肺癌组织中端粒酶基因与端粒酶活性关系的研究

目的 探讨肺癌组织中端粒酶基因hTR、hTERT与端粒酶活性的表达是否与肺癌的发生发展有关，深入了解端粒酶基因对端粒酶活性的调控是在基因水平还是在转录水平。方法 采用TRAP－PCR和RT－PCR方法分别检测68例肺癌组织及相应癌旁肺组织中端粒酶活性、端粒酶基因hTR和hTERP的表达。结果 68例肺癌组织中端粒酶阳性率为79．4％（54／68）,hTR阳性率为98．5％（67／68）,hTERT阳性率为91．2％（62／68）。68例癌旁组织中无一例表达端粒酶阳性，但大多数癌旁组织均表达hTR(62/68,91.2%)，仅7例hTERT表达阳性（10．3％），与hTR相比，hTERT同端粒酶具有更高的一致性，其一致率为89．0％（121／136），而ThTR与端粒酶的一致率为43．4％（59／136）。结论 端粒酶的活性可能在肺癌发生发展中起重要的作用。hTR与hTERT可能是在转录后或翻译后水平对端粒酶进行调控的。     

Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase

Reactivation of telomerase plays an important role in carcinogenesis. Malignant cells almost always possess high activity and expression of telomerase. The aim of this study was to see whether there is any relationship between telomerase activity and expression and hTERT and hTERC gene amplification in acute lymphoblastic leukemia (ALL) and non-lymphoblastic leukemia (ANLL) cells. In addition telomere length was tested in leukemic cells at the time of diagnosis and during remission. Expression of the three components of telomerase (hTERT, hTERC and TP1) as well as telomerase activity was found both in ALL and ANLL cells. Telomerase activity was diminished in patients in remission. The leukemic cells showed considerable heterogeneity of terminal restriction fragments, that is telomere length. ALL cells showed a variable pattern of telomere length in contrast to ANLL cells which produced a predominantly short telomere pattern. Telomere length in the lymphocytes of leukemia patients was shorter in remission as compared to the time of diagnosis. FISH analysis revealed amplification of hTERT and hTERC genes in ALL and ANLL cells. Quantitative analysis showed that leukemic cells possess higher number of hTERT and hTERC copies than the normal PBL. Our results suggest that the activation of telomerase in leukemic cells is connected with amplification of hTERT and hTERC genes. The high expression and activity of telomerase found in leukemic cells may be partially explained by amplified hTERT and hTERC genes. Amplification of the telomerase genes seems to be a common event in carcinogenesis and may play a role in telomerase reactivation leading to cell immortalization.     

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.     

肺鳞癌及腺癌端粒酶逆转录酶表达的原位定量分析及其病理学意义

目的通过定量检测肺癌组织端粒酶hTERT的表达水平,分析肺癌与hTERT表达的定量关系,为从hTERT的表达方面探讨肺癌的诊断问题提供实验依据。方法采用免疫组化阳性单位定量法检测12例肺癌组织癌细胞和9例非肿瘤性肺组织实质细胞端粒酶hTERT的表达水平。结果端粒酶hTERT在肺癌细胞中的表达水平高于肺非肿瘤性肺组织实质细胞(P=0.0001);肺鳞癌癌细胞端粒酶表达水平高于肺非肿瘤组织实质细胞的表达(P=0.0003);肺腺癌癌细胞端粒酶表达水平高于肺非肿瘤组织实质细胞的表达(P=0.0001);肺鳞癌与肺腺癌癌细胞端粒酶表达的阳性单位差异无显著性(P=0.2282)。结论端粒酶hTERT高表达与肺癌有关。肺组织中端粒酶hTERT表达的阳性单位可作为肺癌诊断的参考指标进行研究。     

Telomerase in relation to expression of p53, c-Myc and estrogen receptor in ovarian tumours

Telomerase activity and its subunits (hTERC, hTERT mRNA) were evaluated in ovarian tumours in relation to the expression of p53, c-Myc and estrogen receptor (ER). Furthermore, relations between telomerase activity, hTERC and hTERT with known clinicopathologic prognostic factors and survival in patients with malignant tumours was investigated. Telomerase activity was determined with TRAP, hTERC and hTERT with RT-PCR, while p53, c-Myc and ER expression with immunohistochemistry. Telomerase activity and hTERT mRNA were more frequently observed in malignant ovarian tumours compared to borderline and benign tumours, whereas hTERC was present in all tumour types. p53 and c-Myc were more frequently detected in malignant compared to borderline and benign tumours. Telomerase activity was positively related to hTERT mRNA, p53 and c-Myc expression, but not to hTERC and ER expression. In malignant tumours, hTERC levels were related to tumour stage, while telomerase activity and hTERT mRNA expression were not related to any clinicopathologic feature. Tumour stage, differentiation grade, residual tumour after first laparotomy and presence of ascites were related to (progression free) survival, whereas telomerase activity or its subunits were not. In conclusion, these data suggest that p53 expression (e.g. p53 mutation) as well as c-Myc expression may have a role in regulation of telomerase activity in ovarian tumours.     

肝细胞癌端粒酶活性及hTERT mRNA表达与术后早期复发的关系

目的：研究肝细胞癌端粒酶活性及人端粒酶逆转录酶（hTERT）mRNA表达与肝细胞癌术后早期复发的关系。方法：采用ELISA—TRAP法检测60例肝癌组织及其癌旁组织端粒酶活性，RT—PCR法检测hTERT mRNA表达，5例正常肝脏组织作为对照。分析端粒酶活性及hTERT mRNA表达与临床病理之间的关系。结果：肝癌组织端粒酶活性及hTERT mRNA表达阳性率分别为86．7％（52／60）及90％（54／60），癌旁组织端粒酶活性及hTERT mRNA表达阳性率分别为40％（24／60）及43．3％（26／60）。正常肝脏组织均未检测到端粒酶活性及hTERT mRNA表达。癌旁组织端粒酶活性及hTERT mRNA表达与术后早期复发及包膜浸润、门静脉侵犯、肝内转移等恶性肿瘤的恶性生物学行为有关。结论：癌旁组织端粒酶活性及hTERT mRNA表达可能是肝细胞癌术后早期复发的预后指标。