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c-ABL IS A POSITIVE REGULATOR OF TELOMERASE AND GOVERNS TRANSLOCATION IN DIFFERENT CELLULAR COMPARTMENTS

INTRODUCTION Aim The following study aims to investigate the interaction between c-Abl and hTERT in different cellular compartments and the mechanism by which BCR-ABL regulates hTERT.

Telomere and telomerase Telomeres are highly specialized nucleoprotein complexes located at the end of linear eukaryotic chromosomes8,10,11. These tandem repetitive structures facilitate the capping of chromosomes, without which chromosomes may degrade. Since telomeres cannot be fully replicated by DNA polymerases due to the end-replicating problem, they shorten with each cycle of cell division8,17. When telomeres reach a critically short length, chromosomes become unstable. In order to prevent loss of genetic information due to

telomere shortening, additional repeating DNA sequences must be added. To accomplish this, eukaryotes employ an enzyme called telomerase, a

ribonucleoprotein comprising of (i) a reverse transcriptase subunit (TERT) and (ii) telomerase RNA (TER).

Fig. 1 shows telomere shortening, chromosomal instability and cancer initiation 17.

Elevated levels of telomerase activity have been observed in most human cancers. According to these findings, telomerase play a crucial role in cancer progression by maintaining the telomere length17. How the telomerase activity is regulated, however, remains elusive. Critically short telomeres lose chromosomal capping function, particularly when the physical integrity becomes disrupted8, resulting in activation of DNA damage pathways and double strand breaks (DSB) repair.

Initially, it was proposed that telomere shortening represented a tumor suppression mechanism. However, in line with this proposal was another hypothesis that telomerase re-activation may be the cause of cancer progression. We now know of some telomere and telomerase mechanism; but majority of them are still inadequately understood and as such this research proposal here would help to shed some light and generate certain insights into their complex machinery.

c-Abl tyrosine kinase The ubiquitously expressed protein, c-Abl, is a tightly regulated nonreceptor tyrosine kinase that is able to regulate signaling processes in sub-cellular compartments3,13,15. Perhaps, its tyrosine kinase activity may be best known for its deregulation in chronic myelogenous leukemia (CML).

To further understand the regulation of telomerase, we will first need to study on the regulation of its core component, hTERT. A previous study conducted has shown that the ectopic expression of hTERT is able to promote cell immortalization by blocking telomere shortening. In line with this study, it is reported that c-Abl phosphorylates hTERT and inhibits its activity16.

We are aiming to identify if c-Abl interacts with telomerase because when DNA DSB occur, c-Abl becomes active and the proteins involved in repairing will act in telomere control. In contrast to the previous findings, our preliminary data seems to indicate that c-Abl is a positive regulator of hTERT in normal somatic fibroblast and cancer cells. Moreover, over-expression of the inactive form of Abl (c-Abl kinase dead) decreases the expression of hTERT.

BCR-Abl and CML One of the many hallmarks of cancer includes the disruption of normal regulation of protein kinase activity3. CML is one such cancer characterized by genetic abnormality, where an event of chromosomal translocation takes place2,3,5. In this translocation, there is an exchange of DNA between the c-Abl and the breakpoint cluster (bcr) gene resulting in an abnormally small chromosome termed as Philadelphia

chromosome1,2,5. This defective chromosome carries an aberrant hybrid gene (retaining parts of structural conformation and functionality from both c-Abl gene and bcr gene), eventually giving rise to a fusion oncoprotein, BCR-Abl3,5.

The hyperactive protein activates many of the downstream signaling pathways, resulting in the uncontrolled proliferation of blood cells which accounts for the majority

of CML cases, occurring mainly in adults2,4. In fact, expression of BCR-Abl can also bring about the effects of enhanced proliferation, morphological transformation, or abrogation of growth factors dependence3. How BCR-Abl increases the number of normal mature myeloid cells in CML remains largely unknown.

In this study, we will look into the significance of how tyrosine kinase activity is fundamental to the action of BCR-Abl in the developments of CML and its indication as a possible novel regulation on telomerase. In addition, in-depth understanding of the BCR-Abl induced transformation will enable us to produce more effective treatments for CML.

Therapeutic treatment Medical advancement has led to the discovery of Gleevec (Imatinib Mesylate) or STI571, a drug that has a high affinity for the cleft between the N- and C-lobes of the tyrosine kinase domain of BCR-Abl1,3,4,5. When being used as a treatment, it effectively blocks the growth of BCR-Abl induced cells and in turn, eradicates the expression of BCR-Abl in CML. Extensive clinical studies are carried out to test for the efficacy of the drug in patients afflicted with CML.

When the biology of Gleevec is introduced, mechanism of how STI-571 specifically inhibits the overactive BCR-Abl became the key focus. STI-571 binds to the inactive form of tyrosine kinase Abl domain and competitively inhibits binding of adenosine triphosphate (ATP), thereby resulting in conformational changes 3. Since BCR-Abl is able to activate downstream mitogenic effector pathways, it is likely that STI-571 mediates such inhibition by blocking the BCR-Abl generated signals.

While STI-571 may be approved to be used as an effective drug treatment, it cannot be directed against patients in the blast crisis phase of CML. It was also observed that some human cell lines could develop resistant strains against the drug5. These mutations, apparently, were found only to affect the allosteric binding of STI-571, and confer resistance to the treatment by down regulating the kinase activity of c-Abl. Furthermore, rapid approval of Gleevec meant that there were insufficient studies to be concluded on its long term side effects. To elucidate the mechanism of leukemia resistance in our study, detailed understanding of the resistance towards anticancer therapeutics will be vital for the development of a better and newly improved drug.

