Use of syngeneic monoclonal antibodies in the therapy of disseminated myeloid leukemic cells

A syngeneic monoclonal antibody (MAb) (IC5F5) was successfully used in the immunotherapy of Rauscher-virus-induced myeloid leukemic RMB-I cells. It is directed to a virus-encoded, but aberrantly processed protein, which is expressed on the cell membrane. When applied in vivo, it binds only to RMB-I tumor cells. BALB/c mice were inoculated i.p. or i.v. with 10(7) RMB-I cells and died within 2-3 weeks due to increasing tumor load. Mice inoculated i.p. were completely cured by daily injections of ascites containing IC5F5. Disseminated tumor cells in liver and hemopoietic organs were observed after i.v. inoculation. Daily treatment with MAbs resulted in survival beyond 90 days. No antigenic modulation was observed when tumor tissue was analyzed 2-10 days after treatment. Treatment was successful even when therapy was postponed until day 5 following inoculation of tumor cells. When the number of ascites injections was reduced, survival was identical to that observed among repeatedly treated mice. Ten- and 100-fold dilution of ascites fluid diminished the number of survivors, but still resulted in a median survival time of 38 and 20 days, respectively, as compared to 14 days for untreated mice.     

Syngeneic monoclonal antibodies directed against Rauscher virus-induced myeloid leukemic cells: isolation and characterization

Hybridomas producing syngeneic monoclonal antibodies (MAbs) were prepared by fusion of spleen cells of BALB/c mice, which were immunized with sublethal doses of RMB-I cells. This cell line originates from a Rauscher virus (R-MuLV)-induced myeloid leukemia and forms tumors when re-inoculated into mice. MAbs were characterized as regards their reactivity against virally and non-virally induced cell lines. Two selected MAbs, IC5F5 and 4D2B4, were analyzed further. Their binding to subcellular structures was determined, and so were the properties of the antigens to which they are directed. MAb IC5F5 is of the IgG2A and 4D2B4 of the IgG2b subclass. Both bind to R-MuLV-infected or -transformed cell lines and are not mutually competitive. The antibodies do not react with other murine and human myeloid leukemic cells. As shown by immuno-electron microscopy, these MAbs have affinity to the cell membrane of non-virus producing RMB-I cells. When lysates of purified virus were analyzed, the MAbs were found to be directed to the gag precursor protein Pr65, and one of them (IC5F5) also to be directed to the core protein p12. In RMB-I cells, binding occurs to a 50-kDa glycoprotein and 2 proteins of 26 and 29 kDa. Since RMB-I cells do not produce virus, but express aberrant viral proteins, these MAbs are tumor-specific and useful for immunotherapy.     

Immunoreactivity of leukemic lymphoblasts of T-cell and B-cell precursor origin with monoclonal anti-GD3 and anti-GM3 antibodies.

The glycosphingolipids GD3, GM3, and alpha 2, 3-sialosylparagloboside (SPG) are major gangliosides of lymphoid leukemia cells. The reactivity of two monoclonal anti-ganglioside antibodies, an anti-GD3 (R24) and an antibody cross-reactive to GM3 and SPG (M2590), to blasts of patients with T-cell acute lymphoblastic leukemia (ALL) and B-cell precursor ALL (pre-B-ALL), were compared using indirect immunofluorescence and flow cytometry. Results from 23 patients with T-ALL and eight with pre-B-ALL yielded four subclasses of T-ALL and two subclasses of pre-B-ALL. Blasts from most of the patients with T-ALL were R24+M2590- whereas most of the patients with pre-B-ALL were R24-M2590-. Seven of 23 patients with T-ALL had ganglioside immunophenotypes similar to that of pre-B-ALL, i.e. R24-M2590- or R24-M2590+. These subclasses could not be further characterized by additional cell surface immunophenotypic markers or by gene (immunoglobulin and T-cell receptor) rearrangement analysis. The ratio R24/M2590 was less than 1.0 in all patients with pre-B-ALL, and was greater than 1.0 in all patients with T-ALL who were R24 positive, but was not useful in characterizing the double negative T-ALL subclass. To assess whether cryptogenicity of gangliosides due to cell surface protein could account for the low binding of either R24 or M2590, blasts were treated with trypsin before antibody analysis. Whereas the binding of R24 was unchanged after trypsin treatment, binding of M2590 was increased in a number of samples, particularly in those samples which were originally M2590-positive. The results show that comparative staining of T-ALL and pre-B-ALL cells with both anti-GD3 and anti-GM3/SPG antibodies results in a further subclassification of ALL and provides a quantitative assessment of the expression of tumor-associated gangliosides on the blasts of this disease.     

