端粒酶表达的转录调控和信号传导机制
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摘要
目的：人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)是端粒酶活性调节的限速亚单位，是正常细胞和肿瘤细胞端粒酶活性明显不同的原因。hTERT是通过转录调控的，涉及到转录因子c-Myc,Sp1的转录激活作用；PHA可以诱导Jurkat T细胞端粒酶激活。本文目的在于：探讨转录因子c-Myc，Sp1对端粒酶的转录作用及PHA激活端粒酶的信号传导途径，为更好地理解肿瘤发生的机理及寻找端粒酶特异抑制剂提供理论依据。
方法：用脂质体将Sp1、c-Myc反义寡核甘酸(Oligodeoxynucleotide,ODN)或Sp1、Sp3表达载体转入培养至对数生长期的Jurkat T细胞(2-3×10~6细胞/ml)，预孵育30min后(表达载体预孵育时间为1h)，5％CO2、37℃恒温箱中继续培养36h。用端粒酶PCR—ELISA方法检测端粒酶活性，PCR产物用15％非变性聚丙烯酰胺凝胶分离，并通过银染显示DNA梯度。用逆转录PCR方法检测Sp1、c-Myc和hTERT mRNA水平，用Westernblot检测蛋白水平，用MTT法检测细胞增殖作用；采用脂质体将显性负突变ras、raf表达载体及反义H-ras ODN、反义c-raf ODN转入Jurkat T细胞，或应用信号传导特异抑制剂抑制Jurkat T细胞后，用PHA诱导细胞端粒酶表达，用逆转录PCR方法检测转染效果，并用端粒酶PCR—ELISA方法检测端粒酶活性，PCR产物用15％非变性聚丙烯酰胺凝胶分离，并通过银染显示DNA梯度。
结果：(1)转染反义Sp1 ODN(1μmol·L~(-1))明显抑制Sp1 mRNA和蛋白表达，抑制率分别为44.8％(P＜0.05)和57％(P＜0.01)；转染反义c-MycODN(1μmol·L~(-1))对c-Myc mRNA表达抑制率为36.2％(P＜0.01)；转染1μmol·L~(-1)反义Sp1 ODN、反义c-Myc ODN后，端粒酶活性抑制率
第一军医大学博士学位论文
分另为43．1％（P＜0刀1）和27．2％（P＜0刀＊；在0二5、o．5、1刀、2刀（n mol ＊”‘）
时，反义 SP ODN表现为明显的剂量依赖性端粒酶抑制作用，而反义
O＊c0＊在该剂量范围内，剂量依赖性抑制作用不明显：（2转染1
n mol·L”‘反义＊ ODN、反义 c－Myc ODN后，hTERTmRNA抑制率分
别为 43＊％（P＜0＊1）和 23．4％（P＜0＊5）；（3）Spl、Sp3载体转化 36小时，
Spl、Sp3蛋白水平分别升高59＊％（P＜0＊1）和36．8％（P＜0＊5）；转
染 SpJp3表达载体后，随着即 l表达水平的增加，端粒酶活性和 hTERT
＜ RNRNA水平明显增加，增加率分别为38．5WP＜0刀5）和25．4％0＜0刀5L
而即3表达载体对端粒酶活性和hTERT m洲A水平无明显影响。O）浓
。度从 0二52刀 u mol·L”l时，反义 Spl对细胞的增殖抑制作用随
剂量增加而增强，与反义 C－MyC ODN比较，有较好的量效关系，但各浓
度组的抑制作用比反义 c－Myc ODN低；而 2刀 u m＊·LI浓度的反义
C－MyC、Spl ODN转染 Jurkat T细胞 24h、48h和 72h后，对细胞增殖抑
制作用的时效关系表明，反义C－MyC ODN比反义Spl ODN起效快，作用
强。归）2．5 n g砌l的 PHA刺激 Jurkat T细胞时，端粒酶活性增加了
5080％o叱刀匀；ras负突变表达载体 pZIPrasl7N和 raf负突变表达载体
pCGNraf（l）转染使PHA刺激的端粒酶活性分别降低了38．7％o刃刀5）
和 34．3％（P＜0刀匀。（6）转染 1刀 n mol·L”‘反义 H－rs ODN和反义 c一raf
ODN有效抑制 H－rs mRNA和 c－raf mRNA表达，抑制率分别为
52．l％（P＜0＊1）和 42．8％（P＜0＊1）；不同浓度的反义H－ras ODN和反义c－raf
ODN明显抑制 PHA诱导的端粒酶激活，浓度从 0．5 u mol·L“l至 2刀 u
mol 工’时，抑制率分别从 19＊％升高至68．6％和从20刀％升高至67刀％，
表现出明显的剂量依赖性＜7）用 10 u mol“’的 PD98059J刀卜 mol”
U0126和 5 n mol ．L’l的 Bisindolylmaleimide分别抑制 MEKI、MEKllZ
和 PKC，使 2．5 P g／mlPHA刺激的 JUrkat T细胞端粒酶活性分别降低了
37．2WP＜0＊5）、64．7WP＜0＊1）禾SO．5地P＜0＊1）。
结论：＊）Spl和C－MyC转录因子通过促进hTERT表达水平共同调节
Jurkat T细胞端粒酶活性，但Spl在hTERT转录调控中更为重要八2）SP3
对hTERTInRNA的转录水平虽有一定的抑制作用，但不影响端粒酶活性，
表明即3不是端粒酶活性的负调控因子叶）反义 c－Myc对 Jurkat T细胞
4
第一军医大学博士学位论文
增殖抑制作用不是通过抑制端粒酶活性引起的。O）PHA主要通过
RasKafMEKERK信号途径激活 Jurka T细胞端粒酶／5）PKC对端粒酶
活性的调节作用是双重的，一方面促进端粒酶生成，另一方面增强其功能。
…）反义SPI ODN抑制端粒酶活性，进而抑制肿瘤生长，预示其可作为
一种潜在的抗肿瘤新策略。
Objective Human telomerase reverse transcriptase (hTERT) has been generally acknowledged as a rate-limiting determinant of the telomerase only expressed in immortal or tumor cells, and this may be one of the reason that tumor cells differ from somatic cells. Many observations indicate that transcriptional regulation of the hTERT gene plays a key role in the activation of telomerase, and transcriptional factors of c-Myc and Spl are obviously involved in telomerase expression. In Jurkat T cells, telomerase could be induced by PHA. The purpose of this dissertation is: (1) to investigate the effect of c-Myc and Spl on expression of telomerase and hTERT; (2) to study the signal transduction pathway of telomerase induced by PHA in Jurkat T. Methods Antisense Spl oligodeoxynucleotide (ODN) and antisense c-Myc (ODN) or Spl and Sp3 expression vector(pCMV-Spl, pCMV-Sp3) were delivered to Jurkat T cells by lipofectamin. Telomerase PCR ELISA was used to detect telomerase activity; RT-PCR analysis was used to assess the mRNA expression of Spl ,c-Myc and hTERT; Western blot was used to analyze the levels of Spl and Sp3 protein and the cells proliferation was measured by MTT. The expression vector of dominant negative p21ras (pZIPras17N)and raf-l(pCGNraf(l-130)) or antisense H-ras ODN and antisense c-raf ODN were transferred to Jurkat T cells by lipofectamin, or using selective inhibitors of kinases blocked different signal cascades such as PKC and MEK, and then cells were stimulated with PHA. Telomerase PCR ELISA was