Telomere chromosome integrity in breast cancer 2

CHAPTER INTRODUCTION 1.1: TELOMERES: Hermann Muller and Barbara McClintock first identified that the linear eukaryotic chromosomal ends are capped by a structure termed the telomeres (Muller, 1938; McClintock, 1938; McClintock, 1941). Telomeres are highly regulated, specialized nucleoprotein structures that prevent chromosomal ends from being recognized as DNA double stranded breaks as well as protect the chromosomes from degradation and chromosomal fusions (Greider, 1990; Blackburn, 1991; Greider, 1991). DNA damage checkpoints and repair machinery normally activated by DNA breaks are prevented from acting at the telomeres. Furthermore, telomeres are also thought to function in meiotic and mitotic pairing, and chromosome segregation during meiosis and mitosis (Pandita et al., 2007). Telomeres prevent the loss of coding and regulatory DNA at the chromosomal ends due to the premature replication termination by the end-replication problem (end-replication problem-discussed in section 1.1.2.1). Telomeres also aid in nuclear organization and transcriptional silencing ( Greider, 1990; Greider, 1991; Blackburn, 1991). 1.1.1: Telomeric structure: Telomeres are special nucleoprotein structures composing of a DNA component and telomere associated proteins. The guanine-rich hexameric DNA repeat sequence of the telomeres is non-coding and varies among different organisms ( Greider, 1990; Blackburn, 1991; Greider, 1991; Hahn, 2003). In mammals it is composed of 5’-TTAGGG-3’ repeats (Meyne et al., 1989; Moyzis et al., 1988; Greider, 1990; Blackburn, 1991; Greider, 1991) ranging from to 20 kb. In the classical view, the mammalian telomeres were thought to be linear structures (Figure 1A) with the DNA portion starting as double-stranded structures and terminating as single-stranded 3’G-rich overhangs of variable length ( Greider, 1990; Blackburn, 1991; Greider, 1991;). However, recent electron microscopic studies revealed that the telomere ends can form two loops that contribute to the secondary structure and stabilize the telomeres. This latter model suggests that the C terminal portion of telomeres folds back on itself to form a large telomere loop (T-loop) and the 3' Gstrand binds to the double-stranded telomere repeat sequence of the 5'-end, forming a displacement loop (D-loop) (Figure 1B) (Griffith et al., 1999a). In this way, the Tloop and D-loop mask the overhang structure and cap the telomeres. They play a protective role by sequestering the overhang terminal inside the double strand (Griffith et al., 1999a). A. The classical view B. The new view Figure 1: Classical and new views of telomere structure (A) Classical view of telomere structure displaying 3’G-rich overhang structure. (B) Modern view illustrating the D-loop and T-loop formation that is thought to stabilize the chromosomal ends (Griffith et al., 1999a). Telomere-specific proteins are involved in stabilising the telomeric secondary structure and have been identified at the telomeric ends to form a protein complex called the telosome (Figure 2) (Liu et al., 2004a). Telomere repeat binding factor (TRF1) was the first mammalian telomeric protein identified. It was isolated based on its in-vitro specificity for the double-stranded TTAGGG repeats (Zhong et al., 1992; Chong et al., 1995). Telomere repeat binding factor (TRF2) was identified from the database as a TRF1 paralog (Bilaud et al., 1997; Broccoli et al., 1997). Subsequently, TRF-interacting protein (TIN2) and human repressor activator protein (hRAP1) were found in two-hybrid screens with TRF1 and TRF2, respectively (Kim et al., 1999; Li et al., 2000). POT1 interacting protein TPP1 [TINT1 (Houghtaling et al., 2004), PTOP (Liu et al., 2004b) and PIP1 (Ye et al., 2004b) were identified during the search for TIN2-interacting proteins. Protection of telomeres (POT1), the most conserved component of telosome complex, was identified based on sequence homology to telomere end-binding factors in unicellular eukaryotes (Baumann and Cech, 2001). All the above proteins were found in a single complex in fractionated nuclear extracts (Liu et al., 2004a; Ye et al., 2004a). TRF1 and TRF2 bind directly to the double stranded telomeric DNA, whereas, POT1 binds to the G-rich singlestranded telomeric DNA (Figure 2) (De Lange, 2005a). hRAP1, TIN2 and PTOP1/TPP1/PIP1 are recruited by the directly binding proteins and bind indirectly to the telomeres ( Liu et al., 2004a; Chen et al., 2007;). The telosome complex caps and protects the telomeric ends from being recognized as double stranded breaks (De Lange, 2005a). Figure 2: Telosome complex: The six subunits of telosome complex include the proteins that directly or indirectly bind to the telomeric DNA. The directly binding proteins include those that bind to double-stranded portion (TRF1 and TRF2) or to single-stranded portion (POT1) of telomeric DNA. POT1 can also bind to the duplex telomeric DNA through TPP1. The indirectly binding proteins (TPP1, TIN2 and hRap1) include those that bind to the telomeric DNA through interaction with the directly interacting proteins. TIN2 binds to TRF1 and TRF2. hRap1 binds to TRF2 (Blasco, 2007). 1.1.2: Regulation of telomere function: Telomere function is determined by both its length and the stability of the secondary structure. Telomere length is a balance between the processes that shorten telomeres (end-replication problem) and the processes that stabilize the telomere length (telomerase) (telomerase will be discussed in section 1.1.2.2). Furthermore, the activity and expression of telomere associated proteins regulate telomere length and stability as well as telomerase access. 