Absence of cancer-associated changes in human fibroblasts immortalized with telomerase.

The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long-term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell-cycle checkpoints and karyotypic stability in telomerase-expressing cells are similar to those of untransfected controls. In addition, co-expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Is telomerase a viable target in cancer?

The ideal cancer treatment would specifically target cancer cells yet have minimal or no adverse effects on normal somatic cells. Telomerase, the ribonucleoprotein reverse transcriptase that maintains the ends of human chromosome, is an attractive cancer therapeutic target for exactly this reason [1]. Telomerase is expressed in more than 85% of cancer cells, making it a nearly universal cancer marker, while the majority of normal somatic cells are telomerase negative. Telomerase activity confers limitless replicative potential to cancer cells, a hallmark of cancer which must be attained for the continued growth that characterizes almost all advanced neoplasms [2]. In this review we will summarize the role of telomeres and telomerase in cancer cells, and how properties of telomerase are being exploited to create targeted cancer therapies including telomerase inhibitors, telomerase-targeted immunotherapies and telomerase-driven virotherapies. A frank and balanced assessment of the current state of telomerase inhibitors with caveats and potential limitations will be included.

Telomere dynamics in cancer progression and prevention: fundamental differences in human and mouse telomere biology.

Cells from the telomerase knockout mouse immortalize with an approximately ten million-fold greater frequency than human cells. In this commentary, Wright and Shay discuss the implications of this difference between mice and men and its relationship to cancer.

Time, telomeres and tumours: is cellular senescence more than an anticancer mechanism?

Normal diploid cells, by definition, have a limited life span: they senesce after a set number of divisions both in vivo and in culture. It has been hypothesized that the molecular mechanism that measures the life span of a cell probably involves the shortening of telomeres that occurs with each round of DNA replication. This loss of telomeres is thought to induce antiproliferative signals that result in the induction of cellular senescence. In this article, Woodring Wright and Jerry Shay present a hypothesis for the mechanisms by which telomere shortening regulates cellular physiology and argue that cellular senescence is not only an anticancer mechanism but is also the cause of many of the degenerative changes of aging.

The amplified expression of factors regulating myogenesis in L6 myoblasts.

A strategy for increasing the expression of the factors regulating myogenesis was developed based upon the observation that increased amounts of regulatory factors could overcome the inhibition of differentiation produced by 5-bromodeoxyuridine (BUdR). L6 rat myoblasts were subjected to multiple cycles of cloning in progressively increasing concentrations of BUdR. The first clones to differentiate were picked and replated for the next cycle of selection. After 28 cycles in BUdR, cells were isolated that could differentiate in the presence of 8 microM BUdR. Cell hybrids between myoblasts subjected to 21 cycles of selection (BU21 cells) and differentiation-defective myoblasts exhibited a high probability of differentiation, consistent with the hypothesis that BU21 cells were overproducing factor(s) involved in the decision to differentiate. The selection of cells able to differentiate in the presence of BUdR may provide a general approach for increasing the expression of the regulatory molecules controlling terminal differentiation.

Synthesis of rat myosin light chains in heterokaryons formed between undifferentiated rat myoblasts and chick skeletal myocytes.

The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99% pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin light chains. These results indicate that differentiated chick muscle cells provide some factor that induces L6 myoblasts to synthesize rat myosin light chains. This system provides a model for investigating the processes by which differentiated cell functions are induced.

Induction of muscle genes in neural cells.

The regulation of skeletal muscle genes was examined in heterokaryons formed by fusing differentiated chick skeletal myocytes to four different rat neural cell lines. Highly enriched populations of heterokaryons isolated using irreversible biochemical inhibitors were labeled with [35S]methionine and analyzed on two-dimensional gels. Rat skeletal myosin light chains were induced in three of the four cell combinations. The one exception, the S-20 cholinergic cell line, not only failed to synthesize rat muscle proteins but also suppressed chick myogenic functions. Experiments with heterokaryons between chick myocytes and cells from whole embryonic rat brain cultures demonstrated that rat skeletal myosin light chains are inducible in normal diploid neural cells as well as in established neural cell lines. In contrast, dividing cell hybrids between rat myoblasts and rat glial cells were nonmyogenic. These results demonstrate that although neural cells may contain factors that prevent the decision to differentiate along myogenic lines in cell hybrids, most neural cell lines do not dominantly suppress the expression of muscle structural genes in heterokaryons. Furthermore, the skeletal myosin light chain genes in most neural cell lines are regulated by a mechanism that permits them to respond to putative chick skeletal myocyte-inducing factors. The "open" state of these myogenic genes may explain many of the reports of apparent "transdifferentiation" to muscle in neural cultures and neural tumors.

Control of differentiation in heterokaryons and hybrids involving differentiation-defective myoblast variants.

