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EFFECT OF ANTISENSE hTERT mRNA OLIGODEOXYNUCLEOTIDE ON TELOMERASE ACTIVITY OF LEUKEMIC CELLS
Cell Biology International 2002, Vol. 26, No. 5, 427–431 doi:10.1006/cbir.2002.0875, available online at http://www.idealibrary.com on
EFFECT OF ANTISENSE hTERT mRNA OLIGODEOXYNUCLEOTIDE ON TELOMERASE ACTIVITY OF LEUKEMIC CELLS Y. ZHANG* and D. M. HE Institute of Hematology, Medical College of Jinan University, Guangzhou 510632, P. R. China Received 3 October 2001; accepted 15 February 2002
The effect of antisense hTERT mRNA oligodeoxynucleotide on telomerase activity of leukemia cells was explored and investigated in the present study. Telomerase activity was measured by the telomerase PCR ELISA assay kit (TRAP); hTERT mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) assay and gel-image system, hTERT protein by immunochemistry and flowcytometry. Results showed Incubation of leukemic cells (HL-60 and K562 cell lines) with 10 mol/l AS PS-ODN would significantly reduce the their mRNA levels and in vitro expression of hTERT protein 24 h later, so that the telomerase activity would be significantly down-regulated or inhibited. In conlusion, the hTERT AS PS-ODN is an excellent 2002 Published by Elsevier Science Ltd. inhibitor for telomerase activity. K: antisense; telomerase; leukemia; hTERT (hTERT, human telomerase reverse transcriptase).
INTRODUCTION Recently it was shown that 70–80% tumors, including hematological malignant tumors and leukemia, show very high levels of expression of telomerase, with a few exceptions, normal cells do not (Kim and Piatyszek, 1994). Telomerase is the ribonucleoprotein enzyme complex that elongates telomeric DNA (TTAGGG)n and now is thought to be an important step in the development of cellular immortality and oncogenesis. The most recent and significant advances in this area show that the inhibition or downregulation of telomerase activity would prevent from the malignant proliferation of tumor cells (Feng and Funk, 1995; Meyerson et al., 1997). So that it is interesting research for us to look for inhibiting material of telomerase. Antisense (AS) oligonuleotides in suppressing the expression of certain genes is attractive and have be used to inhibit the telomerase activity by antisense to RNA component of telomerase (Tahara et al., 1995). Human telomerase reverse *To whom correspondence should be addressed: Y. Zhang, Institute of Hematology, Medical College of Jinan University, Guangzhou 510632, P. R. China. Tel: 86-20-8522-6476; Fax: 86-20-8522-1343; E-mail: [email protected] Research was supported from grant of National Natural Science Foundation of China (No. 39870361). 1065–6995/02/$-see front matter
transcriptase (hTERT) is key component of telomerase catalytic activity, evidently, which would be key target attached by the anticancer drug (Takaishi et al., 2000; Ohata et al., 1997; Kavaler et al., 1998; Hoos et al., 1998; Mokbel et al., 1999; Blasco et al., 1997). In the present research, we had shown that incubation of leukemic cells with AS oligonucleotides from hTERT mRNA start site would significantly reduce the in vitro expression of hTERT protein and mRNA levels of HL-60 and K562 leukemic cells, so that their telomerase activity would be significantly down-regulated or inhibited.
MATERIALS AND METHODS Design and synthesis of antisense phosphorothiate oligomers Based on the hTERT gene cDNA sequence, 20 antisense oligomers were synthesized, purified and modified by Shanghai Institute of Biochemistry, from upstream 6-base and downstream 11-base at start code; the antisense phosphorothiate oligodeoxynucleotide sequence (AS PS-ODN) is 5 GGAGCGCGCGGCATCGCGGG-3 , the sense 2002 Published by Elsevier Science Ltd.
