CUEDC2 sensitizes chronic myeloid leukemic cells to imatinib treatment

CUEDC2, a newly reported protein, has been found to be ubiquitously expressed in human tissues and repress NF-κB activity. To study the role of CUEDC2 in chronic myeloid leukemia (CML), we explored the function of CUEDC2 in CML cells through using the CML cell line K562 and its imatinib resistant cells K562/G01. K562 cells expressed a relatively higher level of CUEDC2 compared to K562/G01 cells. Knockdown of CUEDC2 in K562 cells resulted in decreased cell apoptosis after imatinib treatment; when CUEDC2 was overexpressed in K562/G01 cells, imatinib induced more cell apoptosis. By analyzing the activity of NF-κB, the results indicated a negative association between the expression of CUEDC2 and NF-κB signaling pathway in these CML cells. Our data suggested that the expression level of CUEDC2 has an inverse correlation with imatinib resistance and activity of NF-κB signaling pathway in CML cells, CUEDC2 could regulate imatinib sensitivity in CML cells at least partially through NF-κB signaling pathway.     

Factor(s) released from irradiated B-CLL cells induce apoptosis in leukemic lymphocytes

Photon irradiation of peripheral blood lymphocytes from 25 patients with untreated B-chronic lymphocytic leukemia (B-CLL) induced an increase in apoptotic response by 270%. No significant increase in apoptosis was observed after irradiation of peripheral blood mononuclear cells from 15 healthy volunteers. Supernatants (sups) derived from irradiated leukemic cells incubated with non-irradiated autologous cells induced a 75% enhancement in number of apoptotic cells, as compared with sups from non-irradiated CLL cells. The level of tumor necrosis factor alpha, a cytokine known to prevent apoptosis, was reduced in the sups of irradiated CLL cells in comparison to that of non-irradiated lymphocytes. The interleukin (IL)-10 level, an IL reported to induce apoptosis, was similar in the sups of irradiated and non-irradiated lymphocytes from B-CLL patients. No change in IL-2 levels was observed. The significance of these findings and the role of factor(s) in the sups of irradiated leukemic lymphocytes as inducers of apoptosis are discussed.     

p21 Confers resistance to imatinib in human chronic myeloid leukemia cells

Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21^Cip1, initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21^Cip1 expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21^Cip1 in the response to imatinib in CML.     

Study of differentiation of fresh myeloid leukemic cells by physiologic agents that induce a human promyelocytic leukemic line (HL-60) to differentiate

A promyelocytic cell line known as HL-60 can be induced to mature to granulocytes or monocytes after exposure to a variety of physiologic agents including 1-alpha 25 dihydroxy Vitamin D3 (Vit D), retinoic acid, cyclic AMP cell permeant compounds, and stimulators of adenylate cyclase. These compounds were used in primary culture of blast cells from 12 patients with acute nonlymphocytic leukemia. Maturation was assessed by morphology, superoxide production, development of esterase activity and chemotactic peptide receptor expression. Morphologic maturation and superoxide production correlated with chemotactic peptide receptor expression. The majority of blast cells treated with inducer showed no significant change in morphologic or functional markers compared to the blast cells cultured in fresh media alone. Chemotactic peptide receptor expression increased 3 to 30-fold in 13 of 14 cases studied. In 4 patients, the highest receptor expression was without inducer and in 4 patients the highest increase was with dibutyryl cyclic AMP treatment. Our study suggests that physiologic inducers of HL-60 differentiation do not consistently have the same effect on primary suspension culture of freshly isolated human leukemia cells.     

Bleomycin,etoposide and cisplatin (BEP) combination with concurrent imatinib mesylate (GLEEVEC) in chronic myeloid leukemia (CML) patient with mesenchymal tumor

Imatinib is now indicated as the first line therapy for chronic myeloid leukemia (CML). Treatment of CML with imatinib is generally well tolerated and the risk of severe adverse affects is low. Many new drugs including targeted therapy are combined with antineoplastic agents safely. We here report a patient with CML who developed concurrent mesenchymal tumor while undergoing therapy with imatinib and treated with combination chemotherapy including bleomycin, etoposide, and cisplatin, as well as imatinib without severe toxicity.     

