Blueprint for cancer research: Critical gaps and opportunities.

We are experiencing a revolution in cancer. Advances in screening, targeted and immune therapies, big data, computational methodologies, and significant new knowledge of cancer biology are transforming the ways in which we prevent, detect, diagnose, treat, and survive cancer. These advances are enabling durable progress in the goal to achieve personalized cancer care. Despite these gains, more work is needed to develop better tools and strategies to limit cancer as a major health concern. One persistent gap is the inconsistent coordination among researchers and caregivers to implement evidence-based programs that rely on a fuller understanding of the molecular, cellular, and systems biology mechanisms underpinning different types of cancer. Here, the authors integrate conversations with over 90 leading cancer experts to highlight current challenges, encourage a robust and diverse national research portfolio, and capture timely opportunities to advance evidence-based approaches for all patients with cancer and for all communities.

Telomere lengths in women treated for breast cancer show associations with chemotherapy, pain symptoms, and cognitive domain measures: a longitudinal study.

BACKGROUND: Survival rates for breast cancer (BC) have improved, but quality of life post-diagnosis/treatment can be adversely affected, with survivors reporting a constellation of psychoneurological symptoms (PNS) including stress, anxiety, depression, pain, fatigue, sleep disturbance, and cognitive dysfunction. METHODS: To assess a potential relationship between telomere length (TL) and the development/persistence of PNS, we longitudinally studied 70 women (ages 23-71) with early stage BC (I-IIIA) at 5 time-points: prior to treatment (baseline), the mid-point of their chemotherapy cycle, 6 months, 1 year, and 2 years following the initiation of chemotherapy. Measures quantified included assessments of each of the PNS noted above and TL [using both a multiplex qPCR assay and a chromosome-specific fluorescence in situ hybridization (FISH) assay]. RESULTS: Variables associated with qPCR mean TLs were age (p = 0.004) and race (T/S ratios higher in Blacks than Whites; p = 0.019). Significant differences (mostly decreases) in chromosome-specific TLs were identified for 32 of the 46 chromosomal arms at the mid-chemo time-point (p = 0.004 to 0.049). Unexpectedly, the sequential administration of doxorubicin [Adriamycin], cyclophosphamide [Cytoxan], and docetaxel [Taxotere] (TAC regimen) was consistently associated with higher TLs, when compared to TLs in women receiving a docetaxel [Taxotere], Carboplatin [Paraplatin], and trastuzumab [Herceptin] [TCH] chemotherapy regimen [association was shown with both the qPCR and FISH assays (p = 0.036)]. Of the PNS, pain was significantly negatively associated with TL (higher pain; shorter telomeres) for a subset of chromosomal arms (5q, 8p, 13p, 20p, 22p, Xp, Xq) (p = 0.014-0.047). Chromosomal TLs were also associated with 7 of the 8 cognitive domains evaluated, with the strongest relationship being noted for chromosome 17 and the visual memory domain (shorter telomeres; lower scores). CONCLUSIONS: We showed that race and age were significantly associated with telomere length in women treated for early stage BC and that acquired telomere alterations differed based on the woman's treatment regimen. Our study also demonstrated that pain and cognitive domain measures were significantly related to telomere values in this study cohort. Expanding upon the knowledge gained from this longitudinal study could provide insight about the biological cascade of events that contribute to PNS related to BC and/or its treatment.

Restoring SIRT6 Expression in Hutchinson-Gilford Progeria Syndrome Cells Impedes Premature Senescence and Formation of Dysmorphic Nuclei.

