Mechanisms And Biological Significances Of The Regulation Of HTERT Nucleolar Localization

Human telomerase reverse transcriptase (hTERT) is an essential component of telomerase ribonucleoprotein (RNP) complex. It has been established that deregulated expression of hTERT is a key step towards human cellular tumorigenesis and is responsible for telomerase reactivation, telomere-length-maintenance and cellular immortalization in almost all human cancers. Most recently, it has been demonstrated that cellular transformation not only evokes the illegimate hTERT gene expression but also simultaneously alterates the behavior of this protein subcellular localization. It was found that expression of hTERT displays a dominant nucleolar accumulation in normal human somatic cells but a prominent nucleolar exclusive distribution in all cancer cells. Further more, DNA damaging stress has an effect to induce hTERT nucleolar relocalization in cancer cells. These interesting findings indicate that regulations of hTERT nucleolar localization have important implications in human cellular transformation and cancer cellular DNA damage responses. However, the mechanisms and biological significances of the hTERT nucleolar transportation in human cells have not been elucidated yet.Our previous studies have identified a novel nucleolar targeting signal (NTS) within the C-terminal sequence (aa 965-981) of hTERT. Mutational disruption of this NTS activity abrogates hTERT nucleolar localization in both normal and cancerous human cells, emphasizing its crucial role in this process. In this present study, we further investigate the regulatary mechanisms and biological significances involved in the process of hTERT nucleolar localization.A major question about the event of hTERT nucleolar transportation is whether this particular subcellular translocation of hTERT could be an essential step in the processes of the in vivo biogenesis of telomerase RNP and the function of telomerase on telomeric DNA replication. To clarify these issues, the wild-typed GFP-hTERT and the mutated GFP-hTERT-3A, which is defective in nucleolar localization in both the normal and cancerous human cells, were respectively transfected into either the normal diploid human fibroblasts BJ cells or the telomerase-negative U2os cancer cell line for stable expression. TRAP assay indicated that robust telomerase activities were readily detectable in both GFP-hTERT and GFP-hTERT-3A expressing cells, but not in the vector control cells. Our results suggested that nucleolar localization of hTERT is not required for its effect on activating telomerase activity in both normal and cancerous human cells. We next determined whether nucleolar localization of hTERT is required for its functions on telomere-length maintenance and cellular immortalization, the two well-established biological effects of this protein, in the normal BJ fibroblasts. The results demonstrated that loss of nucleolar localization does not affect hTERT abilities on maintaining telomere length and extending cellular proliferative life-span in human cells. Our results thus, for the first time, demonstrate that hTERT nucleolar localization is unrelated to telomerase functions in human normal cells.It has been reported that DNA damage can triggers a reaction of hTERT re-accumulation from the nucleoplasm to the nucleolus in cancer cells, but the mechanisms and the biological significances of such an event are still unclear at present. We firstly investigated the regulatary mechanisms of the DNA damage-induced hTERT nucleolar re-accumulation in tumor cells and have obtained the following findings: (1) induction of hTERT nucleolar re-accumulation is a DNA damage response specifically to double-stranded breaks (DSBs); (2) DNA damage induced hTERT nucleolar re-accumulation is a common phenomenon in tumor cells; (3) the C-NTS also plays an essential role for mediating the inducible hTERT nucleolar accumulation under the DNA damaging stress condition; (4) the inducible hTERT nuleolar accumulation under the DNA damage is via a ATM-dependent but p53-independent signaling transduction pathway; (5) identification of the aa932-953 amino acid sequence of hTERT a responding motif for the DNA damage-induced hTERT nucleolar re-accumulation. The successful identification of this sturctural element will certainly facilitate us to further elucidate the signaling pathway and the molecular mechanisms underlying the inducible hTERT nucleolar relocalization in the cancerous DNA damage response.The biological significances of the inducible hTERT nucleolar localization in tumor cells under the DNA damage stress have also been investigated. We demonstrated that the inducible hTERT nucleolar re-accumulation is unrelated to the regulations of its protein stability or catalytic activity under such a stress condition. Instead, we provide evidences to show that hTERT nucleolar localization is involved in suppressing the phosphorylation activations of ATM, H2AX and 53BP1 DNA damage responding proteins at DNA damage foci and is associated with an effect to promote both the short-termed and the long-termed survival of cancer cells under the DNA damage stress. We therefore for the first time reveal that induction of hTERT nucleolar localization is a inhibitory mechanism of the DNA damage machinery in cancer cells and is in related to the cancer cell survival under the DNA damaging condition.Telomere maintenance plays a crucial role in human cancer development and thus has been proposed as a new tempting target for anti-cancer therapy. Compelling evidences have demonstrated that induction of telomere dysfunction suppress cancer cell growth via activating the cellular DNA damage machinery. We therefore investigated the behavior of hTERT nucleolar relocalization in cancer cellular telomere dysfunction evoked by targetedly suppressing expression of different telomeric protein components. We found that only the telomere dysfunction induced by the TRF2 depletion has the effect of triggering the reaction of hTERT nucleolar relocalization in cancer cells; and again we showed that the event of hTERT nucleolar relocalization is associated with the effects of suppressing the phosphorylation activation of DNA damage responding proteins at telomere foci under the TRF2 depletion-induced telomere dysfunction. Finally, we showed that depletion of hTERT in telomerase-positive cancer cells also raises an acute DNA damage response, providing another evidence to support the suggestion that hTERT has an intrinsic activity to suppress the DNA damage responding machinery and that might account for its pro-survival effects in cancer cells.Based on these results, we demonstrate that hTERT nucleolar localization is unrelated to telomerase functions in human normal cells for the first time and provide evidences supporting the conclusion that hTERT nucleolar localization involve in response of DNA double-strands breaks. Our findings are of importance for understanding the post-translational regulation mechanisms of telomerase in human cells and are also of significance for facilitating the development of the telomerase-targeting anti-cancer therapy.