Bioconjugate Chem. 2000, 11, 153−160
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3′-End Conjugates of Minimally Phosphorothioate-Protected Oligonucleotides with 1-O-Hexadecylglycerol: Synthesis and Anti-ras Activity in Radiation-Resistant Cells Antonina Rait,† Kathleen Pirollo,† David W. Will,‡ Anush Peyman,‡ Vladimir Rait,† Eugen Uhlmann,‡ and Esther H. Chang*,† Georgetown University Medical Center, Departments of Oncology and Otolaryngology, NRB/E420, 3970 Reservoir Road, NW, Washington, D.C. 20007, and Hoechst Marion Roussel Deutschland GmbH, Chemical Research G 838, D-65926 Frankfurt am Main, Germany. Received August 10, 1999; Revised Manuscript Received November 2, 1999
Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with allphosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)modified ODNs having an additional hydrophobic tail at the 3′-end (PPS-C16). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3′- and 5′-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C16 ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C16 ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C16 ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.
INTRODUCTION
Sequence-specificity, nuclease resistance, and efficient cellular uptake are the major determinants of oligonucleotides intended for use as antisense therapeutics (1, 2). Phosphorothioate oligodeoxyribonucleotides (PS ODNs) are the most studied antisense agents and are currently under evaluation in human clinical trials. While these PS ODNs show appropriate serum and intracellular stability, they suffer from poor cellular uptake, and moreover, they demonstrate nonantisense, nonsequencespecific effects (for reviews, see refs 3 and 4). The nonantisense effects, originating primarily from the negative charge on sulfur and a high sulfur polarization, can be decreased by reducing the number of PS internucleoside linkages per ODN, using them only for 3′- and 5′-end-capping (5, 6) and for the protection of linkages adjoining internal pyrimidine nucleosides (7). In serum or medium with cultured cells, such partially phosphorothioated (PPS) ODNs demonstrate a stability which is comparable to uniformly phosphorothioate modified ODNs (7) and, in cell-free and cell-based assays, show fewer nonantisense effects than uniformly PS modified ODN (Rait et al., manuscript in preparation). As summarized by Stein (8), ODNs enter cells via fluid phase endocytosis (pinocytosis) and adsorptive endocy* To whom correspondence should be addressed. Phone: (202) 687-8418. Fax: (202) 687-8434. E-mail: change@ gunet.georgetown.edu. † Georgetown University Medical Center. ‡ Hoechst Marion Roussel Deutschland GmbH.
tosis. The former mechanism markedly contributes to cellular uptake only with a high extracellular ODN concentration. The latter mechanism, mediated by the interaction of negatively charged ODNs with receptorlike cell-surface proteins, seems to be a much more efficient pathway. However, it reaches its limit of capacity at an ODN concentration of around 1 µM. In total, less than 10% of naturally internalized ODNs are released into the cytoplasm with the remainder captured in endosomes and lysosomes (8). Cationic surfactants (called also cationic “lipids”) form supramolecular complexes with ODNs, intensify adsorptive endocytosis by bypassing cellular receptors, and assist the escape of ODNs from endosomes (9-11). However, the cationic lipids were also found to be cytotoxic by damaging the outer and inner cell membranes. Together with the observed instability of the complexes in serum, this may restrict their therapeutic application (12, 13). Another approach for increasing cellular uptake relies on covalent conjugates of ODNs with lipophilic compounds, such as cholesterol, vitamin E, or aliphatic alkyl chains which interact with cellular membranes (14-17). For example, a 3′-ω-hydroxydodecyl-modified nonadeoxyribonucleotide was taken up by T24 human bladder carcinoma cells four times more efficiently than the unmodified oligomer (18). Furthermore, a 5′ undecylmodified antisense undecamer effectively suppressed influenza virus reproduction in MDCK cells, while nonmodified antisense or nonsense oligomers did not (19).
10.1021/bc990106n CCC: $19.00 © 2000 American Chemical Society Published on Web 01/08/2000
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Rait et al. Scheme 1. Reagents: (a) 4,4′-dimethoxytrityl Chloride, Anhydrous Pyridine; (b) Succinic Anhydride, 4,4-(dimethylamino)pyridine, Pyridine; (c) CPG, N-ethylmorpholine, TBTU, DMF
Figure 1. General structure of a hexadecylglycerol-derivatized oligonucleotide.
