Telomerase Immortalization Research Articles

Developing Primary Culture from Human Cholangiocarcinoma

Background: Our goal is to isolate and expand tumor cells in culture and identify the spectrum of malignant attributes. Methods: Fresh tumor tissue from Grade 3, Stage IA, intrahepatic CCA was minced and subjected to enzymatic digestion for 75 minutes at 37°C. Digested contents were filtered through a 40 μm filter and the cells in the filtrate after washing were plated in 6 well collagen coated dishes using Williams E. Media supplemented with 2% FBS and several growth factors. One batch of cells was immortalized by pBABE hTERT retroviral vector. Results: Within 2-3 days, epithelial cells started propagating and cells grew up to 7 passages (P) over the course of two months both with (CCA-telo) or without (CCA) telomerase immortalization. Using qPCR analysis in P6 CCA/CCA-tel lines, expression profiles of liver cell specific markers like HP, ALB, APOA2, SERPINA1, and AFP were found to be very low,compared to HCC line HepG2 but exression of these was similar to lung adenocarcinoma A549 cell line. CCA line showed good telomerase expression however lower than CCA-telo line with exogenous telomerase induction. Conclusions: Cells isolated from human CCA show good viability and propagated for up to 7 passages. Though cells exhibited typical epithelial and mesenchymal morphology, the cells did not show liver epithelial markers. CCA line appears to be spontaneously immortalized, independent of exogenous hTERT induction. The gene expression profile of malignant tissue, if retained by primary culture cells, could facilitate the development and testing of novel molecular targets for cholangiocarcinoma.

Telomerase immortalization of human corneal endothelial cells yields functional hexagonal monolayers.

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness–the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.

Proteome-wide Dysregulation by PRA1 Depletion Delineates a Role of PRA1 in Lipid Transport and Cell Migration

We have previously identified prenylated Rab acceptor 1 (PRA1) as a novel cellular interacting partner for Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1). The intracellular trafficking and full signaling of LMP1 requires its interaction with PRA1. To further explore the role of PRA1 in Epstein-Barr virus-associated nasopharyngeal carcinoma (NPC) cells, we generated several PRA1-knockdown cell clones, which exhibited altered cell morphology and increased cell motility. We identified proteins differentially expressed in the knockdown clones by means of isobaric mass tags labeling coupled with multidimensional liquid chromatography-mass spectrometry. We validated a panel of proteins, which showed consistent up-regulation in PRA1-knockdown clones and participated in regulating lipid homeostasis and cell migration. Immunofluorescence staining further revealed altered localization of these proteins and accumulation of intracellular cholesterol in PRA1-knockdown clones. These effects were phenocopied by treatment with a cholesterol transport inhibitor, U18666A. Moreover, overexpressed PRA1 was able to alleviate the dysregulation of these affected proteins either from PRA1 knockdown or U18666A treatment, implying a role for PRA1 in regulating the levels of these affected proteins in response to altered cholesterol homeostasis. We further demonstrated that LMP1 expression caused PRA1 sequestration in NPC cells, leading to a consequence reminiscent of PRA1 knockdown. Finally, the immunohistochemistry showed a physiological relevance of the PRA1-associated proteome-wide changes in NPC biopsy tissues. In sum, our findings delineated novel roles of PRA1 in lipid transport and cell migration, and provided additional insights into the molecular basis of NPC morphogenesis, namely a consequence of LMP1-PRA1 interaction.

Telomerase immortalization of principal cells from mouse collecting duct

Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models.

Human Embryonic Stem Cell Telomere Length Impacts Directly on Clonal Progenitor Isolation Frequency

The pluripotentiality of human embryonic stem cells is expected to yield an abundance of clinically useful cell types. Using physiologic oxygen culture systems, we show that it is possible to isolate highly proliferative clonal progenitor cells from partially differentiated human embryonic stem cells. These progenitors have similar, though not identical, immunophenotypes with a resemblance to bone marrow-derived adherent stem cells. Through telomere length analysis of multiple early senescing clones, we were able to show that the starting telomere length of a human embryonic stem cell line impacts on the proliferative potential of clonally isolated partially differentiated mortal progeny. Proliferative clones undergo growth arrest with telomere lengths consistent with telomere-driven replicative senescence. To bypass this phenomenon, we transduced progenitor cells with ectopic hTERT (the limiting catalytic component of telomerase). This enabled telomerase immortalization without affecting differentiation potential or immunophenotype. In summary we describe the derivation of clonal progenitor cells from human embryonic stem cells and the relevance of parental cell telomere length to the frequency of highly proliferative clone isolation.