1.1.2.1: Telomeric end-replication problem: In normal human somatic cells, telomeres shorten with each cell division due to loss of terminal sequences due to the end-replication problem which was first hypothesised by Olovnikov and Watson in the early 1970s (Olovnikov, 1971; Watson, 1972; Olovnikov, 1973). About 50-150 bps from the telomeres are lost with each cell division due to the end-replication problem (Harley et al., 1990; Hastie et al., 1990; Levy et al., 1992). Semi-conservative DNA replication is bidirectional and starts at one or more concurrent sites. However, DNA polymerases function unidirectionally, which initiate replication from a primer at the 3' end and run towards the 5' end of the template. The synthesis of the leading strand is towards the replication fork, whereas the synthesis of the lagging begins at the replication fork (consisting of Okazaki fragments). When the synthesis is complete, the primers are degraded and gaps are formed at each site of replication. These gaps between the newly replicated fragments of the lagging strand are filled by the action of DNA ligases. However, the terminal gaps left by the primer at the end of both strands, are not filled ((Olovnikov, 1971; Watson, 1972; Olovnikov, 1973; Harley et al., 1990; Hastie et al., 1990) reviewed by (Hug and Lingner, 2006)). The terminal gap is further enlarged by the action of a putative 5' to 3' exonuclease, which degrades 130210 nucleotides (Hug and Lingner, 2006) (Figure 3). Figure 3: The DNA end replication problem: DNA polymerases only elongate in the 5′ to 3′ direction. Each fork contains a leading (continuous) and a lagging (discontinuous) strand. However, the synthesis of the lagging strand cannot be completed because the removal of primers causes net loss of sequence on the lagging strand (Hug and Lingner, 2006). Further, Olovnikov proposed that the continual telomeric loss induced by the end-replication problem would eventually result in replicative senescence (Olovnikov, 1971; Olovnikov, 1973). The telomeres are therefore suggested to function as mitotic clocks. Normal human somatic cells have a finite replicative potential in culture and enter a stage of replicative senescence when the telomeres are short. This stage is termed as the Hayflick limit (Mortality stage (M1) or replicative senescence), where the cells stop dividing and are arrested at G0 phase (Wright and Shay, 1992). The senescence can however be bypassed in these cells by the inactivation of the tumour suppressors, p53 and retinoblastoma (Rb) (Dyson et al., 1989; Werness et al., 1990). The inactivation of these proteins allow cells to progress through another 20–30 population doublings till the telomeres are critically shortened, resulting in telomere dysfunction and associated genomic instability. The cells then enter into the second stage of massive cell death termed as cellular crisis or Mortality stage (M2) (Wright and Shay, 1992) (Figure 4). The crisis stage is the potential barrier for immortal cell growth in culture. Cells which escape crisis acquire a feature termed immortalization, the ability to divide limitlessly (Wright and Shay, 1999). A rare, one in ten million cells overcome crisis by triggering telomere-maintenance mechanisms, most commonly by reactivating a special reverse transcriptase enzyme called telomerase (Shay and Wright, 2005). Telomere Length M1 (Senescence) M2 (Crisis) Immortalization Telomerase re-activation Population doublings Figure 4: Telomere-telomerase hypothesis: Telomere length decreases after some population doublings and cells with shortened telomeres enter mortality stage 1(M1). If the cells bypass this stage and continue to divide, the cells enter a stage of crisis (Mortality stage or M2) when the telomeres are critically short and apoptosis is triggered. The cells can emerge out of crisis by re-activating telomerase and can become immortal (adapted from Wright and Shay 1999). 1.1.2.2: Regulation of telomere length by telomerase: Telomerase is a ribonucleoprotein complex discovered in 1985 in the holotrichous ciliate Tetrahymena thermophila by Carol Greider and Elizabeth Blackburn (Greider and Blackburn, 1985). The telomerase complex is composed of a RNA moiety (hTR or hTERC) ( Greider and Blackburn, 1987; Feng et al., 1995) with a sequence complementary to telomeric repeats (Greider and Blackburn, 1989), and a catalytic component, the telomerase reverse transcriptase (hTERT) enzyme (Nakamura et al., 1997). Using its integral RNA component (which contains an 11 bp sequence complementary to the telomeric single stranded overhang) as a template for reverse transcription ( Greider and Blackburn, 1989; Yu et al., 1990), TERT reverse transcribes and adds hexanucleotide telomeric repeats onto the 3’end (Greider and Blackburn, 1989; Shippen-Lentz and Blackburn, 1990; Yu et al., 1990) (Figure 5). The extension of the 3’end allows for the replication of the C-rich strand by DNA polymerase, thus compensating for the end-replication problem. The two subunits of the enzyme are coded for by two different genes. The hTERT gene is mapped on chromosome 5p15 (Bryce et al., 2000) and hTERC on 3q26 (Soder et al., 1997). 