Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA onto chromosomal ends to compensate for sequence loss during replication. It has been detected in a variety of human malignancies, suggesting that such activity may play a role in the tumorigenic process. To determine whether telomerase is reactivated in malignant fibrous histiocytoma, 12 tissue samples with this tumor were analyzed for the telomerase activity by a radioactive PCR-based TRAP (telomeric repeat amplification protocol) assay. All of the tumors were further investigated for the expression of human telomerase RNA (hTR) by an in situ hybridization (ISH). Telomerase activity was detected in one (8.3%) sample. Expression of hTR was demonstrated in 7 (58.3%): one telomerase-positive and six telomerase-negatives. These data indicate that the reactivation of telomerase is an uncommon event and not an important factor involved in tumorigenesis in malignant fibrous histiocytoma. It is noteworthy that 50% of the patients with grade 2 tumors expressed hTR, suggesting that telomerase RNA may be useful as a marker for identifying tumor aggressiveness earlier than the conventional histopathologic grading scale.
Carcinogenesis ; DNA ; Histiocytoma, Malignant Fibrous* ; Humans ; In Situ Hybridization ; RNA* ; RNA-Directed DNA Polymerase ; Telomerase*

PURPOSE: Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA onto chromosome ends to compensate for sequence loss during DNA replication. It has been detected in 85~90% of all primary human cancers, implicating that its apparent reactivation in tumors may play a role in the tumorigenic process. The purpose of this study was to evaluate telomerase activity in stomach cancer, and to determine whether methacarn-fixed paraffin-embedded tissues can replace frozen tissue sections for the telomerase (TRAP) assay. MATERIALS AND METHODS: Frozen and corresponding methacarn-fixed paraffin-embedded tissue samples were obtained from 51 patients with gastric adenocarcinoma and analyzed for telomerase activity by using a TRAPeze ELISA kit. RESULTS: Telomerase activity was detected in 37 (73%) frozen samples, and in 13 (25%) methacarn-fixed paraffin blocks. Telomerase activity was well correlated with depth of invasion (p=.037) and tumor differentiation (p=.022). CONCLUSION: These data suggest that reactivated telomerase may play a significant role in the tumorigenesis of gastric cancer and may reflect the malignant potential of the tumor. It is noteworthy that methacarn- fixed tissue cannot as yet substitute for the frozen tissue in the TRAP assay.
Adenocarcinoma* ; Carcinogenesis ; DNA ; DNA Replication ; Enzyme-Linked Immunosorbent Assay ; Humans ; Paraffin ; RNA-Directed DNA Polymerase ; Stomach Neoplasms ; Telomerase*

PURPOSE: Sarcomas have rarely been analyzed for telomerase, which is an RNA-dependent DNA polymerase to maintain telomeres and prevent telomere shortening. This study was undertaken to determine telomerase activity and the expression of the telomerase subunits human telomerase RNA (hTR) and telomerase reverse transcriptase (TERT) in soft tissue sarcomas. MATERIALS AND METHODS: Twenty three sarcomas were analyzed for the telomerase activity by a radioactive PCR-based TRAP assay. All of the samples were further investigated for the expression of hTR by in situ hybridization and for TERT and p53 by immunohistochemistry. RESULTS: Telomerase activity was detected in four (17%) samples. Expression of hTR was demonstrated in 11 (48%) cases, whereas TERT was expressed in 20 (87%).Of the four telomerase-positive tumors, three were positive for both hTR and TERT, and one was positive only for TERT. p53 overexpression was observed in nine (39%) tumors. The frequency of p53 expression increased as the tumor grade advanced (p= .064). CONCLUSION: These data indicate that the reactivation of telomerase is an uncommon event in human soft tissue sarcomas. The high frequency of the expression of hTR and TERT in these tumors suggests that telomerase activity may be regulated at the transcriptional level and an additional event leading to telomerase activation exist.
Humans* ; Immunohistochemistry ; In Situ Hybridization ; RNA ; RNA-Directed DNA Polymerase ; Sarcoma* ; Telomerase* ; Telomere ; Telomere Shortening

BACKGROUND: Telomerase is a ribonucleoprotein complex with RNA-dependent DNA polymerase which is necessary in maintenance of the length of chromosome, and therefore, in preventing genomic instability. Its activity is regarded as an indicator of cell immortalization. So far, there is no comprehensive answer on which step the telomerase activity is required; in some studies, telomerase activity has been found in benign, premalignant, and malignant conditions equally, which means it affects early stage of carcinogenesis, but in other studies, it has been found in malignant conditions at a higher rate. OBJECTIVE: This study was performed to examine telomerase activity in normal and skin cancer tissues and to assess the role of telomerase in the development of malignant transformation of skin cancer by examining benign, premalignant, and malignant conditions together. METHODS: Telomerase activities in four benign skin tumors, five precancerous lesions, and 17 skin cancer tissues of the skin were measured by a method telomeric repeat amplification protocol(TRAP). TRAP assay was also performed on normal control tissue of the same patients and eight skin tissues of the healthy volunteers. RESULTS: Telomerase activity was detected in 50% of benign tumors, 100% of precancerous, and 82% of malignant tissues. Among them, three out of four BCC tissues were shown to contain telomerase activity, whereas normal tissue of the same patients were not. No telomerase activity was detected in all of the eight skin samples of the healthy volunteers. CONCLUSION: Telomerase activities may be required at early stage of tumorigenesis as these activities are required for further steps down of the oncogenesis.
Carcinogenesis ; Genomic Instability ; Healthy Volunteers ; Humans ; Ribonucleoproteins ; RNA-Directed DNA Polymerase ; Skin Neoplasms ; Skin* ; Telomerase*

