MOLT-4F Cell Research Articles

Azoxystrobin Induces Apoptosis and Cell Cycle Arrest in Human Leukemia Cells Independent of p53 Expression.

Azoxystrobin (AZOX), a methoxyacrylate derivative, has potent antimicrobial and antitumor activities. Here, we report the anticancer effects of AZOX on the p53-negative human myelogenous leukemia cell line HL-60RG and the p53 positive human T-cell leukemia cell line MOLT-4F. Using both leukemia cells, the anticancer effect of AZOX treatment was analyzed throughout the cell cycle. AZOX damaged both cell lines dose-dependently, and the cell damage rates were almost the same in both lines. Cell cycle distribution analysis showed that the treated MOLT-4F cells arrested at the S phase, whereas HL-60RG cells increased during the subG1 phase, suggesting that cell death was occurring. AZOX-induced cell death in HL-60RG was inhibited with the addition of uridine, which is used as a substrate for the salvage pathway of pyrimidine nucleotides. AZOX has p53-independent anticancer effects in leukemia cells, but the mechanisms underlying the damage differ between cell lines.

Evaluation of the Anti-Proliferative Activity of Rare Aldohexoses against MOLT-4F and DU-145 Human Cancer Cell Line and Structure-Activity Relationship of D-Idose.

D-Allose (D-All), a C-3 epimer of D-glucose (D-Glc), is a naturally rare monosaccharide, which shows anti-proliferative activity against several human cancer cell lines. Unlike conventional anticancer drugs, D-All targets glucose metabolism and is non-toxic to normal cells. Therefore, it has attracted attention as a unique “seed” compound for anticancer agents. However, the anti-proliferative activities of the other rare aldohexoses have not been examined yet. In this study, we evaluated the anti-proliferative activity of rare aldohexoses against human leukemia MOLT-4F and human prostate cancer DU-145 cell lines. We found that D-All and D-idose (D-Ido) at 5 mM inhibited cell proliferation of MOLT-4F cells by 46 % and 60 %, respectively. On the other hand, the rare aldohexoses at 5 mM did not show specific anti-proliferative activity against DU-145 cells. To explore the structure–activity relationship of D-Ido, we evaluated the anti-proliferative activity of D-sorbose (D-Sor), 6-deoxy-D-Ido, and L-xylose (L-Xyl) against MOLT-4F cells and found that D-Sor, 6-deoxy-D-Ido, and L-Xyl showed no inhibitory activity at 5 mM, suggesting that the aldose structure and the C-6 hydroxy group of D-Ido are important for its activity. Cellular glucose uptake assay and western blotting analysis of thioredoxin-interacting protein (TXNIP) expression suggested that the anti-proliferative activity of D-Ido is induced by inhibition of glucose uptake via TXNIP-independent pathway.

Development of a d-allose-6-phosphate derivative with anti-proliferative activity against a human leukemia MOLT-4F cell line

d-Allose, a C-3 epimer of d-glucose, is a naturally occurring rare monosaccharide that shows anti-proliferative activity against several human cancer cell lines. However, d-allose requires a relatively high concentration for the activity to be observed. Thus, developing more potent derivatives is needed for application. In cells, d-allose is converted to d-allose-6-phosphate (A6P), which is responsible for the anti-proliferative activity of d-allose. In this study, we synthesized A6P derivative 1 with biodegradable protecting groups, which showed higher anti-proliferative activity than A6P against a MOLT-4F human leukemia cell line. Similarly protected derivative of d-glucose-6-phosphate (G6P) (2) and tetraacetyl-A6P (3) showed weaker and less activity compared with 1, respectively, suggesting that both A6P moiety and the protecting group on the phosphate group are responsible for the activity. In addition, significantly weaker induction of thioredoxin-interacting protein (TXNIP) expression by 1 compared with d-allose suggests that 1 exhibited cytotoxicity through the synergetic effect of inducing TXNIP expression and other mechanisms.

Anti-proliferative activity of 6-O-acyl-D-allose against the human leukemia MOLT-4F cell line.

The anti-proliferative activities of the 6-O-acyl derivatives of D-allose against the human leukemia MOLT-4F cell line were examined. The activity of the 6-O-dodecanoyl derivative (3) was approximately 30 times stronger than that of D-allose. An evaluation of the derivatives of 3 that occurred in a furanose form revealed the pyranose forms of 3 to be important for the anti-proliferative activity.