10 CHAPTER APPENDIX 7.1: List of selected amplified genes in different grades of breast cancer: The list of amplified genes were functionally annotated using the DAVID gene functional annotation module (Dennis, Jr. et al., 2003) ito different classes. Few of the functional classes like the signal transduction, cellular processes were chosen and the list of genes amplified in different grades is given below. 7.1.1: List of the amplified genes in the grade II tumours, involved in the function of signal transduction: DGKE SHD CNIH3 ESR2 IL10 CARHSP1 DAPK3 STAP2 DIRAS1 PSEN2 ECM1 GADD45B STMN4 ZNF675 CNIH4 MKNK2 ARHGAP1 KSR2 F2 CALCR RP1L1 IL20 CDC42BPA LITAF ARID1A MATK GNA15 diacylglycerol kinase, epsilon 64kda src homology domain containing transforming protein d cornichon homolog (drosophila) estrogen receptor (er beta) interleukin 10 calcium regulated heat stable protein 1, death-associated protein kinase signal-transducing adaptor protein-2 diras family, gtp-binding ras-like presenilin extracellular matrix protein growth arrest and dna-damage-inducible, beta stathmin-like zinc finger protein 675 cornichon homolog (drosophila) map kinase interacting serine/threonine kinase rho gtpase activating protein kinase suppressor of ras coagulation factor ii (thrombin) calcitonin receptor retinitis pigmentosa 1-like interleukin 20 cdc42 binding protein kinase alpha (dmpk-like) lipopolysaccharide-induced tnf factor at rich interactive domain 1a (swi- like) megakaryocyte-associated tyrosine kinase guanine nucleotide binding protein (g protein), alpha 15 (gq class) 152 APBA3 amyloid beta (a4) precursor protein-binding, family a, member 7.1.2: List of the amplified genes in grade III tumours, involved in the function of signal transduction: GRLF1 STK24 GABRB2 CD2AP ADORA2B GIP ITPKB OR4C3 ADCYAP1R1 CAMTA2 GUCY1A2 NTRK3 TNK2 PDE8A ELMO1 BMPR2 TCF7L2 OR2L13 SPG7 TIAM1 VAC14 TG CD38 IL1RAPL1 ITGA1 GABRG3 TGFBR3 EPS15 MYO9B AKAP13 GPR133 THRAP1 KRIT1 PRKCH MAP3K5 GLI2 EPHA2 glucocorticoid receptor dna binding factor serine/threonine kinase 24 (ste20 homolog, yeast) gamma-aminobutyric acid (gaba) a receptor, beta cd2-associated protein adenosine a2b receptor gastric inhibitory polypeptide inositol 1,4,5-trisphosphate 3-kinase b olfactory receptor, family 4, subfamily c, member adenylate cyclase activating polypeptide (pituitary) receptor type i calmodulin binding transcription activator guanylate cyclase 1, soluble, alpha neurotrophic tyrosine kinase, receptor, type tyrosine kinase, non-receptor, phosphodiesterase 8a engulfment and cell motility bone morphogenetic protein receptor, type ii (serine/threonine kinase) transcription factor 7-like (t-cell specific, hmg-box) olfactory receptor, family 2, subfamily l, member 13 cell matrix adhesion regulator t-cell lymphoma invasion and metastasis tax1 (human t-cell leukemia virus type i) binding protein thyroglobulin cd38 antigen (p45) interleukin receptor accessory protein-like integrin, alpha gamma-aminobutyric acid (gaba) a receptor, gamma transforming growth factor, beta receptor iii (betaglycan, 300kda) epidermal growth factor receptor pathway substrate 15 myosin ixb lymphoid blast crisis oncogene g protein-coupled receptor 133 thyroid hormone receptor associated protein krit1, ankyrin repeat containing protein kinase c, eta mitogen-activated protein kinase kinase kinase gli-kruppel family member gli2 eph receptor a2 153 TSHR GP1BA RIN2 GRIK1 CMKLR1 NF1 GLRA2 MAPK11 DGKE OR9Q1 FSHB TNFRSF11A CELSR1 VIPR2 OR5AP2 FGF14 SORCS3 SLIT2 PLEKHG5 KCNIP1 OR8G1 WISP1 TRAF1 PAK2 GNA13 GRIN2D MAPK1 ERG OR5V1 ANXA3 PIK4CA ADAMTSL3 PAFAH1B1 TNFRSF8 PDE10A NTRK1 KCNH5 SPAG5 PNOC RPS6KA2 thyroid stimulating hormone receptor glycoprotein ib (platelet), alpha polypeptide ras and rab interactor glutamate receptor, ionotropic, kainate chemokine-like receptor neurofibromin (neurofibromatosis, von recklinghausen disease, watson disease) glycine receptor, alpha mitogen-activated protein kinase 11 diacylglycerol kinase, epsilon 64kda olfactory receptor, family 9, subfamily q, member follicle stimulating hormone, beta polypeptide tumor necrosis factor receptor superfamily, member 11a, nfkb activator cadherin, egf lag seven-pass g-type receptor (flamingo homolog, drosophila) vasoactive intestinal peptide receptor olfactory receptor, family 5, subfamily ap, member fibroblast growth factor 14 sortilin-related vps10 domain containing receptor slit homolog (drosophila) pleckstrin homology domain containing, family g (with rhogef domain) member kv channel interacting protein olfactory receptor, family 8, subfamily g, member wnt1 inducible signaling pathway protein tnf receptor-associated factor p21 (cdkn1a)-activated kinase guanine nucleotide binding protein (g protein), alpha 13 glutamate receptor, ionotropic, n-methyl d-aspartate 2d mitogen-activated protein kinase v-ets erythroblastosis virus e26 oncogene like (avian) olfactory receptor, family 5, subfamily v, member annexin a3 phosphatidylinositol 4-kinase, catalytic, alpha polypeptide adamts-like platelet-activating factor acetylhydrolase, isoform ib, alpha subunit 45kda tumor necrosis factor receptor superfamily, member phosphodiesterase 10a neurotrophic tyrosine kinase, receptor, type potassium voltage-gated channel, subfamily h (eag-related), member sperm associated antigen prepronociceptin ribosomal protein s6 kinase, 90kda, polypeptide 154 RAB8B PSTPIP1 SHC4 GLRA1 SUPT6H CCL3 RASD1 VAV2 PLXNC1 KALRN WNT5A POMC GRIK4 RAPGEF4 GNGT2 GSK3B RALGPS1 HGS KSR2 CAMTA1 ADCY9 PTPRT ARID1A ALK GRK5 GRM7 EPHA10 rab8b, member ras oncogene family proline-serine-threonine phosphatase interacting protein shc (src homology domain containing) family, member glycine