OBJECTIVETo investigate the feasibility of life span extension of transformed human embryonic tendon cells (THETC) by reconstitution of the telomerase activity.

METHODSTHETC were transfected by pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) cNDA in vitro by molecular cloning technique. The biological characteristics of transfected cells were detected and compared by morphological observation, plate cloning efficiency, soft agar culture, growth curve of cells cultured in different conditions, immunohistochemistry, telomerase activity assay by telomeric repeat amplification protocol (TRAP).

RESULTSThe THETC transfected by pGRN145 plasmid (telT) could express the telomerase activity with extension of life span. The telT maintained the original characteristics of temperature-dependant and serum-dependant, as well as secretion of type I collagen normally and without tendency of malignant transformation.

CONCLUSIONSThe life span of THETC can be prolonged by reconstitution of telomerase activity, which provides the novel experimental methods to establish the standard cells line.

Cell Line ; Cell Survival ; Embryo, Mammalian ; Humans ; Plasmids ; genetics ; RNA-Directed DNA Polymerase ; Telomerase ; genetics ; metabolism ; Tendons ; cytology ; enzymology ; Transfection

BACKGROUND AND OBJECTIVES: Vasospastic angina (VA) is a specific type of coronary artery disease and develops as a result of coronary artery spasm. Recently, a few studies have revealed that VA caused by coronary artery spasm is related to genetic traits. The objective of this study was to use the recently developed technique of array comparative genomic hybridization (CGH) to screen the genetic aberrations of VA. SUBJECTS AND METHODS: To identify candidate genes that might be causally involved in the pathogenesis of VA, genomic deoxyribonucleic acids were extracted from whole blood of 28 patients with VA who presented at Department of Cardiology at Seoul St. Mary's Hospital, Seoul, Korea. The copy number profiles of these patients was then analyzed using array CGH and reverse transcriptase (RT) quantitative polymerase chain reaction (PCR). RESULTS: Array CGH revealed gains in 31 different regions, with losses in the 4q35.2, 7q22.1, 10q26.3, 15q11.2, 16p13.11, 17p11.2 and 19q13.3 regions (more than 32% aberration in these regions). Several loci were found to be frequently including gains of 5p and 11q (50% of samples). The most common losses were found in 7q (54% of samples). Copy number aberrations in chromosomal regions were detected and corresponding genes were confirmed by RT quantitative PCR. The fold change levels were highest in the CTDP1 (18q23), HDAC10 (22q13.33), KCNQ1 (11p15.5-p15.4), NINJ2 (12p13.33), NOTCH2 (1p12-p11.2), PCSK6 (15q26.3), SDHA (5p15.33), and MUC17 (7q22.1) genes. CONCLUSION: Many candidate chromosomal regions that might be related to the pathogenesis of VA were detected by array CGH and should be systematically investigated to establish the causative and specific genes for VA.
Cardiology ; Coat Protein Complex I ; Comparative Genomic Hybridization ; Coronary Artery Disease ; Coronary Vessels ; DNA ; Humans ; Korea ; Polymerase Chain Reaction ; RNA-Directed DNA Polymerase ; Spasm

OBJECTIVE: Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA. It has been detected in a variety of human malignancies, suggesting that it's activity may play a role in the tumorigenic process. Also, maintenance of telomerase activity is associated with increased resistance to apoptosis. Caspase-3 activation has been found to be essential components of the apoptotic pathway. METHODS: To determine whether telomerase is involved in carcinogenesis of uterine cervix and to analyze the relationship between telomerase RNA and caspase-3 expression according to cervical cancer stage, we performed in situ hybridization for telomerase RNA and immunohistochemistry for caspase-3. The materials were 10 normal cervical tissues, 12 low grade intraepithelial lesions (LSIL), 20 high grade intraepithelial lesions (HSIL), 17 microinvasive carcinomas, 19 invasive carcinomas. RESULTS: Telomerase RNA was weakly expressed in a few basal cells of normal squamous epithelium in uterine cervix. But, high expression rate was noted in squamous intraepithelial lesions and invasive carcinoma groups. Expression of telomerase RNA was demonstrated 5 (41.6%) of LSIL, 7 (35.0%) of HSIL, 6 (35.2%) of microinvasive carcinoma, and 11 (57.8%) of invasive carcinoma. Expression of caspase-3 was demonstrated 0% of LSIL, 13 (65.0%) of HSIL, 13 (76.4%) of microinvasive carcinoma, and 7 (36.8%) of invasive carcinoma. Relationship between telomerase RNA and caspase-3 expression according to stage was not seen. Telomerase RNA and caspase-3 expression showed weakly inverse correlation in invasive carcinoma group. Telomerase RNA and caspase-3 expression was not correlated with clinico-pathologic factors, including stage, tumor differentiation, invasion depth, and lymph node metastasis (p>0.05). But, weak correlation between telomerase RNA expression and tumor size was noted (p=0.05). CONCLUSION: These data indicate telomerase might be involved in carcinogenesis of uterine cervix. Distinct relationship between telomerase RNA and caspase-3 was not seen according to stage. Expression of telomerase RNA and caspase-3 had no correlation with clinico-pathologic factors.
Apoptosis ; Carcinogenesis ; Caspase 3* ; Cervix Uteri ; DNA ; Epithelium ; Female ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Lymph Nodes ; Neoplasm Metastasis ; RNA* ; RNA-Directed DNA Polymerase ; Telomerase* ; Uterine Cervical Neoplasms

