Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells

STI571, an Abl-specific tyrosine kinase inhibitor, selectively kills Bcr-Abl-containing cells in vitro and in vivo . However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P-glycopro-tein (P-gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [¹²⁵l]azidoagosterol A-photolabeling of P-gp, but not that of MRP1. K562/MDR cells that overexpress P-gp were 3.67 times more resistant to STI571 than the parental Philadelphia-chromosome-positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB-G2 cells that overexpress P-gp, but not Bcr-Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP-16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB-G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P-gp, but is less efficiently transported by P-gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P-gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P-gp-expressing KB-G2 cells.     

K562多药耐药细胞系中肿瘤干细胞样细胞对伊马替尼耐药机制的初步研究

Objective： To characterize a novel chronic myeloid leukemia （CML） cell line and to further elucidate the mechanisms of resistance to STI571. Methods： A novel K562 cell line （K562NP16） was achieved after exposure of the K562 cells to VP16. A small subpopulation （K562NP16 SP） that was capable of excluding Hoechst 33342 in the K562NP16 cell line was isolated by fiow cytometry sorting. The rest of the K562NP16 cells were classified as non-SP K562NP16. The mechanisms involved in K562NP16 SP cells which became resistant to STI571 were studied. Results： The levels of Bcr-Abl and Abl proteins were similar in the K562 cell line and in non-SP K562NP16 and K562NP16 SP cells. The multidrug-resistant gene 1 （MDR1） expression of the 170 kDa P-glycoprotein （P-gp） was detected in K562NP16 non-SP and K562NP16 SP cells but not in K562 cells. The expression levels of P-gp in the two K562NP16 cell lines were similar. Compared with non-SP K562/ VP16, the K562NP16 SP cells were more resistant to STI571. This resistance could hardly be reversed by many multidrug resistance inhibitors. In addition, in vivo study showed that the K562NP16 SP cells induced tumorigenesis in mice, while the K562NP16 non-SP cells failed to do so. Conclusion： A novel K562 cell line, K562NP16, was generated. A small side population K562NP16 SP cells, had high resistance to STI571 treatment and more tumorigenic than the K562 cells. It may represent the cancer stem cells of the K562NP16 cell line.     

Aminopeptidase inhibitors inhibit proliferation and induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta pathways

A tyrosine kinase inhibitor, STI571, has been demonstrated to be effective for the treatment of chronic myelogenous leukemia (CML). STI571 inhibits tyrosine kinase activity of ABL and induces apoptosis of CML cells. However, drug resistance develops commonly in patients with blast phase CML, and has become a significant therapeutic problem. We examined the effects of aminopeptidase inhibitors on CML cell line (K562) and a STI571-resistant subline of K562. Ubenimex and the more potent aminopeptidase inhibitor, actinonin, inhibited proliferation of both K562 cells and STI571-resistant K562 cells and also induced their apoptosis in dose- and time-dependent manners. Ubenimex and actinonin induced the activation of caspase-3, and the induction of apoptosis was inhibited by pan-caspase inhibitor, indicating this apoptosis is caspase-dependent. We found that serine phosphorylation of both MAPK and glycogen synthase kinase-3β were suppressed by aminopeptidase inhibitors in parent K562 and STI571-resistant K562 cells. The expression level of cyclin D1 protein was also reduced by ubenimex and actinonin in both cell lines. These results indicated STI571-resistance does not confer the cross-resistance to aminopeptidase inhibitors in K562 cells and revealed the new findings of aminopeptidase inhibitor-induced intracellular signaling pathways.     

