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Experimental Therapeutics, Preclinical Pharmacology |
Antisense Inhibition of PC3M Prostate Cancer Cell Growth: Bcl-2 Hyperphosphorylation, Bax Up-Regulation, and Bad-Hypophosphorylation
Cellular Biochemistry Section, Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-1750 [Y. S. C., M-K. K., L. T., Y. S. C-C.]; Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201 [R. S.]; and Hybridon, Inc., Cambridge, Massachusetts 02139 [S. A.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit of cyclic AMP (cAMP)-dependent protein kinase is enhanced in human cancer cell lines, primary tumors, and cells after transformation. Using an antisense strategy, we have shown that RI has a role in neoplastic cell growth in vitro and in vivo. In the present study, we have investigated the sequence- and target-specific effects of exogenous RI antisense oligodeoxynucleotides (ODNs) and endogenous antisense gene on tumor growth, apoptosis, and cAMP signaling in androgen-insensitive prostate cancer cells, both in vitro and in nude mice. Here, we show that an RI antisense, RNA/DNA mixed backbone ODN exerts a reduction in RI expression at both the mRNA and protein levels, up-regulation of both the RIIß subunit of cAMP-dependent protein kinase or protein kinase A and c-AMP-phosphodiesterase IV expression, and inhibition of cell growth. Growth inhibition was accompanied by changes in cell morphology and the appearance of apoptotic nuclei. In addition, Bcl-2 hyperphosphorylation; increase in the proapoptotic proteins Bax, Bak, and Bad; and Bad hypophosphorylation occurred in the antisense-treated cells. These effects of exogenously supplied antisense ODN mirrored those induced by endogenous antisense gene overexpression. The RI antisense ODNs, which differed in sequence or chemical modification, promoted a sequence- and target-specific reduction in RI protein levels and inhibited tumor growth in nude mice. These results demonstrate that in a sequence-specific manner, RI antisense, via efficient depletion of the growth stimulatory molecule RI, induces growth inhibition, apoptosis, and phenotypic (cell morphology) changes, providing an innovative approach to combat hormone-insensitive prostate cancer cell growth. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary mediator of cAMP action in mammalian cells is PKA (3)
. Two types of PKA, type I (PKA-I) and type II (PKA-II), share a common catalytic subunit but contain distinct regulatory subunits, RI and RII (4)
. Expression of the RI subunit of PKA increases in various human tumors and cell lines, including cancers of the breast (5, 6, 7, 8)
, ovary (9
, 10)
, lung (11)
, and colon (12, 13, 14)
. Overexpression of the RI subunit of PKA correlates with poor prognosis and survival of cancer patients (6
, 7
, 9
, 10
, 15)
, and conversely, specific inhibition of RI expression by an antisense ODN inhibits growth and modulates cAMP signaling in cancer cells (16, 17, 18, 19, 20)
.
In the present study, we examined the downstream biological effects of RI antisense in the androgen-insensitive prostate cancer cells in the in vitro and in vivo tumor models.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP was purchased from ICN (Costa Mesa, CA). Antibodies against the C, RI, RII, and RIIß subunits of PKA and Bad were obtained from Transduction Laboratories (Lexington, KY). Anti-Bcl-2, Bax, Bak, and phospho-Bad antibodies were from Upstate Biotechnology (Lake Placid, NY). Antiactin antibody was obtained from Oncogene Research Products (Cambridge, MA).
Oligonucleotides.
The oligonucleotides used in the present study include the human RI antisense PS-ODN (16)
targeted against codons 8–13 of human RI. The second-generation RNA/DNA MBO PS-ODN contains four 2'-O-methyl ribonucleotides (RNA) at both the 5' and 3' ends (20)
and the mouse RI antisense PS-ODN targeted against codons 8–13 of mouse RI (16)
. A four-base mismatched ODN and random sequence RNA/DNA MBO ODN (20)
were used as control ODNs. These oligonucleotides were synthesized as described previously (18)
.
Cell Culture and Oligonucleotide Treatment.