OBJECTIVES: Mice overexpressing SIRT6 live longer than wild-type mice while SIRT6 knockout mice exhibit similar degenerative phenotypes as individuals with Hutchinson-Gilford progeria syndrome (HGPS). Thus, we sought to test whether levels of SIRT6 are reduced in cells from individuals with HGPS and whether restored SIRT6 expression may impede premature aging phenotypes. METHODS: Levels of endogenous SIRT6 and progerin in HGPS and normal fibroblasts were assessed by Western blotting and immunofluorescence. A tetracycline-inducible system was utilized to test whether progerin causes a rapid reduction in SIRT6 protein. SIRT6 was overexpressed in HGPS cells via lentiviral infection with biological endpoints including senescence-associated ß-galactosidase (SA-ß-gal) positivity, frequency of nuclear atypia, the number of 53BP1-positive DNA damage foci and growth rates. RESULTS: Typical HGPS fibroblasts express lower levels of SIRT6 than fibroblasts from normal and atypical HGPS donors. Experimental induction of progerin did not cause a detectable reduction of SIRT6 protein. However, overexpression of SIRT6 in HGPS cells was associated with a reduced frequency of SA-ß-gal positivity, fewer misshapen nuclei, fewer DNA damage foci, and increased growth rates. CONCLUSIONS: Typical HGPS fibroblasts exhibit reduced levels of SIRT6 protein via a mechanism that remains to be elucidated. Our findings suggest that restoring SIRT6 expression in HGPS cells may partially impede senescence and the formation of dysmorphic nuclei. © 2015 S. Karger AG, Basel.

Telomere length: a review of methods for measurement.

BACKGROUND: The exciting discovery that telomere shortening is associated with many health conditions and that telomere lengths can be altered in response to social and environmental exposures has underscored the need for methods to accurately and consistently quantify telomere length. OBJECTIVES: The purpose of this article is to provide a comprehensive summary that compares and contrasts the current technologies used to assess telomere length. DISCUSSION: Multiple methods have been developed for the study of telomeres. These techniques include quantification of telomere length by terminal restriction fragmentation-which was one of the earliest tools used for length assessment-making it the gold standard in telomere biology. Quantitative polymerase chain reaction provides the advantage of being able to use smaller amounts of DNA, thereby making it amenable to epidemiology studies involving large numbers of people. An alternative method uses fluorescent probes to quantify not only mean telomere lengths but also chromosome-specific telomere lengths; however, the downside of this approach is that it can only be used on mitotically active cells. Additional methods that permit assessment of the length of a subset of chromosome-specific telomeres or the subset of telomeres that demonstrate shortening are also reviewed. CONCLUSION: Given the increased utility for telomere assessments as a biomarker in physiological, psychological, and biobehavioral research, it is important that investigators become familiar with the methodological nuances of the various procedures used for measuring telomere length. This will ensure that they are empowered to select an optimal assessment approach to meet the needs of their study designs. Gaining a better understanding of the benefits and drawbacks of various measurement techniques is important not only in individual studies, but also to further establish the science of telomere associations with biobehavioral phenomena.

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites.

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.

Elevated TRF2 in advanced breast cancers with short telomeres.

Telomere repeat binding factor 2 (TRF2) binds directly to telomeres and preserves the structural integrity of chromosome ends. In vitro models suggest that expression of TRF2 protein increases during mammary cancer progression. However, a recent study has reported that TRF2 mRNA levels tend to be lower in clinical specimens of malignant breast tissue. Here, we conduct the first large-scale investigation to assess the levels and cellular localization of the TRF2 protein in normal, pre-malignant and malignant breast tissues. Breast tissue arrays, containing normal, ductal carcinoma in situ (DCIS) and invasive carcinoma specimens, were used to assess the expression and localization of TRF2 protein. Telomere lengths were semi-quantitatively measured using a pantelomeric peptide nucleic acid probe. A mixed effects modeling approach was used to assess the relationship between TRF2 expression and telomeric signal scores across disease states or clinical staging. We demonstrate that TRF2 is exclusively nuclear with a trend toward lower expression with increased malignancy. More case-to-case variability of TRF2 immunostaining intensity was noted amongst the invasive carcinomas than the other disease groups. Invasive carcinomas also displayed variable telomere lengths while telomeres in normal mammary epithelium were generally longer. Statistical analyses revealed that increased TRF2 immunostaining intensity in invasive carcinomas is associated with shorter telomeres and shorter telomeres correlate with a higher TNM stage. All immortalized and cancer cell lines within the array displayed strong, nuclear TRF2 expression. Our data indicate that elevated expression of TRF2 is not a frequent occurrence during the transformation of breast cancer cells in vivo, but higher levels of this telomere-binding protein may be important for protecting advanced cancer cells with critically short telomeres. Our findings also reinforce the concept that serially propagated cancer cells, although tumor-derived, may not model all types of authentic tumors especially those demonstrating genetic heterogeneity.

