OLIGONUCLEOTIDES 14:1–9 (2004) © Mary Ann Liebert, Inc.
Rapid Screening Method for Antisense Oligonucleotides Against Human Growth Factor Receptor p185erbB-2 ANKE ROHMANN,1,2 DIRK LOCHMANN,2 JÖRG WEYERMANN,2 WOLF M. BERTLING,3 THEODOR DINGERMANN,1 and ANDREAS ZIMMER4
ABSTRACT The aim of this study was the development of an indirect cell proliferation assay as screening tool for antisense oligonucleotides. Unmodified and phosphorothioate-modified oligonucleotides with different amounts of sulfur in the DNA backbone were examined for biologic activity. The human growth factor receptor p185erbB-2 was chosen as cellular target. High-level expression of this protein can be related to an early event in tumor development and cell proliferation. We correlated the expression of p185erbB-2 with the cell proliferation of BT-474. Additionally a control cell line (MCF-7) with very low p185erbB-2 expression was cultivated. Antisense oligonucleotides were transfected as a liposome formulation (Lipofectin®, GIBCO-BRL, Eggenstein, Germany). Cell count was correlated with a total protein quantification assay (BCA method). Stability against nuclease digestion was determined with a DNase I assay. Sequence-specific antisense effects on the p185erbB-2 protein level were determined by Western blot. An antisense phosphorothioate oligonucleotide was identified to inhibit the cell proliferation in comparison to a random control and a negative control oligonucleotide sequence. The comparison of fully thioated, partly thioated, and unmodified oligonucleotides verified the correlation between the enzymatic stability and the biologic activity of the different modifications. Using the unstable oligonucleotides, more treatments were necessary to achieve an antiproliferative effect. In our study, the indirect proliferation assay was found to be a reliable and potent tool for an antisense oligonucleotide screening by targeting the p185erbB-2 protein.
INTRODUCTION
H
1993). Thereby, AS-ODN can inhibit specifically the translation of a single target gene and provide a major tool in anticancer therapy. p185erbB-2, one of four growth factor receptors, is overexpressed in 25%–30% of breast cancer. This overexpression is correlated with a poor prognosis in cancer remission and overall survival (Pegram et al., 1998; Slamon et al., 1987). The reduction of p185erbB-2 has an antiproliferative effect in overexpressing cell lines (Roh et al., 1998). In this study, p185erbB-2 was used as target to investigate sequence-specific antisense effects. In the 1980s, antisense therapy was adopted as a magic
IGH-LEVEL PROTEIN EXPRESSION of
different proteins, such as growth factor receptors, appears to be an early event in tumor development (deFazio et al., 2000; Roh et al., 1998; Slamon et al., 1987; Tzahar and Yarden, 1998). This overexpression can be due to gene amplification or increased transcription rates or both. Antisense oligonucleotides (AS-ODN) bind to the RNA transcript (mRNA) in a sequence-specific manner and partly induce the degradation of the mRNA of the mRNA/DNA duplex by cellular RNase H (Nellen and Lichtenstein,
1 Institute for Pharmaceutical Biology and 2 Institute for Pharmaceutical Technology, Johann Wolfgang Goethe-University, 60439 Frankfurt am Main, Germany. 3 November AG, 91056 Erlangen, Germany. 4 Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, Karl-Franzens-University, 8010 Graz, Austria.
1
2
ROHMANN ET AL.