Cytogenetic analysis of human acute and chronic myeloid leukemic cells cloned in agar culture

Karyotypic analysis was performed on agar cultures of blood or bone marrow from 12 patients with acute or chronic myeloid or myelomonocytic leukemia in whom karyotypic markers were present. The granulocytic colonies and clusters which developed on culture were shown to be derived from representative cells of the leukemic population. In two patients with acute leukemia in remission, normal colonies with a normal karyotype were grown from marrow cells but in two patients with chronic myeloid leukemia in remission the Ph¹ abnormality persisted in colony cells. The agar culture technique appears to be ideal for following the emergence and disappearance of leukemic and normal granulopoietic populations in patients with these types of leukemia.     

Alkylphosphocholines induce apoptosis in HL-60 and U-937 leukemic cells

Alkylphosphocholines (APC) represent a new group of ether-lipid-related compounds with remarkable activity against transformed cells in vitro and good tolerability in vivo. Their mechanism of action remains unknown. The aim of the present study was to investigate the effects of a series of APC on three human leukemic cell lines: K-562, HL-60, and U-937. The tetrazolium dye-reduction (MTT) assay and cell counting were used to determine the cytotoxicity of the APC used. DNA gel electrophoresis and enzyme-linked immunosorbent assay (ELISA) detection of oligonucleosomes were performed to identify and quantify DNA fragmentation. Electron and phase-contrast microscopy were used to detect morphologic changes specific for programmed cell death. HL-60 and U-937 cells were found to be sensitive, but K-562 cells were relatively resistant to APC exposure. APC with long alkyl chains exerted stronger cytotoxicity than did those with short alkyl chains. DNA fragmentation was found after treatment with APC in HL-60 and U-937 cells but not in K-562 cells. In HL-60 cells the increase in mono- and oligonucleosome formation as measured by ELISA was correlated with the length of the alkyl chains at 14 h of exposure to APC but plateaued at 20 h. The morphologic alterations in HL-60 and U-937 cell lines, such as cell shrinkage, chromatin condensation, and formation of apoptotic bodies, confirmed the induction of apoptosis after APC exposure. It is concluded that programmed cell death plays an important role in the cytotoxicity of APC against certain human leukemic cell lines. The antineoplastic profiles of APC with long alkyl chains render them attractive for further therapeutic application. Received: 1 September 1996 / Accepted 4 July 1997     

Clinical results with imatinib in chronic myeloid leukaemia

Since its introduction 5 years ago, imatinib mesylate has shown remarkable efficacy in treating patients with chronic myeloid leukaemia. Here we shall review the clinical results seen with imatinib at all stages of the disease, current views on the best way to monitor patients' responses and potential ways of predicting response to treatment. We shall also briefly cover the reasons why quiescent stem cells pose a theoretical threat to successful treatment.     

Potassium antimonyl tartrate induces reactive oxygen species-related apoptosis in human myeloid leukemic HL60 cells

Potassium antimonyl tartrate (PAT), like arsenic trioxide (As2O3), has recently been shown to exert cytotoxicity towards acute promyelocytic leukemia (APL) cells. In the present study, we demonstrated that PAT treatment also inhibited cell growth of four acute myeloid leukemia (AML) cell lines, i.e., HL60, K562, KG1a and U937, that do not derive from APL. PAT, like As2O3, was further shown to induce apoptosis in HL60 cells as assessed by Hoechst 33342 staining and microscopical detection. Such an apoptotic process was associated with loss of mitochondrial potential and enhanced cellular production of reactive oxygen-related species; it was potentiated by co-treatment with buthionine sulfoximine, a pro-oxidant compound acting through inhibition of glutathione synthesis, and abolished in response to the antioxidant N-acetylcysteine, thus outlining that the toxicity of PAT, similarly to that of As2O3, is modulated by the cellular redox status. Pan-caspase inhibitors failed to inhibit PAT-triggered apoptosis of HL60 cells whereas they fully blocked that linked to As2O3, suggesting that PAT, unlike As2O3, does not require caspase activity for inducing apoptosis. PAT and As2O3 also differently affected intracellular pH, a key parameter commonly altered during apoptotic processes. Such data therefore indicate that PAT can exert cytotoxicity towards AML cells not deriving from APL such as HL60 cells, through inducing an apoptotic process which exhibits some similarities and some differences with that triggered by As2O3.     

