A lymphocyte blastogenesis inhibitory factor (LBIF) reversibly arrests a human melanoma cell line, A375, at G1 and G2 phases of cell cycle

A lymphocyte blastogenesis inhibitory factor, LBIF, has been found in the culture supernatant of a human macrophage-like cell line, U937. The factor has been purified by fast protein liquid chromatography. Partial amino acid sequencing analysis showed that LBIF was a novel immunoregulatory factor. Recent study has demonstrated that LBIF possesses a remarkable tumor growth inhibitory activity. In this study, the cell growth inhibitory activity of LBIF was characterized on the proliferation of a human melanoma cell line A375 in vitro. LBIF strongly inhibits the proliferation of A375 cells. The inhibitory activity was cytostatic and reversible by Day 5 although the lethal effect became apparent at Day 7. Cell cycle analysis by flow cytometry showed that LBIF arrested A375 cells at both G1 and G2/M phases. Mitotic index analysis indicated that A375 cells were arrested in G1 and G2 phases. LBIF function was not attributed to the elevation of intracytoplasmic cyclic-AMP levels. Thus, these results suggest that LBIF plays an important role in controlling cell cycle and there is a similarity between the mechanisms of G1 and G2 arrests in eukaryotic cell proliferation. LBIF-induced reversible cell-cycle arrest of A375 cells can be a useful system to analyze the signal transduction for cell proliferation and cell-cycle arrest.     

A sensitive bioassay for lipase using bacterial bioluminescence

A new bioassay for lipase utilizes a dim mutant of luminous bacteria which emit light upon the addition of long chain fatty acids, especially myristic acid. The luminescence response is proportional to the amount of added myristic acid over a 100-fold range, down to 10 nM. Trimyristin was used as a substrate for lipase and the hydrolyzed myristic acid was determined by the response of the luminous bacteria either on a continuous basis in the same reaction mixture or alternatively, when the hydrolytic stage is done separately followed by the independent detecting system. Using these procedures it is possible to assay lipase activity at rate corresponding to a release of as low as 10 pmol myristic acid per min.     

A simple bioassay for abscisic acid using cucumber hypocotyls

A simple bioassay based on the inhibition by abscisic acid (ABA) of cucumber (Cucumis sativus L., cv. National Pickling) hypocotyl elongation was developed. Sections of 3-day-old dark-grown cucumber hypocotyl taken from 0–5 mm immediately below the cotyledon were used for the assay. A dark incubation period of 20 h was followed by an exposure to light for 24 h. Under these conditions, the inhibition of hypocotyl elongation is proportional to the abscisic acid applied. The minimum detectable level of abscisic acid was 10^–9 M, and the range of linear response to abscisic acid was between 10^–7 and 10^–3 M. This assay is 10 times more sensitive than the cucumber cotyledon greening bioassay for abscisic acid.     

M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3

We have isolated a subline of the M-07 human megakaryoblastic leukemia cell line, designated M-07e, that requires either interleukin-3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) for growth, even in the presence of fetal calf serum. This cell line will not grow long term in any other cytokine although it responds slightly to IL-2, IL-4, IL-6, IL-9, and interferon-gamma. We have used the M-07e subline to develop a quantitative bioassay for the measurement of levels of either GM-CSF or IL-3. This assay is as sensitive to either factor as the human bone marrow colony assay (CFU-GM) or the chronic myelogeneous leukemic (CML) blast cell proliferation assay for these factors and is much more convenient and reliable than either. With this assay, as little as 25-50 pg/ml of either IL-3 or GM-CSF can be detected, a level that should render the assay useful for analysis of these molecules in samples from patients undergoing colony-stimulating factor therapy and from conditioned media from natural sources of the factors. In these cases, neutralizing antisera to each cytokine are required to demonstrate the specificity of the assay. This assay, in combination with quantitative immunoassays, should greatly facilitate the analysis of the roles of IL-3 and GM-CSF in regulating hematopoiesis both in patients and in natural sources of the cytokines.     