PC3M cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mM MEM nonessential amino acids (pH 7.4), and antibiotic-antimycotic in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For cell growth assays, PC3M cells were plated at 2 x 105 cell density in 60-mm culture dishes. After 12 h of seeding, cells were washed with fresh medium, and antisense and control ODNs were added to the culture medium at varying concentrations. To increase the uptake of ODN into the cells, the transfection reagent N-[1-(2, 3- Dioleoyloxy) propyll-N,N,N-trimethylammonium methyl sulfate was used according to the manufacturer’s instructions (Boehringer Mannheim, Indianapolis, IN). The mismatched or random sequence ODNs were used as controls. Cells were harvested at the indicated times and counted using a ZI Coulter counter (Coulter Co., Miami, FL). Results are expressed as the mean cell number per dishes ±SD. Each assay was performed in triplicate.
Construction of Antisense RI Vector and Production of Stable Transfectants.
The 210-bp fragment of human PKA RI cDNA was amplified by PCR using the upstream primer (5'-GCGCGGATCCATGGAGTCTGGCA-3', nucleotide positions 1–13) and downstream primer (5'-GCGCGAATTCTTTCTGCAGATTC-3', nucleotide positions 198–210). The PCR product was cut with BamHI/EcoRI and cloned into the EcoRI/BamHI site of the OT1529 retroviral vector (21)
. OT1529 contains the mouse metallothionein (MT-1) promoter as the internal promoter and a gene encoding neomycin phosphotransferase, which confers G418 resistance and allows selection for stable transfectants. Cells were transfected with MT-antisense RI vector using the lipofectin method (Life Technologies, Inc.). Stably transfected cells were selected by growing cells in the presence of G418 (400 µg/ml; Life Technologies, Inc.). To induce expression of the antisense RI gene, cells were treated with 60 µM ZnSO4 for 5 days.
RNA Preparation and Northern Blot Analysis.
Total RNA was prepared using the Rneasy Midi kit (Qiagen), and 20 µg of each RNA sample were loaded onto an agarose/formaldehyde gel. PKA R- and C-specific probes were generated as described previously (21)
. Northern analyses were performed as described previously (21)
.
Tumor Growth and Antisense Treatment.
PC3M human prostate carcinoma cells (2 x 106 cells) were inoculated s.c. into the left flank of nude mice. When tumors became palpable, antisense or control ODN (0.1 mg/0.1 ml saline/mouse, daily) or saline (0.1 ml/mouse) was injected i.p. into the mice. Tumor volumes were obtained from daily measurement and calculation as described in (16)
. At each indicated time, animals were sacrificed, and tumors, livers, and spleens were removed, weighed, immediately frozen in liquid N2, and stored at -80°C until used.
Western Blot Analysis.
PC3M cells were seeded at a density of 1 x 106 cells/100-mm plate and treated with antisense or control ODN (100 nM) for 2 days. Cells were washed twice with ice-cold PBS, lysed in Buffer 10 [Ref. 21
; 20 mM Tris/HCl, 100 mM NaCl, 5 mM MgCl2, 1% NP40, 0.5% sodium deoxycholate, 100 µM pepstatin, 100 µM antipain, 100 µM chymostatin, 10 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml trypsin inhibitor, and 1 mM benzamidine (pH 7.5)], and placed on ice for 15 min. Tumor and liver extracts were prepared as described in Ref. 16
. Protein concentration was determined by the Bradford assay using the Bio-Rad Protein Assay Kit (Bio-Rad, Richmond, CA). Cell extracts (40 µg) were subjected to SDS-PAGE, and Western analysis was performed as described previously (22)
.
PKA Assay.
Cells were lysed with Buffer 10 (see Western blotting method) on ice. PKA activity was measured by adding 10 µl of cell extract (10 µg of protein) to 40 µl of reaction buffer [50 mM Tris (pH 7.5), 20 µM Kemptide (a Ser peptide; Leu-Arg-Arg-Ala-Ser-Leu-Gly; Life Technologies, Inc.), 1.2 µM (-32P) ATP (100–200 cpm/pmol), 10 mM MgCl2, and 1 mM DTT] in the presence or absence of cAMP (5 µM) or protein kinase inhibitor protein (20 µM) and incubated for 5 min at 30°C. The reaction mixture (40 µl) was spotted onto phosphocellulose discs, then washed three times with 1.5% phosphoric acid. Filters were air dried, then counted using liquid scintillation counter (Beckman, Columbia, MD). One unit of PKA activity is defined as that amount of enzyme that transfers one pmol of 32P from [-32P] ATP to the recovered protein in 5 min at 30°C in the standard assay system.