bullet to heal every disease (Stein and Cheng, 1993). Many years of studies in both in vitro and in vivo models have brought different results. It has become clear that effects of AS-ODNs are not as simple as they were thought to be. Each drug has different effects. Some are expected and others are not. Although antisense effects result from specific binding via hydrogen bonding, obviously nonantisense effects may exist. These nonantisense effects result, for example, from sequence-independent direct binding to proteins (Guvakova et al., 1995), G-quartet formation (Agrawal et al., 1997; Xu et al., 2001), or sequence-related binding (Boggs et al., 1997; Krieg et al., 1996). Sequencerelated binding indicates that the sequence is partly responsible for the binding but that the binding is not due to hydrogen bonding, as with antisense binding. The therapeutic use of AS-ODNs requires an effective cell transfer and a high enzymatic stability. ODNs, as all nucleic acids, are negatively charged, and, therefore, their cell membrane permeability is very low. In addition, ODNs are very unstable against enzymatic digestion (Uhlmann et al., 1997). In our study, a lipid-based transfection reagent (Lipofectin,® GIBCO-BRL, Eggenstein, Germany) was used as a commercially available standard preparation to transfer ODNs into cells. The improvement in chemical stability could be reached with different DNA modifications and mixed modification ODN design, which is now available by an automated ODN synthesis (Stec et al., 1984). Although the enzymatic stability could be tremendously increased by phosphorothioated modifications, for example, the resulting biologic effects were discouraging. The increasing stability, often goes along with an increasing unspecific effect (Milligan et al., 1993; Levin, 1999; Stein, 1996). Therefore, it became clear that optimization of the required biophysical data alone cannot predict the biologic activity. To improve the target specificity and effectiveness of AS-ODN, in most cases, a set of multiple, different sequences and modifications have to be screened for biologic activity. Determination of such specific antisense effects requires target protein quantification. Direct, specific protein quantification methods, such as Western blot, are complicated, expensive, and time consuming and are less useful for fast high throughput screening. The aim of this study was to investigate the ef-
fect of cell proliferation as a reliable and fast tool to screen the biologic activity of AS-ODN against p185erbB-2 expression. Comparison with Western blot analysis demonstrated how an indirect screening assay can be combined to optimize the test design.
MATERIALS AND METHODS Chemicals and oligonucleotides The unmodified ODNs, partly thioated ODNs (MixODN; three modifications on the 39- side and 59 side), and fully thioated ODNs (PTO) were purchased from MWG Biotech (Ebersberg, Germany) and were checked by mass spectrometry (MALDI-TOF MS documentation). The ODN sequences are shown in Table 1. The antisense sequence is complementary to the translation initiation side of c-erbB-2 (Vaughn et al., 1995, 1996), whereas all control sequences do not have any known target in the human genome, verified by a BLAST search (Altschul et al., 1997). All other chemicals were obtained from Sigma (Taufkirchen, Germany) in the highest available quality, except where indicated.
Cell culture Cultivation of two different human adenocarcinoma cell lines was done in RPMI 1640 medium with glutamine (GIBCO-BRL) supplemented with 10% fetal bovine serum (FBS) (GIBCO-BRL) and 1% penicillin/ streptomycin (PAA Laboratories, Cölbe, Germany) at 37°C with 5% CO2. BT-474 cells overexpress p185erbB-2 (Lasfargues et al., 1978), whereas the human adenocarcinoma cell line MCF-7 shows a very low p185erbB-2 expression level (Trempe, 1976).
Liposome preparations Lipofectin liposomes were prepared according to the manufacturer’s protocol. The ODNs were complexed with Lipofectin in serum-free RPMI 1640 medium. The applied mass ratio ODN/Lipofectin was found to be optimized at 1:2.2 (mass ratio). At this ratio, total complexing between ODN and cationic lipids was achieved.
TABLE 1. OLIGONUCLEOTIDE SEQUENCES USED Oligonucleotide Antisense Scrambled control Random conrol Negative control a
Abbreviation (AS) (SC) (RC) (NC)
Sequence 59-CTC CAT 59-CGC CTT 59-ATC TAC 59-ACG TTC
CpG motifs of the scrambled sequence are underlined.
GGT GCT CAC-39 ATC CGT AGC-39a TGG CTC CAT-39 CTC CTG CGG GAA-39
3
RAPID ANTISENSE SCREENING
DNase stability assays For assay, 100 ml of 5 mM ODN, Mix-ODN, and PTO were incubated with 30 IU DNase I (Roche Diagnostics, Mannheim, Germany) in 400 ml buffer (21 mM Tris, 5 mM MgCl2, pH 7.4) for 45 minutes at 600 rpm, 37°C on an Eppendorf thermomixer (Zobel et al., 1997). The reaction was stopped by adding 100 ml of 1 M EGTA. To verify the nuclease activity, 10 mg of each ODN batch was treated under the same conditions. Each sample (50 ml) was injected into a strong anion exchange high-performance liquid chromatography system (HPLC), using a DNApac PA-100 (4 3 250 mm) column (Dionex, Idstein, Germany) and a Merck-Hitachi LaChrom HPLC system (Darmstadt, Germany) equipped with a diode array detector (DAD). Column temperature was set to 60°C. The gradient mobile phase was based on (A) 25 mM sodium hydroxide and (B) 25 mM sodium hydroxide and 800 mM sodium perchlorate in doubledistilled water. A linear gradient with a 1 ml/min flow rate followed 0% (B) for 1.5 minute, 0–30% (B) for 15 minutes, 30%–100% (B) for 5 minutes, and 100% (B) for 5 minutes. The initial setup was reached after 100–0% (B) in 2 minutes. The DAD signal between 250 and 270 nm was recorded.
