Tumor Cell Immortalization Research Articles

Microarray analysis of gene expression in the ovarian cancer cell line HO-8910 with silencing of the ZNF217 gene

Zinc-finger protein 217 (ZNF217), which is overexpressed during cancer progression, can promote tumor cell immortalization. To examine the function of ZNF217, a global expression profile was carried out using Affymetrix Gene Chip analysis with HG-U133 plus 2.0 arrays in the ovarian cancer cell line HO-8910 after silencing of the ZNF217 gene. The results were analyzed using the Gene Ontology program to investigate the functional network affected by ZNF217 in ovarian cancer cells. Changes in the mRNA expression of the affected genes were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the ZNF217 gene is a key regulator of ovarian cancer, as the silencing of ZNF217 resulted in significant down-regulation by at least 8-fold of 164 genes in the HO-8910 cell line compared to non-silenced control cells. Among these down-regulated genes were ALOX15, CD1D, FXYD3, GAS6, KRT4, LIN7B, MMP-24, PDZK1, PEX6, PRSS8, SLC2A9, STRN and WFDC2. Down-regulation was confirmed by real-time RT-PCR after the silencing of ZNF217 (p<0.05). The results suggest that ZNF217 plays a central role in malignant processes in ovarian cancer.

Spontaneous immortalization of oligodendroglial cells derived from an SV40 T antigen-positive human glioblastoma multiforme

The polyoma group of viruses, including SV40, is known to be oncogenic in certain species. Here we report for the first time naturally occurring, immortalized tumor cells from a patient with glioblastoma multiforme (GBM); the cells were shown to be oligodendroglia; cells had developed remarkable chromosomal changes and were positive for SV40 T antigen. Therefore, we postulated that the main cause of immortalization of these cells was the expression of SV40 T antigen gene and protein. Since the cells are naturally generated, they will provide a useful model to study the function of oligodendroglial cells and the development of GBM.

Clinical Trials of Stroke Therapy

Despite recent advances in stroke prevention and acute stroke therapy, few treatments exist for reversal of fixed neurological deficits after stroke. Cellular therapy offers the possibility of improving neurological deficits months or even years after stroke. Animal studies provide evidence that such treatment may be effective. Many questions, however, remain unanswered regarding cellular therapy for stroke. In view of the significant differences between animal models and human stroke, some questions might be best addressed with clinical trials. Completed clinical trials of cellular therapy for stroke are summarized in the Table. Kondziolka et al reported the results of a Phase I study of 12 patients with completed stroke involving the basal ganglia treated with human neuronal cells derived from an immortalized tumor cell line (LBS cells).1 Either 2 or 6 million cells were implanted stereotactically between 6 months and 6 years after stroke. Patients were followed clinically as well as with MRI and positron emission tomography scans. The same group subsequently performed a Phase II study with randomization of 18 patients to either implantation of 5 or 10 million cells and rehabilitation (14 patients) or rehabilitation alone (4 patients).2 Follow-up neurological evaluations were blinded to treatment status. Savitz et al reported the results of transplantation of fetal porcine cells implanted stereotactically in 5 patients with basal ganglia stroke 3 months to 10 years after onset.3 The study was discontinued due to adverse events in 2 patients. In a study by Bang et al, autologous mesenchymal stem cells were given intravenously to 5 patients with middle cerebral artery territory strokes 4 to 5 weeks and again at 7 to 9 weeks after onset.4 An additional 25 patients served as control subjects. All patients were followed for functional outcome and adverse events. View this table: Table. Clinical Trials of Cellular Therapy …

Bone Morphogenetic Protein-7 Inhibits Telomerase Activity, Telomere Maintenance, and Cervical Tumor Growth

Telomere maintenance is critical in tumor cell immortalization. Here, we report that the cytokine bone morphogenetic protein-7 (BMP7) inhibits telomerase activity that is required for telomere maintenance in cervical cancer cells. Application of human recombinant BMP7 triggers a repression of the human telomerase reverse transcriptase (hTERT) gene, shortening of telomeres, and hTERT repression-dependent cervical cancer cell death. Continuous treatment of mouse xenograft tumors with BMP7, or silencing the hTERT gene, results in sustained inhibition of telomerase activity, shortening of telomeres, and tumor growth arrest. Overexpression of hTERT lengthens telomeres and blocks BMP7-induced tumor growth arrest. Thus, BMP7 negatively regulates telomere maintenance, inducing cervical tumor growth arrest by a mechanism of inducing hTERT gene repression.