428
phosphorothiate oligodeoxynucleotide sequence (S PS-SODN) is 5 -CCCGCGATGCCGCGCGCT CC-3 as a control. Through the Blask soft examination from the Internet, there is no homologue for the antisense sequence with other genes except hTERT cDNA. Leukemic cell lines Leukemic cell lines (HL-60 and K562 cell lines) were incubated in the RPMI-1640 medium supplemented with 5% new born bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin and under the conditions of 5% CO2 in air, saturation humidity and 37C with 24-well plastic culture plate, each well of which contained 1105/ml. Sub-culture was performed 3–4 days later and the cells in logarithm growth phrase were used in all experiments. The experiments were divided into three groups of AS PS-ODN, S PS-SODN and normal control groups, each group consisted of repeat three wells on 24-well plate and oligomer was added into wells at last concentration of 10 mol/l, normal control group with culture medium of same volume. At 12, 24, 48, 72 h later for incubation, the cell telomerase activity was measured. Telomerase activity assays Telomerase activity was measured by the telomerase PCR ELISA assay kit (Boehringer Mannheim). Briefly, the cell extract was prepared at different time of oligomers treatment. The positive and negative control group were established in each experiment; cell extract was heated to 65C for 10 min as a negative control, and extract of 293 cell line having telomerase activity as a positive control (from kit). Telomeric repeat amplification protocol (TRAP reaction): (1) primer elongation: transfer 25 l reaction mixture into a tube suitable for PCR amplification, then add 2 l cell extract and sterile water to a final volume of 50 l; transfer the tubes to thermal cycler and perform one cycle at 25C for 30 min; (2) telomerase inactivation: perform one cycle at 94C for 5 min; (3) amplification: 50 l reaction mixture contains dNTP, Taq polymerase, the biotin-labled P1-TS primer and P2 primer. Perform 30 cycles for amplification, then at 94C for 30 s for denaturation, at 50C for 30 s for annealing, at 72C for 90 s for polymerization, then at 72C for 10 min for balance. Hybridization and ELASA procedure: transfer 20 l denaturation reagent into suitable tube, then add 5 l amplification product into it and incubate
Cell Biology International, Vol. 26, No. 5, 2002
at 20C for 10 min, add 225 l hybridization buffer (containing DIG-POD) and mix thoroughly by vortexing briefly. Transfer 100 l mixture on the nuclease-free microtiter plate and shake it at 37C for 2 h. Add 100 l anti-DIG-POD at room temperature for 30 min. At last, add 100 l TMB substrate solution and incubate it for 10 min at room temperature for color development, and then add 100 l stop reagent to stop the reaction. We determine the A (Absorbance) value of wavelength of 450–655 nm to calculate A=A450–A655. Detection of hTERT mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) assay Total RNA was extracted from oligomer-treatment cells with Trizol RNA kit (Life Technologies, U.S.A.). The hTERT cDNA was synthesized with 1 l cell extract, 200 U MMLV reverse transcriptase (Gibco), 6 oligomers of TTAGGG repeats. The primers for hTERT gene to be amplified are that hTERT upstream primer is 5 -CGG AAGAGTGTCTGGAGCAA-3 , hTERT downstream primer is GGATGAAGCGGAGTCTG GA-3 (Shanghai Institute of Biochemistry); actin upstream primer: 5 -GTGGGGCGCCCC AGGCACCA-3 ; -actin downstream primer: 5 GTCCTTAATGTCACGCACGATTTC-3 (-actin primer from Gibco). The conditions of PCR were: 90C 30 s for denaturation; 60C 1 min for annealing; 72C 1 min for extension, 30 cycles. PCR products were assayed by polyacrylamide gel electrophoresis and levels of bands on gel were measured with gel-image system. Determination of hTERT protein by flowcytometry Cells (5105) were collected and fixed with 70% formoldehyde at 4C for 15 min, then cell suspension was washed and resuspended for two times in specific PBS (s-PBS) (contained 1% human AB serum, 1% tween from Boehringer Mannheim, pH 7.2), which is able to perforate on cells surface so as to let antibody into cell inside easily. The hTERT protein antibody of 50 l was added into the cell suspension, which was incubated at 4C for 1 h. The cell suspension was washed two times with s-PBS and 50 l FITC-IgG of rabbit anti-goat was added and incubated at 4C for 30 min. At last, the cells were washed two times with PBS, and then hTERT protein was detected by flowcytometry. Statistical analysis The data were shown with xs, and processed with software of SPSS; one-way analysis of
Cell Biology International, Vol. 26, No. 5, 2002
429
Table 1. Effect of Antisense hTERT oligodeoxynucleotides on hTERT mRNA expression of leukemic cells of HL-60 and K562 (value in table is ratio of gel bands scanned with gel-image system) (xs) Time (h)
24 48
HL-60
K562
Control
S PS-ODN
AS PS-ODN
Control
S PS-ODN
AS PS-ODN
0.710.19 0.750.20
0.620.14 0.570.12
0.320.07* 0.080.02*
0.830.24 0.870.25
0.740.21 0.790.18
0.410.11* 0.230.05*
Abbreviations: S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. *Shown that P<0.01, the value of AS PS-ODN groups compared with the controls or S PS-ODN groups separately. There is no significant difference for the value of S PS-ODN groups compared with the controls separately.