In vitro effects of STI 571-containing drug combinations on the growth of Philadelphia-positive chronic myelogenous leukemia cells

BACKGROUND: Chronic myelogenous leukemia (CML) is characterized by a molecular aberration, a fusion BCR-ABL gene encoding for aberrant tyrosine kinase activity, which is crucial in the pathogenesis of CML. In vitro, inhibition of BCR-ABL protein tyrosine kinase activity by a tyrosine kinase inhibitor, Imatinib mesylate (STI571; formerly CGP57148B), successfully suppressed proliferation/survival of the BCR-ABL positive clones. In clinical studies, hematologic and cytogenetic remissions have been achieved in most patients with chronic phase CML; in accelerated and blastic phases of CML, STI571 appeared less effective. In the current study, the authors tested combinations of STI571 and cytarabine and homoharringtonine (HHT), drugs with documented activity in CML. METHODS: The single agents and their combinations were studied for in vitro effect on proliferation of BCR-ABL positive cell lines KBM5 and KBM7 by 3(4,5-dimethylthiazol-2yl)-2,5 diphenyl-tetrazolium bromide assay and on primary patient-derived BCR-ABL cells by clonogenic assays. The in vitro additive, synergistic, or antagonistic effects of cytarabine and HHT with STI571 were then investigated by computer-assisted analysis using the CalcuSyn software. RESULTS: STI571 consistently suppressed BCR-ABL positive cell proliferation with a dose-effect correlation. In the model system used, STI571/cytarabine and STI571/HHT combinations were more effective in inhibiting KBM5 and KBM7 cell growth than each drug as single agent. These results were also verified in primary CML-derived clonogenic cells in semisolid cultures. CONCLUSIONS: In this experimental system, our studies documented additive or synergistic effects with STI571 plus cytarabine or HHT, supporting the future use of STI571 combinations in clinical trials in patients with Philadelphia chromosome-positive leukemias.     

Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells.

The ABL-specific tyrosine kinase inhibitor STI571 (formerly CGP57148B) induced cytogenetic remissions in 33% of chronic myelogenous leukemia (CML) patients in a phase I trial (Druker et al 1999). Combination therapy may increase this proportion. We tested whether combinations of STI571 and cytarabine or other chemotherapeutic agents such as hydroxyurea, mafosfamide or etoposide would display synergistic activity in BCR-ABL-positive chronic myelogenous leukemia (CML) cell lines derived from patients in blast crisis. In addition, the toxicity of these combinations on BCR-ABL-negative cells was investigated. A tetrazolium-based MTT assay was used to quantity growth inhibition after 48 h of exposure to cytotoxic agents alone and in simultaneous combination with STI571. The drug interactions were analyzed using the median-effect method of Chou and Talalay. The combination index (CI) was calculated according to the classic isobologram equation. At growth inhibition levels of over 50%, STI571 + cytarabine as well as STI571 + etoposide were significantly synergistic (CI < 1, P < 0.05) in the BCR-ABL-positive cell lines evaluated. At 60% inhibition or higher, a similar synergistic pattern became apparent for STI571 + mafosfamide (P < 0.05), while STI571 + hydroxyurea showed ambiguous, cell line-dependent synergism (BV173), additivity (EM-3) or antagonism (K562) in CML cell lines. Furthermore, the BCR-ABL-negative HL-60, KG1a and normal CD34+ progenitor cells were not affected by 0.8 microM STI571, a concentration which produced more than 50% growth inhibition in all BCR-ABL-positive cells tested, and no potentiation of growth inhibition was observed in these BCR-ABL-negative cells when STI571 was combined with chemotherapeutic agents. Our in vitro data with CML blast crisis cell lines strongly suggest that combinations of STI571 with cytarabine or etoposide be rapidly considered for clinical testing.     

Reversal of P-glycoprotein mediated multidrug resistance by a newly synthesized 1,4-benzothiazipine derivative,JTV-519

A newly synthesized 1,4-benzothiazipine derivate, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2,3,4,5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) mediated multidrug resistance (MDR) in K562/MDR and KB/MRP cells, respectively. JTV-519 at 3 microM reversed the resistance of K562/MDR cells to vincristine (VCR), taxol, etoposide (VP16), adriamycin (ADM) and actinomycin D and at 0.5 or 1 microM reversed their resistance to STI571. JTV-519 at 10 microM enhanced the accumulation of ADM in K562/MDR cells to the level in parental K562 cells and inhibited the efflux of ADM from K562/MDR cells. Photoaffinity labeling of P-gp with 3H-azidopine was almost completely inhibited by 500 microM JTV-519. JTV-519 at 3 microM also partially reversed the resistance of KB/MRP cells to VCR and at 500 microM partially inhibited the photoaffinity labeling of MRP1 with (125)I-II-azidophenyl agosterol A (125I-azidoAG-A). These results suggest that JTV-519 reversed the resistance to the anti-cancer agents in P-gp and MRP1 overexpressing multidrug-resistant cells by directly binding to P-gp and MRP1, and competitively inhibiting transport of the anti-cancer agents.     