Randomized, Placebo-Controlled Analysis of the Knee Synovial Environment Following Platelet-Rich Plasma Treatment for Knee Osteoarthritis.

BACKGROUND: Platelet-rich-plasma (PRP) is used to treat knee osteoarthritis; however, mechanistic evidence of PRP effectiveness for pain relief is limited. OBJECTIVE: To assess molecular biomarkers and mesenchymal stem cells (MSCs) in synovial fluid during PRP treatment of the osteoarthritic knee joint. DESIGN: Single blinded, randomized, placebo controlled pilot study. SETTING: Veterans Affairs Medical Center. PARTICIPANTS: Seventeen participants with mild to moderate knee osteoarthritis were randomized in a 2:1 placebo-controlled ratio, receiving PRP or saline (placebo) intra-articular injection into the knee joint. METHODS: Knee synovial fluid was analyzed before the respective injections and again 10 days following injection. Participants were followed up to 12 months completing visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaires at intervals over that period. MAIN OUTCOME MEASURES: The effects of PRP on synovial protein and MSC gene expression levels were measured by multiplex enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. RESULTS: Novel biomarkers including levels of interleukin (IL)-5, IL-6, IL-10, and tumor necrosis factor-α were measured in synovial fluid 10 days after PRP treatment. Altered gene expression profiles in MSCs from patients treated with PRP were observed for matrix metalloproteinases and inflammatory markers (IL-6, IL-8, CCL2, TNF-α). A2M protease was significantly increased following PRP treatment (P = .005). WOMAC scores declined for up to 3 months from baseline levels and remained low at 6 and 12 months in the PRP group. In contrast, WOMAC scores for patients receiving the saline injection were relatively unchanged for up to 12 months. CONCLUSIONS: We report significant changes for the biomarker A2M (P = .005) as well as differences in expression of cellular markers and postulate that PRP modulates the local knee synovial environment by altering the inflammatory milieu, matrix degradation, and angiogenic growth factors. The PRP treatment group had less pain and stiffness and improved function scores.

Multipotent adipose stromal cells and breast cancer development: Think globally, act locally.

It has long been appreciated that stromal cells within the breast tumor microenvironment contribute to mammary carcinogenesis. However, to date, very little is known regarding the role of local adipose-derived stromal cells (ASCs) in the development of breast cancer. Based on pathological, epidemiological and experimental data, we postulate that breast-derived ASCs are unique mesenchymal stem-like cells that play a critical role in the development of breast cancer and discuss the global implications of this working model in terms of breast cancer prevention, early detection, and new targeted therapies.

Induction of nitric oxide synthase-dependent telomere shortening after functional inhibition of Hsp90 in human tumor cells.

In most cancer cells, the lengths of telomeres, the functional DNA-protein complexes located at chromosome ends, are maintained by the ribonucleoprotein telomerase. Hsp90 facilitates the assembly of telomerase and remains associated with the functional complex, implying a direct involvement of Hsp90 in telomere length regulation. In an effort to elucidate the effects of Hsp90 inhibition on function and viability of human prostate cancer cells, both pharmacological (radicicol) and genetic (small interfering RNA) approaches were utilized to target Hsp90. Depletion of functional Hsp90 caused dramatic telomere shortening followed by apoptosis. Of particular significance, these cells exhibit a high level of nitric oxide synthase (NOS)-dependent free radical production, and simultaneous treatment of cells with the NOS inhibitor L-NAME resulted in telomere elongation and prevention of apoptosis. In addition, we observe significant DNA damage assessed by telomere dysfunction, although in the absence of a classical DNA damage response. Overall, our data suggest a novel mechanism whereby inhibition of Hsp90 disrupts free radical homeostasis and contributes directly to telomere erosion, further implicating Hsp90 as a potential therapeutic target for cancer cells.

Tumor cell escape from therapy-induced senescence.

