Human Telomerase Catalytic Subunit Research Articles

Cloning of human telomerase catalytic subunit gene promoter and studying on its specific transcriptional activities in human lung cancer cells

To clone DNA sequence of human telomerase reverse transcriptase (hTERT) promoter, and study its transcriptional activities in various human lung cancer cells and normal lung cell. hTERT promoter of 1.1 kb was amplified with polymerase chain reaction (PCR) method, utilizing DNA of 293 cell as template. After DNA sequencing with correct result, the hTERT promoter was inserted into luci-ferase reporter vectors (pGL3-Basic) to reconstruct a recombinant plasmid named pGL3-hTERTp. Then the reconstructed plasmid was transiently transfected into lung cancer cell lines A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC, GLC-82, A2 and human embryonic pulmonary fibroblast cell line MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection. Electrophoresis demonstrated that cloned hTERT promoter was about 1.1 kb, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between 1 126 bp and 43 bp in upstream of the transcription start site ATG and the length was 1 086 bp. The recombinant plasmid pGL3-hTERTp was confirmed by double digestion and PCR method with correct results. Luciferase assay showed there were different transcriptional activities of hTERTp in various lung cancer cell lines, but not in the MRC-5 cell line. The hTERT promoter cloned in this study has transcriptional activities in various lung cancer cell lines but not in normal cell. It may act as control element in tumor-targeting gene therapy with hTERT.

The hTERT Gene Is Embedded in a Nuclease-resistant Chromatin Domain

Normal human cells rarely undergo spontaneous immortalization. Given that ectopic expression of the human telomerase catalytic subunit hTERT leads to cellular immortalization, the endogenous hTERT gene is likely constitutively repressed. Hence, we have examined the chromatin structure of the native hTERT locus and the neighboring loci, CRR9 and Xtrp2, in normal human fibroblasts and a set of immortal lines. Using generalized DNase I sensitivity assays, we revealed that the entire hTERT gene was embedded in a chromatin domain that was as resistant to the nuclease as the well studied beta-globin loci in both telomerase-positive and -negative cells. This condensed domain was at least 100 kb in size and contained the intergenic region 5' to the hTERT gene and the downstream Xtrp2 locus. A transition from the nuclease-sensitive CRR9 locus to the condensed region appeared near the 3'-end of the CRR9 gene. hTERT transcription was associated with the appearance of a major DNase I-hypersensitive site positioned around the hTERT transcription start site and several minor hypersensitive sites. In telomerase-negative cells, the inhibition of histone deacetylases by trichostatin A led to the opening of this chromatin domain, accompanied by transcription from the hTERT gene but not the Xtrp2 gene. In contrast, the inhibition of protein synthesis by cycloheximide induced transcription from both the hTERT and Xtrp2 genes, indicating that histone deacetylases and labile factors coordinate to silence this chromosomal region. Taken together, our data suggest a novel mechanism of hTERT regulation at the chromatin level and have important implications for studying telomerase expression.

Detection of Human Telomerase Reverse Transcriptase (hTERT) Protein Expression in Bone Marrow Cells of Hematological Disorder by Flow Cytometry.

Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. Recently, it has been reported that telomerase activity level was correlated with the progression and prognosis of hematopoietic malignancies, thus, it is important to analyze telomerase activity. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and unable to isolate specific cell sub-populations. We developed immuno-fluorescence flow cytometry based assay to detect hTERT expression using a monoclonal antibody. This method allows sub-population of cells to be separated according to hTERT expression more easily, combining antibody against cell surface antigen. Expression level of hTERT in T cell leukemia cell line Jurkat and myeloma cell line RPMI8226, KMS12PE, KMS28PE, were very high and well correlated in between this method and real time quantitative PCR. Human mature granulocytes supposed to have no telomerase activity did not show any hTERT expression in this flow-cytometric assay. Immuno-histochemistry demonstrated the specific nuclear expression of hTERT. Bone marrow samples were obtained from 8 MDS patients (5 RA, 1 RAEB, 2 RAEB-t) and analyzed hTERT isolating MDS blast cells. In the blast population, cells expressing hTERT were 33.1% (21.4–40.3) in RA patient sample, while those in RAEB and RAEB-t patients sample were significantly higher at 75.1% (68.2–81.9). When CD34 positive hematopoietic stem cells were analyzed, the cells expressing hTERT were higher in RAEB and RAEB-t than in RA. Mature granulocyte population in bone marrow cells did not show any positivity. Our result suggests that hTERT expression and telomerase activity in the MDS blasts or CD34 positive stem cells were up-regulated during the disease progression and the high expression level of hTERT and telomerase activity were not due to the blast expansion. This technique allows us to easily analyze hTERT expression in hematopoietic malignancy even though the malignant cells were very small in bone marrow sample, e.g. MDS, MM or MGUS of which telomerase activity were supposed to be correlated with the disease progression and prognosis.

Ex Vivo Production of Transfusable Red Blood Cells at Large Scale from Cord Blood CD34+ Cells Using a Coculture System with Human Telomerase Catalytic Subunit (hTERT)-Transfected Human Stromal Cells at Early Phase and with Acitivated Macrophage from Cord Blood CD34+ Cells at Late Phase.

New sources of red blood cells (RBC) would improve the transfusion capacity of blood centers. Several investigators have previously reported that erythroblasts could be obtained from hematopoietic stem cells including those of cord blood (CB) by in vitro culture. However, transfusion of erythroblasts may not be suitable for supplementation of acute blood loss because it should need some time lag until hemoglobin (RBC) boost in circulation due to the fact that transfused erythroblasts once lodged at bone marrow where they undergo maturation into RBCs which are bound to be released into circulation. We have developed a culture system for producing large quantity of enucleated RBCs (e-RBCs) as well as erythroblasts from CB in vitro: one unit e-RBCs (2 x 1012 RBCs) was obtained from one standard CB unit (corresponding to 2 x 106 CD34+ cells) using a coculture system with hTERT-transfected human stromal cells at early phase followed by with activated macrophage in liquid culture (American Society of Hematology 45th Annual Meeting, SanDiego, 2003). In the present study, we first analyzed the function of those manufactured e-RBCs in comparison of that of adult peripheral blood RBCs (PB-RBCs) or that of eryhthroblasts. The hemoglobin (Hb) content of the e-RBCs quantified by photometric determination was almost equivalent to that of adult PBRBC. A Hb A/Hb F ratio of e-RBC analyzed by high-performance liquid chromatography (HbA: HbF = 35: 65) was between those of CB RBCs (10: 90) and adult PB-RBC (99: 1). Oxygen dissociation curves of e-RBCs measured by Hemox-Analyzer was comparable to that of fresh adult PB-RBCs. The erythroblasts showed adhesive property to stromal cells in vitro but e-RBC did not. When we injected e-RBCs into NOD/SCID mice, they were detectable in circulation while erythroblasts were not. In conclusion, the e-RBCs produced by large-scale culturing system from CB CD34+ cells may be useful for acute blood loss.

Effect of daidzein on cell growth, cell cycle, and telomerase activity of human cervical cancerin vitro

Phytoestrogens are some plant compounds exhibiting estrogen-like activities. However, some studies have shown that they also affect the growth of some nonhormone-dependent diseases. In this study, daidzein – one of the most common phytoestrogens – was used to investigate its effects on human cervical cancer cells HeLain vitro. First, the cell growth was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Then, the distributions of cell cycle and apoptosis were analyzed with the help of flow cytometry. Finally, the telomerase activity was detected by using real-time quantitative reverse transcription-polymerase chain reaction. The results showed that at the concentrations from 6.25 to 100 μmol/l, daidzein inhibited the growth of HeLa cells. Flow cytometric analysis showed that cancer cells were arrested at G0/ G1or G2/ M phase with daidzein. The inductive effects of apoptosis were more obviously observed in low-concentration groups. After HeLa cells were treated with daidzein, the expression of human telomerase catalytic subunit mRNA decreased. These meant that daidzein affected human nonhormone-dependent cervical cancer cells in several ways, including cell growth, cell cycle, and telomerase activityin vitro.

