478‹105 M s (Fig 6B)using the method of 15 À 1 À 1 Hendry et al, and a k cat /k m =3671‹78 M s (Fig 7B) applying the calculation proposed by Heidenreich and Eckstein.14 The calculation of the values of the cleavage Figure 6. Experimental determination of the reaction parameters À 4 constant k for RzM2 resulted in k =5.79Â10 k cat /k m of the anti-cMOAT ribozymes, RzM1 and RzM2, according obs obs 15 ‹0.27Â10À 4 s À 1 (Fig 8B)according to the method of to Hendry et al. The values of k cat /k m correspond to the ascent of Hendry et al 15 and in k =4.81Â10 À 4‹0.23Â10 À 4 s À 1 the apparent linear beginning of the curves (small inserted figures). obs All experiments were performed at least in triplicate. A: Curve (Fig 9B)using the equation given by Heidenreich et al. 16 obtained by using RzM1 and substrate-2. The value of k cat /k m was The values of the kinetic parameters of both anti-cMOAT calculated to be k /k =9307‹312 M À 1 s À 1. B: Reaction curve hammerhead ribozymes are presented in comparison with cat m obtained by using RzM2 and substrate-1. The value of k cat /k m was À 1 À 1 that of other published ribozymes in Table 1. determined to be k cat /k m =4478‹105 M s .

Cancer Gene Therapy, Vol 8, No 3, 2001 182 MATERNA, HOLM, DIETEL, ET AL: CMOAT RIBOZYME

A further possible problem in ribozyme design is the phenomenon that ribozymes which are found to be catalytically active to cleave short RNA substrates often show poor potency when applied to long RNA sub- strates.14,19 To circumvent this difficulties, the ribozymes were characterized using long truncated RNA substrate molecules (200±233 nt). Large RNA molecules can form complex secondary and tertiary structures that can interfere with ribozyme recognition and cleavage sites. Very likely, the rate-limiting step is the bimolecular association of the hammerhead ribozyme with the target sequence. Addition- ally, tertiary interactions can slow down product release and interfere with turnover. For these reasons, only a few hammerhead ribozymes, which show a high catalytic cleavage activity with long RNA substrate molecules, were designed.12,20,23,24 The catalytic parameters of both anti- cMOAT hammerhead ribozymes, RzM1 and RzM2, show

Figure 7. Determination of the reaction parameters k cat /k m of the anti-cMOAT ribozymes, RzM1 and RzM2, according to Heidenreich 14 and Eckstein. The values of k cat /k m correspond to the ascent of the apparent linear beginning of the curves (small inserted figures). All experiments were performed at least in triplicate. A: Reaction curve obtained by using RzM1 and substrate-2. The value of k cat /k m was À 1 À 1 calculated to be k cat /k m =7719‹284 M s . B: Curve obtained by using RzM2 and substrate-1. The value of k cat /k m was determined to À 1 À 1 be k cat /k m =3671‹78 M s . and a drug-resistant phenotype as well as potential molecules with therapeutic intent. Difficulties in the successful design and application of hammerhead ribozymes can arise because various potential ribozyme cleavage sites in a particular mRNA molecule may not be accessible for base pairing and because cleavable sites often are not foreseeable.19 Thus, practic- able target sites of hammerhead ribozymes often are determined by trial and error. Basing on own experi- ence,10,12,20 ±23 the anti-cMOAT ribozymes, RzM1 and Figure 8. Experimental determination of the observed reaction RzM2, were designed after computer-based secondary constants k obs of the hammerhead ribozymes, RzM1 and RzM2, 15 structure analyses of the target mRNA molecules. The according to Hendry et al. The values of k obs correspond to the decisive criterion for ribozyme design was the fact that ascent of the apparent linear beginning of the curves (small inserted they interact with the predicted cleavage sites in putative figures). All experiments were performed at least in triplicate. A: Reaction curve obtained by using RzM1 and substrate-2. The value nonbase-pairing containing RNA structures. Here it could of k was calculated to be k =8.73Â10 À 4 ‹0.33Â10 À 4 s À 1. B: be demonstrated that this strategy rapidly reveals catalytic obs obs Curve obtained by using RzM2 and substrate-1. The value of k obs À 4 À 4 À 1 active molecules. was determined to be k obs =5.79Â10 ‹0.27Â10 s .