Imatinib resistance in multidrug-resistant K562 human leukemic cells

The multidrug resistance phenotype (MDR) is one of the major causes of failure in cancer chemotherapy and it is associated with the over-expression of P-glycoprotein (P-gp or MDR1) in tumor cell membranes. A constitutive NF-kappaB activity has been observed in several haematological malignancies and this is associated with its anti-apoptotic role. In the present work, the relationship between NF-kappaB and MDR phenotype was evaluated in wild type K562 human leukemic cells (K562-WT) and in its vincristine-resistant counterpart, K562-Vinc cells. These data showed that K562-Vinc cells, which express an active P-gp, exhibited MDR phenotype. The resistant indexes (IC(50)(K562-Vinc)/IC(50)(K562-WT)) for structurally unrelated drugs like imatinib, doxorubicin and colchicine were 8.0+/-0.3, 2.8+/-0.4 and 44.8+/-8.8, respectively. The imatinib resistance was reversed by P-gp blockade suggesting the involvement of P-gp in imatinib transport. We observed that NF-kappaB was constitutively activated in both cell lines but in a lesser extent in K562-Vinc. The inhibition of NF-kappaB with BAY 11-7082 increased the cytotoxicity of imatinib in K562-Vinc cells but not in K562-WT. Further, the co-administration of imatinib and BAY 11-7082 sensitized multidrug-resistant K562 cells to cell death as detected by increased percentage of annexin V positive cells. The induced cell death in K562-Vinc cells was associated with activation of caspases 9 and 3. Finally, we provide data showing that BAY 11-7082 down-regulates the expression of P-gp suggesting that the activity of NF-kappaB could be functionally associated to this protein in K562 cells. Our results indicate that the vincristine-resistant K562 cells which developed MDR phenotype, exhibited resistance to imatinib associated with a functional P-gp over-expression. This resistance could be partially overcome by the inhibition of NF-kappaB pathway.     

Inositol lipid metabolism accompanies erythroid differentiation of K562 human leukemic cells.

The role of hemin induction of K562 in inositol phospholipid metabolism has not been previously studied. K562 cells were induced to synthesize hemoglobin upon addition of bovine hemin to the culture media. The phospholipid content of K562 was determined before and after the addition of hemin. The results of this study demonstrated significant differences in the phosphoinositides between induced and non-induced cells. Phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-inositol-4,5-bisphosphate (PIP2) levels increased upon induction, and remained above control levels. Phosphatidylinositol (PI) levels decreased 15 min after hemin addition, then increased to control levels by 1 h. From 2-8 h PI levels then remained depressed below control levels. These data suggest that hemoglobin induction in K562 cells occurs concomitantly with inositol phospholipid turnover.     

Ionic currents in multidrug resistant K562 human leukemic cells

In this study, the expression and functional characterization of currents through the CFTR (cystic fibrosis transmembrane regulator) and ORCC (outwardly rectifying chloride channels) were determined in wild-type K562 chronic human leukemia cells (K562-WT) and in its resistant counterpart, the vincristine resistant cell line (K562-Vinc). Expression of the CFTR and MDR1 (multidrug resistant) gene products was determined by a semi-quantitative RT-PCR protocol. The amplified products in K562-WT and K562-Vinc showed two bands corresponding to CFTR and MDR1. MDR1 mRNA increased by 20-fold in K562-Vinc whereas no change in CFTR mRNA levels was observed. CFTR and ORCC channel activity were measured with a whole cell configuration of the patch clamp technique. Forskolin (40 microM n activator of adenylate cyclase, added to the extracellular side increased the current in both cell lines. A fraction of the activated whole cell currents was inhibited by 500 microM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and subsequent addition of 500 microM diphenylamine-2-carboxylate (DPC plus DIDS) further inhibited the remaining currents. The levels of forskolin-activated currents and subsequent blockade were similar in both cell lines. The effect of forskolin was prevented in cells previously exposed to 500 microM DPC. The effects of DIDS and DPC on the forskolin-activated whole cell currents support the idea that both CFTR and ORCC are generating a significant fraction of these currents with DIDS inhibiting ORCC currents and DPC inhibiting CFTR currents when the blockers are added one after another to the extracellular side. Finally, we show that exposure of K562 cells to vincristine which results in the over expression of MDR1 is not accompanied by a significant down regulation of CFTR as in other cells.     