Table 2. The average intensity of fluorescein from hTERT protein of HL-60 and K562 cells by flow cytometric analysis (xs) Time (h)
24 48 72
HL-60
K562
Control
S PS-ODN
AS PS-ODN
Control
S PS-ODN
AS PS-ODN
211.2434.32 303.4746.36 223.1537.50
193.7629.46 200.5228.54 217.1230.39
65.037.57* 52.415.07* 30.457.58*
247.4340.34 267.1942.86 348.4550.57
300.1444.78 380.6554.74 360.3250.58
90.466.57* 45.476.03* 34.745.98*
Abbreviations: S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. *Shown that P<0.01, the value of AS PS-ODN groups compared with the one of controls or S PS-ODN groups separately. There no significant difference for the value of S PS-ODN groups compared with the one controls separately.
variance was used to determine the likelihood of a significant difference (P<0.05). RESULTS Effect of AS PS-ODN on hTERT mRNA expression of leukemic cells of HL-60 and K562 The number of HL-60 or K562 cells did not decline after exposure to AS PS-ODN for 48 h. However, the expression level of cell hTERT mRNA was gradually decreased with time elongation, the ratio of density was only 0.08 for HL-60 and 0.23 for K562 at 48 h. It show that expression of hTERT mRNA was significantly downregulated or inhibited by AS PS-ODN; it was also observed the slight degression without the significant effect for S PS-ODN group.
sure to AS PS-ODN for 24, 48 and 72 h, which show that hTERT protein expression of HL-60 and K562 was gradually dropped down. The level of hTERT protein expression for K562 cells was about one-tenth at 72 h, and about one-seventh for HL-60 at 72 h. However, there exist a slight inhibition of S PS-ODN to protein expression of hTERT for HL-60, without this phenomena for K562. The effect of antisense oligonucleotides on hTERT gene protein expression was agreement with ones of them on mRNA. Effect of hTERT AS PS-ODN on telomerase activity of HL-60 and K562 leukemic cells. As shown in Figure 1 and Figure 2, telomerase activity of HL-60 and K562 was measured by the telomerase PCR ELISA assay kit.
Protein expression of hTERT gene
DISCUSSION
The average intensities of fluorescein from hTERT protein of HL-60 and K562 cells by flow cytometric analysis were gradually dropped down after expo-
The human telomerase reverse transcriptase (hTERT) is a critical part of telomerase activity, and hTERT expression correlates well with
430
Cell Biology International, Vol. 26, No. 5, 2002
Table 3. Percentage of positive cells of hTERT protein expression from HL-60 and K562 cells by flow cytometric analysis (xs) Time (h)
24 48 72
HL-60
K562
Control
S PS-ODN
AS PS-ODN
Control
S PS-ODN
AS PS-ODN
99.285.21 99.134.20 98.152.15
99.4610.16 96.649.06 97.178.76
93.469.12* 48.744.96* 28.156.26*
99.246.34 99.195.86 99.055.57
99.489.14 98.698.07 96.177.56
92.458.73* 46.983.56* 32.892.26*
Abbreviations: S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. *Shown that P<0.01, the value of AS PS-ODN groups compared with the control or S PS-ODN groups separately. There no significant difference for the value of S PS-ODN groups compared with the one control separately.
