Telomerase Telomerase Subunit
Telomerase Telomerase Subunit 2001 12
1 3 7 1 7 2 7 7 MTT 8 8 Senescence-associated â-galactosidase stain 9 9 TRAP assay telomerase 10 RT-PCR telomerase subunit (htert, hter) 11 Real-time PCR htep, c-myc, Mad1 12 Southern blot hybridization terminal restriction fragment (TRF) 13
16 1 MTT assay AZT 16 2 16 3 19 4 21 5 TRAP assaytelomerase 23 6 RT-PCR telomerase subunit 26 7 Real-time PCR htep, c-myc, Mad1 29 8 Southern blot TRF 32 9 AZT 32 35 40 41 50
Figure 1 Dosage determinant by MTT assay 16 Figure 2 Growth curve of MCF-7 and MDA-MB-231 18 Figure 3 Expression of senescence by senescence associated (SA) â-galactosidase stain in breast cancer cells 20 Figure 4 Apoptosis detection by dtd enzyme in breast cancer cells 22 Figure 5 Telomerase activity of MCF-7 by TRAP assay 24 Figure 6 Telomerase activity of MDA-MB-231 by TRAP assay 25 Figure 7 Expression of htert and hter mrna of MCF-7 by RT -PCR 27 Figure 8 Expression of htert and hter mrna of MDA-MB-231 by RT-PCR 28 Figure 9 Expression of telomerase subunits mrna by real-time PCR 31 Figure 10 Length of Terminal restriction fragments (TRFs) by Southern blot hybridization 33 Figure 11 Time points of change pattern in biological and genetic phenotype 34
Table 1 Primer oligonucleotide sequence 13 Table 2 Growth inhibition rate in MCF-7 and MDA-MB-231 17
Telomerase Telomerase Subunit telomere Telomere telomerase telomerase 3 -azido-2,3 - dideoxythymidine (AZT)telomerase telomere telomere telomerase subunit telomere, telomerase AZT X- gal, TRAP assay telomerase telomerase, telomere RT-PCRreal-time PCR telomerase subunit, htertazt telomerase htert c- MychTERT, htermad1, htep AZT telomerase subunit htertc-myc, telomerase subunit telomerase telomere
: Telomerase, Telomere, htert, hter, htep, c-myc, Mad1,,
Telomerase Telomere Subunit (senescence) telomere telomere, 1, 'mitotic clock' telomere DNA (TTAGGG) n
2 Telomere DNA, 3-10 DNA polymerase 'end-replication problem' telomere 50-200 nucleotide 11,12 telomere, 13-15 Telomere duplex telomere TTAGGG-repeat binding factortrf1 16 TRF2 5 telomere telomere Single strand telomere telomerase reverse transctriptase (TERT), 17 telomere associated protein 1 (TEP1), 18,19 hnrnp A1 20 telomere telomere telomere Telomeretelomerase telomerase 21 RNA telomerettaggg telomere Telomerase telomere RNA (TER)catalytic TERT htertelomere telomere template domainpseudoknot structure, CR4-CR5 domain, H/ACA boxcr7 domain 22,23 telomerase hter telomerase associated protein 1 (TEP1) htert 24,25 htert1132 htert T-motif 6motif telomerase htert promoter alternative splicing, 26 htert htert
c-myc 27 Mad 28 htert mrnaalternative splicing domain A 12 htert α, domain Adomain B 182 htert β, α β 26 protein kinase htert telomerase 29 telomere Hayflick limit 30, (transformation) telomerase crisis telomerase (immortalization) telomerase, 31,32 telomere 33-36 31 Telomerase telomerase telomerase, hter hter anti-sense nucleotide, 37-39 hammerhead ribozyme, catalytic component htert TelomeraseRNA (reverse transcriptase, RT) htert telomerase Dideoxyguanosine (ddg), dideoxyinosine (ddi), 3 -azido-2,3 -dideoxythymidine (AZT), AZT- 5' triphosphate (AZT-TP) retrovirus 40,41 AZT ADIS AZT thymidine kinase AZT-MP,
thymidylate kinase AZT-DP, nucleoside diphosphate kinase AZT-TP, 42 AZT-TP DNAthymidine DNA AZT-MP DNA DNA templateprimer 43-46 AZT, 47, 48 AZT 49,50 AZT 51, AZTtelomeric 40,52-56 DNA telomerase telomere in vitro AZT telomerase telomerase subunit telomere, telomerase telomerase