Retrovirally mediated telomerase immortalization of neural progenitor cells

Traditional methods of generating immortalized lines of both somatic cells and their progenitors have relied on the use of oncogenes. However, the resulting lines are typically anaplastic in vitro and tumorigenic in vivo, and hence of limited utility. The overexpression of telomerase, as mediated by the induced overexpression of human telomerase reverse transcriptase (hTERT), has permitted the generation of stable, non-oncogenic lines of a variety of cell types. This strategy for immortalization has found special utility in the central nervous system, as few stable lines are available for the study of either human neural progenitor cells, or of neurons or glia of restricted phenotype. We describe the use of retroviral hTERT overexpression for generating lines of immortalized human neural progenitor cells, whose neuronal progeny are phenotypically restricted, post-mitotic and functionally competent. Although we focus here on telomerase immortalization of spinal neural progenitors, this is a broadly applicable protocol for using hTERT to immortalize human fetal neural progenitors of any pre-selected phenotype and for characterizing the cell lines thereby generated.

Oxidative Stress Response in Telomerase‐Immortalized Fibroblasts from a Centenarian

It has been reported that cells with ectopic expression of telomerase are more resistant to apoptotic cell death than their normal counterpart. However, controversial results were obtained when the cellular response to oxidative stress was analyzed. The present research was therefore aimed at defining the effect of the oxidative stress induced by tert-butylhydroperoxide (tBOOH) and 2-deoxy-D-ribose (D-ribose) in human fibroblasts from a centenarian (cen3) and, in parallel, on the same cells after telomerase immortalization (cen3tel cells). By studying different parameters of apoptosis in situ (i.e., chromatin condensation, phosphatidylserine externalization, and DNA fragmentation), we found that both tBOOH and D-ribose induce apoptosis to a greater extent in cen3 than in cen3tel cells, suggesting a protective role of telomerase toward apoptotic death. However, monitoring the cell number during treatment with the drugs, we found a decrease in cell number; since this reduction was lower in cen3 fibroblasts compared to cen3tel cells, it is likely that telomerase does not fully protect cells from drug toxicity.

Telomerase immortalization of human mammary epithelial cells derived from a BRCA2 mutation carrier

A novel human mammary epithelial cell line, HME348, was established from benign breast tissue from a 44-year-old germ-line BRCA2 mutation carrier with a history of stage 1 breast cancer. Mutation analysis showed that the patient had a known 6872del4 BRCA2 heterozygous mutation. The human mammary epithelial cells passaged in culture exhibited cellular replicative aging as evidenced by telomere shortening, lack of telomerase activity, and senescence. Ectopic expression of telomerase (hTERT) reconstituted telomerase activity in these cells and led to the immortalization of the cells. When grown on glass, the majority of immortalized HME348 cells expressed ESA and p63 with a small population also expressing EMA. In three-dimensional Matrigel culture, HME348 cells formed complex branching acini structures that expressed luminal (EMA, CK18) and myoepithelial (p63, CALLA, CK14) markers. Three clones derived from this culture were also p63(+)/ESA(+)/EMA(+/-) on glass but formed similar acinar structures with both luminal and myoepithelial cell differentiation in Matrigel confirming the mammary progenitor nature of these cells. Additionally, the experimentally immortalized HME348 cells formed acini in cleared mammary fat pads in vivo. As this is the first report establishing and characterizing a benign human mammary epithelial cell line derived from a BRCA2 patient without the use of viral oncogenes, these cells may be useful for the study of BRCA2 function in breast morphogenesis and carcinogenesis.

Épidémiologie, histoire naturelle et pathogenèse du carcinome hépatocellulaire

Hepatocellular carcinoma is one of the most prevalent tumors worldwide and its incidence is increasing due to hepatitis C virus infection. Other etiologic factors are hepatitis B virus infection, alcoholic liver disease and hemochromatosis. This tumor mainly develops in cirrhotic livers that are true precancerous states. Although mechanisms of hepatocarcinogenesis remain badly known, some signaling pathways are frequently deregulated: inactivation of the p53 tumor suppressor factor in 25% of HCC, activation of the Wnt signaling and the telomerase immortalization enzyme in most of tumors. Hepatitis viruses play a direct oncogenic role by interaction between viral proteins and cellular ones, which control cell homeostasis, or by integration of hepatitis B virus genome into the host genome. Furthermore, hepatitis viruses play an indirect oncogenic role by chronic inflammation and hepatocyte regeneration related to viral hepatopathy. In a near future, a better understanding of virus-specific oncogenic mechanisms should allow us to set up innovative preventive and curative therapeutic strategies.

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20 J/m 2 UVC (LD50 = 5.6 J/m 2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21 CIP1/WAF1, and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6 h and from the non-transcribed strand was 73% complete in 24 h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.

Telomerase immortalization of neuronally restricted progenitor cells derived from the human fetal spinal cord.

