Mechanism. Pot1 In Tumor And Normal Cell Chromosome End Protection And Its Significance In Anti-tumor Therapy

Telomeres are the DNA-protein complexes"Shelterin"that make up the natural ends of eukaryotic chromosomes. Shelterin is a six-protein complex consisting of Pot1, TPP1, Tin2, TRF1, TRF2 and RPA. This protein complex binds to the end of chromosome through binding of single-stranded telomeric DNA by the Pot1 protein and binding of double-stranded telomeric DNA by TRF1/TRF2. The instability of telomere DNA–protein function complex at the end of chromosome will cause DAN damage, and then leads to severe cell biology consequences including cell senescence, cell death, end-to-end fusion of the chromosome, genomic instability and so on. Thus, the telomere structure and function play an important role in the cell's life activities.So far, in Shelterin complex, only Pot1 has been found to be a 3'single-stranded telomeric DNA binding protein. The research demonstrats that Pot1 can protect telomere by inhibiting the activation of chromosome ends DNA damage, and the inhibition of the expression of Pot1 might initiate ATR-dependent DNA damage response. However, the telomere protection mechanism by Pot1 is still unknown. So, this paper focuses on the following research on the telomere protection function of Pot1 in human tumor and normal cells.1. The telomere protection function of Pot1 in tumor and normal cellsTo investigate whether there is difference existed in Pot1 ATR-dependent telomere protection function between tumor cells and normal cells, firstly, the Pot1 RNAi experiments were carried out to analyze the induced DNA damage signal pathway in tumor and normal cells. The results suggests that Pot1 has the telomere protection function by inhibiting the ATR in both tumor and normal cells.Secondly, a N-term OB-domain deleted Pot1 mutant was studied. The experiment data indicates that the single-stranded telomeric DNA binding ability was not necessary for Pot1 telomere positioning and telomere protection in tumor cells, which was opposite to that of in normal cells. Thus, Pot1 has telomere protection function in both tumor and normal cells, yet, the relationship of Pot1 structure and function essentially changed.TPP1 is one of the intracellular constitutive telomere proteins which interact with Pot1. In tumor cells, the Pot1-TPP1 interaction can mediate Pot1 nuclear import, telomere positioning, and regulate telomerase level in telomere ends for prolonging telomere DNA and maintaining telomere length homeostasis. The function mechanism of Pot1-TPP1 on telomere of human somatic cells was unknown. The effect of Pot1-TPP1 interaction on telomere of tumor and normal cell was comparatively studied. First of all, the fragment (named pot1-C3) of Pot1 interaction with TPP1 was confirmed by yeast two-hybrid system and Immunoprecipitation. Then, the key amino acid residues 486-491aa of Pot1 interaction with TPP1 was identified by substitution mutation. Furtherly, the effect of Pot1 and TPP1 interaction on mediating Pot1 nuclear import and protecting telomere in tumor and normal cells was studied. Mutated Pot1 (Pot1_M) without TPP1 binding ability was constructed, and expressed in tumor and normal cells. The results showed that Pot1_M could not enter into nuclear, and protect telomere by inhibiting ATR in both tumor and normal cells. These data demonstrated that the interaction of Pot1/TPP1 mediated Pot1 nuclear import, and regulated Pot1 function. To investigate whether the telomere protection function of Pot1 was still controlled by TPP1 after nuclear import, nuclear import sequence was added to N-term of Pot1_M(Pot1_MN). Pot1_MN was highly expressed in tumor and normal cells. It was found that Pot 1_MN in tumor cell can be imported to the nuclear, but Pot 1_MN has no telomere positioning and telomere protection function. However, in normal cells, Pot 1_MN has the similar telomere positioning and telomere protection function compared to that of wild-type Pot1. This important phenomenon indicates that the regulation mechanism of Pot1 on telomere positioning after nuclear import changed significantly.2. The effect of targeted pot1 and TPP1 nuclear interaction on the specific anti-tumor therapyThe above study suggests that Pot1 telomere protection is significantly different between tumor and normal cells. In this part of study, POT1-C3 was used to discuss whether the specific inhibition of Pot1-TPP1 nuclear interaction would be a new pathway for specific anti-tumor therapy. The high expression of POT1-C3 fragment competitively inhibited the Pot1 and TPP1 interaction by immunoprecipitatio, and this inhibition changed the telomere positioning in telomerase positive tumor cells, but not in normal cells. Besides, the highly expressed POT1-C3 caused ATR-dependent telomere damage in telomerase positive tumor cell, but not in telomerase negative tumor cells and normal cells. Therefore, POT1-C3 could block Pot1-TPP1 nuclear interaction, and then induce telomere damage in telomerase positive tumor cell. Whether Pot1-TPP1 interaction inhibition would be a new specific anti-tumor therapy pathway was further discussed. 6 different tissue-derived human telomerase positive tumor cells were transfected with Adv-POT1-C3 respectively, also transfected with empty vectors as controls. The results show that Adv-POT1-C3 inhibited the rapid growth of these cells. This growth inhibition was caused by the cell apoptosis in Adv-POT1-C3 transfected telomerase positive tumor cells by Hoechst staining and Western Blot for detecting the marker molecule PARP degradation. However, the growth of telomerase negative tumor cell U2os and 4 normal human fibroblast cells transfected with Adv-POT1-C3 was not inhibited,and there was on cell apoptosis detected in these cells by Hoechst staining and Western Blot. These data suggested that Adv-POT1-C3 was specfictively inhibited the growth of telomerase positive tumor cells, and also indicated that Adv-POT1-C3 has certain safety in anti-tumor therapy.After the Adv-POT1-C3 in vitro pharmacodynamics study,the in vivo anti-tumor efficiency of Adv-POT1-C3 was further studied. 4 representative telomerase positive tumor cells Hela, SMMC7721, HepG2 and BEL7402 was used for nude mice tumor transplant mode construction. Adenovirus was multi-injected within and around the tumor for therapy, then, nude mice were killed to get the tumor. Compared to the negative controls, Adv-POT1-C3 obviously inhibited the tumor growth in these 4 nude mice tumor transplant modes, and the total inhibition efficiency was above 80%. Different administration routes also were assessed on the effect of tumor growth. Adv-POT1-C3 inhibited the tumor growth via either local drug delivery or intraperitoneal injections. However, the effect of intraperitoneal injection was not as good as local drug delivery due to no target marker in virus. The above in vitro and in vivo anti-tumor pharmacodynamics studies indicates that Adv-POT1-C3 significantly inhibited the growth of telomerase positive cells, and it had certain safety.Conclusions:1. This paper demonstrates that Pot1 functions differently on telomere positioning and telomere protection between tumor and normal cells.2. Targeted Pot1-TPP1 intracellular interaction provides a new pathway for anti-tumor therapy.