1 MCF-7 (ATCC HTB 22)MDA-MB-231 (ATCC HTB 26) Primer oligonucleotidegenset (Singapore Biotech Pte Ltd, Singapore), Amersham Pharmacia (Piscataway, NJ, USA), Sigma (St Louis, MO, USA) 2 MCF-7MDA-MB- 231minimum essential medium (MEM, GIBCO BRL, Grand Island, NY, USA) 10% (FBS, GIBCO BRL, Grand Island, NY, USA)penicillin 100 unit/ streptomycin 100 /(GIBCO BRL, Grand Island, NY, USA) 37 o C, 5% CO 2 2-4 hemocytometer 5 3 passage 025% Trypsin -EDTA (GIBCO BRL, Grand Island, NY, USA) -70 population doubling (PD) 2 PD = (dilution factor) log 2 PD = log (dilution factor)
PD = log (dilution factor) / log 2 = log 5/ log 2 = 232 PD = 232 x (passage number) MTT MTT 57 MCF-7MDA-MB-231 025% Trypsin-EDTA, 10% FBS MEM Trypan blue, 180 5 x 10 3 96-well plate 37 o C, 5% CO 2 16 AZT, Plate4 2 /MTT 50 well 4 plate450 x g 10 formazan 150 dimethyl sulfoxide (DMSO) 37 o C 10 formazan ELISA reader540 58 inhibitory concentration, 10% (IC 10 ), IC 20 (%) = x 100 5 x 10 4
culture plate 24 6 hemocytometer 3well log phage (doubling time, DT) 59 Senescence-associated â galactosidase stain â galactosidase 60 24-well plate5 x 10 4 16 37 o C, 5% CO 2 plate phosphate bufferedsaline(pbs) 2% formaldehyde / 02% glutaraldehyde ( 3 % formaldehyde) 5 PBS senescence-associated β- galactosidase stain [ 40 mm citric acid/sodium phosphate buffer (ph60), 150 mm NaCl, 5 mm potassium ferrocyanide, 5 mm potassium ferricyanide, 2 mm MgCl 2 ] 20 /X- Gal / 37 16 PBS 100 5 DNA Apoptaq Peroxidase In Situ Apoptosis Detection Kit (Intergen company, Purchase, NY, USA) 1 x 10 4 96-well plate 16 PBS
1% paraformaldehyde 10 ethanol : acetic acid 2:15-20 Kit dtd 37, 1 Peroxidase substrate diaminobenzidine (DAB, DAKO Corporation, Carpinteria, CA, USA)30 05% methyl green 15 100 5 TRAP assay telomerase Telomerase telomeric repeat-amplification protocol (TRAP) assay 61 PCR telomerase substrate TS oligonucleotide primertelomerase TTAGGG ACX primerreverse primer PCR Telomerase telomerase 293 (ATCC CRL 1573) CHAPS lysis buffer TRAP Taq polymerase 62,63 PCR 36 internal control primer PCR PBS 1 TRAP [10 mm HEPES-KOH (ph75), 15 mm MgCl 2, 10 mm KCl, 1 mm dithiothreitol] 10000 x g, 4 1 lysis buffer [10 mm Tris-HCl (ph75), 1 mm MgCl 2, 1 mm EGTA, 01 mm PMSF, 5 mm β- mercaptoethanol, 10% glycerol] 50 30 14000 x g4 30 Bradford method
31 05 01 TS, 01 BSA, 625µM deoxynucleotide triphosphates (dntps) TRAP [20 mm Tris-HCl (ph83), 15 mm MgCl 2, 63 mm KCl, 0005% Tween 20, 1 mm EGTA] 20 30 10 PCR 15 mm MgCl 2, 40 nm dntp, 4 ng NT primer, 32 x 10-21 M TSNT pr imer, 50 ng ACX primer, 125 unit Taq polymerase (GIBCO BRL, Grand Island, NY, USA), 25 µci [α- 32 P]dCTP (Amersham Pharmacia, Piscataway, NJ, USA) PCR Thermocycle 94 o C 3 94 o C 30, 60 o C 30 27 PCR 10 12% polyacrylamide gel autoradiography primer oligonucleotide Table 1 telomerase R8 control template(r8) total product generated(tpg) TPG = ( T B ) / CT (R8 B) / CR8 x 100 T : total intensity of telomerase- mediated bands from tested extract B : intensity from the negative control (background) CT : intensity from IC of tested extract R8 : intensity from R8 control CR8 : intensity from IC of R8 RT-PCR telomerase subunits (htert, hter) TRIzol-reagent (GIBCO BRL, Grand Island, NY, USA) RNA cdna First Strand cdna Synthesis Kit (MBI Fermentas, Vilnius, Lithuania) 16 RNA1 mm dntps, 02 random hexamer primer, 20 units ribonuc lease