Effect of Citrullus colocynth fruit extract on pro and anti apoptotic molecules in human melanoma cell line (A375)

Melanoma remains a major cause of death worldwide. Chemotherapy might be used to treat advanced Melanoma which is associated with some side effects. Hence, providing proper treatment or proper employment of lower toxic substances is essential. Citrullus colocynthis, a medicinal plant, seems a potential anticancer herbal medicine against Melanoma via various efficient compounds. Therefore, it is of interest to analyze the effect of Citrullus colocynthis fruit extract on pro and anti apoptotic molecules in the human melanoma cell line (A375). Cell viability test was done using MTT assay. mRNA expression of Bax and Bcl2 was done by real-time PCR. The data was analyzed statistically by one way analysis of variance and Duncan multiple range test with graph prism version 5. p<0.05 was considered significant. Citrullus colocynthis extract caused a marked increase in cell death in a dose-dependent manner. At the end of 48 hours, maximum inhibition (50%) was at 400 and 500µg/ml. The study demonstrated that the effect of Citrullus colocynthis extract has elevated the Bax mRNA expression on human melanoma cell lines. Data shows that Citrullus colocynthis fruit extract has an anticancer effect on the human melanoma cell line (A375) by increasing the expression of Bax and Bcl2 and this could be because of the phytochemicals present in it.     

Activation of two distinct anti-proliferative pathways, apoptosis and p38 MAP kinase-dependent cell cycle arrest, by tumor necrosis factor in human melanoma cell line A375

The proliferation of human melanoma cell line A375-6 cells is inhibited by several cytokines, including interleukin-1 (IL-1). A375-R8 cells, a subclone of A375-6, are resistant to IL-1-induced growth inhibition. The proliferation of both cell lines is inhibitable by tumor necrosis factor (TNF). In this study, we characterized the mechanisms of TNF-induced growth inhibition. TNF-induced growth inhibition in both cell lines was partially suppressed by a selective p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), whereas a combination of SB203580 and Z-VAD-fmk, an inhibitor for a wide range of caspases, completely blocked TNF-induced growth inhibition, indicating that TNF-induced growth inhibition is mediated by both p38 MAPK and caspases. However, Z-VAD-fmk alone suppressed TNF-induced growth inhibition in A375-R8, but not A375-6, cells, suggesting that there may exist a TNF-induced anti-apoptotic mechanism in A375-6 cells which is lost or mutated in A375-R8 cells. Evidence in support of this notion includes (1) TNF-induced apoptosis only in A375-R8, but not A375-6 cells; (2) cycloheximide enabled TNF to induce apoptosis even in A375-6 cells; and (3) somatic hybrid cells between A375-6 and A375-R8 cells are resistant to TNF-induced apoptosis. Since TNF-induced NF-kappa B activation, cell cycle arrest, RB dephosphorylation, and E2F downregulation are indistinguishable in both cell lines, none of these factors is likely to be involved in the TNF-induced anti-apoptotic mechanism in A375-6 cells. Our results indicate that TNF activates two distinct anti-proliferative pathways including p38 MAPK-dependent cell cycle arrest and caspase-mediated apoptosis, as well as an anti-apoptotic mechanism in melanoma cells.     

A lymphocyte blastogenesis inhibitory factor (LBIF) reversibly arrests a human melanoma cell line, A375, at G1 and G2 phases of cell cycle

