hTERT-positive Cells Research Articles

Genome-Protective Topoisomerase 2a-Dependent G2 Arrest Requires p53 in hTERT-Positive Cancer Cells.

Topoisomerase 2a (Topo2a)-dependent G2 arrest engenders faithful segregation of sister chromatids, yet in certain tumor cell lines where this arrest is dysfunctional, a PKCε-dependent failsafe pathway can be triggered. Here we elaborate on recent advances in understanding the underlying mechanisms associated with this G2 arrest by determining that p53-p21 signaling is essential for efficient arrest in cell lines, in patient-derived cells, and in colorectal cancer organoids. Regulation of this p53 axis required the SMC5/6 complex, which is distinct from the p53 pathways observed in the DNA damage response. Topo2a inhibition specifically during S phase did not trigger G2 arrest despite affecting completion of DNA replication. Moreover, in cancer cells reliant upon the alternative lengthening of telomeres (ALT) mechanism, a distinct form of Topo2a-dependent, p53-independent G2 arrest was found to be mediated by BLM and Chk1. Importantly, the previously described PKCε-dependent mitotic failsafe was engaged in hTERT-positive cells when Topo2a-dependent G2 arrest was dysfunctional and where p53 was absent, but not in cells dependent on the ALT mechanism. In PKCε knockout mice, p53 deletion elicited tumors were less aggressive than in PKCε-replete animals and exhibited a distinct pattern of chromosomal rearrangements. This evidence suggests the potential of exploiting synthetic lethality in arrest-defective hTERT-positive tumors through PKCε-directed therapeutic intervention.

Expression of human telomerase reverse transcriptase protein in oral epithelial dysplasia and oral squamous cell carcinoma: An immunohistochemical study.

Background:Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA sequences and almost universally provides the molecular basis for unlimited proliferative potential. The telomeres become shorter with each cycle of replication and reach a critical limit; most cells die or enter stage of replicative senescence. Telomere length maintenance by telomerase is required for all the cells that exhibit limitless replicative potential. It has been postulated that reactivation of telomerase expression is necessary for the continuous proliferation of neoplastic cells to attain immortality. Use of immunohistochemistry (IHC) is a useful, reliable method of localizing the human telomerase reverse transcriptase (hTERT) protein in tissue sections which permits cellular localization. Although there exists a lot of information on telomerase in oral cancer, little is known about their expression in oral epithelial dysplasia and their progression to oral squamous cell carcinoma (OSCC) compared to normal oral mucosa. This study addresses this lacuna.Aims:To compare the expression of hTERT protein in oral epithelial dysplasia and OSCC with normal oral mucosa by Immunohistochemical method.Subjects and Methods:In this preliminary study, IHC was used to detect the expression of hTERT protein in OSCC (n = 20), oral epithelial dysplasia (n = 21) and normal oral mucosa (n = 10). The tissue localization of immunostain, cellular localization of immunostain, nature of stain, intensity of stain, percentage of cells stained with hTERT protein were studied. A total number of 100 cells were counted in each slide.Statistical Analysis:All the data were analyzed using SPSS software version 16.0. The tissue localization, cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity was compared across the groups using Pearson's Chi-square test. The mean percentage of cells stained for oral epithelial dysplasia, OSCC and normal oral mucosa were compared using analysis of variance (ANOVA). A P < 0.05 was considered to be statistically significant.Results:The mean hTERT positive cells in the study groups were as follows, 62.91% in normal oral mucosa samples, 77.06% in oral epithelial dysplasia cases, and 81.48% in OSCC. In 61.9% of oral epithelial dysplasia and 65% of OSCC in our study, staining was visualized within the nucleus predominantly in the dot like pattern. There was a statistically significant difference in the nature of nuclear stain between oral epithelial dysplasia and OSCC (P = 0.023).Conclusions:Our results suggests that the mean percentage of cells showing hTERT expression steadily increased from normal oral mucosa to oral epithelial dysplasia to OSCC. The steady trend of increase in the percentage of cells was evident in different grades of oral epithelial dysplasia group and OSCC. The nature of hTERT staining did show variations among the three groups and promise to be a potential surrogate marker for malignant transformation. Further studies using IHC on larger sample size and clinical follow-up of these patients will be ascertaining the full potential of hTERT as a surrogate marker of epithelial transformation.

HTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

BackgroundActivation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.MethodsUsing luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter.ResultsWe found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity.By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls.ConclusionsMethylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive biomarker for early detection of cervical cancer.

Tumor-Targeted Radionuclide Imaging and Therapy Based on Human Sodium Iodide Symporter Gene Driven by a Modified Telomerase Reverse Transcriptase Promoter

Human telomerase reverse transcriptase (hTERT) is highly active in most cancer cells and, thus, could be used for tumor targeting. The human sodium iodide symporter (hNIS) gene is being actively researched as a potential radioactive iodine (radioiodine) gene therapy. In this study, we investigated the possibilities of using the hNIS gene driven by the hTERT promoter for molecular imaging and radioiodine gene therapy. Stable cell lines of hTERT-positive cells (Hep3B hepatoma) expressing hNIS, under the control of the 5mmTERT promoter, were generated using a retroviral system. Radioiodine uptake and efflux tests were performed, and a clonogenic assay was used to evaluate the in vitro cytotoxicity of 131I. Finally, scintigraphic, biodistribution, and radioiodine therapy studies were performed in vivo. Radioiodine uptake by 5mmTERT-NIS-transfected Hep3B cells was 22 times higher than by nontransfected Hep3B cells, and 5 times that of 5mmTERT-NIS-transfected U2-OS cells (p < 0.05). Clonogenic assays demonstrated that the survival rate of Hep3B-5mmTERT-NIS cells after 131I incubation was significantly lower than that of Hep3B cells (p < 0.001), and radioiodine accumulations in Hep3B-5mmTERT-NIS tumors were significantly higher than in wild-type tumors. In addition, technetium- 99m scintigraphy clearly visualized Hep3B-5mmTERT-NIS tumors. Moreover, after being treated with 111 MBq of 131I-labeled Hep3B-5mmTERT-NIS, tumor growth was retarded, whereas Hep3B tumor growth progressed. hTERT-positive tumors were successfully targeted by the NIS gene under the control of the 5mmTERT promoter. The described system could be useful for targeted molecular imaging and as a radioiodine gene therapy for cancer.

Tumor targeted radionuclide imaging and therapy using human sodium iodide symporter gene driven by a modified telomerase reverse transcriptase promoter

Human telomerase reverse transcriptase (hTERT) is highly active in most cancer cells and, thus, could be used for tumor targeting. The human sodium iodide symporter (hNIS) gene is being actively researched as a potential radioactive iodine (radioiodine) gene therapy. In this study, we investigated the possibilities of using the hNIS gene driven by the hTERT promoter for molecular imaging and radioiodine gene therapy. Stable cell lines of hTERT-positive cells (Hep3B hepatoma) expressing hNIS, under the control of the 5mmTERT promoter, were generated using a retroviral system. Radioiodine uptake and efflux tests were performed, and a clonogenic assay was used to evaluate the in vitro cytotoxicity of 131I. Finally, scintigraphic, biodistribution, and radioiodine therapy studies were performed in vivo. Radioiodine uptake by 5mmTERT-NIS-transfected Hep3B cells was 22 times higher than by nontransfected Hep3B cells, and 5 times that of 5mmTERT-NIS-transfected U2-OS cells (p < 0.05). Clonogenic assays demonstrated that the survival rate of Hep3B-5mmTERT-NIS cells after 131I incubation was significantly lower than that of Hep3B cells (p < 0.001), and radioiodine accumulations in Hep3B-5mmTERT-NIS tumors were significantly higher than in wild-type tumors. In addition, technetium- 99m scintigraphy clearly visualized Hep3B-5mmTERT-NIS tumors. Moreover, after being treated with 111 MBq of 131I-labeled Hep3B-5mmTERT-NIS, tumor growth was retarded, whereas Hep3B tumor growth progressed. hTERT-positive tumors were successfully targeted by the NIS gene under the control of the 5mmTERT promoter. The described system could be useful for targeted molecular imaging and as a radioiodine gene therapy for cancer.

