Differential Alterations of Dihydrofolate Reductase Gene in Human Leukemia Cell Lines Made Resistant to Various Folate Analogues

In order to clarify a molecular mechanism of folate resistance in leukemia cells, we studied alterations of the dihydrofolate reductase (DHFR) gene in a human leukemia cell line, MOLT-3, and its sublines made resistant to methotrexate (MTX), trimetrexate (TMQ) and N¹⁰-propargyl-5,8-dideazafolic acid (CB3717), alone or in combination. Major alterations of the DHFR gene were examined by Southern analysis of high-molecular-weight DNA. The presence of a base change (T→C) at nucleotide position 91 of the DHFR gene, which is reported to be responsible for the reduced affinity of the enzyme for MTX in an MTX-resistant human colon carcinoma cell, was examined by allele-specific oligonucleotide hybridization. In a 10,000-fold MTX-resistant subline (MOLT-3/MTX_10,000), the normal allele of DHFR gene had been amplified. In contrast, a 200-fold TMQ-resistant subline (MOLT-3/TMQ₂₀₀) and a 30-fold CB3717-resistant subline selected from MOLT-3/TMQ₂₀₀ (MOLT-3/TMQ₂₀₀-CB-3717₃₀) were shown to have the mutant allele. Furthermore, the mutant allele had been amplified in a 500-fold MTX-resistant subline, which was established by the continuous exposure of the MOLT-3/TMQ₂₀₀ cells to stepwise increases of drug concentration and designated as MOLT-3/TMQ₂₀₀-MTX₅₀₀. On the other hand, a 40-fold-resistant subline to CB3717 alone (MOLT-3/CB3717₄₀) showed the normal allele without amplification. These data suggest that complex alterations of the DHFR gene are involved in the molecular mechanisms of folate resistance that can be differentially introduced into leukemia cells by exposure to various folate analogues, alone or in combination.     

Electron Microscopic Study of Cell Lines Established from Adult T-Cell Leukemia--Coexistence of Adult T-Cell Leukemia Virus and Epstein-Barr Virus

Six cell lines established from five patients with adult T-cellleukemia (ATL) were studied by electron microscopy. From onepatient two cell lines were established, an interleukin 2-dependentline and a nondependent line. The interleukin 2-dependent T-cellline had only ATL virus (ATLV) particles. The interleukin 2-nondependentB-cell line had both ATLV particles and Epstein-Barr virus (EBV)particles. In two other B-cell lines and one undetermined cellline, both ATLV particles and EBV particies were seen. In oneB-cell line only a few EBV particles were seen. These findingssuggest that (I) interleukin 2 is necessary for the growth ofleukemic T-cells from ATL tissue samples, and (2) ATLV can infectnot only T-cells but also B-cells.     

Interleukins and Colony Stimulating Factors in Human Myeloid Leukemia Cell Lines

The present review has summarized the expression, production and effects of the human interleukins (IL) 1-11 and myelopoietic colony stimulating factors (CSF) in the established myeloid leukemia cell lines and in cells from patients with acute myeloid leukemia as well as the oncogene expression reported in these myeloid leukemia cell lines. The genetic dissection of leukemic myelopoiesis may provide new perspectives for the control of myeloid leukemias. Based on their expression of phenotypic markers (e.g., surface antigens, cytochemical staining, etc.), myeloid cell lines can be further subdivided into myelogenous, monocytic, erythroid and megakaryoblastic leukemia cell lines. Due to the close relationship of erythroid and megakaryoblastic progenitor cells and to the existence of a probably common precursor cell giving rise to these two different cell lineages, many megakaryoblastic cell lines express erythroid markers (e.g., expression of hemoglobin or glycophorin A) and conversely cell lines with a predominant erythroid profile might display megakaryoblastic features (e.g., platelets peroxidase or glycoproteins CD41, CD42b or CD61). The recent cloning of the specific cytokine: thrombopoietin (TPO) and its receptor generated a strong interest in these particular myeloid cell lines that are discussed in more detail in the present review. Both normal and leukemic megakaryocytopoiesis are stimulated by granulocyte-macrophage colony stimulating factor (GM-CSF), IL-3, GM-CSF/IL-3 fusion protein, IL-6, IL-11 and TPO but inhibited by IL-4, interferon-alpha (IFN-alpha) and IFN-gamma. Human megakaryoblastic leukemia cell lines have common biological features: high expression of the megakaryocytic specific antigen (CD41); high expression of early myeloid antigens (CD34, CD33 and CD13); constitutive expression of IL-6 and platelet-derived growth factor; a complex karyotype picture; expression of c-kit (the stem cell factor receptor); growth-dependency or-stimulation by IL-3 and/or GM-CSF; and in vivo tumorigenicity in mice associated with marked fibrosis. Whereas numerous chemical and biologic agents induce granulocytic and/or monocytic differentiation of myeloid leukemia cell lines, only a few agents including phorbol myristate acetate, vitamin D3, IFN-alpha, IL-6 and thrombin have been reported to induce megakaryocytic differentiation in the megakaryoblastic leukemia cells.     