A lymphocyte blastogenesis inhibitory factor, LBIF, has been found in the culture supernatant of a human macrophage-like cell line, U937. The factor has been purified by fast protein liquid chromatography. Partial amino acid sequencing analysis showed that LBIF was a novel immunoregulatory factor. Recent study has demonstrated that LBIF possesses a remarkable tumor growth inhibitory activity. In this study, the cell growth inhibitory activity of LBIF was characterized on the proliferation of a human melanoma cell line A375 in vitro. LBIF strongly inhibits the proliferation of A375 cells. The inhibitory activity was cytostatic and reversible by Day 5 although the lethal effect became apparent at Day 7. Cell cycle analysis by flow cytometry showed that LBIF arrested A375 cells at both G₁ and G₂/M phases. Mitotic index analysis indicated that A375 cells were arrested in G₁ and G₂ phases. LBIF function was not attributed to the elevation of intracytoplasmic cyclic-AMP levels. Thus, these results suggest that LBIF plays an important role in controlling cell cycle and there is a similarity between the mechanisms of G₁ and G₂ arrests in eukaryotic cell proliferation. LBIF-induced reversible cell-cycle arrest of A375 cells can be a useful system to analyze the signal transduction for cell proliferation and cell-cycle arrest.     

A human cell line sensitive to mutation by particle-borne chemicals

A human lymphoblastoid cell line with ability to perform oxidative metabolism of various chemicals is mutated by the direct addition of an intact particulate soot. This experiment demonstrates that materials associated with combustion-generated particulates are biologically available and able to cause genetic changes in metabolically competent human cells.     

Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification

New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the Sph I-5 PCR amplicon ( Sph I-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactions were carried out under isothermal conditions at 65°C for 60 min. The detection limit of both LAMP was six copies, equal to the modified Sph I-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. Sph I-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider Sph I-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections.     

Altered regulation of cell cycle machinery involved in interleukin-1-induced G(1) and G(2) phase growth arrest of A375S2 human melanoma cells

Interleukin-1 (IL-1) inhibits the growth of A375S2 human melanoma cells by arresting them at G(1) and G(2) phases of the cell cycle. The arrests are preceded by a rapid decrease in kinase activities of cyclin E-Cdk2 and cyclin B1-Cdc2, which are critical for G(1)-S and G(2)-M progression, respectively. IL-1 quickly enhances the protein expression of the CDK inhibitor p21(cip1). The induced p21 binds preferentially to cyclin E-Cdk2, and the increase in p21 binding parallels the decrease in cyclin E-Cdk2 activity. Thus, p21 is likely to be responsible for the inhibition of cyclin E-Cdk2 activity and G(1) arrest. Coinciding with the decrease in cyclin B1-Cdc2 activity, there is an increase in tyrosine phosphorylation of Cdc2, suggesting that an increase in the inactive Tyr-15-phosphorylated form of Cdc2 is involved in the decrease in cyclin B1-Cdc2 activity and G(2) arrest. Furthermore, we found that IL-1 causes rapid dephosphorylation of p107, but not of pRb or p130, while the total protein levels of p130 are increased. Thus, IL-1 may exert its growth-arresting effects via p107 and p130 pathways rather than through pRb.     

Antiproliferative effect of IL-1 is mediated by p38 mitogen-activated protein kinase in human melanoma cell A375.

The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375-C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up-regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down-regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.     

Toxicogenomics of A375 human malignant melanoma cells treated with arbutin

Although arbutin is a natural product and widely used as an ingredient in skin care products, its effect on the gene expression level of human skin with malignant melanoma cells is rarely reported. We aim to investigate the genotoxic effect of arbutin on the differential gene expression profiling in A375 human malignant melanoma cells through its effect on tumorigenesis and related side-effect. The DNA microarray analysis provided the differential gene expression pattern of arbutin-treated A375 cells with the significant changes of 324 differentially expressed genes, containing 88 up-regulated genes and 236 down-regulated genes. The gene ontology of differentially expressed genes was classified as belonging to cellular component, molecular function and biological process. In addition, four down-regulated genes of AKT1, CLECSF7, FGFR3, and LRP6 served as candidate genes and correlated to suppress the biological processes in the cell cycle of cancer progression and in the downstream signaling pathways of malignancy of melanocytic tumorigenesis.     

