The Study Of GAB Regulating Telomerase Assembly And Cancer Cell Senescence

Cell senescence is a relatively stable state of growth arrest after the cells in an active growth state leave the cell cycle and irreversibly lose their proliferative capacity, and is the inevitable end of normal cells. This kind of phenomenona generally exists. Cell senescence can be divided into replicative senescence and stress induced premature senescence. Our research focuses on discussing cell replicative senescence. Many changes have occurred in the process of cell replicative senescence, such as cell proliferation inhibition, cell cycle arrest, telomere shortening, DNA damage and so on, and telomere shortening is one of the most important changes, which is considered to be the underlying cause of cell replicative senescence. Currently, the cells, which are capable of inhibiting cell senescence, preventing telomere from shortening and promoting the telomere elongation, were mostly found in the stem cell and cancer cell. There are two mechanisms of telomere elongation. One is by telomerase, and another one is by ALT which exists in those cells that lack h TERT but enrich in h TR. These cells extend the telomere by recombination. But the cells rich in h TERT extend the telomere by telomerase. There are many ways to regulate telomerase activity, which can regulate the h TERT, h TR, Dyskerin and other components of the telomerase complex at the transcriptional or translational level, and can also regulate the assembly of the holoenzyme. In our study, we found that GAB located in the nucleolus can regulate the telomerase activity by regulating the assembly of telomerase holoenzyme, followed by telomere shortening and cell replicative senescence.The present molecular mechanism of telomerase assembly isn’t elaborated clearly. New regulatory protein and mechanism remain to be found, and their function and significance need to be define.Not too long ago, researchers in our laboratory found that with the increase of cell passage, cell proliferation was gradually inhibited and cell gradually became larger and flatter in GAB-knock down stable cell line when they studied GAB’s function. Hence, we hypothesized that the cells might be senescing. Therefore, we knocked down GAB in many telomerase positive cells, and it was found that the cell growth rate has slowed down, but in telomerase negative U2 OS cells, overexpressing GAB or h TERT or co-expressing GAB and h TERT did not change the cell growth rate. At the same time, we carried out the Brd U labling experiment on the telomerase positive tumor cells, and found that the cell cycle arrested in the G1/S phase. Later, we constructed long-term and GAB- knockdown monoclonal cell line in MCF-7 cells. SA-β-Gal staining showed that cell senescence occurred after long-term stable knockdown of GAB, but never occurred in MCF-7 cells of long-term stable overexpression of GAB. Since GAB was related to cell growth, cell cycle and cell senescence, then was GAB related to DNA damage? We detected several markers(γH2AX, p-atm, 53BP1, p-Chk1 and p-Chk2) of DNA damage and found that they formed Foci, so DNA damage was formed. Immunofluorescence co-staining of γH2AX and HP1α indicated that DNA damage occurred, and the cell of DNA damage is the senescent cell. To illuminate that whether cell suffered replicative senescence. Southern Blotting demonstrated that the length of telomere was shortened and the cell replicative senescence occurred after the long-term GAB-knockdown in HT1080 and MCF-7 cells.So, how do GAB regulate the length of telomere? Co-IP, GST Pull-Down, RIP and EMSA demonstrated that GAB could directly bind to h TERT and h TR. Further research showed that GAB’s BRCT domain(311-415) could directly bind to h TERT’s 500-900 domain. GAB could also bind to h TERT’s 234-500 domain as well as Dyskerin through the mediation of RNA, and bind to h TR’s 60-360 domain by its 221-322 domain. Since GAB can interact with telomerase complex, we determined whether GAB can regulate telomerase activity. TRAP showed that GAB promoted the telomerase activity mainly via its BRCT domain not only in vivo but also in vitro. Moreover, GAB-IP complex had a relatively high telomerase activity. Moreover, we demonstrated that the telomerase activity in GAB-transgenic mice was higher than wild type mice.Next, we investigated the specific mechanism of GAB regulating telomerase activity. Firstly, by WB and real time PCR, we found that GAB did not regulate the expression level of h TR, h TERT and Dyskerin. However, RIP, GST Pull-Down, His Pull- Down and EMSA showed that GAB utilized BRCT domain to promote the interaction between h TR and h TERT in vivo and in vitro. Si GAB in vivo changed the interactions of telomerase assembly complex components. For example, Si GAB inhibited the binding between Dyskerin and h TR as well as between Dyskerin and h TERT, but did not change the binding between TCAB1(that is responsible for the transportation of telomerase complex) and h TERT. As telomerase assembly is beneficial for h TERT locating to the nucleolus, we hypothesized that GAB can change h TERT’s nucleolar localization properties. Further evidence showed that GAB was associated with telomerase assembly. At present, it has been found that the protein factors regulating interaction between h TERT and h TR are a pair of ATP enzymes, and IP and TRAP indicated that GAB did not interact with Pontin and Reptin and had no association with Pontin and Reptin in regulating telomerase activity at all. So, the telomerase complex that GAB participates in is different from the complex that Pontin and Reptin participate in.Lastly, we detected the role of p53 in the regulation of telomerase activity by GAB. We found by TRAP that overexpression of p53 could promote telomerase activity in ZR75-1 cells. Moreover, we also found by TRAP that GAB regulating telomerase activity depended partly on p53. GST Pull-Down indicated that GAB could directly interact with p53. RIP demonstrated that si GAB could inhibit the interaction between h TERT and h TR in HCT116 p53+/+ cell line. And si GAB does not change the interaction between h TERT and h TR.In summary, we, for the first time, found a new telomerase assembly complex that GAB participates in. At the same time, we also analyze GAB’s position in the telomerase complex and mechanism in regulating telomerase assembly, as well as functionally main domain. In addition, we also found that GAB can regulate the replicative senescence, which lays the foundation on further understanding of telomerase and cell replicative senescence.