Proteomic Analysis Of The Molecular Mechanism Of Anti-cyclin D1Intracellular Antibody

Cancer is a serious disease that harm to people’s health. Cell cycle disorder is oneof the main causes for malignant proliferation of tumor cells. As first synthesizedcyclin in cell cycle, cyclin D1can form complex with CDK4/6and thenphosphorylate the substrates, such as RB protein. Cyclin D1palys an important rolein G1to S transition. The cyclin D1gene is overexpressed in many human cancers.This overexpression leads to the activated state of CDK4/6, then cause disruption ofcell cycle control, ultimately cause over-proliferation of tumor cells. So cyclin D1is apotential target for cancer therapy.With the development of hybridoma, monoclonal antibody and antibodyengineering, the intracellular antibody technique that uses antibodies as effectormolecules has been emerged in recent years. It can make the target protein inactiveefficiently and specifically. The intracellular antibody opens up a new way for tumorgene therapy. Single chain variable fragment (single chain Fv, scFv) has been widelyapplied to the intracellular antibody preparation because of its simple structure, lowermolecular weight, easy design.In our previous study, an endoplasmic reticulum-retained anti-cyclin D1scFv(ER-ADκ) has been constructed. Stable expression of ER-ADκ in MCf-7cell cansignificantly inhibit the growth and metastasis of tumor cells and lead to cell cyclearrest and cell apoptosis. But the molecular mechanisms of ER-ADκ in cell growthinhibition has not been clear. Proteomics techniques was used in this study toinvestigate the molecular mechanism of anti-cyclin D1scFv in cancer cell proliferation inhibition due to its advantages in cancer biomarkers screening,molecular mechanism study and understanding of intracellular signal transductionpathways.The specific research works are as follows:1. The method improvement of protein extraction of MCF-7cells fortwo-dimensional electrophoresis (2-DE)Five published methods of protein extraction for2-DE have been tested in thisstudy. The SDS-PAGE and2-De results showed that there are remains of saline ionsin the protein extracted by these methods, which disturbs the analysis of2-DE. Inorder to solve the above problem, the improved TCA/Acetone method was set up inthis study. This improved method was based on traditional TCA/Acetone method andcombined with SDS Dense extraction. This improved method can remove remainsaline ions from the protein products, make2-DE gels background more clearly, sothat942spots were detected in the2-DE gel. Meanwhile this method can reducehorizontal streaking in the low molecular weight and high isoelectric point regions of2-DE gels and has high repeatability and resolution ratio. These advantages of theimproved method lay the foundation for analysis of differential proteome betweenMCF-7/pBg-ER-ADκ and MCF-7/plpBg cell lines.2. Analysis and identification of differential proteome between MCF-7/pBg-ER-ADκand MCF-7/plpBg cell lines43differential expression protein spots have been found by2-DE analysis ofMCF-7/pBg-ER-ADκ and MCF-7/plpBg cell lines. Then24protein spots of theseproteins were identified by using mass-spectrometric technique. The identifiedproteins are involved in metabolism, transcription and translation, protein transport,electron transfer and cytoskeleton and so on. This study provides the preliminarydifferential proteomic data for molecular mechanisms of ER-Adκ.