Clones of differentiation-defective myoblasts were isolated by selecting clones of L6 rat myoblasts that did not form myotubes under differentiation-stimulating conditions. Rat skeletal myosin light chain synthesis was induced in heterokaryons formed by fusing these defective myoblasts to differentiated chick skeletal myocytes. This indicates that the structural gene for this muscle protein was still responsive to chick inducing factors and that the defective myoblasts were not producing large quantities of molecules that dominantly suppressed the expression of differentiated functions. The regulation of the decision to differentiate was then examined in hybrids between differentiation-defective myoblasts and differentiation-competent myoblasts. Staining with antimyosin antibodies showed that the defective myoblasts and homotypic hybrids formed by fusing defective myoblasts to themselves could in fact differentiate, but did so more than a thousand times less frequently than the 64% differentiation achieved by competent L6 myoblasts or homotypic competent X competent L6 hybrids. Heterotypic hybrids between differentiation-defective myoblasts and competent L6 cells exhibited an intermediate behavior of approximately 1% differentiation. A theoretical model for the regulation of the commitment to terminal differentiation is proposed that could explain these results by invoking the need to achieve threshold levels of secondary inducing molecules in response to differentiation-stimulating conditions. This model helps explain many of the stochastic aspects of cell differentiation.

Regulation of rat myosin light-chain synthesis in heterokaryons between 5-bromodeoxyuridine-blocked rat myoblasts and differentiated chick myocytes.

Terminal cell differentiation in a variety of model systems is inhibited by the thymidine analogue 5-bromodeoxyuridine (BUdR). We investigated the mode of action of BUdR by forming heterokaryons between undifferentiated BUdR-blocked rat myoblasts and differentiated chick skeletal myocytes. We analyzed newly synthesized proteins on two-dimensional polyacrylamide gels. The induction of rat skeletal myosin light-chain synthesis was reduced fivefold, as compared with controls, when chick myocytes were fused to BUdR-blocked rat myoblasts. This indicates that plasma membrane effects cannot be the proximate cause for the inhibition of myogenesis by BUdR, since BUdR is able to block the effect of chick inducing factors even when a differentiated chick myocyte is in direct cytoplasmic continuity with the BUdR-blocked rat nucleus. The observation that chick cells required an 80% substitution of BUdR for thymidine to block myogenesis, whereas L6 rat myoblasts required only a 20% substitution led to a hypothesis involving a DNA-mediated action of BUdR. This model yielded three testable predictions: (a) putative chick inducing molecules should be present in limiting quantities, (b) exploiting gene-dosage effects to increase the quantity of putative chick inducing factors might overcome the inhibition produced in the rat myoblasts by a 35% BUdR for thymidine substitution, and (c) these gene-dosage effects should be abolished by increasing the level of BUdR substitution in the rat myoblast to 60-80%. All three of these predictions have been verified, providing strong indirect evidence that the inhibition of myogenesis produced by BUdR is a direct result of its incorporation into cellular DNA.

MyoD, myogenin independent differentiation of primordial myoblasts in mouse somites.

The accumulation of two myogenic regulatory proteins, MyoD and myogenin, was investigated by double-immunocytochemistry and correlated with myosin heavy chain expression in different classes of myoblasts in culture and during early myogenesis in vivo. During in vitro differentiation of fetal myoblasts, MyoD-positive cells were detected first, followed by the appearance of cells positive for both MyoD and myogenin and finally by the appearance of differentiated myocytes and myotubes expressing myosin heavy chain (MHC). A similar pattern of expression was observed in cultures of embryonic and satellite cells. In contrast, most myogenic cells isolated from newly formed somites, expressed MHC in the absence of detectable levels of myogenin or MyoD. In vivo, the appearance of both myogenin and MyoD proteins was only detected at 10.5 d postcoitum (d.p.c.), when terminally differentiated muscle cells could already be identified in the myotome. Parasagittal sections of the caudal myotomes of 10.5-d-old embryos showed that expression of contractile proteins preceded the expression of myogenin or MyoD and, when coexpressed, MHC and myogenin did not co-localize within all the cells of the myotome. In the limb bud, however, many myogenin (or MyoD) positive/MHC negative cells could be observed in the proximal region at day 11. During further embryonic development the expression of these proteins remained constant in all the muscle anlagens examined, decreasing to a low level during the late fetal period. Western and Northern analysis confirmed that the myogenin protein could only be detected after 10.5 d.p.c. while the corresponding message was clearly present at 9.5 d.p.c., strongly suggesting a posttranscriptional regulation of myogenin during this stage of embryonic development. These data show that the first myogenic cells which appear in the mouse myotome, and can be cultured from it, accumulate muscle structural proteins in their cytoplasm without expressing detectable levels of myogenin protein (although the message is clearly accumulated). Neither MyoD message or protein are detectable in these cells, which may represent a distinct myogenic population whose role in development remains to be established.