H460 non-small cell lung, HCT116 colon and 4T1 breast tumor cell lines induced into senescence by exposure to either etoposide or doxorubicin were able to recover proliferative capacity both in mass culture and when enriched for the senescence-like phenotype by flow cytometry (based on ß-galactosidase staining and cell size, and a senescence-associated reporter, BTG1-RFP). Recovery was further established using both real-time microscopy and High-Speed Live-Cell Interferometry (HSLCI) and was shown to be accompanied by the attenuation of the Senescence-Associated Secretory Phenotype (SASP). Cells enriched for the senescence-like phenotype were also capable of forming tumors when implanted in both immunodeficient and immunocompetent mice. As chemotherapy-induced senescence has been identified in patient tumors, our results suggest that certain senescence-like phenotypes may not reflect a terminal state of growth arrest, as cells that recover with self-renewal capacity may ultimately contribute to disease recurrence.

Recovery from stress is a function of age and telomere length.

Cells are constantly exposed to a wide variety of stimuli and must be able to mount appropriate physiological responses in order to maintain proper form and function. Cells from every organism have evolved highly conserved mechanisms to cope with environmental changes, including the widely studied heat shock response (HSR), which is induced by a variety of cellular stresses such as heavy metal ion exposure. It has long been known that as organisms and individual cells age, their ability to appropriately cope with environmental stress is attenuated. Here, we examine the ability of two heavy metal ions (ZnCl(2), SnCl(2)) to induce the HSR in human fibroblasts by assessing the expression of heat shock proteins (Hsp90, Hsp70, and p23) and the ability of the cells to recover over time. We demonstrate that the induction and recovery of chaperone levels is attenuated with age and that cells immortalized with the human telomerase reverse transcriptase component of the telomerase enzyme do not attenuate their HSR as their replicative age increases. Our data suggest that the recovery of normal human cells from an HSR is related in part to age and the cell's overall telomere length.

Upregulation of telomerase function during tissue regeneration.

Telomerase plays a primary role in the maintenance of telomeres in immortal, germ, and tumor cells in humans but is lacking in most somatic cells and tissues. However, many species, including fish and inbred mice, express telomerase in most cells and tissues. Little is known about the expression of telomerase in aquatic species, although the importance of telomerase for longevity has been suggested. We compared telomerase activity and telomere lengths among a broad range of tissues from aquatic species and found telomerase at significant levels in both long- and short-lived aquatic species, suggesting constitutive telomerase expression has an alternative function. Telomere lengths in these aquatic species were comparable to those observed in normal human tissues and cell strains. Given that a host of aquatic species with short life spans have telomerase and a tremendous capacity to regenerate, we tested the hypothesis that telomerase upregulation is important for tissue regeneration. During regeneration, telomerase activity was upregulated and telomere lengths are maintained with the shortest telomeres being elongated, indicating the importance for maintaining telomere length and integrity during tissue regeneration. Thus, the expression of telomerase in aquatic animals is likely not related to longevity but to their ability to regenerate injured tissue.

Overexpression of telomerase-associated chaperone proteins in prostatic intraepithelial neoplasia and carcinomas.

Over 90% of prostate cancers express telomerase activity. In an experimental model, hsp90 and p23, which are necessary for telomerase assembly and function, dramatically increase during tumorigenic conversion. We immunohistochemically analyzed 60 prostate carcinomas, 50 prostatic intraepithelial neoplasias (PIN) and 25 benign prostatic tissues to determine whether hsp90/p23 expression correlates with advancing stage and whether chaperone distribution overlaps with hTERT, the catalytic component of telomerase. Strong expression of hsp90/p23 was detected in approximately 95% of PIN and carcinomas without relationship to Gleason score. While hsp90/p23 immunostaining was predominantly diffuse and cytoplasmic, nuclear immunoreactivity was observed in several moderate-to-high grade carcinomas, and those carcinomas with nuclear chaperone staining exhibited detectable hTERT. Our data suggest enhanced chaperone-mediated telomerase assembly as a mechanism for increased activity in advanced prostate carcinomas, stable association between chaperones and telomerase in vivo, and utility for chaperone immunostaining to identify focal PIN in the context of widespread hyperplasia.