Different telomere maintenance mechanisms in alveolar and embryonal rhabdomyosarcoma

The activation of a telomere maintenance mechanism (TMM) is crucial for the immortalization of tumor cells. Most human cancers apply telomerase-dependent TMM but some use a mechanism called alternative lengthening of telomeres (ALT). The latter was suggested to be mainly characterizing sarcomas with nonspecific complex karyotypes, whereas telomerase activation is typical of sarcomas generated by specific translocations. In this study, we investigated the TMM and its association with survival in rhabdomyosarcoma (RMS), which is characterized by two major subtypes: one that is harboring a specific translocation (alveolar) and one that has a nonspecific karyotype (embryonal). Telomerase activity (TA), using telomerase repeat amplification protocol (TRAP) assay, and telomere length (TRF), using Southern blotting, were analyzed in tumor samples from 31 patients (16 embryonal and 15 alveolar). Alveolar RMS tumors exhibited no ALT phenotype and the majority presented TA. Some embryonal tumors exhibited an ALT or "ALT-like" phenotype which lacked TA, whereas others expressed telomerase-dependent TMM, and neither TA nor ALT correlated with outcome. The average TRF length of the embryonal tumors was significantly higher than that of the alveolar tumors (10.8 vs. 7.2 kb, P = 0.003). Interestingly, some tumors of both subtypes presented no TMM. These observations suggest that alveolar RMS predominantly use telomerase-dependent TMM, whereas in embryonal tumors both telomerase and ALT may play a role. These findings have important implications for understanding the role of TMM in the development of RMS tumors, and for future designing adapted treatment strategies.

Photodynamic effect in medulloblastoma: downregulation of matrix metalloproteinases and human telomerase reverse transcriptase expressions

Tumor invasion and immortality are the most unfavorable drawbacks after cancer treatment. In this study, we focus on determining the photodynamic modulation of the proteolytic enzymes, matrix metalloproteinases (MMP); and a core catalytic subunit of telomerase, the human telomerase reverse transcriptase (hTERT) in medulloblastoma (MED) cell line (TE-671). Hexvix (ALA-H) mediated photodynamic therapy (PDT) demonstrated greater efficacy than 5-aminolevulinic acid (5-ALA) in terms of drug uptake and anti-proliferative effect. Both MMP-2 and hTERT expression are down-regulated quantitatively using ELISA and reverse-transcriptase-PCR (RT-PCR) respectively at post-treatment for this cell line. The MMP-9 expression remains unchanged after treatment. Further, there is a statistically significant inhibition of cell migration at 24 h post-ALA-H-PDT at LD(50) (0.01 mM, 2 J cm(-2); p < 0.001) in MED TE-671 cells. Evidently, MMP-2 and hTERT mRNA expressions can be the targets for the photodynamic intervention on tumor cell migration and immortality. Hence, PDT may be an alternate cancer regime for medulloblastoma.

Mouse mammary gland reconstitution with extrinsic gene-transferred mammary epithelial cells in vitro and in vivo

Cancer stem cells (CSCs) comprise a minor population of self-renewing, immortal tumor cells capable of differentiating into the various cell types found within the bulk tumor. However, whether CSCs are derived from normal stem or progenitor cells and the underlying mechanism whereby they are generated remain to be elucidated. To answer these queries and to investigate the process of carcinogenesis, we have developed a novel strategy to introduce genetic changes into a subset of mammary stem/progenitor cells in vitro and in vivo. We used retroviral vectors to introduce the enhanced green fluorescence protein (EGFP) reporter gene into the bulk of mouse mammary epithelial cells, and evaluated the tissue regenerative potential of these cells in vitro and in vivo. We showed that EGFP-transduced primary mouse mammary epithelial cells could regenerate duct-alveolar unit-like structures in 3-D Matrigel culture. However, we could not confirm clonogenic outgrowths of ectopic EGFP-expressing transplanted mammary epithelial cells in the recipient mouse. We present the preliminary results of these studies and discuss the significance of this model.

