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Inhibition of Anchorage-Independent Growth ofras-Transformed Cells on PolyHEMA Surface by Antisense Oligodeoxynucleotides Directed Against K-ras
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
231, 735–737 (1997)
RC976179
Inhibition of Anchorage-Independent Growth of rasTransformed Cells on PolyHEMA Surface by Antisense Oligodeoxynucleotides Directed Against K-ras Manabu Kawada, Hidesuke Fukazawa, Satoshi Mizuno, and Yoshimasa Uehara1 Department of Bioactive Molecules, National Institute of Health, 1-23-1 Toyama, Shinjuku-Ku, Tokyo 162, Japan
Received January 20, 1997
We examined the effect of antisense oligodeoxynucleotides (AS ODNs) directed against v-Ki-ras on the anchorage-independent growth of v-Ki-ras-transformed rat fibroblasts using plates coated with an antiadhesive polymer, poly(2-hydroxyethyl methacrylate) (polyHEMA). Among AS ODNs tested, 17mer AS ODN centered around codon 12 of v-Ki-ras inhibited the vKi-Ras expression and v-Ki-Ras-induced anchorage-independent growth, while its sense sequence did not affect it. Using polyHEMA plates, the direct addition of AS ODNs to the cells growing anchorage-independently and various treatment schedules of AS ODNs are now available to identify the most desirable conditions for AS ODNs treatment. q 1997 Academic Press
Growth of oncogenic transformed cells is anchorageindependent: the cells can proliferate without substrate attachment. Their growth ability correlates well with their tumorigenicity (1, 2). Thus, inhibition of the anchorage-independent growth should be an effective strategy for cancer chemotherapy. While antisense oligodeoxynucleotides (AS ODNs) have been examined for their potential as target-specific anti-cancer drugs (3, 4), it has been necessary to pretreat cells with AS ODNs before the culture was put into soft-agar or methylcellulose-based gel to analyze the effect on anchorage-independent growth (5, 6). Thus, it has been difficult to add AS ODNs directly to cells growing anchorage-independently. We have recently developed a method to quantitate anchorage-independent growth using plates coated with an antiadhesive polymer, polyHEMA (7), and in this study describe the efficacy of polyHEMA plate in evaluating the effect of AS ODNs effect on this growth. 1 To whom correspondence should be addressed. Fax: 81-3-52851150; E-mail: [email protected].
MATERIALS AND METHODS Cell culture. pMAM-ras-REF is a cloned Fischer rat embryo fibroblast cell line transformed with pMAMneo-v-Ki-ras, a plasmid in which v-Ki-ras is linked to the dexamethasone (Dex)-inducible MMTV-LTR promoter (7). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg/ml kanamycin at 377C with 5% CO2 . PolyHEMA-coated plates were prepared as described (7, 8). Phosphorothioate oligodeoxynucleotides. Antisense phosphorothioate oligodeoxynucleotides (PS-ODNs) directed against the initiation codon or codon 12 of v-Ki-ras mRNA were designed using published v-Ki-ras sequence (9) and were purchased from Takara Biomedicals (Japan). The sequences of three antisense PS-ODNs used in this study are shown in Fig. 1. The corresponding sense (S) sequences were used as controls. Cell growth. Cells (51103) were cultured in polyHEMA-coated 96-well plates with or without Dex for 4 days. The cell growth was determined using 3-[4,5-dimethylthiazoil-2yl]-2,5-diphenyl-tetrazolium bromide (MTT) as described previously (7). Lipofectin (GIBCOBRL) was used according to the recommended method at a ratio of PS-ODNs (1 mM)/Lipofectin (5 ml/ml). Western blotting. Cells (2.51104) were cultured in polyHEMAcoated 24-well plates with or without Dex for 3 days. They were washed twice with ice-cold PBS containing 100 mM Na3VO4 and then lysed in a lysis buffer (20 mM Hepes [pH 7.5], 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 50 mM NaF, 50 mM bglycerophosphate, 1 mM Na3VO4 , and 25 mg/ml each of antipain, leupeptin, and pepstatin). Equal protein extracts were separated by SDS-PAGE, and transferred onto Immobilon PVDF membrane (Millipore); the membrane was then blotted with anti-Ras (Ras10; Oncogene Science), anti-tubulin (Oncogene Science), or anti-actin (Amersham) antibody. Enhanced chemiluminescence (Amersham) was used to visualize the immunoblot signals.