Cell proliferation assay bicinchoninic acid [BCA] assay The cell lines were cultured for 24 hours (0.2 3 105 cells/ml) in 24-well plates (Nunc, Roskilde, Denmark). After medium replacement, the cells were incubated with a Lipofectin preparation containing 1 mM PTO in serum-
free medium for 4 hours. A second treatment was necessary for Mix-ODN after 48 hours. For ODN, a third cycle after 72 hours was required. After the incubation period, the ODN incubation mixture was replaced by standard medium, and the cells were cultured for 120 hours altogether. Finally, all cells were lysed with a 2% sodium dodecylsulfate aqueous solution (w/v) (SDS), and the total protein content was measured with a commercially available BCA assay (Uptima, Montluçon, France), which was correlated to the number of cells per well. Cells were stained with trypan blue and counted using a Neubauer cell counting chamber.
Western blot BT-474 cells were cultured for 24 hours (5 3 105 cells/ml) in 6-cm dishes (Nunc). PTO, ODN, and MixODN incubation was carried out along with the proliferation assay. The cells were incubated with a Lipofectin preparation containing 1 mM ODN in serum-free medium for 4 hours in a total volume of 2.5 ml. The incubation mixture was replaced by standard medium, and the cells were cultured for a further 48 hours. Floated and detached cells were combined. Pooled cells were washed twice with phosphate buffer containing 136.9 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO 4, 1.47 mM KH 2PO4, pH 7.4 (PBS), lysed with 2% SDS solution, separated on SDS-PAGE, blotted onto a nitrocellulose membrane, and visualized using alkaline phosphataselabeled antibody (Calbiochem, Bad Soden, Germany). The spots were stained with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Ap-
FIG. 1. Correlation between cell number and BCA assay. The cell number of BT-474 cells correlated with the BCA assay. The increase in adsorption (lmax 540 nm) was linearly related to the cell number (n 5 3).
4
ROHMANN ET AL.
plichem, Darmstadt, Germany) substrates. The membrane was scanned and quantified with Gel Scan V4.0 software. For control reasons, the amount of Lamin B in the samples was quantified and correlated with the target protein amount.
RESULTS Characterization of proliferation assay In all experiments, cell proliferation is expressed as percent of total protein content determined by BCA assay readout, which was found to be proportional to the number of cells (Fig. 1).
ODN DNase stability DNase I treatment of ODN caused a complete fragmentation. Mix-ODN showed a number of different discrete peaks including the full-length ODN, representing incomplete digestion. As expected, the PTO-digested sample showed no fragmentation in the HPLC chromatogram, demonstrating high enzymatic stability (Fig. 2).
Antisense effects in proliferation Treatment with 1 mM liposomally delivered phosphorothioate AS-ODNs (AS-PTO) against the p185erbB-2
target protein showed a strong antiproliferative effect of about 44% in the p185erbB-2-overexpressing cell line BT-474. A random control sequence (RC-PTO) showed only a modest reduction of about 18%. Additionally, to prove the reliability of the BT-474 proliferation system, another independent PTO control, the negative control sequence (NC-PTO), was introduced to verify sequence-specific inhibition of the AS sequence. The cellular target specifity was investigated with the low p185erbB-2-expressing cell line MCF-7. As expected, the total protein reduction resulting from AS and RC sequences was found to be negligible in this control (Fig. 3A).