receptor, alpha (startle disease/hyperekplexia, stiff man syndrome) suppressor of ty homolog (s. cerevisiae) chemokine (c-c motif) ligand ras, dexamethasone-induced vav oncogene plexin c1 kalirin, rhogef kinase wingless-type mmtv integration site family, member 5a proopiomelanocortin (adrenocorticotropin/ beta-lipotropin/ alphamelanocyte stimulating hormone/ beta-melanocyte stimulating hormone/ beta-endorphin) glutamate receptor, ionotropic, kainate rap guanine nucleotide exchange factor (gef) guanine nucleotide binding protein (g protein), gamma transducing activity polypeptide glycogen synthase kinase beta ral gef with ph domain and sh3 binding motif hepatocyte growth factor-regulated tyrosine kinase substrate kinase suppressor of ras calmodulin binding transcription activator adenylate cyclase protein tyrosine phosphatase, receptor type, t at rich interactive domain 1a (swi- like) anaplastic lymphoma kinase (ki-1) g protein-coupled receptor kinase glutamate receptor, metabotropic eph receptor a10 7.1.3: List of the amplified genes in the grade II tumours, involved in the regulation of cellular processes: BCL2L1 TCF3 PTK2B DMBT1 KLF16 RUNDC2A FZR1 bcl2-like transcription factor (e2a immunoglobulin enhancer binding factors e12/e47) ptk2b protein tyrosine kinase beta deleted in malignant brain tumors kruppel-like factor 16 run domain containing 2a homolog of yeast cdh1/hct1 155 MEF2B IL10 SIRT6 TLE6 CREB3L3 ZNF92 AES APC2 GTSE1 MBD3 GATAD1 ZNF138 HMG20B ECM1 ZNF556 ZNF434 SLAMF1 ZNF555 NHLH1 BCL2 ZNF675 TLE2 NOG EBI3 ZNF597 CIITA TSHZ1 CDC42BPA LITAF ZNF117 PEA15 ZBTB7A CDK6 TAF6L ESR2 SFN NFIC CRADD ZNF91 MCL1 ZBTB3 PIAS4 DAPK3 mads box transcription enhancer factor 2, polypeptide b (myocyte enhancer factor 2b) interleukin 10 sirtuin (silent mating type information regulation homolog) (s. cerevisiae) transducin-like enhancer of split (e(sp1) homolog, drosophila) camp responsive element binding protein 3-like zinc finger protein 92 (htf12) amino-terminal enhancer of split adenomatosis polyposis coli g-2 and s-phase expressed methyl-cpg binding domain protein gata zinc finger domain containing zinc finger protein 138 high-mobility group 20b extracellular matrix protein zinc finger protein 556 zinc finger protein 434 signaling lymphocytic activation molecule family member zinc finger protein 555 nescient helix loop helix b-cell cll/lymphoma zinc finger protein 675 transducin-like enhancer of split (e(sp1) homolog, drosophila) noggin epstein-barr virus induced gene zinc finger protein 597 class ii, major histocompatibility complex, transactivator serologically defined colon cancer antigen 33 cdc42 binding protein kinase alpha (dmpk-like) lipopolysaccharide-induced tnf factor zinc finger protein 117 (hpf9) phosphoprotein enriched in astrocytes 15 zinc finger and btb domain containing 7a cyclin-dependent kinase taf6-like rna polymerase ii, p300/cbp-associated factor (pcaf)associated factor, 65kda estrogen receptor (er beta) stratifin nuclear factor i/c (ccaat-binding transcription factor) casp2 and ripk1 domain containing adaptor with death domain zinc finger protein 91 (hpf7, htf10) myeloid cell leukemia sequence (bcl2-related) zinc finger and btb domain containing protein inhibitor of activated stat, death-associated protein kinase 156 NR0B2 CARHSP1 KCNH1 NFATC1 VDR ZNF407 ZNF77 RAXL1 CTNNBIP1 ZNF554 FLJ14981 ZNF588 MKNK2 ZNF680 CASZ1 CHAF1A ZNF174 F2 ZNF273 PACSIN3 ZNF408 IL20 ARID1A GNG7 MATK nuclear receptor subfamily 0, group b, member calcium regulated heat stable protein 1, 24kda potassium voltage-gated channel, subfamily h (eag-related), member nuclear factor of activated t-cells, cytoplasmic, calcineurindependent vitamin d (1,25- dihydroxyvitamin d3) receptor zinc finger protein 407 zinc finger protein 77 (pt1) retina and anterior neural fold homeobox like catenin, beta interacting protein zinc finger protein 554 hypothetical protein flj14981 zinc finger protein 588 map kinase interacting serine/threonine kinase zinc finger protein 680 castor homolog 1, zinc finger (drosophila) chromatin assembly factor 1, subunit a (p150) zinc finger protein 174 coagulation factor ii (thrombin) zinc finger protein 273 protein kinase c and casein kinase substrate in neurons zinc finger protein 408 interleukin 20 at rich interactive domain 1a (swi- like) guanine nucleotide binding protein (g protein), gamma megakaryocyte-associated tyrosine kinase 7.1.4: List of the amplified genes in the grade III tumours, involved in the regulation of cellular processes: STX18 STK24 LRP1B MCCC1 ARPC5L CNOT6 ZNRF1 HDGFRP3 ATBF1 ARSB PCSK6 MAST2 APPBP2 syntaxin 18 serine/threonine kinase 24 (ste20 homolog, yeast) low density lipoprotein-related protein 1b (deleted in tumors) methylcrotonoyl-coenzyme a carboxylase (alpha) actin related protein 2/3 complex, subunit 5-like ccr4-not transcription complex, subunit hypothetical protein dkfzp434e229 hepatoma-derived growth factor, related protein at-binding transcription factor arylsulfatase b proprotein convertase subtilisin/kexin type microtubule associated serine/threonine kinase amyloid beta precursor protein (cytoplasmic tail) binding protein 157 FEZ1 fasciculation and elongation protein zeta (zygin i) OR4C3 olfactory receptor, family 4, subfamily c, member ADCYAP1R1 adenylate cyclase activating polypeptide (pituitary) receptor type i ADCK1 aarf domain containing kinase GUCY1A2 guanylate cyclase 1, soluble, alpha KCNK1 potassium channel, subfamily k, member BRIP1 brca1 interacting protein c-terminal helicase NTRK3 neurotrophic