Courtesy of Ying Zou
Telomerase, a cellular ribonucleoprotein (RNP) reverse transcriptase, is not detected in most normal human tissues but is almost universally expressed in human cancers. Telomerase is transcriptionally silenced during human development, except for a subset of cells in highly proliferative tissues such as the germ line, blood, skin, and intestine. Even in these tissues telomeres progressively shorten with increased age. It has generally been thought that the exclusive function of telomerase was to prevent telomere shortening, but recently a number of reports have suggested a role for telomerase in addition to maintaining telomere length. Results indicate that, even in the absence of limiting telomere length, added telomerase still promotes tumorigenesis and cell proliferation. No specific mechanism by which telomerase might do this has been proposed. We think it is premature to ascribe nontelomere maintenance functions to telomerase. Various caveats exist and specific controls still need to be addressed.
Courtesy of Jerry W. Shay
Jerry W. Shay (left) is the vice chairman of the department of cell biology at the University of Texas Southwestern Medical Center in Dallas and the associate director of the Simmons Comprehensive Cancer Center. He and his long-time colleague, Woodring Wright (right), also a professor in the department of cell biology, have been interested in the relationships between aging and cancer, and have made seminal contributions to the emerging field of telomere and telomerase study.
Unlike humans, inbred laboratory mouse strains possess long telomeres (see image opposite page) and, in early-generation telomerase-deficient mice, the telomere reserve is sufficient to prevent telomere-based checkpoint responses that might lead to growth arrest. The observation that neither the template-RNA (mTR) nor catalytic-telomerase (mTERT) knockout strains shows obvious premature aging or cancer-prone changes in early generations, suggests that neither component serves an essential nontelomere maintenance function.
The table on the opposite page outlines experiments indicating that telomerase may actively promote tumor growth and/or cell survival even when telomeres are sufficiently long. But each of these results could have alternate interpretations. Immortal cell lines with an alternative-lengthening-of-telomere (ALT) pathway appear to require telomerase for malignant transformation, for example. But telomerase-silent ALT tumors, as reviewed by Sandy Chang and Ron DePinho at Harvard, are in a constant state of crisis (with some very short and some very long telomeres).1 TERT may simply function to elongate the shortest telomeres, thus removing their DNA-damage signaling activity and indirectly permitting better tumor growth by an entirely telomere-dependent mechanism.
ALT cells use a recombination-based pathway to maintain their telomeres. Robert Weinberg and colleagues showed that a human ALT immortal cell line was not tumorigenic when high levels of H-RAS were expressed, in contrast to a similar cell line that expressed wild-type telomerase and H-RAS and made robust tumors.2 Stewart then showed expression of a catalytically active TERT-HA fusion protein that failed to maintain telomeres in normal cells (presumably because it could not be recruited to act on telomeres) could impart a tumorigenic phenotype. This result appears on the surface to provide persuasive evidence of TERT functions independent of telomere maintenance.
But it's uncertain whether or not the catalytically active TERT-HA actually has some ability to elongate very short telomeres. The use of in situ hybridization to telomeres could potentially provide some insights into this issue. In its absence, one could argue that while this "telomere-maintenance defective" TERT vector is insufficient to elongate telomeres in a normal setting, in an ALT setting this crippled TERT vector may be able to maintain the shortest telomeres sufficiently to survive (especially when it only has to work on a single or a few critically shortened telomeres entering crisis). Using a catalytically inactive TERT mutant instead of the TERT-HA vector may address this central issue. Thus, the conclusion that telomerase possesses protumorigenic activities that extend beyond its classical role in telomere-synthesis remains an open question.
The TERT transgenic experiments almost always use a strong promoter (e.g., keratin 5, β-actin), establish only one or two founder strains, and do not consider the possibility that effects observed may be due to random insertional mutagenesis. Another important caveat derives from the observations that overexpressed telomerase induces hundreds to thousands of changes, including upregulation of the EGF receptor.3 Endogenous telomerase is present at extremely low abundance: approximately one coding mRNA molecule per typical cultured cancer cell. But retroviral hTERT mRNA is present at thousands of copies per cell. Since hTERT (and many telomere-binding factors) interact with multiple partners including DNA repair factors, it is easy to imagine many nonphysiological effects of such huge overexpression. In the case of breast tumors occurring in TERT transgenic mice, the tumor abundance is low and appears at long latency (e.g., very late in life), suggesting secondary events may be causative, and could easily reflect nonphysiological gene-expression changes due to overexpression.4
Courtesy of Ying Zou
Mouse telomeres on interphase cells and metaphase chromosomes using digital fluorescence microscopy.
BIG HEARTS AND NEUROPROTECTION
In the study on mouse cardiac muscle hypertrophy, Michael Schneider's group at Baylor College of Medicine established that forcible expression of TERT delayed cardiac cell-cycle exit and thus produced bigger hearts with more myocytes.5 These authors showed that a dramatic decrease in telomere length occurred during the first few postnatal days, orders of magnitude greater than could be explained by proliferation. It is known that oxidative damage can cause rapid telomere shortening. One hypothesis is that oxidative damage in the neonatal heart causes some telomeres to become denuded and contribute to growth arrest. Exogenous telomerase could elongate these telomeres and retard the onset of growth arrest until the developmental cardiac program is fully engaged, thus resulting in a beneficial hypertrophy. Thus, these results could reflect the normal telomere-maintenance functions of telomerase.
A similar explanation could be operating in the neuroprotective effects of telomerase, in which telomere loss due to oxidative or other responses to ischemia could have occurred.6 A missing control in many if not most of these experiments is the failure to use a catalytically dead TERT to eliminate the possibility that telomere maintenance is involved.
Observations Suggesting Extra-Telomeric Roles of Telomerase
• Reconstitution of telomerase is required for efficient growth of mouse ALT tumors as metastatic nodules in lung,1 and TERT is required for malignant transformation of human ALT fibroblasts.2
• Transcriptional alteration in a subset of genes in the presence/absence of telomerase.38
• TERT transgenic expression in mouse promotes breast cancer.4
• TERT transgenic expression in mouse cardiac muscle promotes cell proliferation, hypertrophy, and survival.5
• TERT transgenic expression in mouse protects against brain injury resulting from ischemia and drug-induced neurotoxicity.6
• Telomerase enhances survival and promotes proliferation even when telomeres are long.79
• TERT transgenic expression in mouse skin predisposes to papillomas.10
• TERT enhances genomic stability and DNA repair.11
Several studies suggest that the mTR component of telomerase may have telomerase-independent functions. The fact that normal telomerase-negative cells constitutively express hTR is certainly consistent with this concept. Jun-Ping Liu's group at Monash University took cells with telomerase and expressed antisense RNA (directed against the RNA component of telomerase, hTR) and a dominant-negative catalytically inactive TERT mutant and compared the effects on cell survival and apoptosis in breast cancer cell lines.7 Lowering hTR expression induced apoptosis independently of telomere shortening.
An hTERT mutant lacking telomerase enzymatic activity rescued the cells with lowered telomerase activity from undergoing apoptosis. The interpretation is that TERT operates to regulate cell survival over and above the catalytic activity of telomerase on telomeric DNA by distinct interactions beyond the normal telomerase RNP complex. Using shRNA to the telomerase template RNA, Elizabeth Blackburn's group at the University of California, San Francisco, induced tumor-cell death that was too rapid to be due to inhibiting telomerase activity and inducing telomere shortening.8 These results are not easily explained unless hTR is serving an as yet unknown function, or unless this particular shRNA is producing target-independent toxicity.
There are several additional anecdotal reports of extracurricular activities of telomerase independent of telomere maintenance, but definitive experiments are still lacking. While we are certainly willing to entertain the possibility of novel functions of telomerase, it is important to note that at present the verdict is still out.