inhibitor, 40 units M- MLV reverse transcriptase [250 mm Tris-HCl (ph 83), 250 mm KCl, 20 mm MgCl 2, 50 mm DTT] 20 37 1 70 10 cdna cdna 5 15 µci [α- 32 P]dCTP, 5 mm dntp, 15 units Taq polymerase PCR htertpcr 5 pm primer TERT 1784S, TERT 1928A house-keeping gene â-actin Internal control primer 774, 77525 pm 25 PCR 94 3 9430, 6030, 7230 30 hterpcr 25 pm primer TR-46S, TR-148A5 pm â-actin internal control primer 5899, 5900 94 3 94 30, 6030, 7230 22 PCR PCR 5% polyacrylamide gel autoradiography 64 sample â-actin internal control PCR bandintensity Real-time PCR htep, c-myc, Mad1 cdna telomerase subunits real time PCR htert, hter, htep, c-myc, Mad1 primertable 1 cdnaquantitect SYBR Green PCR Kit (QIAGEN, Santa Clarita, CA, USA) Kit HotstarTaq DNA polymerase, QuantiTect SYBR Green PCR buffer, dntp mix including dutp, SYBR Green, ROX (passive reference dye), 5 mm MgCl 2 QuantiTect SYBR Green PCR Master Mix PCR primer 05 µm 20 PCR
Rotor-Gene 2072D (Corbett Research, Australia) 95 15 94 15, 60 20, 72 20 45 PCR Table 1 Primer oligonucleotide sequence Primer TS 5 -AATCCGTCGAGCAGAGTT-3 ACX 5 -GCGGCGGCTTACCCTTACCCTTACCCTA-3 TSNT 5 -AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT -3 NT 5 -ATCGCTTCTCGGCCTTTT-3 R8 5 -AATCCGTCGAGCAGAGTTAG[GGTTAG] 7-3 TERT 1784S 5 -CGGAAGAGTGCTCTGGAGCAA-3 TERT 1928A 5 -GGATGAAGCGGAGTCTGGA-3 â-actin 774 5 -GGGAATTCAAAACTGGAACGGTGAAGG-3 â-actin 775 5 -GGAAGCTTATCAAAGTCCTCGGCCACA-3 TR-46S 5 -CTAACCCTAACTGAGAAGGGCGTAG-3 TR-148A 5 -GAAGGCGGCAGGCCGAGGCTTTTCC-3 â-actin 5899 5 -CAGGTCATCACCATTGGCAATGAGC-3 â-actin 5900 5 -CGGATGTCCACGTCACACTTCATGA-3 c-myc-s 5 -AAGTCCTGCGCCTCGCAA-3 c-myc-as 5 -GCCTGTGGCCTCCAGCAGA-3 Mad1-S 5 -TTCAGACTTGGACTGTGTCA-3 Mad1-AS 5 -ACGCTGAGAGATGAAGTTGT-3 htep-s 5 -TCAAGCCAAACCTGAATCTGAG-3 htep-as 5 -CCCGAGTGAATCTTTCTACGC-3
Southern blot hybridization terminal restriction fragment (TRF) 300 /Proteinase K50 /RNase A DNA lysis buffer, phenol/chloroform ethanol DNA 10 DNA Hinf (Promega, Madison, WI, USA) 15 unit DNA 05 /ethidium bromide (EtBr) 08% agarose gel DNAgel loading buffer UV-transilluminatorgel DNA size markersize Gel EtBrgel loading buffer (05 M NaOH, 15 M NaCl)gel 30 2 gel (15 M NaCl, 05 M Tris-Cl, ph 80) 30 GelDNA Hybond-N membrane (Amersham Pharmacia, Piscataway, NJ, USA) transfer capillary transfer 10X SSC (15 M NaCl, 015 M sodium citrate) 16 DNA transfer Transfer UV transilluminater transfer membranedna UV cross linking membrane42 1 pre-hybridization Pre-hybridization lable oligonucleotide probe 42 16 hybridization Pre-hybridization Rapid-Hyb buffer (Amersham Pharmacia, Piscataway, NJ, USA) hybridization Probetelomere 5 -[TTAGGG] 6-3 [ã- 32 P]ATP(3000 Ci/mM)end-lable 30 pm [TTAGGG] 6 50 pm [ã- 32 P]ATP 20 unitt4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA) 37 1 01 mm EDTA(pH 80) MicroSpin TM G- 25 column (Amersham Pharmacia, Piscataway, NJ, USA) spin column chromatography
Hybridization membrane 1 (2X SSC, 02% SDS) 15 2 2 (01X SSC, 01% SDS) 42 10 01X SSCautoradiography TRF 11 TRF(mean) = (ODi x Li) / (ODi) ODi : signal intensity over interval i Li : kilobase size at the middle of interval i
1 MTT assay AZT AZT MTT assay AZT 10% (10% growth inhibitory concentration, IC 10 )20% (IC 20 ) AZT(low dose; DL)MCF-7 20 ìm, MDA-MB-23125 ìm AZT(high dose; DH)MCF-7 70 ìm, MDA-MB231 50 ì M (Fig1) (CT) Figure 1 Dosage determinant by MTT assay (A) MCF-7, (B) MDA-MB-231 2 AZT
MCF-7 7PD PD, 53PD high dose low dose (60PD) MDA-MB-231 42PD low dosehigh dose 90PD 50%, 66% (Fig 2) PDAZT (Table 2) Table 2 Growth inhibition rate in MCF-7 and MDA-MB-231 +: AZT treatment, -: removal of AZT
Figure 2 Growth curve of MCF-7 and MDA-MB-231 (A) MCF-7, (B) MDA- MB-231 CT(): control, DL(): low dose, DH():high dose, PD: population doubling
3 AZT telomerase X-gal MCF-7 low dosehigh dose PD PD 30PD (28PD: 2%, low dose 5%, high dose 13%) 53PD low dose 12%, high dose 15% 28% 4, 5 AZT, 77PD low dose, high dose MDA-MB-231 42PD 2% low dose, high dose 54%, 58% 25 56PD 10 PD (Fig 3)
AZT (+) (-) Figure 3 Expression of senescence by SA â-gal stain in breast cancer cells (A,C) MCF-7, (B,D) MDA-MB-231 CT(): control, DL(): low dose, DH(): high dose, PD: population doubling, +: AZT treatment, -: removal of AZT
