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Objective:To observe the difference of the human telomeres RNA component (hTERC) genes’amplification in the cervical tissue by applying the environment-friendly fixative poly hydroxy acrylic acid and the transparent dewaxing solution Van-clear separately or jointly to replace the traditional fixative 4% (volume fraction)neutral buffered formalin and the conventional transparent dewaxing solu-tion xylene in the use of fluorescence in situ hybridization (FISH)for detection.Methods:In the study, 255 cases of cervical tissue specimens submitted by the Department of Gynecology in Zhongshan Boai Ho-sipital were collected from Mar.2013 to Apr.2015.Four samples were taken from the same lesion site. All the cases were divided into 4 groups and named group A,B,C,and D.Group A used 4% neutral buffered formalin fixed and xylene dewaxing to make slices.Group B used poly hydroxy acrylic fixed and xylene dewaxing to make slices.Group C used 4% neutral buffered formalin fixed and Van-clear trans-parent to make slices.Group D used poly hydroxy acrylic fixed and Van-clear transparent dewaxing to make slices.The amplification of hTERC genes in the four groups of cervical specimens was also detected by FISH technique.Results:When the hTERC genes were detected by FISH method under the fluore-scence microscope,it was obvious that the tissue profile and the background of group A,B,C and D were all clear.The probe was fixed in the accurate position so that the bright red or green fluorescence signals were easily found in these four groups.Compared with the positive rate of group A,there was no statistical significance in that of group B,C and D (P>0.05).At the same time,the coincidence rate of the FISH results was high,which showed that the new environment-friendly reagent had no significant difference in the detection of cervical hTERC genes by FISH technique.Conclusion:It is possible for the environment-friendly reagent poly hydroxy acrylic acid and Van-clear to replace 4%neutral buffered for-malin and xylene separately or jointly to detect the cervical hTERC genes by adopting FISH technique.
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Objective To explore the relationship between the expression of human telomerase RNA component (TERC) gene , human papilloma virus (HPV) infection and mutation of chromosome 3 number with cervical lesions .Methods 81 women received the treatment in the Gynecology Department of the Second Affiliated Hospital of Kunming Medical University from June 2008 to February 2009 ,including the healthy group(normal pathological examination ,20 cases) ,CIN1 group(28 cases) ,CIN2 group(12 ca‐ses) ,CIN3 group(9 cases) and cervical cancer group(12 cases) .The TERC gene expression in uterine epithelial exfoliated cells was detected by using the fluorescence in situ hybridization (FISH) method ,meanwhile the HPV infection was detected by using the real time fluorescence quantitative polymerase chain reaction (FQPCR) technology .The correlation between cervical cancer with TERC gene and HPV was analyzed .At the same time the number of chromosome 3 mutations in 81 cases was recorded .Results In the cervical lesion detection ,the detection positive rate had no statistical difference between the TERC gene detection and HPV detec ‐tion (P> 0 .05) ,their positive rates in the CIN 1 ,CIN2 ,CIN3 and cervical cancer groups were significantly higher than that in the healthy group (P 0 .05) , while between the CIN3 group and the cervical cancer group had statistical significance (P< 0 .05) ,the higher the malignant degree , the higher the positive rate .The abnormal mutation rate of chromosome 3 number was 0% in the healthy group and the CIN1 group ,16 .7% in the CIN2 group ,66 .7% in the CIN3 group and 100 .0% in the cervical cancer group ,the positive rate in the CIN3 group and the cervical cancer group was significantly higher than that in the healthy group ,CIN1 group and CIN2 group ,the differ‐ences were statistically significant (P< 0 .05) .Conclusion The TERC abnormal gene expression ,high risk HPV infection and mutation of chromosome 3 number could play an important synergistic effect during the process of occurrence and progression of cervical cancer .
