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THE ROLE OF NON-CANONICAL FUNCTION OF TELOMERASE ENZYME IN THE DEVELOPMENT OF KERATINOCYTE CARCINOGENESIS.

Byline: JOHARIA AZHAR SAADAT and SAEEDA ABDULLAH

ABSTRACT

The objective of this study was to test the hypothesis that the non-canonical function of telomer- ase renders cells resistant to cell death thus enhancing tumorogensis. To demonstrate this telomerase positive cells were treated with a Phorbol ester TPA to induce apoptosis as it has documented in lit- erature that TPA induces cell death in normal but not initiated cells. After these treatments the cells were assayed for cloning efficiency. Normal keratinocytes was transfected with TERT gene and after treatments with TPA were assayed again for cloning efficiency. Furthermore to observe the process of apoptosis cells treated with TPA and cis-platinum were assayed for caspase and 7 by performing immunocytochemistry and a biochemical assay for caspase /7. It was demonstrated that TERT does not appear to promote cancer by making cells resistant to TPA. Immunocytochemistry gave some evidence that at least a subset of cells show caspase activity after twenty four hour application of TPA. Therefore our study demonstrates that the non-canonical function does not appear to enhance tumorogensis.

Key Words: Non-Canonical. Telomerase. Carcinigensis Keratinocytes. INTRODUCTION

Telomeres are nucleotide sequence of TTAGGG which is repeated a number of times to form a protec- tive cap at the end of the chromosomes.1 Its functions are to prevent DNA strand ends recognition as DNA strand breaks by the polymerase enzyme so that the repair machinery will not endeavour to repair these.2

They also constitute the internal clock like mechanism that the cell maintains to record the number of times it has divided. These telomeres are maintained in the cell by an enzyme called as the telomerase.4 The canonical function of telomerase enzyme is to protect the cells from telomere erosion and senescence by re- peatedly adding nucleotide sequence TTAGGG at the chromosomal ends with each cell division.56

Recently it was observed that low levels of telomer- ase are present in the s phase of cell cycle of somatic cells at levels that are not adequate to sustain telomeres lengths.7 Furthermore it was observed that ectopic expression of telomerase causes stem cell division8 mobilization and migration9 increased wound repair8 and an increased tumor burden.10 Since mouse cells have very long telomeres so this was unlikely to be due to the lengthening of the telomere repeats. Based on these observations it was established that telomerase has at least one non-canonical and elongation-inde- pendent function.11 Certain studies involving mouse models for two stage tumorigensis showed that TERT over expression promoted papilloma formation.8 There was also wide spread speculation that mice deficient in telomerase and having short telomeres were resis- tant to tumorigensis.

It was therefore theorized that the non-canonical function of telomerase renders cells resistant to cell death thus enhancing tumorogensis. The possibility of utilization of telomerase inhibition in cancer therapeutics has since long haunted the cancer biologists. The idea of having a miracle drug that will only targets the telomerase positive cancer cells while miraculously sparing the normal somatic cells can become a reality. However in the highly unpre- dictable world of cancer research the fact of telomerase presence and contribution to almost 90% of the cancers cannot have straight forward implications. A lot of work is required to understand the role of telomerase in carcinogenesis especially regarding its non-canon- ical pathways before it can become actively involved in cancer therapeutics. This is the basic vision behind this project.

The purpose of this study was to see:

1. whether the non-canonical telomerase functions enhance tumorogensis by rendering cells resistant to cell death. 2. whether the mechanism of TPA induced cell death is apoptosis.

METHODOLOGY

All procedures requiring aseptic conditions were carried out in a class II Biomat(R) Microbiology Safety Cabinet (Medical Air Technologies) except for the ret- roviral transduction which was done in the category II containment facility. All products and materials used were supplied by SIGMA (Sigma Aldrich Company Ltd UK) unless mentioned otherwise. The cell lines used were HaCaT cells (Three genetically altered HaCaT derived cell lines were used provided most generously by Prof. E. K. Parkinson namely HaCaT TERT HaCaT TERT-HA HaCaT PURO) SCC-25 cells HEK-125 cells HEK 127 cells and N-HEK cells.

