Permanent Human Cell Lines Research Articles

Co-cultivation of mastocytes and cumulus cells in polycystic ovary syndrome

The purpose of the study was to study the cytokine-producing function of cumulus cells of the ovaries in PCOS during co-cultivation of cumulus cells with mast cells to identify their mutual influence in the immunopathogenesis of PCOS. The study was approved by the Interdisciplinary Ethics Committee of the FSBEI of HE of the Ministry of Health of the Russian Federation. A permanent human mast cell line HMC-1 (Human mast cell, ATCC, USA) and a primary culture of cumulus cells were used. The condition of cumulus cells and mastocytes was determined using fluorescent dyes followed by flow cytometry before co-cultivation and after 7 days co-cultivation. The levels of IL-6, IL-10, and IFNã were studied on days 1, 3, and 7 of the experiment. In monoculture and during co-culture of cells, the synthesis of cytokines IL-6, IL-10, and IFNã continues, but to varying degrees of severity. In the permanent mast cell line, an increase in IL-6, IL-10, and IFNã is observed on the 1st, 3rd, and 7th days of cultivation. On the first day, cytokine levels of donor cumulus cells and mastocytes did not differ significantly (p 0.05). In the culture of donor cumulus cells, the synthesis of IL-6, IL-10, and IFNã progressively increases by the 7th day of the experiment (p 0.05). On the first day of the experiment, the balance of Th1/Th2 cytokines in PCOS was twice as high as in healthy women. The ratio increased, and on the 7th day reached 3.83 times higher than the ratio of Th1/Th2 cytokines in the control group. Hyperproduction of IFNã by mastocytes was most significant when they were co-cultured with cumulus cells, in monoculture mast cells synthesized excessively cytokine twice from the initial values of IFNã, and the monoculture cumulus cells in PCOS it practically did not contain. The studied cytokines are regulators of the function of ovarian cumulus cells and factors that increase the competence of the oocyte, indicating the need for their correction, which will allow a real influence on the links of pathogenesis in PCOS in the future.later on.

SMAD4\u2013201 transcript as a putative biomarker in colorectal cancer

BackgroundTranscripts with alternative 5′-untranslated regions (UTRs) result from the activity of alternative promoters and they can determine gene expression by influencing its stability and translational efficiency, thus executing complex regulation of developmental, physiological and pathological processes. Transcriptional regulation of human SMAD4, a key tumor suppressor deregulated in most gastrointestinal cancers, entails four alternative promoters. These promoters and alternative transcripts they generate remain unexplored as contributors to the SMAD4 deregulation in cancer. The aim of this study was to investigate the relative abundance of the transcript SMAD4–201 in colorectal cell lines and tissues in order to establish if its fluctuations may be associated with colorectal cancer (CRC).MethodsRelative abundance of SMAD4–201 in total SMAD4 mRNA was analyzed using quantitative PCR in a set of permanent human colon cell lines and tumor and corresponding healthy tissue samples from patients with CRC.ResultsThe relative abundance of SMAD4–201 in analyzed cell lines varied between 16 and 47%. A similar relative abundance of SMAD4–201 transcript was found in the majority of analyzed human tumor tissue samples, and it was averagely 20% lower in non-malignant in comparison to malignant tissue samples (p = 0.001). Transcript SMAD4–202 was not detectable in any of the analyzed samples, so the observed fluctuations in the composition of SMAD4 transcripts can be attributed to transcripts other than SMAD4–201 and SMAD4–202.ConclusionThe expression profile of SMAD4–201 in human tumor and non-tumor tissue samples may indicate the translational potential of this molecule in CRC, but further research is needed to clarify its usability as a potential biomarker for early diagnosis.

U-DCS: characterization of the first permanent human dendritic sarcoma cell line

A dendritic cell sarcoma cell line, U-DCS, was established from a dendritic cell sarcoma in a 53-year-old Caucasian male patient. Since its establishment, U-DCS has maintained stable phenotypic characteristics in vitro and has a doubling time of approximately 2 days under standard culture conditions. U-DCS is growing with typical dendritic cell morphology in tissue and expresses the dendritic cell sarcoma immunophenotypic markers S100 protein, MHCI, MHCII, and vimentin. Expression analysis revealed transcripts for the toll-like receptors TLR3, -4, -9 and DDX58 (RIG-I), but not for TLR2. U-DCS shows functional features of dendritic cells with the ability of phagocytosis and antigen-specific T cell stimulation. Karyotype-, CGH-, and mFISH analysis point to a chromosomal instability and a hypotetraploid karyotype with approximately 130 chromosomes. U-DCS is the first immortalized human dendritic cell sarcoma cell line and has some morphological and functional features of dendritic cells without dependency on growth factors.

