Characterization of three new serous epithelial ovarian cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Cell lines constitute a powerful model to study cancer, and here we describe three new epithelial ovarian cancer (EOC) cell lines derived from poorly differentiated serous solid tumors (TOV-1946, and TOV-2223G), as well as the matched ascites for one case (OV-1946).</p> <p>Methods</p> <p>In addition to growth parameters, the cell lines were characterized for anchorage independent growth, migration and invasion potential, ability to form spheroids and xenografts in SCID mice.</p> <p>Results</p> <p>While all cell lines were capable of anchorage independent growth, only the TOV-1946 and OV-1946 cell lines were able to form spheroid and produce tumors. Profiling of keratins, p53 and Her2 protein expression was assessed by immunohistochemistry and western blot analyses. Somatic <it>TP53 </it>mutations were found in all cell lines, with TOV-1946 and OV-1946 harboring the same mutation, and none harbored the commonly observed somatic mutations in <it>BRAF</it>, <it>KRAS </it>or germline BRCA1/2 mutations found to recur in the French Canadian population. Conventional cytogenetics and spectral karyotype (SKY) analyses revealed complex karyotypes often observed in ovarian disease.</p> <p>Conclusion</p> <p>This is the first report of the establishment of matched EOC cell lines derived from both solid tumor and ascites of the same patient.</p

BMP-2 signaling in ovarian cancer and its association with poor prognosis

BACKGROUND: We previously observed the over-expression of BMP-2 in primary cultures of epithelial ovarian cancer (EOC) cells as compared to normal epithelial cells based on Affymetrix microarray profiling [1]. Here we investigate the effect of BMP-2 on several parameters of ovarian cancer tumorigenesis using the TOV-2223, TOV-1946 and TOV-112D EOC cell lines. METHODS: We treated each EOC cell line with recombinant BMP-2 and assayed various parameters associated with tumorigenesis. More specifically, cell signaling events induced by BMP-2 treatment were investigated by western-blot using anti-phosphospecific antibodies. Induction of Id1, Snail and Smad6 mRNA expression was investigated by real time RT-PCR. The ability of cells to migrate was tested using the scratch assay. Cell-cell adhesion was analyzed by the ability of cells to form spheroids. We also investigated BMP-2 expression in tissue samples from a series of EOC patients. RESULTS: Treatment of these cell lines with recombinant BMP-2 induced a rapid phosphorylation of Smad1/5/8 and Erk MAPKs. Increased expression of Id1, Smad6 and Snail mRNAs was also observed. Only in the TOV-2223 cell line were these signaling events accompanied by an alteration in cell proliferation. We also observed that BMP-2 efficiently increased the motility of all three cell lines. In contrast, BMP-2 treatment decreased the ability of TOV-1946 and TOV-112D cell lines to form spheroids indicating an inhibition of cell-cell adhesion. The expression of BMP-2 in tumor tissues from patients was inversely correlated with survival. CONCLUSION: These results suggest that EOC cell secretion of BMP-2 in the tumor environment contributes to a modification of tumor cell behavior through a change in motility and adherence. We also show that BMP-2 expression in tumor tissues is associated with a poorer prognosis for ovarian cancer patients

p21Waf1 expression is regulated by nuclear intermediate filament vimentin in neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Human neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes. We have previously reported a strong correlation between protein expression levels of vimentin, an S-subtype marker, and the p21^Waf1cyclin-dependent kinase inhibitor. We here investigated whether this correlation extend to the mRNA level in NB cell lines as well as in patients' tumors. We also further explored the relationship between expression of vimentin and p21, by asking whether vimentin could regulate p21 expression.</p> <p>Methods</p> <p>Vimentin and p21 mRNA levels in NB cell lines as well as in patients' tumors (<it>n </it>= 77) were quantified using Q-PCR. Q-PCR data obtained from tumors of high risk NB patients (<it>n </it>= 40) were analyzed in relation with the overall survival using the Log-rank Kaplan-Meier estimation. siRNA-mediated depletion or overexpression of vimentin in highly or low expressing vimentin cell lines, respectively, followed by protein expression and promoter activation assays were used to assess the role of vimentin in modulating p21 expression.</p> <p>Results</p> <p>We extend the significant correlation between vimentin and p21 expression to the mRNA level in NB cell lines as well as in patients' tumors. Overall survival analysis from Q-PCR data obtained from tumors of high risk patients suggests that lower levels of p21 expression could be associated with a poorer outcome. Our data additionally indicate that the correlation observed between p21 and vimentin expression levels results from p21 transcriptional activity being regulated by vimentin. Indeed, downregulating vimentin resulted in a significant decrease in p21 mRNA and protein expression as well as in p21 promoter activity. Conversely, overexpressing vimentin triggered an increase in p21 promoter activity in cells with a nuclear expression of vimentin.</p> <p>Conclusion</p> <p>Our results suggest that p21 mRNA tumor expression level could represent a refined prognostic factor for high risk NB patients. Our data also show that vimentin regulates p21 transcription; this is the first demonstration of a gene regulating function for this type III-intermediate filament.</p

