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Expression Pattern of Alternative Splicing Variants of Human Telomerase Reverse Transcriptase (hTERT) in Cancer Cell Lines Was not Associated with the Origin of the Cells.

Khosravi-Maharlooei M, Jaberipour M, Hosseini Tashnizi A, Attar A, Amirmoezi F, Habibagahi M - Int J Mol Cell Med (2015)

Bottom Line: Relative assessment of hTERT expression showed greater expression of the α-deleted variant in the telomerase negative cells (P= 0.04).Those cells possessed the α/β-deleted variant to a smaller extent when compared to the cells with telomerase activity.In spite that, the regulatory roles of hTERT on telomerase activity is still a potential to be utilized as targets for cancer therapies.

View Article: PubMed Central - PubMed

Affiliation: Student Research Committee, Cell and Molecular Medicine Research Group, Shiraz University of Medical Sciences, Shiraz, Iran.

ABSTRACT
Telomerase and systems controlling their activity have been of great attention. There are controversies regarding the role of the alternative splicing forms of the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Therefore, the correlation between telomerase enzyme activity, the abundance of alternatively spliced variants of hTERT and doubling time of a series of cancer cell lines originated from hematopoietic, breast, colorectal, neural, ovarian, lung, kidney, bladder, prostate and head and neck cancers were investigated. Expression levels of four different variants of hTERT (the full length, α-deletion, β-deletion and α/β-deletion) were quantitatively measured by real time PCR. Telomerase activity was determined by the telomerase repeat amplification protocol (TRAP) while doubling time of the cells measured by plotting growth curves. Results showed high diversity in the relative proportions of hTERT transcripts while the majority of the cells expressed the full length variant as the main transcript. Telomerase activity could not be detected in all cells. Relative assessment of hTERT expression showed greater expression of the α-deleted variant in the telomerase negative cells (P= 0.04). Those cells possessed the α/β-deleted variant to a smaller extent when compared to the cells with telomerase activity. Greater association between full length spliced variant and β-variant expression was observed in cells presenting telomerase activity (P= 0.0007, r= 0.74). High degrees of variation among the studied cells regarding the pattern of hTERT expression were present. In spite that, the regulatory roles of hTERT on telomerase activity is still a potential to be utilized as targets for cancer therapies.

No MeSH data available.


Related in: MedlinePlus

Deletion sites of different variants of hTERT Alpha deletion site is located on exon 6 while beta deletion site is on exons7 and 8. Arrows indicate the position of specific primers used to analyze the expression of full length hTERT and three alternatively spliced variants i.e. α-, ß- and αß- by real time PCR
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Figure 1: Deletion sites of different variants of hTERT Alpha deletion site is located on exon 6 while beta deletion site is on exons7 and 8. Arrows indicate the position of specific primers used to analyze the expression of full length hTERT and three alternatively spliced variants i.e. α-, ß- and αß- by real time PCR

Mentions: Total RNA was extracted by TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions. The quality and quantity of the extracted RNA samples were determined by spectrophotometry. RNA samples were treated with DNase I (Fermentas, Lithuania) to remove genomic DNA contamination. cDNA was synthesized from 5 μg of the total RNA with the RevertAid H minus First Strand cDNA synthesis kit (Fermentas, Lithuania). The hTERT variant transcripts were measured through real time PCR using specific primers (Table 2) for each variant (α-deletion, β-deletion, α/β-deletion and full length variants) and SYBER Green I as reporter dye (Applied Biosystems, USA) on Chromo4 Detector thermal cycler (Bio-Rad, USA). The expression of β-actin was used as a housekeeping gene in all analyzes. Primer-Blast online freeware (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design gene specific primers. Figure 1 shows the schematic location of hTERT gene deletions (∆αand ∆β) and also the binding sites of the designed primers on hTERT cDNA. Duplicated PCR reactions were set up in a final volume of 20 μL contained master mix, 0.5 µg of cDNA and 100 nM of each primer. The amplification program comprised an initial denaturation at 95 °C for 10 min and 45 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 50 s, extension at 78 °C for 30 s. Fluorescence emission was collected at the end of the extension periods. The relative expression of hTERT variants was calculated by 2-ΔCt equation. Melting curves were used to confirm the specificity of the fluorescent emission from the target sequences and rule out possible contamin-ation.