The conjugation of 1,2-di-O-hexadecyl-rac-glycerol to the 5′-end of otherwise unmodified ODN resulted in compounds which inhibited viral proliferation in cell culture, however, with poor sequence specificity (20). To compare a PPS ODN and its 3′-end conjugate with 1-O-hexadecylglycerol (PPS-C16 ODN; Figure 1) for the first time directly, we synthesized the corresponding undecamers complementary to the initiation codon region of human Ha-ras along with a variety of noncomplementary control sequences and tested these compounds in lipofectin-mediated and lipofectin-free cell treatment. In contrast to the 5′-conjugates of bis-hexadecyl ether of glycerol with phosphodiester ODNs reported earlier (20), we used ODN-3′-conjugates of the mono-hexadecyl ether of glycerol, which are more stable to endo- and 3′exonucleases present in serum. Interestingly, we found that our 1-O-hexadecylglycerol undecamer conjugate no longer required a cationic uptake enhancer for optimal sequence-specific biological activity in cell culture. We also demonstrated that the antisense PPS-C16 ODN could reduce the radiation resistance of T24 tumor cells that carry a mutant Ha-ras gene. More significantly, when combined with radiation treatment, intratumorally injected PPS-C16 effectively inhibited the growth of Haras-transformed mouse tumors. MATERIALS AND METHODS
Reagents and solvents were obtained from Sigma (St. Louis, MO), Aldrich Chemical Co. (Milwaukee, WI), or Fluka Chemical Corp. (Milwaukee, WI). Three radiationresistant, Ha-ras-transformed cell lines were used in this study. RS485 is a NIH/3T3-derived cell line, transformed by multiple copies of normal human c-Ha-ras activated by a viral long terminal repeat (21). RS504 is an NIH/ 3T3-derived cell line transformed by mutated (at 12th codon) human c-Ha-ras (22). The human bladder carcinoma cell line T24 has the Ha-ras gene activated by a G f T transversion in the 12th codon (23). All cells were grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Flow Laboratories, McLean, VA) supplemented with 10% heat inactivated fetal calf serum (FCS), antibiotics (50 µg/mL each of penicillin, streptomycin, and neomycin), and 2 mM of L-glutamine. Synthesis of a Hexadecylglycerol-Derivatized Solid Support. 1-O-Hexadecyl-rac-glycerol (1) (1 mmol; Sigma) was dried by coevaporation with and dissolved in anhydrous pyridine (3 mL). The solution was cooled to 0 °C, and 4,4′-dimethoxytrityl chloride (1.15 mmol; Fluka) was added. The mixture was stirred overnight, the reaction was quenched by the addition of water (100
µL), and the mixture was evaporated to dryness. The residue was suspended in dichloromethane (20 mL) and extracted three times with 10 mL of 0.1 M phosphate buffer, pH 7. The organic phase was dried over sodium sulfate, filtered, and evaporated. The residue was dried by coevaporation with and dissolved in anhydrous pyridine (2 mL). 4,4-(Dimethylamino)pyridine (1.4 mmol; Aldrich) and succinic anhydride (2.1 mmol; Fluka) were added. The mixture was stirred overnight, evaporated to dryness, and coevaporated twice with toluene/dichloromethane (1:1, v:v). The residue was dissolved in dichloromethane (20 mL), extracted with 10% citric acid (11 mL) and washed three times with water (11 mL). Two drops of triethylamine were added to the organic phase that was then dried over sodium sulfate, filtered, and evaporated. The product was purified by silica gel chromatography using a gradient of methanol (1-4%) in dichloromethane containing 1% triethylamine. Succinic acid mono-(1-O-dimethoxytrityl-3-O-hexadecyl-rac-glycerol)-ester (2) was obtained as a clear oil in 510 mg yield (62%). The succinic acid mono-ester (0.056 mmol), Nethylmorpholine (0.07 mmol; Aldrich) and N,N,N′,N′tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU; 0.056 mmol; Sigma) were dissolved in DMF (2 mL) and added to a 550 Å aminopropyl controlled-pore glass (CPG; 500 mg; Fluka). The mixture was shaken for 4 h, and the resulting C16-derivatized CPG 3 (Scheme 1) was recovered by filtration and washed with methanol and dichloromethane. Finally, the solid support was capped for 1 h using acetic anhydride/N-methyl imidazole in THF, filtered, and washed with methanol, dichloromethane, THF, and diethyl ether. After drying in vacuo, the loading of dimethoxytrityl groups was determined to be 80 µmol/g. Oligodeoxynucleotide Synthesis. The ODNs listed in Table 1 were synthesized using standard phosphoramidite chemistry on a 394 DNA synthesizer (Applied
Anti-ras PPS Conjugates
Bioconjugate Chem., Vol. 11, No. 2, 2000 155
Table 1. Oligonucleotides Used In Studya sequence, 5′ f 3′
description, abbreviation
partially phosphorothioated ODNs TsAsToToCsCoGoTsCsAsT AsTsGoAoCsGoGoAsAsTsA partially phosphorothioated, 3′ C16-modified ODNs TsAsToToCsCoGoTsCsAsT-C16 AsTsGoAoCsGoGoAsAsTsA-C16 TsTsAoToAsCoGoTsCsCsT-C16 AsTsCoToTsAoCoGsTsTsC-C16
PPS antisense, AS PPS sense, S PPS PPS-C16 antisense, AS PPS-C16 sense, S PPS-C16 mismatch scrambled
Tm (°C)b
calcd
molecular weight measured
36.9
3378.58 3485.68
3378.1 ( 0.03 3484.9 ( 0.53
37.2
3757.05 3864.14 3757.05 3757.05
3756.47 ( 0.15 3863.51 ( 0.14 3756.24 ( 0.16 3778.17 ( 0.24c
a s ) phosphorothioate internucleoside linkage, o ) phosphodiester internucleoside linkage. b T of duplex formed with r(AUGACGm GAAUA). c M + Na+.