Molecular characterization of the human lens epithelium-derived cell line SRA01/04

Cataract-associated gene discovery in human and animal models have informed on key aspects of human lens development, homeostasis and pathology. Additionally, in vitro models such as the culture of permanent human lens epithelium-derived cell lines (LECs) have also been utilized to understand the molecular biology of lens cells. However, these resources remain uncharacterized, specifically regarding their global gene expression and suitability to model lens cell biology. Therefore, we sought to molecularly characterize gene expression in the human LEC, SRA01/04, which is commonly used in lens studies. We first performed short tandem repeat (STR) analysis and validated SRA01/04 LEC for its human origin, as recommended by the eye research community. Next, we used Illumina HumanHT-12 v3.0 Expression BeadChip arrays to gain insights into the global gene expression profile of SRA01/04. Comparative analysis of SRA01/04 microarray data was performed using other resources such as the lens expression database iSyTE (integrated Systems Tool for Eye gene discovery), the cataract gene database Cat-Map and the published lens literature. This analysis showed that SRA01/04 significantly expresses >40% of the top iSyTE lens-enriched genes (313 out of 749) across different developmental stages. Further, SRA01/04 also significantly expresses ~53% (168 out of 318) of cataract-associated genes in Cat-Map. We also performed comparative gene expression analysis between SRA01/04 cells and the previously validated mouse LEC 21EM15. To gain insight into whether SRA01/04 reflects epithelial or fiber cell characteristics, we compared its gene expression profile to previously reported differentially expressed genes in isolated mouse lens epithelial and fiber cells. This analysis suggests that SRA01/04 has reduced expression of several fiber cell-enriched genes. In agreement with these findings, cell culture analysis demonstrates that SRA01/04 has reduced potential to initiate spontaneous lentoid body formation compared to 21EM15 cells. Next, to independently validate SRA01/04 microarray gene expression, we subjected several candidate genes to RT-PCR and RT-qPCR assays. This analysis demonstrates that SRA01/04 supports expression of many key genes associated with lens development and cataract, including CRYAB, CRYBB2, CRYGS, DKK3, EPHA2, ETV5, GJA1, HSPB1, INPPL1, ITGB1, PAX6, PVRL3, SFRP1, SPARC, TDRD7, and VIM, among others, and therefore can be relevant for understanding the mechanistic basis of these factors. At the same time, SRA01/04 cells do not exhibit robust expression of several genes known to be important to lens biology and cataract such as ALDH1A1, COL4A6, CP, CRYBA4, FOXE3, HMX1, HSF4, MAF, MEIS1, PITX3, PRX, SIX3, and TRPM3, among many others. Therefore, the present study offers a rich transcript-level resource for case-by-case evaluation of the potential advantages and limitations of SRA01/04 cells prior to their use in downstream investigations. In sum, these data show that the human LEC, SRA01/04, exhibits lens epithelial cell-like character reflected in the expression of several lens-enriched and cataract-associated genes, and therefore can be considered as a useful in vitro resource when combined with in vivo studies to gain insight into specific aspects of human lens epithelial cells.

Apolipoprotein M induces inhibition of inflammatory responses via the S1PR1 and DHCR24 pathways.