Antisense effects in Western blot Western blot analysis was employed in our study to determine the specific p185erbB-2 protein content in addition to the total protein readout obtained by the BCA method. To compensate for unspecific blotting effects, the p185erbB-2 level was given as a p185erbB-2/Lamin B (internal standard) ratio. A correlation was found between the antiproliferative effect and the p185erbB-2 level in the Western blot. The p185erbB-2 content was reduced by about 53% after treatment with the AS-PTO sequence, whereas the RC-PTO sequence did not alter p185erbB-2 protein expression (Fig. 3B).
FIG. 2. Digestion of ODNs with DNase I. Unmodified (ODN), partly thioated (Mix-ODN), and fully thioated (PTO) ODNs (30 mg of each) were incubated with 30 IU of DNase I under serum-free conditions for 45 minutes at 37°C. The same amounts of the ODNs and digested samples were analyzed by HPLC.
5
RAPID ANTISENSE SCREENING
A
B
FIG. 3. (A) Cell line-dependent ODN effects on cell proliferation. The p185erbB-2-overexpressing cell line BT-474 and the control cell line MCF-7 were treated with antisense (AS-PTO), negative control (NC-PTO), and random control (RC-PTO) phosphorothioates under serum-free conditions for 4 hours. After medium replacement, cells were cultured for 120 hours. The BCA total protein content is given as a value for the cell count. Cell control was set to 100%. (B) PTO effects on p185erbB-2 expression determined by Western blot. BT-474 cells were treated with AS-PTO, scrambled (SC-PTO), and RC-PTO, and Western blot was performed for p185erbB-2 and Lamin B.
Influence of phosphorothioate ODN modification Treatment with 1 mM liposomally delivered unmodified oligonucleotides (AS-ODN) did not change the proliferation of BT-474 cells. The AS-Mix-ODN reduced the total protein amount to about 15%, and AS-PTO sequences achieved a 44% reduction (Fig. 4). More detailed studies on the AS-ODN effect on BT474 cells confirmed that this effect is concentration dependent. BT-474 cells were treated with phosphorothioate ODNs as liposomes in a concentration range of 100–1000 nM (Fig. 5). The protein content was reduced to about 50% with AS-PTO concentrations .500 nM, whereas RC-PTO had almost no effect. Astonishingly, a PTO scrambled control (SC-PTO) sequence also achieved a strong concentration-dependent protein reduction, comparable to the antisense se-
quence. In contrast to these results, the SC-PTO did not change the p185erbB-2 protein level in the Western blot (Fig. 3B).
Influence of application regimen In this report, ODN were less stable against nuclease degradation compared with Mix-ODN and PTO. Thus, different application regimens were investigated for intracellular stability of the ODNs. The unmodified ODNs have to be applied three times at a 1-mM dosage to cause approximately 32% inhibition in cell proliferation. Mix-ODN reduced the protein content to 48% by adding 1 mM Mix-ODN twice. In comparison to SCODN and RC-ODN sequences, the AS-ODN exclusively achieved an antiproliferative effect after three treatments. After a single treatment, the AS-PTO
6
ROHMANN ET AL.
FIG. 4. Phosphorothioate modification-dependent effects on cell proliferation. BT-474 cells were treated with ODN, Mix-ODN, and PTO antisense (gray columns) and random sequences (dark gray) under serum-free conditions for 4 hours. After medium replacement, cells were cultured for 120 hours. The BCA total protein content is given as a value for the cell count.
achieved a strong protein reduction compared with the RC-ODN, which had almost no effect on cell proliferation (Fig. 6A). In addition to the fast proliferation screening assay by BCA, Western blot analysis verified a correlation between ODN modification vs. nuclease stability and treatment cycles vs. ODN concentration. According to the
proliferation assay, the Mix-ODNs have to be given twice in order to reach a significant decrease in the p185erbB-2 protein level, as visualized by Western blot analysis. A single treatment with the 1 mM liposomal Mix-ODN had no effect on the p185erbB-2 content, and, again, a sequence-specific p185erbB-2 reduction was determined only with the AS-PTO (Fig. 6B).
FIG. 5. Dose-response correlation of ODNs on cell proliferation. BT-474 cells were treated with different concentrations (100–1000 nM) of RC-PTO (dark gray), AS sequence (gray columns), and SC-PTO (diagonally striped columns) under serum-free conditions for 4 hours. After medium replacement, cells were cultured for 120 hours. The BCA total protein content is given as a value for the cell count.