4 AZT dtd apoptotic -body MCF-7, PD 1% PD low dose 28PD 22% PD (08%) 3 high dose(28%) PD, 42PD (07%) low dose(3%), high dose(37%) AZT MDA-MB-231, PD, 50PD low dose, high dose 49PD (08%) low dose(26%), high dose(29%) 3, PD (Fig 4)
AZT (+) (-) Figure 4 Apoptosis detection by dtd enzyme in breast cancer cells (A,C) MCF-7, (B,D) MDA-MB-231 CT(): control, DL(): low dose, DH(): high dose, PD: population doubling, +: AZT treatment, -: removal of AZT
5 TRAP assaytelomerase Telomerase TRAP assay 293 telomerase, lysis buffer PCR TRAP band IC band quality control TRAP assay, PD telomerase, AZT telomerase MCF-7, low dosehigh dose PD 42PD 53PD AZT telomerase, AZT low dosehigh dose telomerase (Fig 5) MDA-MB-231 low dose, high dose telomerase MCF-7 low dose, high dose 42PD Telomerase PD MCF-7 (Fig 6) MDA- MB-231MCF-7 telomerase MCF-7 AZT telomerase
Figure 5 Telomerase activity of MCF-7 by TRAP as say IC: internal control, R8: R8 control template, 293: 293 cell, lysis B: lysis buffer, +: AZT treatment, -: removal of AZT
Figure 6 Telomerase activity of MDA-MB-231 by TRAP assay IC: internal control, R8: R8 control template, +: 293 cell, -: lysis buffer
6 RT-PCR telomerase subunits Telomerase htert hter RT-RCR, PCR â-actin primer telomerase htert AZT MCF-7 low dose 10PD htert, PD High dose 53PD AZT htert hter htert MCF-7 low dose 28PD, AZT High dose (Fig7) MDA-MB-231 htert low dose 20PD PD High dose PD htert PD hter low dosehigh dose 42PD hter (Fig8)
Figure 7 Expression of htert and hter mrna of MCF-7 by RT-PCR CT(): control, DL(): low dose, DH(): high dose, +: AZT treatment, - : removal of AZT
Figure 8 Expression of htert and hter mrna of MDA-MB-231 by RT- PCR CT(): control, DL( ): low dose, DH( ): high dose
7 Real-time PCR htep, c-myc, Mad1 Real-time PCR telomerase RNA RT-PCR htert, hter real-time PCR RT-PCR real-time PCR RT-PCR RT-PCR MCF-7 low dose hterthter RT-PCR c-myc AZT htert Mad1 PD c-myc, AZT c-myc htep PD, 40PD high dose RT-PCR hterthter, htert AZT hter htert htert c-myc, low dosec-myc Mad1hTERT c-myc Mad1 htep low dose PD MCF-7 low dosehigh dose htert c-myc hter, htep Mad1hTERTc-Myc MDA-MB-231 low dose htert
, htert hter20pd, PD c-myc htert, htert Mad1, htep PD PD High dose low dose htert, hter Mad1, htep10pd c-myc htert MDA-MB-231 htert,, c-myc hter, Mad, htep (Fig 9)
Figure 9 Expression of telomerase subunits mrna by real-time PCR (A)(B) MCF-7, (C)(D) MDA-MB-231 PD: population doubling, - : control, : htert, : hter, : c-myc, : Mad1, : htep
8 Southern Blot TRF Telomere Southern Blot MCF-7 PD telomere low dosehigh dose telomere 35PD 35PD telomere AZT telomere MDA-MB-231 telomere low dosehigh dose 42PD PD (Fig 10) 9 AZT AZT (,, ) (telomerase, telomerase subunits, telomere ) (Fig 11)
Figure 10 Length of terminal restriction fragments (TRFs) by Southern blot hybridization (A) MCF-7, (B) MDA-MB-231 CT: control, DL: low dose, DH: high dose
Figure 11 Time points of change pattern in biological and genetic phenotype (A) MCF-7, (B) MDA-MB-231 Remove the AZT at 53PD of low dose and high dose cells in MCF-7
telomere telomerase subunits AZTADIS telomere 42-46 47,48 IC 10, IC 20 AZT MTT assay AZT AZT,, AZT AZT AZT, AZT telomere G 0 AZT telomeredna DNA cell cycle DNA
DNA AZT telomerase telomere, telomere telomerase telomerase, AZT AZT AZT DNA PD AZTtelomerase telomere 65-68 AZT AZT G 0 AZT TRAP assay telomerase, PD AZT telomerase, AZT AZT telomerase AZT telomerase
