Analysis of human leukemia/lymphoma cell lines with monoclonal antibodies BA-1, BA-2 and BA-3

A panel of monoclonal antibodies (BA-1, BA-2 and BA-3) were made against the pre-B acute lymphoblastic leukemia (ALL) cell line NALM-6. BA-3 binds to the 100,000 mol. wt common ALL antigen (gp100/CALLA), BA-2 binds to a 24,000 mol. wt (p24) cell surface structure and BA-1 binds to a currently undefined antigen. These antibodies were analysed for their reactivity with 67 human leukemia/lymphoma cell lines. The cell lines studied included T ALL, non-T ALL. surface immunoglobulin⁺ leukemias/lymphomas, and myeloid/monocytoid leukemias. The results indicate that all three antibodies react primarily (but not exclusively) with malignant cells early in B cell development. This pattern of reactivity was similar to previous results obtained with fresh, non-cultured malignant cells. BA-1, BA-2 and BA-3 are useful tools for analysing the developmental options of normal lymphoid cells and for “cleaning up” leukemia/lymphoma cells in selected cases of autologous bone marrow transplantation.     

Combined chemoseparation and immunoseparation of clonogenic T lymphoma cells from human bone marrow using 2'-deoxycoformycin, deoxyadenosine, 3A1 monoclonal antibody, and complement

Chemoseparation and immunoseparation techniques have been combined to eliminate malignant clonogenic T lymphoma cells from human bone marrow. Incubation with 5 microM 2'-deoxycoformycin and 500 microM deoxyadenosine has eliminated 2 logs of HSB-2 T lymphoma cells from a 20-fold excess of irradiated human bone marrow. Multiple incubations with 3A1 antibody and rabbit complement eliminated approximately 2 logs of HSB-2 cells from similar mixtures. Used in combination, the 2 techniques eliminated up to 4 logs of T lymphoma cells. Incubation of normal human bone marrow under similar conditions failed to affect growth of granulocyte-macrophage colony-forming cell units, burst-forming erythroid units, or multipotential erythroid-granulocyte-megakaryocyte-macrophage colony-forming hematopoietic progenitor cells units.     

Imaging of human leukemic T-cell xenografts in nude mice by radiolabeled monoclonal antibodies and F(ab')2 fragments

Monoclonal antibodies (MoAb) that react with the T-lymphocyte markers called cluster of differentiation CD5 and CD2 were labeled with iodine 131 (131I) and were injected intravenously in nude mice bearing solid subcutaneous xenografts derived from the human T-cell leukemia line Ichikawa. Both MoAb anti-CD5 and anti-CD2 yielded favorable mean tumor to whole-body ratios of 3.8 and 5.1, respectively. These ratios were further increased up to 10.0 for MoAb anti-CD5 and 15.5 for MoAb anti-CD2 by using their F(ab')2 fragments. The tumors could be imaged clearly by external scanning after injection of F(ab')2 fragments from both MoAb. F(ab')2 fragments from MoAb anti-CD2 and of a third MoAb recognizing the clonotypic determinant (Ti) of the antigen receptor expressed by the human T-cell line Jurkat were injected in mice bearing intrasplenic Jurkat xenografts. A selective localization of both fragments in tumor tissue was demonstrated with mean tumor to whole-body ratios of 7.5 and 4.1 for MoAb anti-CD2 and anti-Ti, respectively. These in vivo experimental results may provide useful information for the potential use of radiolabeled MoAb and fragments in the diagnosis and treatment of patients with T-cell lymphoma and different other forms of T-cell malignancies.     

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。     

New data on the cytolytic effects of natural killer cells (Kurloff cells) on a leukemic cell line (guinea pig L2C)

L2C leukemia is a leukemia that occurs in strain two guinea pigs. The L2C cells are natural killer-sensitive. The Kurloff cell (KC), a guinea pig NK cell, develops a 3-fold increase in lysosomal enzyme activity and the number of KC cells increases during leukemogenesis, leading to KC cell-mediated L2C cytolysis. This paper shows that conjugates are produced by incubating KC and L2C for 4 h, with 34% of L2C showing chromatin compaction and shrinkage of the cytoplasm. There was also a reorientation of the KC cytoplasmic organelles to face the target cell and an elongation of the KC to produce arms that engulfed the L2C. The L2C had either necrotic or apoptotic characteristics. L2C DNA fragmentation was demonstrated in situ with the comet and the TUNEL assays. 22.2% of the viable L2C lost their membrane asymmetry during KC-L2C conjugation as shown by incubation with Annexin V-FITC. These results provide new evidence that the death of L2C is due, at least partly, to apoptosis. The cytolytic effect of the NKKC might be a model of the cytological changes that occur in NK cell-leukemic cell conjugates.     