Pivanex, a histone deacetylase inhibitor, induces changes in BCR-ABL expression and when combined with STI571, acts synergistically in a chronic myelocytic leukemia cell line

Chronic myelogenous leukemia (CML) is associated with the high TK activity chimeric protein BCR-ABL, known to contribute to cell tumorogenicity, resistance to apoptosis and differentiation. STI571, the TK inhibitor, is the current treatment for CML. One possible approach to overcome STI571 resistance appearing in some cases, involves the combination of histone deacetylase inhibitors (HDI) and STI571. We demonstrated that in K562, the CML cell line, pivaloyloxymethyl butyrate (Pivanex)-induced apoptosis, differentiation and reduced BCR-ABL protein levels and that the combination of Pivanex with STI571 acted synergistically. These data suggest the possible benefit of combining this HDI with STI571 for treatment of CML.     

雷公藤红素逆转K562/A02细胞多药耐药的实验研究

目的探讨雷公藤红素逆转人慢性粒细胞白血病红白血病急变细胞株K562/A02多药耐药的效果。方法采用CCK-8法测定细胞的药敏性及耐药逆转性,应用流式细胞术检测细胞内ADM浓度、P-gp蛋白表达。结果雷公藤红素对K562/A02、K562的半数抑制率浓度（IC50）分别为（295.58±23.288）μmol/L、（411.59±26.551）μmol/L。K562/A02细胞对ADM的耐药性是K562细胞的79.78倍。细胞毒剂量的雷公藤红素作用后,ADM对K562/A02细胞的IC50显著下降（P〈0.05）,逆转倍数为117.860倍。细胞毒剂量（IC50）和非细胞毒剂量（IC10）的雷公藤红素处理后的K562/A02细胞内的ADM浓度显著增加（P〈0.05）,增加倍数分别为1.537倍和1.102倍。雷公藤红素能明显下调K562/A02细胞的P-gp表达。结论雷公藤红素对逆转K562/A02细胞的耐药性有一定的作用,其机制可能与下调P-gp表达有关。     

Phosphatidylinositol-3 kinase inhibitors enhance the anti-leukemia effect of STI571

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph(1))-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). STI571 (Gleevec), a novel anti-leukemia drug targeting BCR/ABL kinase can induce remissions of the Ph(1)-positive leukemias. STI571 was recently combined with the standard cytostatic drugs to achieve better therapeutic results and to overcome emerging drug resistance mechanisms. We decided to search for a more specific partner compound for STI571. Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells (Blood, 86:726,1995). Therefore the anti- Ph(1)-leukemia effect of the combination of BCR/ABL kinase inhibitor STI571 and PI-3k inhibitor wortmannin (WT) or LY294002 (LY) was tested. We showed that STI571+WT exerted a synergistic effect against the Ph(1)-positive cell lines, but did not affect the growth of Ph(1)-negative cell line. Moreover, the combinations of STI571+WT or STI571+LY were effective in the inhibition of clonogenic growth of CML-chronic phase and CML-blast crisis patient cells, while sparing normal bone marrow cells. Single colony RT-PCR assay showed that colonies arising from the mixture of CML cells and normal bone marrow cells after treatment with STI571+WT were selectively depleted of BCR/ABL-positive cells. Biochemical analysis of the CML cells after the treatment revealed that combination of STI571+WT caused a more pronounced activation of caspase-3 and induced massive apoptosis, in comparison to STI571 and WT alone. In conclusion, combination of STI571+WT or STI571+LY may represent a novel approach against the Ph(1)-positive leukemias.     

Resistance to daunorubicin-induced apoptosis is not completely reversed in CML blast cells by STI571.