telomerase subunit htert, hter, htep telomerase htert PD telomerase Telomerase htert PD, telomerase htert AZT htert, telomerase telomerase htert, telomerase htert htert telomerase htertazt hter htert hterazt htert AZT hter htert hterhtert telomerase 22,69 hter telomerase, telomerase 70,71 hterttelomerase 6,72-74 26,29,75,76 htert telomerase htert telomerase telomerase, hter telomerase, AZT htert htert hter hter htert
telomerase subunits telomerase, telomerase htert hterhtert hterthter telomerase subunithteptelomerase c-myc, Mad1 htep htepazt c-myc htert, htert c-mychtert 27 AZTc-Myc c-myc htert Mad1 AZT htert, htert AZT telomere telomere AZT telomere, AZT telomere Telomere htert telomerase, AZT htert telomerase telomere MDA-MB231 40PD AZT telomerase MDA-MB-231MCF-7,
IC 20 AZT, MCF-7MDA-MB-231 telomerase MDA-MB-231MCF-7, telomere MDA-MB-231 AZT telomerase 20% 80% telomerase TRAP assay AZT IC 20 MCF-7 population doubling, MDA-MB-231 telomerase telomere telomere MCF-7 AZT MCF-7, telomerase subunits telomerase, telomere, MDA-MB-231
Telomerase AZT Telomerase AZT htert, c-myc hter, htep telomerase subunits telomerase telomere
1 Harley CB Telomere loss: mitotic clock or genetic time bomb? Mutat Res 1991; 256: 271-282 2 Blackburn EH Structure and function of telomeres Nature 1991; 350: 569-573 3 Zakian VA Structure and function of telomeres Annu Rev Genet 1989; 23: 579-604 4 Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, et al Telomere shortening associated with chromosome instability is arrested in immortal cells with express telomerase activity EMBO J 1992; 11: 1921-1929 5 van Steensel B, Smogorzewska A, de Lange T TRF2 protects human telomeres from end-to-end fusions Cell 1998; 92: 401-413 6 Zakain VA Structure, function and replication of Saccharomyces cerevisiae telomeres Annu Rev Genet 1996; 30: 141-172 7 Greider CW Telomere length regulation Annu Rev Biochem 1996; 65: 337-365 8 McClintock B The stability of broken ends of chromosomes in Zea mays Genetics 1941; 26: 234-282 9 Gilson E, Laroche T, Gasser SM Telomeres and the functional architecture of the nucle us Trends Cell Biol 1993; 3: 128-134 10 Gottschling DE, Aparicio OM, Billington BL, Zakian VA Position effect at
S cereviasae telomeres: reversible repression of Pol transcription Cell 1990; 63: 751-762 11 Harley CB, Futcher AB, Greider CW Telomere shorten during aging of human fibroblasts Nature 1990; 345: 458-460 12 Allsopp RC, Vaziri H, Patterson C, Goldstei S, youglai EV, Futcher AB, et al Telomere length predicts replicative capacity of human fibroblast Proc Natl Acad Sci USA 1992; 89: 10114-10118 13 Faragher RG, Kipling D How might replicative senescence contribute to human ageing? Bioessays 1998; 20: 985-991 14 Campisi J The biology of replicative senescence Eur J Cancer 1997; 33: 703-709 15 Norsgaard H, Clark BF, Rattan SI Distinction between differentiation and senescence and the absence of increased apoptosis in human keratinocytes undergoing cellular aging in vitro Exp Gerontol 1996; 31: 563-570 16 van Steensel B, de Lange T Control of telomere length by the human telomeric protein TRF1 Nature 1997; 385: 740-743 17 Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, et al Telomerase catalytic subunit homologs from fission yeast and human Science 1997; 277: 955-959 18 Harrington L, McPhail T, Mar V, Zhou W, Oulton R, Bass MB, et al A mammalian telomerase-associated protein Science 1997; 275: 973-977 19 Nakamura J, Salito M, Nakamura H, Matsuura A, Ishikayawa F TLP1: a gene encoding a protein component of mammalian telomerase is a novel member of WD repeats family Cell 1997; 88: 875-884