Comparative studies on the in vitro killing of human normal and leukemic clonogenic cells (CFUc) by daunorubicin,daunorubicinol, and daunorubicin-DNA complex

Summary The sensitivity of normal and acute nonlymphocytic leukemia-derived granulocytic stem cells (colony-forming units in culture, CFUc), exposed to daunorubicin, daunorubicinol, or daunorubicin-DNA complex in short-term suspension culture in vitro was studied. Normal CFUc were found to be considerably more sensitive to daunorubicin than to daunorubicinol. Killing of normal marrow CFUc by daunorubicin was an exponential function of the dose within the time and dose range tested. The C₅₀ varied between 100 and 230 ng/ml medium, LD₃₇ between 53 and 79 ng/ml medium. The response of CFUc to daunorubicin-DNA complex was exponential with somewhat larger individual variations (C₅₀=100–330 ng/ml, LD₃₇=46–878 ng/ml). The in vitro sensitivity of leukemia-patient derived CFUc to daunorubicin showed a greater variation (C₅₀=127–454 ng/ml, LD₃₇=74–640 ng/ml) than that of normal-control derived CFUc. In comparative studies with the leukemia-derived CFUc, daunorubicin-DNA complex was more or as effective as the free drug in killing CFUc derived from some patients, but in killing the CFUc derived from other patients was far less effective than the free drug. The results indicate greater individual variations in the response of leukemia-derived than normal CFUc both to daunorubicin and to daunorubicin-DNA complex in vitro. The therapeutic relevance of the results is discussed.     

Circulating blood group-related antigen(s) in cancer patients detected by the monoclonal antibodies produced against hepatocellular carcinoma cell line

Two murine monoclonal antibodies (MAbs) S1 and S3 produced against a human hepatoma cell line were found to recognize both H (type II) antigen and difucosyl type II structure (Y-antigen). Both MAbs reacted with hepatoma tissues obtained from surgical resection. A double determinant enzyme immunoassay (DDEI) employing these MAbs successfully detected the corresponding antigen(s) in the supernatant which is either released or shed from the immunizing hepatoma cell line. DDEI also revealed that these antibodies reacted with the corresponding antigen(s) in the sera from cancer patients. Among the sera from O blood group donors, individuals with primary hepatoma showed markedly higher levels of antigen concentration than the normal control group. Several cases with gall bladder, lung and pancreas carcinomas, which are of O blood type, also had significantly higher levels of antigen. One case with ovarian cancer of B blood type showed higher antigen concentration than normal O blood group donors suggesting a change in the carbohydrate structure of the corresponding antigen(s) in the serum. These data suggest that the release or the shedding of the antigen(s) from the cells may increase due to the malignant transformation, resulting in higher amounts of the antigen(s) in the serum of certain cancer patients.     

PAI-1在鼻咽癌细胞系(CNE-2Z)及其克隆株(CNE-2Z-H5,CNE-2Z-H5-9)中表达和意义

目的:探讨尿激酶激活剂抑制剂-1(Plasminogenactivatorinhibitor-1,PAI-1)在不同淋巴道转移能力的鼻咽癌细胞系(CNE-2Z)及其克隆株(CNE-2Z-H5,CNE-2Z-H5-9)中的表达和意义。方法:应用逆转录聚合酶反应(RT-PCR)方法检测PAI-1在基因转录水平的表达;使用异质粘附、侵袭抑制实验观察PAI-1在鼻咽癌细胞侵袭和转移过程中的作用。结果:PAI-1在CNE-2Z,CNE-2Z-H5中均有转录,但无显著性差异;PAI-1在CNE-2Z-H5-9中无转录。抗PAI-1抗体对CNE-2Z-H5-9异质粘附、侵袭无影响。结论:PAI-1可能抑制鼻咽癌的侵袭和转移。     

Induction of apoptosis in leukemic cells by the reversible microtubule-disrupting agent 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1 -one: protection by Bcl-2 and Bcl-X(L) and cell cycle arrest