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.     

Isofenphos induced metabolic changes in K562 myeloid blast cells

The organophosphate pesticide, isofenphos, is associated with human myeloid leukemia. In this study we describe metabolic changes in K562 myeloid blast cells from exposure to varying concentrations of isofenphos using the stable [1,2-13C(2)]glucose isotope as the single tracer and biological mass spectrometry. Isofenphos (1, 10, 100 microg/ml/72 h) treated K562 cells showed increases of 10.7, 33.8 and 39.7% in lactate production as well as a 14.2% increase (1 microg/ml/72 h) in 13C incorporation into nucleic acid ribose from glucose. Concomitantly, we observed a decrease in glucose oxidation and the synthesis of glutamate, palmitate and stearate from glucose. Our results demonstrate that this organophosphate pesticide exerts a leukemogenic effect by the recruitment of glucose carbons for nucleic acid synthesis thus promoting proliferation simultaneous with poor differentiation. The imbalanced metabolic phenotype with a severe defect in glucose oxidation, lipid and amino acid synthesis concurrent with de novo synthesis of nucleic acids in response to isofenphos treatment conforms to the invasive proliferating phenotype observed in TGF-beta treated lung epithelial carcinoma cells.     

Inhibition of collagenolytic activity in human leukemic K562 cells by tamoxifen

Presence of a collagenolytic activity has been demonstrated in the human leukemic cell line K562. Among various effectors studied, tamoxifen, a well-known antiestrogenic compound, exhibited a strong inhibitory effect. After 3 days of culture in the presence of 10⁻⁷M of tamoxifen, 75% of the collagenolytic activity was inhibited. Hydroxytamoxifen and N-desmethyltamoxifen were equally potent inhibitors though devoid of the direct cytotoxic effect. Cis-tamoxifen was less efficient. K562 cells have no binding sites for estrogens but they possess high affinity binding sites for ³H-tamoxifen (295 fmol/mg of proteins, K_D=0.25 × 10⁻⁹ M). Tamoxifen had no effect on cellular differentiation or enzyme secretion. Anticollagenolytic activity of tamoxifen (10⁻⁷-10⁻⁶ M) could be related to its inhibitory action on plasmin and plasminogen activator.     

Effects of herbimycin A derivatives on growth and differentiation of K562 human leukemic cells.

Herbimycin A, a specific tyrosine kinase inhibitor, induced erythroid differentiation of human myelogenous leukemia K562 cells with a high level of bcr/abl tyrosine kinase. Several derivatives of herbimycin A were synthesized and their effects on cell proliferation and differentiation of K562 cells were examined. Of the compounds tested, 19-allylaminoherbimycin A was the most effective in inducing differentiation of K562 cells. However, the parent compound was the most potent growth inhibitor, suggesting that chemical modification of herbimycin A reduces the growth-inhibiting activity. The sensitivities of K562 cells to herbimycin derivatives were different from those of a rat kidney cell line infected with Rous sarcoma virus (v-src), suggesting that bcr/abl kinase may differ in sensitivity from other tyrosine kinases. These results indicate that a specific inhibitor of bcr/abl kinase could be an effective antitumor agent against chronic myelogenous leukemia.     

Monoclonal antibodies raised against K562 cells reacted with human haematopoietic pluripotent stem cells

We describe the properties of three monoclonal antibodies (McAbs) (21H73, 37G7 and 49C12) against K562 cell surface antigens correlated with differentiation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Each of the McAbs immunoprecipitated K562 cell surface antigen with molecular weight (MW) of approximately 51 kD, 82 kD or 92 kD, respectively. The antigens detected by McAbs 21H73 and 37G7 were not immunoprecipitated from K562 cells differentiated into monocyte-macrophages by TPA (K562-TPA). On the other hand, 49C12 immunoprecipitated an antigen with MW of 92 kD from K562-TPA cells, but not from undifferentiated K562 cells. To examine the distribution of these antigens among human haematopoietic stem cells, bone marrow cells were separated by the panning method using these McAbs and subjected to colony-forming assays. The McAb 21H73 reacted with CFU-mix and BFU-E but with neither CFU-E nor CFU-GM. CFU-mix and BFU-E were enriched approximately 6.2-14.7-fold and 2-fold by the panning procedure using 21H73, respectively. On the other hand, 37G7 reacted only with BFU-E, and 49C12 reacted with CFU-GM but not with any other haematopoietic progenitor cells. We also examined the reactivity of these McAbs with leukaemia cells freshly isolated from 26 patients. The antigen defined by 21H73 was not expressed on any leukaemia cells from patients except for cells from an acute lymphocytic leukaemia (ALL, L3) and a CML in blastic crisis. The McAb 37G7 reacted with several types of leukaemia cells. The antigen defined by 49C12 was expressed on almost all leukaemia cells isolated from patients. These results suggest that 21H73 allows purification and enrichment of normal haematopoietic pluripotent stem cells from both normal and leukaemia patients' bone marrow specimens, especially following the step to remove leukaemia cells and haematopoietic progenitor cells other than CFU-mix by using 37G7 and/or 49C12.     