Conclusion According to our research findings and initial results, it is evident that telomerase dependent telomere is essential for cell immortalization, cancer progression as well as disease metastasis. It is also true that both telomere shortening and telomerase reactivation can play a dual role in cancer initiation and progression. While telomere shortening appears to be associated with chromosomal instability, other evidence also suggests that tumor progression requires re-activation of telomerase in order to prevent chromosomal instability associated with telomere induced crisis.

In the study we conducted, we found that c-Abl is a positive regulator of hTERT in normal human somatic fibroblast cells. And from this, we can safely draw up the conclusion that c-Abl plays a dual role in regulating telomerase.

The chimeric BCR-Abl protein associated with about as much as 95% of CML is likely to act through phosphorylation of downstream substrates, resulting in the activation of various cellular pathways thereby conferring resistance to the Gleevec treatment.

Because BCR-Abl is the leading cause for leukemia, we will look further into how cAbl regulates telomerase and likewise whether BCR-Abl can interact and/or regulate telomerase in vivo and in vitro.

Further discussion and experiments will enable us to understand the importance of studying telomerase regulation by c-Abl and BCR-Abl in cancer cells. This finding will allow us to design a more improved therapeutic drug to fight against CML.

MATERIALS AND METHODS Materials Drugs and chemicals Imatinib (Gleevec/STI-571)

Cells and cultures K562 human chronic myeloid leukemia cells with BCR-Abl over-expression HL60 human promyelocytic leukemia cells expressing normal levels of BCRAbl BJ normal human foreskin fibroblasts MCF-7 human breast adenocarcinoma cells HeLa human cervical epitheloid carcinoma cells IMR90 human diploid lung fibroblast cell line isolated from a female fetus Jurkat cell human T lymphocyte cell line without BCR-Abl over-expression

Animals, plasmids and Abs Retrovirus vector expressing c-Abl, inactive form of c-Abl and BCR-Abl (Clonetech) C-Abl knockout mice

Methods Western blotting Luciferase assay DNA micro-array RT-PCR cRNA amplification Confocal assay

Telomere length assay C-Abl kinase assay

TIME SCHEDULE

WEEK 1 - 7/9/09 to 13/9/09

1. Write up on "Telomere and telomerase in breast cancer and breast cancer stem cells" 2. Learn tissue culture protocols e.g. cell maintenance, cell freezing, primary culture etc 3. Plasmid maxiprep 4. Purify genomic DNA 5. Learn confocal microscopy for live cell imagine 6. Learn nanodrop 1. Western blotting 2. Freeze cells for storage 3. Culture primary breast tumor and normal tissues 4. Learn confocal microscopy for live cell imagine 5. Isolate RNA for various samples (breast pair, colon normal and lung tumor) 1. Maintain cells 2. Western blotting 3. Prepare medium 4. Draw up table on telomeric binding proteins 5. RT-PCR on lung tissue using 4 primers 1. Western blotting 2. Read up on Rap1 knockout mice construct 3. Prepare summary of primary breast cultures 1. Western blotting 2. Prepare RIPA lysis buffer 3. Extract DNA 4. Research on human Rap1 gene sequence (NCBI) 5. Amplify RNA (Illumina Totalprep) 6. RT-PCR 7. Freeze and maintain cells 8. Measure for nanodrop

WEEK 2 - 14/9/09 to 20/9/09

WEEK 3 - 21/9/09 to 27/9/09

WEEK 4 - 28/9/09 to 4/10/09

WEEK 5 - 5/10/09 to 11/10/09

WEEK 6 - 12/10/09 to 18/10/09

1. RT-PCR 2. Prepare slides for viewing under confocal microscope 3. Capture live cell images using confocal microscope 4. Cast gel (7%) for western blotting 5. Western blotting 6. Prepare RIPA lysis buffer 1. Western blotting 2. Repeat western blotting 3. Read up scientific article: Generation of knockout mice 4. Learn live cell imaging using confocal micrscope 5. Measure Luciferase activity 5. Submit brease cancer updates (in powerpoint) 1. Prepare Rap1 protein and knockout mice information (in powerpoint) 2. Cast gel (7%), quantitate protein and transfer gel onto membrane 3. Prepare microarray for DNA copy number information (in powerpoint) 4. Western blotting 1. Read up on journal articles related to breast cancer 2. Prepare micro-array kit informtation (in powerpoint) 3. Submit hRap1 amino acid sequence 4. Western blotting 5. Cell count 1. Western blotting 2. Luciferase assay 3. Luciferase tranfection 1. Western blotting 2. Cell count 3. Culture cell

WEEK 7 - 19/10/09 to 25/10/09

WEEK 8 - 26/10/09 to 1/11/09

WEEK 9 - 2/11/09 to 8/11/09

WEEK 10 - 9/11/09 to 15/11/09

WEEK 11 - 16/11/09 to 22/11/09

WEEK 12 - 23/11/09 to 29/11/09

1. Western blotting 2. Live cell imaging using confocal microscopy 3. Micro-array assay 1. Western blotting 2. Micro-array assay 1. Western blotting 2. Micro-array assay 1. Western blotting 1. Western blotting 2. Prepare buffers 3. RT-PCR 4. RNA isolation

WEEK 13 - 30/11/09 to 6/12/09

WEEK 14 - 7/12/09 to 13/12/09

WEEK 15 - 14/12/09 to 20/12/09 WEEK 16 - 21/12/09 to 27/12/09

WEEK 17 - 28/12/09 to 3/1/09

1. Western blotting 2. Collate results 1. Western blotting 2. Collate results 1. Collate results 2. Prepare poster 1. Collate results 2. Prepare report

WEEK 18 - 4/1/09 to 10/1/09

WEEK 19 - 11/1/09 to 17/1/09

WEEK 20 - 18/1/09 to 24/1/09

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