Efficient isolation of human metapneumovirus using MNT‐1, a human malignant melanoma cell line with early and distinct cytopathic effects

Isolation of human metapneumovirus (HMPV) from clinical specimens is currently inefficient because of the lack of a cell culture system in which a distinct cytopathic effect (CPE) occurs. The cell lines LLC‐MK2, Vero and Vero E6 are used for isolation of HMPV; however, the CPE in these cell lines is subtle and usually requires a long observation period and sometimes blind passages. Thus, a cell line in which an early and distinct CPE occurs following HMPV inoculation is highly desired by clinical virology laboratories. In this study, it was demonstrated that, in the human malignant melanoma cell line MNT‐1, obvious syncytium formation occurs shortly after inoculation with HMPV‐positive clinical specimens. In addition, the growth and efficiency of isolation of HMPV were greater using MNT‐1 than using any other conventional cell line. Addition of this cell line to our routine viral isolation system for clinical specimens markedly enhanced isolation frequency, allowing isolation‐based surveillance. MNT‐1 has the potential to facilitate clinical and epidemiological studies of HMPV.

     

A rapid,sensitive, simple plate assay for detection of microbial alginate lyase activity

Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2–3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay.     

Constitutive and PMA-induced interleukin-1 production by the human astrocytoma cell line T24

Astrocytes and glial cells of different species produce interleukin-1 (IL-1) in vitro. In all cases, however, the evidence relied on the detection of IL-1 activity in biological assays. In this report we describe a human astrocytoma cell line (T24) which produces IL-1 constitutively and upon induction with phorbol myristate acetate in vitro. The IL-1 activity was detected in the culture supernatant by a modified assay measuring IL-1-dependent induction of IL-2 production by EL-4 cells. The active molecule had a molecular weight of 17 kDa on gel filtration and an isoelectric point of 5.2. The activity was not neutralized by a goat antibody reacting against pI 7 IL-1. In contrast, rabbit antibody reacting against pI 5 and pI 7 IL-1 neutralized all the IL-1 activity. Cell-associated IL-1 activity was detected in the supernatant of freeze-thawed cell lysates. Serological evidence as well as isoelectric point determination further supported that the predominant form of IL-1 synthesized was of the pI 5 type, and immunoprecipitation of 35S-labeled cell lysate with monospecific polyclonal antibody to IL-1 alpha or IL-1 beta detected only IL-1 alpha precursor. However, Northern blot analysis of astrocytoma cells indicated that mRNA encoding for both IL-1 species were present. These results, therefore, provide unequivocal evidence that human astrocytoma cells synthesize both forms of IL-1 message and yet only activity corresponding to the pI 5 form is detectable inside and outside these cells, suggesting that the inactive pI 7 IL-1 precursor, if made, is not processed to the mature active 17-kDa form.     

Active interaction of human A375 melanoma cells with the lymphatics in vivo

We have used the avian chorioallantoic membrane (CAM) to study the interaction of tumor cells with the lymphatics in vivo. The vascular endothelial growth factor-C (VEGF-C) has been shown to be lymphangiogenic. We have therefore grown VEGF-C-expressing human A375 melanoma cells on the CAM. These tumors induced numerous lymphatics at the invasive front, and compressed or destroyed VEGF receptor (R)-3-positive lymphatics were observed within the solid tumors. The lymphatics in the CAM and in the A375 melanomas could also be demonstrated with an antibody against Prox 1, a highly specific marker of lymphatic endothelial cells. Proliferation studies revealed a BrdU labeling index of 11.6% of the lymphatic endothelial cells in the tumors and at their margins. A great number of melanoma cells invaded the lymphatics. Such interactions were not observed with VEGF-C-negative Malme 3 M melanoma cells. Lymphangiogenesis was inhibited to some extent when A375 melanoma cells were transfected with cDNA encoding soluble VEGFR-3 (sflt4), and the BrdU labeling index of the lymphatics in these tumors was 3.9%. Invasion of lymphatics and growth of blood vascular capillaries were not inhibited by the transfection. Therefore, tumor-induced lymphangiogenesis seems to be dependent to some extent on VEGF-C/flt4 interactions, but invasion of lymphatics seems to be a distinct mechanism.