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1 Telomerase Telomerase Subunit

2 Telomerase Telomerase Subunit

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5 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

6 16 1 MTT assay AZT TRAP assaytelomerase 23 6 RT-PCR telomerase subunit 26 7 Real-time PCR htep, c-myc, Mad Southern blot TRF 32 9 AZT

7 Figure 1 Dosage determinant by MTT assay 16 Figure 2 Growth curve of MCF-7 and MDA-MB 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

8 Table 1 Primer oligonucleotide sequence 13 Table 2 Growth inhibition rate in MCF-7 and MDA-MB

9 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

10 : Telomerase, Telomere, htert, hter, htep, c-myc, Mad1,,

11 Telomerase Telomere Subunit (senescence) telomere telomere, 1, 'mitotic clock' telomere DNA (TTAGGG) n

12 2 Telomere DNA, 3-10 DNA polymerase 'end-replication problem' telomere nucleotide 11,12 telomere, 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

13 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 Telomerase telomerase telomerase, hter hter anti-sense nucleotide, 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,

14 thymidylate kinase AZT-DP, nucleoside diphosphate kinase AZT-TP, 42 AZT-TP DNAthymidine DNA AZT-MP DNA DNA templateprimer AZT, 47, 48 AZT 49,50 AZT 51, AZTtelomeric 40,52-56 DNA telomerase telomere in vitro AZT telomerase telomerase subunit telomere, telomerase telomerase

15 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 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)

16 PD = log (dilution factor) / log 2 = log 5/ log 2 = 232 PD = 232 x (passage number) MTT MTT 57 MCF-7MDA-MB % Trypsin-EDTA, 10% FBS MEM Trypan blue, x 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 reader inhibitory concentration, 10% (IC 10 ), IC 20 (%) = x x 10 4

17 culture plate 24 6 hemocytometer 3well log phage (doubling time, DT) 59 Senescence-associated â galactosidase stain â galactosidase well plate5 x 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 / PBS DNA Apoptaq Peroxidase In Situ Apoptosis Detection Kit (Intergen company, Purchase, NY, USA) 1 x well plate 16 PBS

18 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 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] 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] x g4 30 Bradford method

19 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] PCR 15 mm MgCl 2, 40 nm dntp, 4 ng NT primer, 32 x 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 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

20 inhibitor, 40 units M- MLV reverse transcriptase [250 mm Tris-HCl (ph 83), 250 mm KCl, 20 mm MgCl 2, 50 mm DTT] 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, pm 25 PCR , 6030, hterpcr 25 pm primer TR-46S, TR-148A5 pm â-actin internal control primer 5899, , 6030, 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

21 Rotor-Gene 2072D (Corbett Research, Australia) , 60 20, 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 GGGAATTCAAAACTGGAACGGTGAAGG-3 â-actin GGAAGCTTATCAAAGTCCTCGGCCACA-3 TR-46S 5 -CTAACCCTAACTGAGAAGGGCGTAG-3 TR-148A 5 -GAAGGCGGCAGGCCGAGGCTTTTCC-3 â-actin CAGGTCATCACCATTGGCAATGAGC-3 â-actin 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

22 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 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) mm EDTA(pH 80) MicroSpin TM G- 25 column (Amersham Pharmacia, Piscataway, NJ, USA) spin column chromatography

23 Hybridization membrane 1 (2X SSC, 02% SDS) (01X SSC, 01% SDS) X SSCautoradiography TRF 11 TRF(mean) = (ODi x Li) / (ODi) ODi : signal intensity over interval i Li : kilobase size at the middle of interval i

24 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 ìm AZT(high dose; DH)MCF-7 70 ìm, MDA-MB ì M (Fig1) (CT) Figure 1 Dosage determinant by MTT assay (A) MCF-7, (B) MDA-MB AZT

25 MCF-7 7PD PD, 53PD high dose low dose (60PD) MDA-MB PD 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

26 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

27 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 PD 2% low dose, high dose 54%, 58% 25 56PD 10 PD (Fig 3)

28 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

29 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)

30 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

31 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

32 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

33 Figure 6 Telomerase activity of MDA-MB-231 by TRAP assay IC: internal control, R8: R8 control template, +: 293 cell, -: lysis buffer

34 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)

35 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

36 Figure 8 Expression of htert and hter mrna of MDA-MB-231 by RT- PCR CT(): control, DL( ): low dose, DH( ): high dose

37 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

38 , 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)

39 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

40 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)

41 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

42 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

43 telomere telomerase subunits AZTADIS telomere ,48 IC 10, IC 20 AZT MTT assay AZT AZT,, AZT AZT AZT, AZT telomere G 0 AZT telomeredna DNA cell cycle DNA

44 DNA AZT telomerase telomere, telomere telomerase telomerase, AZT AZT AZT DNA PD AZTtelomerase telomere AZT AZT G 0 AZT TRAP assay telomerase, PD AZT telomerase, AZT AZT telomerase AZT telomerase

45 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, ,29,75,76 htert telomerase htert telomerase telomerase, hter telomerase, AZT htert htert hter hter htert

46 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,

47 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

48 Telomerase AZT Telomerase AZT htert, c-myc hter, htep telomerase subunits telomerase telomere

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58 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

59 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