RESULTS AND DISCUSSION In the absence of Dex pMAM-ras-REF cells were unable to grow on polyHEMA surface, but the expression of v-Ki-Ras following the addition of Dex induced the anchorage-independent growth (Fig. 2). Using 0.01 mM Dex to induce v-Ki-Ras, we examined the effect of AS ODNs directed against v-Ki-ras on this growth. The expression of mutated Ha-Ras reportedly can be suppressed selectively by AS ODNs directed against the
735
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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691c$$1021
02-07-97 01:17:31
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Vol. 231, No. 3, 1997
FIG. 1. v-Ki-ras.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sequences of antisense PS-ODNs directed against
region of the Ha-ras mRNA carrying a point mutation in codon 12 (10, 11). We used antisense phosphorothioate oligodeoxynucleotides (AS PS-ODNs), which were nuclease-resistant (3, 12), targeting codon 12 of the vKi-ras sequence as well as the initiation codon (Fig. 1). Among the PS-ODNs examined, antisense 17mer PS-ODN centered around codon 12 (Ser17mer-AS) inhibited the v-Ki-Ras-induced anchorage-independent growth dose-dependently with an IC50 of 4 mM, while its sense sequence (Ser17mer-S) did not affect it (Fig. 3). The most desirable effect of Ser17mer PS-ODNs was observed when the initial dose was added on day 0, and then second and third doses (50% of the initial dose) were added on day 1 and day 2 (/1/1/2/1/2). Furthermore, Ser17mer-AS (/1/1/2/1/2), but not Ser17mer-S, inhibited the Dex-induced v-Ki-Ras expression dose-dependently, while they did not inhibit tubulin expression used as an internal control (Fig. 4). When the PS-ODNs were added daily at the same dose (/1/1/1), Ser17mer-AS (/1/1/1) inhibited the v-KiRas expression without change of actin more strongly than Ser17mer-AS (/1/1/2/1/2) (Fig. 4). However, its sense sequence Ser17mer-S (/1/1/1) showed a nonsequence-specific growth inhibiting effect (Fig. 3). These results indicated that Ser17mer-AS inhibited
FIG. 2. Anchorage-independent growth induced by v-Ki-Ras. pMAM-ras-REF cells were cultured in polyHEMA-coated plates for 4 days with the indicated concentrations of Dex. Induction of v-KiRas expression by Dex was determined by Western blotting.
FIG. 3. Effect of PS-ODNs directed against v-Ki-ras on anchorage-independent growth induced by v-Ki-Ras. pMAM-ras-REF cells were cultured in polyHEMA-coated 96-well plates in the presence of 0.01 mM Dex for 4 days. Antisense (l) and sense (s) PS-ODNs were added as follows: the indicated dose was added on day 0 and second and third doses (50% of the initial dose) were added on day 1 and day 2 (/1/1/2/1/2); the indicated dose was added daily three times (/1/1/1); the indicated dose was added once with Lipofectin on day 0 (Lipofectin).
the v-Ki-Ras-induced anchorage-independent growth by inhibiting v-Ki-Ras expression. While Lipofectin has been used to deliver PS-ODNs into the cells (12), in this cell line it enhanced the non-sequence-specific effect of PS-ODNs (Fig. 3). Ser15mer-AS also inhibited the v-Ki-Ras-induced anchorage-independent growth (Fig. 3), but Ser15merAS failed to inhibit the v-Ki-Ras expression (Fig. 4), even though it was only 2mer shorter than Ser17mer-
FIG. 4. Effect of PS-ODNs on v-Ki-Ras expression. pMAM-rasREF cells were cultured in polyHEMA-coated plates with 0.01 mM Dex for 3 days. PS-ODNs were added as described in Fig. 3. The cells were lysed and v-Ki-Ras expression was determined by Western blotting. Anti-tubulin or anti-actin antibody was used as an internal control.
736
AID
BBRC 6179
/
691c$$1021
02-07-97 01:17:31
bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AS. Thus, Ser15mer-AS inhibited the growth non-sequence-specifically. It is possible that the length of 15mer is not enough to make a stable complex with vKi-ras mRNA. Although the mechanism of the nonsequence-specific effect of the PS-ODNs used in this study is unknown, many papers have reported the nonsequence-specific effect of ODNs (3, 13, 14). Furthermore, antisense PS-ODNs directed against the initiation codon (AUG-AS) did not inhibit the anchorageindependent growth (Fig. 3). Thus, it is suggested that codon 12 of v-Ki-ras mRNA is a sensitive region for antisense ODNs as is Ha-ras mRNA (10, 11). These results have shown that polyHEMA-coated plates offer the following advantages to analyze the effect of AS ODNs on anchorage-independent growth: i) there is no need to pretreat the cells with AS ODNs before culture; and ii) various treatment schedules can be easily tested to find desirable conditions for AS ODNs. ACKNOWLEDGMENTS We thank Dr. T. Maekawa (Dept. Blood Transfusion, Inst. Medical Sci., Tokyo Univ.) for helpful advice and Dr. Y. Murakami for helpful discussion. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan, and by the Social Insurance Agency Contract Fund of the Japan Health Sciences Foundation.