Biosystems, Foster City, CA). The PPS-C16 ODNs were prepared starting with the C16-derivatized CPG support 3. Where the introduction of phosphorothioate linkages was required, sulfurization was performed immediately after coupling using 0.075 M Beaucage reagent (Aldrich) in acetonitrile (24), followed by capping with 10% acetic anhydride, 10% 2,6-lutidine, and 16% N-methylimidazole in THF. All ODNs were deprotected by treatment with concentrated ammonia at 55 °C for 16 h and then purified by electrophoresis through a denaturing 19% polyacrylamide gel. After elution with 2% SDS, 75 mM EDTA, 0.3 M NaCl in 0.2 M Tris-HCl buffer, pH 7.6, and ethanol precipitation, the ODNs were dissolved in deionized water. The purity and integrity of all ODNs were found to be greater than 95% by HPLC on Waters Gen-Pak FAX column using a 0.1-1.5 M NaCl gradient in 20% acetonitrile containing 10 mM NaH2PO4. The ODNs were further analyzed by negative ion electrospray mass spectroscopy (Fisons Bio-Q, U.K.) that, in all cases, confirmed the correct mass (Table 1). Thermal Denaturation of Oligomer Heteroduplexes. ODNs were mixed with the complementary oligoribonucleotides at 1 µM of each strand in 10 mM sodium phosphate buffer, pH 7.4, containing 140 mM KCl and 0.1 mM EDTA. Absorbance vs temperature profiles (15-85 °C, heating rate 0.5 °C/min) were recorded at 260 nm using a 8452A diode array spectrophotometer (Hewlett-Packard). All samples were premelted at 8590 °C and then allowed to thermally equilibrate. In Table 1, the Tm values are the mean values of at least three measurements. Preparation of ODN-Lipofectin Complexes and ODN Treatment of Cells. ODN-lipofectin complexes were prepared as recommended by the manufacturer using Lipofectin Reagent (1 mg/mL; Life Technologies, Gaithersburg, MD), a 1:1 (w/w) mixture of N-[1-(2,3dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleyl phosphatidylethanolamine. Briefly, 7 µL of lipofectin was diluted into 100 µL of DMEM and allowed to stand at room temperature for 30 min. The diluted lipofectin was gently mixed with 100 µL of 0-50 µM ODN solution in DMEM and incubated for an additional 15 min. Finally, the mixture was combined with 800 µL of DMEM. RS485 or RS504 cells were seeded in 6-well plates (105 cells/well) and, 18-24 h later at 40-60% confluence, treated with ODNs. The medium was removed, 1 mL of fresh, serum-supplemented DMEM containing ODNs at various concentrations was added to each well, and the plates were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 48 h. In lipofectin-mediated treatment, the RS485 cells were washed twice with serum-free medium and then overlayed with 1 mL of DMEM containing an ODN-lipofectin complex. After 6 h, 1 mL of DMEM supplemented with
4 mM L-glutamine and 20% FCS was added, and incubation was continued for an additional 42 h. Postincubational 5′-End 32P-Labeling of Oligonucleotides. The unlabeled ODNs were incubated in media with RS504 cells for 24 h. After incubation, the media with ODNs was removed from the cell monolayer, heated to 95 °C for 5 min, and immediately cooled on ice. The 5′-end 32P-labeling was performed with [γ-32P]ATP, 6000 Ci/mmol, (NEN, Boston, MA) and T4 polynucleotide kinase in a reaction buffer provided by New England Biolabs (Beverly, MA) as recommended (25). The reaction mixtures were separated on 19% polyacrylamide/urea gels and bands visualized via autoradiograhpy using Biomax MS film (Kodak). Preparation of Cell Lysates and Western Blot Analysis of Ras Protein. For total cellular protein determination, the cells were washed twice with phosphate-buffered saline (PBS), overlayed with 0.5 mL of RIPA buffer (PBS, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), and agitated gently for 60 min at room temperature. Total protein in the lysate was determined using the Pierce Micro BCA protein assay reagent kit (Pierce, Rockford, IL). For Western analysis, the cells were washed with PBS, harvested with trypsin, collected by centrifugation at 1000g for 7 min at 4 °C, and washed once more with cold PBS. The cells were resuspended in 50-100 µL of cold RIPA buffer, containing 0.1 mg/mL PMSF, 30 µg/mL aprotinin, and 