20 LaBranche H, Dupuis S, Ben-David Y, Bani MR, Wellinger RJ, Chabot B Telomere elongation by hnrnp A1 and a derivative that interacts with telomeric repeats and telomerase Nat Genet 1998; 19: 199-202 21 Blackburn EH Telomerase Annu Rev Biochem 1992; 61: 113-129 22 Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chin CP, et al The RNA component of human telomerase Science 1995; 269:1236-1241 23 Romero DP Blackburn EH A conserved secondary structure for telomerase RNA Cell 1991; 67: 343-353 24 Nugent CI, Lundblad V The telomerase reverse transcriptase: components and regulation Genes Dev 1998; 12: 1073-1085 25 Harrington L, Zhou W, McPhail T, Oulton R, Yeung DS, Mar V, et al Human telomerase contains evolutionalarily conserved catalytic and structural subunits Genes Dev 1997; 11: 3109-3115 26 Kilian A, Bowtell DD, Abud HE, Hime GR, Venter DJ, Keese PK, et al Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types Hum Mol Genet 1997; 6: 2011-2019 27 Wang J, Xie LY, Allan S, Beach D, Hannon GJ Myc activates telomerase Genes Dev 1998; 12: 1769-1774 28 Oh S, Song YH, Yim J, Kim TK Identification of Mad as a repressor of the human telomerase (htert) gene Oncogene 2000; 19: 1485-1490 29 Li h, Zhao LL, Funder JW, Liu JP Protein phosphatase 2A inhibits nuclear telomerase activity in human breast cancer cells J Biol Chem 1997; 272: 669-677
30 Hayflick L The cell biology of human aging N Engl J Med 1976; 295: 1302-1308 31 Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, et al Specific association of human telomerase activity with immortal cells and cancer Science 1994; 266: 2011-2015 32 Wright WE, Shay JW The two-stage mechanism controlling cellular senescence and immortalization Exp Gerontol 1992; 27: 383-389 33 Colgin LM, Reddel RR Telomere maintenance mechanisms and cellular immortalization Curr Opin Gen Dev 1999, 9; 97-103 34 Bryan TM, Englzou I, Gupta J, Bacchetti S, Reddel RR Telomere elongation in immortal human cells without detectable telomerase activity EMBO J i995; 14: 4240-4248 35 Murnane JP, Sabatier L, Mardur BA, Morgan WF Telomere dynamics in an immortal human cell line EMBO J 1994; 13: 4953-4962 36 Bryan TM, Englezou A, Dalla -Pozza L, Dunham MA, Reddel RR Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines Nature Med 1997; 3: 1271-1274 37 Kondo Y, Kondo S, Tanaka Y, Haqqi T, Barna BP, Cowell JK Inhibition of telomerase increases the susceptibility of human malignant glioblastoma cells to cisplatin-induced apoptosis Oncogene 1998; 16: 2243-2248 38 Mata JE, Joshi SS, Palen B, Pirruccello SJ, Jackson JD, Elias N, et al A hexameric phosphorothioate oligonucleotide telomerase inhibitor arrests growth Burkitt s lyphoma cezls in vitro and in vivo Toxicol Appl Pharmacol 1997; 144: 189-197
39 Kondo S, Tanaka Y, Kondo Y, Hitomi H, Baroett GH, Ishizakd Y, et al Antisense telomerase trea5ment: induction of mwo distinct pathways, apoptosis and8difkerentiation FASEB J 1998; 12:(801-811 40 Strahh C, Blackburn EH Effects of reverse transcriptase inhibitors on telomere length and telomarase activity in two immortalized huan cell lines Mol Cell Biol 1996; 16: 53-65 41 Strahl C, Blackburn EH The effects of nucleoside analogues on telomerase and telomeres in Tetra hymena Nucleic Acds Res 1994; 22: 893-900 42 Furman PA, Fyfe JA, St Clair MH, Weinhold K, Rideout JL, Freeman GA, et al Phosphorylation of 3 -azido-3 -deoxythymidine and selective interaction of the 5 -triphosphate with humanrimmunodeficiency virus reverse transcriptàse Proc Natl Acad Sci USA 1986; 83: 8333-8337 43 Agarwal RP, Mian AK Thymidine and zidovudine metabolism in chro ically zidovudije -exposed cells in vitro Bio;0em Pharmacol 1991; 42: 905-911 44 Sommadossi JP, Carlisle R, Zhou Z Cellular Pharmacology of 3 -azido- 3 - deoxythymidine#with evidence of incorporation into DNA of human bone marrow cells Mol Pharmacol 1989; 36: 9-14 45 Furman PA, Barry DW Spectrum of antiviral activity and mechanism of action of zidovudine Am J Med 1988; 85: 176-181 46 Heidenreich O, Kruhoffer M, Grosse F, Eckstein F Inhibition of human immunodeficiency virus 1 reverse transcriptase by 3 -azidothymidine triphosphate Eur J Biochem 1990; 192: 621-625 47 Yarchoan R, Mitsuya H, Myers CE, Broder S Clinical pharmacology of 3 - azido-2, 3 -dideoxythymidine (zidovudine) and related dideoxynucleosides