Morphological Determination of Apoptotic Nuclei.
Cells were grown on ethanol-sterilized glass coverslips and treated with antisense or control ODN (100 nM) for 2 days. To examine whole cell morphology, cells were washed with PBS, fixed with 70% methanol for 5 min, and stained with Giemsa (Bio-Rad) for 30 min. Coverslips were rinsed with PBS, mounted on slides with 80% glycerol in PBS, and photographed using a Zeiss Axiovert 25 CFL inverted microscope. Morphological changes characteristic of apoptosis were determined by staining cell nuclei with Hoechst 33258 (Sigma Chemical Co.). After treatment, the coverslips were rinsed gently with PBS, fixed with 3.7% formaldehyde for 10 min, and stained with 1 µM Hoechst 33258 in PBS for 15 min. Coverslips were rinsed with PBS and mounted with SloFade antifade mounting medium (Molecular Probes, Eugene, OR). The slides were observed under a Zeiss Axiovert 25 CFL inverted microscope.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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antisense ODN, we first analyzed the proliferation of PC3M cells in monolayer culture in response to increasing concentrations of antisense ODNs. To address the possibility of nonspecific cytotoxicity on exposure of cells to the ODN, a 4-base mismatched ODN or a scrambled sequence ODN were used as a control. Antisense ODN treatment inhibited growth of PC3M cells in a concentration-dependent (Fig. 1A)
and time-dependent (Fig. 1B)
manner. In contrast, the mismatched control ODN had no effect (Fig. 1, A and B)
. We next examined whether endogenous overexpression of an antisense gene could also inhibit growth in PC3M cells. As shown in Fig. 1C
, the growth of cells transfected with the RI antisense gene was inhibited, compared with parental nontransfectants. Cells transfected with the expression vector alone exhibited no growth retardation (Fig. 1C)
. Thus, both the exogenously supplied antisense ODN and the endogenously overexpressed antisense gene were capable of promoting growth inhibition in PC3M cells.
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, Up-Regulation of RIIß, and Induction of cAMP-PDE IV. protein levels in both antisense ODN-treated cells and antisense gene transfectants, whereas the mismatched ODN had no effect (Fig. 2A)
. Moreover, RI antisense, but not mismatched ODN, induced RIIß protein expression (Fig. 2A)
, although the levels of RII and the C subunit did not change in RI antisense ODN-treated cells or antisense gene transfectants (Fig. 2A)
.
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antisense treatment inhibited cell growth and decreased RI protein levels, we examined whether antisense also influenced RI mRNA levels (Fig. 2B)
. Both RI antisense ODN treatment and antisense gene overexpression markedly reduced RI mRNA levels (Fig. 2B)
. By comparison, the mismatched ODN had no effect (Fig. 2B)
. Both the antisense ODN and the antisense gene increased RIIß mRNA levels without changing RII and C mRNA levels (Fig. 2B)
. Thus, the reduction in the levels of RI protein and mRNA resulting from antisense treatment correlated with an increase in RIIß mRNA and protein levels and a decrease in cell growth.
We have shown previously that the cAMP-inducible enzyme, PDE IV, increases in RI antisense ODN-treated LS-174T colon cancer cells and in LNCaP prostate cancer cells (20)
. Fig. 2B
shows that in cells exposed to RI antisense, either through ODN treatment or gene overexpression, the PDE IV mRNA levels were markedly increased. Thus, RI antisense induced PDE IV, a cAMP-inducible enzyme.
Increase in PKA Activity Ratio.
The induction of PDE IV, which contains a CRE enhancer in its promoter, suggests the activation of PKA. Therefore, we determined a PKA activity ratio, a measure of how much PKA is in its active form (free/total PKA), in antisense ODN-treated cells. As shown in Table 1
, antisense ODN treatment substantially increased the PKA activity ratio in a time-dependent manner.
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. When cells were evaluated for morphology, only RI antisense ODN-exposed cells or cells overexpressing the antisense gene displayed a unique morphology that is different from untreated control (saline treated; Fig. 3
) and mismatched ODN-treated cells (data not shown). The control and mismatched ODN-treated cells exhibited an elongated shaped morphology with a higher ratio of nucleus-to-cytoplasm intensity (Fig. 3A)
. Cells exposed to endogenous or exogenous RI antisense, however, exhibited a flat phenotype and an increased cytoplasm:nucleus ratio, and they grew sparingly (Fig. 3A)
.