Fibronectin fibrils regulate TGF-ß1-induced Epithelial-Mesenchymal Transition.

Epithelial-Mesenchymal Transition (EMT) is a dynamic process through which epithelial cells transdifferentiate from an epithelial phenotype into a mesenchymal phenotype. Previous studies have demonstrated that both mechanical signaling and soluble growth factor signaling facilitate this process. One possible point of integration for mechanical and growth factor signaling is the extracellular matrix. Here we investigate the role of the extracellular matrix (ECM) protein fibronectin (FN) in this process. We demonstrate that inhibition of FN fibrillogenesis blocks activation of the Transforming Growth Factor-Beta (TGF-ß) signaling pathway via Smad2 signaling, decreases cell migration and ultimately leads to inhibition of EMT. Results show that soluble FN, FN fibrils, or increased contractile forces are insufficient to independently induce EMT. We further demonstrate that inhibition of latent TGF-ß1 binding to FN fibrils via either a monoclonal blocking antibody against the growth factor binding domain of FN or through use of a FN deletion mutant that lacks the growth factor binding domains of FN blocks EMT progression, indicating a novel role for FN in EMT in which the assembly of FN fibrils serves to localize TGF-ß1 signaling to drive EMT.

Evasion of a single-step, chemotherapy-induced senescence in breast cancer cells: implications for treatment response.

PURPOSE: The purpose of this study is to define the mechanistic basis for recovery of proliferative capacity in breast tumor cells after chemotherapy. Here, we test the hypothesis that evasion of senescence confers resistance to chemotherapeutic drugs and ionizing radiation. EXPERIMENTAL DESIGN: MCF-7 cells were treated with a single, clinically relevant dose (0.75-1.0 micromol/L) of Adriamycin. Two weeks following induction of senescence, clonal outgrowths were expanded and characterized in terms of senescence-associated beta-galactosidase activity, gene expression profiles (Affymetrix U95 probe sets, Affymetrix, Santa Clara, CA) with confirmatory Western analyses, and telomerase activity following a second drug treatment. Levels of intracellular Adriamycin, as well as cross-resistance to other therapeutic agents, were also determined to define the resistance phenotype. RESULTS: A senescence-resistant (SR) clone (clone 2) was identified that was largely refractory to both Adriamycin-induced and gamma-irradiation-induced senescence. Clone 2 continued to proliferate and maintain high levels of telomerase activity following a second drug treatment, when treated parental cells expressed very low levels of telomerase and many positive cell cycle regulators. SR clone 2 also expressed substantially more cdc-2 than parental cells and undetectable levels of MDR1, showed an intact p53 checkpoint and only a modestly lower level of intracellular drug accumulation, while exhibiting cross-resistance to other topoisomerase inhibitors. CONCLUSIONS: SR clone 2 is intrinsically resistant to DNA damage-induced senescence perhaps through an ability to prevent down-regulation of cdc-2. Telomerase is a marker of proliferative recovery for breast cancer cells after chemotherapy exposure. Evasion or escape from a single-step, drug-induced senescence may represent a unique and previously unrecognized drug-resistance phenotype.

Is Senescence Reversible?

Senescence was originally identified by the finite lifespan of normal cells that is a consequence of telomere shortening with each cycle of DNA replication. Cells undergoing replicative senescence display pronounced morphological and biochemical changes such as flattening and/or enlargement, increases in p21(WAF1) and/or p16(INK4A), a senescence-associated secretory phenotype, and often senescence-associated heterochromatic foci. Senescence also occurs in tumor cells in response to various forms of chemotherapy or radiation (therapy-induced senescence), which could be the basis for prolonged or (ideally) permanent growth arrest. Alternatively, therapy-induced senescence could represent a means whereby tumor cells evade the potential toxicity of chemotherapy and radiation, allowing for the eventual re-emergence or escape from senescence that could lead to disease recurrence. This review discusses the experimental data in the literature that support the premise that senescence is potentially reversible through the inactivation of p53, p16(INK4A) and/or Rb, over-expression of Cdc2/cdk1 and survivin, the development of polyploidy, the survival of cancer stem cells and/or restoration of the nuclear landscape. If senescence is truly reversible, then the re-emergence of tumor cells from senescent arrest induced by chemotherapy or radiation could represent a barrier to the development of effective and curative cancer therapies.