Molecular Cytogenetic Analysis of 17 Renal Cancer Cell Lines: Increased Copy Number at 5q31-33 in Cell Lines from Nonpapillary Carcinomas

Comparative genomic hybridization (CGH) was used to screen for genomic imbalances in cell lines derived from 13 nonpapillary renal-cell carcinomas (RCCs), two papillary RCCs, one renal squamous-cell carcinoma, and one transitional-cell carcinoma of the renal pelvis. Aberrations were found in all 17 lines. The most frequent changes in nonpapillary RCC cell lines were gains of 5q (85%), 7q (69%), 8q (69%) and 1q (54%) and losses of 3p (92%), 8p (77%), 4q (62%) and 14q (54%). High-level gains (HLGs) were detected at 4q12, 5p, 5q23-33, 7q22-qter, 8q23-24, 10q21-qter, 12p and 12q13-22. By means of fluorescence in situ hybridization (FISH) we narrowed the smallest common region involving 5q gains to the genomic segment between D5S642 and D5S673, and found that the HLG at 4q12 possibly involved amplifications of c-kit and PDGFRA . Two papillary RCC cell lines showed gains of entire chromosomes 7, 12 and 17. The CGH data reported here should help to facilitate the choice of individual renal-tumor cell lines for exploring target genes in regions of interest.     

Frameshift Mutations of the hMSH6 Gene in Human Leukemia Cell Lines

Defects in DNA mismatch repair mechanisms, including frameshift mutations of the hMSH6 and hMSH3 genes at their (C)₈ and (A)₈ tracks, respectively, have been shown to be associated with human malignancies. To clarify the possible involvement of these mutations in hematopoietic malignancies, we screened a total of forty-four human leukemia and lymphoma cell lines for mutations in the hMSH6 and hMSH3 genes, as well as in other genes required for DNA replication or repair, by polymerase chain reaction single-strand conformation polymorphism analysis and sequencing analysis. Frameshift mutations at the (C)₈ track of the hMSH6 gene were detected in two cell lines established from lymphoid leukemias. These two cell lines had no wild-type alleles, and both of them showed microsatellite instability. This is the first report that describes mutations and inactivation of the hMSH6 gene in hematological malignancies, suggesting that defects of the hMSH6 gene may be associated with development of hematological malignancies.     

Mechanism of the Discrepant Effect of a Combination of Methotrexate plus Dipyridamole on Human Hematologic Cell Lines

Mechanisms of the discrepant effect of methotrexate and dipyridamoleon human hematologic cultured cell lines were investigated byanalyzing intracellular methotrexate levels and thymidine incorporationthrough the salvage pathway, since the combination of methotrexateand dipyridamole has different effects according to cell type:additive effects on ML-1 and THP-1 (myelomonocytoid cells);reduced effects on MOLT-3, SKW-3, P32/ish and BL-TH (lymphoidcells). Dipyridamole reduced the toxicity of methotrexate bydiminishing intracellular methotrexate levels in MOLT-3 andBL-TH (lymphoid cells), in which the reduction of intracellularmethotrexate affected more than just the blocking of the salvagepathway required for growth by dipyridamole. On the other hand,dipyridamole enhanced the toxicity of the combination by blockingthe salvage pathway in an ML-1 (myelo-monocytoid cell) and ina methotrexate-resistant subline of BL-TH/MTX (lymphoid cell),in which the salvage pathways were considered activated. Dipyridamolecould prove to be a useful drug for reversing the drug resistancecaused by the activation of the salvage pathway.     