Genetic Alterations in the Tyrosine Kinase Transcriptome of Human Cancer Cell Lines

Protein tyrosine kinases (PTKs) play a critical role in the manifestation of cancer cell properties, and respective signaling mechanisms have been studied extensively on immortalized tumor cells. To characterize and analyze commonly used cancer cell lines with regard to variations in the primary structure of all expressed PTKs, we conducted a cDNA-based sequence analysis of the entire tyrosine kinase transcriptome of 254 established tumor cell lines. The profiles of cell line intrinsic PTK transcript alterations and the evaluation of 155 identified polymorphisms and 234 somatic mutations are made available in a database designated "Tykiva" (tyrosine kinome variant). Tissue distribution analysis and/or the localization within defined protein domains indicate functional relevance of several genetic alterations. The cysteine replacement of the highly conserved Y367 residue in fibroblast growth factor receptor 4 or the Q26X nonsense mutation in the tumor-suppressor kinase CSK are examples, and may contribute to cell line-specific signaling characteristics and tumor progression. Moreover, known variants, such as epidermal growth factor receptor G719S, that were shown to mediate anticancer drug sensitivity could be detected in other than the previously reported tumor types. Our data therefore provide extensive system information for the design and interpretation of cell line-based cancer research, and may stimulate further investigations into broader clinical applications of current cancer therapeutics.

Telomere Shortening in Human HL60 Cells by Treatment with 3′-Azido-2′,3′-Dideoxynucleosides and Telomerase Inhibition by Their 5′-Triphosphates

Telomerase is thought to play an important role in the mechanism of tumor cell immortalization by maintenance of telomere length. To obtain information on the susceptibility of telomerase to nucleoside analogues, the effects of base-modified 3′-azido-2′,3′-dideoxynucleoside triphosphates on the enzyme were investigated. It is suggested that the 2-amino group of the nucleotide purine nucleus is important for the inhibitory activity. Telomere shortening caused by long-term treatment with these nucleosides is also described.

Increased Sensitivity of Radiated Murine Cervical Cancer Tumors to E7 Subunit Vaccine–driven CTL-mediated Killing Induces Synergistic Anti-tumor Activity

The development of therapeutic vaccines has important implications for the treatment of cancer patients. Here we investigate whether human papillomavirus (HPV) E7 subunit vaccines can enhance tumor radioresponse using an established cervical cancer animal model. Radiation plus E7 subunit vaccines improved complete response, cure, and recurrence rates of tumors dramatically compared with single therapy alone. In particular, both components of the E7 subunit vaccines (E7 protein and CpG-oligodeoxynucleotide) were required for the induction of antigen (Ag)-specific cytotoxic T-lymphocyte (CTL) responses and for therapeutic synergy with radiotherapy. Moreover, with combined therapy the radiation dose could be reduced by 16 Gy to achieve an equivalent anti-tumor efficacy to radiation treatment alone. This therapeutic synergy was found to be mediated by CD8(+) CTLs and was concomitant with histological changes (presence of apoptotic bodies and multinucleated giant cells; heavy infiltration of lymphocytes), as determined by in vivo T-cell depletion and histological analysis. Finally, phenotypic changes of radiated tumors and their increased sensitivity to CTL-mediated killing appeared to be responsible for therapeutic synergy. These results show that E7 subunit vaccines act as a potent enhancer of tumor radioresponse and that this is mediated by increased sensitivity of radiated tumors to CTL-mediated killing. This study further suggests that E7-targeted therapeutic vaccines have the potential to improve radiotherapy in patients with cervical cancer.