tyrosine kinase, receptor, type PHF20 phd finger protein 20 WRN werner syndrome PEMT phosphatidylethanolamine n-methyltransferase TNK2 tyrosine kinase, non-receptor, PCDH17 protocadherin 17 PDE8A phosphodiesterase 8a ACE angiotensin i converting enzyme (peptidyl-dipeptidase a) ELMO1 engulfment and cell motility BMPR2 bone morphogenetic protein receptor, type ii (serine/threonine kinase) CDK5RAP2 cdk5 regulatory subunit associated protein CNGB1 cyclic nucleotide gated channel beta KCNQ3 potassium voltage-gated channel, kqt-like subfamily, member SFRS14 splicing factor, arginine/serine-rich 14 USP48 hypothetical protein flj11328 SPG7 cell matrix adhesion regulator TIAM1 t-cell lymphoma invasion and metastasis PRKACG protein kinase, camp-dependent, catalytic, gamma TG thyroglobulin SLC39A11 solute carrier family 39 (metal ion transporter), member 11 C21orf29 chromosome 21 open reading frame 29 NXN nucleoredoxin NFATC3 nuclear factor of activated t-cells, cytoplasmic, calcineurindependent IL1RAPL1 interleukin receptor accessory protein-like ITGA1 integrin, alpha GABRG3 gamma-aminobutyric acid (gaba) a receptor, gamma TGFBR3 transforming growth factor, beta receptor iii (betaglycan, 300kda) EPS15 epidermal growth factor receptor pathway substrate 15 MYO9B myosin ixb SPPL2B signal peptide peptidase-like 2b ELP3 elongation protein homolog (s. cerevisiae) TXNRD2 thioredoxin reductase NEFH neurofilament, heavy polypeptide 200kda THRAP1 thyroid hormone receptor associated protein PRKAG2 protein kinase, amp-activated, gamma non-catalytic subunit XRN2 5'-3' exoribonuclease 158 IFI16 MAP3K5 POLR3F CHD5 PDHA1 NUP155 GLI2 RBBP6 VTI1A TSHR NRXN3 UBE2E2 PIR GP1BA TCF4 TPST1 GRIK1 CMKLR1 CA10 NF1 ASH1L DLGAP4 TRIP12 NUP160 TMPRSS2 NEDD4L RUNX1 MTHFD1 PAN3 NKTR ACADSB TNFRSF11A FSHB PFN1 OR5AP2 BTAF1 FZR1 FGF14 interferon, gamma-inducible protein 16 mitogen-activated protein kinase kinase kinase polymerase (rna) iii (dna directed) polypeptide f, 39 kda chromodomain helicase dna binding protein pyruvate dehydrogenase (lipoamide) alpha nucleoporin 155kda gli-kruppel family member gli2 retinoblastoma binding protein vesicle transport through interaction with t-snares homolog 1a (yeast) thyroid stimulating hormone receptor neurexin ubiquitin-conjugating enzyme e2e (ubc4/5 homolog, yeast) pirin (iron-binding nuclear protein) glycoprotein ib (platelet), alpha polypeptide transcription factor tyrosylprotein sulfotransferase glutamate receptor, ionotropic, kainate chemokine-like receptor carbonic anhydrase x neurofibromin (neurofibromatosis, von recklinghausen disease, watson disease) ash1 (absent, small, or homeotic)-like (drosophila) discs, large (drosophila) homolog-associated protein thyroid hormone receptor interactor 12 nucleoporin 160kda transmembrane protease, serine neural precursor cell expressed, developmentally down-regulated 4-like runt-related transcription factor (acute myeloid leukemia 1; aml1 oncogene) methylenetetrahydrofolate dehydrogenase (nadp+ dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase pabp1-dependent poly a-specific ribonuclease subunit pan3 natural killer-tumor recognition sequence acyl-coenzyme a dehydrogenase, short/branched chain tumor necrosis factor receptor superfamily, member 11a, nfkb activator follicle stimulating hormone, beta polypeptide profilin olfactory receptor, family 5, subfamily ap, member btaf1 rna polymerase ii, b-tfiid transcription factor-associated, 170kda (mot1 homolog, s. cerevisiae) homolog of yeast cdh1/hct1 fibroblast growth factor 14 159 RANBP17 HCN1 RLBP1 NCOR1 ADAMTS19 KCNIP1 RFPL1 NF2 ATP9B LSM8 SCRN1 CFB ERGIC2 IRF2 GRIN2D GPAM CDH20 FUT8 OR5V1 TAOK1 CDS2 ANXA3 PIK4CA NOG FHIT ATP5J2 PAFAH1B1 ADAMTSL3 CTPS MKKS KCNH5 VGLL4 FLJ10815 SEC14L1 PRDM16 NLGN1 SPAG5 SLCO3A1 SDF2 SCNN1D AGC1 ran binding protein 17 hyperpolarization activated cyclic nucleotide-gated potassium channel retinaldehyde binding protein nuclear receptor co-repressor adam metallopeptidase with thrombospondin type motif, 19 kv channel interacting protein ret finger protein-like neurofibromin (bilateral acoustic neuroma) atpase, class ii, type 9b lsm8 homolog, u6 small nuclear rna associated (s. cerevisiae) secernin complement factor b ergic and golgi interferon regulatory factor glutamate receptor, ionotropic, n-methyl d-aspartate 2d glycerol-3-phosphate acyltransferase, mitochondrial cadherin 20, type fucosyltransferase (alpha (1,6) fucosyltransferase) olfactory receptor, family 5, subfamily v, member tao kinase cdp-diacylglycerol synthase (phosphatidate cytidylyltransferase) annexin a3 phosphatidylinositol 4-kinase, catalytic, alpha polypeptide noggin fragile histidine triad gene atp synthase, h+ transporting, mitochondrial f0 complex, subunit f2 platelet-activating factor acetylhydrolase, isoform ib, alpha subunit 45kda adamts-like ctp synthase mckusick-kaufman syndrome potassium voltage-gated channel, subfamily h (eag-related), member vestigial like (drosophila) amino acid transporter sec14-like (s. cerevisiae) pr domain containing 16 neuroligin sperm associated antigen solute carrier organic anion transporter family, member 3a1 stromal cell-derived factor sodium channel, nonvoltage-gated 1, delta aggrecan (chondroitin sulfate proteoglycan 1, large aggregating proteoglycan, antigen identified by monoclonal antibody a0122) 160 GLRA1 NSF SGCD PLXNC1 VAV2 BACH1 RG9MTD3 WNT5A RAPGEF4 ALB CFTR SCD5 EIF4G3 GNGT2 FLJ31196 USP7 TRIM29 RALGPS1 PC ZSCAN5 CDH13 USP25 PPAPDC1A KSR2 EFEMP2 CAMTA1 ADCY9 NR1H3 PCQAP PTPRT NRG1 ARID1A SENP5 