Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

Human tissue factor pathway inhibitor-2 induces caspase-mediated apoptosis in a human fibrosarcoma cell line

Human TFPI-2 is an extracellular matrix-associated Kunitz-type serine proteinase inhibitor. We previously demonstrated that a human fibrosarcoma cell line, HT-1080, does not express TFPI-2, but genetic restoration of TFPI-2 expression in these cells markedly inhibited their growth and metastasis in vivo. In the present study, either full-length recombinant TFPI-2, or its mutated first Kunitz-type domain (R24K KD1), were offered to HT-1080 cells, and the degree of apoptosis assessed by nuclear fragmentation, ethidium bromide and acridine orange staining, fluorescence activated cell sorting, immunoblotting and gene expression profiling. R24K KD1 induced apoptosis in 69% of HT-1080 cells in a 48 h period compared to 39% for TFPI-2, while a KD1 preparation lacking a reactive site arginine/lysine residue (R24Q KD1) produced only an 18% apoptosis rate, suggesting that the observed apoptosis was related to proteinase inhibition. Immunoblotting experiments indicated increased caspase 3 and 9 activation, up-regulation of pro-apoptotic Bax and suppression of anti-apoptotic Bcl-2 protein. Finally, microarray analyses of R24K KD1-treated cells indicated elevated expression of several pro-apoptotic genes and under-expression of anti-apoptotic genes. Collectively, our results demonstrate that treatment of HT-1080 cells exogenously with either TFPI-2 or R24K KD1 activates caspase-mediated, pro-apoptotic signaling pathways resulting in apoptosis.     

Properties of growth-related acetylcholinesterase in a cell line of fibroblastic origin

We have previously reported the presence and regulation of an acetylcholine-hydrolyzing enzyme in high density suspension cultures of WRL-10A fibroblasts where its activity increases 100-fold when growth is arrested. Substrate specificity, substrate inhibition, and product identification studies indicate that this enzyme is acetylcholinesterase (AChE, EC 3.1.1.7). Treatment of whole cells with 5 mM diazotized sulfanilic acid revealed that most of the AChE is located on the external surface of the cell membrane. It was also found that the enzyme is released in the medium at a rate of 0.5 U/h/mg cell protein and that within a 24-h period the de novo synthesized and liberated AChE is equivalent to 90% of the activity associated with the cells. No similar synthesis of AChE was found in six order fibroblastic cell lines examined. These and related findings indicating that acetylcholine is also present in high density populations of WRL-10A cells suggest that this unique phenotype may be used profitably in exploring further the relationship between components of the cholinergic system and non-neuronal cell growth.     

Functional and biochemical studies of CD9 in fibrosarcoma cell line

CD9, a member of the tetraspanin family, plays important roles in a variety of cell activities. Fibrosarcoma is a malignant tumor that arises from fibroblasts. Low CD9 expression is found in fibrosarcoma tumor, but function of CD9 in fibrosarcoma has been rarely studied. In this study, stable cell lines for CD9 overexpression and vector were generated in HT1080, a human fibroscarcoma cell line, and cellular functions were widely investigated. In CD9-HT1080 cells, CD9 mainly localized in the membrane and co-localized with F-actin in the filopodia of cell surface. In functional assays, we demonstrated that CD9 could up-regulate total and active caspase-3 expression and induce cell apoptosis, but cell proliferation remained unchanged. CD9 overexpression inhibited HT1080 cell adhesion to FN but promoted cell spreading on FN. We also observed CD9 reduced cell migration using FN a chemoattractant and inhibited cell colony formation in soft agar medium. To explore the biochemical mechanism for functional changes, we investigated the effects of CD9 overexpression on cellular pathways and protein association. CD9 overexpression induced Akt phosphorylation on FN but did not change total Akt expression. Phosphorylation of p38 but not ERK was increased by CD9 overexpression, total p38 and ERK were not affected. CD9 overexpression did not affect the expression of TGFα, EGFR, β1, and EWI-2, but EWI-F expression was up-regulated. Moreover, CD9 could associate with TGFα, EGFR, β1, EWI-2, and EWI-F in HT1080 cell line. Take together, CD9 overexpression had promoting effects on cell apoptosis and cell spreading, but had inhibitory effects on cell adhesion, migration, and cell colony formation. These effects might be ascribed to CD9 associations with EWI-2/EWI-F/β1 complex and EGFR pathway, and the activation of Akt and p38 signalings as well.     