used to detect telomerase activity. Results (1) Treatment of Jurkat T with Spl antisense inhibitorC 1 μ mol ·L-1)resulted in dramatically reduced Spl mRNA and protein levels. The inhibition rate were 44.8%(P<0.05) and 57% (P<0.01) respectively. Treatment of Jurkat T with c-Myc antisense inhibitor (1 u mol -L-1) also resulted in significantly reduced c-Myc mRNA with an inhibition rate of 36.2%(P<0.01). Following the transcriptional factor Spl and c-Myc functionally altering, the telomerase activity were significantly suppressed with a 43.1% (P<0.01) and a 27.2% (P<0.01) inhibition rate respectively. From 0.25-2.0 n mol -L-l, a dose-dependent inhibition of telomerase activity by antisense Spl ODN was discovered, but the similar dose-dependent inhibition did not occurred by antisense c-Myc ODN. (2) Treatment of Jurkat T with 1 μmol·L-1 Spl antisense ODN or c-Myc ODN, hTERT mRNA expression were significantly decreased by 43.6% (P<0.01) and a 23.4% (P<0.05) respectively. (3) Treatment of Jurkat T cells with Spl or Sp3 expressing vectors for 36h result in significantly increase of Spl and Sp3 protein levels by 59.6%( P<0.01) and 36.8% (P<0.05) respectively. Enhancement of Spl expression obviously increased telomerase activity and hTERT mRNA levels with a rate of 38.5%(P<0.05) and 25.4% (P<0.05) respectively, whereas Sp3 had no significant effect on both telomerase activity and hTERT mRNA levels. (4) Dose-effect relationship of cells survival rate indicated a dose-dependent survival decreasing after treated with 0.25, 0.5, 1.0, 2.0(μmol·L-l) antisense Sp1 ODN for 48h, the similar dose-dependent inhibition did not occurred by antisense c-Myc ODN, but antisense c-Myc ODN of different concentrations inhibited cells proliferation more significantly than antisense Spl ODN. Time-effect relationship of cells survival rate indicated that antisense c-Myc ODN had an earlier and more powerful effect on cells proliferation than antisense Spl ODN. (5) Telomerase activity was greatly stimulated after exposure to PHA, and treatment of Jurkat T cells with dominant negative p21ras and raf-1 for 36h result in significantly decrease of telomerase by 38.7% (PO.05) and 34.3% (P<0.05) respectively. (6) Transferring to Jurkat T with 1 v mol -L-l H-ras or c-raf antisense inhibitor resulted in dramaticallyreduced H-ras mRNA and c-raf mRNA by 52.1%(P<0.05) and 42.8% (P<0.01) respectively. From 0.5-2.0 μmol·L-1, a dose-dependent inhibition of telomerase activity by antisense H-ras ODN and antisense c-raf ODN. (7) The increase of telomerase activit
方法：用脂质体将Sp1、c-Myc反义寡核甘酸(Oligodeoxynucleotide,ODN)或Sp1、Sp3表达载体转入培养至对数生长期的Jurkat T细胞(2-3×10~6细胞/ml)，预孵育30min后(表达载体预孵育时间为1h)，5％CO2、37℃恒温箱中继续培养36h。用端粒酶PCR—ELISA方法检测端粒酶活性，PCR产物用15％非变性聚丙烯酰胺凝胶分离，并通过银染显示DNA梯度。用逆转录PCR方法检测Sp1、c-Myc和hTERT mRNA水平，用Westernblot检测蛋白水平，用MTT法检测细胞增殖作用；采用脂质体将显性负突变ras、raf表达载体及反义H-ras ODN、反义c-raf ODN转入Jurkat T细胞，或应用信号传导特异抑制剂抑制Jurkat T细胞后，用PHA诱导细胞端粒酶表达，用逆转录PCR方法检测转染效果，并用端粒酶PCR—ELISA方法检测端粒酶活性，PCR产物用15％非变性聚丙烯酰胺凝胶分离，并通过银染显示DNA梯度。
结果：(1)转染反义Sp1 ODN(1μmol·L~(-1))明显抑制Sp1 mRNA和蛋白表达，抑制率分别为44.8％(P＜0.05)和57％(P＜0.01)；转染反义c-MycODN(1μmol·L~(-1))对c-Myc mRNA表达抑制率为36.2％(P＜0.01)；转染1μmol·L~(-1)反义Sp1 ODN、反义c-Myc ODN后，端粒酶活性抑制率
第一军医大学博士学位论文
分另为43．1％（P＜0刀1）和27．2％（P＜0刀＊；在0二5、o．5、1刀、2刀（n mol ＊”‘）
时，反义 SP ODN表现为明显的剂量依赖性端粒酶抑制作用，而反义
O＊c0＊在该剂量范围内，剂量依赖性抑制作用不明显：（2转染1
n mol·L”‘反义＊ ODN、反义 c－Myc ODN后，hTERTmRNA抑制率分
别为 43＊％（P＜0＊1）和 23．4％（P＜0＊5）；（3）Spl、Sp3载体转化 36小时，
Spl、Sp3蛋白水平分别升高59＊％（P＜0＊1）和36．8％（P＜0＊5）；转
染 SpJp3表达载体后，随着即 l表达水平的增加，端粒酶活性和 hTERT
＜ RNRNA水平明显增加，增加率分别为38．5WP＜0刀5）和25．4％0＜0刀5L
而即3表达载体对端粒酶活性和hTERT m洲A水平无明显影响。O）浓
。度从 0二52刀 u mol·L”l时，反义 Spl对细胞的增殖抑制作用随
剂量增加而增强，与反义 C－MyC ODN比较，有较好的量效关系，但各浓
度组的抑制作用比反义 c－Myc ODN低；而 2刀 u m＊·LI浓度的反义
C－MyC、Spl ODN转染 Jurkat T细胞 24h、48h和 72h后，对细胞增殖抑
制作用的时效关系表明，反义C－MyC ODN比反义Spl ODN起效快，作用
强。归）2．5 n g砌l的 PHA刺激 Jurkat T细胞时，端粒酶活性增加了
5080％o叱刀匀；ras负突变表达载体 pZIPrasl7N和 raf负突变表达载体
pCGNraf（l）转染使PHA刺激的端粒酶活性分别降低了38．7％o刃刀5）