P16, cyclin D1, and human telomerase reverse transcriptase expression in cervical premalignant and malignant lesions

ABSTRACT Background and aim: Although morphological criteria are important for diagnosis of cervical intraepithelial neoplasia, it is not possible to differentiate those that will regress from those that will progress into invasive cancer. We evaluate the usefulness of p16, cyclin D1, and human telomerase reverse transcriptase (hTERT) as diagnostic and prognostic biomarkers in cervical lesions. Methods: We studied 12 cases of benign lesion, 15 cases of low‐grade squamous intraepithelial lesion, 46 cases of high‐grade squamous intraepithelial lesion, and 13 cases of squamous cell carcinoma by immunohistochemistry. Results: The p16 expression was significantly correlated with high risk‐human papillomavirus (HR‐HPV) status, lesion grade, and decreased cyclin D1 expression. The decreased cyclin D1 expression was significantly correlated with HR‐HPV status, lesion grade, and p16 expression. The positive predictive values for high grade lesion of p16 expression, HR‐HPV status, and decreased cyclin D1 expression were 93.8%, 81.3%, and 51.2%, respectively. The distribution pattern of hTERT positive cells was correlated with morphologically dysplastic cells. Conclusions: A good correlation was observed between cervical lesion grade and p16 expression, decreased cyclin D1 expression, and HR‐HPV status. p16 expression could be used as a diagnostic and prognostic biomarker for risk of developing cervical cancer in women infected with HPV.

Telomerase and Markers of Cellular Proliferation Are Associated With the Progression of Cervical Intraepithelial Neoplasia Lesions

The expression of the catalytic subunit of telomerase protein (human telomerase reverse transcriptase [hTERT]), which is associated with telomerase activity, was evaluated as a potential marker of the high-grade premalignant cervical intraepithelial neoplasia (CIN 2/3) lesions. For comparison, cases of normal cervical squamous mucosa, low-grade CIN1 lesion, and cervical squamous cell carcinoma were included. The hTERT expression was also compared with Ki-67 and topoisomerase II-alpha (TPII-alpha) to determine the proliferative activity of the hTERT-positive dysplastic cells by a quantitative immunohistochemical staining method and was classified as follows: negative, 5% or less; moderate, 6% to 50%; or high, greater than 50% of the positive cells. The hTERT-positive cells were detected in a patchy pattern in the lower parabasal layers and in much of the basal layer in normal squamous mucosa. A similar frequency of Ki-67- or TPII-alpha-positive cells was observed, with the exception of the basal layer cells that were mostly negative. It is worthy to note that the recognizable intact basal layer cells in cases of CIN lesions were also consistently positive for the expression of hTERT, but rarely for Ki-67 or TPII-alpha. The expression of hTERT was detected in a less patchy pattern at a high or moderate percentage of the dysplastic epithelial cells each in 28.5% of cases of CIN1 lesions. A similar frequency, high and moderate percentage combined, of the TPII-alpha-positive dysplastic cell was also observed. In contrast, a high percentage of the hTERT-positive dysplastic cells were detected as diffuse basal or full-length thickness in 87.5% or 95% of cases of CIN2 or CIN3, respectively. A similar frequency of Ki-67 or TPII-alpha expression was observed in the dysplastic cells of CIN3 lesions. The pattern of hTERT-positive malignant cells in squamous cell carcinoma and dysplastic cells in the high-grade CIN lesions, to a greater extent, and dysplastic cells in the low-grade CIN lesion, to a lesser extent, was distinct from that of the normal cervical squamous mucosa. The results suggest that the progressive increase in the hTERT expression, together with the proliferative activity of the dysplastic epithelial cells of the high-grade CIN lesions, represents an early genetic abnormality in cervical pathogenesis.