Lineage-restricted progenitors of the central nervous system (CNS) are not readily expandable because their mitotic competence is limited. Here we used retroviral overexpression of human telomerase reverse transcriptase (hTERT) to immortalize progenitors from human fetal spinal cord. The hTERT-immortalized cells divided in basic fibroblast growth factor (bFGF) expressed high telomerase activity, and gave rise to phenotypically restricted subpopulations of either glia or neurons. The latter included a prototypic line, hSC11V-TERT, that gave rise only to neurons. These included both chx10(+) interneurons and Islet1(+)/Hb9(+)/ChAT(+) motor neurons; the latter were recognized by green fluorescent protein (GFP) driven by the Hb9 enhancer. The neurons were postmitotic and achieved electrophysiologic competence. Upon xenograft to both fetal rat brain and injured adult spinal cord, they matured as neurons and survived for 6 months, with no evident tumorigenesis. The cells have survived >168 doublings in vitro, with karyotypic normalcy and without replicative senescence. hTERT overexpression thus permits the generation of progenitor lines able to give rise to phenotypically restricted neurons.

Telomerase immortalization of human neural progenitor cells

It is necessary to expand human neural progenitor cells in vitro to obtain large numbers for research purposes and cell transplantation. A potential obstacle to in vitro expansion, however, is that neural progenitor cells have a limited replication life-span and gradually lose their differentiation potential. We report here that ectopic expression of the catalytic subunit of human telomerase (hTERT) gene in neural progenitor cells could induce telomerase activity, stabilize telomeres and extend their replicative life-spans. The telomerase-immortalized cells (hNPC-TERT) maintained the normal diploid karyotype, expressed the markers of human neural progenitor cells and meanwhile held the differentiation potential in vitro for up to 120 population doublings. This study provides a new approach for obtaining unlimited quantities of normal phenotypic and homogeneous human neural progenitor cells in vitro.

Telomerase immortalization of human myometrial cells.

Several strategies have been described for the primary culture of human myometrial cells. However, primary cultures of myometrial cells have a limited life span, making continual tissue acquisition and cell isolation necessary. Recent studies have demonstrated that cell culture life span is related to chromosomal telomere length, and cellular senescence results from progressive telomere shortening and the lack of telomerase expression. Transfection of cells with expression vectors containing the human telomerase reverse transcriptase (hTERT) maintains telomere length and effectively gives normal cells an unlimited life span in culture. In addition, hTERT extends the life span of cultured cells far beyond normal senescence without causing neoplastic transformation. In the present study, we developed a cell line from hTERT-infected myometrial cells (hTERT-HM). Cells were isolated from myometrial tissue obtained from women undergoing hysterectomy, and retroviral infection was used to express the catalytic subunit of telomerase in myometrial cells. Cells expressing hTERT have been in continuous culture for >10 mo, whereas the control culture senesced after approximately 2 mo. Telomerase activity was monitored in cells with a polymerase chain reaction-based telomerase activity assay. Telomerase-expressing cells contained mRNA for alpha smooth muscle actin, smoothelin, oxytocin receptor, and estrogen receptor alpha, but the estrogen receptor beta receptor was lost. Immunoblotting analysis identified the expression of calponin, caldesmon, alpha smooth muscle actin, and oxytocin receptor. Although estrogen receptor expression was below the level of detection with immunoblotting, transfection experiments performed with reporter constructs driven by estrogen response elements demonstrated estrogen responsiveness in the hTERT-HM. In addition, treatment of hTERT-HM with oxytocin caused a concentration-dependent increase in intracellular calcium levels, confirming the presence of functional oxytocin receptors. Myometrial cells immortalized with hTERT retained markers of differentiation that are observed in primary cultures of smooth muscle cells. The expression of various smooth muscle/myometrium cell markers suggests that these cells may be an appropriate model system to study certain aspects of human myometrial function.

Human telomerase reverse transcriptase-immortalized MRC-5 and HCA2 human fibroblasts are fully permissive for human cytomegalovirus.

MRC-5 cells are a well-characterized human diploid fibroblast cell line approved for vaccine production and favoured for the routine propagation of human cytomegalovirus (HCMV). Ectopic expression of telomerase in fibroblasts is capable of overcoming replicative senescence induced by telomere shortening. Following delivery of the hTERT gene to MRC-5 cells using a retrovirus vector three clones were generated that (i) expressed functional telomerase activity, (ii) exhibited telomere extension and (iii) were sustained for >100 population doublings. Immortalized MRC-5-hTERT and also HCA2-hTERT human fibroblasts were both fully permissive for HCMV as determined by plaque assay, studies of virus growth kinetics and measurement of virus yields. Furthermore, telomerase-immortalized HCA2 cells proved capable of supporting the stable maintenance of an EBV-based episomal vector with efficient transgene expression when driven by the HCMV immediate early promoter. An indicator cell line suitable for the efficient detection of HCMV infection was also generated using an episome containing a reporter gene (lacZ) under the control of the HCMV beta-2.7 early promoter. Telomerase immortalization of human fibroblasts will thus facilitate the growth and detection of HCMV and also the generation of helper cell lines for the propagation of HCMV deletion mutants. Immortalization of fibroblasts by telomerase does not affect cell morphology or growth characteristics. The MRC-5-hTERT clones may therefore be suitable for additional applications in virology, cell biology, vaccine production and biotechnology.