Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line

Background: A broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but few studies examined cytotoxicity in these cell systems. Therefore, we compared four cytotoxicity assays in the zebrafish liver cell line, ZFL, to test four differently acting model compounds. The tests comprised two colorimetric assays (MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity) and two fluorometric assays (alamarBlue® using resazurin, and CFDA-AM based on 5-carboxyfluorescein diacetate acetoxymethyl ester). Model compounds were the pharmaceutical Tamoxifen, its metabolite 4-Hydroxy-Tamoxifen, the fungicide Flusilazole and the polycyclic aromatic hydrocarbon Benzo[a]pyrene. Results: All four assays performed well in the ZFL cells and led to reproducible dose-response curves for all test compounds. Effective concentrations causing 10% or 50% loss of cell viability (EC10 and EC50 values) varied by a maximum factor of 7.0 for the EC10 values and a maximum factor of 1.8 for the EC50 values. The EC values were not statistically different between the four assays, which is due to the assessed unspecific effects of the compounds. However, most often, the MTT assay and LDH assay showed the highest and lowest EC values, respectively. Nevertheless, the LDH assay showed the highest intra- and inter-assay variabilities and the lowest signal-to-noise ratios. In contrast to MTT, the other three assays have the advantage of being non-destructive, easy to handle, and less time consuming. Furthermore, AB and CFDA-AM can be combined on the same set of cells without damaging the cells, allowing later on their use for the investigation of other endpoints. Conclusions: We recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.JRC.H.5-Rural, water and ecosystem resource

The small cell lung cancer antigen cluster-4 and the leukocyte antigen CD24 are allelic isoforms of the same gene (CD24) on chromosome band 6q21.

Cluster-4 and CD24 cDNA's have recently been cloned from the small cell lung carcinoma (SCLC) cell line SW2 and from the erythroleukemia cell line K562, respectively. The only difference in the coding sequence, between cluster-4 and CD24 antigens is the substitution of a single base pair leading to a substitution of Val by Ala near the putative glycosylphosphatidylinositol (GPI) anchorage sites of the mature protein. Here we demonstrate that the nucleotide substitution which distinguishes the cluster-4 and CD24 antigen genes is due to an allelic polymorphism on chromosome band 6q21. In addition, we identified by Southern blotting and PCR of DNA from somatic human x hamster hybrid cell lines homologues of cluster-4/CD24 on the Y chromosome and chromosome 15. We suggest, however, that the gene on 6q21 is the active locus since the mRNA of cell lines always represents the allelic variants found on chromosome 6. The distribution pattern of this allelic polymorphism in SCLC cell lines and leukocytes of healthy donors did not reveal any obvious relationship with disease. However, it is noteworthy that homozygosity for cluster-4 was found in only one case whereas heterozygosity and homozygosity for CD24 both contribute up to 50% of the samples examined

CD24 promotes tumor cell invasion by suppressing tissue factor pathway inhibitor-2 (TFPI-2) in a c-Src-dependent fashion.

CD24 is a glycosyl-phosphatidylinositol-anchored protein with mucin-type structure that resides exclusively in membrane microdomains. CD24 is often highly expressed in carcinomas and correlates with poor prognosis. Experimentally, the over-expression or depletion of CD24 alters cell proliferation, adhesion, and invasion in vitro and tumor growth in vivo. However, little is known about the mechanisms by which CD24 mediates these cellular effects. Here we have studied the mechanism of CD24-dependent cell invasion using transient CD24 knock-down or over-expression in human cancer cell lines. We show that CD24 depletion reduced tumor cell invasion and up-regulated expression of Tissue Factor Pathway Inhibitor 2 (TFPI-2), a potent inhibitor of extracellular matrix degradation that can block metastases formation and tumor cell invasion. Over-expression of CD24 in A125 cells resulted in reduced TFPI-2 expression and enhanced invasion. We provide evidence that the activity of c-Src is reduced upon CD24 knock-down. The silencing of c-Src, similar to CD24, was able to enhance TFPI-2 expression and reduce tumor cell invasion. An inverse expression of CD24 and TFPI-2 was observed by immunohistochemical analysis of primary breast cancers (N = 1,174). TFPI-2 expression was highest in CD24 negative samples and lowered with increasing CD24 expression. Patients with a CD24 low/TFPI-2 high phenotype showed significantly better survival compared to CD24 high/TFPI-2 low patients. Our results provide evidence that CD24 can regulate cell invasion via TFPI-2 and suggests a role of c-Src in this process