PURPOSE: The control of tuberculosis is seriously threatened worldwide by the recently emerging multidrug-resistant Mycobacterium tuberculosis. As a result, early detection of drug resistant M.tuberculosis strain has become very important but conventional laboratory methods are time consuming and delayed results often affect patients adversely in controlling tuberculosis. The authors studied the usefulness of the line probe assay to determine the mutaion in rpoB gene of rifampin resistant M.tuberculosis and to find out if this method can substitute conventional methods in the detection of resistant strain. METHODS: This study employed 40 clinical samples of M.tuberculosis which had been determined by culture and drug sensitivity test. After amplification of rpoB-the gene for the B subunit of the RNA polymerase-by PCR, the amplified products were hybridized with specific oligonucleotide probes immobilized on nitrocellulose strip and direct DNA sequencing was also performed. The results were compared with those of the classical susceptibility test. RESULTS: Among the 40 samples, 10 were identified as drug resistant strain by classical drug susceptibility test. Three of the ten resistant samples were rifampin resistant strains, which were identified by either method. All mutations were clustered within the region of 69bp of rpoB and all were single nucleotide mutations. Two isolates had a TCG->TTG(serine->leucine) mutation in codon 522. One isolate had a CAC->CTC(histidine->leucine) mutation in codon 526. CONCLUSION: In contrast to culture and sensitivity tests, line probe assay is an easy and speedy method for detecting rifampin resistant M.tuberculosis in clinical samples as well as a helpful tool for choosing antituberculosis drug in children.
Child ; Codon ; Collodion ; DNA-Directed RNA Polymerases* ; Early Diagnosis* ; Humans ; Mycobacterium tuberculosis* ; Mycobacterium* ; Oligonucleotide Probes ; Polymerase Chain Reaction ; Rifampin ; RNA Polymerase II* ; RNA* ; Sequence Analysis, DNA ; Tuberculosis

BACKGROUND AND OBJECTIVES: Telomeres are specialized structures found at the ends of eukaryotic chromosomes. Telomeres stabilize the chromosome and protect DNA from illegitimate recombination. Telomerase is a ribonucleoprotein, a RNA dependent DNA polymerase, and acts as a reverse transcriptase-like enzyme, which maintains telomere length by adding telomeric repeat units of TTAGGG to the telomeric end. These telomeric repeat units have been found only in cells with unlimited replicative potential such as sperm cells, immortalized cell lines and cancer tissues, but not in normal somatic cells. Telomerase is believed to be characteristic of and may be the best indicator of cell immortality. This study was performed to indentify the role of telomerase activity in the multistep carcinogenesis of oral squamous cell carcinoma. MATERIALS AND METHODS:We performed a telomeric repeat amplification protocol assay in 10 oral leukoplakia, 5 tongue cancers and 10 normal oral mucosa tissues. RESULTS: All the five tongue cancer tissues showed telomerase activity. Although telomerase activity was detected in 9 of 10 oral leukoplakia tissues and in 9 of 10 normal oral mucosa tissues, the activity of telomerase was low compared to that of cancer tissues by semiquantitative analysis. CONCLUSION: These findings suggest that telomerase maybe play a key role in multistep carcinogenesis of oral malignancy. Telomerase activity was detectable in normal oral mucosa with renewal activity suggested that this enzyme might be an indicator of cell proliferation.
Carcinogenesis ; Carcinoma, Squamous Cell ; Cell Line ; Cell Proliferation ; DNA ; Leukoplakia, Oral* ; Mouth Mucosa ; Mouth Neoplasms ; Recombination, Genetic ; Ribonucleoproteins ; RNA-Directed DNA Polymerase ; Spermatozoa ; Telomerase* ; Telomere ; Tongue Neoplasms

No abstract available.
Antiretroviral Therapy, Highly Active* ; Humans ; RNA-Directed DNA Polymerase*