Apolipoprotein M (ApoM) is a type of apolipoprotein. It is well known that high‑density lipoprotein (HDL) decreases inflammatory responses via the apoM‑sphingosine‑1‑phosphate (S1P) pathway. The present study further investigated the importance of ApoM in the inhibitory effects of HDL on inflammation. Mice with an apoM gene deficiency (apoM‑/‑) were employed to investigate the effects of ApoM on the expression of interleukin‑1β (IL‑1β), monocyte chemotactic protein‑1 (MCP‑1), S1P receptor‑1 (S1PR1) and 3β‑hydroxysterol Δ‑24‑reductase (DHCR24), as compared with in wild‑type mice (apoM+/+). Furthermore, cell culture experiments were performed using a permanent human hybrid endothelial cell line (EA.hy926). Cells were cultured in the presence of recombinant human apoM (rec‑apoM) or were induced to overexpress apoM (apoMTg); subsequently, cells were treated with tumor necrosis factor‑α (TNF‑α), in order to investigate the effects of ApoM on IL‑1β and MCP‑1. The results demonstrated that the mRNA expression levels of IL‑1β and MCP‑1 were significantly higher in the liver following administration of lipopolysaccharide in apoM‑/‑ mice compared with in apoM+/+ mice. In cell culture experiments, when cells were pre‑cultured with rec‑apoM or were engineered to overexpress apoM (apoMTg), they exhibited decreased expression levels of IL‑1β and MCP‑1 following TNF‑α treatment compared with in normal apoM‑expressing cells (apoMTgN). Furthermore, the mRNA expression levels of IL‑1β and MCP‑1 were significantly elevated following addition of the S1PR1 inhibitor W146, but not by the scavenger receptor class B type I inhibitor, block lipid transport‑1 (BLT‑1), in apoMTg cells prior to TNF‑α treatment. Conversely, there were no differences in these inflammatory biomarkers under the same conditions in apoMTgN cells. The mRNA expression levels of DHCR24 were significantly reduced by the addition of BLT‑1 prior to TNF‑α treatment in apoMTg cells; however, there was no difference in the expression of this inflammatory biomarker in apoMTgN cells. In conclusion, ApoM displayed inhibitory effects against the inflammatory response invivo and in vitro; these effects may be induced via the S1PR1 and DHCR24 pathways.

Metamizole (dipyrone) – cytotoxic and antiproliferative effects on HeLa, HT-29 and MCF-7 cancer cell lines

Cancer pain treatment is a big challenge for healthcare providers and patients as well. The wide range of non-steroidal anti-inflammatory drugs (NSAIDs) used as painkillers in cancer patients, requires in-depth characterization of their effect on the disease process. The effects of NSAIDs have been widely studied over the last decades as preventive drugs in some oncological diseases. Metamizole is an NSAID belonging to the non-narcotic analgesics group and is highly recommended in oncology either alone or in combinations with opioid analgesics. There is a dearth of information regarding the cytotoxicity profile of metamizole and hence the present study evaluated the potential anticancer activity of metamizole in some permanent human tumour cell lines: HeLa, human cervical cancer cells; HT-29, a human colorectal adenocarcinoma cell line; and МСF-7, human breast adenocarcinoma cells. The studied tumour cells were sensitive to metamizole at doses higher than 25 μg/mL. Metamizole induced a statistically significant decrease in the viability of HeLa, HT-29 and MCF-7 cells in in vitro tests as measured by the MTT assay; the highest effect was observed at the 48th hour of the treatment. Metamizole could induce cell death by apoptosis. Metamizole also suppressed the migration of the three tumour cell lines. This was most clearly pronounced in HeLa cells. The results obtained indicate that metamizole is a suitable choice for the treatment of cancer pain and has prospects for further in-depth studies.

Preclinical human models and emerging therapeutics for advanced systemic mastocytosis.

Mastocytosis is a term used to denote a group of rare diseases characterized by an abnormal accumulation of neoplastic mast cells in various tissues and organs. In most patients with systemic mastocytosis, the neoplastic cells carry activating mutations in KIT. Progress in mastocytosis research has long been hindered by the lack of suitable in vitro models, such as permanent human mast cell lines. In fact, only a few human mast cell lines are available to date: HMC-1, LAD1/2, LUVA, ROSA and MCPV-1. The HMC-1 and LAD1/2 cell lines were derived from patients with mast cell leukemia. By contrast, the more recently established LUVA, ROSA and MCPV-1 cell lines were derived from CD34+ cells of non-mastocytosis donors. While some of these cell lines (LAD1/2, LUVA, ROSAKIT WT and MCPV-1) do not harbor KIT mutations, HMC-1 and ROSAKIT D816V cells exhibit activating KIT mutations found in mastocytosis and have thus been used to study disease pathogenesis. In addition, these cell lines are increasingly employed to validate new therapeutic targets and to screen for effects of new targeted drugs. Recently, the ROSAKIT D816V subclone has been successfully used to generate a unique in vivo model of advanced mastocytosis by injection into immunocompromised mice. Such a model may allow in vivo validation of data obtained in vitro with targeted drugs directed against mastocytosis. In this review, we discuss the major characteristics of all available human mast cell lines, with particular emphasis on the use of HMC-1 and ROSAKIT D816V cells in preclinical therapeutic research in mastocytosis.

