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Anaplastic carcinoma of thyroid is uncommon and is associated with a poor prognosis. an effective treatment of this carcinoma has not been found. We have examined the inhibition of promotion by 1, 25(OH)2D3 and its analogue without hypercalcemia, 22-oxacalcitriol (OCT) in the thyroid anaplastic carcinoma cell line. TTA-1, thyroid anaplastic carcinoma cell line, and TAC-1, thyroid anaplastic carcinoma cells. TPC-1,2,3,4, which are all papillary carcinoma of thyroid were used as control. Tumor growth was measured by MTT assay. 1,25(OH)2D3 additive cell growth was observed in diphasic pattern in 3/4 papillary carcinoma cell lines. OCT was more effective, showing dose-dependent inhibition of cell growth in anaplastic carcinoma cell lines, than that in papillary carcinoma cells. Conclusively, OCT might be useful for the inhibition of growth in anaplastic thyroid cancer.
Int J Mol Med 1999 Dec
PMID:Antineoplastic activity of 1,25(OH)2D3 and its analogue 22-oxacalcitriol against human anaplastic thyroid carcinoma cell lines in vitro. 1056 71

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.
J Mol Endocrinol 1999 Dec
PMID:Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems. 1060 76

Reactive oxygen species (ROS) are implicated as agents of cellular damage in pulmonary oxygen toxicity. Glutathione (GSH) and GSH-dependent antioxidant enzymes protect against damage by ROS, and recycling of glutathione disulfide (GSSG) to GSH by glutathione reductase (GR) is essential for the optimum functioning of this system. Exposure to hyperoxia inhibits lung development in newborn animals and humans, and attenuates cell growth in proliferating cell cultures. Considerable evidence supports a role for ROS as growth-altering molecules. Previously, we have observed that gene transfer of GR to mitochondria in H441 cells, using a vector containing a mitochondrial leader sequence (LGR), protected these cells against t-BuOOH-induced cytotoxicity. The present studies tested the hypothesis that gene transfer of LGR would attenuate the cytostatic effects of hyperoxia exposure in H441 cells. H441 cells (0.9 x 10(6) cells/plate) transfected with adenovirus containing LGR or the complementary DNA (cDNA) for manganese superoxide dismutase in reverse orientation (DOS) as a control construct, and untransfected cells (CON) were maintained in 21% oxygen (normoxia) or 95% oxygen (hyperoxia) for 48 h, and cell growth was assessed by cell counts and by reduction of the tetrazolium dye 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan. Cells maintained in normoxia achieved normal growth (CON, 1.98; DOS, 1.91; LGR, 2.0 x 10(6) cells/plate). Hyperoxia inhibited cell growth and the reduction of MTT; however, cells transfected with LGR had greater mitochondrial GR activities (CON, 16+/-2; DOS, 19+/-3; LGR, 322+/-18 mU/mg of protein), sustained more normal growth patterns (CON, 1.25+/-0.12; DOS, 1.24 +/-0.21, LGR, 1.8+/-0.25 x 10(6) cells/plate), and had less inhibition of MTT reduction (CON, 29; DOS, 27; LGR, 16% inhibition, P<0.01) after exposure to hyperoxia for 48 h than was observed in cells transfected with DOS or in control cells not infected with virus. In addition, resistant cells had higher mitochondrial GSH levels and maintained mitochondrial GSH/GSSG ratios in hyperoxia, suggesting that maintaining mitochondrial GSH homeostasis determined critical aspects of cell division in these studies. The mechanisms for sustaining cell growth during hyperoxia in H441 cells with enhanced mitochondrial GR activities are unknown, but similar effects in infants exposed to supplemental oxygen could be highly beneficial.
Am J Respir Cell Mol Biol 2000 Jun
PMID:Attenuation of hyperoxia-induced growth inhibition in H441 cells by gene transfer of mitochondrially targeted glutathione reductase. 1083 71

Tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, produced by maternal inflammatory cells, may compromise trophoblast survival at the trophoblast-maternal interface and notably in the placental bed which is invaded by trophoblast. Extracellular matrix components, e.g. fibronectin, may enhance trophoblast survival. A possible protective effect of fibronectin against toxic effects of TNF-alpha and IFN-gamma was investigated in cultured trophoblasts isolated from six human term placentas, grown on uncoated and fibronectin-coated plastics. IFN-gamma and increasing doses of TNF-alpha resulted in decreasing viability of trophoblast on uncoated as well as fibronectin-coated dishes, as shown by 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays, but for each TNF/IFN treatment condition viability on fibronectin was higher (P < 0.001). Epidermal growth factor (EGF), a growth factor reported to protect against TNF-alpha/IFN-gamma induced toxicity, resulted in further increased viability, but not if IFN-gamma was included in the treatment. EGF caused increased fibronectin secretion into the medium (P < 0.001), and double cytokeratin/fibronectin immunostaining confirmed the trophoblastic nature of fibronectin secreting cells. We conclude that fibronectin increases viability, but does not completely abolish the cytotoxic action of TNF-alpha and IFN-gamma on trophoblast. The protective effect of EGF may be related to stimulation of fibronectin secretion by trophoblast.
Mol Hum Reprod 2000 Jul
PMID:Cytotoxic effects of tumour necrosis factor (TNF)-alpha and interferon-gamma on cultured human trophoblast are modulated by fibronectin. 1087 51