以microRNA-17为靶点的反义核酸对白血病细胞K562的作用

目的 研究以microRNA-17(miR-17)为靶点的反义核酸对人类白血病K562细胞的生长抑制作用.方法 人工合成miR-17的反义核酸,硫代修饰,用Lipofectamine 2000转入K562细胞.四甲基偶氮唑蓝(MTT)比色法检测转染反义核酸后K562细胞的增殖活性,流式细胞术(FCM)检测细胞的凋亡水平,实时荧光定量聚合酶链反应(PCR)检测反义核酸作用后K562细胞内miR-17的相对表达水平.结果 MTT结果显示,转染反义核酸后的24、48、72 h,K562细胞的增殖活性分别为0.872±0.001、0.710±0.002、0.551±0.001,与随机核酸序列组(随机对照组)(增殖活性分别为1.001±0.002、1.009±0.003、1.211±0.003;t值分别为182.58、269.77、660.40)、空白对照组(增殖活性分别为1.113±0.001、1.114±0.001、1.101±0.001;t值分别为537.98、571.20、1230.51)相比较差异有统计学意义(均P＜0.05);FCM检测结果表明,在转染反义核酸48 h后细胞的凋亡率为(20.14±0.01)％,与随机对照组的(3.54±0.02)％及空白对照组的(1.98±0.01)％比较差异有统计学意义(t分别为2347.6、2568.2,均P＜ 0.01);实时荧光定量PCR结果证实,在反义核酸作用后,K562细胞内miR-17的相对表达水平(0.07)明显下降,与随机组(1.00)、空白组(1.01)相比较差异有统计学意义(t值分别为148.63、147.04,均P＜0.05).结论 以miR-17为靶点的反义核酸可抑制K562细胞的增殖活性,并显著促进细胞的凋亡.     

Clonal response of K562 leukemic cells to exogenous p21WAF1

The p21WAF1 protein is involved in the control of cell differentiation and proliferation. We have previously shown that p21WAF1 is upregulated in normal, proliferating hematopoietic cells undergoing differentiation. Exogenous p21WAF1 has been reported to increase colony-formation by normal hematopoietic progenitors. We examined the effects of exogenous p21WAF1 on proliferation, differentiation, gene expression and colony-formation by K562 cells using an inducible p21WAF1 expression construct. Expression of the stathmin (oncoprotein 18) gene decreased within 24 h of p21WAF1 expression; Hox B4 expression increased. Four K562 subclones were derived which differed in their response to equivalent induction of p21WAF1. All four subclones exhibited growth arrest in response to p21WAF1 in liquid culture. Three of four clones developed cytoplasmic granulation and partial morphologic differentiation after p21WAF1 induction. One clone exhibited fewer morphologic features of differentiation following p21WAF1 induction and unlike other clones, colony formation in methlycellulose was not decreased by p21WAF1 expression in this clone. This indicates that additional cell-specific factors influence cellular fate in the presence of elevated p21WAF1.     

Hemin dependent morphological maturation and endogenous porphyrin synthesis by K562 leukemic cells

The alterations in the morphological phenotypes of K562 cells induced by exogenous hemin, and its effect on porphyrin accumulation was analyzed by scanning electron microscopy and flow cytometry. The morphology of the uninduced cells was mainly of microvilli, blebi and ruffle covered cells, while after hemin induction the fraction of the blebi-cells increased and smooth erythroid-like cells appeared. The large variations in cell-volume of the cells were decreased only marginally after hemin treatment. Porphyrin fluorescence revealed a marked accumulation of endogenous porphyrins after induction by hemin.     