Absorbance (A)
2 1.5
NC or PC Control
1
S PS–ODN AS PS–ODN
0.5 0
NC
PC
24 h
48 h
72 h
Fig. 1. Effect of hTERT AS PS-ODN on telomerase activity of HL-60. Abbreviations: NC, negative control; PC, positive control; S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. Absorbance levels of AS PS-ODN were significantly low to ones of controls or S PS-ODN groups 48 h after cells with ODN together for incubation.
Absorbance (A)
2.5 2 NC or PC 1.5
Control S PS–ODN
1
AS PS–ODN 0.5 0
NC
PC
24 h
48 h
72 h
Fig. 2. Effect of hTERT AS PS-ODN on telomerase activity of K562. Abbreviations: NC, negative control; PC, positive control; S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. Absorbance levels of AS PS-ODN were significantly low to ones of controls or S PS-ODN groups 48 h after cells with ODN together for incubation.
telomerase activity in tumors and established cell lines (HL-60, K562 and others). Telomerase activity would be downregulated or inhibited through influencing the hTERT gene expression. In the present research, we have used the hTERT AS PS-ODN to inhibit its expression so as to
influence telomerase activity. From the results shown, we succeed in reducing the hTERT mRNA and protein level, or downregulating or inhibiting hTERT gene expression, so as to drop down the telomerase activity of HL-60 and K562 cells availability (Stein and Cheng, 1993).
Cell Biology International, Vol. 26, No. 5, 2002
According to RT-PCR assays, the inhibiting effect produced by AS PS-ODN to hTERT mRNA of HL-60 cells was much stronger than to K562 cells, one-tenth of HL-60 hTERT mRNA to one-fourth of K562 hTERT mRNA at 48 h compared with control group. However, the lowering level of protein expression was not a conformance with lowering level of hTERT mRNA, and a conformance with lowering level of telomerase for those two cell lines. This phenomena show telomerase activity is based on the hTERT protein level, not the hTERT mRNA level. It was not found that PS-ODN exist non-sequence-specific RNase inhibiting effect to hTERT gene expression in our experiments, because we had used low dose of AS PS-ODN(10 mol/l at day 1, 5 mol/l at day 2, 5 mol/l at day 3), so that the specific inhibiting effect would be displayed significantly. According to our experiments of three days, telomerase inhibition would not affect growth of leukemic cells until their telomeres reach the critical size for entering senescence or apoptosis. This means that during anti-telomerase therapy, leukemic or tumor cells will continue grow 20–30 divisions until the telomeres reach critical size leading to cell senescence or apoptosis. If the one division is 24 h (one day), 20–30 divisions would be 20–30 days (Lavelle, 1999). During 20–30 days, the different strategies will be developed. We speculate if cell characteristics changes during period of telomerase activity inhibited, especially, such as sensitivity to chemotherapy drug or irradiation, which will be needed to study further. Next, effect of inhibition telomerase on sensitivity of chemotherapy drugs or radiation will explored or investigation.