Fas/Fas ligand (FasL) is well known for its role in delivering apoptotic signals; however, it is unclear whether FasL can mediate apoptosis in cardiomyocytes. We hypothesized that apoptosis via Fas/FasL system may be augmented in damaged cardiomyocytes. To determine whether FasL mediates cardiomyocyte apoptosis, recombinant FasL (rFasL) was added to the culture of neonatal rat ventricular myocytes pretreated with and without doxorubicin. Without doxorubicin, high dose of rFasL caused an increase in TUNEL-positive cardiomyocytes and a mild decrease in MTT activities. When cardiomyocytes were pretreated with doxorubicin (0.5 microM), rFasL dramatically augmented TUNEL-positive cardiomyocytes in a concentration-dependent manner, which was accompanied with nuclear fragmentations. The rFasL also caused a concentration-dependent reduction in MTT activities in cardiomyocytes. The rFasL-induced caspase-8 activity was greatly facilitated by pretreatment of doxorubicin. TUNEL-positive nuclei with rFasL was inhibited by Fas-Fc, neutralizing agent of rFasL, and Z-IETD-FMK, caspase-8 inhibitor. Fas mRNA transcript by RT-PCR was up-regulated in cardiomyocytes with doxorubicin. We conclude that FasL can induce cardiomyocyte apoptosis particularly when cardiomyocyte becomes susceptible for Fas-mediated apoptosis.
J Mol Cell Cardiol 2000 Jun
PMID:Apoptosis in rat cardiac myocytes induced by Fas ligand: priming for Fas-mediated apoptosis with doxorubicin. 1088 43

The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene6, and compared it with two other transfection reagents, Tfx-50 and LipoTaxi. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.
Cell Mol Life Sci 2000 Aug
PMID:Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies. 1102 22

Allyl sulfur compounds play a major role in the chemoprevention against carcinogenesis. The present study compared the antiproliferative effects of diallyl sulfide (DAS), diallyl disulfide (DADS) and garlic extract on p53-wild type H460 and p53-null type H1299 non small cell lung cancer cells (NSCLC). The DAS and DADS treatment of both H460 and H1299 cells resulted in the highest numbers of cells in apoptotic state as measured by acridine orange staining, however, garlic extract treatment did not induce any significant apoptotic cells by MTT assay. DADS was found to be more effective in inducing apoptosis on NSCLC. The level of p53 protein in H460 cell was increased following DADS treatment. DAS and garlic extract treatment of H460 cells induced a rise in the level of Bax and a fall of Bcl-2 level. These results demonstrate that DAS, DADS and garlic extract are effective in reduction of anti-proliferative gene in NSCLC and suggest that modulation of apoptosis-associated cellular proteins by DAS, DADS and garlic extract may be the mechanism for apoptosis which merit further investigation as potential chemoprevention agents.
Exp Mol Med 2000 Sep 30
PMID:Effects of allyl sulfur compounds and garlic extract on the expression of Bcl-2, Bax, and p53 in non small cell lung cancer cell lines. 1104 43

An oxidation-reduction (redox) indicator, alamarBlue, was used to measure the bioactivity of interleukin 2 (IL-2). This assay system has several advantages over other bioassays for measuring IL-2. It is a nonradioactive method unlike the conventional tritium-labeled thymidine ([3H]TdR) incorporation assay. The alamarBlue assay is also easier to use than other colorimetric methods, such as the MTT assay, because the alamarBlue assay does not depend on the extraction of insoluble formazan salt, which is time-consuming, error-prone, and cumbersome. Due to its solubility in culture medium and its nontoxicity to cells, alamarBlue provides an easy method to monitor cellular growth using either a fluorescence- or an absorbance-based instrument. The alamarBlue assay is not sample-destructive, unlike the thymidine incorporation and MTT methods. This adds another advantage to the alamarBlue method as the measurement of cellular growth by sample-destructive methods requires as many tubes as time points whereas the alamarBlue method requires only one tube for the entire growth period. In this study, alamarBlue was used to measure the proliferation of the IL-2-dependent cytotoxic T cell line, CTLL-2. The colorimetric change of alamarBlue at 570 nm compared to the reference wavelength, 600 nm, was proportional to the number of viable cells. The sensitivity of the IL-2 assay using alamarBlue was comparable to that of the [3H]thymidine incorporation method. These results demonstrate that the alamarBlue assay is valid for the IL-2 bioassay and that alamarBlue can replace the [3H]thymidine employed in the conventional proliferation assays.
Mol Cells 2000 Oct 31
PMID:A new non-radioactive method for IL-2 bioassay. 1110 Nov 50