和 34．3％（P＜0刀匀。（6）转染 1刀 n mol·L”‘反义 H－rs ODN和反义 c一raf
ODN有效抑制 H－rs mRNA和 c－raf mRNA表达，抑制率分别为
52．l％（P＜0＊1）和 42．8％（P＜0＊1）；不同浓度的反义H－ras ODN和反义c－raf
ODN明显抑制 PHA诱导的端粒酶激活，浓度从 0．5 u mol·L“l至 2刀 u
mol 工’时，抑制率分别从 19＊％升高至68．6％和从20刀％升高至67刀％，
表现出明显的剂量依赖性＜7）用 10 u mol“’的 PD98059J刀卜 mol”
U0126和 5 n mol ．L’l的 Bisindolylmaleimide分别抑制 MEKI、MEKllZ
和 PKC，使 2．5 P g／mlPHA刺激的 JUrkat T细胞端粒酶活性分别降低了
37．2WP＜0＊5）、64．7WP＜0＊1）禾SO．5地P＜0＊1）。
结论：＊）Spl和C－MyC转录因子通过促进hTERT表达水平共同调节
Jurkat T细胞端粒酶活性，但Spl在hTERT转录调控中更为重要八2）SP3
对hTERTInRNA的转录水平虽有一定的抑制作用，但不影响端粒酶活性，
表明即3不是端粒酶活性的负调控因子叶）反义 c－Myc对 Jurkat T细胞
4
第一军医大学博士学位论文
增殖抑制作用不是通过抑制端粒酶活性引起的。O）PHA主要通过
RasKafMEKERK信号途径激活 Jurka T细胞端粒酶／5）PKC对端粒酶
活性的调节作用是双重的，一方面促进端粒酶生成，另一方面增强其功能。
…）反义SPI ODN抑制端粒酶活性，进而抑制肿瘤生长，预示其可作为
一种潜在的抗肿瘤新策略。
Objective Human telomerase reverse transcriptase (hTERT) has been generally acknowledged as a rate-limiting determinant of the telomerase only expressed in immortal or tumor cells, and this may be one of the reason that tumor cells differ from somatic cells. Many observations indicate that transcriptional regulation of the hTERT gene plays a key role in the activation of telomerase, and transcriptional factors of c-Myc and Spl are obviously involved in telomerase expression. In Jurkat T cells, telomerase could be induced by PHA. The purpose of this dissertation is: (1) to investigate the effect of c-Myc and Spl on expression of telomerase and hTERT; (2) to study the signal transduction pathway of telomerase induced by PHA in Jurkat T. Methods Antisense Spl oligodeoxynucleotide (ODN) and antisense c-Myc (ODN) or Spl and Sp3 expression vector(pCMV-Spl, pCMV-Sp3) were delivered to Jurkat T cells by lipofectamin. Telomerase PCR ELISA was used to detect telomerase activity; RT-PCR analysis was used to assess the mRNA expression of Spl ,c-Myc and hTERT; Western blot was used to analyze the levels of Spl and Sp3 protein and the cells proliferation was measured by MTT. The expression vector of dominant negative p21ras (pZIPras17N)and raf-l(pCGNraf(l-130)) or antisense H-ras ODN and antisense c-raf ODN were transferred to Jurkat T cells by lipofectamin, or using selective inhibitors of kinases blocked different signal cascades such as PKC and MEK, and then cells were stimulated with PHA. Telomerase PCR ELISA was used to detect telomerase activity. Results (1) Treatment of Jurkat T with Spl antisense inhibitorC 1 μ mol ·L-1)resulted in dramatically reduced Spl mRNA and protein levels. The inhibition rate were 44.8%(P<0.05) and 57% (P<0.01) respectively. Treatment of Jurkat T with c-Myc antisense inhibitor (1 u mol -L-1) also resulted in significantly reduced c-Myc mRNA with an inhibition rate of 36.2%(P<0.01). Following the transcriptional factor Spl and c-Myc functionally altering, the telomerase activity were significantly suppressed with a 43.1% (P<0.01) and a 27.2% (P<0.01) inhibition rate respectively. From 0.25-2.0 n mol -L-l, a dose-dependent inhibition of telomerase activity by antisense Spl ODN was discovered, but the similar dose-dependent inhibition did not occurred by antisense c-Myc ODN. (2) Treatment of Jurkat T with 1 μmol·L-1 Spl antisense ODN or c-Myc ODN, hTERT mRNA expression were significantly decreased by 43.6% (P<0.01) and a 23.4% (P<0.05) respectively. (3) Treatment of Jurkat T cells with Spl or Sp3 expressing vectors for 36h result in significantly increase of Spl and Sp3 protein levels by 59.6%( P<0.01) and 36.8% (P<0.05) respectively. Enhancement of Spl expression obviously increased telomerase activity and hTERT mRNA levels with a rate of 38.5%(P<0.05) and 25.4% (P<0.05) respectively, whereas Sp3 had no significant effect on both telomerase activity and hTERT mRNA levels. (4) Dose-effect relationship of cells survival rate indicated a dose-dependent survival decreasing after treated with 0.25, 0.5, 1.0, 2.0(μmol·L-l) antisense Sp1 ODN for 48h, the similar dose-dependent inhibition did not occurred by antisense c-Myc ODN, but antisense