CACNB2 ALK GRK5 CLTCL1 GRM7 GRLF1 SALL3 NUP133 EXOC6 LARGE glycine receptor, alpha (startle disease/hyperekplexia, stiff man syndrome) n-ethylmaleimide-sensitive factor sarcoglycan, delta (35kda dystrophin-associated glycoprotein) plexin c1 vav oncogene btb and cnc homology 1, basic leucine zipper transcription factor rna (guanine-9-) methyltransferase domain containing wingless-type mmtv integration site family, member 5a rap guanine nucleotide exchange factor (gef) albumin cystic fibrosis transmembrane conductance regulator, atp-binding cassette (sub-family c, member 7) stearoyl-coa desaturase eukaryotic translation initiation factor gamma, guanine nucleotide binding protein (g protein), gamma transducing activity polypeptide hypothetical protein flj31196 ubiquitin specific peptidase (herpes virus-associated) tripartite motif-containing 29 ral gef with ph domain and sh3 binding motif pyruvate carboxylase zinc finger and scan domain containing cadherin 13, h-cadherin (heart) ubiquitin specific peptidase 25 phosphatidic acid phosphatase type domain containing 1a kinase suppressor of ras fibulin calmodulin binding transcription activator adenylate cyclase nuclear receptor subfamily 1, group h, member trinucleotide repeat containing protein tyrosine phosphatase, receptor type, t neuregulin at rich interactive domain 1a (swi- like) sumo1/sentrin specific peptidase calcium channel, voltage-dependent, beta subunit anaplastic lymphoma kinase (ki-1) g protein-coupled receptor kinase clathrin, heavy polypeptide-like glutamate receptor, metabotropic glucocorticoid receptor dna binding factor sal-like (drosophila) nucleoporin 133kda exocyst complex component like-glycosyltransferase 161 KIAA0100 GABRB2 ITCH MEF2B KCNC2 CD2AP GPHN ADORA2B CREB3L3 GIP ITPKB B4GALNT2 FLJ25006 CAMTA2 RNF167 ABAT PDCD6 PEPD CDH19 PPM1D SLAMF1 HPS4 CPLX1 GKN1 PRDM2 MGAT4A L3MBTL4 CTBP2 TCF7L2 OR2L13 EIF2B3 VAC14 UHMK1 CD38 SLC25A11 DNAH9 FGFR2 ARHGAP17 ZNF79 CARM1 kiaa0100 gamma-aminobutyric acid (gaba) a receptor, beta atrophin-1 interacting protein mads box transcription enhancer factor 2, polypeptide b (myocyte enhancer factor 2b) potassium voltage-gated channel, shaw-related subfamily, member cd2-associated protein gephyrin adenosine a2b receptor camp responsive element binding protein 3-like gastric inhibitory polypeptide inositol 1,4,5-trisphosphate 3-kinase b beta-1,4-n-acetyl-galactosaminyl transferase hypothetical protein flj25006 calmodulin binding transcription activator ring finger protein 167 4-aminobutyrate aminotransferase programmed cell death peptidase d cadherin 19, type protein phosphatase 1d magnesium-dependent, delta isoform signaling lymphocytic activation molecule family member le protein complexin gastrokine pr domain containing 2, with znf domain mannosyl (alpha-1,3-)-glycoprotein beta-1,4-nacetylglucosaminyltransferase, isozyme a l(3)mbt-like (drosophila) c-terminal binding protein transcription factor 7-like (t-cell specific, hmg-box) olfactory receptor, family 2, subfamily l, member 13 eukaryotic translation initiation factor 2b, subunit gamma, 58kda tax1 (human t-cell leukemia virus type i) binding protein u2af homology motif (uhm) kinase cd38 antigen (p45) solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11 dynein, axonemal, heavy polypeptide fibroblast growth factor receptor (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, crouzon syndrome, pfeiffer syndrome, jackson-weiss syndrome) rho gtpase activating protein 17 zinc finger protein 79 (pt7) coactivator-associated arginine methyltransferase 162 RAD51L1 TLN2 PRPF8 ALPK3 GPR133 AKAP13 KRIT1 PRKCH KCNJ6 TRIM25 RABGAP1 MRPS9 EPHA2 SCMH1 SLBP RIN2 SUHW2 UBE2L3 DDX19B USP32 POLN ENPP6 CNTNAP2 MLLT3 LPP SLC9A4 MAPK11 GLRA2 DGKE PCLKC OR9Q1 STEAP2 PARK2 CELSR1 MBTPS2 VIPR2 SORCS3 SLIT2 PLEKHG5 OR8G1 M6PRBP1 KLF2 rad51-like (s. cerevisiae) talin prp8 pre-mrna processing factor homolog (yeast) alpha-kinase g protein-coupled receptor 133 lymphoid blast crisis oncogene krit1, ankyrin repeat containing protein kinase c, eta potassium inwardly-rectifying channel, subfamily j, member tripartite motif-containing 25 rab gtpase activating protein mitochondrial ribosomal protein s9 membrane-associated ring finger (c3hc4) eph receptor a2 sex comb on midleg homolog (drosophila) stem-loop (histone) binding protein ras and rab interactor suppressor of hairy wing homolog (drosophila) ubiquitin-conjugating enzyme e2l dead (asp-glu-ala-as) box polypeptide 19b ubiquitin specific peptidase 32 polymerase (dna directed) nu ectonucleotide pyrophosphatase/phosphodiesterase contactin associated protein-like myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, drosophila); translocated to, lim domain containing preferred translocation partner in lipoma solute carrier family (sodium/hydrogen exchanger), member mitogen-activated protein kinase 11 glycine receptor, alpha diacylglycerol kinase, epsilon 64kda protocadherin lkc olfactory receptor, family 9, subfamily q, member six transmembrane epithelial antigen of the prostate parkinson disease (autosomal recessive, juvenile) 2, parkin cadherin, egf lag seven-pass g-type receptor (flamingo homolog, drosophila) membrane-bound transcription factor peptidase, site vasoactive intestinal peptide receptor sortilin-related vps10 domain containing receptor slit homolog (drosophila) pleckstrin homology domain containing, family g (with rhogef domain) member olfactory receptor, family 8, subfamily g, member mannose-6-phosphate receptor binding protein kruppel-like