7
RAPID ANTISENSE SCREENING
A
B
FIG. 6. (A) Influence of different phosphorothioate ODN modifications on cell proliferation after different treatment regimens. BT-474 cells were treated with an unmodified ODN three times, Mix-ODN two times, and PTO once under serum-free conditions for 4 hours. Treatment of the Mix-ODN and the unmodified ODN was repeated after 48 hours, and treatment for the unmodified ODN was repeated again after 72 hours. The BCA total protein content is given as a value for the cell count after 120 hours. (B) ODN effects on p185erbB-2 expression determined by Western blot. BT-474 cells were treated once and twice with AS-Mix-ODN. Western blot was performed for p185erbB-2 and Lamin B.
DISCUSSION A new screening tool for antisense sequences against p185erbB-2 was developed. In previous studies, proliferation of cells was determined by colorimetric assay based on the intracellular metabolism of tetrazolium salts, for example, MTT, WST, and XTT assays. However, all these assays have the disadvantage that we cannot exclude an influence of ODN as well as of liposomes on the metabolic activity of the cells. Therefore, we correlated the number of cells with the total protein content determined by BCA (Fig. 1). In addition, we studied the stability against nuclease digestion of ODN and exemplary ODN modifications (Mix-ODN and PTO) by HPLC analysis (Fig. 2). As expected, the PTO modification demonstrated the highest stability against enzymatic digestion.
Comparison of the modestly p185erbB-2 -expressing control cell line MCF-7 with the overexpressing BT474 cells verified that the strong antiproliferative effect of the antisense sequence is confined to high p185erbB-2 expression levels (Fig. 3A). The sequence dependency and dose-effect correlation (Figs. 3A and 5) of AS-PTO were examined with an RC-PTO in a proliferation assay. Unexpected results were observed with an additional SC-PTO. This scrambled sequence showed almost the same strong antiproliferative effect as the AS-PTO in BT-474 cells (Fig. 5), although this sequence has no known target in the human genome. This finding indicates that this SC-PTO could be an example of nonsequence-specific ODN activity. These results disagree with earlier studies that did not detect an antiproliferative effect of the SC-PTO
8
sequence in a proliferation assay (Roh et al., 1998). To investigate this phenomenon in more detail, a Western blot was applied to study the ODN effects on the p185erbB-2 protein level. Treatment of BT-474 cells with PTO sequences achieved a p185erbB-2 reduction exclusively with the AS-PTO, and neither the SC-PTO nor the RC-PTO influenced the p185erbB-2 protein level (Figs. 3B and 6B). Therefore, it can be assumed that the antiproliferative effect of the CC-PTO sequence is nonsequence specific and not related to p185erbB-2 inhibition. Further experiments showed a dose-dependent biologic effect of different ODN modifications. Mix-ODNs, which have a lower phosphorothioate content, were applied twice, and pure ODNs have to be treated three times to cause a significant antisense effect (Fig. 6A). This antiproliferative effect is correlated with specific p185erbB-2 protein reduction (Fig. 6B) and also corresponds to the enzymatic stability of these DNA modifications (Fig. 2). The indirect proliferation assay can be used as a fast and reliable antisense screening tool, but not all antiproliferative effects can be related to the p185erbB-2 level. Additional controls employed in our study, for example, Western blot, can optimize and verify the expressiveness of the indirect proliferation assay by detecting and characterizing unspecific effects. Such unspecific effects can be subdivided into two different groups, the sequenceindependent and sequence-related effects. Sequenceindependent effects are substance dependent and can be associated with a chemical ODN modification. In this context, each modified sequence, for example, phosphorothioates, should have the same unspecific effect. Unspecific PTO effects are well known, in the form of amplified, nonsequence-specific protein binding of thioated ODNs, compared with unmodified ODN (Guvakova et al., 1995; Stein, 1996). Treatment of both cell lines with phosphorothioate control sequences demonstrated for all thioated ODN sequences a modest antiproliferative effect (Fig. 3A) independent of the ODN sequence and the p185erbB-2 expression level of the cell line. Sequence-related effects depend on the ODN sequence but do not result from duplex formation with the target mRNA. A large number of these effects depend on CpG motifs (Hartmann et al., 2000; Krieg, 1999; Krieg et al., 1996; Rothenfusser et al., 2001). CpG effects are caused by the immune response against bacterial infections. CpG motifs are underrepresented in the human genome, and the cytosines are mostly methylated, contrary to the bacterial genome (Hartmann et al., 2000; Kerkmann et al., 2003). Thus, CpG effects combine sequence-independent phosphorothioate effects and sequence-related effects. In our study, the effectiveness of the SC-PTO in the proliferation assay did not correlate with a p185erbB-2 protein reduction in the Western blot (Figs. 3B and 6B). In addition, the SC-PTO has no known target in the hu-
ROHMANN ET AL.