Complement-mediated lysis of K562 human leukemic cells by antibodies to phosphotyrosine and identification of cell surface proteins phosphorylated on tyrosine

Intact K562 human leukemic cells showed bright membrane immunofluorescence after staining with monoclonal antibody to O-phosphotyrosine (PTyr). Up to 60% of the cells were lysed with mouse, rabbit, or human antibodies to PTyr by a complement-mediated mechanism. A new method has been developed for identifying proteins that have PTyr residues on the outside of cell membrane, and at least two species of PTyr-containing proteins with the molecular weights of 45,000 and 36,000 were identified as the most probable candidates of the antigens responsible for the membrane fluorescence and cell lysis.     

Potentiation of cytotoxicity of 1-beta-D-arabinofuranosylcytosine for K562 human leukemic cells by cadeguomycin

The treatment of K562 human myeloblastic leukemia cells and YAC-1 murine lymphoma cells with cadeguomycin at concentrations over 0.6 microM significantly enhanced the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C). The degree of potentiation depended upon the antibiotic concentration. The treatment with 75 microM cadeguomycin for 18 h increased cellular uptake of [3H]ara-C into K562 cells and formation of ara-C nucleotides, as well as incorporation into nucleic acids. The level of the diphosphate of ara-C plus the triphosphate of ara-C was approximately 10 times higher in the cadeguomycin-treated cells than in the untreated cells by 30 min of incubation with [3H]ara-C. The extracts of 15 microM cadeguomycin-treated K562 cells showed increased activity of formation of ara-C nucleotides, resulting in 4- to 5-fold higher formation of the di- and triphosphates of ara-C than the control cell extracts. Cadeguomycin did not significantly change the level of ribonucleotide and deoxyribonucleotide pool in K562 cells. The mechanism of potentiation of ara-C by cadeguomycin was discussed.     

The differential reactivity of cells of the melanocytic lineage with four monoclonal antibodies against IFN-gamma inducible molecules.

The reactivity of four monoclonal antibodies (MAbs) directed against IFN-gamma inducible antigens with melanocytic cells was investigated in the course of local and systemic tumor progression of human malignant melanoma. Frozen sections of histologically defined melanocytic tissues at different stages of progression were stained with these MAbs using an indirect immunoperoxidase technique. The reactivity of MAbs Me15/B3 and Me15/F9, directed against two different epitopes of a 90-kDa molecule, was found to correlate with melanoma progression. Indeed, a significantly lower percentage of small than of advanced primary melanomas or metastases stained positively. A differential staining of nevocytic and dysplastic nevi was further observed for these two MAbs, which were also non-reactive with normal skin melanocytes. The reactivity of MAb Me14/D12, which identifies the intercellular adhesion molecule ICAM-1 and MAb Mel14/F12, directed against a 40-kDa molecule, was found to be independent of the Breslow thickness of primary melanomas. Both the latter MAbs stained a high proportion of nevocytic and dysplastic nevi. The co-expression of the surface molecules defined by MAbs Me14/D12, Me15/B3 and Me15/F9 in the course of melanoma progression was also analyzed. The frequency of this co-expression increased according to the Breslow thickness of primary melanomas. In addition, up to 100% of metastases, as opposed to 20% of dysplastic nevi, were found to be simultaneously stained by these three MAbs. It is therefore conceivable that high-risk melanocytic lesions might be identified by the use of a combination of MAbs directed against IFN-gamma regulated antigens.     

DNA damage response in imatinib resistant chronic myeloid leukemia K562 cells

Abstract Resistance to imatinib in patients with chronic myeloid leukemia can lead to advanced disease and blast crisis. Conventional chemotherapy with DNA damaging agents is then used, alone or in combination with other tyrosine kinase inhibitors (TKIs). Our aim was to assess whether imatinib resistant K562 cells were also resistant to DNA damaging agents. After treatment with H(2)O(2) and doxorubicin, but not camptothecin, cell survival was higher in imatinib resistant cells compared to parental cells. DNA damage, measured by comet and γ-H2AX assays, was lower in imatinib resistant cells. mRNA expression levels of 50 genes of the DNA damage response pathway showed increased expression of the base excision repair (BER) genes MBD4 and NTHL1. Knockdown of MBD4 and NTHL1 expression in resistant cells using siRNA decreased cell survival after treatment with H(2)O(2) and doxorubicin. Our results indicate that imatinib resistant cells display cross-resistance to oxidative agents, partly through up-regulation of BER genes. Expression of these genes in imatinib resistant patients was not significantly different compared to sensitive patients. However, the strategy followed in this study could help identify chemotherapeutic agents that are more effective as alternative agents in cases of resistance to TKIs.