The Effect And The Mechanism Of HER2/neu Overexpression On The Amount Of P53 Protein And Cell Proliferation And Apoptosis In Breast Cancer Cells

IntroductionHER2/neu overexpression occurs in up to 30% breast cancer and several other kinds of tumors, and was correlated with poor prognosis and therapeutic resistance. Recent research indicates Herceptin, a humanized anti-HER2/neu monoclonal antibody can increase the sensitivity of HER2/neu overexpression breast cancer cell to the chemotherapy or irradiation, and improve the suiviving time in the HER2/neu overexpression breast cancer petients. However, the mechanism for these phenomena is complicated and not very clearly known so far. Furthermore, the efficiency of Herceptin therapy is only 15-20%, even in the HER2/neu overexpression breast cancer petients, suggesting some other factors affect the efficacy of Herceptin therapy.The product of tumor suppression gene p53 is crucial in regulating cell growth and apoptosis. p53 is often mutated or deleted in many types of tumors (about 60-70%). In addition, many tumors with a wild-type p53 gene do not have normal p53 function, suggesting some oncogenic signals suppress the function of p53.HER2/neu overexpression has been reported to activate the Akt pathway and to delay p53-mediated apoptosis, more p53 protein and inhibiting of PI3K pathway can be seen in the anti-HER2/neu monoclonal antibody, Herceptin, treated cells. These phenomena suggest HER2/neu overexpression may affect quantity or activity of p53, thus affect cell proliferation and apoptosis through activation of PI3K-Akt pathway.Cells with Ras gene mutation or transformed by Ras become resistant to p53 dependent apoptosis. As the analogues of human epidermal growth factor recptor, HER2/neu overexpression can activate Ras/Raf/MEK/MAPK pathway. However, there is no report on whether it can affect the expression of p53 protein, the cell proliferation and apoptosis through this pathway.In the present study, we investigated the effect of HER2/neu overexpression on the amount of wild p53 protein through PI3K and Ras/Raf/MEK/MAPK pathways. We also detected how the above change affected cell growth and sensitivity to drug and irradiation therapy in human breast tumor cells. In addition, we analysed the possible mechanism about therapeutic resistance in HER2/neu overexpression patients. Our research may have implication for the breast tumor therapy.Part I Preperation of anti-HER2/neu monoclonal antibody and its inhibiting ability on the growth of breast carcinoma cellsPurpose: To prepare specific anti-HER2/neu monoclonal antibody, and select clones which can inhibit the growth of HER2/neu overexpression breast tumor cells.Methods: The HER2/neu protein expressed by prokaryotic vector was used to immunize female Balb/c mice. After cell fusion, ELISA, immunocytofluorescence,immunohistochemistry and MTT were used to select clones.Results: HER2/neu fusion protein was successfully expressed with prokaryotic system. 8 hybridoma cell lines were got which can consistently produce anti-HER2/neu monoclonal antibody. These antibodies can be used in immunohistochemistry detection and 5 clones can secrete antibody inhibiting the growth of HBT133 cell. Conclusions: Anti-HER2/neu antibody was successfully prepared which can be used for immunohistochemistry detection and humanization for the future therapy. PartII Overexpression of HER2/neu on the effect of p53, p21, mdm2 protein in MCF7 breast cancer cellPurpose: To investigate the effect of HER2/neu overexpression on p53, p21 protein and the expression and localization of mdm2 in MCF7 cell.Methods: Lipofectin-mediated gene transfection method was used to establish HER2/neu overexpression MCF7 cell. Western blot was used to detect p53, p21 protein, semi-quantified RT-PCR was used to detect p53 mRNA level; Western blot and immunocytofluorescence were used to detect the expression and localization of mdm2.Results: HER2/neu overexpression MCF7 cell (MCF7-neu3) was established. There was less p53 protein in MCF7-neu3 than that in parental MCF7 cell and empty plasmid transfected MCF7 cell while p53 mRNA remained the same; There was mor...