Bone marrow-derived mesenchymal stem cells migrate to healthy and damaged salivary glands following stem cell infusion / 国际口腔科学杂志·英文版

Xerostomia is a severe side effect of radiation therapy in head and neck cancer patients. To date, no satisfactory treatment option has been established. Because mesenchymal stem cells (MSCs) have been identified as a potential treatment modality, we aimed to evaluate stem cell distribution following intravenous and intraglandular injections using a surgical model of salivary gland damage and to analyse the effects of MSC injections on the recruitment of immune cells. The submandibular gland ducts of rats were surgically ligated. Syngeneic adult MSCs were isolated, immortalised by simian virus 40 (SV40) large T antigen and characterized by flow cytometry. MSCs were injected intravenously and intraglandularly. After 1, 3 and 7 days, the organs of interest were analysed for stem cell recruitment. Inflammation was analysed by immunohistochemical staining. We were able to demonstrate that, after intravenous injection, MSCs were recruited to normal and damaged submandibular glands on days 1, 3 and 7. Unexpectedly, stem cells were recruited to ligated and non-ligated glands in a comparable manner. After intraglandular injection of MSCs into ligated glands, the presence of MSCs, leucocytes and macrophages was enhanced, compared to intravenous injection of stem cells. Our data suggest that injected MSCs were retained within the inflamed glands, could become activated and subsequently recruited leucocytes to the sites of tissue damage.

Well-Differentiated Papillary Mesothelioma of the Tunica Vaginalis: A Case Study and Review of the Literature

Well-differentiated papillary mesothelioma is an uncommon tumor of the testes that usually presents as a hydrocele. Here, we present the case of one patient who did not have a history of asbestos exposure. The tumor was localized in the tunica vaginalis and was composed of three pedunculated masses macroscopically. Microscopically, branching papillary structures with focal coagulative necrosis were present. In addition to immunohistochemistry, simian virus 40 DNA was also tested by polymerase chain reaction. This report presents one case of this rare entity, its clinical and macroscopic features, and follow-up results.

No Detection of Simian Virus 40 in Malignant Mesothelioma in Korea

BACKGROUND: Simian virus 40 (SV40), a polyomavirus, was discovered as a contaminant of a human polio vaccine in the 1960s. It is known that malignant mesothelioma (MM) is associated with SV40, and that the virus works as a cofactor to the carcinogenetic effects of asbestos. However, the reports about the correlation between SV40 and MM have not been consistent. The purpose of this study is to identify SV40 in MM tissue in Korea through detection of SV40 protein and DNA. METHODS: We analyzed 62 cases of available paraffin-blocks enrolled through the Korean Malignant Mesothelioma Surveillance System and performed immunohistochemistry for SV40 protein and real-time polymerase chain reaction (PCR) for SV40 DNA. RESULTS: Of 62 total cases, 40 had disease involving the pleura (64.5%), and 29 (46.8%) were found to be of the epithelioid subtype. Immunostaining demonstrated that all examined tissues were negative for SV40 protein. Sufficient DNA was extracted for real-time PCR analysis from 36 cases. Quantitative PCR of these samples showed no increase in SV40 transcript compared to the negative controls. CONCLUSIONS: SV40 is not associated with the development of MM in Korea.

Detection of Helicobacter pylori in gastric biopsies, saliva and dental plaques of dyspeptic patients from Marília, São Paulo, Brazil: presence of vacA and cagA genes

Helicobacter pylori, a gram-negative bacterium, possesses two important virulence factors: the vacuolating toxin (vacA), and the cytotoxin-associated gene product (cagA). The aim of the present study was to evaluate the presence of H. pylori in the stomach and oral cavity of humans and compare the cagA and vacA genotypes of H. pylori found in different samples (stomach, saliva and dental plaque) from the same patient. Gastric biopsies, saliva and dental plaques were obtained from 62 dyspeptic adults. DNA was extracted and evaluated for the presence of H. pylori and the alleles cagA and vacA. Persons with gastritis had a higher frequency of H. pylori -positive samples in the stomach while positive samples from gastric biopsies were significantly correlated with those from the oral cavity. There was a high H. pylori frequency in patients while the cagA gene was associated with vacA s1 alleles in gastric biopsies. Our results suggest a reservoir of the species in the oral cavity and that, in one patient, more than one H. pylori strain may exist in the saliva, dental plaque and stomach. We found a relationship between gastric infection and the bacterium in the oral cavity, with the cytotoxin genotype varying between saliva and dental plaque.(AU)