431
REFERENCES B MA, L H, H MP, 1997. Telomere shortening and tumor formation by mice cells lacking telomerase RNA. Cell 91: 25–34. F J, F WD, 1995. The RNA component of human telomerase. Science 269: 1236–1241. H A, H HH, K S, K S, A T, B G, W D, 1998. Telomerase activity correlates with tumor aggressiveness and reflects therapy effect in breast cancer. Int J Cancer 79: 8–12. K E, L J, C Y, D MJ, L BC, 1998. Detecting human bladder carcinoma cells in voided urine samples by assaying for the presence of telomerase activity. Cancer 82: 708–714. K NW, P MA, 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266: 2011–2015. L F, R JF, L A, M P, 2000. Telomerase: a therapeutic target for the third millennium? Current Reviews in Oncology/Hematology 34: 111–126. M M, C CM, E EN, 1997. hEST2, the putative human telomerase catalytic subunit gene is upregulated in tumor cells and during immortalization. Cell 90: 785–789. M K, P CN, R R, G M, N RF, 1999. Telomerase activity and prognosis in breast cancer. Eur J Surg Oncol 25: 269–272. O K, K T, M Y, 1997. Telomerase activity in hepatocellular carcinoma as a predictor of postoperative recurrence. J Gastroenterol 32: 791–796. S CA, C YH, 1993. Antisense oligonucleotides as therapeutic agents is the bullet really magical? Science 261: 1004–1007. T H, K H, Y H, Y W, S JW, I T, T E, 1995. Telomerase activity in preneoplastic and neoplastic gastric and colorectal lesions. Clin Cancer Res 1: 1245–1251. T H, K M, T S, 2000. Precancerous hepatic nodules had significant levels of telomerase activity determined by sensitive quantitation using a hybridization protection assay. Cancer 88: 312–317.
EFFECT OF ANTISENSE hTERT mRNA OLIGODEOXYNUCLEOTIDE ON TELOMERASE ACTIVITY OF LEUKEMIC CELLS Y. ZHANG* and D. M. HE Institute of Hematology, Medical College of Jinan University, Guangzhou 510632, P. R. China Received 3 October 2001; accepted 15 February 2002
The effect of antisense hTERT mRNA oligodeoxynucleotide on telomerase activity of leukemia cells was explored and investigated in the present study. Telomerase activity was measured by the telomerase PCR ELISA assay kit (TRAP); hTERT mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) assay and gel-image system, hTERT protein by immunochemistry and flowcytometry. Results showed Incubation of leukemic cells (HL-60 and K562 cell lines) with 10 mol/l AS PS-ODN would significantly reduce the their mRNA levels and in vitro expression of hTERT protein 24 h later, so that the telomerase activity would be significantly down-regulated or inhibited. In conlusion, the hTERT AS PS-ODN is an excellent 2002 Published by Elsevier Science Ltd. inhibitor for telomerase activity. K: antisense; telomerase; leukemia; hTERT (hTERT, human telomerase reverse transcriptase).
INTRODUCTION Recently it was shown that 70–80% tumors, including hematological malignant tumors and leukemia, show very high levels of expression of telomerase, with a few exceptions, normal cells do not (Kim and Piatyszek, 1994). Telomerase is the ribonucleoprotein enzyme complex that elongates telomeric DNA (TTAGGG)n and now is thought to be an important step in the development of cellular immortality and oncogenesis. The most recent and significant advances in this area show that the inhibition or downregulation of telomerase activity would prevent from the malignant proliferation of tumor cells (Feng and Funk, 1995; Meyerson et al., 1997). So that it is interesting research for us to look for inhibiting material of telomerase. Antisense (AS) oligonuleotides in suppressing the expression of certain genes is attractive and have be used to inhibit the telomerase activity by antisense to RNA component of telomerase (Tahara et al., 1995). Human telomerase reverse *To whom correspondence should be addressed: Y. Zhang, Institute of Hematology, Medical College of Jinan University, Guangzhou 510632, P. R. China. Tel: 86-20-8522-6476; Fax: 86-20-8522-1343; E-mail: [email protected] Research was supported from grant of National Natural Science Foundation of China (No. 39870361). 1065–6995/02/$-see front matter
transcriptase (hTERT) is key component of telomerase catalytic activity, evidently, which would be key target attached by the anticancer drug (Takaishi et al., 2000; Ohata et al., 1997; Kavaler et al., 1998; Hoos et al., 1998; Mokbel et al., 1999; Blasco et al., 1997). In the present research, we had shown that incubation of leukemic cells with AS oligonucleotides from hTERT mRNA start site would significantly reduce the in vitro expression of hTERT protein and mRNA levels of HL-60 and K562 leukemic cells, so that their telomerase activity would be significantly down-regulated or inhibited.