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Objective To evaluate the significance of human papillomavirus L1 capsid protein (HPVL1) and human telomerase RNA component (hTERC) gene in the cytologic specimens of cervix which was infected by the high-risk types of human papillomavirus (HR-HPV),and to expose their relationship with cervical lesions.Methods The fluorescence signal of cytologic samples of cervix were detected by interphase FISH in chromosome enumeration double-color DNA probes TERC.The expression of HPVL1 capsid protein was detected by MaxVision immunohistochemistry method.300 samples were analyzed with HR-HPV positive from the cervical biopsy.The diagnoses as normal or chronic inflammation (n =45),cervical intraepithelial lesions Ⅰ grade (CIN Ⅰ,n =95),CIN Ⅱ (n =58),CIN Ⅲ (n =64),and squamous cervical cancer (SCC,n =37).Results The percentage of HPVL1 positive rates in normal or chronic inflammation,CIN Ⅰ,CIN Ⅱ,CIN Ⅲ and SCC groups were 58.70 ％ (27/46),63.16 ％ (60/95),37.93 ％ (22/58),10.94 ％ (7/64) and 0 (0/37),respectively.The percentage of HPVL1 decreased along with the increase of severity of the cervical intraepithelial lesions.Genomic amplification of hTERC positive rates in normal or chronic inflammation,CIN Ⅰ,CIN Ⅱ,CIN Ⅲ and SCC groups were 6.52 ％ (3/46),11.58 ％ (11/95),51.72 ％ (30/58),85.94 ％ (55/64) and 100.00 ％ (37/37),respectively.The percentage of hTERC increased along with the severity of the cervical intraepithelial lesions (rs =0.302,P ＜ 0.01).The percentage of HPVL+/hTERC-was 57.89 ％ in CIN Ⅰ group and 4.69 ％ in CIN Ⅲ group.The percentage of HPVL-/hTERC+ was 6.32 ％ in CIN Ⅰ group and 79.69 ％ in CIN Ⅲ group.Conclusion The detection of HPVL1 and hTERC are important for assisting cervical lesions screening and monitoring of disease progression in the HR-HPV positive cytologic specimens.
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During the progression of cervical intraepithelial neoplasia (CIN) to invasive cervical cancer,human telomerase RNA gene component (hTERC gene) amplification varies with the different stages of cervical lesions.hTERC gene plays an important role in the early diagnosis of cervical lesions and monitoring the development of cervical cancer.hTERC gene is expected to be an effective specific marker in high-risk lesions of cervical screening.
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BACKGROUND: Telomere-associated proteins wil directly affect the function of telomeres, adjust the length of telomeric DNA, which are closely related with cellsenescence and carcinogenesis. OBJECTIVE: To find the key regulatory molecules in the cellsenescence process through observing the telomere-associated factor expression in normal cel replicative senescence process. METHODS: Based on established cel replicative senescence model, reverse transcription-PCR and western blot were used to detect the telomere-associated factor expression on the molecular and protein levels, including the telomere-associated factor human telomere binding protein 1, tankyrase 1, telomerase RNA, telomere protection protein 1 and P53 expressions in the human embryonic lung fibroblast replicative senescence. RESULTS AND CONCLUSION: The results showed that with the cellsenescence, transcription of human telomere binding protein 1 did not changed, while the protein expression of human telomere binding protein 1 was increased gradual y and then decreased rapidly; mRNA and protein expressions of telomere protection protein 1 did not changed; with the human embryonic lung fibroblast replicative senescence, expression of telomere protection protein 1 was decreased gradual y; with cellsenescence, telomerase RNA component showed an increasing trend; protein expression of P53 did not changed. Human telomere binding protein 1, telomere protection protein 1 and telomerase RNA play an important role in cellsenescence.
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Objective To investigate the expression level of human telomerase RNA component (hTERC) and C-myc in cervical lesions.Methods Fluorescence in situ hybridization (FISH) was applied to detect hTERC and C-myc expression in 62 cases of cervical tissue including 35 cases of cervical intraepithelial neoplasia,8 cases of invasive cervical cancer,19 cases of inflammation as controls.Results The positive rates of hTERC on chromosome 3 in chronic cervicitis organizations,CIN Ⅰ,CIN Ⅱ-Ⅲ and cervical cancer were 5.3 ％ (1/19),16.7 ％(2/12),87.0 ％ (20/23),87.5 ％ (7/8) (x2 =36.299,x2 =40.237,P ＜0.01).The positive rates of C-myc in chronic cervical inflammation organization,CIN Ⅰ,CIN Ⅱ-Ⅲ and cervical cancer were 5.3 ％ (1/19),8.3 ％ (1/12),78.3 ％ (18/23),62.5 ％ (5/8) (x2 =30.200,x2 =34.224,P ＜0.01 ).The differences among groups were statistically significant.hTERC had a positively correlation with C-myc expression (r =0.514,P ＜ 0.01).Conclusion The expression of hTERC and C-myc on chromosome 3 is closely related to cervical cancer progression.