The leukemogenic property of BCR-ABL in chronic myeloid leukemia (CML) is critically dependent on its protein tyrosine kinase activity. STI571 inhibits the BCR-ABL kinase activity, the growth and the viability of BCR-ABL expressing cells. In this study, we report the apoptotic effect of STI571 in combination with daunorubicin (DNR) on peripheral blood mononuclear cells from 11 CML patients and four BCR-ABL-positive cell lines: AR230, LAMA84, K562 and KCL22. Primary blast cells were identified by flow cytometry on the basis of their low CD45 expression. Nucleus fragmentation, exposure of phosphatidylserines and decrease in mitochondrial membrane potential were measured using acridine orange, FITC-annexin V and DiOC6(3), respectively, to evaluate apoptosis. On cell lines, the effect of DNR was negligible, whereas STI571 induced 10 to 35% of apoptosis in 18 h. STI571 sensitized AR230, LAMA84 and K562 cells to DNR when apoptosis was measured at the mitochondrial and membrane but not the nuclear levels. On CML blast cells, phosphatidyl serine exposure was significantly induced by both DNR and STI571 and was higher when these drugs were used in combination (P < 0.0003). However, the effects of this drug combination were only additive and no sensitization of blast cells to DNR by STI571 was observed. Interestingly, sensitization was evidenced in CML but not normal lymphocytes. These results suggest that other mechanisms additional to Bcr-Abl tyrosine kinase activity could be responsible for DNR resistance, and further investigations are needed to understand its origin.     

硒酸酯多糖通过下调mdr1/P-gp表达逆转K562/ADM细胞凋亡抑制

目的:研究硒酸酯多糖(Kappa-selenocarrageenan,KSC)对多药耐药K562/ADM细胞的诱导凋亡效应及其分子机制。方法:以白血病多药耐药细胞K562/ADM为KSC作用的靶细胞,用MTT比色法检测细胞增殖活性,形态学、DNA片段化和流式细胞术(FCM)观察细胞凋亡;RT-PCR检测mdr1基因和Caspase-3基因mRNA的表达;FCM测定P-gp蛋白表达水平和Caspase-3活性。结果:KSC显著抑制K562/ADM细胞增殖,KSC诱导后K562/ADM细胞出现典型的凋亡形态学变化、DNA片段化和亚G1期细胞群等特征性改变。KSC下调K562/ADM细胞mdr1基因表达、抑制P-gp合成,并上调caspase-3基因表达、增强caspase-3活性。结论:KSC通过下调mdr1/P-gp表达逆转K562/ADM多药耐药细胞的凋亡抑制。     

Identification of heme oxygenase-1 as a novel BCR/ABL-dependent survival factor in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a stem cell disease in which BCR/ABL promotes the survival of leukemic cells. Heme oxygenase-1 (HO-1) is an inducible stress protein that catalyzes the degradation of heme and has recently been implicated in the regulation of growth and survival of various neoplastic cells. In the present study, we analyzed the expression and role of HO-1 in CML cells. As assessed by Northern and Western blot analysis as well as immunostaining, primary CML cells were found to express HO-1 mRNA and the HO-1 protein in a constitutive manner. Exposure of these cells to the BCR/ABL tyrosine kinase inhibitor STI571 resulted in decreased expression of HO-1 mRNA and protein. In addition, BCR/ABL was found to up-regulate HO-1 promoter activity, mRNA levels, and protein levels in Ba/F3 cells. To investigate the role of HO-1 for survival of primary CML cells, the HO-1 inducer hemin was used. Hemin-induced expression of HO-1 was found to protect CML cells from STI571-induced cell death. In addition, inhibition of HO-1 by zinc-(II)-deuteroporphyrin-IX-2,4-bisethyleneglycol resulted in a substantial decrease of cell viability. Furthermore, overexpression of HO-1 in the CML-derived cell line K562 was found to counteract STI571-induced apoptosis. Together, our data identify HO-1 as a novel BCR/ABL-driven survival molecule and potential target in leukemic cells in patients with CML. The pathogenetic and clinical implications of this observation remain to be elucidated.     

