| The occurrence of cross-resistance to structurally unrelated drugs, designated as multidrug resistance (MDR), is a main cause of failure in the chemotherapy of tumor. Several mechanisms of MDR have been identified, such as up-regulation of drug transporter proteins (P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), etc.), detoxification enzymes (glutathione S transferases (GSTs), etc.) and anti-apoptotic stress protein HSP70, and down-regulation of some of the DNA topoisomerases and cytochrome P450.The identification and elucidation of these mechanisms have provided distinct targets and methods to solve clinical problems, and some of these have already lead to the discovery of drugs of therapeutic significance, e.g. verapamil, a specific inhibitor of P-gp, is an effective MDR reversal agent in chemotherapy. However, all known mechanisms up to date are not enough to fully explain the occurrence of MDR, and other factors may also be associated with MDR, such as the interaction between integrin and extracellular matrix (ECM). Therefore it is of great significance to find out more MDR associated factors to fully understand and solve this issue.In an attempt to identify new MDR-associated factors, we cultured human leukemia cell K562 in adriamycin (ADR)-containing medium in our previous work, mimicking clinical situation. The multidrug resistant K562 (K562/ADR) cells were established with 149-fold increase of resistance to ADR and more than 200-fold increase of resistance to vincristine (VCR) with comparison to the parental K562 cells. Protein expression profiles of K562 and K562/ADR cells were analyzed by 2-dimensional gel-electrophoresis (2D-GE), and differences between the two patterns might provide clues to the molecular basis of MDR phenotype of K562 cells after treatment with ADR. One protein with high level expression in K562/ADR cells but almost absent in K562 cells by silver stain was subject to mass spectrometry analysis, and was identified subsequently as the eukaryotic translation initiation factor 3 subunit 4 (eIF3g).eIF3 is the largest and most complicated eukaryotic translation initiation factor, which is composed of at least 12 subunits: a (p170, p150, eIF3s10), b (pll6, eIF3s9), c (p110), d (p66, eIF3s7), e (p48, int6), f (p47), g (p44, p42, eIF3s4), h (p40, eIF3s3), I (p36), j (p35, eIF3sl), k (p28), 1 (p69). eIF3 functions in the formation of 43s preinrtiation complex and priming the correct binding to mRNA. However, detailed function of each subunit of eIF3 remains unknown.Studies show that eIF3a/p170 is significantly up-regulated in human cancers of the breast, cervix, stomach, esophagus and lung, and the expression level correlates with clinical pathological types. The high level expression may be a marker of early malignancy, and may play an important role in the growth and malignant phenotypes of cancer cells. Another subunit of this complex, eIF3h/p40, also shows an increased expression in some prostate cancer and primary breast cancer. Moreover, eIF3e/int6 is considered as a prognostic parameter of non-small cell lung cancer patients. To our interest, the eIF3e/int6 homologue was found to play a significant role in multidrug resistance in a study with the model system fission yeast.As the subject of this project, there has no report about the roles the subunit eIF3g plays in human diseases including cancer except some of its function in translation initiation to date. In order to investigate the relationship between eIF3g and the multidrug resistance of cancer cells, the full length coding region of eIF3g cDNA was cloned, and GST-eIF3g fusion protein was expressed in E. coli and purified and used as antigen to immunize rabbit to obtain the polyclonal antibody, and a (His)6-tagged eIF3g expression vector was constructed and transfected into Bcap37 cells with successful expression, and subcellular localization of eIF3g was also studied by EGFP-fusion protein strategy. The details are listed below.1. Cloning of the full length coding region cDNA of eIF3gTo get the eIF3g cDNA with full length coding region for further study, a 969 bp human eIF3g fragment was amplified by reverse transcription followed by polymerase chain reaction (RT-PCR) using total mRNA from K562 as template and subsequently cloned into pGEM T-easy vector. The cDNA was verified by DNA sequencing and aligned with sequence data in GenBank database.2. Expression of GST-eIF3g fusion protein and preparation of rabbit anti-eIF3g polyclonal antiserumThe cDNA of human eIF3g obtained from above was cut out from pGEM T-easy vector with restriction enzyme EcoRI and inserted int the pGEX-5X-2, a prokaryotic GST-fusion protein expression vector, in an in-frame manner, and confirmed by Pst I and Xho I double digestion. The plasmid was transformed into E. coli strain DH5a and the expression of the fusion protein was induced by IPTG. SDS-PAGE showed an expected 60 kD GST-eIF3g fusion protein band, in both soluble and insoluble forms. The fusion protein was collected by preparative SDS-PAGE and used as antigen to immunize 2 rabbits. After a total of 4 immunizations, rabbit antisera were tested for the specificity against eIF3g.3. Eukaryotic expression of eIF3gTo establish a stable eIF3g expressing cell for futher study on the role of eIF3g in cancer MDR phenotype, the cDNA of eIF3g was inserted into the pcDNA4/HisMaxB mammalian expression vector at the EcoRI site. The construct was transfected into human breast cancer Bcap37 cells and selected with Zeocin, and single clones were obtained by limited dilution method. PCR and Western blot were used to verify the incorporation of the vector into Bcap37 genome and the expression of exogenous eIF3g. The results showed the successful expression of eIF3g in Bcap37 cells.4. Subcellular localization of eIF3gAs indicated by bioinformatic analysis, eIF3g may have nuclear localization besides its normal cytoplasm distribution. To confirm this and identify sequences responsible for the nuclear localization we constructed several GFP-fusion protein expression vectors with different regions of eIF3g including full length eIF3g (nt 51-nt1013), fragments nt214-nt1013, nt726-nt1013 and nt51-nt725. The vectors were transfected into human colon cancer cell HR8348 with Lipofectamine 2000, and observed after 48 hours under a fluorescence microscope. The results showed both cytoplasm and nuclear distribution of eIF3g.Conclusions1. Rabbit anti-eIF3g antiserum was successfully obtained;2. Exogenous eIF3g was stably expressed in Bcap37 breast cancer cells;3. Both cytoplasm and nuclear distribution of eIF3g were observed. |
