Ectopic Expression Of hTERT Research Articles

Telomerase prevents accelerated senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts

Fibroblasts derived from glucose-6-phosphate dehydrogenase (G6PD)-deficient patients display retarded growth and accelerated cellular senescence that is attributable to increased accumulation of oxidative DNA damage and increased sensitivity to oxidant-induced senescence, but not to accelerated telomere attrition. Here, we show that ectopic expression of hTERT stimulates telomerase activity and prevents accelerated senescence in G6PD-deficient cells. Stable clones derived from hTERT-expressing normal and G6PD-deficient fibroblasts have normal karyotypes, and display no sign of senescence beyond 145 and 105 passages, respectively. Activation of telomerase, however, does not prevent telomere attrition in earlier-passage cells, but does stabilize telomere lengths at later passages. In addition, we provide evidence that ectopic expression of hTERT attenuates the increased sensitivity of G6PD-deficient fibroblasts to oxidant-induced senescence. These results suggest that ectopic expression of hTERT, in addition to acting in telomere length maintenance by activating telomerase, also functions in regulating senescence induction.

277 PLASMID MEDIATED EXPERIMENTAL TELOMERE EXTENSION IN BOVINE EMBRYOS BY ECTOPIC EXPRESSION OF HUMAN TERT

Telomeres are composed of repetitive hexanucleotide sequences, (TTAGGG)n, encompassing several kilobase pairs, and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage and up to the morula stage, telomerase is not active or is found only at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) has 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from a local abattoir, followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. Two plasmid encoding CMV promoter-driven sequences of hTERT and green fluorescent protein (GFP) were coinjected in bovine zygotes, and GFP driven by a muscle specific promoter was injected for mock experiments. The hTERT and GFP were co-injected to allow live separation of embryos. A total of 768 bovine embryos were injected; 468 (61%) of the treated embryos showed specific GFP-fluorescence. Of a total of 132 blastocysts (17%), 45 showed GFP fluorescence (34%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic hTERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. The mRNA level of the ectopic hTERT began to increase from the 4- to the 8-cell stage and remained high up to the morula stage. Embryos at the morula and blastocyst stages were spread on slides and analyzed by quantitative fluorescence in situ hybridization (qFISH). A Cy3-labeled 18-mer peptide nucleic acid (PNA) probe was used to hybridize the telomeres. The resulting spot intensities were quantified by using TFL-Telo software and were statistically analyzed. A modest increase in telomere length was observed in hTERT injected [775 ± 69 fluorescence unit (fu)] group compared to uninjected control (679 ± 75 fu) group at blastocyst stage. In conclusion, this study demonstrates that the ectopic expression of hTERT in embryos results in telomere elongation; overexpression of TERT may facilitate the derivation of bovine embryonic stem cells. Supported by DFG and Goyaike SAACIYF.

Transfection of adult canine Schwann cells and olfactory ensheathing cells at early and late passage with human TERT differentially affects growth factor responsiveness and in vitro growth

Adult canine Schwann cells and olfactory ensheathing cells (OECs) are closely related cell types that are considered attractive candidates for translational studies of neural repair. To establish a reliable cell source by comparing the in vitro properties of immortalized Schwann cells and OECs for transplantation purposes, we transfected both cell types with human telomerase reverse transcriptase (hTERT). Ectopic hTERT expression has been shown to induce immortalization of various cell types without substantial alterations of their phenotypes. Schwann cells and OECs were isolated from adult dogs, transfected with hTERT at early (P4) and late passage (P26), characterized regarding in vitro proliferation, antigenic expression and senescence-associated genes in the presence and absence of fibroblast growth factor-2 (FGF-2). Ectopic hTERT expression in late passage glia treated with but not without FGF-2 prevented the decline in proliferation observed in non-transfected cells. Immortalization did not alter p75 NTR and GFAP but O4 and A2B5 expression. Contrary to this, early passage hTERT transfection significantly reduced proliferation independent of FGF-2 and lowered expression of O4 and GFAP in both cell types. Transfection did not alter mRNA expression of senescence-associated genes such as p53 and p16. No substantial differences were found between Schwann cells and OECs underscoring the close relationship of both cell types. Taken together, we established a stable source of adult canine Schwann cells and OECs and demonstrated that the effects of hTERT expression on in vitro growth and growth factor responsiveness depend on the replicative age.