The Synergistic Inhibitory Effect of STI571 in Combination with Arsenic Trioxide (As2O3) on Multidrug-Resistant Leukemic Cells

OBJECTIVE To study the synergistic effect of STI571, an inhibitor of tyrosine kinase, in combination with arsenic trioxide As2O3 on a multidrug-resistant leukemia cell line expressing bcr-abl.METHODS The cytotoxic effect of STI571 alone or in combination with different concentrations of As2O3 on the bcr-abl and mdr1 -positive leukemia cell line, K562-n/VCR, was examined by the MTT method.RESULTS One μmol/L of STI571 alone had no significant cytotoxic effect on K562-n/VCR cells. However the cytotoxic effect increased markedly when combined with As2O3 at concentrations of 10-5, 10-6, 10-7 and 10-8 mol/L. The IC50 of K562-n/VCR cells in As2O3 group was 1.879 μmol/L, with. Upon addition of STI571, the IC50 decreased to 0.155 μmol/L resulting in a synergistic cytotoxic effect on K562-n/VCR ceils that was increased 12.1 times.CONCLUSION A combination of STI571 with As2O3 has a more powerful inhibitory effect on leukemia cells expressing positive bcr-abl and positive mdrl compared to the effect with As2O3 alone.     

STI571与三氧化二砷对多药耐药bcr-abl阳性白血病细胞的协同抑制作用

目的　研究酪氨酸激酶抑制剂STI5 71与三氧化二砷 (As2 O3 )联合应用对多药耐药bcr abl阳性白血病细胞的协同效应。方法　采用MTT法比较STI5 71单独或与不同浓度的As2 O3 联合应用对bcr abl和mdr1共同阳性的白血病细胞系K5 6 2 n/VCR的抑制作用。结果　 1μmol/L的STI5 71对K5 6 2 n/VCR细胞无明显细胞毒作用 ,与 10 -5,10 -6,10 -7,10 -8mol/L的As2 O3 联合应用 ,细胞毒作用明显增强。As2 O3 单独对K5 6 2 n/VCR细胞的IC50 为 1.879μmol/L ,加STI5 71后 ,IC50 为 0 .15 5 μmol/L ,协同抑制作用为 12 .12倍。结论　STI5 71与As2 O3 联合应用对于bcr abl与mdr1共同阳性的白血病细胞有更强的抑制作用     

Decitabine (5-Aza-2'-deoxycytidine) decreased DNA methylation and expression of MDR-1 gene in K562/ADM cells.

Multidrug resistance (MDR) is a major problem in patients with hematological malignancies. Although drug-resistance is known to be induced by the expression of P-glycoprotein (P-gp) encoded by the MDR-1 gene, little is known about the mechanisms regulating this gene. Herein, we studied the DNA methylation patterns at the enhancer and repressor binding sites of the MDR-1 gene using the human erythroleukemia cell line K562 and its multidrug resistant derivative K562/ADM (adriamycin). Direct DNA sequence analysis demonstrated methylation to be present at the repressor site (minus 110 GC-box) of the MDR-1 gene in K562/ADM cells, but not in parental K562 cells. Methylation-specific PCR (MSP) analysis yielded similar results. Treatment of K562/ADM cells with 5-Aza-2'-deoxycytidine (decitabine; DAC), an inhibitor of DNA methyltransferase, caused demethylation of the repressor binding site of MDR-1 gene, as assessed by MSP, and also decreased P-gp expression, as assessed by flow cytometric and Northern blot analysis. Although it is generally accepted that DAC upregulates gene expression by demethylating the activator binding sites, our present results suggest that DAC induces down-regulation of P-gp expression as a result of demethylation at the repressor binding site in K562/ADM cells. In this regard, methylation-dependent regulation of the MDR-1 gene in K562/ADM cells is unique.     