1 mM sodium orthovanadate, and lysed for 20 min in an ice bath. The lysates were passed through a 21 gauge needle, incubated on ice an additional 30 min, and then centrifuged at 15000g for 20 min at 4 °C. Protein concentrations of the cleared lysates were measured and the lysates aliquoted and stored at -80 °C. Lysate samples, containing 40 µg of total protein, were denatured, separated on a 12.5% SDS-PAGE, and electroblotted onto Protran BA 85 nitrocellulose transfer membrane (Schleicher and Schuell, Keene, NH). To block nonspecific binding, the membrane was incubated at room temperature for 1 h with 5% nonfat dry milk in 10 mM Tris-HCI buffer, pH 8.0, containing 150 mM NaCl and 0.05% Tween 20 (TBST). The blot was probed for 4 h with 0.1 µg/mL human Ras p21-directed primary antibody (C-20, rabbit polyclonal IgG; Santa Cruz Biotechnology, Santa Cruz, CA) and washed three times with TBST. Proteins were detected using 1:1000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology), ECL Western blotting detection reagent, and Hyperfilm ECL (Amersham). Films were scanned and quantified using a PDI scanner, model DNA 35 (Huntington Station, NY), and the Quantity One PDI analysis software. In Vitro Radiobiology. The in vitro response of antiras ODN-treated T24 cells to radiation was evaluated by
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Rait et al.
Figure 2. Representative Western blot analysis of human Ras p21 levels in RS485 cells treated with free (-) or lipofectin-bound (+) antisense PPS or PPS-C16 ODNs. RS485 cells were incubated with ODNs for 48 h. Cell lysates were fractionated by discontinuous gel electrophoresis. Ras p21 was detected using human Ras p21-directed rabbit primary antibody, peroxidase-conjugated goat antirabbit IgG, and ECL Western blotting reagents.
the cell survival assay (26). Briefly, T24 cells were seeded at 3 × 104 cells/well of a 6-well tissue culture plate. Twenty-four hours later, the cells were treated with antisense (or sense) PPS-C16 ODNs at 1 µM concentration in 1 mL of medium (DMEM plus 10% FCS, glutamine, and antibiotics) per well. After 48 h at 37 °C, the cells were harvested, suspended in fresh medium, and irradiated with graded doses of 137Cs γ-rays at a dose rate of 36 Gy/min in a Mark I irradiator (J. L. Shepard and Associates, San Fernando, CA) at room temperature. Irradiated cells were plated at 300-5000 cells/well and incubated in 3 mL of medium per well at 37 °C. Two days after plating, the wells were supplemented with 0.5 mL of FCS plus 5 µg/mL hydrocortisone. Ten to fourteen days later, the plates were stained with 1% crystal violet and colonies of 50 cells or more of normal appearance were scored. D10 (the dose required to reduce survival to 10%) values were calculated from the initial survival data. In Vivo Studies. To evaluate the ability of antisense ODNs to sensitize tumors to radiation in vivo, female athymic nude mice were subcutaneously injected with 5 × 106 radiation-resistant RS504 cells in 50 µL of PBS. Tumors were treated with ODNs when the size of the tumors was between 2 and 6 mm3. A total of 50 µL/tumor of a 5 µM solution of antisense (or sense) PPS-C16 ODN (250 pmol) was injected directly into the tumors. After 24 h, the tumor area only was irradiated with 2.0-2.5 Gy. The irradiation was repeated every 48 h until an accumulated dose of 20 Gy had been delivered. ODN injections were repeated at 48 h (50 µL), 192 h (50 µL), and 240 h (100 µL) following the initial injection. Tumor volume was recorded in mm3. For comparison, some tumors received only the antisense PPS-C16 or only radiation, while one group of tumors was left completely untreated. RESULTS
Synthesis of 1-O-Hexadecylglycerol ODN Conjugates. In the antisense PPS ODN (see Table 1) that targets the initiation codon region 5′ AUG ACG GAA UA 3′ of human Ha-ras mRNA (27), the phosphorothioate pattern was designed according to the “minimal protection” strategy (7). To render the ODN stable against 3′and 5′-exonucleases, it was capped by three and two