c-Myc ODN of different concentrations inhibited cells proliferation more significantly than antisense Spl ODN. Time-effect relationship of cells survival rate indicated that antisense c-Myc ODN had an earlier and more powerful effect on cells proliferation than antisense Spl ODN. (5) Telomerase activity was greatly stimulated after exposure to PHA, and treatment of Jurkat T cells with dominant negative p21ras and raf-1 for 36h result in significantly decrease of telomerase by 38.7% (PO.05) and 34.3% (P<0.05) respectively. (6) Transferring to Jurkat T with 1 v mol -L-l H-ras or c-raf antisense inhibitor resulted in dramaticallyreduced H-ras mRNA and c-raf mRNA by 52.1%(P<0.05) and 42.8% (P<0.01) respectively. From 0.5-2.0 μmol·L-1, a dose-dependent inhibition of telomerase activity by antisense H-ras ODN and antisense c-raf ODN. (7) The increase of telomerase activit
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24.Tanaka H et al. Evidence for a putative telomerase repressor gene in the 3p14.2-p21. 1 region. Genes Chromosomes Cancer. 1998;23(2): 123-33.
25.Cuthbert AP et al. Telomerase repressor sequences on chromosome 3 and induction of permanent growth arrest in human breast cancer cells. J Natl Cancer Inst. 1999;91(1):37-45.
26. Ohmura H et al. Restoration of the cellular senescence program and repression of telomerase by human chromosome 3. Jpn J Cancer Res. 1995;86(10):899-904.
27. Greenberg RA, O'Hagan RC, Deng H, Xiao Q, Hann SR, Adams RR, Lichtsteiner S, Chin L, Morin GB, DePinho RA.elomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation. Oncogene 1999; 18(5): 1219-1226.
28. Park NH, Guo W, Kim HR, Kang MK, Park NH. c-Myc and Sp1/3 are required for transactivation of hamster telomerase catalytic subunit gene promoter. Int J Oncol 2001; 19(4):755-61.
29. Xu D et al. Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells. Oncogene .2000,19(45):5123-5133.
30. Kanaya T, Kyo S, Hamada K, Takakura M, Kitagawa Y, Harada H, Inoue M. Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription. Clin Cancer Res 2000 ;6(4): 1239-47.
31. Oh ST, Kyo S, Laimins LA. Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites. J Virol 2001; 75(12):5559-66.
32. Takakura M, Kyo S, Sowa Y, Wang Z, Yatabe N, Maida Y, Tanaka M, Inoue M. Telomerase activation by histone deacetylase inhibitor in normal cells. Nucleic Acids Res 2001; 29(14):3006-11.
33. Suske G. The Sp-family of transcription factors. Gene, 1999, 238:291-300.
34. Hagen G, Muller S, Beato M, Suske G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J, 1994, 13:3843-51.
35. Buchkovich K J, Greider CW. Telomerase regulation during entry into the cell cycle in normal human T cells. Mol Biol Cell. 1996;7(9):1443-54.
36. Andrea G et al. Mechanism of teiomerase induction during T cell activation. Exp Cell Res. 1996;228:58-64.
37. Yong Q et al.Regulation of lymphotoxin production by the p21ras-raf-MEK-ERK cascade in PHA/PMA-stimulated Jurkat cells. J Immun. 1999,162:3316-3320.
38. Juanita L et al. Sp1 phosphorylation by Erk2 stimulates DNA binding. Biochem Biophy Res Commu. 1999;254:454-461.
39. Chupreta S et al. EGF stimulates gastrin promoter through activation of Sp1 kinase activity. Am J Physiol cell physiol. 2000,278:697-708.
40. Tomokazu O et al. Analysis of the T-cell activation signaling pathway mediated by tyrosine kinase, protein kinase C,and Ras protein, which is modulated by intracellular cyclic AMP. Bioch Biophy Acta. 1996;1310:223-232.
41. Seimiya H, Tsuruo T. Telomerase downregulation during differentiation of leukemia cells. Nippon Rinsho. 1998;56(5): 1121-5.
42. Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrew WH, Lingner J, Harley CB, Cech TR. Telomerase catalytic subunit homologs from flssion yeast and human. Science, 1997, 277:955-59.
43. Jia R et al. Truncation of Sp1 transcription factor by myeloblastin in undifferentiated HL60 cell. J cell Physiol. 1998,175:121-128.