Immortalization of the SD rats' dental follicle cell with simian virus 40 large tumor antigen gene / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct SD rat immortalized dental follicle cells (rDFC) induced by simian virus 40 large tumor antigen (SV40Tag) gene to provide a reliable cell source for periodontal tissue engineering research.</p><p><b>METHODS</b>The rDFC was isolated by tissue mass method combined with enzyme digestion method and evaluated by immunohistochemistry. Cell293 were transfected with plasmid pSSR69/pAmpho containing SV40Tag gene by mediating liposome. Normal rDFC were infected with virus-contained supernate and the successfully transfected cell lines were screened with hygromycin, and positive clones were cultured. While non-transfected cells served as negative controls, the cell morphology was observed, the proliferation characteristics was evaluated by calculating cell population. The expression of SV40Tag gene and telomerase in cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. The biological property of immortalized rDFC was assessed with calculating formation rate of flat cloning, soft agar colony formation test and tumor-forming test.</p><p><b>RESULTS</b>Morphology of immortalized rDFC was not different from that of normal rDFC. The RT-PCR results of SV40Tag revealed amplification band at 357 bp, while no band was seen in the normal cells. The expression of telomerase in immortalized rDFC was higher than that in normal rDFC. The two groups had no significant difference in growth curves, but the immortalized rDFC exhibited stronger proliferative activity. No significant differences of formation rate in flat cloning were observed between the immortalized rDFC [34% (33/96)] and normal rDFC at passage four [22% (21/96)] (χ(2) = 3.71, P > 0.05). No cell cloning was seen in soft agar and the tumor formation was not observed in nude mice.</p><p><b>CONCLUSIONS</b>The rDFC induced by SV40Tag gene could be cultured and passaged in vitro, which retained the stable proliferation and differentiation characteristics and could be used for periodontal tissue engineering research.</p>

Detection of SV40 Large T Antigen in Malignant Lymphomas

BACKGROUND: The association of simian virus 40 (SV40) with certain types of human cancers, including malignant lymphomas, has been a topic of interest for some time. Although the virus is distributed worldwide, its incidences vary according to the specific types of tumors, and the epidemiological areas. The aim of this study was to investigate the frequency of SV40 in malignant lymphomas among Korean patients. METHODS: One hundred seventy three cases of malignant lymphomas were evaluated by immunohistochemical staining for SV40 large T antigen (TAg), using an extremely sensitive, tyramide based, catalyzed signal amplification method. RESULTS: From 158 non-Hodgkin's lymphomas, including 115 diffuse large B-cell lymphomas, and 15 Hodgkin's lymphomas, none of the cases were positive for SV40 TAg. CONCLUSIONS: SV40 does not appear to be related to the pathogenesis of malignant lymphomas among Koreans.

Effect of simian vacuolating virus 40 on development and differentiation of dendritic cells from Rhesus macaque / 病毒学报

To study the effect of simian vacuolating virus 40 (SV40) on development and differentiation of dendritic cells (DC) from rhesus macaque, the peripheral blood-derived dendritic cells from rhesus monkey were pulsed with inactivated SV40 and infective SV40, respectively at the 5th day post DC cultivation. Expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface at the 7th, 9th day post DC cultivation were analyzed by flow cytometry (FCM). The results showed that expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface in the inactivated SV40-pulsed experimental group were higher than those in the infective SV40-pulsed experimental group (P < 0.05). These cell surface molecules represented characteristic development and differentiation phase of DC. Down-regulation of expressions of these cell surface molecules indicated that infective SV40 might hamper differentiation and maturation of dendritic cells from rhesus monkey.

Immortalization of human precartilaginous stem cells by transfecting SV40Tag / 华中科技大学学报（医学）（英德文版）

Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells (IPSCs), which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.