phosphorothioate residues at the 3′- and 5′-ends, respectively. An additional phosphorothioate linkage was also placed between internal pyrimidine nucleosides, major sites of degradation by endonucleases. The position of the phosphorothioate linkages was retained in the structure of the control ODNs (Table 1). Figure 1 shows that PPS-C16 ODNs contain an extra phosphodiester linkage joining the 3′-terminal nucleoside with the 1-O-hexadecylglycerol residue, intended to facilitate permeation of the cell membrane. The 3′-end 1-O-hexadecylglycerol residue might also provide additional resistance to 3′-exonucleases. In the solid-phase synthesis of PPS-C16 ODNs, the general strategy of MacKellar et al. (28) for alcohol end-attachment was adopted. Thus, [1-(4,4′-dimethoxytrityl)-2-succinyl-3hexadecyl]glycerol (2) was first synthesized starting from commercially available 1-O-hexadecyl-rac-glycerol (1), which was regioselectively dimethoxytritylated at the primary hydroxy group and then converted to the corresponding succinate 2 in 62% overall yield (Scheme 1). The succinic acid mono-(1-O-dimethoxytrityl-3-hexadecyl-racglycerol)-ester 2 was then coupled with TBTU/N-ethylmorpholine to a 550 Å aminopropyl-CPG to yield the functionalized solid support 3 (Scheme 1) with a loading of 80 µmol/g. Purified by denaturing PAGE, antisense PPS or PPSC16 undecamers and complementary oligoribonucleotide r(AUGACGGAAUA) formed heteroduplexes with almost identical Tms of 36.9 and 37.2 °C, respectively (Table 1). These values are approximately 4 °C higher than the Tm measured for the corresponding heteroduplex with uniformly phosphorothioated ODN (data not shown). Inhibition of Ras p21 Expression by PPS-C16 ODN Conjugates in RS485 and RS504 Cells. To evaluate the in vitro anti-ras activity of the PPS and PPS-C16 ODNs, the human Ras p21 level was determined in RS485 cells by Western blot analysis after cell treatment with ODNs alone or in combination with lipofectin. The concentration of the ODNs was varied in the 0.25-5 µM range, while the concentration of lipofectin, where included, was kept constant at 7 µg/mL. Representative results of the Western analysis and relating quantitative data are shown in Figures 2-4. The comparison of the dose effect obtained without lipofectin (Figures 3 and 4) shows that, while 5 µM antisense PPS
Anti-ras PPS Conjugates
Figure 3. Ras p21 and total protein levels in RS485 cells versus concentration of free or lipofectin-bound antisense (AS) PPS ODNs. RS485 cells were incubated with ODNs for 48 h. At a fixed ODN concentration, three experiments were run in parallel in three separate wells. Before quantifying, lysates from the wells were combined to obtain an average value. The level of Ras p21 was determined by Western blot analysis.
Figure 4. Ras p21 and total protein levels in RS485 cells versus concentration of free or lipofectin-bound antisense (AS) PPS-C16 ODNs. RS485 cells were incubated with ODNs for 48 h. At a fixed ODN concentration, three experiments were run in parallel in three separate wells. Before quantifying, lysates from the wells were combined to obtain an average value. The level of Ras p21 was determined by Western blot analysis.
ODN was required to reach 50% inhibition of Ras p21, the same level of inhibition was achieved with the PPSC16 at a concentration less than 0.75 µM. Additionally, PPS-C16 ODN exerted its major effect (up to 94% inhibition) in a narrow range of concentrations, 0.5-1.25 µM, although a 5 µM concentration was required to completely supress the human Ha-ras expression in RS485 cells (Figure 4). Interestingly, within the 0.75-5 µM interval, the PPS-C16 ODN without lipofectin was even slightly more efficient in inhibiting Ha-ras expression than either the lipofectin-PPS ODN complex or the lipofectin-PPS-C16 ODN complex (Figures 3 and 4). At 7 µg/mL, a concentration considered to be nontoxic (11), lipofectin alone did not affect Ras p21 synthesis (Figures 3 and 4). In contrast, total cellular protein appeared to be more sensitive to lipofectin treatment, decreasing by 15% in the absence of ODNs. The same decline in total protein synthesis was observed with antisense PPS-C16 ODN treatment at a concentration of 1-1.25 µM, where Ras p21 synthesis was already 8995% inhibited.