Introducing a single‐cell‐derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer

Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chon-drogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

Differentiation of human telomerase reverse transcriptase immortalized human marrow mesenchymal stem cell into chondrocyte

To establish an immortalized marrow mesenchymal stem cell line to facilitate advances in cartilage engineering research. Human telomerase reverse transcriptase (hTERT) cDNA was transferred into primary human marrow mesenchymal stem cells (hMSC) by retroviral vector pLEGFP-C1-hTERT. Subsequently G418 resistant cell clone was screened and expanded for further studies. hMSC biomarkers and hTERT expression were confirmed by examination. Transfected hMSC was induced to differentiate into chondrocyte using TGF-P1 and dexamethasone. Up-regulated hTERT expression was detected in transfected hMSC. hMSC-hTERT cells could be induced to differentiate into chondrocyte. Higher telomerase activity in transfected cells was maintained for 50 population doublings so far. Collagen II could be detected in induced transfected hMSC by immunocytochemical and hybridization in situ. Ectopic expression of hTERT can effectively immortalize hMSC in vitro. Immortalized hMSC can be induced to differentiate into chondrocyte under certain condition. It may be an ideal target of further studies in cartilage engineering.

Impairment of the telomere/telomerase system and genomic instability are associated with keratinocyte immortalization induced by the skin human papillomavirus type 38

The skin human papillomavirus (HPV) types belonging to the genus beta of the HPV phylogenetic tree appear to be associated with nonmelanoma skin cancer. We previously showed that the beta HPV type 38 E6 and E7 oncoproteins are able to inactivate the tumor suppressors p53 and retinoblastoma. Here, both viral proteins were expressed in primary human skin keratinocytes in order to study their effects on the telomere/telomerase system. We show that immortalization of skin keratinocytes induced by HPV38 E6/E7 is associated with hTERT gene overexpression. This event is, in part, explained by the accumulation of the p53-related protein, DeltaNp73. Despite elevated levels of hTERT mRNA, the telomerase activity detected in HPV38 E6/E7 keratinocytes was lower than that observed in HPV16 E6/E7 keratinocytes. The low telomerase activation in highly proliferative HPV38 E6/E7 keratinocytes resulted in the presence of extremely short and unstable telomeres. In addition, we observed anaphase bridges, mitotic multipolarity, and dramatic genomic aberrations. Interestingly, the ectopic expression of hTERT prevents both telomere erosion and genomic instability. Thus, we showed that in HPV38 E6/E7 keratinocytes characterized by unscheduled proliferation, suboptimal activation of telomerase and subsequent extensive telomere shortening result in genomic instability facilitating cellular immortalization.

Induction of claudins in passaged hTERT-transfected human nasal epithelial cells with an extended life span

The epithelial barrier of the upper respiratory tract, such as that of the nasal mucosa, plays a crucial role in host defense. The epithelial barrier is regulated in large part by the apical-most intercellular junctions, referred to as tight junctions. However, the mechanisms regulating of tight junction barrier in human nasal epithelial cells remain unclear because the proliferation and storage of epithelial cells in primary cultures are limited. In the present study, we introduced the catalytic component of telomerase, the hTERT gene, into primary cultured human nasal epithelial cells and examined the properties of the transfectants, including their expression of tight junctions, compared with primary cultures. The ectopic expression of hTERT in the epithelial cells resulted in adequate growth potential and a longer lifespan of the cells. The properties of the passaged hTERT-transfected cells including tight junctions were similar to those of the cells in primary cultures. The barrier function in the transfectants after treatment with 10% FBS was significantly enhanced with increases of integral tight junction proteins claudin-1 and -4. When the transfectants were treated with TGF-beta, which is assosciated with nasal polyposis and chronic rhinosinusitis, upregulation of only claudin-4 was observed, without a change of barrier function. In human nasal epithelial cells, the claudins may be important for barrier function and a novel target for a drug-delivery system. Our results indicate that hTERT-transfected human nasal epithelial cells with an extended lifespan can be used as an indispensable and stable model for studying the regulation of claudins in human nasal epithelium.