Effect of daidzein on cell growth, cell cycle, and telomerase activity of human cervical cancer in vitro.

Phytoestrogens are some plant compounds exhibiting estrogen-like activities. However, some studies have shown that they also affect the growth of some nonhormone-dependent diseases. In this study, daidzein--one of the most common phytoestrogens--was used to investigate its effects on human cervical cancer cells HeLa in vitro. First, the cell growth was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Then, the distributions of cell cycle and apoptosis were analyzed with the help of flow cytometry. Finally, the telomerase activity was detected by using real-time quantitative reverse transcription-polymerase chain reaction. The results showed that at the concentrations from 6.25 to 100 micro mol/l, daidzein inhibited the growth of HeLa cells. Flow cytometric analysis showed that cancer cells were arrested at G(0)/G(1) or G(2)/M phase with daidzein. The inductive effects of apoptosis were more obviously observed in low-concentration groups. After HeLa cells were treated with daidzein, the expression of human telomerase catalytic subunit mRNA decreased. These meant that daidzein affected human nonhormone-dependent cervical cancer cells in several ways, including cell growth, cell cycle, and telomerase activity in vitro.

Bisphenol A from dental polycarbonate crown upregulates the expression of hTERT.

Bisphenol A (BPA) is one of the endocrine-disrupting chemicals (EDCs) that possess estrogen-like biologic activity. Many dental materials have been reported to release BPA. However, there are few reports available on the release of BPA from dental polycarbonates. The purpose of this study was to investigate the release of BPA from dental polycarbonate crowns and to evaluate the estrogenic activity of BPA. Polycarbonate crowns were immersed in five solvents (water, ethanol, n-hepthane, acetic acid, and acetonitrile) at 37 or 65 degrees C for 24 h. The elution from the material was analyzed by high-performance liquid-chromatography (HPLC) and mass-spectrometry (MS) analysis. BPA release was detected corresponding to the degradation of dental polycarbonates under the some storage conditions (ethanol, acetic acid, and acetonitrile). A previous report proved that estrogen increased human telomerase catalytic subunit (hTERT) mRNA, whereas the effect of EDCs on the hTERT promoter has never been reported. The estrogenic activity of BPA was analyzed by luciferase assay with the use of the hTERT promoter. This assay revealed that BPA was a positive regulator of hTERT transcription. In addition, quantitative real-time PCR analysis showed that BPA increased the expression level of hTERT mRNA in MCF7 cells. Herein, it is demonstrated that hTERT is a new target of BPA.

Use of exogenous hTERT to immortalize primary human cells

A major obstacle to the immortalization of primary human cells and the establishment of human cell lines is telomere-controlled senescence. Telomere-controlled senescence is caused by the shortening of telomeres that occurs each time somatic human cells divide. The enzyme telomerase can prevent the erosion of telomeres and block the onset of telomere-controlled senescence, but its expression is restricted to the early stages of embryonic development, and in the adult, to rare cells of the blood, skin and digestive track. However, we and others have shown that the transfer of an exogenous hTERT cDNA, encoding the catalytic subunit of human telomerase, can be used to prevent telomere shortening, overcome telomere-controlled senescence, and immortalize primary human cells. Most importantly, hTERT alone can immortalize cells without causing cancer-associated changes or altering phenotypic properties. Primary human cells that have so far been established by the forced expression of hTERT alone include fibroblasts, retinal pigmented epithelial cells, endothelial cells, oesophageal squamous cells, mammary epithelial cells, keratinocytes, osteoblasts, and Nestin-positive cells of the pancreas. In this article, we discuss the use of hTERT to immortalize of human cells, the properties of hTERT-immortalized cells, and their applications to cancer research and tissue engineering.

