Telomerase is a ribonucleoprotein complex that maintains chromosome length by synthesizing and adding repetitive (TTAGGG)n
DNA sequences to the ends of telomeres [1
]. Its absence from most normal somatic cells is believed to contribute to eventual senescence and limited cellular life span [2
], whereas its reactivation in immortalized cells has been associated with the unlimited growth potential required for malignancy [3–5
]. In particular, progression of melanoma is known to be accompanied by a steady increase in telomerase activity during the transformation of isolated naevi to metastatic disease [6,7
], with virtually all melanoma cell lines exhibiting some degree of telomerase activity [6
]. These discriminating properties have made telomerase an attractive target for cancer therapy [8
], and there has been much emphasis on methods for detecting and determining the regulatory mechanisms of this important enzyme.
Telomerase is made up of two essential components: a constitutively expressed human telomerase RNA (hTR), which acts as a transcription template [9,10
], and a catalytic human telomerase reverse transcriptase (hTERT), whose expression controls enzymatic activity [11,12
]. The transcriptional and posttranscriptional regulation of hTERT is complex and remains to be fully elucidated. Studies in human development have revealed the presence of multiple hTERT RNA transcripts occurring in patterns that are both tissue-specific and gestational stage-dependent [13
]. Such nonrandom alternative splicing is a common method of genetic regulation in eukaryotes [14
], and to date, 10 different splice variants of hTERT have been identified [13,15–17
]. The most widely studied variants involve splicing at two main sites: the α splice site, which produces a 36-bp inframe deletion within the conserved reverse transcript motif A; and the β site, which results in a 183-bp deletion and non-sense mutation that truncates the protein, effectively deleting the remaining three reverse transcriptase motifs [13,18
]. Splicing at either site can occur independently or in combination to produce three variants from the full-length α+/β+: α-/β+, α+/β-, and α-/β-, which have been shown to occur at approximate proportions of 5%, 1%, 80% to 90%, and 5% to 15%, respectively, within various cancer cell lines [19
]. To date, only the α-/β+ variant has been shown to exhibit any regulatory function, acting as a dominant-negative inhibitor of telomerase activity when overexpressed in either normal or tumor cells [20,21
]. It is unclear whether the ratio of full length to spliced hTERT is important in determining telomerase activity [22
], because some studies have shown that the absolute expression of hTERT is well correlated with telomerase activity [23–27
], and still others have found no correlation with either relative or absolute amounts of variant transcripts [28
]. The regulatory functions of various hTERT transcripts may well be cell type-specific; however, the many different methods used to quantify hTERT mRNA have made it difficult to interpret these findings.
Most reverse transcription-polymerase chain reaction (RT-PCR)-based assays for hTERT variants use primers that flank the α and β subunits and thus amplify all transcripts in one reaction (). The products are then scanned by densitometry to give the relative distribution of hTERT variants in each sample [19,29,30
], the results of which are largely biased by the competitive nature of PCR. A few techniques have been developed using dual-labeled probe technology on a real-time PCR platform [31–35
]; however, most of these assays use primers designed to the region downstream from the α and β subunits and, therefore, do not discriminate between the deletion variants [31–33
]. A commercially available Light Cycler kit (TeloTAGGGG hTERT Quantification Kit; Roche Diagnostics, Basel, Switzerland) has proven popular, but the exact primer sequences are not revealed, thus it is not clear which transcripts are measured. Therefore, results from these types of studies need to be interpreted with caution.
Figure 1 hTERT mRNA α and β deletion variants. Relevant hTERT exons are numbered and corresponding location of α and β sequences are illustrated by shaded boxes. Deleted sequences are designated by dashed lines. Locations of PCR (more ...)
The aim of the present study was to establish whether absolute or relative levels of hTERT variant transcripts determine telomerase activity. We developed an SYBR green-based real-time PCR assay as a more affordable option to labeled probes, with the further advantage of enabling melt curves to confirm the presence of specific transcripts. The assay was then used to determine the absolute and relative hTERT variant expression in a series of melanoma cell lines. In doing so, we have revealed the importance of overall levels of full-length hTERT mRNA in determining telomerase expression, and a possible role for the relative amount of β deletion variant in the regulation of telomerase activity.