Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells

<p>Abstract</p> <p>Background</p> <p>Recently, periostin (<it>POSTN</it>), a gene encoding a protein with similarity to the fasciclin family and involved in cell survival and angiogenesis, has emerged as a promising marker for tumor progression in various types of human cancers. There is some controversy regarding both <it>POSTN </it>expression levels and the nature of periostin-producing cells within tumors. In this study, we used quantitative RT-PCR to assess periostin gene expression in normal tissues, primary cell cultures, tumor tissues and tumor cell lines.</p> <p>Results</p> <p>Periostin expression levels are highly variable in both normal tissues and tumors and strong <it>POSTN </it>overexpression is mostly detected in tumors from pancreas and liver. <it>POSTN </it>is not expressed in blood cancers. In melanoma samples, average periostin expression is not increased in primary tumors whereas <it>POSTN </it>overexpression was detected in about 60% of melanoma metastatic tumors in the liver or lymph nodes. Identification of the cellular source of periostin production in melanoma metastases -cancer cells or stroma- was assessed by comparing periostin expression in 23 newly-established melanoma cell lines and matched tumors. In contrast to the reduction by more than 99% of <it>COL6A3 </it>stromal marker mRNA in all cell lines, significant <it>POSTN </it>transcription was maintained in some melanoma cell lines, suggesting that both stromal cells and melanoma cells express periostin. The high level of periostin expression in primary cultures of skin fibroblasts suggests that fibroblasts may contribute for a large part to periostin production in melanoma-associated stroma. On the other hand, periostin expression in melanoma cells is probably acquired during the tumorigenic process as 1) normal melanocytes do not express <it>POSTN </it>and 2) melanoma cells from distinct metastases of the same patient were associated with very different levels of periostin expression.</p> <p>Conclusion</p> <p>Our comparative analysis suggests that, although periostin overexpression is clearly detected in some cancers, it is not a general feature of tumors. In melanoma, our study identifies both stromal and melanoma cells as sources of periostin production and correlates <it>POSTN </it>expression levels with increased primary tumor thickness and metastatic process development.</p

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Overrunning cellular replicative capacities, through telomere maintenance or oncogene activation, alters epigenetic profiles

Telomeres are specialized structures that cap the ends of eukaryotic chromosomes and, together with the subtelomeres, constitute the heterochromatin of chromosomes ends. In most normal somatic cells, telomere length decreases with cell divisions and this progressive shortening eventually results in cell cycle arrest. In cancer cells, telomere loss is prevented by the activation of a telomere maintenance mechanism (TMM) dependent on either telomerase (TEL+ cells) or alternative mechanism(s) (ALT cells). Although it has been established that ALT cells rely on homologous recombination between telomeric sequences to extend telomeres, mechanisms of ALT activation still require elucidation. Our initial goal was to identify genes involved in ALT pathway(s) and/or genes directly or indirectly regulated by telomerase. To this end, we compared gene expression profiles of TEL+ and ALT SV40-immortalized fibroblasts by representational difference analysis (RDA). This approach did not allow us to identify genes implicated in ALT, suggesting that ALT activation may reside at another level, like post-translational or epigenetic modifications. Recent studies from mice suggested that subtelomeric DNA hypomethylation might contribute to the ALT process by facilitating telomeric sister chromatid exchanges (T-SCE). Hence, we wished to investigate the possible link between epigenetic modifications of chromosome ends and ALT process in human cells. We found that ALT/T-SCEhigh tumor cells display low methylation levels at D4Z4 and DNF92 subtelomeric DNA sequences. Surprisingly however, the same sequences retained high methylation level in ALT/T-SCEhigh SV40-immortalized fibroblasts, suggesting that subtelomeric DNA hypomethylation is not required for T-SCE in human ALT cells. We also found that hypomethylation of subtelomeric sequences in ALT tumor cells was correlated with genome-wide hypomethylation. To understand why genome-wide DNA hypomethylation is prevalent in ALT tumor cells and, in a broader perspective, to investigate the mechanisms of DNA demethylation during the tumorigenic process, we relied on an in vitro model of tumorigenesis. Following replicative senescence or oncogene activation, we detected a two-fold reduction in meCpG content of Sat2 DNA, a pericentromeric region of chromosome 1 displaying strong hypomethylation in tumors and reported to be associated with chromosomal translocations. Our preliminary FISH experiments suggested that hypomethylated Sat2 may indeed be associated with chromosomal translocations in RasV12 oncogeneexpressing fibroblasts. In particular, we detected translocations of Sat2 to chromosome ends, a feature also observed in ALT tumor cells, raising the interesting hypothesis that Sat2 sequences may contribute to ALT telomere maintenance. We propose that this extensive Sat2 DNA demethylation may coincide with an overrunning of replication capacities under low DNMT1 level conditions and/or with delocalization of DNMT1 to DNA damage foci induced by excessive replication. In both cases, DNMT1 maintenance activity at genomewide level would be impaired. This hypothesis remains to be further tested and transposed to the understanding of ALT activation during tumorigenesis.(SBIM 3) -- UCL, 201