Breast epithelial cell infiltration in enhanced electrospun silk scaffolds.

In the present study, the effects of air-flow impedance electrospinning and air-flow rates on silk-based scaffolds for biological tissues were investigated. First, the properties of scaffolds obtained from 7% and 12% silk concentrations were defined. In addition, cell infiltration and viability of MCF-10A breast epithelial cells cultured onto these scaffolds were used to determine the biological suitability of these nanostructures. Air-flow impedance electrospun scaffolds resulted in an overall larger pore size than scaffolds electrospun on a solid mandrel, with the largest pores in 7% silk electrospun with an air pressure of 100 kPa and in 12% silk electrospun with an air pressure of 400 kPa (13.4 ± 0.67 and 26.03 ± 1.19 µm, respectively). After 14 days in culture, the deepest MCF-10A cell infiltration (36.58 ± 2.28 µm) was observed into 7% silk air-flow impedance electrospun scaffolds subjected to an air pressure of 100 kPa. In those scaffolds MCF-10A cell viability was also highest after 14 days in culture. Together, these results strongly support the use of 7% silk-based scaffolds electrospun with a 100 kPa air-flow as the most suitable microenvironment for MCF-10A infiltration and viability.

Telomerase as a target for cancer immunotherapy.

Telomerase is the ribonucleoprotein that enables cancer and stem cells to maintain their telomeres, resulting in unlimited proliferative potential. The catalytic component of telomerase in humans, hTERT, is upregulated in nearly 90% of all cancers, making it the most widely expressed marker of malignancy. With the exception of germ cells and stem cells, hTERT is undetectable in somatic human tissues. Together, these properties make telomerase a leading candidate for cancer therapy. Various therapies have been tested in tissue culture and in mouse models utilizing genetic, pharmacological, and immunological approaches. The purpose of this review is to critically examine the role of hTERT in cancer immunotherapy by providing a comparison of the current experiments and a proposal for an innovative method utilizing DNA vaccination.

Telomerase protects cancer-prone human cells from chromosomal instability and spontaneous immortalization.

Studies were conducted to directly test whether the introduction of telomerase protects cancer-prone human mammary epithelial cells from chromosomal instability and spontaneous immortalization. Using a model for Li Fraumeni Syndrome (LFS), infection of human telomerase resulted in maintenance of telomere lengths, extension of in vitro lifespan, and prevention of spontaneous immortalization. In stark contrast to the spontaneously immortalized LFS cells, cells expressing ectopic telomerase displayed a remarkably stable karyotype and even after >150 population doublings, did not express endogenous telomerase. Since the hTERT-infected and spontaneously immortal LFS cells, like the parental cells, exhibit loss of p53 function, our data suggests that telomere shortening is the primary driving force for the genomic instability characteristic of LFS cells, while p53 inactivation is necessary for triggering the spontaneous immortalization event. Collectively, our data indicate that exogenous telomerase prevents chromosomal instability and spontaneous immortalization of LFS cells, suggesting a unique protective role for telomerase in the progression to immortalization.

Retroviral-mediated expression of telomerase in normal human cells provides a selective growth advantage.

Retroviral infection of hTERT, the catalytic component of telomerase, into BJ fibroblasts (population doubling 28) resulted in reconstitution of telomerase activity, telomere maintenance, and extension of in vitro lifespan. The hTERT-infected cells also exhibited increased growth rate and colony forming efficiency relative to controls, while remaining contact-inhibited and maintaining a p53-mediated damage response following gamma-irradiation. All single cell-derived BJ-hTERT clones grew faster than the hTERT mass cultures and maintained telomeres; however, neither telomerase activity levels nor mean telomere length correlated with the growth rate. Introduction of hTERT rescued aged BJ fibroblasts from senescence via a telomere-dependent mechanism and provided renewed proliferative potential. Collectively, our data indicate that both early and late in the cellular lifespan of human cells, ectopic expression of telomerase using a retroviral system provides a growth advantage while maintaining normal cellular characteristics.