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OPTIMIZATION OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) ANALYSIS TO DETECT TERT GENE AMPLIFICATION IN A

CANCER CELL LINE MODEL, K562

by

KALAISEL VI MANVEERAN

Dissertation submitted in partial fulfillment of the requirements for the degree of Bachelor of Health Sciences (Biomedicine)

October 2009

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CERTIFICATE

This is to certify that the dissertation entitled "Optimization of Fluorescence in situ hybridization (FISH) analysis to detect TERT gene amplification in a cancer cell line model K562" is the bonafide record of research work done by Ms Kalaiselvi Manveeran during the period from July 2008 to October 2008 under my supervision.

Supervisor,

ka .

... ... .

long Lecturer

Human Genome Centre School of Medical Sciences, Universiti Sains Malaysia Health Campus

16150 Kubang Kerian Kelantan, Malaysia

Date: ...

1./ ?·: J. ~? ... ·

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ACKNOWLEDGEMENTS

First of all, I am very grateful to God, with His willingness and guidance, I am able to finish my final year project and accomplish a dissertation. I would like to take this opportunity to acknowledge in writing, those who have been very helpful to assist me in completing my fmal year project.

My deepest appreciation and gratitude goes to my supervisor, Dr Sarina Sulong for her guidance, advice, encouragement and patience throughout the entire study period. I really feel grateful to work under her supervision and the opportunity given by her to me to gain new experiences and full freedom in doing this project. My special acknowledgement also goes to USM for funding this project with 'short term grant' (304/PPSP/6139042).

I gratefully acknowledge Ahmad Syibli Othman and Puan Noratifah Mohd Adam for their guidance, technical advice, support and guidance throughout my project. I would like to express my sincere gratitude to all member and staff at Human Genome Centre, PPSP, USM, especially Cytogenetic lab staff, Kak Shima, Kak Cma, Kak Alia and Abang Zaki for their technical support and in particular, the spirit of sharing the knowledge, expertise and equipment that exists among them.

This acknowledgement also goes to Dr See Too Wei Cun from PPSK for being a very dedicated Biomedicine fmal year project coordinator throughout this semester. Thanks for giving your endless support, encouragement and guidance to us in completing our fmal year project.

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From the bottom of my heart, I would like to thank my father, Manveeran Suppiah my mother, Sivagami Kannan, and my siblings for their endless support, that energize me when I'm having obstacles and encourage me to move further. High pleasure is due to my family members for all their love, encouragement and support during my studies.

I am very thankful to all my beloved friends especially Jayapramila Jayamani, Nazirah Abd. Kahar, Aisya Zulkifli, Niswathul Haania Zain Ali and my entire biomedicine batch mate for all their opinions, advices, suggestions, love, optimistic encouragement and everlasting support during the projects progression. Not forgetting those who had contributed to this study direct or indirectly, many thanks.

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TABLE OF CONTENTS

PAGE CERTIFICATE

ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES

LIST OF ABBREVIATIONS ABSTRACT

ABSTRAK

CHAPTER 1 : INTRODUCTION

1.1 General introduction 1.2 Aim of study

CHAPTER2: LITERATURE REVIEW

2.1 Telomerase Reverse Transcriptase (TERT) gene

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2.1.1 Telomerase and its associated protein 6

2.1.2 Involvement ofTERT gene amplification in cancer 8 2.1.2.1 Role ofTERT gene and its regulation ofTelomerase activity 9

2.2 Methods for detection of gene amplification 11

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2.2.1 Fluorescence in situ hybridization (FISH) 2.2.2 Comparative genomic hybridization (CGH) 2.2.3 Digital karyotyping

2.2.4 Quantitative microsatellite analysis (QuMA)

2.3 Importance of gene amplification detection

CHAPTER 3 : MATERIALS AND METHODS 3.1 Materials

3.1.1 General equipment 3.1.2 Chemical reagents 3.1.3 K562 cancer cell line

3.1.4 Peripheral blood sample from healthy donor (to be used for normal control)

3.1.5 Fluorescence in situ hybridization (FISH) probe (from POSEIDON™ REPEAT FREE™)

3.2 General methodology

3 .2.1 Tissue culture

3.2.1.1 Maintenance of cell lines

3 .2.1.2 Assessment of cell viability and cell count 3 .2.1.3 Subculture of cell line