CELL CULTURE

Incubation of cells was carried out in a water saturated atmosphere of 5% CO2/ 95% air at 7C in an incubator. Confluence was determined by visual inspection and the cells were counted using glass hae- mocytometer following the manufacturer's instructions. Two types of media were used in these experiments. HaCaT cells and SCC-25 were cultured in DMEM while KGM MEDIUM was used for the HEK cell lines.

CLONING EFFICIENCY ASSAY

Cloning efficiency assay was performed for pro- liferation assessment of the cells treated with TPA a potent tumor promoter being used here as a death inducing agent. TPA also called as PMA is Phorbol ester 12-myristate 1-acetate. The concentration used was 100ng of TPA /ml of DMSO (dimethyl sulfoxide).

For this assay HEK-125 were used as a positive control as they have been documented in the literature to have been killed by TPA application ( Parkinson et al 1980) while SCC-25 were used as negative control. The main cell lines used were the HaCaTs HaCaT PURO HaCaT TERT and HaCaT TERT-HA. The stock plates of each of these cell lines were trypsinized following the previous protocol and plated in a 5mm well of a six well plate each well representing a single cell line at a concentration of 2A-105. Two such plates were set up. The plates were cultured for 72 hours after which one of the plates was treated with TPA using 2ml of TPA+DMEM/well (2l of TPA stock+ 20ml of DMEM) for the HaCaT and SCC-25 cell lines while 2ml of TPA +KGM/well (2l of TPA stock+ 20ml of KGM) for the HEK-125 cells. The other plate was treated with DMSO using the same concentration in media i.e. 2ml/well.

TPA and DMSO treatment was carried out for 24 hours after which media containing TPA and DMSO was as- pirated out. The plates were trypsinized and replated in duplicates at 5000 cells per well. The four plates two for TPA and two for DMSO were cultured for 72 hours. After this period the media was aspirated. Cells were fixed using formaldehyde solution and stained with haemotoxylin to stain the nuclei. The number of cell colonies in each well that cells had made were counted and recorded. Six such cloning efficiency assays were performed. A six well plate is shown in fig 1.

RETRO-VIRAL TRANSDUCTION

Retro-viral transduction was performed on HEK-125 cells to introduce TERT and TERT-HA constructs in order to determine the cloning efficiency of these cells after TPA application.

HEK-125 cells were seeded at a density of 7.5A-104 in a six well plate and cultured over night. The first transduction using polybrene was carried out follow- ing the manufacturer's instructions. Briefly 5g/ml Polybrene was prepared in KGM (refer to appendix) in a glass bottle was warmed at 7C and vortexed. Culture media was aspirated from the HEK-125 cells and replaced with polybrene 1ml/well after which they were incubated. The viral supernatants containing the DNA constructs were defrosted. These retroviral supernatants were made and titrated by Dr. Caroline Fltchett. There were four different supernatants hav- ing very similar titres namely TERT and TERT-HA which contained the normal telomerase with canon- ical function and the wild type TERT with only the non-canonical function respectively. P-Babe contained the empty vector instead of the TERT gene and P-Sin which had the

GFP (green fluorescent protein) tagged vector to determine the transduction efficiency. Since the titres were similar so the transduction efficiencies were assumed to be equal as well. The presence of GFP allowed the number of cells that had become successfully transduced to be determined. One of the cell lines HEK- MOCKS was being used as the negative control so it was only treated with polybrene and was not exposed to viral particles with DNA vectors. The supernatants were transferred to glass bottles and 5g/ml polybrene (1l polybrene/ml of supernatant) was added to them. Polybrene/media was aspirated and replaced with polybrene/supernatant 2ml/well in the six well plate. Plate was centrifuged for 1 hour at 2C with 50rpm. After one hour polybrene/supernatant was aspirated and cells washed with PBS KGM was then added to them and they were cultured for 24 hours in the Cate- gory II containment room.