HTERT immunopositivity patterns in the normal brain and in astrocytic tumors

Accumulating data about the impact of hTERT in astrocytic tumor carcinogenesis and recent evidence about its association with disease outcome prompt the evaluation of this molecule with methods applicable in routine pathology practice. In this study, we investigated hTERT protein expression with immunohistochemistry (IHC) and the NCL-hTERT antibody in 49 astrocytic tumors. Results were validated with the assessment of hTERT mRNA (relative quantification, identification of splice variants, in situ hybridization). Specific nuclear hTERT immunostaining patterns (IPs) were characterized as patterns As (single large dot) and Am (multiple dots) without nucleoplasm staining and pattern B (nucleoplasm staining with or without dots), corresponding to low and high relative hTERT expression values (P<0.0001). Low- and high-grade astrocytic tumors were found positive for hTERT in 74 and 85% of cases, respectively. Heterogeneity in the distribution of hTERT-positive cells was observed in all tumors. The prevailing nuclear IPs differed significantly between pilocytic astrocytomas (pattern As) and the rest of histologic types up to glioblastoma (patterns Am and B) (P<0.0001). The described nuclear IPs were also observed in non-neoplastic cells. Positive endothelial cells were found in astrocytic tumors of all grades, even when tumor cells showed no hTERT immunoreactivity. A subset of mature normal neurons was positive for hTERT (pattern As), suggesting a role for this molecule in neuronal maintenance in the adult brain. The nuclear hTERT IPs described here may reflect the functional status of non-neoplastic brain and neoplastic astrocytic cells and support the model of a continuum in the development of glioblastomas from diffuse fibrillary astrocytomas.

Transcript Regulation of Human Telomerase Reverse Transcriptase by c-myc and mad1

Telomerase activity is highly positive correlated to most malignant neoplasms. Human telomerase reverse transcriptase (hTERT) is the rate-limiting factor of telomerase activity. Recent studies have shown that the expression of hTERT is mainly determined by its transcript regulation. Among the transcript regulation factors of hTERT, c-myc and mad1 are well known. Here, we constructed c-myc and mad1 eukaryotic expression vectors, then co-transfected them with the wild-type (Tw) or mutant hTERT promoter (Td) luciferase reporter plasmid, which were double-mutated in the E-box sequences from CACGTG to CACCTG of Tw. The change of luciferase activity in different cells was detected. The results showed that Tw was obviously activated in T24 and EJ bladder cancer cells, but not in normal fibrocytes. c-myc and mad1 had positive and negative effects respectively on the Tw transcript in a dose-dependent manner, while the roles of c-myc and mad1 in regulating the Td transcript were reversed. c-myc combined with mad1 can down-regulate Tw but not Td. These observations indicate that c-myc and mad1 can regulate the hTERT transcript in a different manner in hTERT positive cells, but not in normal cells. This may provide an insight into some telomerase-related carcinogenesis mechanisms.

Telomere‐independent cellular senescence in human fetal cardiomyocytes

Fetal cardiomyocytes have been proposed as a potential source of cell-based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT-PCR. Cells were found to senesce after 20-25 population doublings, as determined by growth arrest, morphological changes and senescence-associated beta-galactosidase activity. Using the telomeric repeat amplification protocol assay, telomerase activity was undetectable in primary HFCs. Cells were transduced to express the human reverse transcriptase subunit (hTERT) of telomerase. This resulted in greatly increased telomerase activity, but no significant lifespan extension. Analysis of telomere length in primary HFCs revealed that the senescent phenotype was not accompanied by telomere shortening. Telomeres in hTERT-positive cells were elongated in comparison with primary cells, and elongation was retained in senescent cells. Levels of the tumor suppressor protein p16INK4A increased in all senescent cells whether telomerase-positive or -negative. Senescence was accompanied by a decline in transcript levels of the polycomb gene Bmi-1, Ets1 and Ets2 transcription factors, and Id1, Id2 and Id3 helix-loop-helix proteins, suggesting roles for these genes in maintenance of cardiomyocyte proliferative capacity. In addition to offering novel insights into the behavior of human fetal cardiomyocytes in culture, these findings have implications for the development of a cell-based therapy for cardiac injury using primary fetal heart tissue.