环孢素诱导白血病细胞系Nalm—6细胞凋亡

目的：研究环孢素（环孢霉素A，CsA）在体外对B－细胞性白血病细胞株Nalm-6细胞增殖的影响，并探讨其可能的作用机制。方法：应用细胞形态学、DNA凝胶电泳等方法检测在体外CsA对B－细胞性白血病细胞株Nalm-6细胞增殖的作用。结果：临床耐受剂量CsA能明显抑制Nalm-6细胞增殖，且证实诱导细胞凋亡是CsA抑制增殖的主要机理之一。结论：CsA能有效地通过诱导人类B－细胞性白血病细胞株凋亡而起到抑制增殖的作用。     

Arsenic targets tubulins to induce apoptosis in myeloid leukemia cells

Arsenic exhibits a differential toxicity to cancer cells. At a high concentration (>5 microM), As2O3 causes acute necrosis in various cell lines. At a lower concentration (0.5-5 microm), it induces myeloid cell maturation and an arrest in metaphase, leading to apoptosis. As2O3-treated cells have features found with both tubulin-assembling enhancers (Taxol) and inhibitors (colchicine). Prior treatment of monomeric tubulin with As2O3 markedly inhibits GTP-induced polymerization and microtubule formation in vitro but does not destabilize GTP-induced tubulin polymers. Cross-inhibition experiments indicate that As2O3 is a noncompetitive inhibitor of GTP binding to tubulin. These observations correlate with the three-dimensional structure of beta-tubulin and suggest that the cross-linking of two vicinal cysteine residues (Cys-12 and Cys-213) by trivalent arsenic inactivates the GTP binding site. Furthermore, exogenous GTP can prevent As2O3-induced mitotic arrest.     

Acetylome and phosphoproteome modifications in imatinib resistant chronic myeloid leukaemia cells treated with valproic acid

Chronic myeloid leukaemia has a specific therapy: BCR/ABL inhibitor imatinib. Resistance due to BCR/ABL dependent and independent mechanisms is partially reversible by histone deacetylase inhibitors. We analysed by 2D-electrophoresis and anti-pan-acetylated and anti-phosphotyrosine immunoblots, followed by spot-matching and MALDI-TOF mass spectrometry, which proteome modifications would parallel restoration of sensitivity to imatinib by valproic acid (VPA). VPA plus imatinib significantly increased acetylation of HSP90 and hnRNP L and decreased phosphorylation of HSPs and hnRNPs in imatinib resistant cells. VPA was able to modify profoundly acetylome and phosphoproteome of CML cells, while reverting resistance to imatinib.     

2-Chlorodeoxyadenosine alone and in combination with cyclophosphamide and mitoxantrone induce apoptosis in B chronic lymphocytic leukemia cells in vivo

The purpose of the study was to determine some apoptotic events in mononuclear cells obtained from peripheral blood of patients with B-cell chronic lymphocytic leukemia (B-CLL) during and after therapy with 2-chlorodeoxyadenosine (2-CdA; C), and the combination of 2-CdA with cyclophosphamide (CC), or 2-CdA with mitoxantrone and cyclophosphamide (CMC). Western blot technique was performed to estimate expression/proteolytic degradation of generally accepted apoptotic markers, i.e., Bcl-2 protein, lamin B, PARP-1, and caspase-3 in leukemic cells isolated from blood samples of patients before treatment and subjected to drug(s) administration. The decrease of antiapoptotic protein Bcl-2 expression and proteolytic cleavage of nuclear proteins--lamin B and PARP-1 were observed in leukemic cells of patients treated according to the above therapy protocols, however, each to a different level among the studied groups. The obtained results indicated also that procaspase-3 was cleaved and activated in leukemic cells of three drug(s) treated groups. However, the cleavage of procaspase-3 and the generation of fragments with mol. mass of 17/20 kDa occurred especially effectively among patients treated according to CMC regimen. The changes in expression/proteolytic degradation of the above selected apoptotic markers, are accompanied by the appearance of apoptotic morphology in leukemic cells originated from blood of patients treated with the above drug(s) in comparison to untreated ones.     