Enhancement of DNA vaccine-induced immune responses by a 72-bp element from SV40 enhancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although DNA vaccine is considered as the next generation of vaccine, most DNA vaccine candidates are still suffering from the relatively weak immunogenicity despite the increased dosage of plasmid DNA administered. In order to enhance the immune responses elicited by a codon-optimized HIV gag DNA vaccine, a modified plasmid vector pDRVI1.0 and a booster immunization with replicating Tiantan vaccinia (RTV) strain expressing the same gene were employed.</p><p><b>METHODS</b>Vector pDRVI1.0 was constructed through inserting the 72-bp element from the SV40 enhancer, which was reported promoting nuclear transport of plasmid DNA, to the upstream of cytomegalovirus enhancer/promoter region of the plasmid vector pVR1012. Gene expression levels from expression plasmids based on pDRVI1.0 and pVR1012 were tested. Humoral and cellular immune responses induced by DNA vaccine alone or DNA prime-RTV boost regimen were determined in mice.</p><p><b>RESULTS</b>It was shown that the 72-bp element significantly enhanced the gene expression level in non-dividing cells. gag-specific humoral and cellular immune responses induced by DNA vaccination were both significantly improved, while the Th1/Th2 balance was not obviously affected by the 72-bp element. RTV boosting further significantly enhanced DNA vaccine-primed antibody and T cell responses in a Th1-biased manner.</p><p><b>CONCLUSIONS</b>The 72-bp SV40 enhancer element should be included in the DNA vaccine vector and RTV strain is a very efficient live vector for boosting immunization.</p>

Insertion of liver enriched transcription factor hepatocyte nuclear factor-4 [HNF-4] In a vector which contains simian virus [SV40] Promoter

One way of targeting gene expression in vivo is to control transcription using a tissue-specific regulatory system. Tissue-specific promoters or enhancers are in use in transgenic animals and could be utilized in medicine for gene therapy. At present the usual method for selection of a tissue-specific promoter is to identify a gene, which is expressed at unusually high level in the target tissue, and then to use the promoter for this gene to drive expression of another therapeutic gene in the target tissue. This approach is logical but does not always lead to high levels of gene expression. A second approach is to investigate the scope for discovery of synthetic specific promoters using a target tissue. The objective of the work described in this paper was to use both approaches to design plasmid DNA expression vectors that would carry liver-specific promoter/enhancer linked to a reporter gene [i.e. luciferase]. Then transfect these vectors to both liver-derived and non-liver cell lines. This is followed by evaluation of the liver-specificity of each construct by measuring the basal level expression of the reporter gene [i.e. luciferase activity] in both cell lines. Hepatocyte nuclear factor-4 [HNF-4] is liver-enriched transcription factor used to design new synthetic enhancers by inserting a tandem array of 1', 3' or 5' repeats of the HNF-4 binding site upstream of the SV40 promoter linked to the luciferase reporter gene within an Epstein-Barr virus [EBV]-based vector, p706. The results of transfection revealed that unexpectedly the HNF-4 binding sites in these constructs act as a repressor rather than enhancer of the liver-specific expression of the luciferase gene

Relationship between the malignant mesothelioma and simian virus 40 in China: a study of 17 cases / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate whether simian virus 40 (SV40) was related to patients of malignant mesothelioma in China.</p><p><b>METHODS</b>Paraffin-embeded samples of 17 patients with malignant mesothelioma were collected. After isolation of DNA from paraffin blocks, polymerase chain reaction (PCR) analyses were performed using three different sets of primer for detection of SV40 large T antigen gene. These samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 Tag (Pab101 and Ab-2).</p><p><b>RESULTS</b>Only one of the three primer pairs successfully amplified SV40 genome in three malignant mesothelioma samples. No immunopositive staining for SV40 TAg was found in any of the samples.</p><p><b>CONCLUSIONS</b>The study shows that malignant mesothelioma in China may be independent of SV40 infection.</p>

Immortalization of human umbilical vein endothelial cells by transfected with hTERT and SV40LT / 中华心血管病杂志