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antisense ODN or overexpression of the antisense gene inhibited growth, it was of interest to examine if apoptosis was involved in this process. Treatment of PC3M cells for 3 days with RI antisense ODN (0.2 µM) induced apoptosis, as evident from fragmented nuclei or condensed chromatin (Fig. 3B)
. The antisense treatment induced apoptosis in a concentration- and time-dependent manner, whereas mismatched ODN could not mimic the antisense effect (Fig. 3C)
. Under the experimental conditions, the saline-treated control cells exhibited no appreciable changes in nuclear morphology (Fig. 3C)
. The fragmented nuclear morphology and chromatin condensation observed in the antisense ODN-treated cells were mirrored in cells overexpressing the antisense gene (Fig. 3B)
.
Hyperphosphorylation of Bcl-2 and Up-Regulation of Bax.
Some proteins within the Bcl-2 family, including Bcl-2 and Bcl-Xl, inhibit apoptosis, whereas others, such as Bax, Bak, and Bad, promote apoptosis. It has been shown that the microtubule-damaging drugs induce Bcl-2 hyperphosphorylation and apoptosis in cancer cells and that Bcl-2 hyperphosphorylation is mediated by activated protein kinase A (23)
. Because the above data show that treatment of cells with RI antisense resulted in the activation of PKA and the induction of apoptosis, we examined the effect of RI antisense on the expression of Bcl-2 family proteins. PC3M cells either treated with RI antisense ODN or overexpressing the antisense gene exhibited a marked increase in the proapoptic proteins Bax, Bak, and Bad (Fig. 4)
. In addition, RI antisense induced hyperphosphorylation of the antiapoptic Bcl-2 protein and a decrease in the phosphorylated form of Bad (Fig. 4)
, which is also antiapoptotic (24)
. p53 protein was undetected, and the p21waf1/cip1 protein level was unchanged in the antisense-treated cells (Fig. 4)
. In comparison, the mismatched ODN could not mimic these effects (Fig. 4)
. These data confirm the above findings that RI antisense induces apoptosis in PC3M cells.
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antisense ODN on human prostate tumor growth in vivo was next examined using s.c. implanted PC3M cells in nude mice. To verify the sequence specificity of the antisense effects on in vivo tumor growth, we used three antisense ODNs that differed in sequence or in chemical modification: a PS-ODN, directed against codons 8–13 of human RI (16)
; the immunosuppressive (25)
, less cytotoxic (26)
, and second-generation RNA/DNA MBO PS-ODN (20)
; and a nonimmunostimulatory 5'-CCG-containing (27)
PS-ODN antisense, targeted to codons 8–13 of mouse RI, which can cross-hybridize with human RI (16)
. As shown in Fig. 5
, all three antisense ODNs markedly inhibited growth. By day 9, tumor growth was inhibited by 60–90%, compared with untreated control tumors, and the antisense RNA/DNA MBO ODN exhibited the most potency (Fig. 5B)
. By comparison, the scrambled sequence control ODN produced no effect. Compared with untreated (saline-injected) control tumors, the weights of treated tumors after 4 days of treatment were 42, 63, 56, and 98% for antisense RNA/DNA MBO ODN, PS-ODN, mouse PS-ODN, and control ODN, respectively (Fig. 5A)
. There was no sign of systematic toxicity in ODN-treated animals, and the sizes of the liver and spleen remained unchanged.