Elevated homocysteine levels have been suggested to contribute to various pathological states of the brain. However, the basic mechanisms underlying homocysteine-induced neurotoxicity have not yet been fully elucidated. In the present series of experiments, we investigated the effect of homocysteine on mRNA levels of genes coding for cytoplasmic- or endoplasmic reticulum-resident stress proteins. Primary neuronal cell cultures were exposed to different homocysteine levels for 1-24 h. Cell injury was evaluated using the MTT assay, protein synthesis was studied by measuring the incorporation of L-[4,5-3H]leucine into proteins, mRNA levels of hsp70, gadd153, grp78, and grp94 were evaluated by quantitative PCR, and changes in protein levels of hsp70, grp78 and grp94 were analyzed by immunoblotting. Exposure of cells to 5 or 10 mM homocysteine for 24 h induced marked cell injury (decrease of viability to 58 or 45% of control respectively). After 6 h treatment, gadd153, grp78 and grp94 mRNA levels increased markedly, but only when cells were exposed to levels of homocysteine high enough to induce cell injury. In addition, hsp70 mRNA levels and protein synthesis were significantly reduced. At earlier (1 or 3 h) or later (12 or 24 h) time intervals, homocysteine exposure induced a marked increase in mRNA levels of all genes studied. GRP78 and GRP94 protein levels were increased in cells exposed to 5 mM homocysteine for 24 h but not in cells exposed to 10 mM homocysteine. HSP70 protein levels, in contrast, were decreased in cells exposed to homocysteine for different periods. The expression of genes coding for ER-resident stress proteins is specifically activated under conditions of ER stress. The close relationship between the extent of cell injury and increase in grp78 mRNA levels suggests that ER dysfunction may contribute to the pathological process. The results imply that the ER is an intracellular target of homocysteine toxicity.
Brain Res Mol Brain Res 2000 Dec 08
PMID:Homocysteine-induced changes in mRNA levels of genes coding for cytoplasmic- and endoplasmic reticulum-resident stress proteins in neuronal cell cultures. 1111 29

The potential anti-radical properties and cytoprotective effects of Mg-gluconate were studied. When microsomal membranes were peroxidized by a *O2- driven, Fe-catalyzed oxy-radical system (R* = dihydroxyfumarate + Fe2+), Mg-gluconate inhibited lipid peroxidation (TBARS formation) in a concentration-dependent manner with IC50 being 2.3 mM. For the entire range of .25-2 mM, MgSO4 or MgCl2 were < or = 20% effective compared to Mg-gluconate. When cultured bovine aortic endothelial cells were incubated with the R* for 50 min. at 37 degrees C, 56% loss of total glutathione occurred. Pre-treatment (10 min.) of the cells with 0.25-4 mM Mg-gluconate before R* exposure significantly (p<0.05) prevented the GSH loss to varying degrees; the EC50 was 1.1 mM. In separate experiments, with 30 min. of free radical incubation of endothelial monolayers (approximately 65% confluent), cell survival/proliferation determined by the tetrazolium salt MTT assay, decreased to 38% of control at 24 hrs; Mg-gluconate concentration-dependently attenuated the lost cell survival with EC50 of approximately 1.3 mM. For comparison, the effects provided by MgSO4 or MgCl2 were significantly lower and were < or = 1/3 as potent as that produced by Mg-gluconate. In a Fenton-reaction system consisting of Fe(II)+ H2O2, Mg-gluconate but not other Mg-salts, significantly inhibited the formation of OH radicals as determined by the ESR DMPO-OH signal intensity. Mg-gluconate also dose-dependently inhibited the 'Fe-catalyzed' deoxyribose degradation suggesting that Mg-gluconate could displace Fe from 'catalytic sites' of oxidative damage. These data suggest that Mg-gluconate may serve as a more advantageous Mg-salt for clinical use due to its additional anti-radical and cytoprotective activities.
Cell Mol Biol (Noisy-le-grand) 2000 Dec
PMID:Protective mechanisms of Mg-gluconate against oxidative endothelial cytotoxicity. 1115 79


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