The evidences of human Orphan Receptor COUP-TFII inhibiting telomerase activity through decreasing hTERT transcription

Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells, but the details of regulation of hTERT need to be elucidated. By screening HeLa cDNA library with hTERT promoter-based yeast one-hybrid assay, one of positive clones, which potentially interacted with hTERT promoter, contained the full sequences of COUP-TFII cDNA. EMSA showed that the prepared His-tagged COUP-TFII could firmly bind to −201 to +35 fragment of hTERT promoter. The precise binding sites were confirmed by DNase I footprint analysis and proved to involve the E-box motif of hTERT promoter. Luciferase reporter assays indicated that COUP-TFII could suppress the transcription of hTERT promoter, and the inhibition to some extent could be reversed by co-transfection of c-myc. Stable introduction of COUP-TFII into HeLa cells decreased both endogenous hTERT expression and telomerase activity. The results suggested that the human COUP-TFII could specifically interact with hTERT promoter, in part via the E-box, and suppress the expression of hTERT.

The Establishment of Telomerase-immortalized Tangier Disease Cell Lines Indicates the Existence of an Apolipoprotein A-I-inducible but ABCA1-independent Cholesterol Efflux Pathway

Tangier disease (TD) is a human genetic disorder associated with defective apolipoprotein-I-induced lipid efflux and increased atherosclerotic susceptibility. It has been linked to mutations in the ATP-binding cassette protein A1 (ABCA1). Here we describe the establishment of permanent Tangier cell lines using telomerase. Ectopic expression of the catalytic subunit of human telomerase extended the life span of control and TD skin fibroblasts, and (in contrast to immortalization procedures using viral oncogenes) did not impair apolipoprotein A-I-induced lipid efflux. The key characteristics of TD fibroblasts (reduced cholesterol and phospholipid efflux) were observed both in primary and telomerase-immortalized fibroblasts from two unrelated homozygous patients. Surprisingly, the apolipoprotein-inducible cholesterol efflux in TD cells was significantly improved after immortalization (up to 40% of normal values). In contrast to ABCA1-dependent cholesterol efflux, this efflux was not inhibited by brefeldin A, glybenclamide, or intracellular ATP depletion but was inhibited in the presence of cytochalasin D. Apolipoprotein A-I-dependent cholesterol efflux was inversely correlated with the population doubling number in cell culture and was inhibited up to 40% in near-senescent normal diploid fibroblasts. This inhibition was completely reversed by telomerase. Thus ectopic expression of telomerase is a way to circumvent the lack of critical experimental material and represents a major improvement for studying cholesterol efflux pathways in lipid disorders. Our findings indicate the existence of an ABCA1-independent but cytoskeleton-dependent cholesterol removal pathway that may help to prevent early atherosclerosis in Tangier disease but may also be sensitive to aging phenomena ex vivo and possibly in vivo.

Role of telomerase in chrysotile induced malignant transformation of normal human embryonic lung fibroblasts

To explore the role of telomerase in asbestos dust induced malignant transformation of human embryonic lung fibroblasts in vitro. Human telomerase catalytic subunit (hTERT) was transferred into human embryonic lung fibroblasts (HELF). Chrysotile dust at concentration of 2.5 microg/cm(2) was added to HELF transduced with and without hTERT (HELF-T+), respectively, and their transduced foci were separated. Biological characteristics of the cells, telomerase activity, length of telomere and cell growth curve were observed. Colony forming test was performed on soft agar to evaluate the nature of transformation. The hTERT gene was transferred into HELF steadily, and HELF-T+ was established. Malignant transformation occurred in both HELF and HELF-T+ by asbestos stimulation. Asbestos dusts could induce higher rate of transformations in HELF-T+ [(2.08 +/- 1.08)/utensil] than in HELF [(1.08 +/- 0.10)/utensil], P < 0.05. Telomerase activity in both transformed malignant cells and HELF-T+ was higher, as well as the longer length of telomere in them. Rate of malignant transformation in cells with more activity of telomerase and longer length of the telomere was higher after stimulation with asbestos, indicating telomerase could play an important role in asbestos induced human cells malignant transformation.