We have found that the bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC) induced a dose- and time-dependent apoptotic response in human leukemic cells. MTC and colchicine rapidly disrupted the microtubule integrity and arrested cells at the G2-M phase before the onset of apoptosis. These responses were mediated by microtubule inhibition because 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)propionyl]amino]-2,4,6-cycloh eptatrien-1-one and lumicolchicine, inactive analogues of MTC and colchicine, respectively, were unable to promote microtubule disassembly, cell cycle arrest, and apoptosis. Although 1 microM MTC induced a complete microtubule disruption after 1 h of incubation in human leukemic HL-60 cells that led to an accumulation of cells at the G2-M phase, MTC-induced apoptosis occurred after 9 h of treatment. This indicates the existence of a rather long lag between microtubule disruption and the onset of apoptosis. Unlike colchicine, the removal of MTC during this lag resulted in rapid microtubule repolymerization, followed by restoration of normal cell cycle and cell growth. MTC, but not 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino]-2,4,6-cyclo heptatrien-1-one, induced c-jun expression as well as c-Jun NH2-terminal kinase and caspase activation, indicating that these signaling pathways are triggered by the specific action of MTC on microtubules. Caspase inhibition prevented MTC-induced apoptosis. Overexpression of bcl-2 or bcl-xL by gene transfer in human erythroleukemic HEL cells abrogated MTC-induced apoptosis, but cells remained arrested in G2-M, suggesting that bcl-2 and bcl-xL block the signaling pathway between G2-M arrest and triggering of apoptosis. MTC-treated bcl-2 and bcl-xL-transfected HEL cells recovered their capacity to proliferate after MTC removal. These results indicate that microtubule inhibition induces G2-M arrest and apoptosis in leukemic cells, showing a lag phase between G2-M arrest and the onset of apoptosis, regulated by bcl-2 and bcl-xL, during which MTC displays a reversible action on microtubule depolymerization and G2-M cell cycle arrest. Thus, MTC is a potent apoptotic inducer on human leukemic cells and shows a remarkable reversible action on microtubule network and cell cycle before commitment for apoptosis is reached.     

5(S)-Hydroxy-6,8,11,14-E,Z,Z,Z-eicosatetraenoate stimulates PC3 cell signaling and growth by a receptor-dependent mechanism

5(S)-Hydroxy-6,8,11,14-E,Z,Z,Z-eicosatetraenoate (5-HETE) causes PC3 cells to grow by an unknown mechanism. We find that it also induces the cells to activate extracellular signal-regulated kinases and Akt. Pertussis toxin inhibits both responses. 5-HETE, 5-oxo-6,8,11,14-E,Z,Z,Z-eicosatetraenoate, and 5-oxo-15-hydroxy-eicosatetraenoate are known to stimulate leukocytes by a receptor coupled to pertussis toxin-sensitive G proteins. Their respective relative potencies in leukocytes are 1, 10, and 3. In PC3 cells, however, these values are 10, 1, and 0. PC3 cells, we propose, express a non-leukocyte-type, G protein-coupled, 5-HETE receptor. This novel receptor and the extracellular signal-regulated kinase and Akt pathways it recruits may contribute to the progression of prostate adenocarcinoma.     

Stimulation of proliferation in human colon cancer cells by human monoclonal antibodies against the TF antigen (galactose β1-3 N-acetyl-galactosamine)

In many tissues, the TF (Thomsen-Friedenreich) blood group antigen (Galβ1-3GalNAcα-) behaves as an onco-foetal carbohydrate antigen, showing increased expression in malignancy and hyperplasia. Dietary lectins which bind the TF antigen have marked effects on proliferation of epithelial cells without cytotoxicity. This led us to speculate that anti-TF antibodies, including those that naturally occur in humans, might have similar effects. Five anti-TF antibodies, TF2 (human), TF5 (human), 5A8 (mouse), 8D8 (mouse) and BM22 (mouse), but not TF1 (human) or 49H.9 (mouse), showed marked dose-dependent stimulation (95–192%) of [³H]thymidine incorporation by HT29 human colon cancer cells. Similar stimulation of proliferation of HT29 cells by these monoclonal antibodies (MAbs) was found when cell count assessment was used. Antibody-stimulated proliferation was inhibited by co-incubation with glycoproteins expressing Galβ1-3GalNAcα- (asialo glycophorin or [Galβ1-3GalNAcα-O-p-aminophenyl]_n-human serum albumin). A proliferative effect of these antibodies was also demonstrated on human colon cancer cell lines LS174T and HT29-MTX but not on Caco-2 cells. Although immunoblotting showed similar binding patterns of all the antibodies on HT29 cell membrane extracts, there was little correlation between cell surface binding assessed by immunofluorescence and proliferative response, and internalization of the biotinylated antibody TF5 was demonstrated by confocal microscopy. Our results provide further evidence that cell surface glycoproteins which express TF antigen may play an important role in the regulation of cell proliferation and also suggest that human anti-TF antibodies may have proliferative effects on cells which express TF antigen. Int. J. Cancer 73:424–431, 1997. © 1997 Wiley-Liss, Inc.     

CHARACTERIZATION OF A HUMAN HERPES VIRUS－6（HHV－6） AND EPSTEIN－BARR VIRUS（EBV） ASSOCIATED LEUKEMIC CELL LINE，J6－1

ＣＨＡＲＡＣＴＥＲＩＺＡＴＩＯＮＯＦＡＨＵＭＡＮＨＥＲＰＥＳＶＩＲＵＳ－６（ＨＨＶ－６）ＡＮＤＥＰＳＴＥＩＮ－ＢＡＲＲＶＩＲＵＳ（ＥＢＶ）ＡＳＳＯＣＩＡＴＥＤＬＥＵＫＥＭＩＣＣＥＬＬＬＩＮＥ，Ｊ６－１ＷｕＫｅｆｕ吴克复；ＪａｎｏｓＬｕｋａ；Ｓｈａｎｔ...     