Expression of 11beta-hydroxysteroid dehydrogenase 2 contributes to glucocorticoid resistance in lymphoblastic leukemia cells

Synthetic glucocorticoids (GCs) form a crucial first-line treatment for childhood acute lymphoblastic leukemia (ALL). However prolonged GC therapy frequently leads to GC-resistance with an unclear molecular mechanism. 11β-hydroxysteroid dehydrogenase (11β-HSD) 2 inactivates GCs within cells. Here, we show the association between GC sensitivity and 11β-HSD2 expression in human T-cell leukemic cell lines. 11β-HSD2 mRNA and protein levels were considerably higher in GC-resistant MOLT4F cells than in GC-sensitive CCRF-CEM cells. The 11β-HSD inhibitor, carbenoxolone pre-treatment resulted in greater cell death with prednisolone assessed by methyl-thiazol-tetrazolium assay and caspase-3/7 assay, suggesting that 11β-HSD2 is a cause of GC-resistance in ALL.

가시오갈피 열매 추출물의 생리활성 검정

In this study, we investigated the antioxidant activities, inhibition activity against ACE (angiotensin converting enzyme) and antitumor activity of extract from Acanthopanax senticosus HARMS fruits for development novel functional resources. In order to understand the factors responsible for the potent antioxidant and antihypertensive ability of fruits in A. senticosus, it has been evaluated for anti-oxidative activity using Fenton`s reagent/ethyl linoleate system and for free radical scavenging activity using the l,l-diphenyl-2-picryl hydrazyl free radical generating system. The fruits extract of A. senticosus showed higher antioxidant activities than positive control, -tocopherol at all concentrations, while fruits extract of A. senticosus showed same degree of radical scavenging activity with positive control, -tocopherol. The ability of fruit extracts from A. senticosus to influence the inhibitory activity of angiotensin converting enzyme (ACE) and xanthine oxidase (XOase) has also been discussed. The activity of growth-inhibitory of fruit extracts of A. senticosus was screened by SRB (sulphorhodamine B) method on diverse cancer cells representing different types of cancers. The fruit extracts of A. senticosus showed moderate inhibition on proliferation of LNCaP and MOLT-4F cells and did not inhibit the proliferation of other cancer cells. The fruit extracts of A. senticosus inhibited the proliferation of cancer cells with values ranging from 5 to . This result revealed that the fruit extracts of A. senticosus was expected to be good candidate for development into source of free radical scavengers, antihypertentive, and anti-tumor agent.

Analysis of the inhibitory mechanism of d-allose on MOLT-4F leukemia cell proliferation

D-Allose, the C-3 epimer of D-glucose, is one of the rare sugars found in nature. In the present study, we have elucidated for the first time that various leukemia cell lines have different susceptibility to anti-proliferative activity of D-allose, and that this difference is related to the difference in induction of thioredoxin interacting protein (TXNIP) expression. We examined 5 leukemia cell lines (MOLT-4F, IM-9, HL-60, BALL-1 and Daudi), and found that MOLT-4F (T-cell lymphoblastic leukemia) had the highest susceptibility to D-allose, and that Daudi (Burkitt's lymphoma) had the lowest. D-Allose significantly slowed the cell cycle progression without causing apoptosis of MOLT-4F cells. Intracellular TXNIP expression was specifically and markedly enhanced in MOLT-4F cells by D-allose treatment, and subsequent increase of p27(kip1), a cell cycle inhibitor, was observed. On the other hand, D-allose did not increase TXNIP and p27(kip1) levels at all in Daudi cells. These results indicate that D-allose suppresses MOLT-4F cell proliferation possibly by the inhibition of cell cycle progression via induction of TXNIP expression.

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability.

Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD-negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. The results show that cells stained with FITC-labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions.

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability

Background: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. Materials and Methods: Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. Results: Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD–negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. Conclusions: The results show that cells stained with FITC–labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions. Cytometry 35:64–74, 1999. © 1999 Wiley-Liss, Inc.

