Study On The Up Regulation Of Cyclin A Stability In Etoposide Induced HeLa Cells

Various diseases will arise when the normal cell cycle losing control. Cell cycle over-speeding carried out abnormally, DNA replication uncompletly or in the case of under damage will lead cancer happen, many cancer happen accompanied with cell cycle proteins (cyclins) over-expression. Research and design the role of cell cycle proteins is the target of the anti-cancer drugs development.CyclinA is one of important member of cyclins family. CyclinA is a necessary protein in the cell cycle on S phase and M phase. Overseas study found that in head and neck cancer, lung cancer, esophageal cancer and other malignant tumors there are cyclinA protein over-expression. There is also a study found that in patients with primary liver cancer, cyclin A protein overexpression was about 60%. Cyclin A is implicated in the control of DNA replication because ectopic expression of cyclin A in mammalian cells accelerates the entry of G1 cells into S phase. Likewise, cyclin A can promote DNA replication in cell-free extracts from Xenopus eggs or human cells, and is sufficient to initiate SV40 DNA replication in G1 cell extracts. Microinjection of antisense cyclin A or anti-cyclin A antibodies blocks progression through S phase. Cyclin A is synthesized at the onset of S phase and localizes to the sites of DNA replication. The current model of DNA replication implicates roles of cyclin A-CDK both in the initiation of DNA replication and in the restriction of initiation to only once in a cell cycle. Cyclin B-CDC2 is the classic M phase-promoting factor (MPF) that drives G2-M transition. Cyclin A can also bind CDC2, and microinjection of cyclin A into Xenopus ocytes or mammalian cells stimulates their entry into M phase. Although cyclin A-CDK clearly exhibits MPF activity, the precise involvement of cyclin A in mitosis is obscure. Furthermore, these experiments did not reveal a distinct role of cyclin A in comparison to cyclin B during G2-M. In support of a role of cyclin A in G2-M, microinjection of antibodies against cyclin A into G2 human cells leads to cell cycle arrest before mitosis. Similarly, cells in Drosophila mutant lacking cyclin A are arrested in G2 phase. One caveat is that Drosophila cyclin A only binds CDC2 but not CDK2 and is only important for G2-M but not S phase. Almost all of the tumor cell cycle control mechanisms have led to the destruction of uncontrolled cell growth, differentiation blocked, apoptosis abnomaly.The messenger RNA (mRNA) of cyclin A2 starts to accumulate during S phase and diminishes at mitosis, slightly ahead of cyclin B mRNA. The role of E2F in the regulation of cyclin A2 transcription is particularly well characterized. E2F is inhibited by binding to hypophosphorylated pRb family proteins during G1, but their phosphorylation by cyclin D/E-CDK complexes releases E2F, which is then able to activate the transcription of genes involved in S phase progression (including that of cyclin A2). Cyclin A2 promoter is repressed during the G0 /G1 and is activated at S phase entry.Live cell imaging using cyclin A2-green fluorescent protein fusion proteins shows that human cyclin A2 begins to be degraded in early prometaphase and is completed at metaphase. Degradation of the mitotic cyclins requires a short sequence near their N-terminus called the destruction box (D-box), which acts as a signal for ubiquitin-dependent proteolysis. The major ubiquitinligase in mitosis is the anaphase-promoting complex/cyclosome (APC/C). The APC/C core complex is under complex control via phosphorylation and is activated by binding to targeting subunits including CDC20 and CDH1. CDC20 accumulates during late S phase and mitosis as a result of transcriptional activation. Destruction of CDC20 at the end of mitosis is in part mediated by its own D-box and APC/C. Formation of APC/C CDC20 complexes alone is probably insufficient to trigger degradation of their substrates, and may require the phosphorylation of APC/C by cyclin B-CDC2 and PLK. After APC/C CDC20 is inactivated, APC/C activity is maintained from the end of mitosis to late G1 by binding to CDH1. Interaction between CDH1 and APC/C is inhibited from S phase until the end of mitosis by CDK phosphorylation, most likely through cyclin A-CDK.Etoposide used mainly for the treatment of lung cancer (especially small cell lung cancer) in clinical, a variety of difficult to treat acute leukemia, such as acute lymphoblastic leukemia ALL, AML, malignant lymphoma and many other kinds of malignant tumors. Etoposide can cause DNA strand breaks, but other DNA insets difference is that it does not embed the double helix structure of DNA, or with DNA binding, but because it can interfere with DNA topoisomerase II, DNA breakage reaction to reconnect. Etoposide is a topoisomerase II inhibitor, which form a relatively stable compounds with topoisomerase II and DNA, interfere with DNA topoisomerase II on the DNA phosphodiester key role in the hydrolysis and continuous. The ability selectively reduce the normal DNA topoisomerase II activity, thereby increasing the frequency of DNA breakage and prolong the duration of DNA breakage, which causes DNA double-strand break permanent, eventually leading to cell death. Some studies show that etoposide has some effect on the normal cell cycle: people think that etoposide can stop the cell cycle S phase or early G2 phase, also suggested that the main role of etoposide in M phase. But the specific mechanism of action is unclear.In the present study, we obtained the following results:1. In the process of etoposide induced apoptosis in HeLa cells discovered the protein content of cyclin A specificity increases during the drug treatment. And2. Proved this increased expression is not caused by elevated levels of transcription.3. Proved Etoposide can cause HeLa cells G2/M cell cycle arrest, and that led to the arrest was not due to etoposide caused cyclin A increased.4. Proved that after Etoposide induces HeLa cells, cyclin A combination with ubiquitin reduced, this maybe one of the reasons that cyclin A stability increasing.Studies on the regulation of cell cycle progress will help raise awareness and understanding of the tumor cells in line for chromosome rearrangement, loss, or unequal distribution to progeny cells. Cyclins, CDKs, CKIs such as the level of cell cycle regulators will no doubt be applied to the detection of cancer diagnosis and treatment, and will be an indicator of prognosis. Depth research in this area will also open up new ways for cancer therapy, people can design a cell cycle regulators that control cell growth and division of the drug to prevent cancer cell proliferation. Molecule CDK inhibitors currently used as cancer therapy being studied in clinical trials. Study on cell cycle control mechanism will bring benefits to the cancer prevention.Our study demonstrated that in the process of etoposide induced apoptosis in HeLa cells, cyclin A increase was not due to increased transcription leads to increased synthesis, but increased cyclin A stability lead degradation slow down. And the experiment proved in cyclin A degradation pathway, the combination with ubiquitin had a little affecte. We also proved that the stability of cyclin A up-regular is not due to the cell cycle arrest. These provide a theoretical basis for future degradation mechanism research and a new anti-cancer drugs design.