2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖对人食管癌细胞Eca-109的致凋亡研究

目的探讨2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖是否具有诱导人食管癌细胞Eca-109发生凋亡的作用.方法采用体外细胞培养实验方法,应用MTT法、流式细胞仪检测、DNA琼脂糖凝胶电泳、透射电镜等技术,观察药物对细胞生长的抑制率及细胞的形态学变化,检测凋亡峰及DNA Ladder.结果 MTT法测得细胞抑制率具有显著的时间及浓度依赖性;透射电镜下可见到凋亡典型的形态学变化,流式细胞仪检测显示细胞周期的G1期阻滞及凋亡峰;DNA琼脂糖凝胶电泳显示清晰的DNA梯状电泳.结论 2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖能够明显的抑制人食管癌细胞Eca-109的增殖,并诱导其发生凋亡.     

2-（3-羧基-1-丙酰氨基）-2-脱氧-D-葡萄糖对人食管癌细胞Eta-109的致凋亡研究

目的 探讨2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖是否具有诱导人食管癌细胞Eta-109发生凋亡的作用。方法 采用体外细胞培养实验方法，应用MTT法、流式细胞仪检测、DNA琼脂糖凝胶电泳、透射电镜等技术，观察药物对细胞生长的抑制率及细胞的形态学变化，检测凋亡峰及DNALadder。结果 MTT法测得细胞抑制率具有显著的时间及浓度依赖性；透射电镜下可见到凋亡典型的形态学变化，流式细胞仪检测显示细胞周期的G期阻滞及凋亡峰；DNA琼脂糖凝胶电泳显示清晰的DNA梯状电泳。结论 2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖能够明显的抑制人食管癌细胞Eca-109的增殖，并诱导其发生凋亡。     

铬,汞,镉,镍四种重金属化合物对FL／P4501A1细胞酶活性的影响

利用不同浓度的重金属化合物溴化汞、三氯化铬、硫酸镉、氯化镍处理人羊膜ＦＬ／Ｐ４５０１Ａ１细胞，其半数抑制浓度（ＩＣ５０，ｍｇ／ｍｌ）分别为６．０２６０，３７．５２１３，２４．５４９１和４５．３８４５。结果表明：不同的重金属化合物对ＦＬ／Ｐ４５０１Ａ１细胞的毒性相差很大，溴化汞和硫酸镉的细胞毒性较大。ＦＬ／Ｐ４５０１Ａ１细胞带有Ｐ４５０１Ａ１基因，该基因产物为乙氧基异吩恶唑Ｏ－去乙基酶（ＥＲＯＤ）。     

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. METNODS The cytotoxic effect of STI571 alone or in combination with different concentrations of As2O3 on the bcr-abl and mdrl -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 cells 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.     

Characteristics of human T-lymphotropic virus type-1 (HTLV-1)-infected cell line MT-2, which is not killed by a natural killer cell line NK-92 but is killed by lymphokine-activated killer cells

Natural killer (NK) cell-mediated cytolysis (NK-lysis) is triggered by costimulatory signals of adhesion molecules and is downregulated by negative signals of killer cell inhibitory receptors (KIRs). Recently, a NK cell line, NK-92, was established. This cell line can kill several tumor cells, which possess adhesion molecules CD54 and CD102. However, the NK-92 cannot kill a human T-lymphotropic virus type 1 (HTLV-1)-infected cell line, MT-2, although lymphokine-activated killer (LAK) cells can kill MT-2. In this report we investigated the reason for LAK sensitivity but NK-92 resistance of the MT-2. The MT-2 highly expressed CD54 and CD102, suggesting that the costimulatory signals may be intact. Then we tested the responsibility of the negative signals by determining HLA type of the MT-2 and KIRs of the effector cells. The MT-2 expressed HLA-A24, B40, B51, Cw3, and HLA-G. The NK-92 did not express KIR2DL1, KIR2DL2,3, nor KIR3DL1, but 24% of the cells weakly expressed CD94. The blocking tests against these HLA class I molecules and KIRs did not restore the NK-92 resistance, although blocking against HLA-G slightly increased its lysis. Finally, in order to eliminate the class I molecules from the cell surface, we treated the MT-2 using a buffered citric acid solution (pH 3.8). By using this treatment, the expression of class I molecules and HTLV-1 antigen decreased, and then the MT-2 was killed by the NK-92. These findings suggest that an aberrant class I molecule of the MT-2 transferred a negative signal to the NK-92 and induced the NK-92 resistance. It remains to be elucidated whether or not the HTLV-1 infection contributed to the alteration of the class I molecule.