Expression of human telomerase reverse transcriptase in laryngeal tissues

Problem: Telomerase is believed to play a major role in transforming cells (immortalization) to carcinoma. Its activity has been detected in 90% of human cancers. We evaluated telomerase activity in laryngeal carcinoma patients who underwent surgery for cure through the immunohistochemical detection of human telomerase reverse transcriptase (hTERT). Telomerase activity is correlated to hTERT expression. Methods: Patients were divided into 2 groups (those who were cured and those who had recurrences) and the (height) percentage of the thickness of the epithelium with hTERT positive cells and the percentage of hTERT positive cells per high-powered field (HPF) was compared. Twenty-three carcinoma specimens (14 cured and 9 recurrence) were stained with hTERT antibody. Results: The positive hTERT expression (85.7%) in the cured specimens was not statistically significant when compared to the recurrence specimens (100%) ( P = 0.50). The minimum height and minimum percentage of positive cells per HPF in the cured group was zero. However, the minimum height in the recurrence group was 55.0%, and the minimum percentage of positive cells per HPF was 52.3%. Furthermore, we evaluated the hTERT expression of the normal epithelium surrounding the carcinomas and found no differences between the cured and recurrence groups ( P = 0.46). Conclusion: In conclusion, although hTERT expression could not positively predict the patient’s outcome (cure versus recurrence), those specimens with less than 50% height involvement or percentage of positive hTERT staining cells per HPF tended to be cured from the treatment. Larger sample sizes are necessary to establish the predictive ability of hTERT expression in cancer. Significance: Telomerase activity has been detected in over 90% of all human cancers. hTERT and Telomerase levels may be predictive of cure in cancer. A multi-institutional trial has been started to increase the number of samples to increase the statistical power and possibly arrive at a definitive answer to this important question. Support: None reported.

Expression of human telomerase reverse transcriptase (hTERT) in laryngeal tissues

Problem: Telomerase is a multi-subunit enzyme that preserves the telomeric length in human cells. It is involved in the process of immortalization of cells in many types of carcinoma and has been detected in over 90% of human cancers. This study was conducted to determine whether the presence of telomerase in different laryngeal tissues could be used as a marker for malignancy. Methods: We immunohistochemically stained 4 types of laryngeal specimens for human telomerase reverse transcriptase (hTERT); normal true vocal folds, vocal polyps, squamous atypia, and glottic carcinomas. The hTERT expression in the epithelium of all specimens was analyzed by light microscopy with 2 observers. Positive expression was determined by the presence of intranuclear staining. Results: The percentage of hTERT positive cells in a given high-powered field was 5.09% for normal vocal folds, 12.70% for vocal polyps, 5.87% for squamous atypia, and 72.65% in squamous cell carcinoma. Only the expression in the laryngeal carcinoma was statistically significant compared to the other laryngeal tissues ( P < 0.001). Conclusion: hTERT expression is a good marker for laryngeal carcinoma but is not good at differentiating within benign vocal pathology. Significance: Telomerase expression is an excellent predictor of malignancy in the larynx. It does not assist in distinguishing within benign pathology. Support: None reported.