REFERENCES 1. Macpherson, I., and Montagnier, L. (1964) Virology 23, 291– 294. 2. Shin, S., Freedman, V. H., Risser, R., and Pollack, R. (1975) Proc. Natl. Acad. Sci. USA 72, 4435–4439. 3. Stein, C. A., and Cheng, Y.-C. (1993) Science 261, 1004–1012. 4. Nesterova, M., and Cho-Chung, Y. S. (1995) Nature Med. 1, 528– 533. 5. Skorski, T., Kanakaraj, P., Ku, D.-H., Nieborowska-Skorska, M., Canaani, E., Zon, G., Perussia, B., and Calabretta, B. (1994) J. Exp. Med. 179, 1855–1865. 6. Kimura, S., Maekawa, T., Hirakawa, K., Murakami, A., and Abe, T. (1995) Cancer Res. 55, 1379–1384. 7. Fukazawa, H., Mizuno, S., and Uehara, Y. (1995) Anal. Biochem. 228, 83–90. 8. Folkman, J., and Moscona, A. (1978) Nature 273, 345–349. 9. Tsuchida, N., Ryder, T., and Ohtsubo, E. (1982) Science 217, 937–939. 10. Saison-Behmoaras, T., Tocque´, B., Rey, I., Chassignol, M., Thuong, N. T., and He´le`ne, C. (1991) EMBO J. 10, 1111–1118. 11. Monia, B. P., Johnston, J. F., Ecker, D. J., Zounes, M. A., Lima, W. F., and Freier, S. M. (1992) J. Biol. Chem. 267, 19954–19962. 12. Wagner, R. W. (1994) Nature 372, 333–335. 13. Barton, C. M., and Lemoine, N. R. (1995) Br. J. Cancer 71, 429– 437. 14. Carter, G., Gilbert, C., and Lemoine, N. R. (1995) Int. J. Oncology 6, 1105–1112.
737
AID
BBRC 6179
/
691c$$1021
02-07-97 01:17:31
bbrcg
AP: BBRC
231, 735–737 (1997)
RC976179
Inhibition of Anchorage-Independent Growth of rasTransformed Cells on PolyHEMA Surface by Antisense Oligodeoxynucleotides Directed Against K-ras Manabu Kawada, Hidesuke Fukazawa, Satoshi Mizuno, and Yoshimasa Uehara1 Department of Bioactive Molecules, National Institute of Health, 1-23-1 Toyama, Shinjuku-Ku, Tokyo 162, Japan
Received January 20, 1997
We examined the effect of antisense oligodeoxynucleotides (AS ODNs) directed against v-Ki-ras on the anchorage-independent growth of v-Ki-ras-transformed rat fibroblasts using plates coated with an antiadhesive polymer, poly(2-hydroxyethyl methacrylate) (polyHEMA). Among AS ODNs tested, 17mer AS ODN centered around codon 12 of v-Ki-ras inhibited the vKi-Ras expression and v-Ki-Ras-induced anchorage-independent growth, while its sense sequence did not affect it. Using polyHEMA plates, the direct addition of AS ODNs to the cells growing anchorage-independently and various treatment schedules of AS ODNs are now available to identify the most desirable conditions for AS ODNs treatment. q 1997 Academic Press
Growth of oncogenic transformed cells is anchorageindependent: the cells can proliferate without substrate attachment. Their growth ability correlates well with their tumorigenicity (1, 2). Thus, inhibition of the anchorage-independent growth should be an effective strategy for cancer chemotherapy. While antisense oligodeoxynucleotides (AS ODNs) have been examined for their potential as target-specific anti-cancer drugs (3, 4), it has been necessary to pretreat cells with AS ODNs before the culture was put into soft-agar or methylcellulose-based gel to analyze the effect on anchorage-independent growth (5, 6). Thus, it has been difficult to add AS ODNs directly to cells growing anchorage-independently. We have recently developed a method to quantitate anchorage-independent growth using plates coated with an antiadhesive polymer, polyHEMA (7), and in this study describe the efficacy of polyHEMA plate in evaluating the effect of AS ODNs effect on this growth. 1 To whom correspondence should be addressed. Fax: 81-3-52851150; E-mail: [email protected].