MATERIALS AND METHODS Design and synthesis of antisense phosphorothiate oligomers Based on the hTERT gene cDNA sequence, 20 antisense oligomers were synthesized, purified and modified by Shanghai Institute of Biochemistry, from upstream 6-base and downstream 11-base at start code; the antisense phosphorothiate oligodeoxynucleotide sequence (AS PS-ODN) is 5 GGAGCGCGCGGCATCGCGGG-3 , the sense 2002 Published by Elsevier Science Ltd.
428
phosphorothiate oligodeoxynucleotide sequence (S PS-SODN) is 5 -CCCGCGATGCCGCGCGCT CC-3 as a control. Through the Blask soft examination from the Internet, there is no homologue for the antisense sequence with other genes except hTERT cDNA. Leukemic cell lines Leukemic cell lines (HL-60 and K562 cell lines) were incubated in the RPMI-1640 medium supplemented with 5% new born bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin and under the conditions of 5% CO2 in air, saturation humidity and 37C with 24-well plastic culture plate, each well of which contained 1105/ml. Sub-culture was performed 3–4 days later and the cells in logarithm growth phrase were used in all experiments. The experiments were divided into three groups of AS PS-ODN, S PS-SODN and normal control groups, each group consisted of repeat three wells on 24-well plate and oligomer was added into wells at last concentration of 10 mol/l, normal control group with culture medium of same volume. At 12, 24, 48, 72 h later for incubation, the cell telomerase activity was measured. Telomerase activity assays Telomerase activity was measured by the telomerase PCR ELISA assay kit (Boehringer Mannheim). Briefly, the cell extract was prepared at different time of oligomers treatment. The positive and negative control group were established in each experiment; cell extract was heated to 65C for 10 min as a negative control, and extract of 293 cell line having telomerase activity as a positive control (from kit). Telomeric repeat amplification protocol (TRAP reaction): (1) primer elongation: transfer 25 l reaction mixture into a tube suitable for PCR amplification, then add 2 l cell extract and sterile water to a final volume of 50 l; transfer the tubes to thermal cycler and perform one cycle at 25C for 30 min; (2) telomerase inactivation: perform one cycle at 94C for 5 min; (3) amplification: 50 l reaction mixture contains dNTP, Taq polymerase, the biotin-labled P1-TS primer and P2 primer. Perform 30 cycles for amplification, then at 94C for 30 s for denaturation, at 50C for 30 s for annealing, at 72C for 90 s for polymerization, then at 72C for 10 min for balance. Hybridization and ELASA procedure: transfer 20 l denaturation reagent into suitable tube, then add 5 l amplification product into it and incubate
Cell Biology International, Vol. 26, No. 5, 2002
at 20C for 10 min, add 225 l hybridization buffer (containing DIG-POD) and mix thoroughly by vortexing briefly. Transfer 100 l mixture on the nuclease-free microtiter plate and shake it at 37C for 2 h. Add 100 l anti-DIG-POD at room temperature for 30 min. At last, add 100 l TMB substrate solution and incubate it for 10 min at room temperature for color development, and then add 100 l stop reagent to stop the reaction. We determine the A (Absorbance) value of wavelength of 450–655 nm to calculate A=A450–A655. Detection of hTERT mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) assay Total RNA was extracted from oligomer-treatment cells with Trizol RNA kit (Life Technologies, U.S.A.). The hTERT cDNA was synthesized with 1 l cell extract, 200 U MMLV reverse transcriptase (Gibco), 6 oligomers of TTAGGG repeats. The primers for hTERT gene to be amplified are that hTERT upstream primer is 5 -CGG AAGAGTGTCTGGAGCAA-3 , hTERT downstream primer is GGATGAAGCGGAGTCTG GA-3 (Shanghai Institute of Biochemistry); actin upstream primer: 5 -GTGGGGCGCCCC AGGCACCA-3 ; -actin downstream