The Mechanism Of P21^WAF1/CIP1 Promoter Activation By Chemotherapeutics In P53Negative Cancer Cells

Cancer is one of the leading causes of death in the world. Cancer isthe second cause of death in developed countries just behind heart diseaseand is well on the way to become the number one cause of death. In orderto search for better ways of cancer treatment, the association betweentherapeutic effects and the mechanism of action of cancer drugs hasattracted much attention in the field of molecular oncology. Cancer iswidely believed to be caused by alterations in several key genes. Duringmalignant transformation, these alterations contribute to the deregulationof cell proliferation. Therefore, cancer can also be considered as a cellcycle deregulation disease, therefore controlling cell growth by inhibitingcell proliferation is one of the important strategies for cancer therapy.Cyclin-dependent Kinase inhibitors (CKIs) are important negativeregulators in cell cycle progression. It mainly contains2categories: theINK4family and the CIP/KIP family. p21is the primary member of theCIP/KIP family and the first CKI gene discovered. As a potent cell cycle inhibitor, the biological effects and regulations of p21have been widelyinvestigated. By the inhibition of cyclin-CDK complex, p21suppressescell proliferation and induces cell cycle arrest. Moreover, p21is involvedin cell differentiation, senescence and apoptosis. p21is the target ofmultiple upstream signals related to cell growth and differentiation,therefore, p21plays a central role in the cell growth signaling network.Previous research suggested that p21may be a potential tumorsuppression gene. It is highly conserved in mammalian genome. p21expression was regulated by both p53-denpendent and-independentmechanisms at the transcription level. Many chemotherapy drugs couldup-regulate p21expression through different pathways, and achieve theeffect of tumor growth inhibition. However, half of the human cancers arep53deficient and p21expression is often hard to detect in these tumorcells. In p53deficient cancer cells, p21activation is mainly throughp53-independent pathway. p21research in these cells is mostly focusd onthe promoter region because promoter activity is directly associated withtranscription efficiency.In order to associate therapeutic effect with the mechanisms of p21induction in p53deficient cancer cells, we used human ovarian cancercell line SKOV3for our research. SKOV3is a p53deficient cell linederived from human ovarian cancer and resistant to some commonly usedchemotherapy drugs. We screened a variety of chemotherapy drugs to identify p21inducers in a screening that showed oxaliplatin (Oxa), aplatinum compound, could significantly activate p21promoter and alsoinduced gene expression and S-phase arrest. In the SKOV3xenograftmice, Oxa could also inhibit tumor growth, while chemotherapy drugsthat did not induce p21expression have little effect on xenograft tumorgrowth inhibition in nude mice.To demonstrate the patterns that Oxa activated p21promoter, wefirst constructed a series of mutated promoter-reporter plasmids. Byluciferase assay, we found a cis-element located between-216and-236bpthat was the key sequence responsible for Oxa induced p21activation.Meanwhile, with the help of the bioinformatics, we analyzed the-1to-500bp promoter sequence and discovered a previously unreportedvertebrate-conserved cis-element exists on the promoter. The conservedelement located between-219and-237bp, exactly overlapping theoxaliplatin response element. These data suggested the conserved elementhad important functions in the p21regulation by oxaliplatin.Consequently, our research focused on this conserved oxaliplatinresponsive element. In order to discover the transcription factors bindingto this element, we have performed the DNA-protein binding reaction invitro. Based on the sequence analysis, the conserved element could be thetarget binding sites for Sp1/Sp3and Zic1/Zic2. Therefore, we hypothezedthat oxaliplatin response may associate with these factors. We designed and synthesized biotin-labeled and unlabeled DNA probe named F3corresponding to-213/-242bp of the p21promoter, which contained theconserved oxaliplatin response element. We also synthesized other2probes F1and F2, according to the adjacent promoter sequence. Amongthese probes, only F3could bind proteins that extracted from the cellnuclear. The results further demonstrated the importance of the conservedoxaliplatin-responsive element. We then confirmed that the transcriptionfactors binding on the element were indeed Sp1/Sp3, which was alsoconsistent with the bioinformatics analysis results. In addition, oxaliplatintreatment induced Sp1phosphorylation, and Sp1/Sp3showed higheraffinity with the conserved element. Furthermore, when the Sp1/Sp3binding sites were mutated, transcription factors could no longer bindingto the element.Finally, we investigated the biological roles of F3. We found F3could activate p21promoter in SKOV3cells without oxaliplatintreatment indicating an invivo competition to bind a potential repressor.Immunofluorescence results suggested that p21expression was inducedby F3transfection. Thus, we speculated the conserved element of the p21promoter could form a repressive complex with other Sp1/Sp3sites innormal conditions. The complex negatively regulate p21promoter innormal cells to keep p21expression at a low level for their regularproliferation. When cells were stimulated by DNA damage agents, the conformation of the complex altered through phosphorylation, leading tothe loss of the promoter inhibition effects. This novel conservedcis-element acts as a transcriptional switch for p21activation byoxaliplatin in p53negative cancer cells.These results suggest that the ability of inducing p21expression ispositively related to the efficacy of chemotherapeutics and thatoxaliplatin induction of p21expression through derepression of a novelconserved palindrome sequence of p21promoter. The ability to inducethe expression of p21, therefore, provides a molecular predictor for theefficacy of selected chemotherapeutic drugs in the treatment ofp53-defective cancers. In addition, the newly revealed conservativepalindrome sequence can serve as a key component in a drug screeningsystem that can identify p21-induction compounds in p53defective cells.