Human telomerase represses ROS-dependent cellular responses to Tumor Necrosis Factor-α without affecting NF-κB activation.

In addition to its well-established role in telomere synthesis, telomerase exerts non-canonical functions that may promote cancer and stem cell survival, notably by lowering reactive oxygen species (ROS) levels and acting as transcriptional cofactor in Wnt-β-catenin signaling pathway. We investigated the impact of telomerase on ROS-dependent and -independent cellular responses to Tumor Necrosis Factor-α (TNF-α), a potent inducer of endogenous ROS production and activator of NF-κB signaling pathway. Strikingly, telomerase overexpression in normal human fibroblasts treated with TNF-α strongly repressed ROS-dependent activation of both ERK1/2 mitogen-activated protein kinases and cell death. Telomerase overexpression also considerably diminished TNF-α-induced transcription of SOD2 Superoxide Dismutase 2 gene by reducing ROS contribution to SOD2 gene induction, both in normal fibroblasts and in cancer cells. Conversely, telomerase did not impair TNF-α-induced transcription of various ROS-insensitive NF-κB target genes. These data were in apparent contrast with the striking observation that telomerase overexpression induced strong constitutive nuclear accumulation of NF-κBp65. Accumulated NF-κBp65, however, lacked Ser-536 activating phosphorylation, was not associated with global constitutive NF-κB activation and did not impair subsequent nuclear translocation of phosphorylated NF-κBp65 in response to TNF-α. Our results demonstrate that human telomerase represses ROS-dependent intracellular signaling and gene induction in response to TNF-α

Case Reports in Oncological Medicine Myoepithelioma: A New Rearrangement Involving the Locus in a Case of Multiple Bone and Soft Tissue Lesions.

We report a case of multiple myoepithelioma with synchronous bone and soft tissue tumors, associated with a new genomic alteration of the locus. The lesions occurred in the foot by presenting one lump in the plantar soft tissue, and three lesions were detected in the calcaneus and in the navicular bone. All tumors showed the double immunophenotype of epithelial markers and S100 protein expression. No rearrangement of the and loci was detected as reported in myoepitheliomas. However, molecular karyotyping detected an unbalanced rearrangement of the locus, not involving the locus, which is the most frequent translocation partner observed in benign mesenchymal tumors such as lipomas (of soft tissue as well as parosteal) and pulmonary chondroid hamartoma

Validation of an ELISA-based screening assay for the detection of amphetamine, MDMA and MDA in blood and oral fluid.