<p><b>OBJECTIVE</b>To immortalize human umbilical vein endothelial cells (HUVECs) by ectopic expression of the telomerase reverse transcriptase enzyme (hTERT), and by Simian Virus 40 Large T (SV40LT) antigen without malignant transformation.</p><p><b>METHODS</b>Two different retroviruses that contained hTERT/SV40LT cDNA fragment and drug resistance gene were constructed, and were used to transfect normal primary HUVECs. The transfected cells were screened with 500 microg/ml G418 and 4 microg/ml puromycin. Drug resistance cell clones were selected 3 days after transfection and cultured for further studies. An under inverted microscope and a scanning electron microscope were used to observe the morphology and growth of the cells. The expression of VIII factor and transfected DNA fragments were detected for identification of the endothelial origin and successful transfection. And the expression of E-selectin and endothelial lipase with or without the stimulus of TNF-alpha were also assayed to analyze the biological activity of the transfected cells.</p><p><b>RESULTS</b>The cells were homogenous, closely apposed, large, flat, and polygonal, displayed a characteristic ovoid nucleus with one or two nucleoli and formed monolayer with polygonal shape without overlapping. Immunocytochemical staining showed the existence of VIII factor. SV40LT/hTERT antigen expressed by the transfected cells was detected, while the contrasts had non-expression. Telomerase activity of the cell was detected in the transfected cells, which was 0.36 at 12 th passage and 0.38 at 50 th passage. However, the activity in the normal HUVECs was 1.12 at the first passage and 0.06 at the third passage assayed by PCR-ELISA. Both E-selectin and endothelial lipase were all specific in endothelial cells. The expressions of these two were also detected. And the expression of E-selectin can be up-regulated with the stimulus of TNF-alpha, while the expression of endothelial lipase was not unregulated significantly.</p><p><b>CONCLUSION</b>Ectopic expression of hTERT and SV40LT can effectively immortalize HUVECs without tumorigenesis.</p>

Conditionally immortalized human colorectal crypt cell line / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish human colorectal crypt cell line.</p><p><b>METHODS</b>Colorectal crypt cells were separated from human fetal gut by dispase I digestion, AKP-negative cells from fetal colorectal crypt were collected and cultured on Matrigel matrix. Subsequently the primary cultured cells were transfected with recombinant retrovirus containing human telomerase reverse transcriptase (hTERT) and simian virus 40 large T antigen (SV40 LT) in 48 h. The characterization of immortalized cells was identified after the transfection and cells were screened with antibiotics for 12 approximately 16 weeks and expanded.</p><p><b>RESULTS</b>Mucin, cytokeratin-pan, 8, 19 were presented in immortalized cells by immunohistochemical staining; ectopic expressions of both hTERT and SV40 LT were also found in immortalized cells by Western blotting. Agarose electrophoresis showed that the cells expressed Musashi-1 mRNA. No evidence of carcinogenesis was found in nude mouse experiment and soft-agarose cloning test.</p><p><b>CONCLUSION</b>The immortalized human colorectal crypt cells were characterized and the established cell line may be an ideal target for carcinogenesis study in vitro.</p>

The Role of Nuclear Factor Kappa B on Lens Epithelial Cells after Ultraviolet B Irradiation

PURPOSE: This study was performed to determine the role of nuclear factor kappa B (NF-kappa B) on the lens epithelial cell death after ultraviolet (UV) irradiation. METHODS: Simian virus 40 transfected human lens epithelial cells (HLE B-3 cells) were used in this study. UVB located at 10cm from the bottom was irradiated during 1, 2, 3 and 4 minutes. To measure the cytotoxicity MTT assay was used. Translocation of NF-kappa B was examined by immunocytochemistry with anti NF-kappa B p65 antibody and electrophoretic mobility shift assay (EMSA). To confirm the role of NF-kappa B, the cells were pretreated with sulfasalazine, a specific inhibitor of NF-kappa B, for 30 minutes before irradiation, and cytotoxicity and translocation of NF-kappa B were evaluated. RESULTS: UV irradiation produced a progressive cytotoxic effect in cultured HLE B-3 cells after 1 minute and maximum cytotoxicity was reached after 3 minutes irradiation. When HLE B-3 cells were irradiated with UVB, the translocation of NF-kappa B was observed in immunocytochemistry. These translocations were peaked 6 hours after UV irradiation in EMSA. In HLE B-3 cells pretreated with sulfasalazine, the translocation of NF-kappa B was blocked. The cellular death after UV irradiation was markedly blocked by sulfasalazine. UV irradiation can translocate NF-kappa B and sulfasalazine is a useful blocking agent in this pathway. In addition, sulfasalazine can prevent cellular death after UV irradiation. CONCLUSIONS: These findings suggest that NF-kappa B plays an important role in cellular death after UV irradiation.

