Chantal Autexier

Telomerase and cancer: revisiting the telomere hypothesis

Telomerase is a ribonucleoprotein DNA polymerase that elongates telomeres in eukaryotes. The telomere hypothesis implicates short telomere length and telomerase activation as critical players in cellular immortalization and cancer. In this review, we refine the original telomere hypothesis to address the results of recent studies on telomerase and telomere length regulation.

Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA

Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR. Using this assay, truncations at the 5′ and 3′ ends of hTR identified a functional region of hTR, similar in size to the full‐length telomerase RNAs from ciliates. This region is located between positions 1‐203. Furthermore, we found that residues 1‐44, 5′ to the template region (residues 46–56) are not essential for activity, indicating a minimal functional region is located between residues 44–203. Mutagenesis of full‐length hTR between residues 170–179, 180–189 or 190–199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components.

Telomerase inhibition enhances the response to anticancer drug treatment in human breast cancer cells

Breast cancer is the most common malignancy among women. Current therapies for breast tumors are based on the use of chemotherapeutic drugs that are quite toxic for the patients and often result in resistance. Telomerase is up-regulated in 95% of breast carcinomas but not in adjacent normal tissues. Therefore, it represents a very promising target for anticancer therapies. Unfortunately, the antiproliferative effects of telomerase inhibition require extensive telomere shortening before they are fully present. Combining telomerase inhibition with common chemotherapeutic drugs can be used to reduce this lag phase and induce tumor cell death more effectively. Few studies have analyzed the effects of telomerase inhibition in combination with anticancer drugs in breast cancer cells. In this study, we inhibited telomerase activity in two breast cancer cell lines using a dominant-negative human telomerase reverse transcriptase and analyzed cell viability after treatment with different anticancer compounds. We found that dominant-negative human telomerase reverse transcriptase efficiently inhibits telomerase activity and causes telomere shortening over time. Moreover, cells in which telomerase was suppressed were more sensitive to anticancer agents independently of their mechanism of action and this sensitization was dependent on the presence of shorter telomeres. Altogether, our data show that blocking telomere length maintenance in combination with anticancer drugs can be used as an effective way to induce death of breast cancer cells. [Mol Cancer Ther 2006;5(7):1669–75]

Telomerase and neuronal marker status of differentiated NT2 and SK‐N‐SH human neuronal cells and primary human neurons

Upon treatment with retinoic acid, NTera‐2 (NT2) human teratocarcinoma and SK‐N‐SH neuroblastoma cells can be induced to terminally differentiate into postmitotic neuronal cells. The neuronal cell yield obtained from the NT‐2 cells is partially dependent on the time of differentiation (24–55 days). SK‐N‐SH cells differentiate into a mixed population of neuronal and epithelium‐like cells. Here we report modified protocols that increase the number of differentiated NT‐2 and SK‐N‐SH cells and that establish an enriched neuronal SK‐N‐SH‐derived cell population essentially devoid of nonneuronal cells. Differentiated cells express the cytoskeleton‐associated protein tau and other typical neuronal markers, such as Map2, Ngn1, NeuroD, Mash1, and GluR which are also expressed in primary human fetal neurons. Telomerase activity is down‐regulated in differentiated cells, which is consistent with the telomerase status of primary fetal human neurons. Thus, differentiated NT2 and SK‐N‐SH cells may represent an excellent source for studies investigating the role of telomerase or other survival‐promoting activities in protecting human neuronal cells from cell death‐mediating stresses associated with neurodegenerative diseases.

A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation

The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.

Telomeric function of mammalian telomerases at short telomeres.

Telomerase synthesizes telomeric sequences and is minimally composed of a reverse transcriptase (RT) known as TERT and an RNA known as TR. We reconstituted heterologous mouse (m) and human (h) TERT-TR complexes and chimeric mTERT-hTERT-hTR complexes in vitro and in immortalized human alternative lengthening of telomere (ALT) cells. Our data suggest that species-specific determinants of activity, processivity and telomere function map not only to the TR but also to the TERT component. The presence of hTERT-hTR, but not heterologous TERT-TR complexes or chimeric mTERT-hTERT-hTR complexes, significantly reduced the percentage of chromosomes without telomeric signals in ALT cells. Moreover, heterologous and chimeric complexes were defective in recruitment to telomeres. Our results suggest a requirement for several hTERT domains and interaction with multiple proteins for proper recruitment of telomerase to the shortest telomeres in human ALT cells. Late-passage mTERT−/− mouse embryonic stem (ES) cells ectopically expressing hTERT or mTERT harboured fewer chromosome ends without telomeric signals and end-to-end fusions than typically observed in late-passage mTERT−/− ES cells. The ability of hTERT to function at mouse telomeres and the inability of mTERT to function at human telomeres suggest that mechanisms regulating the recruitment and activity of hTERT at mouse telomeres might be less stringent than the mechanisms regulating mTERT at human telomeres.

POT of gold: modeling dyskeratosis congenita in the mouse

Dyskeratosis congenita (DC) is a rare syndrome, characterized by cutaneous abnormalities and premature death caused by bone marrow failure. In this issue of Genes & Development, Hockemeyer and colleagues (pp. 1773-1785) report a new mouse model that reconstitutes key features of DC. Disease phenotypes are generated by a POT1b deletion in a telomerase-deficient background that accelerates the shortening of telomeres by degradation.

21 DNA Telomerases

Telomerase is a specialized DNA polymerase that synthesizes telomeric DNA sequences onto chromosome ends. Linear DNA genomes pose a special problem for the DNA replication machinery. Because DNA polymerases function in the 5′ to 3′ direction and require a primer, part of the lagging strand is predicted to be lost at each round of division. For most eukaryotic chromosomes, sequence loss is balanced by the de novo addition of telomeric sequence by telomerase (for review, see Blackburn 1991). Telomere DNA sequences in ciliates consist of simple GT-rich tandem repeats, and this motif is generally conserved in eukaryotes, although the exact repeat sequence is species-specific. For example, Tetrahymena contains tandem TTGGGG repeats, whereas mammals have TTAGGG repeats (for review, see Blackburn 1991; Henderson 1995). Telomerase synthesizes these short repeats by using an integral RNA component of the enzyme as a template (Fig. 1) (Greider and Blackburn 1989). Telomerase activity has been identified from Tetrahymena, Euplotes, Oxytricha, yeast, Xenopus, and mammals. In each case, the sequence synthesized by telomerase in vitro corresponds to the telomere sequence of the organism from which it was isolated (Table 1), and the RNA template contains more than one repeat of the sequence (Table 2) (Greider and Blackburn 1985; Zahler and Prescott 1988; Morin 1989; Shippen-Lentz and Blackburn 1989; Prowse et al. 1993; Lingner et al. 1994; Mantell and Greider 1994; Cohn and Blackburn 1995; Lin and Zakian 1995; Lue and Wang 1995). TETRAHYMENA TELOMERASE The telomerase mechanism has been best studied in Tetrahymena, where enzyme activity...