Telomere Maintenance Mechanism And Its Key Protein In Telomerase-inhibited Laryngeal Cancer Cells

Telomere length is associated with the proliferative capacity of cells. Whereas normal cells with reduced proliferative capacity exhibit low telomerase activity and shortened telomeres, cancer cells maintain telomere length primarily by reactivation of telomerase. The telomerase enzyme which adds specific DNA sequences to the ends of chromosomes is composed of three elements:human telomerase reverse transcriptase (hTERT), telomerase RNA, and dyskerin. While the majority of tumors show high telomerase activity, some cancer cells may utilize the alternative lengthening of telomeres (ALT) mechanism. The ALT-mediated elongation of telomeres has been associated with homologous recombination events between sister chromatids at telomeres, known as telomeric sister-chromatid exchange (T-SCE). ALT cells are usually characterized by a remarkable heterogeneous telomere length within a given cell and the presence of promyelocytic leukemia (PML) nuclear bodies that contain telomeric DNA and telomere-binding proteins. While these ALT-associated PML bodies (APBs) may be used to identify tumors which use the ALT pathway, their function is still unknown.It has been hypothesized that cancer cells may activate the ALT pathway when telomerase is inhibited or otherwise rendered non-functional. For example, when cells with active ALT were fused with normal somatic cells or telomerase-positive cancer cells, the ALT activity was suppressed, suggesting that it is normally repressed. Another study demonstrated that human fibroblasts with spontaneous inactivation of telomerase had sustained proliferation in vitro, although these cells did not express APBs or telomere length patterns typical of ALT. When treated with the demethylating agent 5-aza-20-deoxycytidine, these cells reactivated telomerase. Still, other studies suggest that ALT and telomerase can occur together in human cancer cells. Therefore, the environmental conditions which trigger or maintain the ALT pathway are yet to be precisely defined. In a previous study, we reported that some laryngeal cancer cells could still replicate after RNAi-hTERT-based anti-telomerase treatment, but whether the survival of these cells depended on the ALT pathway upon inhibition of telomerase was not clear. In the present study, we collected cells surviving RNAi-hTERT treatment and analyzed their hTERT levels, telomerase activity and APBs expression in order to explore the telomere maintenance mechanisms (TMM) in laryngeal squamous carcinoma (Hep-2) cells when telomerase is inhibited. We also evaluated differences in invasive ability, tumorigenicity, telomere length and T-SCE between Hep-2 cells and the surviving cells. We further performed a proteomic survey of proteins differentially-expressed between the two cell lines by two-dimensional gel electrophoresis (2-DE) and ESI-MS/MS and validated the differentially expressed proteins by immunoblot analysis. These ALT specific proteins may be candidates for further studies as diagnostic, prognostic or therapeutic tools for cancer.Objective:Using RNA interference to inhibit telomerase activity in laryngeal carcinoma cells, collect and study the prognosis and characteristics of surviving cells. Identify the telomere maintenance mechanism and the changes of the tumor characteristics through the study for the telomere dynamics and tumor characteristics. To compare differences proteins between two cell and selected the key protein in telomere maintenance mechanisms, that provide a new target and direction for gene therapy for laryngeal cancer cells.Methods:A plasmid termed pEGFP-shTERT was constructed.After transfected with the plasmids, cells was analyzed and sorted by flow cytometry. Continue to cultivate the cells to detect growth. After stabile passage, we measure the telomerase activity and detect the expression hTERT and APBs bodies to clear the characteristics of surviving cells.To detect the telomere length between untreated laryngeal carcinoma cells and surviving cells by FLOW-FISH and T-SCE levels by CO-FISH. To compare the invasion and tumorigenicity by transwell experiments and tumor formation in vivo. Using two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS) technology to screen and identify the differences protein between the untreated laryngeal cells and the surviving cells in the cell nucleus, and used between the nucleus Westernblot method validation expression.Results:Flow cytometer had sorted the transfected laryngeal carcinoma cells that expressed green fluorescent successfully. Interference early, the proliferation of the cells accompanied by a large number of aging and apoptosis, but there are still a few cells can survive about 15 days, the cells begin to stabilize the growth of passage. Compared with untreated laryngeal carcinoma cells, telomerase activity in surviving cells was inhibited, hTERT expression significantly decreased accompanied by increased expression of APBs bodies, the difference was statistically significant(P<0.05). The result showed that the surviving cells has long telomere length, telomere structure analysis revealed that the ALT mechanism specific T-SCE structure exist in surviving cells. Transwell experiments and tumor formation in vivo confirmed the tumor growth and invasion in surviving cells is more weaker than the untreated cells.Using two-dimensional electrophoresis and mass spectrometry and verified by Westernblot, we found that compared with untreated cells, the proteins of SAGE1, GAS8, PML, SYCE1, NBS1 high express in surviving cells (P<0.05), suggesting that these proteins may be the key proteins for the ALT mechanism. On the contrary, the proteins TERT and SMC1βthat increased expressed in untreated cells (P<0.05) may be the key proteins in the telomerase mechanism.Conclusion:In the surviving cells, telomerase activity was eliminated. Additionally, these cells were enriched for the APBs bodies, suggesting an alternative mechanism for telomere maintenance following telomerase inhibition. These results could have major impacts in designing new cancer treatments.The surviving cells has long telomeres. Similar to ALT cells, the surviving cells showed evidence of ALT telomere homeostasis. Invasiveness and tumorigenicity changes in surviving cells suggest possible changes in the characteristics of the tumor.The proteomics study found that the difference proteins between two cells may be the key protein in telomere maintenance mechanisms. The discovery of these key proteins provide a new target to improve telomere-based therapy for cancer.SummaryHere, we report that surviving Hep-2 cells that survived anti-telomerase treatments showed sustained proliferation in culture with down-regulated hTERT expression and the greatly raised levels of alternative lengthening of telomeres specific promyelocytic leukaemia bodies. Analysis of the telomere length kinetics also demonstrated elevated telomeric sister chromatid exchange (T-SCE) in surviving Hep-2 cells, consistent with their long telomeres. Similar to ALT cells, the surviving cells showed evidence of ALT telomere homeostasis. Furthermore, proteomic analysis identified several proteins differentially expressed between the original Hep-2 cells and surviving cells that may provide new insight for understanding these two telomere maintenance mechanisms. Thus, the findings in this study may help to improve telomerase-based therapy for cancer.