The pc12 cell as a model for studies of the mechanism of induction of peripheral neuropathy by anti-HIV-1 dideoxynucleoside analogs

The ability of DNA polymerase gamma to utilize ddNTPs+ as substrates, incorporating these chain terminators into DNA (see citations in[1]), has led us to propose that AZT-induced bone marrow suppression results from inhibition of mtDNA replication [1]. This could alter metabolism since mtDNA encodes proteins involved in ATP synthesis and mitochondrial ultrastructure. We have shown that AZT and some other ddNs used in AIDS therapy inhibit DNA replication by purified DNA polymerase gamma and by isolated mitochondria [1,2]. Moreover, proliferation of a hemopoietic cell, the Friend erythroleukemic cell, is strongly inhibited by AZT [2], and mitochondria from these cells show impairment of DNA replication++ [2]. Additional evidence comes from studies on the effect of ddC on the Molt-4F cell [3], and on the involvement of mitochondria in AZT-induced myopathy [4].We propose that inhibition of mtDNA replication is also the primary step in ddC-induced [5], ddI-induced [6] and d4T-induced [7] peripheral neuropathy. We have found that a c-Ha-ras transformant of the PC12 cell, GS-ras-1+++, is a promising neuronal cell model for such studies. The PC12 cell line [8] is derived from a pheochromocytoma, an adrenal medullary tumor; cells of the adrenal medulla share their embryological origin with neuronal cells. When the PC12 cell is induced by NGF (or by dexamethasone in GS-ras-1) to differentiate, neurite outgrowth and increased synthesis of acetylcholine and other neuronal markers occur. We describe the effects of some ddNs on both the uninduced and the induced cell.

IMP dehydrogenase and action of antimetabolites in human cultured blast cells

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the K m values for IMP and NAD were 29 and 54 μM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5′-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the K i values of RMP (0.58 μM) and TAD (0.075 μM) were several orders of magnitude lower than those of XMP (85 μM) and NADH (94 μM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.

Delayed cytotoxicity and selective loss of mitochondrial DNA in cells treated with the anti-human immunodeficiency virus compound 2′,3′-dideoxycytidine

The compound 2',3'-dideoxycytidine (ddC) is a potent inhibitor of human immunodeficiency virus replication in vitro and is currently in clinical trials for treatment of acquired immunodeficiency syndrome. The compound was found to exert delayed cytotoxicity against Molt-4F cells, a human T lymphoblastic cell line. At a concentration as low as 0.1 microM, the doubling time of the cells was increased after 8 days of ddC treatment. This concentration is 5-fold lower than plasma levels reached in clinical trials. The cells finally died after a 2-week exposure to 0.1 or 0.2 microM ddC. The delayed cytotoxicity was not due to a greater accumulation of 2',3'-dideoxycytidine triphosphate in cells with longer exposure to the compound. The cellular content of mitochondrial DNA was found to decrease and the rate of glycolysis was found to increase with continuous exposure of cells to ddC. The mitochondrial toxicity and cell growth inhibition were reversed when ddC was removed. The reduction in cellular content of mitochondrial DNA caused by ddC may partially explain the delayed toxicity observed in acquired immunodeficiency syndrome patients treated with the drug.

Leukemia-derived growth factor (non-interleukin-2) produced by murine lymphoma T-cell lines.

Autocrine growth factor activity was found in supernatants of AKR T-cell lymphoma lines cultured in serum-free medium. This factor was designated leukemia-derived growth factor (LDGF). Active supernatants stimulated the growth of the AKR murine T-cell lymphoma line SL 12, its cloned derivatives, and all other murine T-cell lymphoma lines tested. Growth factor activity in conditioned medium was found to be different from interleukin 2 (IL-2) and several other known growth factors. LDGF was able to stimulate growth of the human leukemia T-cells MOLT4f, and the LDGF from MOLT4f cells stimulated the mouse cells. Because mouse T-cell lymphoma lines produced and respond to this factor, it may support the continued proliferation of these cells and could be responsible for their malignant in vivo properties.

Leukemia-derived growth factor (non-interleukin 2) produced by a human malignant T lymphoid cell line.

A growth factor was found in the supernatants of MOLT-4f, a cell line derived from acute T lymphoblastic leukemia. This factor, which we designated leukemia-derived growth factor from MOLT-4f (LDGF-M4), is different from interleukin 2. LDGF-M4 has features of a polypeptide with a molecular weight in the range of 5,000-15,000, as indicated by gel diffusion chromatography. LDGF-M4 does stimulate MOLT-4f and at least two other T cell lines that do not respond to interleukin 2. Because MOLT-4f cells produce and respond to LDGF-M4, this factor may contribute to the independence of MOLT-4f and related T leukemia cell lines.

Detection and partial characterization of human thymus-leukemia antigens.