44. Jackson SP, McDonald JJ, Lees-Miller, Tjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell, 1990, 63:155-65.
45. Ammanamanchi S, Kim SJ, Sun LZ, Brattain, M.G. Induction of transforming growth factor-beta receptor type Ⅱ expression in estrogen receptor-positive breast cancer cells through SP1 activation by 5-aza-2'-deoxycytidine. J. Biol. Chem, 1998, 273: 16527-34.
46. Kennett S B, Udvadia A.J, Horowitz J M. Sp3 encodes multiple proteins that differ in their capacity to stimulate or repress transcription. Nucleic Acids Res, 1997 25:3110-7.
47. Xiaoxing SX et al. A role for c-Raf Kinase and Ha-Ras in cytokine-mediated Induction of cell adhesion molecules. J Biol Chem. 1998,273(50):33230-33238.
48. Duroux Ⅰ et al. Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-ras mRNA in human cells. Nucleic Acids Res. 1995,23(17):3411-3418.
49. Deshpande RV, Moore MA. G-CSF receptor-mediated up-regulation of c-fos but not c-raf mRNA expression in myeloid cells. Mol Cell Biochem. 1998, 178:47-50.
50. Chou WC, Hawkins AL, Barrett JF, Griffin CA, Dang CV. Arsenic inhibition of telomerase transcription leads to genetic instability. J Clin Invest 2001 ;108(10):1541-7.
51. Mitchell PJ, Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 1989; 245:371-75.
52. Patterson C, Wu Y, Lee ME, DeVault JD, Runge MS, Haber E. Nuclear protein interactions with the human KDR/flk-1 promoter in vivo. Regulation of Sp1 binding is associated with cell type-specific expression. J Biol Chem 1997; 272:8410-16.
53. Yasuaki H, Elia D, Kang Z, Gregory SR, Lioyd PA. Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence. J Biol Chem 1998; 273:19294-303.
54. Zhengyu W, Ying Z, Jun L, Shinnshin S, Katya R. Mp1 ligand enhances the transcription of the cyclin D3 gene: a potential role for Sp1 transcription factor. Blood 1999; 93:4208-21.
55. D'Angelo DD, Oliver BG, Davis MG, McCluskev TS, Dom GW. J Biol Chem 1996; 271:19696-704.
56. Noti JD, Reinemann BC, Petrus MN. Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription. Mol Cell Biol 1996; 16:2940-50.
57. Baker DL, Dave V, Reed T Periasamy M. Multiple Sp1 binding sites in the cardiac/slow twitch muscle sarcoplasmic reticulum Ca2+-ATPase gene promoter are required for expression in So18 muscle cells. J Biol Chem 1996; 271:5921-28.
58. Jeffrey DS, Stephen PJ, Mary BA. Developmental expression of Sp1 in the mouse. Mol Cell Biol 1991; 11(4):2189-99.
59. Fischer KD, Haese A, Nowock J. Cooperation of GATA-1 and Sp1 can result in synergistic transcriptional activation or interference. J Biol Chem 1993; 268:23915-23924.
60. Lecointe N, Bernard O, Naert K, Joulin V, Larsen C J, Romeo PH, Mathieu-Mahul D. GATA-and Sp1-binding sites are required for the full activity of the tissue-specific promoter of the tal-1 gene. Oncogene 1994; 9:2623-2632.
61. Block KL, Shou Y, Poncz M. An Ets/Sp1 interaction in the 5'-flanking region of the megakaryocyte-specific alpha IIb gene appears to stabilize Sp1 binding and is essential for expression of this TATA-less gene. Blood 1996; 88:2071-2082.
62. Yang X, Fyodorov D, Deneris ES. Transcriptional analysis of acetycholine receptor alpha 3 gene promoter motifs that bind Sp1 and AP2. J Biol Chem 1995; 270:8514-8520.
63. Karlseder J, Rotheneder H, Wintersberger E. Interaction of Sp1 with the growth-and cell cycle-regulated transcription factor E2F. Mol Cell Bill 1996; 16: 1659-1664.
64. Lin SY, Black AR, Kostic D, Pajovic S, Hoover CN, Azizkhan JC. Cell cycle-regulated association of E2F1 and Sp1 is related to their functional interaction. Mol Cell Biol 1996; 16:1668-1677.
65. Cerezo A et al. Dual regulation of telomerase activity through c-Myc-dependent inhibition and alternative splicing of hTERT. J Cell Sci 2002;115(Pt6):
66. Ulaner GA et al. Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts. Cancer Res, 1998,58:4168-4172.
67. Ulaner GA, Hu JF, Vu TH, Oruganti H, Giudice LC, Hoffman AR. Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium. Int J Cancer 2000;85(3):330-5.
68. Yi X, White DM, Aisner DL, Baur JA, Wright WE, Shay JW. An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity. Neoplasia 2000;2(5):433-40.
69. Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR. Tissue-specific alternate splicing of human telomerase reverse transcriptase (hTERT) influences telomere lengths during human development. Int J Cancer 2001;91(5):644-9.
70. Krams M, Claviez A, Heidom K, Krupp G, Parwaresch R, Harms D, Rudolph P. Regulation of telomerase activity by alternate splicing of human telomerase reverse transcriptase mRNA in a subset of neuroblastomas. Am J Pathol 2001; 159(5): 1925-32.
71. Park NH, Guo W, Kim HR, Kang MK, Park NH. c-Myc and Sp1/3 are required for transactivation of hamster telomerase catalytic subunit gene promoter. Int J Oncol, 2001, 19(4):755-61.
72. Guo W, Okamoto M, Lee YM, Baluda MA, Park NH. Enhanced activity of cloned hamster TERT gene promoter in transformed cells. Biochim Biophys Acta, 2001, 1517(3):398-409.
73. Nozawa K, Maehara K, Isobe K. Mechanism for the reduction of telomerase expression during muscle cell differentiation. J Biol Chem, 2001, 276(25):22016-23.
74. Zhao JQ, Glasspool RM, Hoare SF, Bilsland A, Szatmari I I, Keith WN. Activation of Telomerase RNA Gene Promoter Activity by NF-Y, Sp1, and the Retinoblastoma Protein and Repression by Sp3. Neoplasia, 2000, 2(6):531-9.