Аntimicrobial and anticancer activity of new poly(propyleneamine) metallodendrimers

The synthesis, EPR characterisation and biological evaluation of two new metallodendrimers, i.e. a poly(propyleneamine) dendrimer functionalized at the external surface with 4-bromo-1,8-naphthalimide and conjugated with Cu(II) and Zn(II), was performed with the aim to evaluate their antimicrobial and anticancer activity. The antimicrobial activity was investigated in meat-peptone broth against bacteria B. subtilis and P. aeruginosa, and the yeast C. lipolytica. The results showed that the compounds inhibited effectively the tested pathogens even after their deposition on a textile fabric. Anticancer activity was investigated against three human permanent cell lines from non-small cell lung cancer (A549), triple negative breast cancer (MDA-MB-231) and carcinoma of the uterine cervix (HeLa) in the c = 0.01–30 μM concentration range. The results suggest that these compounds are promising for application in biomedicine as anticancer drugs in the design of new effective preparations. The antimicrobial and anticancer activity may be related to the peculiar structural and dynamical properties revealed for the Cu(II) complexes, by a computer aided analysis of the electron paramagnetic resonance (EPR) spectra. This analysis indicated the formation, at the lowest Cu(II) concentrations, of a flexible rhombic Cu-N4 coordination with the internal amino groups of the dendrimer, which transformed into a Cu2-N4 coordination already at 0.25 equiv. of Cu(II).

Human embryonic stem cells

The establishment of permanent human embryonic stem cell lines (hESCs) was first reported in 1998. Due to their pluripotent nature and ability to differentiate to all cell types in the body, they have been considered as a cell source for regenerative medicine. Since then, intensive studies have been carried out regarding factors regulating pluripotency and differentiation. hESCs are obtained from supernumerary human IVF (in vitro fertilization) embryos that cannot be used for the couple's infertility treatment. Today, we can establish and expand these cells in animal substance-free conditions, even from single cells biopsied from eight-cell stage embryos. There are satisfactory tests for the demonstration of genetic stability, absence of tumorigenic mutations, functionality, and safety of hESCs. Clinical trials are ongoing for age-related macular degeneration (AMD) and spinal cord injury (SCI). This review focuses on the present state of these techniques.

Cytostatic and cytotoxic properties of monensic acid and its biometal(II) complexes against human tumor / non-tumor cell lines

Abstract The anticancer activity of monensic acid (MonH) and its biometal(II) complexes [M(Mon)2(H2O)2](M = Mg, Ca, Mn, Co, Ni, Zn) was evaluated against cultured human permanent cell lines established from glioblastoma multiforme (8MGBA) and cancers of the lung (A549), breast (MCF-7), uterine cervix (HeLa) and liver (HepG2). The viability and proliferation of the non-tumor human embryonic cell line Lep3 was also tested. The investigations were carried out using a thiazolyl blue tetrazolium bromide test, neutral red uptake cytotoxicity assay, crystal violet staining, colony forming method and double staining with acridin orange and propidium iodide. The results obtained reveal that the compounds applied at concentrations of 0.5–25 µg mL−1 for 24–72 h decrease the viability and proliferation of the treated cells in a time- and concentration-dependent manner. The metal(II) complexes studied (especially those of Co(II), Ni(II) and Zn(II)) have been found to express stronger cytotoxic and cytostatic activities than the non-coordinated monensic acid. The non-tumor human cell line showed strong chemosensitivity towards compounds tested comparable to that of cultured human tumor cell lines.