primer: 5 GTCCTTAATGTCACGCACGATTTC-3 (-actin primer from Gibco). The conditions of PCR were: 90C 30 s for denaturation; 60C 1 min for annealing; 72C 1 min for extension, 30 cycles. PCR products were assayed by polyacrylamide gel electrophoresis and levels of bands on gel were measured with gel-image system. Determination of hTERT protein by flowcytometry Cells (5105) were collected and fixed with 70% formoldehyde at 4C for 15 min, then cell suspension was washed and resuspended for two times in specific PBS (s-PBS) (contained 1% human AB serum, 1% tween from Boehringer Mannheim, pH 7.2), which is able to perforate on cells surface so as to let antibody into cell inside easily. The hTERT protein antibody of 50 l was added into the cell suspension, which was incubated at 4C for 1 h. The cell suspension was washed two times with s-PBS and 50 l FITC-IgG of rabbit anti-goat was added and incubated at 4C for 30 min. At last, the cells were washed two times with PBS, and then hTERT protein was detected by flowcytometry. Statistical analysis The data were shown with xs, and processed with software of SPSS; one-way analysis of
Cell Biology International, Vol. 26, No. 5, 2002
429
Table 1. Effect of Antisense hTERT oligodeoxynucleotides on hTERT mRNA expression of leukemic cells of HL-60 and K562 (value in table is ratio of gel bands scanned with gel-image system) (xs) Time (h)
24 48
HL-60
K562
Control
S PS-ODN
AS PS-ODN
Control
S PS-ODN
AS PS-ODN
0.710.19 0.750.20
0.620.14 0.570.12
0.320.07* 0.080.02*
0.830.24 0.870.25
0.740.21 0.790.18
0.410.11* 0.230.05*
Abbreviations: S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. *Shown that P<0.01, the value of AS PS-ODN groups compared with the controls or S PS-ODN groups separately. There is no significant difference for the value of S PS-ODN groups compared with the controls separately.
Table 2. The average intensity of fluorescein from hTERT protein of HL-60 and K562 cells by flow cytometric analysis (xs) Time (h)
24 48 72
HL-60
K562
Control
S PS-ODN
AS PS-ODN
Control
S PS-ODN
AS PS-ODN
211.2434.32 303.4746.36 223.1537.50
193.7629.46 200.5228.54 217.1230.39
65.037.57* 52.415.07* 30.457.58*
247.4340.34 267.1942.86 348.4550.57
300.1444.78 380.6554.74 360.3250.58
90.466.57* 45.476.03* 34.745.98*
Abbreviations: S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. *Shown that P<0.01, the value of AS PS-ODN groups compared with the one of controls or S PS-ODN groups separately. There no significant difference for the value of S PS-ODN groups compared with the one controls separately.
variance was used to determine the likelihood of a significant difference (P<0.05). RESULTS Effect of AS PS-ODN on hTERT mRNA expression of leukemic cells of HL-60 and K562 The number of HL-60 or K562 cells did not decline after exposure to AS PS-ODN for 48 h. However, the expression level of cell hTERT mRNA was gradually decreased with time elongation, the ratio of density was only 0.08 for HL-60 and 0.23 for K562 at 48 h. It show that expression of hTERT mRNA was significantly downregulated or inhibited by AS PS-ODN; it was also observed the slight degression without the significant effect for S PS-ODN group.
sure to AS PS-ODN for 24, 48 and 72 h, which show that hTERT protein expression of HL-60 and K562 was gradually dropped down. The level of hTERT protein expression for K562 cells was about one-tenth at 72 h, and about one-seventh for HL-60 at 72 h. However, there exist a slight inhibition of S PS-ODN to protein expression of hTERT for HL-60, without this phenomena for K562. The effect of antisense oligonucleotides on hTERT gene protein expression was agreement with ones of them on mRNA. Effect of hTERT AS PS-ODN on telomerase activity of HL-60 and K562 leukemic cells. As shown in Figure 1 and Figure 2, telomerase activity of HL-60 and K562 was measured by the telomerase PCR ELISA assay kit.