man genome. Therefore, the antiproliferative effect has to be a sequence-related unspecific effect. Further studies are necessary to characterize the effect of the SC-PTO in detail. Possible reasons for this effect could be related to CpG effects and the rather high dosage of 1 mM ODN. The SC-PTO contains two CpG motifs that exist in neither the antisense nor the random control sequence (Table 1). In conclusion, an indirect proliferation assay was found to be a reliable and potent tool in an antisense screening strategy against p185erbB-2 using different ODN and cell controls. Further application on a high throughput screening platform seems to be possible. However, second-line experiments, such as Western blot or alternative protein techniques, might be necessary to investigate sequence-specific p185erbB-2 protein effects of ODN in more detail.
REFERENCES AGRAWAL, S., TAN, W., CAI, Q., XIE, X., and ZHANG, R. (1997). In vivo pharmacokinetics of phosphorothioate oligonucleotides containing contiguous guanosines. Antisense Nucleic Acid Drug Dev. 7, 245–249. ALTSCHUL, S.F., MADDEN, T.L., SCHAFFER, A.A., ZHANG, J., ZHANG, Z., MILLER, W., and LIPMAN, D.J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. BOGGS, R.T., McGRAW, K.K., CONDON, T., FLOURNOY, S., VILLIET, P., BENNETT, C.F., and MONIA, B.P. (1997). Characterization and modulation of immune stimulation by modified oligonucleotides. Antisense Nucleic Acid Drug Dev. 7, 461–471. DEFAZIO, A., CHIEW, Y.E., SINI, R.L., JANES, P.W., and SUTHERLAND, R.L. (2000). Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines. Int. J. Cancer 87, 487–498. GUVAKOVA, M.A., YAKUBOV, L.A., VLODAVSKY, I., TONKINSON, J.L., and STEIN, C.A. (1995). Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor, inhibit its binding to cell surface receptors, and remove it from low affinity binding sites on extracellular matrix. J. Biol. Chem. 270, 2620–2627. HARTMANN, G., WEERATNA, R.D., BALLAS, Z.K., PAYETTE, P., BLACKSWELL, S., SUPARTO, I., RASMUSSEN, W.L., WALDSCMITT, M., SAJUTHI, D., PURCELL, R.H., DAVIDS, H.L., and KRIEG, A.M. (2000). Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164, 1617–1624. KERKMANN, M., ROTHENFUSSER, S., HORNUNG, V., TOWAROWSKI, A., WAGNER, M., SARRIS, A., GIESE, T., ENDRES, S., and HARTMANN, G. (2003). Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type I IFN synthesis in human plasmacytoid dendritic cells. J. Immunol. 170, 4465–4474.