HTERT inhibits the Epstein‐Barr virus lytic cycle and promotes the proliferation of primary B lymphocytes: Implications for EBV‐driven lymphomagenesis

Transformation of primary B lymphocytes by Epstein-Barr Virus (EBV) requires the establishment of a latent infection, the expression of several latent viral proteins and a sustained telomerase activity. We investigated the interplay between the activation of human telomerase reverse transcriptase (hTERT), the catalytic rate-limiting component of the telomerase complex, and the expression of latent/lytic EBV genes during the establishment of a stably latent EBV infection of normal B lymphocytes. Cell cultures at early passages after EBV infection greatly differed in their timing of hTERT expression and telomerase activation. Induction of hTERT was dependent on the balance between latent and lytic EBV gene expression, being positively associated with a high ratio of latent/lytic isoforms of latent membrane protein 1, and negatively associated with the expression of BZLF1 gene, the main activator of the viral lytic cycle. In turn, hTERT expression was followed by a decrease in EBV lytic gene expression and virus production. Ectopic expression of hTERT in BZLF1-positive B cell cultures resulted in BZLF1 down-regulation, increased resistance to lytic cycle induction, and enhanced in vitro growth properties, whereas hTERT inhibition by siRNA triggered the activation of the EBV lytic cycle. These findings indicate that hTERT contributes by multiple mechanisms to the EBV-driven transformation of B lymphocytes and suggest that hTERT may constitute a therapeutic target for EBV-associated B cell lymphomas.

P53-dependent integration of telomere and growth factor deprivation signals.

Ectopically expressed hTERT enables p16(INK4A)(-) human mammary epithelial cells to proliferate in the absence of growth factors, a finding that has led to the hypothesis that hTERT has growth regulatory properties independent of its role in telomere maintenance. We now show that telomerase can alter the growth properties of cells indirectly through its role in telomere maintenance, without altering growth stimulatory pathways. We find that telomere dysfunction, indicated by 53BP1/phosphorylated histone H2AX foci at chromosome ends, is present in robustly proliferating human mammary epithelial cells long before senescence. These foci correlate with increased levels of active p53. Ectopic expression of hTERT reduces the number of foci and the level of active p53, thereby decreasing sensitivity to growth factor depletion, which independently activates p53. The continuous presence of hTERT is not necessary for this effect, indicating that telomere maintenance, rather than the presence of the enzyme itself, is responsible for the increased ability to proliferate in the absence of growth factors. Our findings provide a previously unrecognized mechanistic explanation for the observation that ectopically expressed hTERT conveys growth advantages to cells, without having to postulate nontelomeric functions for the enzyme.

Immortalization and Characterization of Porcine Ventricular Endocardial Endothelial Cells

Endocardial endothelial cells (EECs), which form the inner lining of the cavities of the heart, are a distinct cell population whose dysfunction can be critical in pathological conditions of heart. Insights into the role and organization of these cells in pathological states of the heart are limited mainly due to a dearth of experimental models. To date no endocardial endothelial cell line is available. The authors attempted to immortalize porcine ventricular EECs by transfecting the cells with human telomerase reverse transcriptase (hTERT). EECs immortalized by ectopic expression of hTERT exhibit phenotypic and functional characteristics similar to primary EECs. The EE cell line could be useful for the study of mechanisms involved in the interaction of EECs with the underlying myocardium and cardiac interstitium and as useful tools in understanding their role in diseased states of heart.

Immortalization of Human HSPC by a Self-Renewal Signal Coupled with Telomere Maintenance.

