The Anti-cancer Strategy Mechanism Study Of Targeting Telomere G-quadruplex

The eukaryotic chromosomes are protected at both ends by telomere, a specialized structure consists of repetitive TTAGGG DNA tracts and associated proteins.Human telomere DNA terminates with a3'single-stranded G-rich overhang that can fold into a four-stranded G-quadruplex structure and inhibit telomerase activity. Telomere length homeostasis is a prerequisite for the unlimited growth potential of cancer cells. In more than85%cancer cells, telomere length homeostasis is maintained by telomerase, a ribonucleoprotein that synthesize telomere repeats onto telomere ends to compensate the loss of telomere sequence in DNA replication. In others15%cancer cells, telomere length homeostasis is maintained by the alternative lengthening of telomere (ALT) mechanism which based on the strands exchange.In1999, H. Hurley groups reported the processivity of human telomerase decreased as K+concentration increased, and the banding patterns of telomerase reaction products in the presence of high K+concentrations are very similar to those generated in the reaction with early G-quadruplex interactive agents,such as TMPYP4and BSU-1051.From then on, reseachers began to connect the IC50of the ligands in the TS-TRAP with their abilities of stabilization on G-quadruplex.I select four reported G-quadruplex interactive agents:BMVC,Zn-TTAPc, TSPc and Cu-TSPc, extending the TS substrate use one wild telomerase and two hTR temple-mutated telomerases(CUA and CAC). This work raises a concern on how much telomere G-quadruplex stabilization by ligands we are exploring can actually contribute to the inhibition of telomerase-mediated telomere extension using the conventional TS-TRAP and Single-tube/Two-color TRAP (ST/TC-TRAP) assay.These observations surprise us for they demonstrating that the ligands are much more than G-quadruplex stabilizers with strong off-target effect and inhibit telomerase via multiple pathways among which stabilization of telomere G-quadruplex may only make a minor or neglectable contribution. This conclusion demonstrates that the IC50of direct primer extension,TS-TRAP and TSG4-TRAP can not be measured as the ability of stabilization of G-quadruplex by ligands.It also cause us to study the anit-cancer pathway of stabilization of telomere G-quadruplex by ligands.And the ST/TC-TRAP can be a useful tool for us to investigate the specific and unspecific inhibition by ligands in the process of telomerase-mediated telomere elongation.bisQMP is a potent, water-soluble and photoinducible DNA cross-linking agent synthesized by Zhou groups.BisQMP possessing a quaternary ammonium group can form o-quinone methide by photoactivation using visible light in aqueous solution. o-QM has been found to have significance for their nucleic acid bases and DNA alkylation. I cross-link the bisQMP with a oligonucleotie complementary with the chromosome telomere. The photoinducible DNA cross-linking probe showed good sequence recognition to the telomeric sequence in the the gel-shift and PCR stop assays in vitro.We expected this probe could specially target and alkylate with the chromosome telomere in Hela cells.And the photo-adduct formed in the telomere can stop the telomere replication or the telomerase miedated telomere elongation. So the rapid loss in telomere can lead to the cell apoptasis. However, after this probe deliveried in the Hela cell, we compared the apoptasis ratio by the photocontrolled probe complementary with telomere and the photocontrolled random probe in the Annexin-V-FITC apoptosis assay, resulting in little difference between them. Considering the nonspecific interactions between oligonucleotides and cellular components, such as cellular proteins or non-target mRNA and DNA sequences, the experiment is still in progress.