615. Feasibility of hTERT-Targeting Trans-Splicing Ribozyme in Tumor-Targeted Gene Therapy

Cancer gene therapy without toxicity is hampered by lack of efficient tumor-directed therapeutic gene expression system. In the present study, we describe a new tumor-targeted gene therapy tool that is based upon trans-splicing ribozymes. Trans-splicing ribozymes are RNA-based catalysts capable of performing both cleavage and ligation reaction in target RNA. Thus a chimeric mRNA can be produced and new gene activities triggered in cells dependent on the presence of the target RNA. We chose hTERT-targeting trans-splicing ribozyme for efficient tumor-directed expression of therapeutic gene by transcriptional targeting and RNA replacement because human telomerase reverse transcriptase (hTERT) is expressed at high levels in 90% of malignant tumors and tumor cell lines but is absent in normal postmitotic tissues. After developing a group I intron-based ribozyme targeting hTERT with high fidelity and specificity, we constructed novel adenoviral vectors containing hTERT-targeting trans-splicing ribozymes with downstream lacZ gene (Ad-ribolacZ) or herpes simplex virus thymidine kinase gene (Ad-ribotk). This hTERT-targeting trans-splicing ribozyme could simultaneously reduce hTERT expression and trigger expression of a new gene, such as lacZ or HSVtk, in cells expressing hTERT. To determine whether new gene activity could be selectively induced in hTERT expressing cells by trans-splicing ribozyme, we transfected Ad-ribolacZ into hTERT-positive and -negative cell lines. Then lacZ expression was analyzed by X-gal staining and β-galactosidase enzyme activity assays. We observed 4 to 9-fold increase of lacZ expression in hTERT-positive cells but not in h-TERT-negative ones. In contrast, induction of lacZ expression by adenovirus harboring lacZ gene driven by CMV promoter (Ad-CMVlacZ) could be observed in both hTERT-negative and-positive cells. Those results suggest that selective induction of transgene activity in hTERT-dependent manner by Ad-ribolacZ was mediated by correct targeting of hTERT with trans-splicing ribozyme in hTERT-positive cells. Given the observation that the trans-splicing ribozyme induced transgene expression selectively in target RNA-expressing cells, we determined whether the ribozyme would trigger selective cytotoxicity in hTERT-expressing cells. After transfecting Ad-ribotk into hTERT-positive and -negative cell lines, selective cytotoxicity was monitored by MTT assays. Ad-CMVtk was used as control. We could observe that Ad-ribotk revealed more than 50% reduction in survival rate of hTERT-positive cells, while not in hTERT-negative ones. In control, reductions of cell survival were observed regardless of hTERT expression. Those observation of highly selective cytotoxicity rendered by Ad-ribotk suggest that hTERT-targeting trans-splicing ribozyme may be one of powerful tools in tumor-targeted gene therapy.

Regulation of telomerase reverse transcriptase gene activity by upstream stimulatory factor.

Upregulation of human telomerase reverse transcriptase (hTERT) transcription accounts for the immortalization of greater than 85% of all human tumor cells. However, the mechanism whereby hTERT expression is activated remains unresolved. Specifically, recent data challenging the role of Myc/Max in E-box-dependent activation of hTERT expression suggests that other E-box-binding proteins regulate hTERT transcription. Indeed, we now demonstrate that two such proteins, upstream stimulatory factor (USF) 1 and 2, readily associate with two E-boxes in the hTERT promoter in vitro and in vivo primarily as heterodimers, whereas Myc/Max does not. The avid binding of USF1/2 heterodimers to these E-boxes occurs in both hTERT-positive and -negative cells. In contrast, USF1/2 activates the hTERT promoter exclusively in hTERT-positive cells in a manner that is enhanced by the coactivator p300 and attenuated upon inhibiting p38-MAP kinase, a known modulator of USF activity. Collectively, our data indicate that USF binding to the hTERT promoter may be transcriptionally neutral, or even repressive, in nonimmortalized hTERT-negative somatic cells, but stimulatory in hTERT-positive cells where USF1/2 contributes to the acquisition and maintenance of immortality.

Development of a Conditional Replication Competent Adenovirus, Controlled by the Human Telomerase Promoter (hTERT).

This study has been planned to generate a replication-competent adenovirus which replicates in a cancer cell-specific manner, thus minimizing the side effects and toxicity of cancer gene therapy. we have generated an E1B 19 kD attenuated recombinant adenoviruses, Ad-TERT-delta19 and Ad-mTERT-delta19, which encode E1A gene driven by the wild type hTERT and modified m-hTERT promoter containing additional c-myc and Sp1 binding sites in the backbone of Ad-deltaE1B19. The in vitro efficacy and specificity of the hTERT and m-hTERT promoter have been evaluated by the comparison of viral replication and cytopathic effect in cancer cells and normal cell lines. To assess anti-tumor effect and safety of hTERT or m-hTERT promoter driven replication competent adenoviruses, tumor regression after subcutaneous injection in subcutaneous C33A xenografts and lacZ expression after systemic injection in organs were examined. The activation of hTERT or m-hTERT promoter was significantly up-regulated only in hTERT-positive cells, but not in hTERT-negative cells. Moreover, the activity of m-hTERT promoter was substantially increased in hTERT-positive cancer cells, but not in hTERT-negative cells. While Ad-TERT-delta19 replicated in and induced cytopathic effect in cancer and in some normal cell lines, Ad-mTERT-delta19 enhanced viral replication and cytopathic effect in cancer cells only. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-mTERT-delta19. S: The use of m-hTERT promoter is not only useful in the regulation of therapeutic gene expression but also that replication-competent oncolytic adenovirus under the control of m-hTERT promoter may be a new promising tool for the treatment of human malignancies.

Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4A/pRb.

Recent studies show that stable expression of the human telomerase catalytic subunit, hTERT, alone can lead several types of normal human somatic cells to bypass replicative senescence and become immortal. The molecular mechanisms by which telomerase immortalizes cells are not fully understood, although a key function of telomerase is to maintain a critical length of telomeres in order to preserve the stability and integrity of the genome. Here we report that stable transfection of hTERT alone was sufficient to allow bovine capillary endothelial (BCE) cells to bypass senescence and acquire immortality. Surprisingly, telomere lengths in these stable transfectants were progressively shortened during an increasing number of population doublings (PDLs), despite high telomerase activity. The expression of the cyclin-dependent kinase inhibitors (CDKIs) p16INK4A and p21CIP1/WAF1 was concomitantly repressed, and the retinoblastoma protein (pRb) was maintained in a hyperphosphorylated state in the telomerase-expressing cells. Re-expression of p16INK4A in these cells by either treatment with a demethylating agent or by adenovirus-mediated expression reinduced a senescence-like phenotype, suggesting that the inactivation of p16INK4A was due to DNA methylation and was crucial for the immortalization process. In agreement with this finding, the expression levels of the prototypic DNA methyltransferase DNMT1 were elevated in the hTERT-positive cells.

Imbalance Between Apoptosis and Telomerase Activity in Myelodysplastic Syndromes: Possible Role in Ineffective Hemopoiesis

The myelodysplastic syndromes (MDS) are a group of disorders characterized by peripheral pancytopenia despite normo- or hyper-cellular bone marrow. This is thought to be due to apoptosis of hematopoietic bone marrow cells, resulting in ineffective hematopoiesis. The heterogeneous nuclear ribonucleoprotein (hnRNP) B1 is involved in pre-mRNA processing and binds to telomeric cDNA repeats. The hnRNP B1 is a marker for early cancer. The aim of our study was to clarify the relationships between prognosis and apoptosis, telomerase activity (TA) and hnRNP expression in the bone marrow. The subjects were 51 patients with MDS, including patients with refractory anemia (RA) <formula>(<italic>n</italic>=32),</formula> refractory anemia with ringed sideroblasts (RARS) <formula>(<italic>n</italic>=1),</formula> refractory anemia with excess blasts (RAEB) <formula>(<italic>n</italic>=7),</formula> refractory anemia with excess blasts in transformation (RAEB-t) <formula>(<italic>n</italic>=8)</formula> and chronic myelomonocytic leukemia (CMMoL) <formula>(<italic>n</italic>=3).</formula> We also studied 6 cases with acute myelogenous leukemia (AML) arising from MDS (AML-MDS) and 10 control subjects. Bone marrow biopsies were stained immunohistochemically for caspase-3 (marker of apoptotic activity) and human telomerase reverse transcriptase (hTERT), and hnRNP B1. Fatal pancytopenia was the cause of death in 19 of the 51 patients. The caspase-3 positive cell rate was higher in MDS (16.3%) than in controls (4.4%) and AML-MDS (0.5%). The percentage of hnRNP B1-positive cells was higher in MDS (15.3%) and AML-MDS (56.3%) than in controls (5.6%). In MDS, hnRNP B1 levels were higher in RAEB and RAEB-t subtypes than in RA and RARS. The percentage of hTERT-positive cells was higher in AML-MDS (50.0%) than in controls (20.2%) and MDS (23.6%). Our findings suggest that activation of apoptosis occurs in MDS in the absence of hTERT expression, implicating high apoptosis in the absence of high TA with ineffective hematopoiesis. Poor prognosis correlated with higher caspase-3 and lower hTERT rates. In MDS, hnRNP B1 activity may be associated with leukemic transformation.