3.2.2 Preparation of Interphase cell for FISH analysis 3.2.2.1 Solution Preparation

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3.2.2.2 Harvesting process and dropping of the cells on slide 23 3.2.3 Fluorescence in situ Hybidization (FISH) method using 24

Poseidon™Repeat Free™ hTERT (5p15) & EGRI (5q31) probe 3.2.3.1 Preparation ofFISH working solution

3 .2.3 .2 Pretreatment of slides

3.2.3.3 Denaturation and Hybridization

3.2.3.4 Post hybridization washing and counterstaining 3.2.4 Visualization and interpretation

CHAPTER 4 : RESULT

4.1 Interpretation of FISH signal visualization

CHAPTER 5 : DISCUSSION

5.1 Application of FISH analysis to detect genetic aberrations 5.2 Optimization factors to achieve good quality of FISH results 5.3 Increase of TERT gene copies in cancer cell

CHAPTER 6 : CONCLUSION

REFERENCES

APPENDIX Appendix A

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LIST OF FIGURES

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PAGE

Figure 3.1 Dual colour probe and the gena location (Poseidon™ Repeat 21 Free hTERT (5P15) and EGRl (5q31) control probe)

Figure 4.1 A, B, C and D show the presence of2 red and 2 green signals 30 representing 2 copies of gene TERT and EGRl. FISH signal

pattern in normal cells visualised using Cyto Vision System.

Figure 4.2 K562 cell line (1st slide) FISH signal visualization using 32 Cyto Vision System. A, B and C each exhibiting 3 green signals

with more than 3 -6 red signals pattern indicating Chromosome 5 trisomy with TERT gene amplified. B shows 3G6R. C shows 2G5R indicating TERT gene amplification.

Figure 4.3 K5 K562 cell line (2nd slide) FISH signal visualization using 33 Cyto Vision System. A and C shows 2 green and more than 3

red signals indicating TERT gene amplification. B shows normal signal pattern with 2G2R signals. D shows 3G6R signals exhibit the Chromosome 5 trisomy with TERT gene amplified.62 cell line (2nd slide) FISH signal visualization using Cyto Vision System

Figure 4.4 K562 cell line FISH signal visualization using Leica system. A 35 shows the merge picture of the signal (signal not clearly seen).

B shows cell line with DAPI counterstain. C shows green signal to visualize EGRl gene as control (clearly seen) and D shows red signal to visualize TERT gene (poorly seen).

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LIST OF TABLES

NO

PAGE

Table 3.1 Suitable excitation and emission range for POSEIDON fluorophores 27

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LIST OF ABBREVIATIONS

BAC Bacterial artificial chromosome

CCNDI CyclinDl

CDK4 Cyclin-dependent kinase 4

eDNA Complementary DNA

CGH Comparative genomic hybridization

CML Chronic myeloid leukemia

CNS Central Nervous system

C02 Carbon dioxide

dmins Double minutes

dH20 Distilled water

DHFR Dihydrofolate reductase

DKCI Dyskerin

DNA Deoxyribonucleic acid

EGFR Epidermal growth factor receptor

ERBB2 Human Epidermal growth factor Receptor 2

ESP End sequence profling

FBS Foetal bovine serum

FISH Fluorescent in situ hybridization

g Gram

HER2 Human Epidermal growth factor Receptor 2 HSR Homogeneously staining region

hTERT Human telomerase reverse transcriptase

hTR Human telomerase RNA

KCl Potassium chloride

M Molar

MB Medulloblastomas

Mg Milligram

mm Minute

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ml p PBS PCR q QuMA RNA RFIO rpm TERC TERT TR TYMS

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Milliliter

Short arm chromosome Phosphate buffered saline Polymerase chain reaction Long arm chromosome

Quantitative microsatellite analysis Ribonucleic acid

RPMI medium supplemented with 10% FBS Revolution per minute

Telomerase RNA component Telomerase Reverse Transcriptase Telomerase RNA

Thymidylate synthetase Volume

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ABSTRACT

TERT gene located on chromosome 5p15.33 is one of the core components oftelomerase enzyme which encodes human telomerase reverse transcriptase (hTERT) and commonly amplified in human cancers. With this regards, the detection ofTERT gene amplification in cancer cell may have useful application in cancer diagnosis and prognosis. Hence, in this study, K562 cell line, a chronic leukemic cell line was used to demonstrate the TERT gene amplification by using Fluorescence in situ hybridization (FISH) method in interphase stage.