Synergetic transactivating effect of HCV core and HBV X proteins on SV40 early promoter/enhancer / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the synergetic transactivating effects of HCV core and HBV X proteins.</p><p><b>METHODS</b>HCV core and HBV X protein-expressing plasmids were constructed with the vector pcDNA3.1(-). The plasmids were transfected into HepG2 cells and cotransfected Hep2 cells with reporter plasmid Psv-lacZ by lipofectamine plus reagents. The virus proteins produced in transient expression system were detected at the transcription and translation levels. The activity of b-galactosidase was detected, which reflected the transactivating function of the proteins.</p><p><b>RESULTS</b>The expression of plasmids were detected in soluble protein cell extracts of transiently transfected HepG2 cells. HCV core protein activated the b-galactosidase expression at a value 4.9 times higher than the control, while HBV X protein activated at a value 3.5 times. It arrived at 9 times transfected with the plasmids simultaneously. The activating effect increased in relation to the amount of plasmids.</p><p><b>CONCLUSIONS</b>The results suggested that the two kinds of virus proteins have transactivating effect on SV40 early promoter/enhancer, and they acted synergistically. These contribute to explain the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection.</p>

Immunohistochemical study of p21 and p53 expression in ameloblastoma

The p53 protein was discovered in 1979 as cellular 53-kD nuclear phosphoprotein bound to the large transforming antigen of SV40 virus. P21WAF1/CIP1, which has been described as the critical downstream mediator of p53, is known to suppress DNA replication and arrest the G1 cell cycle by quaternary complex with cyclin D, cyclin-dependent kinase(CDK) and proliferating cell nuclear antigen(PCNA). In these days, some studies shows that the p21 can be induced by independent pathways. There are various reports about the expression of p21 (67%.82.4%) in oral squamous cell carcinoma. But these studies are mostly done in malignant tumor not in benign tumor. So we decided to study the expression of p21 in ameloblastoma and the relationship between p53 and p21 as a downstream mediator of p53 in ameloblastoma. We investigated the expression of p21 and p53 with the method of immunohistochemistry. We selected 30 cases of ameloblastoma tissue blocks (acanthomatous type: 5 cases, follicular type: 8 cases, plexiform type: 17 cases) imbedded in paraffin. We used 30 cases of normal gingival tissues and 30 cases of squamous cell carcinoma tissues (SCC) respectively and compared their results with those of ameloblastoma. We made slides with the streptavidin-biotin methods and used monoclonal antibody DO-7 (Novocastra, Newcastle, United Kingdom) as p53 antibody and monoclonal antibody M7202 (DAKO, California, U.S.A.) as p21 antibody. We used Pearson's correlation coefficient to analyse the relationship. The results were as follows: 1. p21 was expressed in ameloblastoma about 30% and this is lower than that of normal gingiva and SCC. 2. In normal gingiva and ameloblastoma, p21 expression was correlated with p53 expression. 3. In SCC, p21 were expressed about 83.3% and this is more than that of p53. But there was no correlation between p21 and p53 expression. We confirmed p21 expression and relation with p53 in ameloblastoma. But, to confirm the function of p21, more studies about p21 expression in malignant ameloblastoma and ameloblastic carcinoma are needed.

The Translocation of Nuclaer Factor Kappa B on Corneal Epithelial Cells after Ultraviolet B Irrradiation

PURPOSE: To seek the role of nuclear factor kappa B (NF-kB) on the corneal epithelial cell death after ultraviolet (UV) irradiation. METHODS: Human corneal epithelial cells transfected by Simian Virus 40 were used in this study. UVB(312 nm) located at 10cm distance from bottom (0.6 mW/cm2 ) was irradiated for 10, 20, 30, and 40 seconds. To measure the cytotoxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used. Translocation of NF-KB was examined by immunocytochemistry with anti NF-K B p65 antibody and Electrophoretic Mobility Shift Assay (EMSA). To confirm the role of NF-KB , sulfasalazine, a specific inhibitor of NF-KB (0.5 mmole), was pretreated for 30 minutes before irradiatrion, and cytotoxicity and translocation of NF-KB was evaluated. RESULTS: UV irradiation resulted in a significant decrease in viability of cultured human corneal epithelial cells, especially after 20 second duration. When HCECs were irradiated with UVB, the translocation of N F -KB was observed in immunocytochemistry. These translocation was peaked 2 hours after UV irradiation in EMSA. In HCECs pretreated with sulfasalazine, either the cellular death or the translocation of NF-KB was blocked. CONCLUSION: UV irradiation can translocate NF-KB on the cultured human corneal epithelial cells. The cellular death after UV irradiation was blocked by sulfasalazine, a potent inhibitor of translocation of NF-KB. These findings suggest that NF-KB plays an important role in cellular death after UV irradiation.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.