9
RAPID ANTISENSE SCREENING KRIEG, A.M. (1999). Mechanisms and applications of immune stimulatory CpG oligodeoxynucleotides. Biochim. Biophys. Acta 1489, 107–116. KRIEG, A.M., MATSON, S., and FISHER, E. (1996). Oligodeoxynucleotide modifications determine the magnitude of B cell stimulation by CpG motifs. Antisense Nucleic Acid Drug Dev. 6, 133–139. LASFARGUES, E.Y., COUTINHO, W.G., and REDFIELD, E.S. (1978). Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J. Natl. Cancer Inst. 61, 967–978. LEVIN, A.A. (1999). A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim. Biophys. Acta 1489, 69–84. MILLIGAN, J.F., MATTEUCCI, M.D., and MARTIN, J.C. (1993). Current concepts in antisense drug design. J. Med. Chem. 36, 1923–1937. NELLEN, W., and LICHTENSTEIN, C. (1993). What makes an mRNA anti-sense-itive?, Trends Biochem. Sci. 18, 419–423. PEGRAM, M.D., PAULETTI, G., and SLAMON, D.J. (1998). HER-2/neu as a predictive marker of response to breast cancer therapy. Breast Cancer Res. Treat. 52, 65–77. ROH, H., PIPPIN, J., BOSWELL, C., and DREBIN, J.A. (1998). Antisense oligonucleotides specific for the HER2/ neu oncogene inhibit the growth of human breast carcinoma cells that overexpress HER2/neu. J. Surg. Res. 77, 85–90. ROTHENFUSSER, S., JAHRSDOERFER, B., KRUG, A., ENDRES, S., and HARTMANN, G. (2001). CpG-oligonukleotide: Immuntherapie nach dem Muster der bakteriellen DNA. Deutsches Ärzteblatt 98, A 981–985. SLAMON, D.J., CLARK, G.M., WONG, S.G., LEVIN, W.J., ULLRICH, A., and McGUIRE, W.L. (1987). Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235, 177–182. STEC, W.J., ZON, G., EGA, W., and STEC, B. (1984). Automated solid-phase synthesis, separation and stereochemistry of phosphorothioate analogues of oligodeoxynucleotides. J. Am. Chem. Soc. 106, 6077–6089. STEIN, C.A. (1996). Phosphorothioate antisense oligodeoxynucleotides: Questions of specificity. Trends Biotechnol. 14, 147–149. STEIN, C.A., and CHENG, Y.C. (1993). Antisense oligonucleotides as therapeutic agents—Is the bullet really magical? Science 261, 1004–1012.
TREMPE, G.L. (1976). Human breast cancer in culture. Recent Results Cancer Res. 57, 33–41. TZAHAR, E., and YARDEN, Y. (1998). The ErbB-2/HER2 oncogenic receptor of adenocarcinomas: From orphanhood to multiple stromal ligands. Biochim. Biophys. Acta 1377, M25–37. UHLMANN, E., RYTE, A., and PEYMAN, A. (1997). Studies on the mechanism of stabilization of partially phosphorothioated oligonucleotides against nucleolytic degradation. Antisense Nucleic Acid Drug Dev. 7, 345–350. VAUGHN, J.P., IGELHART, J.D., DEMIRJI, S., DAVIS, P., BABISS, L.E., CARUTHERS, M.H., and MARKS, J.R. (1995). Antisense DNA downregulation of the ERBB2 oncogene measured by a flow cytometric assay. Proc. Natl. Acad. Sci. USA 92, 8338–8342. VAUGHN, J.P., STEKLER, J., DEMIRJI, S., MILLS, J.K., CARUTHERS, M.H., IGELHART, J.D., and MARKS, J.R. (1996). Inhibition of the erbB-2 tyrosine kinase receptor in breast cancer cells by phosphoromonothioate and phosphorodithioate antisense oligonucleotides. Nucleic Acids Res. 24, 4558–4564. XU, X., HAMOUIYA, F., THOMAS, S.D., BURKE, T.J., GIRVAN, A.C., McGREGOR, W.G., TRENT, J.O., MILLER, D.M., and BATES, P.J. (2001). Inhibition of DNA replication and induction of S phase cell cycle arrest by Grich oligonucleotides. J. Biol. Chem. 276, 43221–43230. ZOBEL, H.P., KREUTER, J., WERNER, D., NOE, C.R., KUMEL, G., and ZIMMER, A. (1997). Cationic polyhexylcyanoacrylate nanoparticles as carriers for antisense oligonucleotides. Antisense Nucleic Acid Drug Dev. 7, 483–493.
Address reprint requests to: Prof. Dr. Andreas Zimmer Institute of Pharmaceutical Chemistry and Pharmaceutical Technology Karl-Franzens-University Graz Schubertstrasse 6 A-8010 Graz Austria E-mail:
[email protected] Received July 10, 2003; accepted in revised form December 10, 2003.