羟基磷灰石纳米粒子对人白血病细胞K562毒性作用的研究

目的:探讨羟基磷灰石(HAP)纳米粒子体外抑制人白血病细胞K562的生长增殖作用。方法:采用MTT法检测HAP和联合应用化疗药物对K562细胞的增殖抑制及化疗增敏作用。结果:(1)HAP和抗癌药物对K562细胞增殖均有抑制作用;(2)K562细胞生长抑制率与HAP的浓度和作用时间呈明显正相关性;(3)HAP与化疗药物合用具有更良好的抑癌效果。结论:HAP纳米粒子明显抑制K562细胞生长,具有体外抗白血病作用和化疗增敏作用。     

不同氧化态叶酸对人成淋巴细胞系基因组DNA甲基化水平的影响

目的 比较最高氧化态叶酸（folic acid, FA）与还原态5-甲基四氢叶酸（5-methyltetrahydrofolate,5-MeTHF）对人成淋巴细胞LINE-1和Alu的甲基化效应,从而评价受试物对全基因组DNA甲基化的影响。方法 以含30、60和120 nmol/L FA或5-MeTHF的改良RPMI1640培养液干预培养人成淋巴细胞系GM12593,20 d后提取基因组DNA,用亚硫酸盐修饰测序法（BSP）比较不同浓度和氧化态叶酸对受试细胞Alu和LINE-1甲基化水平的影响。结果 基因组Alu和LINE-1甲基化水平均随FA或5-MeTHF浓度的升高而增加,两目标序列的甲基化水平在120 nmol/L FA或5-MeTHF浓度下显著高于30和60 nmol/L组（P<0.01~0.05）,60和120 nmol/L的5-MeTHF提高LINE-1甲基化水平的能力显著高于同等浓度的FA（P<0.01~0.05）。结论 FA和5-MeTHF浓度与人成淋巴细胞基因组DNA甲基化水平显著正相关,5-MeTHF的基因组甲基化维护效应强于FA。     

Detection of Epidermal Growth Factor Receptor Gene Amplification in Human Squamous Cell Carcinomas Using Fluorescence in situ Hybridization

The gene for epidermal growth factor receptor (EGFR) is associated with development of certain human cancers. In this study, we employed the improved fluorescence in situ hybridization technique to detect EGFR gene amplification in cell lines and tissue sections from human squamous cell carcinomas. We detected multiple distinct signals as arrayed amplicons on metaphase chromosomes and interphase nuclei of tumor cells. Our results provide a basis for rapid and quantitative DMA diagnosis of the EGFR gene amplification in individual cells of tumor specimens.     

Lanreotide-Induced Modulation of 5-Fluorouracil or Mitomycin C Cytotoxicity in Human Colon Cancer Cell Lines: a Preclinical Study

Abstract

The effect on growth of the long-acting somatostatin analogue lanreotide (LAN), alone or in combination with 5-fluorouracil (5-FU) and mitomycin C (MIT), was investigated in three human colon cancer lines. Cell survival inhibition induced by LAN alone, as evaluated by sulforhodamine B assay, ranged from 20% to 40% as a function of cell line and concentration. The IC₅₀, the concentration inhibiting cell survival by 50%, was never reached. The antipro-liferative effect produced by a 48h exposure to 5-FU or MIT was synergistically enhanced in all cell lines by a subsequent 48h exposure to LAN. The synergis-tic interaction was not related to specific cell cycle perturbations or to the somatostatin receptor 2 (sst2) mRNA abundance. In conclusion, our study seems to indicate that LAN is a potentially useful modulating agent for enhancing 5-FU and MIT activity in colorectal cancer patients.     

Cytotoxicity Profiling of Annona Squamosa in Cancer Cell Lines

Objective: In the study our aim was to evaluate the cytotoxic activity of different solvent extracts of Annona squamosa seeds. Methods and materials: The four extracts used were petroleum ether, chloroform, ethyl acetate and methanol were tested using cytotoxicity assays. Results: Among the four extracts tested petroleum ether showed maximum cytotoxicity against a panel of cancer cell lines such as nasopharyngeal cancer (KB) cells, lung cancer (A-549) cells, breast cancer (MCF- 7) cells, leukemic (K-562) cells and inhibited the growth of murine cancer cells such as Dalton’s lymphoma ascites (DLA) and Ehrlich ascites carcinoma (EAC). Conclusion: Petroleum ether extract of Annona squamosa seeds showed cytotoxicity towards a panel of cancer cells meanwhile non-significant cytotoxicity towards normal cells.     

