The Study Of Cell Membrane Proteins And The Underlying Mechanisms Of Drug Resistance In K562 And K562/A02 Cell Lines

The cell membrane, also called the plasma membrane or plasmalemma, is one biological membrane separating the interior of a cell from the outside environment. The cell membrane surrounds all cells and it is selectively-permeable, controlling the movement of substances in and out of cells. The cell membrane is also the channel of materials exchange and signal transduction between cell to cell or cell to the outside environment. The cell membrane proteins are one of important parts for cell membrane, they participate in cell recognition and immune response, signal transduction, the inner materials and energy transmission. It is important for cell's existence, proliferation, separation and differentiation.The proteome is the entire complement of proteins, and proteornics is the large-scale study of proteins, particularly their structures and functions. Plasma membrane proteomics is one part of the proteomics. The researches of plasma membrane proteomics focus on not only the membrane protein composition, expression, modification and enriching the knowledge of the cell signal transduction, the interactions of cell to cell, and materials transmission, but also providing the theoretical basis for clinical application on vaccine and pathology. The plasma membrane protein researches are particularly important in drug discovery. Currently, they account for nearly 70% of all known pharmaceutical drug targets are membrane proteins. And the functions of membrane are controlled by the membrane proteins which account for 20-25% of total cellular proteins.Although the pure membrane proteins are the basic of membrane protein research, they have been underrepresented in proteomics studies, due to their low abundance and poor solubility of many lipid proteins. The post-translational modification also makes the membrane proteins heterogeneous, like glycosylation, phosphorylation, methylation and so on. All of them make it difficult to purify the membrane proteins. To circumvent these problems, in this study, we employed the biotin-avidin system, using the nano-particles as the carrier, and substantially increased the surface area of the carrier and the binding capacity of streptavidin compared with other general beads or particles. This original technology abandons long time ultracentrifugation and improves the efficiency of separation and purification of membrane proteins. We applied this method on tumor cells, mouse embryonic stem cells, human embryonic stem cells and human sperms cell membrane proteome studies. As an example of suspension tumor cells, we found 1294 and 1415 proteins in K562 and K562/A02 cells including 645 and 726 cell membrane or membrane relative proteins. It is one of the most efficient methods in the recent membrane protein researches. Meanwhile, we also found 8 cancer markers in K562 cells and a series of low abundance, function unknown or lipid proteins at the first time. That reflects the high sensitivity and broad applicability of the method for the future study of cell membrane proteins and also provides a reliable technology platform and theoretical basis.Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by the malignant expansion of bone marrow stem cell with an annual incidence of 1 to 2 cases per 100 000 people. K562 cells were the first human immortalized myelogenous leukemia line to be established. K562/A02 is a multi-drug resistant (MDR) cell line which builted up from K562 cells induced by adriamycin (ADM). This MDR cell line can resistant vincristine, homoharringtonine (HHT) and the other antineoplastic drugs which have the anthracene nucleus. The mechanism of MDR includes metabolism, signal transduction, RNA modification, cell proliferation and viability. In this study, we used the proteomics techniques, comparative analysis of the plasma membrane between K562 and K562/A02. Only in K562 cell's membrane proteins, we discovered 114 signaling transmission,119 metabolism,95 channel or transporter,51 vesicle transport,23 adhesion or junction,17 oxidoreductase,26 chaperone,19 cytoskeleton associated,17 modification,27 protein degradation,62 other membrane relative proteins, and 74 uncharacterized proteins. Further more, we categorized and gathered statistics of all the K562/A02 special membrane protein data, and confirmed them. It gave a new direction to find new drug resistance associated proteins.On the base of comparative analysis of membrane protein between K562 and K562/A02, in this study, we used RNAi technique, and found out the relationship between K562/A02 special protein neuronal acetylcholine receptor alpha 3 (nAChRa3) and multi-drug resistance. Utilizing nicotine to stimulate K562/A02, will enhance 1.1 times of the resistant capacity to ADM, compared with the control group(P＜0.01). Further studies revealed the nAChRa3 can promote the expression of ERK, and then increase the cell viability. This is the first time to discover the nAChRa3 in K562/A02 cell line and also the first time to relate this receptor to drug resistance. This consequence gave a new clue to clarify the mechanisms of multi-drug resistance in CML.