Expression of the leukemia-associated fusion protein AML1-ETO (AE) in human hematopoietic stem and progenitor cells (HSPC) results in long-term cytokine-dependent cells that maintain CD34 expression, multipotential differentiation and the ability to engraft NOD/SCID mice. These cells gradually lose telomere length and are not immortal. To determine whether expression of telomerase would cooperate with AE in immortalization of human HSPC, we transduced AE-expressing CD34+ cells with the telomerase gene, hTERT. In four independent AE cell cultures, hTERT expression allowed the cells to proliferate beyond the crisis point. Cells retained B lymphoid, myeloid and erythroid differentiation potential, displayed continuous replating ability in methylcellulose assays and engrafted in NOD/SCID mice. The hTERT-expressing cells had a significant increase in progenitor cell numbers, more cells in S phase, and increased resistance to gamma radiation-induced apoptosis compared to control cells. No genetic changes were identified by G-banding or comparative genomic hybridization during the passage through crisis, implying that these signals are sufficient for the immortalization. Similar results were obtained using a long-term culture established with the related core binding factor (CBF) fusion gene CBFβ-MYH11 (CM). By microarray analysis we identified a number of genes implicated in stem cell self-renewal, including HOXB4, GATA3 and STAT3, that are upregulated in CBF-expressing cells when compared to control CD34+ cells. Ectopic hTERT expression did not affect the level of expression of these self-renewal genes. We hypothesize that the continual rescue of progenitor cells during culture, as a result of telomerase maintenance of telomeres, is responsible for the proliferation and survival phenotype observed in hTERT expressing AE and CM cells. This is supported by our findings that control cultures contained significantly more DNA damage foci as detected by immunofluorescence staining for phosphorylated H2AX. In addition, signal-free chromosome ends were significantly more abundant in control cultures as demonstrated by a telomeric FISH probe when compared to the hTERT cultures.Senescence-associated β-gal staining was prominent in control cells as they slowed proliferation after 7 months of growth, but hTERT-expressing cells did not display SA-β-gal staining and continued proliferating indefinitely, for over two years at this point in time. We conclude that the self-renewal signal supplied by a CBF oncogene cooperates with telomere maintenance initiated by hTERT for the immortalization of human HSPC. This system permits a molecular dissection of the signals involved in HSPC self-renewal and leukemogenesis and in the contribution of hTERT expression to this process.

Mitochondrial localization of telomerase as a determinant for hydrogen peroxide-induced mitochondrial DNA damage and apoptosis

We have previously shown that the protein subunit of telomerase, hTERT, has a bonafide N-terminal mitochondrial targeting sequence, and that ectopic hTERT expression in human cells correlated with increase in mtDNA damage after hydrogen peroxide treatment. In this study, we show, using a loxP hTERT construct, that this increase in mtDNA damage following hydrogen peroxide exposure is dependent on the presence of hTERT itself. Further experiments using a dominant negative hTERT mutant shows that telomerase must be catalytically active to mediate the increase in mtDNA damage. Etoposide, but not methylmethanesulfate, also promotes mtDNA lesions in cells expressing active hTERT, indicating genotoxic specificity in this response. Fibroblasts expressing hTERT not only show a approximately 2-fold increase in mtDNA damage after oxidative stress but also suffer a 10-30-fold increase in apoptotic cell death as assayed by Annexin-V staining, caspase-3 activation and PARP cleavage. Mutations to the N-terminal mitochondrial leader sequence causes a complete loss of mitochondrial targeting without affecting catalytic activity. Cells carrying this mutated hTERT not only have significantly reduced levels of mtDNA damage following hydrogen peroxide treatment, but strikingly also do not shown any loss of viability or cell growth. Thus, localization of hTERT to the mitochondria renders cells more susceptible to oxidative stress-induced mtDNA damage and subsequent cell death, whereas nuclear-targeted hTERT, in the absence of mitochondrial localization, is associated with diminished mtDNA damage, increased cell survival and protection against cellular senescence.