Maintenance and cultivation of the K562 cell line was performed where the cells were grown in tissue culture flask as suspension culture in RF 10 medium. Normal peripheral blood also was used in this study as the normal control. In this study, TERT gene amplification was examined in K562 cell line by using a dual-color probe (Poseidon ™) that covered the genomic region of TERT gene at region 5p15 together with the control DNA probe, EGRI (5q31) gene to facilitate identification of chromosome 5. FISH analysis was successfully performed based on the recommendation protocol provided by the manufacturer with a minor modification. The FISH signals were visualized using fluorescence microscope with Leica system and Cyto Vision system. Amplification involving the TERT gene region showed several red signals, while the control at the chromosome 5q31 region (EGRl) will provide 2 green signals. In normal cells, the FISH signal pattern indicated the presence of2 red (R) 2 green (G) signals. Our fmdings showed that the ratio ofTERT/5q31 signal varied between 1 and 3 per cell. The cells which have 3- 4 red (TERT) signals/cell and two green (5q31) signals/cell were considered to be a low

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grade of amplification. There was also an occurrence of chromosome 5 aneusomy, where there were 3 green signals indicating the presence of 3 copies of chromosome 5 or trisomy

5. Some cells also showed 3 green signals with more than 2 red signals.

Our study suggests that TERT gene amplification was detected in K562 cells but further investigation by observing the frequency of signal pattern in 200 cells or using metaphase FISH or cytospin samples should be done to reveal the pattern ofTERT gene amplification in K562 cells or other types of cancer cells. Additional optimization of FISH technique should be done to ensure cost-effectiveness before utilizing it for routine diagnosis of TERT gene amplification in cancer patients.

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ABSTRAK

Gen TERT yang terletak pada kromosom 5p15.33 merupakan salah satu komponen teras telomerase dimana ia mengkodkan transkriptase membalik telomeras manusia (hTERT) dan biasanya berganda dalam kanser manusia. Oleh itu, pengesanan ampliflkasi gen TERT dalam sel kanser boleh menjadi aplikasi berguna dalam diagnostik dan prognostik kanser.

Maka, dalam kajian ini, titisan sel K562, sel kronik leukemia digunakan untuk menunjukkan ampliflkasi gen TERT dengan mengunakan teknik Fluorescence in situ hybridization (FISH).

Pengkulturan dan penuaian sel K562 telah dilakukan di mana sel dibiakkan dalam kelalang kultur tisu sebagai kultur cairan dalam medium RF10. Darah periferi normal digunakan dalam kajian ini sebagai kawalan normal. Dalam kajian ini, ampliflkasi gen TERT telah diperiksa dalam titisan sel K562 dengan mengunakan prob dwi warna (Poseidon ™) yang meliputi gen TERT dikawasan 5p 15 bersama dengan probe control DNA, gen EGRI ( 5q31) bagi memudahkan pengenalpastian kromosom 5. Analisis FISH berjaya dilakukan berpandukan protokol yang disediakan oleh syarikat pengeluar dengan sedikit modiflkasi.

Isyarat FISH telah diperhati dengan mengunakan mikroskop fluoresen dengan system Leica dan system Cytovision. Ampliflkasi yang meliputi kawasan gen TERT akan menunjukan beberapa isyarat merah, manakala kontrol pada kromosom kawasan 5q31 (EGR1) akan memberi 2 isyarat hijau. Didalam sel normal, isyarat FISH menunjukkan kehadiran 2 isyarat merah (R) dan 2 isyarat hijau (G). Kajian ini, menunjukan nisbah isyarat TERT/5q31 berlainan diantara 1 and 3 dalam satu sel. Sel yang mempunyai 3-4

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isyarat/sel merah (TERT) and dua isyarat hijau (5p31) dianggap mempunyai amplifikasi gred rendah. Terdapat juga kewujudan aneusomy kromosom 5, dimana terdapat 3 isyarat hijau menunjukkan kehadiran 3 salinan kromosom 5 atau trisomi 5. Sesetengah sel juga menunjukkan 3 isyarat hijau dengan lebih dari 2 isyarat merah.

Kajian kami mancadangkan bahawa amplifikasi gen TERT dikenalpasti dalam sel K562 tetepi penyiasatan lanjutan dengan memerhati kekerapan bentuk isyarat dalam 200 sel atau mengunakan sample metafasa FISH atau cytospin harus dilakukan untuk menunjukkan corak amplifikasi gen TERT dalam sel K562 atau jenis sel kanser yang lain. Optimesasi tambahan bagi teknik FISH sepatutnya dilakukan bagi memastikan keberkesanan kos sebelum ia digunapakai dalam untuk diagnosis rutin bagi mengesan amplifikasi gen TER T dalam pesakit-pesakit kanser.

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CHAPTERl

INTRODUCTION

J.l General introduction

Gene amplification is a copy number increase of a restricted region of a chromosome arm.

It is prevalent in some tumors and is associated with overexpression of the amplified

· gene(s). Gene amplification is a relatively rare event in haematological malignancies but it is more common in the development of many solid tumors (Schwab, 1998). The development of tumors is associated with the acquisition of genetic and epigenetic alterations and the corresponding changes in gene expression that modify normal growth control and survival pathways. These changes can be brought about at the genomic level in a variety of ways, including altered karyotypes, point mutations and epigenetic mechanisms. Genomic DNA copy number aberrations are frequent in solid tumors and are expected to contribute to tumor evolution by copy number-induced alterations in gene expression (Donna, 2006). The analysis of amplified DNA in mammalian cell lines and tumors has revealed that it can be organized as extrachromosomal copies, called double minutes; in tandem arrays as head-to-tail or inverted repeats within a chromosome, often forming a cytologically visible homogeneously staining region (HSR); or distributed at various locations in the genome (Donna et al., 2003). The unit of amplified DNA in some cases can involve sequences from two or more regions of the genome, indicating a complex

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process of formation involving multiple chromosomes. The unit of amplified DNA in tumors can range in size from kilobases to tens of megabases (Donna et al., 2003, Donna, 2006).

The TERT gene is one of the main components in telomerase enzyme which encodes human telomerase reverse transcriptase (hTERT) catalytic subunit which is located at human chromosome band 5p15.33. Telomerase is a ribonucleoprotein enzyme complex that adds telomeric repeats to the ends of chromosomes. This enzyme also consists of RNA component known as telomerase RNA (TR) template subunit which is encoded by TERC gene located at chromosome 3q26.3. Chromosomal gains and gene amplifications involving chromosome arms 5p and 3q are among the most frequent in human tumors (Ying et al., 2008). Telomerase activity has been detected in germ cell line and most cancer cells, but in normal human somatic cell it is either undetectable or present at low level. A chronic myeloid leukemic cell line, K562 has been found to express TERT gene amplification (Ying et al., 2008).

The development of fluorescence in situ hybridization (FISH) techniques has allowed the rapid and sensitive detection of single-copy DNA sequences in metaphase and interphase nuclei, thus greatly facilitating gene mapping by allowing the unambiguous assignment of genomic probes to specific chromosomal segments with great precision (Shapiro et al., 1993). Fluorescence in situ hybridization (FISH) provides a simple, fast, and reliable means to assess genetic instability in cancer. Perhaps the simplest method for detecting the genetic instability in cancerous tissue is to examine for abnormal numbers of chromosomes

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(aneusomies) in the affected tissue. Myeloid leukemias, including acute and chronic myeloid leukemia and myelodysplastic syndrome, have long been known to demonstrate the presence of a third copy (trisomy) of chromosome 8 (Fox et al., 1995). Increased knowledge of cancer genetics has led to the development of new assays for the detection of malignancy. One such assay, fluorescence in situ hybridization (FISH), has become a valuable tool for detecting and monitoring cancer FISH utilizes fluorescently labeled DNA probes to assess interphase or metaphase cells for chromosomal alterations. Studies have shown that FISH is able to identify malignant cells in a variety of cytologic specimens such as fine needle aspirates, effusions, and urine (Benjamin et al., 2004).

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1.2 Aim of study

K562 has been found to demonstrate TERT gene amplification. Therefore, this cell line was used as a model to validate TERT gene amplification status using Fluorescent in situ hybridization (FISH) technique in interphase stage. This study may facilitate the optimized FISH method to detect gene amplification in K562 cell line using a commercial dual color probe. The most reliable method for detection of TERT gene may have useful application in cancer patients.

The specific aims of the study were as follows:

1) To optimize detection method for TERT gene amplification in K562 cancer cell line using Fluorescence in situ Hybridization (FISH) in interphase cells.

2) To determine the TERT gene amplification signal pattern using FISH technique in K562 cell line.

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CHAPTER2

LITERATURE REVIEW

2.1 Telomerase Reverse Transcriptase (TERT) gene

The core telomerase components are the telomerase reverse transcriptase {TERT) catalytic subunit, and the telomerase RNA (TR) template subunit. In most cancers, telomerase activity is expressed at levels that are substantially higher than in normal cells. A known consequence of telomerase upregulation which is considered to play a critical role in oncogenesis is maintenance of telomere length, and thus evasion by cancer cells of the normal limits on proliferation that are associated with the steady decrease in telomere length that accompanies proliferation of normal cells. It has also been suggested that telomerase upregulation confers other advantages on cancer cells independent of its enzymatic activity (Ying et al., 2008). The mechanisms responsible for up-regulation of telomerase in cancer are incompletely understood. Here we review evidence suggesting that this frequently results from increased copy number of the genes encoding telomerase components. The TERT gene is located at human chromosome band 5p15.33, and the telomerase RNA component (TERC) gene that encodes TR is at 3q26.3 (Ying et al., 2008).

Increased TERT and TERC gene dosage has been detected frequently in a variety of human cancers and clonal evolution of cells with increased TERT or TERC copy number has been

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observed, suggesting a growth advantage in cells with increased TERT or TERC gene dosage. (Ying et al., 2008).

2.1.1 Telomerase and its associated protein

Telomerase enzyme maintains telomere ends by addition of the telomere repeat TTAGGG.

The enzyme consists of a protein component with reverse transcriptase activity, encoded by this gene, and an RNA component which serves as a template for the telomere repeat.

Telomerase expression plays a role in cellular senescence, as it is normally repressed in postnatal somatic cells resulting in progressive shortening of telomeres. Deregulation of telomerase expression in somatic cells may be involved in oncogenesis. Studies in mouse suggest that telomerase also participates in chromosomal repair, since de novo synthesis of telomere repeats may occur at double-stranded breaks. Alternatively spliced variants encoding different isoforms of telomerase reverse transcriptase have been identified; the full-length sequence of some variants has not been determined. Alternative splicing at this locus is thought to be one mechanism of regulation of telomerase activity.(Ying et al., 2008)

The active human telomerase enzyme is composed of human telomerase reverse transcriptase (hTERT), human telomerase RNA (hTR) and dyskerin (Cohen et al., 2007).

hTERT (encoded by the TERT gene) is the catalytic reverse transcriptase component (Nakamura et al., 1997), hTR (encoded by the TERC gene) serves as the RNA template for

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the addition of telomeric repeats (Feng et al., 1995) and dyskerin (encoded by the DKC1 gene) is an RNA binding protein (Mitchell et al., 1999). Mutations in any of these components may result in dyskeratosis congenita, a human disease syndrome associated with short telomeres (Kirwan and Dokal, 2008).