Localization and modulation of galactosyltransferase during in vitro proliferation of a human ovarian adenocarcinoma cell line

A cell line, IGROV1, originating from a human ovarian cancer, releases a galactosyltransferase activity in its culture medium during proliferation. The proliferating IGROV1 cells appear as two populations: some cells grow in floating clusters whereas the greater part of them adhere to the culture substrate. The study of galactose transfer by intact cells onto an exogenous glycoprotein acceptor (ovomucoid) shows the presence of surface-associated galactosyltransferase onto the two cellular sub-populations. Opposite to intracellular activity, surface-associated and released galactosyltransferase activities depend on cellular adhesion and proliferation.     

Inhibitory effects of p-dodecylaminophenol on the invasiveness of human fibrosarcoma cell line HT1080

Cancer is a major cause of death, and the development of new anticancer drugs is urgently needed. Invasion and metastasis are the primary causes of death due to cancer rather than growth of the primary tumor. In the current study, we examined the anti-invasive effects of p-dodecylaminophenol (1), which was developed based on N-(4-hydroxyphenyl)retinamide (2), a synthetic amide of all-trans-retinoic acid (3). In HT1080 cells 1 inhibited growth, induced apoptosis and arrested the cell cycle in S phase in a dose-dependent manner. In addition, 1 significantly suppressed cell invasion, and the activity and mRNA expression of matrix metalloproteinase-9 (MMP-9). Furthermore, the expression of the reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is a negative regulator of MMP-9, was increased by treatment with 1. These results suggest that 1 could be an effective anti-cancer agent that suppresses cell growth through apoptosis induction and cell cycle arrest, which also inhibits cell invasion by decreasing MMP-9 expression due to an increase in RECK. Compound 1 might be useful clinically as a new and potent anticancer agent that could overcome adverse side effects of the retinoids.     

Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080.

A full length N-ras gene has been cloned from both the human fibrosarcoma cell line HT1080 and from normal human DNA. N-ras isolated from HT1080 will efficiently induce morphological transformation of NIH/3T3 cells in a transfection assay, whereas N-ras isolated from normal human DNA has no effect on NIH/3T3 cells. The coding regions of the normal N-ras gene have been sequenced and the predicted amino acid sequence of the N-ras product is very similar to that of the c-Ha-ras1 and c-Ki-ras2 products. By making chimeric molecules between the two cloned genes the activating alteration in the HT1080 N-ras gene has been localised to a single base change that results in an amino acid alteration at position 61 of the p21 N-ras product.     

Stability of an inverted repeat in a human fibrosarcoma cell.

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.     

Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.     

LET and ion species dependence for cell killing in normal human skin fibroblasts

We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/microm. The RBE of carbon ions at 98 keV/microm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/microm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*2/beta2 (where Z* is the effective charge and beta is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*2/beta2 curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*2/beta2, it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.     

Inhibitory effect of caffeic acid on cancer cell proliferation by oxidative mechanism in human HT-1080 fibrosarcoma cell line

Tyrosine (Y) kinases inhibitors have been approved for targeted treatment of cancer. However, their clinical use is limited to some cancers and the mechanism of their action remains unclear. Previous study has indicated that PP2, a selective inhibitor of the Src family of non-receptor tyrosine kinases (nRTK), efficiently repressed cervical cancer growth in vitro and in vivo. In this regard, our aims are to explore the mechanism of PP2 on cervical cancer cell growth inhibition by investigating the suppressive divergence among PP1, PP2, and a negative control compound PP3. MTT results showed that three compounds had different inhibitory effects on proliferation of two cervical cancer cells, HeLa and SiHa, and PP2 was most efficient in a time- and dose-dependent manner. Moreover, we found 10 μM PP2 down-regulated pSrc-Y416 (P < 0.05), pEGFR-Y845 (P < 0.05), and -Y1173 (P < 0.05) expression levels, while 10 μM PP1 down-regulated pSrc-Y416 (P < 0.05) and pEGFR-Y845 (P < 0.05), but not pEGFR-Y1173; 10 μM PP3 down-regulated only pEGFR-Y1173 (P < 0.05). PP2 could modulate cell cycle arrest by up-regulating p21(Cip1) and p27(Kip1) in both HeLa and SiHa cells and down-regulating expression of cyclin A, and cyclin dependent kinase-2, -4 (Cdk-2, -4) in HeLa and of cyclin B and Cdk-2 in SiHa. Our results indicate that Src pathway and EGFR pathway play different roles in the proliferation of cervical cancer cells and PP2 efficiently reduces cervical cancer cell proliferation by reduction of both Src and EGFR activity.     