Bioconjugate Chem., Vol. 11, No. 2, 2000 157
Figure 5. Western blot analysis of Ras p21 levels in RS504 cells treated by antisense (AS) or sense (S) PPS-C16 ODNs. Lane C corresponds to untreated RS504 cells. RS504 cells were incubated with ODNs for 48 h. Cellular lysates were fractionated by discontinuous gel electrophoresis. Ras p21 was detected using human Ras p21-directed rabbit primary antibody, peroxidase-conugated goat anti-rabbit IgG, and ECL Western blotting reagents.
To estimate the sequence specificity of the inhibition, three control PPS-C16 ODNs were tested at a concentration of 1.25 µM. The relative levels of Ras p21 were 87, 97, and 124% for the sense, mismatched, and scrambled PPS-C16 ODNs, respectively. None of the controls decreased total protein synthesis by more than 6%. The antisense efficacy and sequence specificity of the PPS-C16 ODN were also tested using RS504 cells, another murine transformant expressing mutated human c-Ha-ras (22). Scanning of the bands in Figure 5, we found 16, 73, and 92% inhibition of p21 synthesis at 0.5, 1, and 5 µM, respectively. At the highest tested concentration (5 µM), the control sense PPS-C16 ODN caused only minimal (13%) inhibition of Ras p21 and total protein syntheses. To evaluate serum stability, C16-conjugated and nonconjugated ODNs were incubated for 24 h in complete medium with RS504 cells and then postlabeled with [γ-32P]ATP (Figure 6). No degradation was seen in the case of the antisense PPS-C16 ODN (lane 4 in Figure 6), while the nonconjugated PPS ODN showed at least some cleavage products (lane 3). The bands shown in lane 3 represent degradation products less than 6 nucleotides in length (25). Reversal of Radiation Resistant Phenotype by Antisense PPS-C16 ODN in Vitro and in Vivo. Ras genes are known to be involved in signal-transduction of various factors for growth, differentiation, and oncogenesis (29, 30). Moreover, a strong association has been made between the presence of either a mutated form of, or overexpression of the normal, Ha-ras gene and cellular radiation resistance (26). Treatment of cells with an abnormal Ha-ras gene (RS485 and RS504) with antisense PPS-C16 was able to downregulate Ras expression. Since the failure to respond to radiotherapy is a significant problem in the treatment of human cancer, we wished to determine if the downmodulation could revert a radiation-resistant phenotype, such as the radiationresistant T24 human bladder carcinoma cell line. As shown in Figure 7, there was a decrease in the radiation resistance level of the cells treated with 1 µM antisense PPS-C16 ODN, reducing the D10 level from 5.4 ( 0.2 Gy to 4.5 ( 0.4 Gy, a value in the range considered to be radiosensitive. To examine the possible therapeutic potential of antisense PPS-C16 ODN in treating human cancer, we wished to determine if the radiosensitization observed in vitro translated to the in vivo radiation-induced inhibition of tumor growth in a mouse model. Since T24 cells are poorly tumorigenic, the radiation-resistant NIH/
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Figure 6. Autoradiograph of antisense PPS and PPS-C16 ODNs (lanes 3 and 4, respectively), incubated 24 h with RS504 cells in serum-supplemented medium and then 5′-end 32Plabeled in withdrawn heat-denatured medium. Control antisense PPS and PPS-C16 ODNs are shown in lanes 1 and 2, respectively. ODNs were separated by electrophoresis on 19% polyacrylamide/urea gel. XC, xylene cyanol, and BP, bromophenol blue. These dyes comigrate with ODNs of 22 and 6 nucleotides, respectively (25).
Rait et al.
Figure 8. Effect of combination of sense (S) or antisense (AS) PPS-C16 ODNs and radiation on tumor growth in a xenograft mouse model. RS504 cells were subcutaneously injected on the back of female athymic nude mice. The antisense or sense PPSC16 ODNs (250 pmol in 50 µL) were injected directly into the tumors. Beginning 24 h after the first ODN injection, the tumor area only was exposed to 2-2.5 Gy doses of ionizing radiation (X-rays), as indicated by the arrows, to a total of 20 Gy. (-R) Control untreated tumor; (+R) tumor received only radiation; (AS+R) tumor treated with AS PPS-C16 ODN and radiation; (AS-R) tumor treated with AS PPS-C16 ODN only; (S+R) tumors treated with control S PPS-C16 ODN and radiation. Bold arrows indicate ODN injections; thin arrows indicate doses of irradiation.
experiment are shown in Figure 8. Neither the antisense PPS-C16 ODN nor radiation alone had much effect on tumor growth. Although some nonspecific radiosensitizing effect was observed with the control sense ODN, only the combination of the antisense treatment and radiation was able to significantly inhibit the growth of these tumors. These results demonstrate the considerable potential of the PPS-C16 modified anti-ras ODNs in future clinical studies of radiation resistance reversal. DISCUSSION
Figure 7. Effect of sense (S) or antisense (AS) PPS-C16 ODNs on the radiation resistance level of T24 bladder carcinoma cells in vitro. T24 cells were incubated with antisense or control sense PPS-C16 ODNs at 1 µM for 48 h, and then irradiated with graded doses of 137Cs γ-rays. Radioresistance levels are given as D10 values. Error bars represent SEM of 1-3 values.
