Establishment Of A HeLa Cell Line Expressing Mutated CDK2

Apoptosis,also known as programmed cell death,is a kind of cell death regulated by special genes. Apoptosis is a means to regulate the multicellular organism development, and maintain the stability of the internal environment. Many people found that disordered apoptosis is connected with many diseases, especially with tumors. When apoptosis was restrained, the unwanted cells do not die, it will increase the number of cells that showed growth superiority, and further develop to be tumors. Genes that promote apoptosis such as p53, bax, etc., are found to be inactivated in many tumor cells,however some anti-apoptotic genes such as bcl-2, bcl-xl is overexpressed in tumor cells. These discoveries proved that the formation of tumor has a close connection with the disorder of apoptosis. The current standard treatment for most tumors, such as chemotherapy, radiation therapy, immunotherapy, are considered to kill tumor cells by inducing tumor cells to trigger apoptosis. Therefore, to clarify the exact molecular mechanism of apoptosis, and to active the apoptotic program of tumor cells is considered to be an effective approach for cancer therapy.CDK2 is a member of the cyclins-dependent kinases(CDKs) family.It is activated by combining with CyclinA in S phase to propel the cell cycle. Some studies suggest that CDK2 also plays an important role in apoptosis. In apoptotic progression of hepatoma cells induced by Taxol, CyclinA/CDK2 acticity was significantly raised in the early stage of apoptosis, and it is correlated with the mitochondrial membrane depolarization. Similarly, in the ginsenoside (G-Rh2), TRAIL, TGF-?1, and Etoposide- induced apoptotic progression in tumor cells, upregulation of CDK2 activity was all an essential event for the apoptosis. Overexpression of the dominant negative Cdk2 (Cdk2D145N) can reduce the drug-induced apoptotic cell death. However, under the same conditions overexpression of the dominant negative Cdk1 has no effect on cell survival. Similarly, specific chemical inhibitors of CDK2 can effectively inhibit the drug-induced apoptosis in tumor cells. Shankland found that CyclinA/CDK2 transferred from the nucleus to the cytoplasm and selectivly activited in the UV- induced apoptotic progression of mesangial cells, and this activation and changed subcellular localization promotes apoptosis. Therefore, CDK2 play an essencial role in apoptotic pathway, but the mechanism of the regulation is undefined. Identifying the CDK2 substrate is meaningful to clarify the downstream of the CDK2 signal transduction, and to find the drug target for cancer therapy.Kevan Shokat's laboratory developed a approach to screen kinase substrates. In this technique, the kinase to be studied is mutated by replacing a conserved bulky residue within the ATP-binding pocket with a smaller residue. This creates an enlarged ATP binding pocket that enables the mutant kinase to utilize bulky ATP analogues that cannot be used by wild-type cellular kinases, thereby isolating the activity of the mutant kinase from all other cellular kinases. According to Yong Chi's experiments we know that we can use engineered cyclin A-CDK2(F80A) and N6-(2-Phenylethyl)-ATP (PE-ATP) analogue to label proteins with thiophosphates in cell lysates, and after digestion of the protein mixtures, we employed a single-step chemical enrichment procedure to selectively isolate thiophosphorylated peptides. Then we can get the substrate information of CDK2. We are going to screen the CDK2 substrates in apoptotic cell, expecting to find the apoptosis-related substrates of CDK2.A cell line stably expressing CDK2 (F80A) is the foundation of the screening of CDK2 substrates. We picked CDK2 cDNA from Hela cell cDNA library, and constructed it to pCS4 vector, to obtain myc tagged CDK2. pCS4 vector do not contain the neo resistance gene, so it can not be used to screen the stable cell lines. Therefore, we construct the pCMV CDK2-myc vector. Ultimately we obtained the pCMV-CDK2 (F80A)-myc vector by site directed mutagenesis. The Hela cells were transfected with pCMV-CDK2 (F80A)-myc vector by G418 selection to obtain 6-positive cell lines. We detected CDK2 (F80A)-myc expression by western-blotting, found that four out of six express CDK2 (F80A)-myc. Two of them has the same expression level with the wild-type CDK2, which can serve as a system to screen the substrates of CDK2.However,stable cell lines simultaneously express two sorts of CDK2. The way that CDK2 affect the cell cycle or apoptosis may be changed. Theoretically the wild-type CDK2 may make the phosphorylation sites of the substrates occupied, which will affect the identification of the substrates. To exclude these factors, we also expect to establish a CDK2 (F80A) knock-in cell line. Gene embeding (knock-in), also known as gene replacement, use gene sequences with homologous gene sequences within the replacement gene. We select the long homologous arm and short homologous arm in genome of CDK2, with neo resistance gene between the long arm and short arm. Mutation is in the long arm of the exon.We use genome as a template, PCR get the long arm and short arm. We construct the long arm into the pBluescript SK + vector and mutate the Phe(80) to Ala. We construct the mutated long arm into pPol 2 long neo bpA vector within the short arm. Finally, the TK gene was constructed into the pPol 2 long neo bpA vector to obtain CDK2 (F80A) knock-in targeting vector. The linearized targeting vector was transfected into Hela cells. The long arm and short arm of homologous recombination occurs, which can import mutation into the genome. Under continuous G418 selection, we get 102 positive cell lines which were not successful homologous recombination.by PCR testing.Taken together, we have successfully established a Hela cell line stablely expressing CDK2 (F80A)-myc. We also successfully constructed CDK2 (F80A) knock-in targeting vector, and screened knock-in cell lines. Our work laid a solid foundation for the future identification of CDK2 apoptosis-related substrates. It is meaningful for clarifying the apoptosis pathway and furthermore to find the drug targets for tumor therapy.