Study On The Resistant Mechanisms Of K562 Cells Induced By STI571

Background and objective:Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by the malignant expansion of bone marrow stem cells. Its malignant clonal marker is Philadelphia chromosome (Ph) which harbors the Bcr/Abl fusion gene. The latter encodes a chimeric Bcr/Abl protein, P210~Bcr-Abl, with a deregulated tyrosine kinase activity and identified as having a central role in the pathogenesis of CML.STI571 (also called imatinib mesylate or Gleevec) is a protein tyrosine kinase inhibitor, which can combine competively with the ATP-binding site of P210~Bcr-Abl and inhibit aberrant PTK activity of P210~Bcr-Abl. It inhibits proliferation and induces apoptosis in Bcr/Abl positive cells and has shown remarkable clinical activity in patients with CML. However, a significant proportion of patients treated with STI571 develop resistance and some of them have primitive resistance, which usually are found in patients treated with other chemotherapy drugs. So the potential mechanisms of STI571 resistance become a new focal point.Till now, the resistant mechanisms of STI571 have yet been unknown. The possible mechanisms involve the overexpression of MDR-1, the effect of al-AGP, the amplification of Bcr/Abl fusion gene and its overexpression, the point mutant of abl kinase domain, the effects of protein tyrosine phosphatase (TC-PTP) and other PTK (LYN) and so on. In general, the resistant mechanisms to STI571 are verycomplicated and involve several potential mechanisms.To explore the mechanisms of STI571 resistance, we select a Bcr/Abl(+) cell line, K562, which is induced resistance in vitro by culturing in gradually increased concentrations of STI571.We hope the establishment of the resistance cell line will be conducive to study on STI571 resistance as a new cell model.Materials and Methods:1. The induction of STI571 resistance cell line (K562-R):The wild-type K562 cells (K562-W) were cultured in gradually increased concentrations of STI571.2. The analysis of K562-R characteristics:The growth curve was drawn by counting cells and the percentage of living cells was examined with trypan blue staining. The cytotoxic effects of K562-W and K562-R treated with different concentration of STI571 were analyzed by MTT assay and the apoptosis effects by Hoechest33342 staining.3. The preliminary studies of the resistant mechanisms:MDR-1 expression examining, MTT assay, sequence analysis, interphasal fluorescence in situ hybridization (I-FISH) and cDNA array were used to study the potential mechanisms of acquired resistance of k562-R.Results:1. The establishment of K562-R and its characteristics:We establish a STI571 resistance cell line by culturing K562-W in gradually increased concentrations of STI571 over a period of six months. K562-R shows exponential growth in 0.5uM STI571 and its survival percentage is 98.5% by trypan blue staining. IC50 (50% inhibiting concentration) of K562-R and K562-W are (1.64 +0.13)uM and (3.32 ± 0.05)uM respectively by MTT assay and the resistance to STI571 increases to (2.04+0.16) fold. The apoptosis percentage of K562-R treated with different concentration of STI571 decreases significantly as compared with that of K562-W, and especially with 0.5uM STI571, shows 10 fold decrease.2. The analysis of the induced resistance mechanisms:The MDR-1 expression percentages of K562-R and K562-W with FASC analysis are 2.68% and 1.39% respectively, showing significant difference(P<0.001). However, it is testified that there is no statistic difference after comparing the cytotoxic effects of K562-W and K562-R treated respectively with different concentration of adriamycin(ADR) and harringtonine(HT) by MTT assay. No point mutant in the Bcr/Abl ATP-binding site is detected and the copies of Bcr/Abl fusion gene increase in K562-R by I-FISH analysis(P < 0.001). Analyzing different expression genes of K562-R with cDNA array, we detect different expression of 662 genes. 335 genes of them are up-regulated including hematopoietic cell-specific Lyn substrate 1 (HCLS1) gene and 327 genes down-regulated including receptor type protein tyrosine phosphatase f polypeptide (PPFIA2) gene. In particular, 81 genes down-regulated in K562-W treated with STI571 are up-regulated or no difference in K562-R including signal transduction genes (n=18) and metabolism/protein synthesis genes (n=17). It is possible that these genes participate in the process of resistance to STI571 in K562-R cells.Conclusions:1. In vitro, We establish a resistant cell line(K562-R) with resistance to 0.5uM STI571 and (2.04+0.16) fold resistance as compared with that of K562-W.2. The mechanisms of resistance of K562-R involve amplification of Bcr/Abl fusion gene.3. Comparing gene different expression profile of K562-R with that of K562-W treated with STI571, we conclude that signal transduction genes and metabolism/protein synthesis genes possibly involved the acquisition of STI571 resistance.