Use of Telomerase to Create Bioengineered Tissues

Telomeres are repetitive DNA (TTAGGG) elements at the ends of chromosomes. Telomerase is a ribonucleoprotein complex that catalyzes the addition of telomeric sequences to the ends of chromosomes. The catalytic protein component of telomerase (hTERT) is expressed only in specific germ line cells, proliferative stem cells of renewal tissues, and cancer cells. The expression of hTERT in normal cells reconstitutes telomerase activity and circumvents the induction of senescence. Telomeres shorten with each cell division, eventually leading to senescence (aging), due to incomplete lagging DNA strand synthesis and end-processing events, and because telomerase activity is not detected in most somatic tissues. There are specific tissues and locations in which replicative senescence likely contributes to the decline in human physiological function with increased age and with chronic illnesses. While expressing hTERT in cells results in the maintenance of telomere length and greatly extended life span, blocking replicative aging systemically would be predicted to increase the potential for tumor formation. However, there are many situations in which the transient rejuvenation of cells could be beneficial. Ectopic expression of hTERT has been shown to immortalize human skin keratinocytes, dermal fibroblasts, muscle satellite (stem), and vascular endothelial, myometrial, retinal-pigmented, and breast epithelial cells. In addition, human bronchial, corneal and skin cells expressing hTERT can be used to form organotypic (3D) cultures (bioengineered tissues) that express differentiation-specific proteins, demonstrating that hTERT by itself does not alter normal physiology. The production of hTERT-engineered tissues offers the possibility of producing tissues to treat a variety of chronic diseases and age-related medical conditions that are due to telomere-based replicative senescence.

A balanced transcription between telomerase and the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 in resting, activated, HTLV-1-transformed and Tax-expressing human T lymphocytes

BackgroundThe functional state of human telomeres is controlled by telomerase and by a protein complex named shelterin, including the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 involved in telomere capping functions. The expression of hTERT, encoding the catalytic subunit of telomerase, plays a crucial role in the control of lymphocyte proliferation by maintaining telomere homeostasis. It has been previously found that hTERT activity is down-regulated by the human T cell leukaemia virus type 1 (HTLV-1) Tax protein in HTLV-1 transformed T lymphocytes. In this study, we have examined the effects of Tax expression on the transcriptional profile of telomerase and of shelterin in human T lymphocytes.ResultsWe first provide evidence that the up-regulation of hTERT transcription in activated CD4+ T lymphocytes is associated with a down-regulation of that of TERF1, TERF2 and POT1 genes. Next, the down-regulation of hTERT transcription by Tax in HTLV-1 transformed or in Tax-expressing T lymphocytes is found to correlate with a significant increase of TRF2 and/or Pot1 mRNAs. Finally, ectopic expression of hTERT in one HTLV-1 T cell line induces a marked decrease in the transcription of the POT1 gene. Collectively, these observations predict that the increased transcriptional expression of shelterin genes is minimizing the impact on telomere instability induced by the down-regulation of hTERT by Tax.ConclusionThese findings support the notion that Tax, telomerase and shelterin play a critical role in the proliferation of HTLV-1 transformed T lymphocytes.

Comparison of early passage, senescent and hTERT immortalized endothelial cells

The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far. Therefore, we have immortalized human umbilical vein endothelial cells (HUVECs) by hTERT overexpression and compared them to their normal early passage and senescent counterparts. This study, including a proteomic approach, shows that ectopic hTERT expression leads to a stable growing cell line. Although these cells are highly differentiated, the protein expression profile of the cell line is different to that of normal early passage and senescent cells.

