Telomerase: a promising target for cancer Scott Cohen Sydney - PowerPoint PPT Presentation

Telomerase: a promising target for cancer Scott Cohen Sydney Australia

linear chromosomes have ends! prokaryotic chromosome (circular) eukaryotic chromosome (linear) presence of chromosome “ends” posed new challenges to cellular biology

telomeres: distinguishing “end” from “break” how can the cell distinguish the chromosome end from a deleterious internal DNA break that requires repair? telomeres: the unique DNA/protein complexes at the ends of linear chromosomes

telomeres: unique DNA/protein complex human telomeres consist of tandem repeats of 5’-TTAGGG-3’: 5’-TTAGGGTTAGGGTTAGGG(TTAGGG) n TTAGGG-3’ centromere 3’-A A T C C C A A T C C C ~5-15 kb double-stranded DNA ~300-500 nt single-stranded TTAGGG 3’-overhang

telomeres: unique DNA/protein complex human telomeres consist of tandem repeats of 5’-TTAGGG-3’: 5’-TTAGGGTTAGGGTTAGGG(TTAGGG) n TTAGGG-3’ centromere 3’-A A T C C C A A T C C C Telomeric Repeat Factors 1,2 Protection of Telomeres 1 ( TRF1 , TRF2 ) ( POT1 ) telomeric DNA coated with sequence-specific DNA binding proteins

telomeres: unique DNA/protein complex human telomeres consist of tandem repeats of 5’-TTAGGG-3’: 5’-TTAGGGTTAGGGTTAGGG(TTAGGG) n TTAGGG-3’ centromere 3’-A A T C C C A A T C C C this unique DNA/protein structure distinguishes the telomere from an internal double-strand DNA break

telomeres: a conserved structure in eukaryotes the telomere: •a “cap” protecting the end of the chromosome, essential for genomic stability •a conserved structure in eukaryotic evolution 5’-TTAGGG-3’ 5’-TTGGGG-3’ human Tetrahymena

telomere shortening and cellular lifespan telomere length (kb) 9 8 7 6 5 4 0 20 40 60 80 100 population doublings

telomere shortening and cellular lifespan (arbitrary units) telomere length lymphocytes 0 10 20 30 40 50 60 70 80 90 age (yrs) Rufer et al., (1999) J. Exp. Med. 190 : 157-167.

telomere shortening and cellular lifespan senescence senescence: non-proliferative cellular state – permanent withdrawal from cell cycle telomere length senescence evolved as requisite marginal barrier to unlimited cellular proliferation….. cancer critical population doublings

telomere shortening and cellular lifespan senescence genetic mutations  activation of a telomere maintenance mechanism: telomere length marginal immortalisation critical Telomerase population doublings

specific association of telomerase activity and cancer cancer % telomerase positive breast carcinoma 88 colorectal carcinoma 86 small cell lung carcinoma 100 ovarian carcinoma 91 prostate carcinoma 90 telomerase NOT detected in corresponding normal tissue!!! Kim et al., (1994) Science 266 : 2011-2015.

Telomerase: a specialised reverse transcriptase hTR 3’ hTERT A A U C C C A A U C catalytic protein component: Telomerase Reverse Transcriptase ( hTERT ) TERT RT motifs motifs human Lingner et al., (1997) Science 276 : 561-567.

Telomerase: a specialised reverse transcriptase hTR 3’ hTERT A A U C C C A A U C catalytic protein component: Telomerase Reverse Transcriptase ( hTERT ) TERT RT motifs motifs human Tetrahymena Lingner et al., (1997) Science 276 : 561-567. Bryan et al., (1998) Proc. Natl. Acad. Sci. USA 95 : 8479-8484.

Telomerase: a specialised reverse transcriptase hTR 3’ hTERT A A U C C C A A U C catalytic protein component: Telomerase Reverse Transcriptase ( hTERT ) TERT RT motifs motifs Telomerase RNA ( hTR ) provides an internal template complementary to the telomeric DNA sequence: 5’- TTAGGG -3’

extension of telomere by telomerase: 1) binding to 3’-end hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G- 3’

extension of telomere by telomerase: 1) binding to 3’-end hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G- 3’

extension of telomere by telomerase: 2) nucleotide addition hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G- 3’

extension of telomere by telomerase: 2) nucleotide addition hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G G- 3’

extension of telomere by telomerase: 2) nucleotide addition hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G G G- 3’

extension of telomere by telomerase: 2) nucleotide addition hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G G G T- 3’

extension of telomere by telomerase: 2) nucleotide addition hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G G G T T- 3’

extension of telomere by telomerase: 2) nucleotide addition hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G G G T T A- 3’

extension of telomere by telomerase: 2) nucleotide addition hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G G G T T A G -3’

extension of telomere by telomerase: 3) translocation hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G G G T T A G -3’

extension of telomere by telomerase: 3) translocation hTR 3’ hTERT A A U C C C A A U C centromere TTAGGGTTAGGG T T A G G G T T A G -3’

direct activity assay for human telomerase extension of a telomeric primer with dTTP , dATP , α - 32 P-dGTP hTR 3’ hTERT A A U C C C A A U C 5’-TTAGGGTTAGGG T T A G G G dTTP dATP α - 32 P-dGTP

direct activity assay for human telomerase extension of a telomeric primer with dTTP , dATP , α - 32 P-dGTP hTR 3’ hTERT A A U C C C A A U C 5’-TTAGGGTTAGGG T T A G G G dTTP dATP α - 32 P-dGTP 5’-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG 5’-TTAGGGTTAGGGTTAGGGTTAGGGTTAG 5’-TTAGGGTTAGGGTTAGGGTTAG

my research objective in 2003 •size measurements indicated complex of ~0.7 – 1 MDa: hTERT (1132 aa) = 127 kDa hTR (451 nt) = 153 kDa = 280 kDa •35 distinct proteins proposed to associate with telomerase: total mass ~ 2.5 MDa the precise composition of the core human telomerase enzyme complex remained undefined

my research objective in 2003 •size measurements indicated complex of ~0.7 – 1 MDa: hTERT (1132 aa) = 127 kDa hTR (451 nt) = 153 kDa = 280 kDa •35 distinct proteins proposed to associate with telomerase: total mass ~ 2.5 MDa PURIFICATION

telomerase: the rarest enzyme in the human cell? ~20 – 50 molecules per cell ~1 part in 10 9 ~1 ng per 300,000,000 HEK293 cells

telomerase: a collaborative effort of CMRI & CSIRO George Lovrecz Tram Phan Louis Lu

telomere-substrate affinity purification hTR 3’ hTERT A A U C C C A A U C Avidin t 1/2 > 10 h TTAGGGTTAGGG T T A G G G bead

dissociation of telomeric DNA substrate 5’-TTAGGGTTAGGGTTAGGG 5’-TTAGGGTTAGGGTTAGGGT 5’-TTAGGGTTAGGGTTAGGGTT 5’-TTAGGGTTAGGGTTAGGGTTA 5’-TTAGGGTTAGGGTTAGGGTTAG 5’-TTAGGGTTAGGGTTAGGGTTAGG K d and k off between enzyme and substrate change as a function of the nucleotide at the 3’-end

dissociation of telomeric DNA substrate 5’-TTAGGGTTAGGGTTAGGG 5’-TTAGGGTTAGGGTTAGGGTTA K d and k off between enzyme and substrate change as a function of the nucleotide at the 3’-end

activity-dependent elution of telomerase hTR 3’ hTERT t 1/2 > 10 h A A U C C C A A U C TTAGGGTTAGGG T T A G G G hTR 3’ hTERT t 1/2 < 5 min A A U C C C A A U C TTAGGGTTAGGG T T A G G G T T A

activity-dependent elution of telomerase hTR 3’ hTERT t 1/2 > 10 h A A U C C C A A U C Avidin TTAGGGTTAGGG T T A G G G bead

activity-dependent elution of telomerase hTR 3’ hTERT t 1/2 > 10 h dTTP A A U C C C A A U C Avidin dATP TTAGGGTTAGGG T T A G G G bead hTR 3’ hTERT t 1/2 < 5 min A A U C C C A A U C Avidin TTAGGGTTAGGG T T A G G G T T A bead

activity-dependent elution of telomerase 110 100 k = 0.25 ± 0.05 min -1 relative activity 90 80 70 60 +dNTP 50 -dNTP 40 30 20 k = < 0.002 min -1 10 0 0 10 20 30 40 50 60 t (min)

mass spectrometry hTERT: dyskerin: TPVGQGSWAHPGR HGKPTDSTPATWK RVGDDVLVHLLAR TTHYTPLACGSNPLKR HRVTYVPLLGSLR QGLLDKHGKPTDSTPATWK RPGLLGASVLGLDDIHR KSLPEEDVAEIQHAEEFLIKPESK LVDDFLLVTPHLTHAK KWGLGPK AWNHSVR LLTSHKR RSPGVGCVPAAEHR SQQSAGKEYVGIVR ARPAEEATSLEGALSGTR ARRPGLLGASVLGLDDIHR 100 amino acids = 19% RGAAPEPERTPVGQGSWAHPGR PERTPVGQGSWAHPGR 168 amino acids = 15%

core human telomerase enzyme complex observed size: ~670 kD hTERT: 127 kD dyskerin: 58 kD hTR: 153 kD total mass: 338 kD

core human telomerase enzyme complex observed size: ~670 kD 2 x hTERT: 254 kD 2 x dyskerin: 116 kD 2 x hTR: 306 kD total mass: 676 kD

core human telomerase enzyme complex 2 x hTERT 2 x hTR 2 x dyskerin 315: 1850-1853 (2007)

telomerase over-expression in 293T cells hTR hTERT dyskerin • transient >>> stable •293 T >>> 293, 293 F • ~500-fold activity over endogenous •scalable to 20-L WAVE ( Tram, Louis )