Antibacterial Properties of the Mammalian L-Amino Acid Oxidase IL4I1

L-amino acid oxidases (LAAO) are flavoproteins that catalyze the oxidative deamination of L-amino acids to a keto-acid along with the production of H(2)O(2) and ammonia. Interleukin 4 induced gene 1 (IL4I1) is a secreted LAAO expressed by macrophages and dendritic cells stimulated by microbial derived products or interferons, which is endowed with immunoregulatory properties. It is the first LAAO described in mammalian innate immune cells. In this work, we show that this enzyme blocks the in vitro and in vivo growth of Gram negative and Gram positive bacteria. This antibiotic effect is primarily mediated by H(2)O(2) production but is amplified by basification of the medium due to the accumulation of ammonia. The depletion of phenylalanine (the primary amino acid catabolized by IL4I1) may also participate in the in vivo inhibition of staphylococci growth. Thus, IL4I1 plays a distinct role compared to other antibacterial enzymes produced by mononuclear phagocytes

IL4I1 bactericidal effect.

<p>Bacteria were grown 24 hours in serial dilutions of conditioned medium of THP1 or THP1-IL4I1 cells and OD measured. The lowest OD for both conditions was selected (t = 0). New DMEM/F12 containing 1% FCS was added, then bacterial re-growth was measured after 7 and 24 hours and compared between bacteria pre-cultured in THP1 and THP1-IL4I1 conditioned medium. Data are given as mean ± SEM from five independent experiments performed in duplicate. *<i>p</i><0.05 and **<i>p</i><0.01, Mann-Whitney test.</p

Susceptibility of bacteria to IL4I1 substrates and catabolites.

<p>Bacteria were serially diluted in DMEM/F12, with: (<b>A</b>) no Phe, 35.49 or 500 µg/ml Phe and/or no Trp, 9.02 or 500 µg/ml Trp; (<b>B</b>) ten folds dilutions of phenylpyruvate from 1 µM to 10 mM; (<b>C</b>) ten folds dilutions of H₂O₂ from 1 µM to 1 mM or 64 µg/ml glutathione with or without 1 mM H₂O₂; (<b>D</b>) ten folds dilutions of NH₃ from 5 µM to 50 mM or 15 mM HEPES with or without 50 mM NH₃. After 24 hours, bacterial growth was monitored at an OD of 595 nm. Data are given as mean ± SEM from five independent experiments performed in duplicate. *<i>p</i><0.05 **<i>p</i><0.01 ***<i>p</i><0.001, Mann-Whitney test in comparison to DMEM/F12 without Phe and Trp (<b>A</b>), without phenylpyruvate (<b>B</b>) or according to the bars on the graph (<b>C</b> and <b>D</b>).</p

IL4I1 inhibitory growth effect.

<p>(<b>A</b>) IL4I1 activity in conditioned medium from THP1 and THP1-IL4I1 cells (0.5×10⁶ cells/ml cultured in DMEM/F12+1% FCS, 48 hours at 37°C). Results are expressed as pmol H₂O₂ produced in the presence of phenylalanine per hour per ml of conditioned medium. (<b>B</b>) Bacteria were diluted in conditioned medium (CM) from THP1 containing different ratios of IL4I1-conditioned medium. After 24 hours, bacterial growth was monitored at an OD of 595 nm. Data are given as mean ± SEM from five independent experiments performed in duplicate. Mean OD in 100% CM THP1 was 0.227 for <i>E. coli</i>, 0.266 for B2599, 0.450 for MSSA and 0.516 CNS, respectively. These OD were considered as 100% bacterial growth and the OD measured in the other culture conditions were calculated as percentages with respect to this control. Statistical analyses were performed using the Mann-Whitney test in comparison to 100% CM THP1. ***<i>p</i><0.001 for all the bacterial strains at 100% CM THP1-IL4I1; **<i>p</i><0.01 for <i>E. coli</i>, B2599 and MSSA and ^#<i>p</i><0.05 for CNS at 75%; **<i>p</i><0.01 for <i>E. coli</i> and B2599 at 50%; NS, not significant for MSSA and CNS at 50% and for all the strains at 25%.</p