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Objective:To investigate the amplification of the telomerase RNA component (TERC) gene in cervical cancer and cervical intraepithelial neoplasia (CIN) using dual-color interphase fluorescence in situ hybridization (FISH) and its significance in screening of cervical lesion. Methods:Cervical cast-off cell specimens were screened from 120 outpatients with cervical lesion, including normal cell from liquid based cytology (20 cases), CIN Ⅰ , Ⅱ , Ⅲ and cervical cancer cell from liquid based cytology (each of 25 cases). The amplification of TERC gene in cervical cast-off cells was detected by dual-color interphase FISH. The results of normal samples were used to set up the threshold. Results:①The percentages of abnormal cells in CIN Ⅰ , Ⅱ , Ⅲ and cervical cancer were significantly higher than threshold value ( P<0.05), and the percentage of abnormal cells was increased with the serious degree of pathological grade (P<0.01) ; ②The percentagesof 2:3,2:4,2:5 and great than or equal to 4:4 types in CIN Ⅰ , Ⅱ , Ⅲ and cervical cancer were significantdifferences (all P<0.01). The percentages of 2:4,2:5 and great than or equal to 4:4 types in cervical cancer were significant higher than that in CIN Ⅰ (P<0.001) while the percentage of 2:3 type was significant lower than that in it (P<0.001);③The percentage of abnormal cells was increased with the serious degree of cytological grade (P<0.01) in different cytological grades. The percentages of 2:3,2:5 and great than or equal to 4:4 types in atypical squamous cell of undetermined significance (ASCUS), low-grade squamous intraepithe-lial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL) were significant differences (all P <0.01), and the percentages of 2:5 and great than or equal to 4:4 types in HSIL were significant higher than that in ASCUS( P <0.01) while the percentage of 2:3 type was significant lower than that in it ( P <0.01);④The detection rates of LSIL and HSIL with cytological examination in low grade and high grade CIN were 40% (10/25) and 62% (31/50) respectively, which were significant lower than the detection rates of FISH which was 100%(P<0.05).Conclusions:There is an abnormal amplification of TERC gene in CIN Ⅰ , Ⅱ , Ⅲ and cervical cancer, and its copy numbers are increased with the serious degree of pathological and cytological grades. Detection of amplification of TERC gene in cervical cast-off cells with FISH has a certain value for screening cervival lesion and prediction of lesion progression.
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the severity of cytological and histological grade. The evidence of hTERC, with or without amplification, might serve as a prognostic indicator to measure the grade of lesion.
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Objective:To investigate the growth inhibition of nasopharyngeal carcinoma cell strain and effect on its tumorigenic ability by AS-ODN targeting telomerase RNA.Methods:Telomerase activity was analysed by PCR-ELISA technique.Proliferation of HNE-1 cells was examined by MTT test and clone formation test.Ultrastructure changes were examined by electron microscope.AS-ODN treated HNE-1 cells were implanted subcutaneously into nude mice to observe tumor growths.Results:When HNE-1 cells were incubated with the AS-ODN targeting telomerase RNA,telomerase activities of HNE-1 cells were significantly inhibited;proliferation of HNE-1 cells was significantly inhibited,which showed a dose-dependent and time-dependent correlation and was sequential specific;swelling or necrosis was found in a small proportion of HNE-1 cells by electron microscopy but no apoptosis and large necrosis area could be found.AS-ODN reduced the tumorigenic ability of HNE-1 cell strain.The tumor formation time was prolonged and tumor growth was slowed down.Conclusion:Through inhibiting the telomerase activity of HNE-1 cell strain,the AS-ODN targeting telomerase RNA can inhibit its growth and reduce its tumorigenic ability.
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Telomerase activity appears to be associated with cell immortalization and malignant progression. Understanding how telomerase activity is regulated in vivo is important not only for understanding the molecular biology of telomerase but also for the potential clinical application of anticancer drugs. This study evaluated telomerase activity and quantified the expression of human telomerase reverse transcriptase (hTERT) mRNA and human telomerase RNA (hTR) using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method before and after the exposure of cisplatin and 5-fluorouracil (5-FU) in two head and neck squamous cell carcinoma (HNSCC) cell lines. Two human HNSCC cell lines (PNUH-12 and SNU-899) were studied. Cell cytotoxicity, the change of telomerase activity, and hTERT mRNA and hTR expression by 5-FU and cisplatin exposure were assessed by MTT assay, TRAP assay, and real-time RT-PCR, respectively. In two cell lines, after cisplatin exposure, the telomerase activity and hTERT mRNA expression decreased, but hTR expression in- creased according to the concentration of drug. However, in both cell lines, the telomerase activity and hTR did not show any significant change after 5-FU treatment, but the expression of hTERT mRNA decreased. These results suggest that there may be other important regulating mechanism except hTERT mRNA as the regulation factor of telomerase activity in HNSCC cell lines.