Human TERT subunit (hTERT) is a protein of 1132 amino acids with a highly conserved domain homologous to reverse transcriptase (RT) (Nakamura et al., 1997). In contrast to hTR, hTERT is only expressed in a limited number of normal tissues, such as germ cells, stem cells, and activated lymphocytes, but is highly expressed in immortalized cells and most tumor tissues (Yinhua et al., 2002). Telomerase activation requires the interaction between TERT and TR. In vitro studies showed that telomerase activity could be reconstituted using only hTR and hTERT although a variety of additional factors may participate in regulating or promoting in vivo activity (Yinhua et al., 2002). Mouse fibroblast cells lacking the RNA component of telomerase exhibited progressive telomere shortening and chromosomal instability (Hande et al., 1999). Late generations of TR knockout mice had shorter telomeres and an increased incidence of spontaneous tumors (Rudolph et al., 1999). All these data show that the combination of both subunits is necessary for telomerase function. However, where and exactly how these two parts are assembled to form the active RNP complex (Telomerase RNP) in mammalian cells remains unknown (Yinhua et al., 2002).

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2.1.2 Involvement of TERT gene amplification in cancer

Telomerase activity has been detected in more than 85% of human tumors (Kim eta/., 1994) whereas in normal human somatic cells it is either undetectable or present at low levels. In normal cells, telomeres shorten with every cell division, and this eventually results in senescence, a state characterized by permanent withdrawal from the cell division cycle. The increased telomerase activity found in cancers prevents telomere shortening, and allows cancer cells to escape the normal limits on cellular proliferation. (Ying et a/., 2008).

Several studies demonstrated copy number increases of the TERT gene in multiple tumors or immortalized cell lines (Ying eta/., 2008). Previous study (Anju eta/., 2000), shows that FISH analysis using a probe that covered the genomic region encoding TERT together with a specific sequence at 5q31 as a marker probe detected two TERT gene copies located on band 5p15.33 and a 1:1 ratio ofTERT/5q31 signal in normal cells. However, only 5 of 26 human tumor cell lines from different origins and 28 of 58 human primary tumors carried two TERT and two 5q31 marker copies. The remainder of the cell lines and primary tumors had more than two copies of TERT and a TERTI 5q31 ratio 2:1. Some of these (50% of cell lines and 22% of primary tumors) displayed 3-4 TERT copies/cell while 31%

of cell lines and 30% of human primary tumors had 2:5 copies of TERT per cell (Anju et a/., 2000). These included cell lines derived from neuroblastomas (Lan2, Lan5 and SHEP1) and carcinomas of breast (578T), cervix (HeLa and CaSki) and lung (H125, U1285, U1752, SHP77/97, H1688, Colo677/97, H446/97, BEN and H209). (Anju eta/., 2000) (Zhang et al., 2002) (Saretzki et a/., 2002). Cell lines derived from bladder and epidelli).al carcinomas (5637 and A431) were reported to have 2:3 copies ofhTERTper cell (Bryce et 8

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a/., 2000). In pnmary tumors, increased TERT copy number has been detected in neuroblastomas (12%), CNS embryonal tumors (42%), hepatocellular carcinomas (22%) and cancers of the lung (30-63%), cervix (24-30%), breast (26%) and colon (48%). In addition, FISH analysis revealed 2-60 copies of TERT in leukemic cells (Nowak eta/., 2006).

2.1.2.1 Role of TERT gene and in regulation of Telomerase activity

Most human somatic cells do not produce active telomerase and do not maintain stable telomere length with proliferation. Most or all do have telomerase RNP, which raises the possibility of a second telomerase function independent of DNA synthesis. The significance of telomerase function in any given cell is dependent on both telomere length and the number of future cycles of proliferation. It becomes more difficult to interpret the importance of telomerase regulation in settings where this is means something other than just the constitutive absence or overabundance of activity (Kathleen Collins and Mitchell,

2002).

In humans, telomerase activity detectable at the blastocyst stage and in most embryonic tissues before 20 weeks of gestation is subsequently lost (Wright et a/., 1996). Some correlation can be made between telomerase inactivation, differentiation and increased rate of telomere loss with time (Ulaner et a/., 2001 ). Several levels of regulation combine to determine the amount of active telomerase in any given cell.

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