75. Izquierdo M, Leevers S J, Marshall CJ, Cantreil D. p21ras couples the T cell antigenreceptor to extracellular signal-regulated kinase 2 in T lymphocytes. J Exp Med 1993;178(4):1199-208
76. Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993; 268: 14553-14556.
77. Cano E, Mahadevant LC. Parallel signal processing among mammalian MAPKs. TIBS. 1995;20(3):117-123.
78. Seger R, Krebs EG. The MAPK signaling cascade. FASEB J. 1995;9(9):726-735.
79. Cobb MH, Gold Smith EJ. How MAP kinase are regulated. J Bioi Chem. 1995;270:1483-86.
80. Aronheim A et al. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell. 1994;78(6):949-961.
81. Rosen LB et al. Stimulation of growth factor receptor signal transduction by activation of voltage-sensitive calcium channels. Proc Natl Acad Sci USA.1996;93:l 113-1118.
82. Marshall CJ, Lloyd AC, Morris JD, Paterson H, Price B, Hall A. Signal transduction by p21ras. Int J Cancer Suppl 1989;4:29-31.
83. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. B iochem J 2000;351(Pt1):95-105.
84. He Li, Linlin Zhao, Zhiyong Yang, John W. Funder Telomerase is controlled by protein kinase Cα in Human Breast Cancer Cells. J Biol Chem. 1998. 273(50):33436-33442.
85. Chuang CF, blg SY. Functional divergence of the MAP kinase pathway. ERK1 and ERK2 activate specific transcription factors. FEBS Lett 1994;346(2-3):229-34.
86. Seimiya H et al. Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells. Biochem Biophys Res Commun. 1999;260(2):365-70.
87. Loke SL et al. Characterization of oligonucleotide transport into living cells. Proc Natl Acad Sci USA. 1989,86:3474-3478.
88. Sambrook JEF et al. Molecular Cloning: A Lboratory Manual. 1989,Cold Spring Hartor Laboratory, Cold Spring Harbor NY, P117.
89. Stein CA et al. Physicochemical properties of phosphrothiote oligodeoxynucleotides. Nucleic Acids Res. 1988; 16:3209-3221.
2.J un-ping L. Studies of the molecular mechanisms in the regulation of telomerase activity. FASEB J, 1999,13:2091-2104.
3.H elder MN et al. Telomerase and telomeres: from basic biology to cancer treatment. Cancer Invest. 2002;20(1):82-101.
4.Perry PJ et al. Telomerase inhibitors for the treatment of cancer: the current perspective. Expert Opin Investig Drugs. 2001; 10(12):2141-56.
5.Hodes R. Molecular targeting of cancer: telomeres as targets. Proc Natl Acad Sci U S A. 2001 ;98(14):7649-51.
6.Bearss DJ et al. Telomere maintenance mechanisms as a target for drug development. Oncogene. 2000 27; 19(56):6632-41.
7.Rowley PT et al. Telomerase inhibitors. Anticancer Res. 2000;20(6B):4419-29.
8.White LK et al. Telomerase inhibitors. Trends Biotechnol. 2001 Mar;19(3):114-20.
9.Koyanagi Y et al. Telomerase activity is down regulated via decreases in hTERT mRNA but not TEP1 mRNA or hTERC during the differentiation of leukemic cells. Anticancer Res. 2000;20(2A):773-8.
10.Ducrest AL, Szutorisz H, Lingner J, Nabholz M. Regulation of the human telomerase reverse transcriptase gene. Oncogene 2002;21 (4):541-52.
11.Ducrest AL, Amacker M, Mathieu YD, Cuthbert AP, Trott DA, Newbold RF, Nabholz M, Lingner J. Regulation of human telomerase activity: repression by normal chromosome 3 abolishes nuclear telomerase reverse transcriptase transcripts but does not affect c-Myc activity. Cancer Res 2001 ;61 (20):7594-602.
12.Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol 1998; 8:279-82.
13.Takakura M, Kyo S, Kanaya T, Hirano H, Takeda J, Yutsudo M, Inoue M. Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells. Cancer Res 1999; 59:551-59.
14.Kyo S, Inoue M. Complex regulatory mechanisms oftelomerase activity in normal and cancer cells: how can we apply them for cancer therapy? Oncogene 2002;21 (4): 688-97.
15.Aisner DL, Wright WE, Shay JW. Telomerase regulation: not just flipping the switch.. Curr Opin Genet Dev 2002;12(1):80-85.
16.Fujimoto K et al. Telomerase activity in human leukemic cell lines is inhibited by antesense pentade-cadeoxynucleotides targeted against c-myc mRNA. Biochem Biophys Res Commun, 1997,241:775-781.
17.Wang J et al. Myc activates telomerase. Genes Dev, 1998,12:1769-1774
18.Satoru K et al. SP1 cooperates with c-Myc to activate transcription of the human telomerase reverse teanscriptase gene (hTERT). Nucleic Acids Res,2000,28(3):669-677
19.Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrew WH, Lingner J, Harley CB, Cech TR.Telomerase catalytic subunit homoiogs from flssion yeast and human. Science 1997; 277:955-59.
20.Sangtaek O et al. Identification of Mad as a repressor of the human telomerase (hTERT) gene. Oncogene,2000,19:1485-1490
21.Gunes C, Lichtsteiner S, Vasserot AP, Englert C. Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1. Cancer Res 2000 Apr 15;60(8):2116-2121.
22.Mehle C et al. Loss of heterozygosity at chromosome 3p correlates with telomerase activity in renal cell carcinoma. Int J Oncol. 1998;13(2):289-95.
23.Horikawa I et al. Repression of the telomerase catalytic subunit by a gene on human chromosome 3 that induces cellular senescence. Mol Carcinog. 1998;22(2):65-72.