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Humans , Antineoplastic Agents/pharmacology , Base Sequence , Carcinoma, Squamous Cell/drug therapy , Cell Line, Tumor , Cisplatin/pharmacology , DNA Primers/genetics , Fluorouracil/pharmacology , Gene Expression/drug effects , Head and Neck Neoplasms/drug therapy , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction , Telomerase/geneticsABSTRACT
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.
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Female , Humans , Apoptosis , Carcinogenesis , Caspase 3 , Cervix Uteri , DNA , Epithelium , Immunohistochemistry , In Situ Hybridization , Lymph Nodes , Neoplasm Metastasis , RNA , RNA-Directed DNA Polymerase , Telomerase , Uterine Cervical NeoplasmsABSTRACT
To detect the expression of telomerase subunits human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA) in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect telomerase subunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100 % and 25 %, respectively. The former was significantly higher than the latter (χ2 =26.4, P＜0.01). The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100 % and 91.7 %, respectively and no significant difference was found between them (χ2 =2.1, P＞0.05). The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100 % and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.
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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.
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Humans , Immunohistochemistry , In Situ Hybridization , RNA , RNA-Directed DNA Polymerase , Sarcoma , Telomerase , Telomere , Telomere ShorteningABSTRACT
Objective To investigate the expression of telomerase-associated genes and c-myc protein before and after eradication of Helicobacter pylori(H.pylori) and to elucidate the possible correlation between human telemerase catalytic subunit(hTERT) and human telomerase RNA(hTR) and c-myc protein. Methods Thirty nine H.pylori positive and 21 negative patients were enrolled. The expression of hTR,hTERT and c-myc protein before and after H.pylori eradication were compared. The expression of hTR was determined by in situ RNA hybridization whereas hTERT and c-myc protein were detected by immunohistochemical staining. Results Before treatment,the positive expression of hTR,hTERT and c-myc protein in H.pylori -positive group were significantly higher than those in H.pylori -negative group(51.3% vs. 19.0%,53.8% vs. 23.8% ,53.8% vs. 28.6%,P 0.05). Conclusions H.pylori infection may upregulate the expression of hTERT by inducing the overexpression of c-myc protein,which can cause telomerase activation and gastric carcinogenesis. The expression of telomerase-associated genes and c-myc protein may decrease or disappear after H.pylori eradication,which can lower the risk of gastric carcinogenesis.
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To study the expression of human telomerase RNA(hTR) in different grades of bladder transitional cell carcinoma and its relation to its prognosis. With in situ hybridization, the expression of hTR was observed in 67 cases of bladder transitional cell carcinoma specimens, to be compared with specimens of 10 normal bladder tissue and 10 benign lesions. The RNA probe used in hybridization was telomerase reverse transcript (TERT), and the marker was digoxin. There was a significent correlation between the expression of hTR and grading and prognosis. hTR is considered as an important and independent prognostic factor in this carcinoma.
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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.
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Humans , Carcinogenesis , DNA , Histiocytoma, Malignant Fibrous , In Situ Hybridization , RNA , RNA-Directed DNA Polymerase , TelomeraseABSTRACT
Objective:To investigate the inhibitory effect of antisense human telomerase RNA(hTR) gene on implanted hepatocellular carcinoma in nude mice.Methods: HepG2 cells were subcutaneously inoculated into BALB/c nude mice at the axilla to establish implanted hepatocellular carcinoma model.The retrovirus plasmid containing antisense telomerse RNA(PLXSN-hTR-BamHⅠ) was injected into the tumor(0.2 ml every time,5 times).Retrovirus plasmid containing sense telomerase RNA(PLXSN-hTR-EcoRⅠ) and normal saline were inoculated as control groups.Tumor volume was determined and the inhibitory rate was calculated.Tumor necrosis was observed by histological analysis and cell apoptosis was analyzed by terminal transferase dUTP nick end labeling(TUNEL).Results: Tumor growth in antisense hTR group was significantly inhibited compared with the two control groups.The tumor inhibitory rate(26.78%) of antisense hTR group was significantly higher than that of sense hTR group(1.93%,P