STI571治疗慢性髓系白血病的临床疗效与毒副作用观察

背景与目的:bcr-abl融合基因翻译的蛋白产物P210bcr-abl的酪氨酸激酶(proteintyrosinekinase,PTK)活性异常增高被认为是导致慢性髓系白血病(chronicmyeloidleukmeia,CML)发病的根本原因。STI571能高效特异性抑制P210bcr-abl的PTK活性,在临床应用中获得了显著的疗效,但对急变期患者的治疗效果维持时间短。本研究观察和比较了STI571治疗慢性期与加速/急变期CML患者的临床疗效和所发生的不良反应,并从细胞遗传学的角度对急变期患者STI571耐药机制进行初步的分析。方法:选择接受STI571治疗的CML患者22例,其中慢性期6例,加速/急变期16例。按照血液学缓解和细胞遗传学缓解的标准,结合骨髓细胞形态学分析、骨髓细胞G显带技术分析和间期荧光原位杂交检测结果,对患者STI571治疗前和治疗3个月后的血液学和细胞遗传学缓解情况进行分析,并对3个月内出现耐药复发的患者进行核型演化分析。同时密切观察各系统发生的不良反应及严重程度。结果:6例(100%)慢性期CML患者获血液学完全缓解和细胞遗传学缓解,4例(25%)加速/急变期CML患者获血液学完全缓解,8例(50%)获不同程度的细胞遗传学反应。获血液学完全缓解和细胞遗传学反应的百分率两组比较均有统计学差异(P<0.05)。3例急变期CML患者出现耐药复发,其中2例可见2Ph和其它新     

三氧化二砷联合甲磺酸伊马替尼、咖啡因对K562白血病细胞的协同作用

 目的 探讨三氧化二砷（Arsenic trioxide,As2O3）与酪氨酸激酶抑制剂甲磺酸伊马替尼（实验药物代号STI571）以及细胞周期调节剂咖啡因联合诱导K562细胞凋亡的作用及机制,为寻找克服K562细胞对As2O3抵抗的有效手段提供实验依据。方法 以As2O3与STI571、咖啡因联合作用于K562细胞,采用MTT方法检测细胞增生活性,PI染色流式细胞仪检测细胞周期,PI和Annexin V双染色流式检测细胞凋亡,Western blot法检测细胞周期相关调节蛋白的表达。结果 5.0 μmol／L浓度的咖啡因对K562细胞增生无抑制作用,亦不能增强As2O3的抑制作用,单独以STI571 1.0 μmol／L处理,即可有效抑制K562细胞的生长,与As2O3联用能明显增加其抑制增生的效应;咖啡因与As2O3联合作用,不增加As2O3诱导的K562细胞凋亡率[（14.7±3.6）%vs（15.3±3.3）%,P＞0.05]; STI571具有轻度诱导K562细胞凋亡的作用[（18.3±4.5）%],与As2O3合用可显著增加诱导凋亡率[（14.7±3.6）%vs（42.8±4.2）%,P＜0.01],并明显降低As2O3诱导的G2／M期细胞比例。与单用As2O3比较,As2O3 + STI571明显抑制cdc2、cdc2-p及survivin蛋白表达,而As2O3与咖啡因合用不能诱导survivin蛋白表达下调,对cdc2、cdc2-p蛋白的表达无明显影响。结论 周期调节药物咖啡因对As2O3诱导K562细胞凋亡无增敏作用;酪氨酸激酶抑制剂STI571能协同As2O3诱导K562细胞凋亡,下调抗凋亡蛋白survivin的表达可能是其机制之一,值得深入研究其临床应用效果。     

A novel pyridopyrimidine inhibitor of abl kinase is a picomolar inhibitor of Bcr-abl-driven K562 cells and is effective against STI571-resistant Bcr-abl mutants.