24.Tanaka H et al. Evidence for a putative telomerase repressor gene in the 3p14.2-p21. 1 region. Genes Chromosomes Cancer. 1998;23(2): 123-33.
25.Cuthbert AP et al. Telomerase repressor sequences on chromosome 3 and induction of permanent growth arrest in human breast cancer cells. J Natl Cancer Inst. 1999;91(1):37-45.
26. Ohmura H et al. Restoration of the cellular senescence program and repression of telomerase by human chromosome 3. Jpn J Cancer Res. 1995;86(10):899-904.
27. Greenberg RA, O'Hagan RC, Deng H, Xiao Q, Hann SR, Adams RR, Lichtsteiner S, Chin L, Morin GB, DePinho RA.elomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation. Oncogene 1999; 18(5): 1219-1226.
28. Park NH, Guo W, Kim HR, Kang MK, Park NH. c-Myc and Sp1/3 are required for transactivation of hamster telomerase catalytic subunit gene promoter. Int J Oncol 2001; 19(4):755-61.
29. Xu D et al. Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells. Oncogene .2000,19(45):5123-5133.
30. Kanaya T, Kyo S, Hamada K, Takakura M, Kitagawa Y, Harada H, Inoue M. Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription. Clin Cancer Res 2000 ;6(4): 1239-47.
31. Oh ST, Kyo S, Laimins LA. Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites. J Virol 2001; 75(12):5559-66.
32. Takakura M, Kyo S, Sowa Y, Wang Z, Yatabe N, Maida Y, Tanaka M, Inoue M. Telomerase activation by histone deacetylase inhibitor in normal cells. Nucleic Acids Res 2001; 29(14):3006-11.
33. Suske G. The Sp-family of transcription factors. Gene, 1999, 238:291-300.
34. Hagen G, Muller S, Beato M, Suske G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J, 1994, 13:3843-51.
35. Buchkovich K J, Greider CW. Telomerase regulation during entry into the cell cycle in normal human T cells. Mol Biol Cell. 1996;7(9):1443-54.
36. Andrea G et al. Mechanism of teiomerase induction during T cell activation. Exp Cell Res. 1996;228:58-64.
37. Yong Q et al.Regulation of lymphotoxin production by the p21ras-raf-MEK-ERK cascade in PHA/PMA-stimulated Jurkat cells. J Immun. 1999,162:3316-3320.
38. Juanita L et al. Sp1 phosphorylation by Erk2 stimulates DNA binding. Biochem Biophy Res Commu. 1999;254:454-461.
39. Chupreta S et al. EGF stimulates gastrin promoter through activation of Sp1 kinase activity. Am J Physiol cell physiol. 2000,278:697-708.
40. Tomokazu O et al. Analysis of the T-cell activation signaling pathway mediated by tyrosine kinase, protein kinase C,and Ras protein, which is modulated by intracellular cyclic AMP. Bioch Biophy Acta. 1996;1310:223-232.
41. Seimiya H, Tsuruo T. Telomerase downregulation during differentiation of leukemia cells. Nippon Rinsho. 1998;56(5): 1121-5.
42. Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrew WH, Lingner J, Harley CB, Cech TR. Telomerase catalytic subunit homologs from flssion yeast and human. Science, 1997, 277:955-59.
43. Jia R et al. Truncation of Sp1 transcription factor by myeloblastin in undifferentiated HL60 cell. J cell Physiol. 1998,175:121-128.
44. Jackson SP, McDonald JJ, Lees-Miller, Tjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell, 1990, 63:155-65.
45. Ammanamanchi S, Kim SJ, Sun LZ, Brattain, M.G. Induction of transforming growth factor-beta receptor type Ⅱ expression in estrogen receptor-positive breast cancer cells through SP1 activation by 5-aza-2'-deoxycytidine. J. Biol. Chem, 1998, 273: 16527-34.
46. Kennett S B, Udvadia A.J, Horowitz J M. Sp3 encodes multiple proteins that differ in their capacity to stimulate or repress transcription. Nucleic Acids Res, 1997 25:3110-7.
47. Xiaoxing SX et al. A role for c-Raf Kinase and Ha-Ras in cytokine-mediated Induction of cell adhesion molecules. J Biol Chem. 1998,273(50):33230-33238.
48. Duroux Ⅰ et al. Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-ras mRNA in human cells. Nucleic Acids Res. 1995,23(17):3411-3418.
49. Deshpande RV, Moore MA. G-CSF receptor-mediated up-regulation of c-fos but not c-raf mRNA expression in myeloid cells. Mol Cell Biochem. 1998, 178:47-50.
50. Chou WC, Hawkins AL, Barrett JF, Griffin CA, Dang CV. Arsenic inhibition of telomerase transcription leads to genetic instability. J Clin Invest 2001 ;108(10):1541-7.
51. Mitchell PJ, Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 1989; 245:371-75.
52. Patterson C, Wu Y, Lee ME, DeVault JD, Runge MS, Haber E. Nuclear protein interactions with the human KDR/flk-1 promoter in vivo. Regulation of Sp1 binding is associated with cell type-specific expression. J Biol Chem 1997; 272:8410-16.
53. Yasuaki H, Elia D, Kang Z, Gregory SR, Lioyd PA. Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence. J Biol Chem 1998; 273:19294-303.
54. Zhengyu W, Ying Z, Jun L, Shinnshin S, Katya R. Mp1 ligand enhances the transcription of the cyclin D3 gene: a potential role for Sp1 transcription factor. Blood 1999; 93:4208-21.
55. D'Angelo DD, Oliver BG, Davis MG, McCluskev TS, Dom GW. J Biol Chem 1996; 271:19696-704.
56. Noti JD, Reinemann BC, Petrus MN. Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription. Mol Cell Biol 1996; 16:2940-50.
