Detection Of PTP Activity And Sequencing Of ATP Binding Domain Of Bcr-Abl Gene In Chronic Myeloid Leukemia Resistant To STI571

Background and objective:Chronic myeloid leukemia (CML) is a malignant disorder arising from hematopoietic stem cell. As the tumor marker of CML, Philadelphia chromosome (Ph) leads to the formation of BCR-ABL fusion gene which encodes a cytoplasmic protein, P210^BCR-ABL . The protein tyrosine kinase(PTK) activity of P210^BCR-ABL is aberrantly regulated, which is considered as the sufficient and necessary factor in the pathogenesis of CML. The phosphorylation degree of tyrosine in cytoplasmic protein is regulated by the coordination of two enzyme families, including PTKs and protein tyrosine phosphatases(PTPs).To inhibit the PTK activity of P210^BCR-ABL and induce apoptosis of tumor cells is a new strategy in the treatment of CML. STI571(Gleevec) is a new approved PTK inhibitor. By targeting the P210^BCR-ABL directly, STI571 can inhibit the proliferation and induce apoptosis of tumor cells by disturbing the downstream signal pathway. Our former data found that some family members of PTPs upregulated in k562 cells after the treatment of STI571, which indicated some potential PTPs members should have participated in the apoptosis induction of K562 cells. In anotherway, more and more clinic data indicated that STI571 resistance is a critical problem in the treatment of CML. The resistance mechanism is extensive, including the expression of MDR, genetic aberrance of bcr-abl gene itself and the activation of other important PTKs.In this article, we try to find the potential PTPs members related to the STI571 resistance in a resistant cell line, K562/G01, with expression profile michroarray and FTP activity analysis. In addition, we detected the activity of PTP in CML patients treated with STI571, and analyzed the DNA sequence of the ATP binding loop of Abl gene at the same time. Materials and Methods:1. Biologic characteristics assay of K562/G01:(1) cytotoxic effect of STI571 on K562/G01 were measure by MTT assay;(2) apoptosis inducing effect of STI571 on K562/G01 was measured with Hoechest33342 staining.2. An expression profile cDNAmicroarray, BioStarH40s, was used to dectect the potential differential expression genes of PTP members. And the results were validated by semi-quantitative RT-PCR technique. Furthermore, the total PTP activity of tumor cells from CML patients and of K562/G01 were detected with the Tyrosine Phosphatase Assay System.3. With the PCR products, the DNA sequence of ATP binding domain was analyzed, in order to detect the potential DNA mutation mechanisms of acquired resistance ofSTI571inCML.Results:1 STI571 resistance assay with MTT: the IC50 of K562/ G01(5.67+/-0.10)uM is ten times high of that of K562-W (0.48+/-0.20)uM. Apoptosis percentage assay with Hoechst 33324 staining found only 1.5% being apoptosis cells, which indicatesK562/G01 is resistant to the apoptosis inducing effect of STI571.2. Among 566 differential expression genes found with BioStarH40s, 297 down-regulated and 269 up-regulated. With further analysis we found one PTPs member, PTPRF(PPFIA4), down-regulated and one PTKs member up-regulated. The semi-quantitative RT-PCR validated the results of microarray detection.3. The PTP activity of K562/G01 were lower than that of k562/W. And the same phenomenon was found between the mononuclear cells of STI571 resistant CML cells and non-resistant cells.4. No point mutant in the bcr/abl ATP-binding site was detected in K562/ GOl.This single nucleotide C-*T change results in a substitution of threonine to isoleucine at position 315 (Thr315-*Ile; T315I) of c-Abl kinase in two acquired STI571-resistant patients o This single nucleotide A-*G change results in a lysine to Arg substitution at position 1070 (Lys — ArgI) of c-Abl kinase in a CML-CP patient.Conclusions:1. The mechanisms of STI571-resistance of K562-R involved the expression of PTP member.2. ATP binding domain mutants of Abl kinase were one of the major mechanism of resistance to imatinib.