Two monkey antisera against human thymocytes after absorption with human erythrocytes and peripheral blood leukocytes were shown to detect human thymus-leukemia (HTL)-like antigens. These sera were cytotoxic for thymocytes (> 90% lysis at a 1:10 dilution) but were nonreactive with enriched peripheral blood T- and B-lymphocytes or with cells from myeloid or B-cell lymphoid leukemias. Most (16/17) sheep erythrocyte rosette-forming acute lymphoblastic leukemia (ALL) cells reacted with these sera. Cells from patients with T-cell chronic lymphocytic leukemia, lymphoblastic lymphoma (LBL), and thymoma were also positive. Three of 4 T-cell lymphoblastoid lines derived from ALL patients reacted with these sera. Absorption of the sera with MOLT-4F cells, thymocytes, or LBL cells removed the reactivity against all types of cells tested. However, sera absorbed with the T-cell line HSB remained cytotoxic for thymocytes, MOLT-4F, and most (6/9) T-cell cancers tested. The peripheral blood cell-absorbed sera precipitated a molecule with an apparent molecular weight of 48,000 from lactoperoxidase-labeled thymocytes but not from similarly labeled peripheral blood lymphocytes. The ability of the sera to precipitate this antigen was decreased by absorption with thymocytes, MOLT-4, or LBL cells but not by absorption with HSB, SB, or non-T, non-B ALL cells. Sequential precipitation studies suggested that the HTL antigen was not associated with beta 2 microglobulin.

Immunodiagnosis of mesothelioma. Use of antimesothelial cell serum in an indirect immunofluorescence assay

Cells isolated from human serous effusions were cultured in vitro. Monolayers of large multipolar cells were established. Antisera to the cultured cells were prepared in rabbits and rats. The antisera were absorbed with human red cells, liver powder and MOLT-4F cell line lymphocytes. Specificity of the absorbed antisera for human mesothelial cells were demonstrated in an indirect immunofluorescence assay. The antisera were used to confirm the diagnosis of mesothelioma in two cases. In both the patients, the morphologically identifiable malignant cell populations in the effusions stained positively with the antimesothelial cell serum thus establishing their mesothelial origin. Normal nonmesothelial tissue and known nonmesothelial tumors failed to react with the antisera thus confirming the specificity of the antisera.

Ribonucleotide reductase isolated from human cells: Heterogeneity among the sources

The amount of ammonium sulfate required to precipitate the ribonucleotide reductase activities derived from histocytic lymphoma cells obtained from patients or from KB or Connaughton cultured human cells is different than for reductase activity derived from Molt-4F and HeLa-S3 cells. The reductase activity of Molt-4F cells sedimented in a sucrose density gradient at a faster rate than the enzyme activity from KB cells; CDP and ADP reductase activity from either source co-sedimented. Enzymes from both cell lines sedimented at a faster rate in the presence of CDP and ATP than in the absence of these compounds, with CDP and ADP reductase still co-sedimenting. In addition, the enzymes obtained from the two sources have different stabilities at 37°. Hydroxyurea, 1-formylisoquinoline thiosemicarbazone (IQ-1), 1,10-phenanthroline and potassium chloride inhibit the enzyme activity derived from either Molt-4F or KB cells. However, a difference in the ratio of CDP to ADP reductase activity in Molt-4F and KB cells and a difference between the sensitivities of the CDP and the ADP reductase activities from the same cell line to various concentrations of inhibitors was observed.

Radioimmunoassay for human T-cell associated antigens on MOLT 4 cultured cells.

We have detected antigens associated with two malignant T-cell lines by use of a sensitive radioimmunoassay. Baboon antisera to a cultured malignant T cell line (MOLT 4F) were prepared and the gammaG-anti-MOLT 4F was enriched by specific adsorption onto, and elution from, MOLT 4F cells followed by repeated absorption with pools of 21 established, long-term cultured B cell lines. Purified 125I-gammaG-anti-MOLT 4F preparation was tested for relative binding to autologous (MOLT 4F) and allogenetic (Sommer-RPMI 8402) T cells as well as B cells, including Sommer B cells (RPMI 8392). The degree of enrichment of antibody to malignant T cell associated antigens, expressed in terms of the ratios of the amount of globulin bound by T cells relative to the amount bound by B cells, were 6.3 and 5.0 for MOLT 4F and Sommer T cells, respectively.