The use of amphetamine and 'ecstasy' (MDMA) has increased exponentially in many European countries since the late nineties, leading to a rapid growth in the number of clinical and forensic analyses. Therefore, a rapid screening procedure for these substances in biological specimens has become an important part of routine toxicological analysis in forensic laboratories. The objective of this study was to evaluate the Cozart amphetamine enzyme-linked immunosorbent assay (ELISA) for the screening of plasma samples and oral fluid samples (collected with the Intercept device). Authentic plasma samples from drivers (n=360) were screened, using an 1:5-fold dilution. True positive, true negative, false positive and false negative results were determined relative to the in-house routine GC-MS analysis. Samples consisted of 144 amphetamine-only positives, 141MDMA/MDA-only positives, and 74 negatives when using the limit of quantitation as the cut-off level for confirmation (10 ng/mL). Using these results, receiver operating characteristic (ROC) curves were generated and optimal cut-off values for the screening assay were calculated. Analysis showed that the ELISA is able to predict the presence of either amphetamine or *MDMA/MDA (*MDMA as its metabolite MDA) in plasma samples with 98.3% sensitivity and 100% specificity at a cut-off value of 66.5 ng/mL d-amphetamine equivalents. A similar analysis was conducted on 216 oral fluid specimens collected from a controlled double blind study. Subjects received placebo or a high (100 mg) or low (75 mg) dose of MDMA. Oral fluid samples were collected at 1.5 and 5.5h after administration. Combined results of the analysis of the high and low dose oral fluid samples indicated a screening cut-off of 51 ng/mL d-amphetamine equivalents with both a sensitivity and specificity of 98.6% (using a LC-MS/MS confirmation cut-off of 10 ng/mL). In conclusion, these data indicate that the Cozart AMP EIA plates constitute a fast and accurate screening technique for the identification of amphetamine and MDMA/MDA positive plasma samples and oral fluid specimens (collected with Intercept. It should be emphasized that method validation should be performed for each type of biological matrix

Cancer-linked satellite 2 DNA hypomethylation does not regulate Sat2 non-coding RNA expression and is initiated by heat shock pathway activation.

Epigenetic dysfunctions, including DNA methylation alterations, play major roles in cancer initiation and progression. Although it is well established that gene promoter demethylation activates transcription, it remains unclear whether hypomethylation of repetitive heterochromatin similarly affects expression of non-coding RNA from these loci. Understanding how repetitive non-coding RNAs are transcriptionally regulated is important given that their established upregulation by the heat shock (HS) pathway suggests important functions in cellular response to stress, possibly by promoting heterochromatin reconstruction. We found that, although pericentromeric satellite 2 (Sat2) DNA hypomethylation is detected in a majority of cancer cell lines of various origins, DNA methylation loss does not constitutively hyperactivate Sat2 expression, and also does not facilitate Sat2 transcriptional induction upon heat shock. In melanoma tumor samples, our analysis revealed that the HS response, frequently upregulated in tumors, is probably the main determinant of Sat2 RNA expression in vivo. Next, we tested whether HS pathway hyperactivation may drive Sat2 demethylation. Strikingly, we found that both hyperthermia and hyperactivated RasV12 oncogene, another potent inducer of the HS pathway, reduced Sat2 methylation levels by up to 27% in human fibroblasts recovering from stress. Demethylation occurred locally on Sat2 repeats, resulting in a demethylation signature that was also detected in cancer cell lines with moderate genome-wide hypomethylation. We therefore propose that upregulation of Sat2 transcription in response to HS pathway hyperactivation during tumorigenesis may promote localized demethylation of the locus. This, in turn, may contribute to tumorigenesis, as demethylation of Sat2 was previously reported to favor chromosomal rearrangements

Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells-0

<p><b>Copyright information:</b></p><p>Taken from "Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells"</p><p>http://www.molecular-cancer.com/content/6/1/80</p><p>Molecular Cancer 2007;6():80-80.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222651.</p><p></p>s calculated as [(cDNA /cDNA ) × 10]. A. expression in normal tissues. Error bars represent standard deviations (SD). The table below the graph gives the average POSTN/ACTB × 10± SD values for each sample. B. expression in primary cultures of human embryonic stem cells at day 5 (HUES), fetal lung fibroblasts (IMR90), newborn foreskin fibroblasts (HFF2) and adult skin fibroblasts (LB2924). Error bars represent SD

Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells-6

<p><b>Copyright information:</b></p><p>Taken from "Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells"</p><p>http://www.molecular-cancer.com/content/6/1/80</p><p>Molecular Cancer 2007;6():80-80.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222651.</p><p></p>s calculated as [(cDNA /cDNA ) × 10]. A. expression in normal tissues. Error bars represent standard deviations (SD). The table below the graph gives the average POSTN/ACTB × 10± SD values for each sample. B. expression in primary cultures of human embryonic stem cells at day 5 (HUES), fetal lung fibroblasts (IMR90), newborn foreskin fibroblasts (HFF2) and adult skin fibroblasts (LB2924). Error bars represent SD