Cytotoxic and Apoptogenic Potential of Red Microalgal Polysaccharides

ABSTRACTThe efforts of scientists are aimed at finding anti-cancer agents of natural origin which have high biological activity, low toxicity and broad spectrum of therapeutic activity. This study was designed to determine the cytotoxic properties of polysaccharides derived from the red microalgae Dixoniella grisea and Porphyridium cruentum and to elucidate the mechanism of their action on two permanent human cell lines MCF-7 (breast adenocarcinoma) and HeLa (cervical carcinoma), as well as on primary culture from Graffi myeloid tumor in hamsters. Our investigations indicated that both algal polysaccharides showed high cytotoxic and apoptogenic activities on cancer cells and may be a promising alternative to synthetic substances.

No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

BackgroundThe large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation.MethodsWe evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines.ResultsDerivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line.ConclusionEven very poor quality embryos with few cells in the ICM can give origin to hESC lines.

Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line, FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization

BackgroundPleomorphic malignant fibrous histiocytoma (MFH) is one of the most frequent malignant soft tissue tumors in adults. Despite the considerable amount of research on MFH cell lines, their characterization at a molecular cytogenetic level has not been extensively analyzed.Methods and resultsWe established a new permanent human cell line, FU-MFH-2, from a metastatic pleomorphic MFH of a 72-year-old Japanese man, and applied multicolor fluorescence in situ hybridization (M-FISH), Urovysion™ FISH, and comparative genomic hybridization (CGH) for the characterization of chromosomal aberrations. FU-MFH-2 cells were spindle or polygonal in shape with oval nuclei, and were successfully maintained in vitro for over 80 passages. The histological features of heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original tumor. Cytogenetic and M-FISH analyses displayed a hypotriploid karyotype with numerous structural aberrations. Urovysion™ FISH revealed a homozygous deletion of the p16INK4A locus on chromosome band 9p21. CGH analysis showed a high-level amplification of 9q31-q34, gains of 1p12-p34.3, 2p21, 2q11.2-q21, 3p, 4p, 6q22-qter, 8p11.2, 8q11.2-q21.1, 9q21-qter, 11q13, 12q24, 15q21-qter, 16p13, 17, 20, and X, and losses of 1q43-qter, 4q32-qter, 5q14-q23, 7q32-qter, 8p21-pter, 8q23, 9p21-pter, 10p11.2-p13, and 10q11.2-q22.ConclusionThe FU-MFH-2 cell line will be a particularly useful model for studying molecular pathogenesis of human pleomorphic MFH.

Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas

Glial fibrillary acidic protein (GFAP) is an intermediate filament expressed in glial cells that stabilizes and maintains the cytoskeleton of normal astrocytes. In glial tumors, GFAP expression is frequently lost with increasing grade of malignancy, suggesting that GFAP is important for maintaining glial cell morphology or regulating astrocytoma cell growth. Most permanent human glioma cell lines are GFAP negative by immunocytochemistry. Given that the GFAP gene is not mutated in human glioma specimens or glioma cell lines, we considered epigenetic mechanisms, such as promoter methylation, as a cause of silencing of GFAP in these tumors. In this study, we treated known GFAP-negative glioma cell lines with 5-aza-2'-deoxycytidine to examine GFAP promoter hypermethylation. Additionally, we performed bisulfite sequencing on primary glioma samples and glioma cell lines and showed an inverse relationship between GFAP promoter methylation status and GFAP expression. Using a gene reporter assay with the GFAP promoter cloned upstream of a luciferase gene, we showed that methylation of the GFAP promoter downregulates the expression of the luciferase gene. Our results suggest that epigenetic silencing of the GFAP gene through DNA methylation of its promoter region may be one mechanism by which GFAP is downregulated in human gliomas and glioma cell lines.

The Bionas technology for anticancer drug screening

Background: The Bionas 2500® analyzing system is an advanced label-free technology using a cell-based multi-sensor array, which is commercially available. Data on metabolism, respiration, adhesion, cell proliferation and cell death rates, as well as ligand–receptor interactions (multi-parametric) can be acquired and statistically evaluated. Noteworthy is the possibility of analyzing later after effects and/or recovery after drug treatment. In addition, Bionas supports all conceivable drug application modes (one, two or more drugs). Specimens for drug screening with Bionas consist of human permanent cell lines, primary cell cultures as well as ‘tumor slices’ obtained from biopsies and surgery material. Objective/methods: Examples of measurements are presented and discussed. Conclusion: Although drug throughput is modest, the high quality of the information allows in-depth evaluation.

Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC50). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100-1,000, and the corresponding IC50 values were in the range from 2.2nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC50 values were 4.4-10.8nmol/l for T-2 toxin, 7.5-55.8mol/l for HT-2 toxin, 600-4,900nmol/l for DON, 300-2,600nmol/l for NIV, and 2.2-18.3nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC50 values ranging from 16.5nmol/l (T-2 toxin) to 4,500nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.

A novel L1 retrotransposon marker for HeLa cell line identification

The HeLa cell line is the oldest, most widely distributed, permanent human cell line. As a nearly ubiquitous inhabitant of laboratories using tissue culture techniques, its aggressive growth characteristics make it a problematic contaminant that can overgrow less robust cell lines. Consequently, HeLa contamination is common in both the research laboratory and cell line repository contexts, and its detection is hampered by the lack of a rapid, sensitive and robust assay. Here we report the development of a HeLa-specific DNA diagnostic test: a single duplex detection PCR assay targeting an L1 retrotransposon insertion. All HeLa clones from a geographically diverse panel were positive by this assay, and the particular L1 insertion we identified appears to be unique to the HeLa cell line. The assay can detect very low levels of HeLa contamination (<1%), and can be performed on un-purified cell pellets, allowing rapid routine screening.

The cytotoxicity of orthodontic metal bracket immersion media

The purpose of the present study was to evaluate the cytotoxic effects of four different orthodontic metal bracket immersion media on primary human oral gingival fibroblasts (HGFs) and one permanent human osteogenic sarcoma cell line (U2OS). Four different metal brackets (Unitek, Tomy, Ormco, and Dentaurum) were immersed in buffer solutions of NaHNO(3) (1 mM) with a pH of 4 or 7, as well as artificial saliva. The concentrations for the experiments were 0.01, 0.1, and 1.0 mul/ml. At the end of the period of bracket immersion, morphological observations were conducted using light microscopy. The tetrazolium reduction assay was used to detect the survival rate of the target cells. Statistical analysis was conducted using one-way analysis of variance. The results showed microscopically no morphological changes of the HGF or U2OS cells exposed to the metal bracket immersion media. At pH 4, the survival rates of the U2OS cells and the HGFs differed statistically for the Unitek (P = 0.003) and Ormco (P = 0.000) groups. At pH 7, the survival rate for the HGFs and the U2OS cells differed statistically for the Dentaurum (P = 0.021) and Unitek (P = 0.03) groups. The results demonstrate that differing cells exhibit various cellular reactions on exposure to metal bracket immersion media, although the four types of brackets appear to be biocompatible with HGF and U2OS cells.

Alternatives in pharmaceutical toxicology: Global and focussed approaches - Two case studies

Safety testing of potential drugs has been and will continue to be a challenging task for the toxicologist in the pharmaceutical industry. We present two examples for the use of target-specific cell models to detect and assess species-specific toxicity. In the first example, adrenal models based on primary cells as well as a permanent human adrenal cell line were used. Both cell systems enabled a good prediction of adrenal effects in rodents, non-rodents and humans. The second example made use of primary hepatocytes. In this project, a potential drug candidate showed unexpected toxicity in vitro as well as species-specific cytochrome P450 (CYP) induction in vivo. We therefore analysed CYP induction and gene expression signatures in rat and human hepatocytes as well as in samples from in vivo animal toxicity studies. By this approach, the rat hepatocyte model correctly predicted the effects observed in rats and the in vitro/in vivo comparison enabled a solid extrapolation of consequences in humans. These examples demonstrate that an intelligent testing strategy, using alternative methods, can enable a meaningful safety assessment for humans by adding a ''tailor-made'' range of technologies to ''classic'' toxicological methods.

Establishing tumor cell lines from aggressive telomerase-positive chordomas of the skull base

Permanent cell cultures are invaluable tools for understanding the biological characteristics of tumors. In the present study the authors report on the establishment of permanent human cell lines from three cases of aggressive chordomas of the clival region. All of the parental tumors showed telomerase activity. Cultured chordoma cells had a doubling time of 5 to 7 days and grew as a monolayer of cells that retained both the immunophenotype and the p53 status of the parental tumor. In vitro, chordoma cells overexpressed telomerase, supporting the hypothesis that this enzyme is required for the immortalization process.