Protein expression of hTERT gene
DISCUSSION
The average intensities of fluorescein from hTERT protein of HL-60 and K562 cells by flow cytometric analysis were gradually dropped down after expo-
The human telomerase reverse transcriptase (hTERT) is a critical part of telomerase activity, and hTERT expression correlates well with
430
Cell Biology International, Vol. 26, No. 5, 2002
Table 3. Percentage of positive cells of hTERT protein expression from HL-60 and K562 cells by flow cytometric analysis (xs) Time (h)
24 48 72
HL-60
K562
Control
S PS-ODN
AS PS-ODN
Control
S PS-ODN
AS PS-ODN
99.285.21 99.134.20 98.152.15
99.4610.16 96.649.06 97.178.76
93.469.12* 48.744.96* 28.156.26*
99.246.34 99.195.86 99.055.57
99.489.14 98.698.07 96.177.56
92.458.73* 46.983.56* 32.892.26*
Abbreviations: S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. *Shown that P<0.01, the value of AS PS-ODN groups compared with the control or S PS-ODN groups separately. There no significant difference for the value of S PS-ODN groups compared with the one control separately.
Absorbance (A)
2 1.5
NC or PC Control
1
S PS–ODN AS PS–ODN
0.5 0
NC
PC
24 h
48 h
72 h
Fig. 1. Effect of hTERT AS PS-ODN on telomerase activity of HL-60. Abbreviations: NC, negative control; PC, positive control; S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. Absorbance levels of AS PS-ODN were significantly low to ones of controls or S PS-ODN groups 48 h after cells with ODN together for incubation.
Absorbance (A)
2.5 2 NC or PC 1.5
Control S PS–ODN
1
AS PS–ODN 0.5 0
NC
PC
24 h
48 h
72 h
Fig. 2. Effect of hTERT AS PS-ODN on telomerase activity of K562. Abbreviations: NC, negative control; PC, positive control; S PS-ODN, sense phosphorothioate oligodeoxynucleotide; AS PS-ODN, antisense phosphorothioate oligodeoxynucleotide. Absorbance levels of AS PS-ODN were significantly low to ones of controls or S PS-ODN groups 48 h after cells with ODN together for incubation.
telomerase activity in tumors and established cell lines (HL-60, K562 and others). Telomerase activity would be downregulated or inhibited through influencing the hTERT gene expression. In the present research, we have used the hTERT AS PS-ODN to inhibit its expression so as to
influence telomerase activity. From the results shown, we succeed in reducing the hTERT mRNA and protein level, or downregulating or inhibiting hTERT gene expression, so as to drop down the telomerase activity of HL-60 and K562 cells availability (Stein and Cheng, 1993).
Cell Biology International, Vol. 26, No. 5, 2002
According to RT-PCR assays, the inhibiting effect produced by AS PS-ODN to hTERT mRNA of HL-60 cells was much stronger than to K562 cells, one-tenth of HL-60 hTERT mRNA to one-fourth of K562 hTERT mRNA at 48 h compared with control group. However, the lowering level of protein expression was not a conformance with lowering level of hTERT mRNA, and a conformance with lowering level of telomerase for those two cell lines. This phenomena show telomerase activity is based on the hTERT protein level, not the hTERT mRNA level. It was not found that PS-ODN exist non-sequence-specific RNase inhibiting effect to hTERT gene expression in our experiments, because we had used low dose of AS PS-ODN(10 mol/l at day 1, 5 mol/l at day 2, 5 mol/l at day 3), so that the specific inhibiting effect would be displayed significantly. According to our experiments of three days, telomerase inhibition would not affect growth of leukemic cells until their telomeres reach the critical size for entering senescence or apoptosis. This means that during anti-telomerase therapy, leukemic or tumor cells will continue grow 20–30 divisions until the telomeres reach critical size leading to cell senescence or apoptosis. If the one division is 24 h (one day), 20–30 divisions would be 20–30 days (Lavelle, 1999). During 20–30 days, the different strategies will be developed. We speculate if cell characteristics changes during period of telomerase activity inhibited, especially, such as sensitivity to chemotherapy drug or irradiation, which will be needed to study further. Next, effect of inhibition telomerase on sensitivity of chemotherapy drugs or radiation will explored or investigation.
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