Cytostatic Effects of Dolastatin 10 and Dolastatin 15 on Human Leukemia Cell Lines

We studied in vitro the cytostatic/cytotoxic effects of the peptides dolastatin 10 and dolastatin 15 on various human leukemia cell lines, peripheral blood mononuclear cells (PBMNC), and tonsillar mononuclear cells (tonsillar MNC). On leukemia cell lines both drugs proved to be highly potent cvtostatic agents; however, the cells were not killed even at concentrations of maximum growth-inhibition. Growth-suppression was not paralleled by induced differentiation as exposure to dolastatins did not significantly alter the immunophenotype, cytochemistry or morphology of HL-60 cells. Growth-inhibitory effects of dolastatins were reversed following the removal of the agents from the culture medium. The dolastatins inhibited proliferation of leukemia cell lines at lower concentrations than those which inhibited the growth of stimulated tonsillar MNC. The growth-inhibiting properties of the dolastatins regarding leukemia cell lines and their low toxicity towards normal MNC indicate that these agents might be promising new drugs for future experiments examining their effects on primary leukemic cells.     

Schedule-dependent Synergism and Antagonism between Raltitrexed ("Tomudex") and Methotrexate in Human Colon Cancer Cell Lines in vitro

The folate-dependent enzymes are attractive targets for cancer chemotherapy. Methotrexate (MTX), which inhibits dihydrofolate reductase, has been widely used for the treatment of solid tumors and hematological cancers. Raltitrexed ("Tomudex"), which inhibits thymidylate synthase, is a novel anticancer agent active against colorectal cancer and some other solid tumors. We studied the optimal schedule of raltitrexed and MTX in combination against four human colon cancer cell lines Colo201, Colo320, LoVo, and WiDr. These cells were simultaneously exposed to raltitrexed and MTX for 24 h, or sequentially exposed to raltitrexed for 24 h followed by MTX for 24 h, or vice versa. Cell growth inhibition after 5 days was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentrations of drug that produced 80% and 50% cell growth inhibition (Icg₈₀ and IC₅₀) were analyzed by the isobologram method (Steel and Peckham, 1979). Cytotoxic interactions between raltitrexed and MTX were schedule-dependent. The simultaneous exposure to raltitrexed and MTX showed additive effects in Colo201, LoVo and WiDr cells and antagonistic effects in Colo320 cells. The sequential exposure to raltitrexed followed by MTX produced additive effects in all four cell lines. The sequential exposure to MTX followed by raltitrexed produced synergistic effects in Colo201, LoVo and WiDr cells and additive effects in Colo320 cells. These findings suggest that the sequential administration of MTX followed by raltitrexed produces more than the expected cytotoxicity and may be the optimal schedule at the cellular level. Further in vivo and clinical studies will be necessary to determine the toxicity and to test the antitumor effects of sequential administration of MTX followed by raltitrexed proposed on the basis of the in vitro synergism.     

Potentiation by Tumor Necrosis Factor of Mitoxantrone Cytotoxicity to Human Ovarian Cancer Cell Lines

The cytotoxic activity of human recombinant tumor necrosis factor (rHuTNF) (from 0.01 to 10000 U/ml) was assayed on six human ovarian cancer cell lines and one human cervical carcinoma cell line using a crystal violet assay. rHuTNF was cytotoxic to four cell lines (A2780, A2774, SW626, PAD, while 3 cell lines (IGROV1, SKOV3, Mel80) were marginally sensitive to its activity. However, under the same experimental conditions rHuTNF markedly enhanced the cytotoxicity of mitoxantrone, a chemotherapeutic drug targeted at DNA topoisomerase II, in six cell lines. The potentiation of mitoxantrone cytotoxicity was not caused by increased drug accumulation after rHuTNF treatment. No significant increase in cytotoxicity to Me180 cell line was seen when rHuTNF was added to mitoxantrone.     

Reactivity of Human Monoclonal Antibody Campath-1 with Human Leukemia Lymphoma Cell Lines of Varying Maturation

Human leukemia-lymphoma cell lines reflecting hematopoietic clones at various stages of differentiation were examined for binding and complement mediated lysis by Campath-1. Expression of the cell surface antigen was determined with fluorescein isothiocyanate conjugated Campath-1, by ultraviolet microscopy and with a fluorescent activated cell sorter (FACS). The results indicate that there is a correlation between Campath-1 binding and the stage of lymphoid-cell differentiation. The null and T lines bound Campath-1 weakly and the fluorescence intensity was low. In the B lines there was a gradual increase in labelling correlating with the stage of differentiation. The redistribution pattern of Campath-1 on the membrane of null and T lines was in the form of rings and small caps whereas that on the B lines was that of moderate patches. Complement mediated cytolysis occurred with the majority of the cell lines, but did not correlate with the extent of surface antibody binding nor with the stage of lymphoid differentiation. The recovery and proliferative capacity of the residual cells after treatment with Campath-1 and complement was low for the null and T lines. This was variable for the B lines, but some did not regain proliferative capacity, whereas others recovered after treatment. The present results suggest that Campath-1 may have considerable utility in marrow depletion of residual. leukemic cells, more specifically of null and T-cell origin, prior to autologous transplantation. Determination of both the sensitivity of the patient's leukemic: cells to cytolysis and the recovery of proliferative capacity of Campath-1 resistant cells may contribute essential information concerning the possible efficacy of purging with this antibody in patients with significant marrow involvement prior to transplantation.     