Establishment and characterization of a goat synovial membrane cell line susceptible to small ruminant lentivirus infection

Primary goat synovial membrane (GSM) cells are widely used to study small ruminant lentiviruses (SRLV), i.e. maedi visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV), but their limited life-span of 15–20 passages in vitro is problematic. Here, we report that ectopic expression of the catalytic subunit of human telomerase (hTERT) was sufficient to immortalize primary GSM cells. Cultures of hTERT-transfected GSM cells have been passaged for 2 years without showing any phenotypic difference from the original primary GSM cells. The hTERT-transfected cells continued to grow beyond a population doubling number of 250, while no net telomere lengthening was observed for these cells. Moreover, the immortalized GSM cells were susceptible to infection by both CAEV and MVV and were able to propagate theses viruses. Such cell line provides a useful source of standard and robust cells for both research and veterinary purposes.

Reduction in mortalin level by its antisense expression causes senescence‐like growth arrest in human immortalized cells

Human normal cells have active p53 and pRB tumor suppressor pathways and undergo telomere shortening at each cell division. When immortalized, these exhibit functional inactivation of one or both tumor suppressor pathways and activation of telomere-maintaining mechanisms. Regulation of immortalization promoting molecular pathways by other genes is poorly understood and is an essential component of cancer therapeutics. In the present study, we have immortalized human normal cells by functional inactivation of p53 and pRB tumor suppressor proteins and simultaneous activation of telomerase function. We demonstrate that when the expression of mortalin (a member of hsp70 family proteins) was suppressed in such genetically defined immortal cells they underwent a permanent growth arrest. WI-38 cells were transfected with expression plasmids encoding papilloma virus E6 and E7 proteins and a catalytic subunit of human telomerase enzyme. The derivative cells were compromised for p53 and pRB senescence pathways, showed telomerase activity and were immortalized. These cells were transfected with antisense expression plasmid for mortalin. The derivative clones were analyzed for mortalin expression, proliferation and telomerase activity. WI-38 cells were immortalized with E6, E7 and the catalytic subunit of human telomerase. The derivative-immortalized cells when suppressed for mortalin expression underwent senescence-like growth arrest. The data demonstrate that suppression of mortalin is sufficient to induce growth arrest in human immortalized cells that have compromised p53 and pRB functions and possess telomerase activity.

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.

Stress-induced Premature Senescence in hTERT-expressing Ataxia Telangiectasia Fibroblasts

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.

Telomerase-mediated lifespan extension of human bronchial cells does not affect hexavalent chromium-induced cytotoxicity or genotoxicity.

Hexavalent chromium (Cr(VI)) is a metal of increasing public health concern, as exposure to it is widespread and it is a well-established cause of human bronchial carcinomas and fibrosarcomas. The water-insoluble Cr(VI) salts are potent carcinogens compared to the water soluble salts; yet the genotoxic mechanisms of both may be mediated by soluble Cr(VI) ions. Currently, these mechanisms are poorly understood. Emerging evidence suggests that initial cell culture models used to study the general toxicity of Cr(VI) may be suboptimal for investigating mechanisms specific to human bronchial cells. Accordingly, we have developed a new model system of human bronchial cells by introducing hTERT, the catalytic subunit of human telomerase, into primary human bronchial fibroblasts (PHBF). We have isolated a stable, clonally derived cell line, WHTBF-6, that demonstrate reconstitution of telomerase activity and maintenance of telomere lengths with increasing culture age. WHTBF-6 has been characterized as having an extended in vitro lifespan, a normal growth rate, a normal diploid karyotype that is maintained over time, and exhibits serum-dependent contact-inhibited anchorage-dependent growth. Moreover, we find that both particulate and soluble hexavalent chromium induce a pattern and degree of cytotoxicity and clastogenicity in WHTBF-6 that is similar to the parental PHBF cells. Because telomerase does not compromise growth or the response to Cr(VI), our results indicate that this is an excellent system for studying the mechanisms of Cr(VI) and potentially other carcinogens implicated in the development of lung cancer.