N Engl J Med 1989; 321: 726-738 48 Sommadossi JP, Carlisle R Toxicity of 3 -azido-3 -deoxythymidine and 9- (1,3- dihydroxy- 2-propoxymethyl) guanine for normal human hematopoietic progenitor cells in vitro Antimicrob Agents Chemother 1987; 31: 452-454 49 Ayers KM, Clive D, Tucker WE Jr, Hajian G, de Miranda P Nonclinical toxicology studies with zidovudine: Genetic toxicity tests and carcinogenicity bioassays in mice and rats Fundam Appl Toxicol 1996; 32: 148-158 50 Olivero OA, Beland FA, Fullerton NF, Poirier MC Vaginal epithelial DNA damage and expression of preneoplastic markers in mice during chronic dosing with tumorigenic levels of 3 -azido-2, 3 -dideoxythymidine Cancer Res 1994; 54: 6235-6242 51 Olivero OA, Poirier MC Preferential incorporation of 3 -azido 2, 3 - dideoxythimidine into telomeric DNA and Z DNA-containing regions of Chinese hamster ovary cells Mol Carcinog 1993; 8: 81-88 52 Falcone A, Lencioni M, Brunetti I, Pfanner E, Allegrini G, Antonuzzo A, et al Maximum tolerable doses of intravenous zidovudine in combination with 5-fluorouracil and leucovorin in metastatic colorectal cancer patients Clinical evidence of significant antitumor activity and enhancement of zidovudine-induced DNA single strand breaks in peripheral nuclear blood cells Ann Oncol 1997; 8: 539-545 53 Posner MR, Darnowshi JW, Calabresi P, Brunetti I, Corvese D, Curt G, et al Oral zidovudine, continuous- infusion fluorouracil, and oral leucovorin calcium: a Phase 1 study J Natl Cancer Inst 1990; 82: 1710-1714 54 Posner MR, Darnowski JW, Weitberg AB, Dudley MN, Corvese D, Cummings FJ, et al High-dose intravenous zidovudine with 5-fluorouracil
and leucovorin Cancer 1992; 70: 2929-2934 55 Melana SM, Holland JF, Pogo BG Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by 3 -azido- 3 -deoxythymidine Clin Cancer Res 1998; 4: 693-696 56 Murakami J, Nagai N, Shigemasa K, Ohama K Inhibition of telomerase activity and cell proliferation by a reverse transcriptase inhibitors in gynaecological cancer cell lines Eur J Cancer 1998; 35: 1027-1034 57 Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB Evaluation of a tetrazolium- based semiautomated colorimetric assay: assessment of radiosensitivity Cancer Res 1987; 47: 943-946 58 Mosmann T Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays J Immunol Methods 1983; 65: 55-63 59 McAteer JA, Davis J Basic cell culture technique and the maintenance of cell line, In: J M Davis (ed), Basic cell culture: A Practical Approach, 93-145 New York: IRL press at Oxford University Press 60 Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelly C, et al A biomarker that identifies senescence human cells in culture and in aging skin in vivo Proc Natl Acad Sci USA 1995; 92: 9363-9367 61 Kim NW, Wu F Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP) Nucleic Acids Res 1997; 25: 2595-2597 62 Piatyszek MA, Kim NW, Weinrich SL, Hiyama K, Hiyama E, Wright WE, et al Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP) Methods Cell Sci 1995; 17:
1-15 63 Hiyama E, Yokoyama T, Tatsumoto N, Hiyama K, Imamura Y, Murakami Y, et al Telomerase activity in gastric cancer Cancer Res 1995; 55: 3258-3262 64 Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR 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 65 Goldstein S Replicative senescence: The human fibroblast comes of age Science 1990; 249: 1129-1133 66 Stein GH Dulic V Origins of G 1 arrest in senescent human fibroblasts Bioessays 1995; 17: 537-543 67 Campisi J, Dimri GP, Nehlin JO, Testori A, Yoshimoto K Coming of age in culture Exp Gerontol 1996; 31: 7-12 68 Campisi J Replicative senescence : An old lives tale? Cell 1996; 84: 497-500 69 Ogoshi M, Le T, Shay JW, Taylor RS In situ hybridization analysis of the expression of human telomerase RNA in normal and pathologic conditions of the skin J Invest Dermatol 1998; 110: 818-823 70 Wen J, Cong YS, Bacchetti S Reconstitution of wild- type or mutant telomerase activity in telomerase-negative immortal human cells Hum Mol Genet 1998; 7: 1137-1141 71 Mitchell JR, Wood E, Collins K A telomerase component is defective in the human disease dyskeratosis congenita Nature 1999; 402: 551-555
72 Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al hest2, the putative human telomerase catalytic subunit gene is upregulated in tumor cells and during immortalization Cell 1997; 90: 785-795 73 Nakayama J, Tahara H, Tahara E, Saito M, Ito K, Nakamura H, et al Telomerase activation by htrt in human normal fibroblasts and hepatocellular carcinomas Nat Genet 1998; 18: 65-68 74 Takakura M, Kyo S, Kanaya T, Tanaka M, Inoue M Expression of human telomerase subunits and correlation with cervical cancer Cancer Res 1998; 58: 1558-1561 75 Ku WC, Cheng AJ, Wang TC Inhibition of telomerase PKC inhibitors in human nasopharyngeal cancer cells in culture Biochem Biophys Res Commun 1997; 241: 730-736 76 Kang SS, Kwon T, Kwon DY, Do SI Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit J Biol Chem 1999; 274: 13085-13090
The dynamics of the telomerase activity and the telomerase subunit genes with reverse transcriptase inhibitor treatment in human cancer cells Hyun Jung Ji Brain Korea 21 project for Medical Sciences The Graduate School, Yonsei University (Directed by Professor Hyun Cheol Chung) Shortening of the telomeric DNA at chromosome ends is postulated to limit the life span of human cell In contrast, activation of telomerase, the reverse transcriptase that synthesized telomeric DNA, is proposes to be essential step in cancer cell immortalization and cancer progression Several reports have described about attempts to inhibit telomerase activity using reverse transcriptase inhibitors 3 -azido-2,3 -dideoxythymidine (AZT), a reverse transcriptase inhibitor, was reported to incorporate in telomeric sequences immortalized cells in culture and shown to suppress the activity of telomerase and inhibit the cell proliferation In this study, we induced cancer cell senescence after long-term treatment of AZT with IC 10, IC 20 dose After inducing senescence, we investigated the dynamics of telomerase subunit (htert, hter, htep), htert transcription
factors (c-myc, Mad1), telomerase activity, and finally, telomere length in MCF-7 and MDA-MB-231 cell lines We demonstrated evidences of senescence, apoptosis and growth delay after AZT treatment Also, AZT-treated cancer cells have shown inhibition of telomerase activity and shortening of telomere length in a dose- and durationdependent way Among telomerase subunits, htert and c-myc were the first factors which changed after AZT treatment followed by the changes of hter, Mad1 and htep In conclusion, the suppression of the htert and c-myc by AZT treatment was the initial genetic phenomenon followed by the change of hter, Mad1 and htep Key Words : telomerase, telomere, reverse transcriptase inhibitor, senescence, htert, hter, htep, c-myc, Mad1