氨甲蝶呤对映体耐药A549细胞株的建立及其生物学特征

 目的 诱导并建立耐氨甲蝶呤对映体的A549细胞株并观察耐药细胞系（L-(+)-MTX/A549、D-(-)-MTX/A549）的生物学特性。 方法 以MTX对映体为诱导剂,采用浓度递增结合低剂量持续诱导方法诱导A549细胞株,建立MTX不同对映体耐药细胞系;倒置相差显微镜观察细胞形态变化;MTT法绘制细胞生长曲线;MTT法检测耐药细胞株的耐药指数;流式细胞仪检测细胞周期和细胞的分裂增殖能力。 结果 L-(+)-MTX/A549、D-(-)-MTX/A549耐药指数分别为6.0的和20.2。倒置相差显微镜观察细胞形态发生了改变;细胞生长曲线显示D-(-)-MTX/A549的增殖略慢于亲本细胞,而L-(+)-MTX/A549的增殖最慢;流式细胞仪检测细胞周期结果显示L-(+)-MTX/A549、D-(-)-MTX/A549耐药细胞株S期细胞数量减少(P<0.05),G₀/G₁期细胞增多(P<0.05);CFSE检测A549、L-(+)-MTX/A549、D-(-)-MTX/A549的MFI分别为(6.08±0.55)、(7.72±0.30)、(6.90±0.18)。两对映体细胞株间有明显手性差异。 结论 本研究建立了MTX两种对映体耐药细胞株,为进一步研究其耐药机制提供了一种实验模型。     

Simultaneous Expression of T-Cell and Myeloid Cell Phenotypes in Eight Newly Established HTLV-I-positive T-Cell Lines

Eight cell lines were established from patients with adult T-cell leukemia, and from normal adults, by cocultivation with human T-cell leukemia virus type I(HTLV-I)-producer cell lines in the presence of interleukin-2. All of these cell lines harbored HTLV-I and showed T-cell markers CD2, CD3 and CD4, hut not B-cell markers. Unexpectedly, all eight cell lines expressed a myeloid marker CD13 and three of the eight lines also expressed another myeloid marker CD33. Dual staining showed the simultaneous expression of CD3 and CD13 on the same cells. Thus, evidence was obtained for the expression of myeloid antigens on HTLV-I-harboring T cells.     

Cytotoxicity of Trichoderma spp. Cultural Filtrate Against Human Cervical and Breast Cancer Cell Lines

Trichoderma spp. are known as a rich source of secondary metabolites with biological activity belonging to a variety of classes of chemical compounds. These fungi also are well known for their ability to produce a wide range of antibiotic substances and to parasitize other fungi. In search for new substances, which might act as anticancer agents, the overall objective of this study was to investigate the cytotoxic effects of Trichoderma harzianum and Trichoderma asperellum cultural filtrates against human cervical and breast cancer cell lines(HeLa and MCF-7 cells respectively). To achieve this objective, cells were exposed to 20, 40, 60, 80 and 100 mg/ ml of both T. harzianum cultural filtrate (ThCF) and T. asperellum cultural filtrate (TaCF) for 24h, then the cell viability and the cytotoxic responses were assessed by using trypan blue and 3-(4,5-dimethylthiazol-2yl)-2,5-biphenyl tetrazolium bromide (MTT) assays. Morphological changes in cells were investigated by phase contrast inverted microscopy. The results showed that ThCF and TaCF significantly reduce the cell viability, have cytotoxic effects and alter the cellular morphology of HeLa and MCF-7 cells in a concentration dependent manner. A concentration of 80 and 100mg/ml of ThCF resulted in a sharp decline in the cell viability percentof HeLa and MCF-7 respectively (25.2%, 26.5%) which was recorded by trypan blue assay. The half-maximal inhibitory concentrations (IC50) of ThCF and TaCF in HeLa and MCF-7 were recorded as 16.6, 12.0, 19.6 and 0.70mg/ml respectively by MTT assay. These results revealed that ThCF and TaCF have a substantial ability to reduce the viability and proliferation of human cervical and breast cancer cells.