Telomere position effect in human cells.

In yeast, telomere position effect (TPE) results in the reversible silencing of genes near telomeres. Here we demonstrate the presence of TPE in human cells. HeLa clones containing a luciferase reporter adjacent to a newly formed telomere express 10 times less luciferase than do control clones generated by random integration. Luciferase expression is restored by trichostatin A, a histone deacetylase inhibitor. Overexpression of a human telomerase reverse transcriptase complementary DNA results in telomere elongation and an additional 2- to 10-fold decrease in expression in telomeric clones but not control clones. The dependence of TPE on telomere length provides a mechanism for the modification of gene expression throughout the replicative life-span of human cells.

Specific association of human telomerase activity with immortal cells and cancer.

Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.

Extension of life-span by introduction of telomerase into normal human cells.

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

CASTing for multicomponent DNA-binding complexes.

Degenerate oligonucleotides and polymerase chain reaction-based reiterative selection techniques have been used to define the consensus binding sites for an increasing number of transcription factors. The use of crude nuclear extracts rather than purified proteins permits multicomponent complexes to form, and allows the technique to generate information about the combinatorial interactions involved in gene regulation.

Muscle basic helix-loop-helix proteins and the regulation of myogenesis.

Significant progress has been made in defining the structural motifs that distinguish the muscle-specific from other basic helix-loop-helix proteins. Evidence is accumulating for multiple levels of regulation of the expression and action of the muscle basic helix-loop-helix factors.

Cellular senescence as a tumor-protection mechanism: the essential role of counting.

The term 'cellular senescence' has often been applied indiscriminately to any form of growth arrest of cultured cells that occurs either after some period in culture or following insults such as the overexpression of oncogenes. Recent reports have suggested there may be many mechanisms of cellular senescence. Our increasing understanding of the role of telomere shortening in the replicative aging of cultured fibroblasts now permits a re-examination of what may reasonably be called cellular senescence, and what most likely represents artifacts of the culture environment and/or specific cell-cycle control mechanisms.

Telomere positional effects and the regulation of cellular senescence.

Normal cells have a limited capacity to proliferate but the molecular clock that regulates the onset of cellular senescence remains unidentified. The ends of chromosomes--telomeres--have been shown to shorten progressively with age in normal cells. Here, we present a working model of how telomeric shortening may induce programmed changes in the regulation of cellular proliferation.

Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site.

The consensus binding site for the muscle regulatory factor myogenin was determined from an unbiased set of degenerate oligonucleotides using CASTing (cyclic amplification and selection of targets). Stretches of totally random sequence flanked by polymerase chain reaction priming sequences were mixed with purified myogenin or myotube nuclear extracts, DNA-protein complexes were immunoprecipitated with an antimyogenin antibody, and the DNA was amplified by polymerase chain reaction. Specific binding was obtained after four to six cycles of CASTing. The population of selected binding sites was then cloned, and a consensus was determined from sequencing individual isolates. Starting from a pool with 14 random bases, purified myogenin yielded a consensus binding site of AACAG[T/C]TGTT, while nuclear extracts retrieved the sequence TTGCACCTGTTNNTT from a pool containing 35 random bases. The latter sequence is consistent with that predicted from combining an E12/E47 half-site (N[not T]CAC) with the purified myogenin half-site ([T/C] TGTT). The presence of paired E boxes in many of the sequences isolated following CASTing with nuclear extracts proves that myogenin can bind cooperatively with other E-box-binding factors.

Regulation of telomerase activity in immortal cell lines.

Telomerase is a ribonucleoprotein whose activity has been detected in germ line cells, immortal cells, and most cancer cells. Except in stem cells, which have a low level of telomerase activity, its activity is absent from normal somatic tissues. Understanding the regulation of telomerase activity is critical for the development of potential tools for the diagnosis and treatment of cancer. Using the telomeric repeat amplification protocol, we found that immortal, telomerase-positive, pseudodiploid human cells (HT1080 and HL60 cells) sorted by flow repressed in quiescent cells. This was true whether quiescence was induced by contact inhibition (NIH 3T3 mouse cells), growth factor removal (bromodeoxyuridine-blocked mouse myoblasts), reexpression of cellular senescence (the reversibly immortalized IDH4 cells), or irreversible cell differentiation (HL60 promyelocytic leukemia cells and C2C12 mouse myoblasts). Taken together, these results indicate that telomerase is active throughout the cell in dividing, immortal cells but that its activity is repressed in cells that exit the cell cycle. This suggests that quiescent stem cells that have the potential to express telomerase may remain unaffected by potential antitelomerase cancer therapies.