Inhibition of the constitutively active Bcr-abl tyrosine kinase(TK) by STI571 has proven to be a highly effective treatment for chronic myelogenous leukemia (CML). However, STI571 is only transiently effective in blast crisis, and drug resistance emerges by amplification of or development of mutational changes in Bcr-abl. We have screened a family of TK inhibitors of the pyrido [2,3-d]pyrimidine class, unrelated to STI571, and describe here a compound with substantial activity against STI-resistant mutant Bcr-abl proteins. This compound, PD166326, is a dual specificity TK inhibitor and inhibits src and abl in vitro with IC(50)s of 6 and 8 nM respectively. PD166326 inhibits the growth of K562 cells with IC(50) of 300 pM, leading to apoptotic G(1) arrest, whereas non-Bcr-abl cell types require >1000 times higher concentrations. We tested the effects of PD166326 on two of the clinically observed STI571-resistant Bcr-abl mutants. PD166326 potently inhibits the E255K mutant Bcr-abl protein and the growth of Bcr-ablE255K-driven cells. The T315I mutant Bcr-abl protein, which is mutated within the ATP-binding pocket, is resistant to PD166326; however, the growth of Bcr-ablT315I-driven cells is partially sensitive to this compound, likely through the inhibition of Bcr-abl effector pathways. These findings show that TK drug resistance is a structure-specific phenomenon and can be overcome by TK inhibitors of other structural classes, suggesting new approaches for future anticancer drug development. PD166326 is a prototype of a new generation of anti-Bcr-abl compounds with picomolar potency and substantial activity against STI571-resistant mutants.     

K562多药耐药细胞系中肿瘤干细胞样细胞对伊马替尼耐药机制的初步研究

 目的 进一步阐明一些高表达P-糖蛋白（P-gp）的慢性粒细胞白血病细胞对伊马替尼耐药的机制。方法 经过对K562细胞系长期的足叶乙苷（VP16）诱导和克隆筛选,建立一株耐药细胞系K562／VP16;利用干细胞高效能将Hoechst 33342 荧光染料泵出细胞的特性,采用流式细胞术,从K562／VP16细胞系中分选出一小群细胞,即边缘细胞（SP）,称为K562／VP16 SP细胞,并初步探讨其抗伊马替尼的机制。结果 bcr／abl和abl 蛋白在K562细胞、K562／VP16 SP细胞及非K562／VP16 SP细胞（non-SP K562／VP16）中的表达水平差异无统计学意义;P-gp在K562细胞中不表达,在K562／VP16 SP及non-SP K562／VP16细胞中均高表达且表达水平一致;与non-SP K562／VP16细胞比较,K562／VP16 SP细胞对伊马替尼的耐药性更强,并且这种抗性几乎不能被多种多药耐药逆转剂逆转;另外,体内外实验显示,K562／VP16细胞的致瘤性几乎全部来源于K562／VP16 SP细胞。结论 bcr／abl基因的扩增、过度表达和多药耐药基因及其蛋白表达产物P-gp的高表达,可能不是白血病细胞产生对伊马替尼临床耐药的重要机制;白血病细胞对伊马替尼具有一定的抗性,可能与数量极少的白血病干细胞有直接的关系。因此,这类数量极少的干细胞样的肿瘤细胞应当成为有效治疗肿瘤的靶细胞。     

CD3AK细胞对耐药白血病细胞的体外净化作用

目的 :观察CD3 AK细胞对耐药的白血病细胞系及慢性髓细胞白血病 (chronicmyelogenousleukemia ,CML)急变患者原代肿瘤细胞的体外净化作用。方法 :采用固化的抗CD3单克隆抗体联合小剂量IL 2诱导CD3 AK细胞 ;MTT法观察CD3 AK细胞对K5 6 2、HL6 0及其耐药株的细胞毒活性 ;肿瘤细胞集落培养 (tumorcolonyassay ,TCA)观察CD3 AK细胞对K5 6 2、HL6 0及其耐药株集落形成的抑制作用 ;流式细胞仪 (flowcytometry ,FCM)检测耐药的CML急变患者原代细胞经CD3 AK细胞净化后Pgp阳性细胞的比例变化。结果 :MT法显示CD3 AK细胞在体外对K5 6 2细胞、HL6 0细胞及其耐药株有相似的杀伤作用 ;集落培养观察CD3 AK细胞对HL6 0细胞株及其耐药株的集落形成均有较强的抑制作用 ;FCM结果显示耐药CML原代细胞经CD3 AK细胞净化后Pgp阳性细胞比例下降 2 3 2 0 %。结论 :CD3 AK细胞在体外对耐药白血病细胞株及耐药白血病原代细胞均有较强的净化作用。