Expression And Purification P53^N239S Protein And Mouse Suffered Lung Cancer Was Caused By Oncogene Kras^G12D And P53^N236S

In tumor tissue, mutant p53 is a common mutation. Most of mutant p53 proteins gain function of promoting tumor progression. Hence, mutant p53 has become a target of cancer therapy. In tumorus aging model cell lines, we found a same p53 mutation, named p53S (p53N236S, in human, p53N239S). In early studies, we found that p53S protein lost the ability of binding DNA and regulating downstream genes. p53S protein could cooperate with Ras, leading to tumorigenesis. In early stage, p53S knock-in mouse could have a risk of malignant tumor, indicated that p53S protein got function of carcinogenesis, and it might be a key target for cancer therapy. Because p53S has directly related to aging induced tumorigenesis, the research of mutation characteristics of p53S will contribute to understand the relationship between aging and tumorigenesis. Cancer molecular epidemiology comfirmed that p53S mutation existed in breast cancer, bladder cancer, colorectal cancer, gastric cancer, brain tumors, indicated the important role of p53S in tumorigenesis, and p53S may be one of targets for cancer treatment.Therefore, first part of this study aims to obtain purified p53S protein to lay foundation for p53S crystal preparation. We used site-directed mutagenesis to construct pGEX-4T-1-p53S prokaryotic expression plasmid. Then we transformed pGEX-4T-1-p53S plasmid into Escherichia coli BL21 (DE3), and used IPTG to induce the GST-p53S fusion protein overexpression. Next, we used affinity chromatography to obtain purified GST-p53S fusion protein by GST tag. Last, we used hydrophobic interaction chromatography to eliminate thrombin and other proteins, obtain purified p53S protein.We found that p53S protein was not stable in room temperature. With the extension of time, p53S protein was gradually degradated. p53S protein was also sensitive to pH. At pH6.3, p53S protein was stable. Higher pH or lower pH will increase the dissociation and degradation of p53S protein. GST-p53S protein was more stable than p53S protein, but with the extension of time, GST-p53S protein was also gradually degradated.Because we obtained the p53S protein in prokaryotic system, the protein’s structure may different from human p53S protein. However, this results will provide support for small molecule drug discovery, molecular simulation and etc.In other hand, we aims to study the interaction between KrasG12D and p53N236S in mouse model. The mutation of oncogene Ras will respond to tumor suppressor gene p53 and induce cell senescence and apoptosis. When p53 is inhibited or mutated, it will lose the normal monitoring of Ras, leading to excessive cell proliferation and tumorigenesis. Some p53 mutations even gains the potential of oncogene. When such kind of mutant p53 cooperate with mutant Ras, it makes organisms more sensitive to tumor, and these tumors are more malignant. In early studies, we found that p53S could cooperate with Ras to promote tumorigenesis. When we injected p53-/-+p53S+HrasG12V MEF into SCID mouse, SCID mouse suffered from tumor, indicating that oncogenic function of p53S was enhanced by HrasG12V. Because these results were obtained in HrasG12V overexpression and in vitro, they couldn’t fully reflect the interaction of oncogene Ras and p53S in vivo. So we design to obtain KrasG12D/+:p53S mouse to detect the interaction between KrasG12D and p53S in vivo.In this study, we used 3 genetically engineered mice, Cre mouse (Cre recombinase protein could be induced by tamoxifen), KrasG12D/+mouse (LSL was in front of KrasG12D and could be excised by Cre recombinase), p53S mouse, to obtain Cre KrasG12D/+p53S mouse. We designed to inject tamoxifen (1 week, continuously) to Cre KrasG12D/+p53S mouse in 4-6 weeks. After 6-8 weeks, we would get lung tissue and analysis the pathological condition of mouse by H&E staining and IHC. At the same time, we would get all tissues and analysis the expression of KrasG12D and p53S by WB.We found that the birth rate of Cre KrasG12D/+p53S mouse was very low, and they were smaller than other mouses. LSL was still exist in 4 weeks. However, the mouse suffered from lung cancer in 8 weeks, but we hadn’t injected tamoxifen. We detected LSL in lung tissue and found LSL was excised. This phenomenon indicated that KrasG12D/+was activated. The results of IHC and WB also comfirmed the activation of KrasG12D/+. However, we have not known which mechanism activate KrasG12D/+.In this study, we found that mouse suffered from lung cancer because of the abnormal activation of KrasG12D/+, but the mechanism had not been known. In cells, exploring the interaction between KrasG12D/+ and p53S was preliminary. Subsequently, we will study the molecular mechanism in detail.