Fibrinolytic activity in a human fibrosarcoma cell line and evidence for the induction of plasminogen activator secretion during tumor formation.

Seven clones were isolated from the HT1080 human fibrosarcoma cell line using a fibrinagarose overlay technique. Three of these clones induced lysis of the fibrin overlay, whereas four did not. The extracellular and intracellular levels of protease were then measured using 125I-fibrin plates incubated with acid-treated human serum. The extracellular protease can be directly assayed in the medium from cells incubated with 10% fetal calf serum. Although there were large differences in the amounts of protease secreted by these two sets of clones, the intracellular levels of protease were similar. No significant differences were found between the abilities of the cells to grow in soft agar or as tumors in immunosuppressed hamsters. However, cells grown from tumors derived from all the low secretors of protease showed an increase in the amount of protease secreted. It appeared, therefore, that the secretion of protease might be selected for or induced during tumor growth. Further detailed studies with one of the low secreting clones (clone E) suggested an inductive rather than a selective mechanism for this increase in extracellular plasminogen activator.     

DNA methyltransferase inhibition in normal human fibroblasts induces a p21-dependent cell cycle withdrawal

Maintenance of methylation patterns in the mammalian genome by DNA (cytosine-5) methyltransferases (DNAMeTase) is required for normal cell and tissue function. Inhibition of DNAMeTase in cultured cells induces the expression of p21, a cyclin-dependent kinase (Cdk) inhibitor critical for cells to enter replicative senescence. We investigated the effects of DNAMeTase inhibition in normal human fibroblasts and found that it induces an irreversible growth arrest. Cells arrested by DNAMeTase inhibition became enlarged and had a flat morphology, exhibited an increased expression of collagenase and p21, and the DNA synthesis block could be overcome by the introduction of the SV40 large T antigen, all characteristics of senescent cells. In contrast, normal human fibroblasts lacking a functional p21 gene fail to undergo cell cycle arrest following DNAMeTase inhibition, indicating that p21 is an essential component of this arrest. Furthermore, DNAMeTase activity was reduced as cells approached the end of their proliferative potential. These data suggest that DNAMeTase could be an integral part of the mechanisms by which cells count the number of cell divisions completed and initiate a signaling cascade that ultimately results in the senescent phenotype.     

Metabolic fate of the major cell surface protein of normal human fibroblasts

The major cell surface protein of a strain of human fetal lung fibroblasts is a 220,000 dalton glycoprotein termed fibronectin. Fibronectin is released from fibroblasts into the culture medium with a half-time of about 25 hours. The release occurs with an initial (0–3 hours) rapid phase followed by a second (3–48 hours) slower phase. Release of fibronectin occurs in a stoichiometric fashion. All of the fibronectin released from the cells is quantitatively recovered from the culture media as a similar sized soluble glycoprotein. Thus, release of fibronectin from normal human fibroblasts results from mechanisms other than extensive degradation of the protein.     

Increase in activity of acetylcholinesterase by 20-OH-ecdysone in a Chironomus tentans cell line

Summary An epithelial cell line from Chironomus tentans exhibits acetylcholinesterase activity (specific activity 0.05–0.2 nkat/mg protein), which rises 30– to 40-fold after addition of 10^–6M 20-OH-ecdysone. The first visible increase occurs after 4 days of incubation with hormone. The enzyme has an apparent K_m of 2.3±0.2×10^–4M for acetylthiocholine iodide as substrate and is inhibited by eserine and BW284 C51 (50% inhibition at 5×10^–7M for both inhibitiors) as well as by high concentrations of substrate, but not by tetraisopropylpyrophosphamide. The sensitivity against inhibitors is the same in extracts from hormone-treated cells and from controls. The cholinesterase activity correlates with morphological changes (shape and cell arrangement) and is indepenent of neuronal differentiation. We therefore propose a function for this activity during morphogenesis.     

Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.