Cancer Gene Therapy, Vol 8, No 3, 2001 MATERNA, HOLM, DIETEL, ET AL: CMOAT RIBOZYME 183

exhibit a high catalytic cleavage activity. However, on the basis of the more advantageous kinetic values of v ini and k cat /k m calculated for the anti-cMOAT hammerhead ribozyme RzM1, this catalytic molecule appears to be the better suitable candidate for a potential application. The application of hammerhead ribozymes has been proposed to be a tool in analyzing gene expressions, to modulate the malignancy of a tumor, or to reverse the drug- resistant phenotype of cancer cells. Our experiments demonstrate that both anti-cMOAT hammerhead ribozymes may provide potential help in cancer research and may serve as potential modulators of cisplatin resistance applying gene therapeutic strategies. However, the reported data were obtained in a cell-free system, and the relevance in tumor cisplatin-resistant tumor cells remains to be demonstrated. We are currently making efforts to demonstrate these effects in a cellular model.

ACKNOWLEDGMENTS

This work was supported by a grant of ``Berliner Krebsgesellschaft'' to H. L. We thank Anke Wichert, Petra Kowalski, and Klaus Mantwill for numerous fruitful discussions and technical help.

REFERENCES

1. Fichtinger-Schepman AM, van der Veer JL, den Hartog JH, Lohman PH, Reedijk J. Adducts of the antitumor drug cis- diaminedichloroplatinum(II)with DNA: formation, identifica- tion, and quantitation. Biochemistry. 1985;24:707±713. Figure 9. Determination of the cleavage constant k obs of the anti- 2. Taniguchi K, Wada M, Kohno K, et al. A human canalicular cMOAT ribozymes, RzM1 and RzM2, according to Heidenreich et multispecific organic anion transporter (cMOAT)gene is al.16 The values of k correspond to the ascent of the apparent obs overexpressed in cisplatin-resistant human cancer cell lines linear beginning of the curves (small inserted figures). All experi- with decreased drug accumulation. Cancer Res. 1996;56: ments were performed at least in triplicate. A: Reaction curve 4124±4129. obtained by using RzM1 and substrate-2. The value of k was obs 3. Keppler D, KoÈnig J. Hepatic canalicular membrane 5: calculated to be k =7.66Â10 À 4 ‹0.29Â10 À 4 s À 1. B: Curve obs expression and localization of the conjugate export pump obtained by using RzM2 and substrate-1. The value of k obs was À 4 À 4 À 1 encoded by the MRP2 (cMRP/cMOAT)gene in liver. FASEB determined to be k obs =4.81Â10 ‹0.23Â10 s . J. 1997;11:509±516. 4. KoÈnig J, Nies AT, Cui Y, Leier I, Keppler D. Conjugate export values that are comparable with some of these ribozymes pumps of the multidrug resistance protein (MRP)family: (Table 1)indicating that the target sites are very accessible localization, substrate specificity, and MRP2-mediated drug for both anti-cMOAT ribozymes and that both ribozymes resistance. Biochim Biophys Acta. 1999;1461:377±394.

Table 1. Catalytic Efficiencies of Different Hammerhead Ribozymes Targeted on Synthetic Substrates

À 1 À 1 À 1 À 1 Target Length of substrate (nt) v ini (fmol s ) k cat /k m (M s ) k obs (s ) Reference cMOAT/RzM1 233 0.127 9307*, 7719y 8.73Â10 À 4*, 7.66Â10 À 4z this study cMOAT/RzM2 200 0.086 4478*, 3671y 5.79Â10 À 4*, 4.81Â10 À 4z this study GPC3 234 0.155 10,600*, 4700y ND 12 BCRP 343 2.21 55,000*, 24,180y 2.68Â10 À 2*, 1.17Â10 À 2z 23 HIV-1 LTR 985 ND 22±500y ND 14 Pit-950 950 ND 500y ND 19 S17 60 ND 4166y ND 19 KruÈppel 13 ND 166,667* 1.48Â10 À 1* 15 TAT 13 ND 5333* 4.0Â10 À 3* 15 *Calculated according to Hendry et al.15yCalculated according to Heidenreich and Eckstein.14zCalculated according to Heidenreich et al.16

Cancer Gene Therapy, Vol 8, No 3, 2001 184 MATERNA, HOLM, DIETEL, ET AL: CMOAT RIBOZYME

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Cancer Gene Therapy, Vol 8, No 3, 2001