factor (lung) 163 WISP1 CLCA2 ZNF148 LSAMP TRAF1 ST3GAL1 PAK2 ZNF306 GNA13 SLC4A10 ABI3 CARD8 ALDOC ATP5H TMEM118 MAPK1 ERG SLIT3 PIGL E2F3 PCYT1B ZNF597 TNFRSF8 PDE10A NTRK1 ENO3 EIF4ENIF1 ABCB8 DHODH SCPEP1 PHF21A PMF1 ALG3 PNOC SLC22A18 RPS6KA2 FBXO42 PDZD2 CAV1 KCNMB3 RAB8B PSTPIP1 wnt1 inducible signaling pathway protein chloride channel, calcium activated, family member zinc finger protein 148 (phz-52) limbic system-associated membrane protein tnf receptor-associated factor st3 beta-galactoside alpha-2,3-sialyltransferase p21 (cdkn1a)-activated kinase zinc finger protein 306 guanine nucleotide binding protein (g protein), alpha 13 solute carrier family 4, sodium bicarbonate transporter-like, member 10 abi gene family, member caspase recruitment domain family, member aldolase c, fructose-bisphosphate atp synthase, h+ transporting, mitochondrial f0 complex, subunit d transmembrane protein 118 mitogen-activated protein kinase v-ets erythroblastosis virus e26 oncogene like (avian) slit homolog (drosophila) phosphatidylinositol glycan, class l e2f transcription factor phosphate cytidylyltransferase 1, choline, beta zinc finger protein 597 tumor necrosis factor receptor superfamily, member phosphodiesterase 10a neurotrophic tyrosine kinase, receptor, type enolase 1, (alpha) eukaryotic translation initiation factor 4e nuclear import factor atp-binding cassette, sub-family b (mdr/tap), member septin dihydroorotate dehydrogenase serine carboxypeptidase phd finger protein 21a polyamine-modulated factor asparagine-linked glycosylation homolog (yeast, alpha-1,3mannosyltransferase) prepronociceptin imprinted polyspecific membrane transporter ribosomal protein s6 kinase, 90kda, polypeptide f-box protein 42 pdz domain containing caveolin 1, caveolae protein, 22kda potassium large conductance calcium-activated channel, subfamily m beta member rab8b, member ras oncogene family proline-serine-threonine phosphatase interacting protein 164 SHC4 PAM FARP1 CCL3 SUPT6H RASD1 PMS1 RUVBL2 KALRN GALNT2 HGF POMC GRIK4 H6PD MACF1 GSK3B HGS GBF1 DAO PGA5 AFF3 BCHE NME6 SLC38A1 UNC119 RBM17 EPHA10 shc (src homology domain containing) family, member peptidylglycine alpha-amidating monooxygenase ferm, rhogef (arhgef) and pleckstrin domain protein (chondrocyte-derived) chemokine (c-c motif) ligand suppressor of ty homolog (s. cerevisiae) ras, dexamethasone-induced pms1 postmeiotic segregation increased (s. cerevisiae) ruvb-like (e. coli) kalirin, rhogef kinase udp-n-acetyl-alpha-d-galactosamine:polypeptide nacetylgalactosaminyltransferase (galnac-t2) hepatocyte growth factor (hepapoietin a; scatter factor) proopiomelanocortin (adrenocorticotropin/ beta-lipotropin/ alphamelanocyte stimulating hormone/ beta-melanocyte stimulating hormone/ beta-endorphin) glutamate receptor, ionotropic, kainate hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) microtubule-actin crosslinking factor glycogen synthase kinase beta hepatocyte growth factor-regulated tyrosine kinase substrate golgi-specific brefeldin a resistance factor d-amino-acid oxidase pepsinogen 4, group i (pepsinogen a) af4/fmr2 family, member butyrylcholinesterase non-metastatic cells 6, protein expressed in (nucleosidediphosphate kinase) solute carrier family 38, member unc-119 homolog (c. elegans) rna binding motif protein 17 eph receptor a10 7.2: List of selected deleted genes in different grades of breast cancer: The list of deleted genes were functionally annotated using the DAVID gene functional annotation module (Dennis, Jr. et al., 2003) ito different classes. Genes involved in the regulation of cellular processes were chosen and the list of genes deleted in different grades is given below. 165 7.2.1: List of the deleted genes in the grade II tumours, involved in the regulation of cellular processes: DNAH8 ABCB5 HNRPH1 NEGR1 ITIH5 TIMM23 EEFSEC RAD51L1 IMMP2L SUCLA2 RANBP17 PTPRD DSCR1L1 RBED1 GPR116 ODZ1 SLC25A27 RPE65 SUPT3H RORC COG7 CLIC5 MEP1A FHOD3 ENPP4 PIK3CD C14orf106 CDH8 TP73L TMOD1 GLO1 SCNN1B RUNX2 MAP3K13 DLGAP4 EXOC4 ENPP5 HIST1H2AI SPRED1 BTBD9 dynein, axonemal, heavy polypeptide atp-binding cassette, sub-family b (mdr/tap), member heterogeneous nuclear ribonucleoprotein h1 (h) neuronal growth regulator inter-alpha (globulin) inhibitor h5 translocase of inner mitochondrial membrane 23 homolog (yeast) eukaryotic elongation factor, selenocysteine-trna-specific rad51-like (s. cerevisiae) imp2 inner mitochondrial membrane peptidase-like (s. cerevisiae) succinate-coa ligase, adp-forming, beta subunit ran binding protein 17 protein tyrosine phosphatase, receptor type, d down syndrome critical region gene 1-like rna binding motif and elmo/ced-12 domain g protein-coupled receptor 116 odz, odd oz/ten-m homolog 1(drosophila) solute carrier family 25, member 27 retinal pigment epithelium-specific protein 65kda suppressor of ty homolog (s. cerevisiae) rar-related orphan receptor c component of oligomeric golgi complex chloride intracellular channel meprin a, alpha (paba peptide hydrolase) formin homology domain containing ectonucleotide pyrophosphatase/phosphodiesterase (putative function) phosphoinositide-3-kinase, catalytic, delta polypeptide putative protein p243 which interacts with transcription factor sp1 cadherin 8, type tumor protein p73-like tropomodulin glyoxalase i sodium channel, nonvoltage-gated 1, beta (liddle syndrome) runt-related transcription factor mitogen-activated protein kinase kinase kinase 13 discs, large (drosophila) homolog-associated protein exocyst complex component ectonucleotide pyrophosphatase/phosphodiesterase (putative function) histone 1, h2ai sprouty-related, evh1 domain containing btb (poz) domain containing 166 CYP39A1 NFATC2 cytochrome p450, family 39, subfamily a, polypeptide nuclear factor of activated t-cells, cytoplasmic, calcineurindependent 7.2.2: List of the deleted genes in the grade III tumours, involved in the regulation of cellular processes: AKAP13 CAMKK2 RPS6KA5 ASB6 TIAM2 RRP22 APOL3 PRKCZ lymphoid blast crisis oncogene calcium/calmodulin-dependent protein kinase kinase 2, beta ribosomal protein s6 kinase, 90kda, polypeptide likely ortholog of mouse ankyrin repeat and socs box-containing protein t-cell lymphoma invasion and metastasis ras-related on chromosome 22 tnf-inducible protein cg12-1 protein kinase c, zeta 167 [...]