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and Up-Regulation of RIIß in Tumors in Vivo. expression. Western blotting demonstrated sharp down-regulation of RI protein levels in antisense-treated tumors, whereas random sequence ODN had no effect on RI levels (Fig. 6A)
. Moreover, RI antisense ODN, but not the control, increased RIIß protein levels (Fig. 6A)
. The host livers contained a low level of RI and high levels of RIIß, and RI antisense had no appreciable effect on any of the R and C subunits of PKA (Fig. 6B)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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regulatory subunit of protein kinase A. This antisense inhibition of cell growth correlated with the down-regulation of RI and the up-regulation of RIIß, at both the mRNA and protein levels, without affecting the RII or C subunits of protein kinase A. Thus, the increased expression of the differentiation-inducible RIIß (28
, 29)
, which was not detected in the untreated control cells, compensated for the loss of RI resulting from antisense treatment as was shown previously in other cancer cells (16
, 17
, 20
, 30)
. Despite the often compensatory up-regulation of RIIß, the PKA activity ratio, which measures the degree of PKA activation, increased in RI antisense-treated cells (Table 1)
. This might have occurred because the rate of RI loss exceeded that of RIIß increase and because the C subunit was rescued from rapid proteolysis by the continued up-regulation of RIIß in the cell. In fact, in such cells as LS-174T colon carcinoma and LNCaP prostate cancer cells, in which both PKA-I and PKA-II were expressed, the compensatory up-regulation of RIIß occurred on RI antisense treatment, and the C subunit was stable (20)
. However, in HCT-15 multidrug-resistant colon cancer cells, in which PKA-I is primarily expressed, antisense-directed loss of RI was not balanced by an increase in RIIß, and C subunit degradation increased because of a decreased half-life (20)
. Thus, the RI down-regulation, the up-regulation of RIIß, and the PKA activation show strong target specificity of RI antisense and the biochemical adaptation of the cell to the loss of RI.
Demonstration of such specificity is usually considered to be the most important criterion for determining the true antisense mechanism underlying the biological hallmarks, such as effects on cell growth, cell death, and cell differentiation (16
, 31
, 32) . Importantly, our results show that the target specificity of RI antisense extended beyond that of direct target down-regulation, leading to the effect on the regulation of the cAMP signaling cascade. The antisense-induced activation of PKA (Table 1)
led to an induction of PDE IV (Fig. 2B)
, a cAMP-responsive enzyme that contains CRE enhancer (33)
. Thus, the loss of RI resulted in activation of cAMP signaling through activation of PKA, bypassing adenylate cyclase/cAMP. This was shown previously in other cancer cells (20)
. We have interpreted these results in the context of CRE-directed transcription. PKA is known to activate the transactivation activity of CREB (34)
by phosphorylating Ser 133 (35)
. On the other hand, it has been shown that the phosphorylation of CREB at Ser 133 is also critical for growth factor induction of c-fos transcription (36
, 37)
. Thus, in transformed cells, the growth factor-mediated phosphorylation of CREB may supersede that mediated by PKA and stimulate cell growth. On RI antisense treatment, the activated PKA (free C subunit) may cause a switch in the mechanism of CREB phosphorylation from one mediated by growth factors to one mediated by PKA. This would inhibit the growth factor signals that stimulate cell proliferation and, thus, promote growth inhibition, consistent with a possible mechanism for the RI antisense inhibition of tumor cell growth.
Antisense RI not only blocked cell proliferation but also promoted apoptosis in PC3M cells. It has been shown that inactivation of the antiapoptotic function of Bcl-2 by phosphorylation occurs after treatment with the microtubule-damaging anticancer drug, paclitaxel, or the phosphatase inhibitor okadaic acid (23)
. It has also been shown that PKA is involved in Bcl-2 hyperphosphorylation and apoptosis induced by microtubule-damaging drugs (23)
. Our present study shows that an antisense ODN targeted against the RI subunit of PKA induces Bcl-2 hyperphosphorylation and apoptosis (Fig. 3, B and C)
. These results suggest that the activated PKA (free C subunit) arising from the antisense-mediated depletion of RI may be responsible for the Bcl-2 hyperphosphorylation observed in the antisense-treated cells.
It has been shown previously that Bcl-2 is phosphorylated on serine residues, and of the 17 serine residues present in Bcl-2, several could be the potential sites of phosphorylation by different kinases (38 , 39) . Recently, it has been shown that angiotensin type 2 receptor binding dephosphorylates Bcl-2 by activating the mitogen-activated protein kinase phosphatase 1 and induces apoptosis in PC12 cells (40) . Furthermore, interleukin-3, erythropoietin, or the protein kinase C activator byrostatin-1 hyperphosphorylates Bcl-2 and suppress apoptosis in murine interleukin-3-dependent National Science Foundation/N1.H7 cells (41 , 42) . These studies show that Bcl-2 phosphorylation involves multiple kinases and that specific sites of phosphorylation may determine whether Bcl-2 loses or gains its antiapoptotic function. Bcl-2 phosphorylation by mitogen-activated protein kinase and protein kinase C may promote antiapoptotic activity of Bcl-2, leading to suppression of apoptosis, whereas Bcl-2 phosphorylation by PKA may suppress Bcl-2 function, leading to promotion of apoptosis.