MATERIALS AND METHODS Cell culture. pMAM-ras-REF is a cloned Fischer rat embryo fibroblast cell line transformed with pMAMneo-v-Ki-ras, a plasmid in which v-Ki-ras is linked to the dexamethasone (Dex)-inducible MMTV-LTR promoter (7). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg/ml kanamycin at 377C with 5% CO2 . PolyHEMA-coated plates were prepared as described (7, 8). Phosphorothioate oligodeoxynucleotides. Antisense phosphorothioate oligodeoxynucleotides (PS-ODNs) directed against the initiation codon or codon 12 of v-Ki-ras mRNA were designed using published v-Ki-ras sequence (9) and were purchased from Takara Biomedicals (Japan). The sequences of three antisense PS-ODNs used in this study are shown in Fig. 1. The corresponding sense (S) sequences were used as controls. Cell growth. Cells (51103) were cultured in polyHEMA-coated 96-well plates with or without Dex for 4 days. The cell growth was determined using 3-[4,5-dimethylthiazoil-2yl]-2,5-diphenyl-tetrazolium bromide (MTT) as described previously (7). Lipofectin (GIBCOBRL) was used according to the recommended method at a ratio of PS-ODNs (1 mM)/Lipofectin (5 ml/ml). Western blotting. Cells (2.51104) were cultured in polyHEMAcoated 24-well plates with or without Dex for 3 days. They were washed twice with ice-cold PBS containing 100 mM Na3VO4 and then lysed in a lysis buffer (20 mM Hepes [pH 7.5], 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 50 mM NaF, 50 mM bglycerophosphate, 1 mM Na3VO4 , and 25 mg/ml each of antipain, leupeptin, and pepstatin). Equal protein extracts were separated by SDS-PAGE, and transferred onto Immobilon PVDF membrane (Millipore); the membrane was then blotted with anti-Ras (Ras10; Oncogene Science), anti-tubulin (Oncogene Science), or anti-actin (Amersham) antibody. Enhanced chemiluminescence (Amersham) was used to visualize the immunoblot signals.
RESULTS AND DISCUSSION In the absence of Dex pMAM-ras-REF cells were unable to grow on polyHEMA surface, but the expression of v-Ki-Ras following the addition of Dex induced the anchorage-independent growth (Fig. 2). Using 0.01 mM Dex to induce v-Ki-Ras, we examined the effect of AS ODNs directed against v-Ki-ras on this growth. The expression of mutated Ha-Ras reportedly can be suppressed selectively by AS ODNs directed against the
735
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 6179
/
691c$$1021
02-07-97 01:17:31
bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
FIG. 1. v-Ki-ras.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sequences of antisense PS-ODNs directed against
region of the Ha-ras mRNA carrying a point mutation in codon 12 (10, 11). We used antisense phosphorothioate oligodeoxynucleotides (AS PS-ODNs), which were nuclease-resistant (3, 12), targeting codon 12 of the vKi-ras sequence as well as the initiation codon (Fig. 1). Among the PS-ODNs examined, antisense 17mer PS-ODN centered around codon 12 (Ser17mer-AS) inhibited the v-Ki-Ras-induced anchorage-independent growth dose-dependently with an IC50 of 4 mM, while its sense sequence (Ser17mer-S) did not affect it (Fig. 3). The most desirable effect of Ser17mer PS-ODNs was observed when the initial dose was added on day 0, and then second and third doses (50% of the initial dose) were added on day 1 and day 2 (/1/1/2/1/2). Furthermore, Ser17mer-AS (/1/1/2/1/2), but not Ser17mer-S, inhibited the Dex-induced v-Ki-Ras expression dose-dependently, while they did not inhibit tubulin expression used as an internal control (Fig. 4). When the PS-ODNs were added daily at the same dose (/1/1/1), Ser17mer-AS (/1/1/1) inhibited the v-KiRas expression without change of actin more strongly than Ser17mer-AS (/1/1/2/1/2) (Fig. 4). However, its sense sequence Ser17mer-S (/1/1/1) showed a nonsequence-specific growth inhibiting effect (Fig. 3). These results indicated that Ser17mer-AS inhibited
FIG. 2. Anchorage-independent growth induced by v-Ki-Ras. pMAM-ras-REF cells were cultured in polyHEMA-coated plates for 4 days with the indicated concentrations of Dex. Induction of v-KiRas expression by Dex was determined by Western blotting.