A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells

Anti-MY9 is an IgG2b murine monoclonal antibody selected for reactivity with immature normal human myeloid cells. The MY9 antigen is expressed by blasts, promyelocytes and myelocytes in the bone marrow, and by monocytes in the peripheral blood. Erythrocytes, lymphocytes and platelets are MY9 negative. All myeloid colony-forming cells (CFU-GM), a fraction of erythroid burst-forming cells (BFU-E) and multipotent progenitors (CFU-GEMM) are MY9 positive. This antigen is further expressed by the leukemic cells of a majority of patients with AML and myeloid CML-BC. Leukemic stem cells (leukemic colony-forming cells, L-CFC) from most patients tested were also MY9 positive. In contrast, MY9 was not detected on lymphocytic leukemias. Anti-MY9 may be a valuable reagent for the purification of hematopoietic colony-forming cells and for the diagnosis of myeloid-lineage leukemias.     

Multidrug resistance gene (MDR1) polymorphisms correlate with imatinib response in chronic myeloid leukemia

The human multidrug resistance gene (MDR1, ABCB1) codes for P-glycoprotein (P-gp) that affects the pharmacokinetics of many drugs. MDR1 single nucleotide polymorphisms (SNPs) are associated with drug clearance. Imatinib is a substrate of P-gp-mediated efflux. We investigated the MDR1 T1236C, G 2677T/A, and C3435T polymorphism in 52 patients with chronic myeloid leukemia treated with imatinib. The distribution of MDR1 1236, 2677, or 3435 genotypes was significantly different between the resistance patients and sensitivity patients. The resistance incidence correlated with the number of T alleles at locus 1236 and 3435. Resistance was higher for patients homozygous for the 1236T allele when compared to patients with CT/CC genotype groups (75% vs. 31.3%, P = 0.004). For the G2677T/A polymorphism, a better complete cytogenetic remission was observed for patients with genotype AG/AT/AA, when compared to other genotype groups (TT/GT/GG, P = 0.02). Patients with 3435 TT/CT genotypes showed a higher resistance when compared with patients with CC genotype (59.4% vs. 25%, P = 0.023). In conclusion, determination of 1236T, C3435T, and G2677T MDR1 polymorphisms might be useful in response prediction to therapy with imatinib in patients with CML.     

Butyrate-induced differentiation in leukemic myeloid cells - in-vitro and in-vivo studies

A study of the effects of three differentiating agents, butyric acid, retinoic acid and cytosine arabinoside on proliferation and differentiation of primary cultures, obtained from sixteen patients with myelo-proliferative disorder was conducted. The results showed that BA was an effective inhibitor of cell proliferation and inducer of cytodifferentiation. An acute non-lymphoblastic leukemia patient was treated with sodium butyrate. A temporary increase in differentiation-associated parameters were noted. However, the effects of SB were short-lived. The lack of clinical response led to the development of a BA prodrug pivaloyloxymethylbutyrate (AN-9). This prodrug was more potent in vitro than BA in the induction of cytodifferentiation and inhibition of cell proliferation.     

慢性粒细胞白血病患者血浆伊马替尼浓度检测与临床疗效分析

 目的　探讨慢性粒细胞白血病（CML）患者服用伊马替尼治疗后,伊马替尼血浆浓度在个体间的差异以及与临床疗效的关系。方法　2005年7月至2008年2月开始服用伊马替尼治疗的CML患者共51例纳入研究,其中男 34例,女 17例,服用剂量300 mg／d 9例、400 mg／d 37例,600 mg／d 5例;采用高效液相色谱法（HPLC）测定患者空腹伊马替尼血浆谷浓度;SPSS13.0软件进行统计分析。结果　伊马替尼血浆谷浓度与服用剂量有关,且个体之间差异较大,为（342～4688）ng／ml;300 mg／d剂量组的伊马替尼血浆谷浓度为（1037±514）ng／ml,低于400 mg／d剂量组的（2123±1016）ng／ml（t＝2.34,P＝0.032）;300 mg／d剂量组的治疗有效率为66.67 %（6／9）,低于400 mg／d剂量组的89.19 %（33／37）（χ2＝7.14,P＝0.008）;在300、400 mg／d剂量组中,39例治疗有效,伊马替尼血浆谷浓度高于治疗效果不理想患者,差异有统计学意义（t＝2.25,P＝0.037）;受试者工作特征曲线（ROC曲线）结果提示伊马替尼血浆谷浓度低于1050 ng／ml者,其临床疗效可能较差,敏感度为84.6 %,特异度为71.1 %。结论　CML患者服用伊马替尼治疗后药物血浆浓度与服用剂量有关,不同个体间差异较大,血浆谷浓度低于1050 ng／ml提示其临床疗效可能较差。