57. Baker DL, Dave V, Reed T Periasamy M. Multiple Sp1 binding sites in the cardiac/slow twitch muscle sarcoplasmic reticulum Ca2+-ATPase gene promoter are required for expression in So18 muscle cells. J Biol Chem 1996; 271:5921-28.
58. Jeffrey DS, Stephen PJ, Mary BA. Developmental expression of Sp1 in the mouse. Mol Cell Biol 1991; 11(4):2189-99.
59. Fischer KD, Haese A, Nowock J. Cooperation of GATA-1 and Sp1 can result in synergistic transcriptional activation or interference. J Biol Chem 1993; 268:23915-23924.
60. Lecointe N, Bernard O, Naert K, Joulin V, Larsen C J, Romeo PH, Mathieu-Mahul D. GATA-and Sp1-binding sites are required for the full activity of the tissue-specific promoter of the tal-1 gene. Oncogene 1994; 9:2623-2632.
61. Block KL, Shou Y, Poncz M. An Ets/Sp1 interaction in the 5'-flanking region of the megakaryocyte-specific alpha IIb gene appears to stabilize Sp1 binding and is essential for expression of this TATA-less gene. Blood 1996; 88:2071-2082.
62. Yang X, Fyodorov D, Deneris ES. Transcriptional analysis of acetycholine receptor alpha 3 gene promoter motifs that bind Sp1 and AP2. J Biol Chem 1995; 270:8514-8520.
63. Karlseder J, Rotheneder H, Wintersberger E. Interaction of Sp1 with the growth-and cell cycle-regulated transcription factor E2F. Mol Cell Bill 1996; 16: 1659-1664.
64. Lin SY, Black AR, Kostic D, Pajovic S, Hoover CN, Azizkhan JC. Cell cycle-regulated association of E2F1 and Sp1 is related to their functional interaction. Mol Cell Biol 1996; 16:1668-1677.
65. Cerezo A et al. Dual regulation of telomerase activity through c-Myc-dependent inhibition and alternative splicing of hTERT. J Cell Sci 2002;115(Pt6):
66. Ulaner GA et al. Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts. Cancer Res, 1998,58:4168-4172.
67. Ulaner GA, Hu JF, Vu TH, Oruganti H, Giudice LC, Hoffman AR. Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium. Int J Cancer 2000;85(3):330-5.
68. Yi X, White DM, Aisner DL, Baur JA, Wright WE, Shay JW. An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity. Neoplasia 2000;2(5):433-40.
69. Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR. Tissue-specific alternate splicing of human telomerase reverse transcriptase (hTERT) influences telomere lengths during human development. Int J Cancer 2001;91(5):644-9.
70. Krams M, Claviez A, Heidom K, Krupp G, Parwaresch R, Harms D, Rudolph P. Regulation of telomerase activity by alternate splicing of human telomerase reverse transcriptase mRNA in a subset of neuroblastomas. Am J Pathol 2001; 159(5): 1925-32.
71. Park NH, Guo W, Kim HR, Kang MK, Park NH. c-Myc and Sp1/3 are required for transactivation of hamster telomerase catalytic subunit gene promoter. Int J Oncol, 2001, 19(4):755-61.
72. Guo W, Okamoto M, Lee YM, Baluda MA, Park NH. Enhanced activity of cloned hamster TERT gene promoter in transformed cells. Biochim Biophys Acta, 2001, 1517(3):398-409.
73. Nozawa K, Maehara K, Isobe K. Mechanism for the reduction of telomerase expression during muscle cell differentiation. J Biol Chem, 2001, 276(25):22016-23.
74. Zhao JQ, Glasspool RM, Hoare SF, Bilsland A, Szatmari I I, Keith WN. Activation of Telomerase RNA Gene Promoter Activity by NF-Y, Sp1, and the Retinoblastoma Protein and Repression by Sp3. Neoplasia, 2000, 2(6):531-9.
75. Izquierdo M, Leevers S J, Marshall CJ, Cantreil D. p21ras couples the T cell antigenreceptor to extracellular signal-regulated kinase 2 in T lymphocytes. J Exp Med 1993;178(4):1199-208
76. Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993; 268: 14553-14556.
77. Cano E, Mahadevant LC. Parallel signal processing among mammalian MAPKs. TIBS. 1995;20(3):117-123.
78. Seger R, Krebs EG. The MAPK signaling cascade. FASEB J. 1995;9(9):726-735.
79. Cobb MH, Gold Smith EJ. How MAP kinase are regulated. J Bioi Chem. 1995;270:1483-86.
80. Aronheim A et al. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell. 1994;78(6):949-961.
81. Rosen LB et al. Stimulation of growth factor receptor signal transduction by activation of voltage-sensitive calcium channels. Proc Natl Acad Sci USA.1996;93:l 113-1118.
82. Marshall CJ, Lloyd AC, Morris JD, Paterson H, Price B, Hall A. Signal transduction by p21ras. Int J Cancer Suppl 1989;4:29-31.
83. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. B iochem J 2000;351(Pt1):95-105.
84. He Li, Linlin Zhao, Zhiyong Yang, John W. Funder Telomerase is controlled by protein kinase Cα in Human Breast Cancer Cells. J Biol Chem. 1998. 273(50):33436-33442.
85. Chuang CF, blg SY. Functional divergence of the MAP kinase pathway. ERK1 and ERK2 activate specific transcription factors. FEBS Lett 1994;346(2-3):229-34.
86. Seimiya H et al. Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells. Biochem Biophys Res Commun. 1999;260(2):365-70.
87. Loke SL et al. Characterization of oligonucleotide transport into living cells. Proc Natl Acad Sci USA. 1989,86:3474-3478.
88. Sambrook JEF et al. Molecular Cloning: A Lboratory Manual. 1989,Cold Spring Hartor Laboratory, Cold Spring Harbor NY, P117.
89. Stein CA et al. Physicochemical properties of phosphrothiote oligodeoxynucleotides. Nucleic Acids Res. 1988; 16:3209-3221.