The next day the cells were visually checked for green fluorescence and the second transduction carried out following the same protocol as described. After 24 hours the tranduced cells were seeded in duplicates at a concentration of 20000 cells per well and then allowed to grow for 72 hours. A six well plate with the cell lines is shown in Fig. 2. CLONING EFFICIENCY AFTER RETRO VIRAL TRANSDUCTION

The P-Sin wells of the four plates were checked for green fluorescence and after confirming that the transduction has been successful the main cells i.e. Mock P-Babe Tert and Tert-ha were prepared to undergo the cloning efficiency assay. The same proto- col was followed as already described and two of the plates were treated with TPA+KGM (2l TPA in 20ml KGM) while the other two treated with DMSO+KGM (2l DMSO in 20ml KGM). Cells were left for 24 hours in TPA and DMSO after which they were replated and then left to form colonies for 72 hours. At the end of this period they were fixed using formaldehyde stock solution and stained with haemotoxyline. The total numbers of colonies made by the cells were then counted and the readings recorded.

IMMUNOCYTOCHEMISTRY

Immunocytochemistry was performed using the cleaved caspase antibody (supplied by cell signal- ing Catalog # 9661S with a concentration of 1:50). In this experiment TPA was again used to induce cell death DMSO was used as the negative control while CIS-Platinum (SIGMA product no. P494) was used as the positive control for induction of apoptosis. The cells used were HEK-127 which were grown on LabTek(R) glass culture chamber slides. The cells were treated with TPA and DMSO in the concentrations already described while the final concentration of cis-platin in DMSO was 20M (refer appendix) .Cells were left with TPA DMSO and CIS-Platinum for 24 hours after which the used medium was removed and the cells washed in PBS. They were then fixed using acetone at room temperature for 10 minutes after which they were rinsed in the PBS. The blocking serum from the Vector Laboratories ABC Kit diluted 1:10 was added for 20 minutes at room temperature.

The slides were then drained (not rinsed).

The primary caspase anti-body was applied at the dilution of 1:50 overnight at 4 degree C. Slides were washed three times with gentle rocking in washing buffer (Refer to Appendix) 10 minutes each. Second- ary anti-body from the kit was added at the dilution of 1:200 for one hour at room temperature. The slides were washed again three times with gentle rocking in washing buffer. ABC reagent was added for one hour (at room temperature). Once again gentle rocking in washing buffer applied. Finally DAB reagent was added in PBS (7 minutes at room temperature in the dark). The cells were then washed with PBS and light- ly counter stained with haematoxylin and mounted using E Z mount. All incubations were carried out in a humidified box to prevent drying. All dilutions were made in sterile PBS 0.1% weight/volume bovine serum albumin fraction V pH 7.6.

RESULTS

CLONING EFFICIENCY OF HaCaT's

The HaCaTs proved to be quiet resistant to TPA. Six such cloning efficiency assays were performed and while the positive and negative controls behaved as expected HaCaTs appeared to be resistant giving a hint of rather being stimulated to grow with 24 hour TPA application. So it was concluded that HaCaTs are resistant to TPA and at least in the case of HaCaT cells the TERT was not acting according to our hypothesis. Figures show the relative values of number of colonies formed by each cell line after treatment with TPA and DMSO.

TRANSDUCTION OF HEK CELLS

In this experiment the main cell lines virally transduced with TERT and TERT-HA gene were HEK- TERT and HEK-TERT-HA. PBabe was transduced with empty vector and was synonymous with PURO used in the cloning efficiency assay. HEK-MOCK was the negative control which was not treated with viral particles containing vectors. P-Sin was treated with the GFP (green fluorescent protein) tagged vector to determine the transduction efficiency which showed 79% results. Fig 9 and 10 show the transduction effi- ciency as represented by the number of cells expressing green fluorescence.

CLONING EFFICIENCY AFTER THE TRANSDUCTION

The following pictures represent the morphological differences between transduced HEK cells that were treated with TPA and DMSO. As it can be easily ob- served from the above pictures that 24 hour exposure to TPA was enough to kill the HEK cells. Their cloning efficiency was greatly decreased as compared to the ones that had been treated with DMSO. Further more many of the cardinal signs of cell death could be seen like shrinkage of the cell condensation of cytoplasm and chromatin detachment of cell from neighbouring cells rounded up margins and in some cases membrane blebing.