FIG. 3. Effect of PS-ODNs directed against v-Ki-ras on anchorage-independent growth induced by v-Ki-Ras. pMAM-ras-REF cells were cultured in polyHEMA-coated 96-well plates in the presence of 0.01 mM Dex for 4 days. Antisense (l) and sense (s) PS-ODNs were added as follows: the indicated dose was added on day 0 and second and third doses (50% of the initial dose) were added on day 1 and day 2 (/1/1/2/1/2); the indicated dose was added daily three times (/1/1/1); the indicated dose was added once with Lipofectin on day 0 (Lipofectin).
the v-Ki-Ras-induced anchorage-independent growth by inhibiting v-Ki-Ras expression. While Lipofectin has been used to deliver PS-ODNs into the cells (12), in this cell line it enhanced the non-sequence-specific effect of PS-ODNs (Fig. 3). Ser15mer-AS also inhibited the v-Ki-Ras-induced anchorage-independent growth (Fig. 3), but Ser15merAS failed to inhibit the v-Ki-Ras expression (Fig. 4), even though it was only 2mer shorter than Ser17mer-
FIG. 4. Effect of PS-ODNs on v-Ki-Ras expression. pMAM-rasREF cells were cultured in polyHEMA-coated plates with 0.01 mM Dex for 3 days. PS-ODNs were added as described in Fig. 3. The cells were lysed and v-Ki-Ras expression was determined by Western blotting. Anti-tubulin or anti-actin antibody was used as an internal control.
736
AID
BBRC 6179
/
691c$$1021
02-07-97 01:17:31
bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AS. Thus, Ser15mer-AS inhibited the growth non-sequence-specifically. It is possible that the length of 15mer is not enough to make a stable complex with vKi-ras mRNA. Although the mechanism of the nonsequence-specific effect of the PS-ODNs used in this study is unknown, many papers have reported the nonsequence-specific effect of ODNs (3, 13, 14). Furthermore, antisense PS-ODNs directed against the initiation codon (AUG-AS) did not inhibit the anchorageindependent growth (Fig. 3). Thus, it is suggested that codon 12 of v-Ki-ras mRNA is a sensitive region for antisense ODNs as is Ha-ras mRNA (10, 11). These results have shown that polyHEMA-coated plates offer the following advantages to analyze the effect of AS ODNs on anchorage-independent growth: i) there is no need to pretreat the cells with AS ODNs before culture; and ii) various treatment schedules can be easily tested to find desirable conditions for AS ODNs. ACKNOWLEDGMENTS We thank Dr. T. Maekawa (Dept. Blood Transfusion, Inst. Medical Sci., Tokyo Univ.) for helpful advice and Dr. Y. Murakami for helpful discussion. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan, and by the Social Insurance Agency Contract Fund of the Japan Health Sciences Foundation.
REFERENCES 1. Macpherson, I., and Montagnier, L. (1964) Virology 23, 291– 294. 2. Shin, S., Freedman, V. H., Risser, R., and Pollack, R. (1975) Proc. Natl. Acad. Sci. USA 72, 4435–4439. 3. Stein, C. A., and Cheng, Y.-C. (1993) Science 261, 1004–1012. 4. Nesterova, M., and Cho-Chung, Y. S. (1995) Nature Med. 1, 528– 533. 5. Skorski, T., Kanakaraj, P., Ku, D.-H., Nieborowska-Skorska, M., Canaani, E., Zon, G., Perussia, B., and Calabretta, B. (1994) J. Exp. Med. 179, 1855–1865. 6. Kimura, S., Maekawa, T., Hirakawa, K., Murakami, A., and Abe, T. (1995) Cancer Res. 55, 1379–1384. 7. Fukazawa, H., Mizuno, S., and Uehara, Y. (1995) Anal. Biochem. 228, 83–90. 8. Folkman, J., and Moscona, A. (1978) Nature 273, 345–349. 9. Tsuchida, N., Ryder, T., and Ohtsubo, E. (1982) Science 217, 937–939. 10. Saison-Behmoaras, T., Tocque´, B., Rey, I., Chassignol, M., Thuong, N. T., and He´le`ne, C. (1991) EMBO J. 10, 1111–1118. 11. Monia, B. P., Johnston, J. F., Ecker, D. J., Zounes, M. A., Lima, W. F., and Freier, S. M. (1992) J. Biol. Chem. 267, 19954–19962. 12. Wagner, R. W. (1994) Nature 372, 333–335. 13. Barton, C. M., and Lemoine, N. R. (1995) Br. J. Cancer 71, 429– 437. 14. Carter, G., Gilbert, C., and Lemoine, N. R. (1995) Int. J. Oncology 6, 1105–1112.
737
AID
BBRC 6179
/
691c$$1021
02-07-97 01:17:31
bbrcg
AP: BBRC