3T3 transformant cell line RS504 was used in this experiment. RS504 was obtained by transfection of NIH/ 3T3 with genomic DNA from T24 cells and consequently carries the point mutated human Ha-ras gene (22). As shown in Figure 5, treatment of RS504 with antisense PPS-C16 ODN in vitro was able to decrease synthesis of Ras p21. Subcutaneous RS504 tumors were induced in female nude mice. Once the tumors were palpable, they were intratumorially injected with 250 pmol of the antisense PPS-C16 ODN or, as control, sense PPS-C16 ODNs. Radiation treatment was started 24 h later. Radiation was administered every other day. A total of four injections of the ODN (days 0, 2, 8, and 10) and a total of 20 Gy (days 1, 3, 5, 7, 9, 11, 13, 15, and 17) were administered over the course of 18 days. The results of this
The antisense efficacy and sequence-specificity of ODNs, bearing 3′-terminally attached acyclic hydrocarbons, has been the subject of only a few studies. Encouraging results were obtained with an antisense nonamer carrying a dodecandiol tail at the 3′-end which was directed against codon 12 point mutated Ha-ras (18). This derivative, designed as a control for a more sophisticated 3′,5′-disubstituted construct with acridine at the 5′-end, was used in both cell-free and cell-based translation assays. The former assay showed that the concentration needed for 50% inhibition of targeted mRNA translation was 1.5 µM, i.e., 15 times lower than was the case with the nonmodified parent ODN and 3 times higher than the double terminally modified construct. Although no data was shown, it was mentioned that the dodecandiol tail caused a 4-fold increase in the uptake of the nonamer by T24 cells (18). Quantitative, and even qualitative, results of cellular uptake and biological activity of lipophilic conjugates may vary significantly depending on the cells employed, the antisense ODN backbones, and/or the lipophilic group position in a conjugate structure. This has been demonstrated by a recent comparison of methylphosphonate chimeric ODNs, all 3′-labeled with fluorescein and 5′modified with lipophilic groups, such as dodecanol,
Anti-ras PPS Conjugates
cholesterol, or vitamin E (31). None of the conjugated substances, as well as lipofectin, improved delivery of the chimeric 18-mer or displayed molecular effects in human leukemic KYO1 cells. Moreover, the ODN, when conjugated to cholesterol or vitamin E, caused lysis of the target cells if its concentration exceeded 2 µM. The remainder of the currently available data was obtained using viral assays where the host cell survival was not the principal issue. A 5′ undecyl-modified antisense undecamer was used to inhibit synthesis of the influenza virus polymerase 3 (and, consequently, virus reproduction) in infected MDCK cells (19). It has been shown semiquantitatively that the obtained suppression was sequence specific and modification dependent. However, suppression of virus reproduction was achieved at conjugate concentrations of 50-200 µM. A more comprehensive study, including evaluation of cellular uptake and cellular toxicity, was carried out with phosphodiester ODNs, 5′-end conjugated to 1,2-di-Ohexadecyl-rac-glycerol, complementary to the initiation codon regions of the N, NS, and G protein mRNAs of vesicular stomatitis virus (20). In comparison to the PPS-ODN-3′-glycerol-hexadecyl-mono-ether conjugate of this work, the 5′-glycerol-bis-hexadecylesters phosphodiester ODN conjugates of Shea et al. (20) should be even more lipophilic, but metabolically much less stable, since they are neither protected against 3′-exonucleases nor against endonucleases present in serum. These glycerolbis-hexadecyl ether conjugated ODNs associated 8-10 times more efficiently with uninfected L929 cells than either phosphodiester or phosphorothioate ODNs. Although these conjugates showed enhanced antiviral activity, they suffered from poor sequence discrimination (20), an observation also made with lipophilic antisense conjugates in other viral assay systems (32). Furthermore, no adequate solution to the proof of antisense mechanism in viral replication studies is available (33). In our study, the “minimal protection strategy” (7) was applied to minimize the number of PS bonds to render the ODN resistant to nuclease degradation and at the same time retain sequence specificity. In addition, a 1-Ohexadecylglycerol was attached to the 3′-terminal phosphate group of the antisense and control ODNs, which confers additional stability against 3′-exonucleases, rendering them practically indigestible in complete medium with RS504 cells. In