... proteins, including TIN2 (Kim et al., 20 04), TPP1 (Houghtaling et al., 20 04; Liu et al., 20 04b; Ye et al., 20 04b), POT1 (Baumann and Cech, 20 01; Loayza and De Lange, 20 03), hRAP1 (Li et al., 20 00) and tankyrase1 (TRF1-interacting ankyrin related ADPribose polymerase) and tankyrase2 to ensure proper telomere maintenance ( Kaminker et al., 20 01; Cook et al., 20 02) TIN2 was found to bind TRF1 and TRF2 simultaneously... Singapore cancer registry reported that breast cancer is the most common cancer among the women in Singapore, making up about 28 % of the women with cancer It is estimated that in Singapore, every year, about 1000 women are affected with breast cancer and around 25 0 -27 0 women die of breast cancer This has doubled over the past 20 years and is increasing at a rate of 3% annually From 1968 to 20 02, Singapore... experienced an almost three-fold increase in breast cancer incidence (Sim et al., 20 06) The breast cancer incidence pattern in Singapore is rapidly becoming similar to populations in the West (Chia et al., 20 02) 1.4.1: Types of Breast Cancer: Most breast cancers develop in the glandular tissue and are classified as adenocarcinoma There are several histological classifications of breast cancers The WHO classification... gene containing 24 exons with a coding region of 5.5Kb and its mRNA covering 8kb ( Miki et al., 1994; Boulton, 20 06) It encodes a large 22 0-kDa nuclear phosphoprotein with 1863 amino acids in humans (Miki et al., 1994) and 18 12 amino acids in mice (Lane et al., 1995) It has three important structural motifs, including a highly conserved amino-terminal RING (Really Interesting New Gene) finger motif,... discussed briefly in the following sections 22 1 .2. 1.1: Role of BRCA1 in DNA repair: One of the mechanisms of BRCA1 in maintaining genome integrity was thought to be through its roles in DNA damage repair (Figure 7) Increasing evidence has implicated role of BRCA1 in homologous recombination repair (HRR) (Moynahan et al., 1999), non homologous end joining (NHEJ) (Zhong et al., 20 02) and nucleotide... proteins MSH2, MSH6 and MLH1, the Bloom syndrome helicase BLM, the ATM kinase, DNA replication factorC (RFC) and PCNA (Wang et al., 20 00) This also suggests a role for BRCA1 in coordinating various functions of DNA replication that are important for maintaining genomic integrity in the cell (Starita and Parvin, 20 03) 23 BRCA1 has been shown recently to be important in facilitating NHEJ, while inhibiting... through its role in maintaining genome integrity, instead of directly inhibiting cell proliferation (Deng, 20 01b; Deng, 20 01a; Deng and Scott, 20 00b; Venkitaraman, 20 02) The role of BRCA1 in tumour suppression (Smith et al., 19 92) has been attributed to its role in genome surveillance through its role in multiple cellular functions as mentioned earlier (Deng and Brodie, 20 00; Deng and Scott, 20 00) Some of... alternative lengthening of telomere (ALT) mechanism involving homologous recombination (Bryan et al., 1995) 1.1 .2. 3: Regulation of telomeres by telomere binding proteins: In addition to telomerase, the telosome complex functions in regulating the telomere length as well as stabilising its secondary structure TRF1 plays a primary 12 role in telomere length control TRF1 consists of 439 amino acid residues... According to this classification, the two major groups of breast cancers are the non-invasive or invasive types In- situ carcinoma is characterised by growth within the ducts without penetration of the basement membrane and thus without ducts invading the stroma Non-invasive carcinomas include lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS) The most common type of breast cancer, invasive... telomere mediated genomic instability has never been explored Understanding the interconnections between DNA damage checkpoints and telomere integrity is important because both play important roles in preserving genomic stability and preventing cancer 28 1.3: TELOMERE MEDIATED GENOMIC INSTABILITY IN CANCER: The specialised function of telomeres in chromosomal end-protection was first defined by Hermann Muller . indirectly binding proteins (TPP1, TIN2 and hRap1) include those that bind to the telomeric DNA through interaction with the directly interacting proteins. TIN2 binds to TRF1 and TRF2. hRap1 binds. protein 1 TPP1 [TINT1 (Houghtaling et al., 20 04), PTOP (Liu et al., 20 04b) and PIP1 (Ye et al., 20 04b) were identified during the search for TIN2-interacting proteins. Protection of telomeres. damage (Mao et al., 20 07). TRF1and TRF2 interact with a number of other proteins, including TIN2 (Kim et al., 20 04), TPP1 (Houghtaling et al., 20 04; Liu et al., 20 04b; Ye et al., 20 04b), POT1 (Baumann