In accordance with this, our present data show that the antisense-induced Bcl-2 hyperphosphorylation occurs along with the up-regulation of the proapoptotic proteins Bax, Bak, and Bad and with the hypophosphorylation of Bad (Fig. 4)
. Although the mode by which Bcl-2 affects the process of cell death is not fully understood, recent studies indicate that the Bcl-2 protein binds to other proteins with which it has amino acid sequence homology, including Bax, Bcl-Xl, Bcl-Xs, Mc1–1, Bak, Bik, and Bad (43, 44, 45)
. The functional significance of many of these Bcl-2 family protein-protein interactions remains unclear. However, the heterodimerization of Bcl-2 with Bax appears crucial in preventing Bax-mediated apoptosis (44)
; hyperphosphorylated Bcl-2 is less able to form heterodimers with Bax (23
, 46)
. On the other hand, the phosphorylated Bad releases Bcl-2 from mitochondria to the cytoplasm leading to a Bcl-2-promoted cell survival pathway (24)
. Thus, dephosphorylated Bad remains bound with Bcl-2 in the mitochondria, preventing the Bcl-2 promotion of cell survival. Our present data about RI antisense promotion of Bcl-2 hyperphosphorylation, along with up-regulation of Bax and hypophosphorylation of Bad, clearly favor apoptosis over cell survival.
The results observed for three RI antisense ODNs that differ in sequence or chemical modification show highly sequence-specific antisense effects on the inhibition of in vivo tumor growth. The RNA/DNA MBO antisense was the most potent, and the human RI antisense PS-ODN exhibited the least potency in growth inhibition. RI antisense inhibition of tumor growth accompanied the target specificity of the antisense illustrated by RI down-regulation and the compensatory up-regulation of RIIß. However, in host livers, which express very low levels of RI and high levels of RIIß currently beyond the experimental limits of detection, antisense treatment brought about no appreciable changes in these protein levels. One possible explanation for these differential effects between tumor and liver is the different basal levels of RI and RIIß in these tissues. A second explanation would be the difference in the half-lives of RI and RIIß, both at the mRNA and protein level. Finally, the tissue specificity between tumor and liver, with regard to ODN uptake, pharmacokinetics, stability, and tissue retention time, may be another factor. Nevertheless, the differential effects of antisense RI observed between tumors and host livers are striking and may have important implications for the clinical development of this antisense.
Our present study, which demonstrates target-specific and sequence-specific growth inhibitory effect by antisense RI, clearly supports a use for this antisense ODN in combating the androgen-independent growth of prostate cancer and other cancers in which expression of the protein kinase A RI is increased.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at National Cancer Institute, Building 10, Room 5B05, 9000 Rockville Pike, Bethesda, MD 20892-1750. Phone: (301) 496-4020; Fax: (301) 496-2443; E-mail: chochung{at}helix.nih.gov 
2 The abbreviations used are: CRE, cyclic AMP responsive element; cAMP, cyclic AMP; PKA, cyclic AMP-dependent protein kinase; ODN, oligodeoxynucleotide; PS-ODN, phosphorothioate oligodeoxynucleotide; MBO, mixed backbone; PDE IV, phosphodiesterase IV; CREB, cyclic AMP response element binding protein.
Received 4/18/01; revised 10/ 2/01; accepted 10/ 9/01.
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) or cells treated with RNA/DNA MBO antisense ODN (AS ODN; •) or mismatched ODN (Control ODN; 
) for 2 days at the indicated concentrations. B, time dependence. Cells were treated with ODNs as in A, at a concentration of 200 nM for the indicated times. In C, parental nontransfectants (
, PS-ODN, at (
) mouse PS-ODN was injected daily into mice (see "Materials and Methods"). A, tumor weights determined on day 5 and tumor volumes measured daily 
day 9 (B; see "Materials and Methods"). Data represent average values ± SD of five tumors per group.