The relative cloning efficiency of the HEK cells that have been transduced can be seen from fig 15: This graph represents the cloning efficiency with TPA as a percentile of cloning efficiency with DMSO. Here TERT seemed to be doing a little to make the HEK cells somewhat more resistant to cell death by TPA but the values of the HEK TERT-HA were inconsistent with the rest of the data. Further more this experiment was only performed once on account of unavailability of early passage keratinocytes and hence represents a sole attempt at determining the cloning efficiency after transduction. This experiment needs to be repeated a number of times to optimize the protocol and to get statistically relevant reproducible results. IMMUNOCYTOCHEMISTRY FOR CASPASE

Antibody against caspase was used to detect the number of cells if any that were undergoing apopto- sis. After the completion of these experiments it was observed that in the case of HEK-127 cells treated with TPA for 72 hours rare cells showed definite caspase activity which could be seen by the presence of cells showing brown staining. Brown stain represent cells that had been treated with cis-platin for 24 hours the positive control in this experiment which strange- ly enough didn't stain positive for caspase activity although the cells did show morphological changes consistent with those seen in apoptosis namely cyto- plasmic and chromatin condensation in some cases fragmentation of the cell. The cis-platin dose used was very high which might have induced cell death earli- er then the 24 hours when cells were tested and this may explain the reason of absence of active caspase activity.

CONCLUSIONS

TERT does not appear to promote cancer by making cells resistant to TPA. The cloning efficiency assay done both on HaCaTs and HEKs after transduction gives sufficient data to support this notion. Although in the cloning efficiency assay after transduction TERT appeared to be doing something on a small scale but further experiments need to be performed to confirm this.

HaCaT cells are resistant to TPA. The levels of TPA that were sufficient to induce cell death in normal keratinocytes did not affect HaCaT. On the contrary they appeared to be stimulating HaCaTs on account of the large number of colonies that cells formed after TPA treatment. In the HEK Cells treated with TPA at least a subset of cells undergoes caspase activity.

REFERENCES

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2 Sheila A. Stewart William C. Hahn Benjamin F. O'Connor Elisa N. Banner Ante S. Lundberg Poonam Modha Hana Mizuno Mary W. Brooks Mark Fleming Drazen B. Zimonjic Nicholas C. Popescu and Robert A. W. Telomeras contributes to tumorogensis by a telomere length-independent mechanism. PNAS 2002; 99: 12606-12611.

Bruce Alberts Dennis Bray Karen Hopkin Alexander Johnson Julian Lewis Martin Raff Keith Roberts and Peter Walter. Essential cell biology. Second edition. Pg 201-208. 2004.

4 Maria A. Blasco. Telomerase beyond telomeres. Nature Reviews/ Cancer. 2002; 2: 1-6.

5 Maria A. Blasco and William c. Hahn. Evolving views of telo- merase and cancer. Trends in cell biology 200; 1: 289-94.

6 Maria A Blasco. Telomeres and Cancer. Current opinion in genetics and development. 200; 1 70-76.

7 Kenkichi Masutomi Richard Possemato Judy M. Y. Wong Jennifer L. Currier Zuzana Tothova Judith B. Manola Shridar Ganesan Peter M. Lansdorp Kathleen C. and William C. Hahn. The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. PNAS 2005; 102: 8222-8227.

8 Eva Gonzalez-Suarez Enrique Samper Juana M. Flores and Maria A. Blasco. Telomerase-deficient mice with short telomeres are resistant to skin tomorigensis. Nature Genetics 2000; 26: 114-17.

9 Ignacio Flores Maria L. Cayuela Maria A. Blasco. Effects of telomerase and telomere length on epidermal stem cell be- haviour. Science Express 2005; 1126: 1-10.

10 Cayuela M Flores J Blasco M. The telomerase RNA compo- nent Terc is required for the tumour-promoting effects of Tert overexpression. EMBO Rep. 2005; 6: 268-74.

11 Rahman R Latonen L and Wiman KG. hTERT antagonizes p5-induced apoptosis independently of telomerase activity. Oncogene. 2005; 24(8): 120-27.
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Publication:Pakistan Oral and Dental Journal
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2014
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