Establishment of human fibroma cell lines from a MEN1 patient by introduction of either hTERT or SV40 early region

Establishment of tumor cell lines as model systems for studying tumor biology or as a part of immunotherapeutic anti-cancer strategies is of high importance, whereby the highest possible preservation of the original tumor cell phenotype is a prerequisite for these aims. Since overexpression of the catalytic subunit of human telomerase (hTERT) is known to minimally alter the cellular phenotype, we focused on the establishment of cell lines derived from human fibroma from a MEN1 patient by ectopic expression of hTERT. Additionally, a cell line was generated by introduction of the early region of SV40 (SV40 ER). Both approaches resulted in continuous cell lines, and neither T1-LOHG (hTERT) nor SV1-LOHG (SV40 ER) showed a transformed phenotype. While SV40 ER-transfected cells underwent dramatic changes in morphology and growth characteristics, hTERT-expressing cells indeed retained a phenotype highly similar to the parental cells. Nevertheless, hTERT overexpression resulted in increased growth rates after about 70 population doublings (PD) and alterations of mRNA levels of genes associated with tumor pathogenesis. Thus, our data suggest that ectopic hTERT expression leads to immortalization of LOHG-F, sustaining many characteristics of the non-transfected counterparts, but continuous growth in vitro is associated with changes of the cellular phenotype.

Heavy ion induced transformation of telomerase immortalized human small airway epithelial cells

The tumorigenic effects of galactic cosmic rays (GCR) have become a major concern to the members of the international space station program. Normal human small airway epithelial cells were immortalized by ectopic expression of hTERT. The hTERT-expressing cells were exposed to 56Fe ions which simulate GCR irradiation. The irradiated cells underwent a stepwise transformation process. They showed phenotypic alterations and loss of some chromosomes compared with control hTERT-expressing cells.

Telomerase Prolongs the Lifespan of Normal Human Ovarian Surface Epithelial Cells Without Inducing Neoplastic Phenotype

The aim of this study was to determine the effects of exogenous expression of the catalytic subunit of telomerase (hTERT) on the lifespan, growth characteristics, and tumorigenicity of normal human ovarian surface epithelial (OSE) cells. Low-passage primary cultures of normal human OSE cells were transfected with hTERT and the resulting cell lines were characterized. The ectopic expression of hTERT stabilized the telomeres of the OSE cultures above 8 kb. The hTERT-transfected OSE cell lines grew beyond the normal lifespan seen in OSE cells and propagated in culture for more than 40 passages before senescing. Moreover, the hTERT-transfected cells demonstrated extensive proliferative capacity as evidenced by their ability to continuously grow even when seeded at low dilutions. The morphologic features and normal differentiation patterns seen in normal OSE cells were likewise retained by the hTERT-transfected cells. In addition, the cultures remained responsive to physiologic concentrations of epidermal growth factor and transforming growth factor-beta. Changes associated with neoplastic transformation like anchorage-independent growth, tumorigencity and karyotypic instability were not observed. We were able to show that the ectopic expression of hTERT in normal human OSE: 1) resulted in cultures with greater growth potential and longer lifespan and 2) did not induce a transformed phenotype previously seen in viral oncogene-transfected OSE cells. The established cell lines would not only provide sufficient material for comprehensive studies to investigate the normal physiology of OSE cells, but could also help in the understanding of the early steps of ovarian carcinogenesis.

Immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase.

Human telomerase reverse transcriptase (hTERT) is catalytic subunit of human telomerase. We studied the immortalization of a series of human dental and periodontal cells by ectopic expression of hTERT and co-expression of hTERT with human papilloma virus 16 (HPV16) or simian virus 40 (SV40). Differentiation abilities of the established cell lines were studied in terms of the mineralized matrix formation and gene expression. We established immortalized gingival fibroblasts by hTERT, dental papilla and periodontal ligament cells by hTERT and HPV16, and pulp cells by hTERT and SV40. The papilla and pulp cells showed mineralization and dentin sialophosphoprotein (DSPP) expression when cultured in the presence of beta-glycerophosphate. The immortalized periodontal ligament cells did not show mineralization or DSPP expression, although expressions of alkaline phosphatase, osteopontin and osteocalcin were detected. These cell lines will be useful tools for studying the repair and regeneration of dental and periodontal tissues and various diseases including odontogenic tumors.

Generation and Characterization of Telomerase-Transfected Human Lymphatic Endothelial Cells with an Extended Life Span

The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin alpha9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tube-like structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro.