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

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.

Keywords: Alternative splicing; cancer cell line; hTERT variants; proliferation capacity; telomerase activity.

Fig. 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

Fig. 2

Doubling time of cancer cell lines Doubling time of different cancer cell lines were estimated by plotting the corresponding growth curves. The calculated time was used as index of proliferative capacity of the cell lines

Fig. 3

Relative expression levels of different hTERT transcript variants in the studied cancer cell lines. The expression of (A) full length, (B) α-deleted variant, (C) β-deleted variant and (D) α/β-deleted variants of hTERT were measured by real time PCR. The expression of the transcripts was compared to β-actin as internal housekeeping gene. All the studied cancer cell lines expressed a mixed pattern of all four hTERT variants

Fig. 4

Telomerase enzyme activity in the cells was quantified by TRAP assay. The majority of the cells showed varying degrees of telomerase enzyme activity; however, four cell lines of different tissue origins failed to do that

Fig. 5

Correlation between the expression of full length hTERT variants and other alternative forms . The Spearman’s correlation coefficient was used to find statistical relation between the expression of full length hTERT transcript and the three spliced variants. The expression of full length transcript in the cells with telomerase activity was found in significant association with both β-variant (■ symbol, sold line, P= 0.0007, r= 0.74) and then α-variant (● symbol, broken line, P= 0.001, r= 0.7) but not α/β-deleted variant (▲ symbol, dotted line, P= 0.002, r= 0.68). There was no statistically significant correlation between the expression of the full length variant and other variants in the telomerase negative cells (P< 0.3, r< 0.3).