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer.

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.

Antisense oligonucleotide targeting at the initiator of hTERT arrests growth of hepatoma cells.

To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells. The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with as-hTERT at the concentration of 10 micromol/L. After 72 h, these cells were obtained for detecting growth inhibition, telomerase activity using the methods of MTT, TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline. Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo. Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570-A630 was found between cells treated with as-hTERT and control (P<0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced, the value of A450 nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro. As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.

Immortalization of human preadipocytes

Primary human preadipocytes in culture are characterized by a low proliferative capacity associated with a rapid decline of differentiation ability during subculturing; thereby limiting their use as cellular model. Cellular immortalization constitutes an interesting approach for establishing cell lines presenting an unlimited life span and a maintained differentiation capacity. Different procedures for developing immortalized human preadipocytes are discussed in this review. Transformation of human preadipocytes with the simian virus 40 large T-antigen (SV40 T-Ag) permitted the development of immortalized cells; however these cells could not maintain their capacity to differentiate into adipocytes. This limitation may be explained by the ability of SV40 T-Ag to inhibit transcriptional factors involved in the differentiation of preadipocyte. Reconstitution of the telomerase activity by stable expression of the hTERT (human telomerase catalytic subunit) gene was able to partially extend the lifespan of primary preadipocytes but not to promote cellular immortalization. However, a combined expression of hTERT and the E7 oncoprotein of human papillomavirus type 16, generated human preadipocytes with both an unlimited life span and a preserved adipogenic potential. This approach appears to be an effective method for establishing human preadipose cell lines for studying adipocyte differentiation and metabolism.

Inhibition of the Epidermal Growth Factor Receptor Suppresses Telomerase Activity in HSC-1 Human Cutaneous Squamous Cell Carcinoma Cells

Activation of telomerase, which stabilizes the telomere length of chromosomes, is crucial for the continued growth or progression of cancer cells. In a previous study, we showed that telomerase is frequently activated in skin tumors. Because epidermal growth factor plays an important role during the tumorigenesis of epithelial tissue, we have now examined the role of epidermal growth factor signaling in regulating telomerase activity using HSC-1 human cutaneous squamous cell carcinoma cells. Treatment of HSC-1 cells with AG 1478, an inhibitor of the epidermal growth factor receptor, or with a neutralizing antibody to the epidermal growth factor receptor, significantly suppressed their telomerase activity, in association with inhibiting their growth. The suppression of telomerase activity was obvious at day 3 and was maximal at day 5 after treatment with AG 1478. The suppression of telomerase activity correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. The expression of c-Myc and of Sp1 proteins, transcription factors for hTERT, were also suppressed by AG 1478 in HSC-1 cells, but the expression of Ets-2 protein, another transcription factor, was not affected. The expression of Mad-1, a competitor of c-Myc, was increased. Inhibition of ERK, Src, or Akt suppressed telomerase activity in HSC-1 cells, but to a lesser extent than did treatment with AG 1478. Serum starvation suppressed telomerase activity, but addition of epidermal growth factor or transforming growth factor alpha did not increase it, indicating the involvement of other epidermal growth factor receptor ligands in the activation of telomerase in HSC-1 cells. These data indicate that blockade of the epidermal growth factor receptor might be effective in inhibiting telomerase activity of squamous cell carcinomas, which would lead to the suppression of tumor growth.