Oxidative stress pathways highlighted in tumor cell immortalization: association with breast cancer outcome

An improved understanding of cell immortalization and its manifestation in clinical tumors could facilitate novel therapeutic approaches. However, only rare tumor cells, which maintain telomerase expression in vitro, immortalize spontaneously. By expression-profiling analyses of limited-life primary breast tumor cultures pre- and post-hTERT transduction, and spontaneously immortalized breast cancer cell lines, we identified a common signature characteristic of tumor cell immortalization. A predominant feature of this immortalization signature (ImmSig) was the significant overexpression of oxidoreductase genes. In contrast to epithelial cells derived from low histologic grade primary tumors, which required hTERT transduction for the acquisition of ImmSig, spontaneously immortalizing high-grade tumor cultures displayed similar molecular changes independent of exogenous hTERT. Silencing the hTERT gene reversed ImmSig expression, increased cellular reactive oxygen species levels, altered mitochondrial membrane potential and induced apoptotic and proliferation changes in immortalized cells. In clinical breast cancer samples, cell-proliferation-pathway genes were significantly associated with ImmSig. In these cases, ImmSig expression itself was inversely correlated with patient survival (P=0), and was particularly relevant to the outcome of estrogen receptor-positive tumors. Our data support the notion that ImmSig assists in surmounting normal barriers related to oxidative and replicative stress response. Targeting a subset of aggressive breast cancers by reversing ImmSig components could be a practical therapeutic strategy.

Photodynamic therapy‐generated tumor cell lysates with CpG‐oligodeoxynucleotide enhance immunotherapy efficacy in human papillomavirus 16 (E6/E7) immortalized tumor cells

Immunotherapy with photodynamic therapy (PDT) offers great promise as a new alternative for cancer treatment; however, its use remains experimental. In this study, we examined the immunotherapeutic significance of human papillomavirus (HPV)-immortalized tumor cell lysates induced by PDT with CpG-oligodeoxynucleotide (ODN). PDT-cell lysates were generated by irradiating Radachlorin (5 microg/mL) preloaded TC-1 cells carrying HPV 16 E7. PDT-cell lysates plus ODN coinjection for protection against E7-expressing tumors as well as specific immune responses were evaluated with the following tests: heat shock protein 70 (HSP70) enzyme-linked immunosorbent assay, in vitro and in vivo tumor growth inhibition, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) assay, cytotoxic T-lymphocyte assay, and fluorescence activated cell sorting (FACS) analysis. PDT-cell lysates plus ODN coinjection showed a significant suppression of tumor growth at both prophylactic and therapeutic levels, compared to PDT (or F/T)-cell lysates or ODN alone. In addition, we evaluated the level of the immune response with the coinjection. HSP70, an important regulator of inflammatory and immune response, was observed in abundance in the PDT-cell lysates. IFN-gamma production and cytotoxic T lymphocytes (CTL) responses were induced by PDT-cell lysates plus ODN injection. The coinjection resulted in PDT-cell lysate-specific antibodies (IgG1, IgG2a, IgG2b, and IgG3) and T-helper cell responses significantly higher than PDT-cell lysates alone. Moreover, IFN-gamma production and CTL responses were significantly induced in the PDT-cell lysate plus ODN immunized groups. These enhanced immune responses appeared to be mediated by CD8+ T cells only. These data suggest that PDT-cell lysates plus ODN injection may be an effective approach to induce CTL immune responses as a possible immunotherapeutic strategy for cancer therapy.

Mutation, replicative infidelity of DNA and aneuploidy sequentially in the formation of malignant pleomorphic tumors.

Mutations are thought to be involved in tumor formation because (i) tumor cells transmit their abnormalities to their descendants; and (ii) many carcinogens are mutagens. Aneuploidy is thought to be involved in tumor formation because (i) it is a common phenomenon, especially among malignant neoplasms; (ii) certain particular types of tumors are associated with specific karyotypic changes; and (iii) many immortal tumor cell lines are hyperploid. In recent years, acquired somatic cell replicative infidelity of DNA ("mutator phenotype") has been suggested as a mechanism of tumor formation, because more somatic genomic events occur in malignant tumor cells than could be caused by repeated exogenous mutagenic insults. Previously, theories of the genomic pathogenesis of tumors have involved these mechanisms individually. Here it is suggested that all three mechanisms may play roles in the formation of certain tumor types. For example, a sequence could occur such that first, a mutation affects genomic elements for control of growth, and for replicative fidelity of DNA, leading to "mutator phenotype". Second, when replicative infidelity of DNA results in mutation of genomic elements for mitotic-and-chromosomal stability, aneuploidy develops. Third, an asymmetric mitosis (in the course of the aneuploid stage) could produce occasional cells in which the "bad copy" is lost (or an extra "good copy" is gained) of the original genomic element which had supported replicative fidelity of DNA. These resulting cells would regain fidelity of replication of DNA, and hence could give rise to populations which are relatively genomically stable, hyperploid and immortal.

Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.

Correlations of telomere length, telomerase activity, and telomeric‐repeat binding factor 1 expression in colorectal carcinoma

Telomere maintenance has been proposed as an essential step for tumor cell immortalization. The objectives of the current study were to investigate the mechanisms implicated in telomere length in colorectal carcinoma (CRC) and to evaluate the prognostic impact of telomere status. Ninety-one colorectal carcinoma samples that were obtained from patients who underwent surgery were analyzed to investigate the factors related to telomere function. The authors studied telomerase activity, terminal restriction fragment (TRF) length, and telomeric-repeat binding factor (TRF1) expression and analyzed the prognostic implications of those factors. Most tumors (81.3%) displayed telomerase activity. Overall, telomeres in CRC specimens were significantly shorter compared with telomeres in normal, adjacent specimens (P=0.02). Moreover, tumors that demonstrated shortened telomeres displayed higher TRF1 levels than tumors without telomere shortening. In relation to patient prognosis, a significantly poor clinical course was observed in the group of patients who had tumors with longer telomeres (P=0.02), and this finding emerged as an independent prognostic factor in a Cox proportional hazards model (P=0.04; relative risk, 6.48). Among patients with tumors classified as telomerase-positive, telomere length ratios (the ratio of tumor tissues to normal tissues)<or=0.66 or TRF1 over-expression conferred a favorable outcome (P=0.03 and P=0.05, respectively). The majority of CRC specimens in the current study displayed telomerase reactivation. However, only those tumors that displayed telomere elongation conferred a poor prognosis. Conversely, colorectal tumors that over-expressed TRF1 demonstrated telomere shortening, and patients with those tumors had a better clinical course.

Influence of long-term treatment with cytosine arabinoside on telomere length in human HL60 cells

Immortal tumor cells employ a telomere length maintenance mechanism to escape the normal limits on proliferation. We investigated whether treatment with cytosine arabinoside (AraC), whose triphosphate derivative AraCTP might partially inhibit the synthesis of C-rich telomere strands, is effective for inducing telomere shortening in human HL60 cells. Long-term treatment with 0.01 microM AraC was found to cause significant telomere lengthening, but had no marked effects on cell population doubling rates or morphology.

Preclinical efficacy, safety, and ADME of GRN163L, a novel telomerase inhibitor developed for the treatment of cancer

3168 Background: Telomerase maintains telomeric DNA and plays a critical role in tumor cell immortality. Human telomerase is repressed or transiently active in normal somatic cells and telomeres gradually shorten over decades. In most cancers, telomeres are short, but maintained by telomerase. A correlation between tumor progression and telomerase activation makes the inhibition of telomerase a promising target for the treatment of cancer. GRN163L is a lipid-conjugated thio-phosphoramidate (N3’-P5’) oligonucleotide, developed as a potent and specific telomerase inhibitor for the treatment of cancer. Methods: GRN163L was administered systemically as a single agent or in combination with other chemotherapeutics in tumor models to assess activity of the molecule. IND-supporting preclinical toxicology, pharmacodynamic and ADME studies were carried out in multiple species to characterize the safety, pharmacokinetics and activity of GRN163L, and to determine the desired dose and schedule to be used in initial clinical trials for the treatment of cancer. Results: GRN163L administration in vivo inhibited telomerase activity and tumor growth in tumor xenografts. Models studied included melanoma, myeloma, hepatocellular, ovarian, and lung cancer. Combination studies of GRN63L with melphalan, Velcade, or taxol in various tumor models demonstrated increased efficacy of the combination regimens versus single agent treatment. ADME, pharmacodynamic and preclinical safety studies showed wide biodistribution and support an intermittent schedule and dose that should achieve efficacious levels of GRN163L in patients with an acceptable safety profile. Conclusions: Sufficient GRN163L has been manufactured under GLP and GMP conditions to support IND-enabling preclinical and Phase I clinical studies. The efficacy, PD, and ADME data have identified a target dose range and schedule for GRN163L, and preclinical safety studies have shown a robust safety margin that should allow attainment of this dose and schedule in the clinic. Additional studies to support the clinical development strategy are ongoing. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Geron Geron

Recent advances in telomere biology: implications for human cancer

Research into the basic biology of telomeres continues to reveal details relevant to fundamental aspects of human cancer. The goal of this review is to highlight discoveries made within the last year, with emphasis on their relevance to cancer prevention, diagnosis, prognostics, and treatment. Increasing evidence indicates that dysfunctional telomeres likely play a causal role in the process of malignant transformation, in at least a fraction of human cancers, by initiating chromosomal instability. Telomeres form protective capping structures composed of telomeric DNA complexed with a multitude of associated proteins, the loss of which can have profound effects on telomeric stability. Critical telomeric shortening can lead to telomere "uncapping" and may occur at the earliest recognizable stages of malignant transformation in epithelial tissues. The widespread activation of the telomere synthesizing enzyme telomerase in human cancers not only confers unlimited replicative potential but also prevents intolerable levels of chromosomal instability. Several details regarding telomere structure and telomerase regulation have recently been elucidated, providing new targets for therapeutic exploitation. Various therapeutic strategies aimed at either telomerase or its telomeric substrate are showing promise and may synergize with established anti-cancer agents. Further support for anti-telomerase approaches comes from recent studies indicating that telomerase may possess additional functions, beyond telomere maintenance, that support the growth and survival of tumor cells. Substantial progress has been made in understanding the complex relationships that exist between telomeres and cancer. However, important issues, such as transient activation of telomerase in normal cells and the potential for tumor cell immortalization via telomerase independent means, remain to be clarified.

Molecular cloning of the feline telomerase reverse transcriptase (TERT) gene and its expression in cell lines and normal tissues.

Telomerase is a kind of reverse transcriptase which synthesizes and elongates telomeres. Telomerase activity is detected in many naturally occurring tumors and its expression appears to play an important role in the immortalization of tumor cells. In this study, cDNA encoding the feline telomerase reverse transcriptase (TERT) gene was cloned partially from a feline lymphoma cell line. The clone obtained in this study was 237 bp long including a reverse transcriptase motif 2, and was shown to have amino acid sequence similarity of 81.0% and 58.2% with human and mouse TERT cDNAs, respectively. TERT mRNA expression was detected in telomerase-positive cells (FL74, FT-1, 3201, FKNp, FONp, and FYMp), and was not detected in telomerase-negative cells (normal fibroblasts and CRFK). TERT mRNA was detected in various normal tissues including the spleen, pancreas, stomach, cerebrum, testis, bone marrow, lymph node and thymus, and relatively high-level expression was observed in the small bowel and large bowel. No expression of TERT mRNA was detected in the liver, adrenal gland, urinary bladder and lung. The TERT cDNA clone and the results obtained in this study will be useful for further investigation of feline tumors.

Multiple levels of telomerase regulation.

Normally, cell division leads to shortening of telomeres, the nucleoprotein complexes located at the ends of linear chromosomes. When telomeres reach a critically short length, cells cease to divide. However, immortal tumor cells display stable telomere lengths and are able to maintain their proliferative state. Wong and colleagues have found that telomerase is sequestered by nucleoli during certain stages of the cell cycle, decreasing the likelihood of telomerase access to chromatin until the late S phase. Additionally, they demonstrate that ionizing radiation tends to keep telomerase sequestered in nucleoli, whereas cell transformation leads to telomerase translocation into the nucleoplasm, where, presumably, it can catalyze the lengthening of telomeres at appropriate and inappropriate sites. The sequestration of telomerase thus imposes a newly identified level of regulation on telomerase activity, implicating telomerase localization as a potentially useful target for pharmacotherapy.