principle, antisense oligonucleotides with a free 3′-hydroxy group could serve as primers for DNA polymerases and reverse transcriptases in vivo. Clearly, a 3′-modification fully deprives the conjugates of priming properties. Due to the decreased content of PS groups, both antisense PPS and PPS-C16 ODNs formed more thermostable hybrids with complementary RNA than the uniformly PS-modified ODN (∆Tm ) +4.2 °C). Moreover, the attached lipophilic fragment did not affect the duplex stability. In preliminary studies, we have also investigated the uptake of the PPS-ODN-3′-glycerol-hexadecylmonoether with a fluorescence label at its 5′-end by fluorescence microscopy. A 1 µM ODN concentration was used for incubation of NIH/3T3 cells in the absence of uptake enhancers. After 4 h, strong fluorescence could be observed inside the cells, concentrated mostly in the nucleus. No punctate distribution was observed as is frequently reported. The same FITC-labeled ODN without the 3′-glycerol-hexadecyl-monoether did not show any significant uptake under the same conditions (E. Unger, G. Gothe, M. Schwerdel, and E. Uhlmann, unpublished results). In summary, the fluorescence microscopy study showed us that the lipophilic 3′-end modification does not
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only enhance cell uptake of the ODN, but appears also to influence the intracellular distribution in favor of nuclear localization. Comparison of the effect of antisense PPS and PPSC16 ODNs on target human Ras p21 synthesis in RS485 or RS504 cells clearly demonstrated a dramatic increase in anti-Ras activity caused by the 3′-end hexadecylglyceryl group. The dose of PPS-C16 ODN necessary to decrease p21 expression by 50% in these ras-transformed cells in culture was 8- to 10-times lower than that of PPS-ODN. The addition of lipofectin had no significant effect on the delivery and inhibitory effect of the PPSC16 ODN. More than 90% inhibition was reached at a concentration of 1 µM. This ability to reach high levels of specific inhibition at low concentrations of oligonucleotide in the absence of lipofectin has the benefit of reducing the toxic side effects sometimes seen with lipofectin treatment. In our studies, the PPS-C16 oligonucleotide at concentrations ranging from 0.25 to 1.0 µM inhibits cell growth by only 6 to 16%. Of particular importance to future applications of antisense oligonucleotide treatment of tumors is the efficacy of the PPS-C16 anti-ras oligonucleotide in reversing the radiation resistance associated with tumors that arise through activation of the ras oncogene. Our experiments show that ras-transformed T24 cells in culture and RS504-induced tumors in nude mice (which contain an activated ras) are more sensitive to 137Cs γ-rays following introduction of PPS-C16 oligonucleotides into the cells and injection into the tumors. CONCLUSION
In conclusion, (i) a synthetic route for obtaining 3′-end conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol was designed; the route is compatible with standard phosphoramidite chemistry and the solid-phase strategy; (ii) 3′-end C16 modification does not alter the hybridization properties of ODNs and increases nuclease resistance and antisense potency of partially phosphorothioated oligonucleotides without affecting their sequence specificity; (iii) in rastransformed cells in culture, the addition of a C16 alkyl group provides the ODN with the ability to exert high inhibitory activity without the need of uptake enhancers; (iv) treatment of T24 tumor cells with partially phosphorothioated C16-modified anti-ras undecamer reverts the radiation resistant phenotype of the cells in vitro; (v) the combination of intratumorial injection of C16-modified anti-ras undecamer and radiation treatment inhibits the growth of RS504 xenograft tumors in a synergistic manner due to reversal of radio-resistance. ACKNOWLEDGMENT
This work was supported in part by National Foundation for Cancer Research, Grant HU0001 (E.H.C). We are indebted to S. Hein, L. Hornung, and C. Weiser for excellent technical assistance and to Dr. A. Schaefer and M. Girg for measuring the ESI mass spectra. LITERATURE CITED (1) Cook, P. D. (1993) Medicinal Chemistry Strategies for Antisense Research. In Antisense Research and Applications (S. T. Crooke and B. Lebleu, Eds.) pp 149-187, CRC Press, Boca Raton, FL. (2) Uhlmann, E., and Peyman, A. (1990) Antisense Oligonucleotides: A New Therapeutic Principle. Chem. Rev. 90, 543584. (3) Agrawal, S., and Zhao, Q. (1998) Antisense Therapeutics. Curr. Opin. Chem. Biol. 2, 519-528.
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