Expression Pattern of Alternative Splicing Variants of Human Telomerase Reverse Transcriptase (hTERT) in Cancer Cell Lines Was not Associated with the Origin of the Cells.

Khosravi-Maharlooei M, Jaberipour M, Hosseini Tashnizi A, Attar A, Amirmoezi F, Habibagahi M - Int J Mol Cell Med (2015)

Deletion sites of different variants of hTERT Alpha deletion site is located on exon 6 while beta deletion site is on exons7 and 8. Arrows indicate the position of specific primers used to analyze the expression of full length hTERT and three alternatively spliced variants i.e. α-, ß- and αß- by real time PCR
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499573&req=5

Figure 1: Deletion sites of different variants of hTERT Alpha deletion site is located on exon 6 while beta deletion site is on exons7 and 8. Arrows indicate the position of specific primers used to analyze the expression of full length hTERT and three alternatively spliced variants i.e. α-, ß- and αß- by real time PCR
Mentions: Total RNA was extracted by TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions. The quality and quantity of the extracted RNA samples were determined by spectrophotometry. RNA samples were treated with DNase I (Fermentas, Lithuania) to remove genomic DNA contamination. cDNA was synthesized from 5 μg of the total RNA with the RevertAid H minus First Strand cDNA synthesis kit (Fermentas, Lithuania). The hTERT variant transcripts were measured through real time PCR using specific primers (Table 2) for each variant (α-deletion, β-deletion, α/β-deletion and full length variants) and SYBER Green I as reporter dye (Applied Biosystems, USA) on Chromo4 Detector thermal cycler (Bio-Rad, USA). The expression of β-actin was used as a housekeeping gene in all analyzes. Primer-Blast online freeware (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design gene specific primers. Figure 1 shows the schematic location of hTERT gene deletions (∆αand ∆β) and also the binding sites of the designed primers on hTERT cDNA. Duplicated PCR reactions were set up in a final volume of 20 μL contained master mix, 0.5 µg of cDNA and 100 nM of each primer. The amplification program comprised an initial denaturation at 95 °C for 10 min and 45 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 50 s, extension at 78 °C for 30 s. Fluorescence emission was collected at the end of the extension periods. The relative expression of hTERT variants was calculated by 2-ΔCt equation. Melting curves were used to confirm the specificity of the fluorescent emission from the target sequences and rule out possible contamin-ation.

Bottom Line: Relative assessment of hTERT expression showed greater expression of the α-deleted variant in the telomerase negative cells (P= 0.04).Those cells possessed the α/β-deleted variant to a smaller extent when compared to the cells with telomerase activity.In spite that, the regulatory roles of hTERT on telomerase activity is still a potential to be utilized as targets for cancer therapies.

View Article: PubMed Central - PubMed

Affiliation: Student Research Committee, Cell and Molecular Medicine Research Group, Shiraz University of Medical Sciences, Shiraz, Iran.

ABSTRACT
Telomerase and systems controlling their activity have been of great attention. There are controversies regarding the role of the alternative splicing forms of the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Therefore, the correlation between telomerase enzyme activity, the abundance of alternatively spliced variants of hTERT and doubling time of a series of cancer cell lines originated from hematopoietic, breast, colorectal, neural, ovarian, lung, kidney, bladder, prostate and head and neck cancers were investigated. Expression levels of four different variants of hTERT (the full length, α-deletion, β-deletion and α/β-deletion) were quantitatively measured by real time PCR. Telomerase activity was determined by the telomerase repeat amplification protocol (TRAP) while doubling time of the cells measured by plotting growth curves. Results showed high diversity in the relative proportions of hTERT transcripts while the majority of the cells expressed the full length variant as the main transcript. Telomerase activity could not be detected in all cells. Relative assessment of hTERT expression showed greater expression of the α-deleted variant in the telomerase negative cells (P= 0.04). Those cells possessed the α/β-deleted variant to a smaller extent when compared to the cells with telomerase activity. Greater association between full length spliced variant and β-variant expression was observed in cells presenting telomerase activity (P= 0.0007, r= 0.74). High degrees of variation among the studied cells regarding the pattern of hTERT expression were present. In spite that, the regulatory roles of hTERT on telomerase activity is still a potential to be utilized as targets for cancer therapies.

No MeSH data available.


Related in: MedlinePlus