Alternative Lengthening of Telomeres is characterized by reduced compaction of telomeric chromatin.

Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric RNA species. These results offer new insight on telomeric chromatin properties in ALT cells and support the hypothesis that telomeric chromatin decondensation is important for ALT pathway.

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-ß-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.

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

BACKGROUND: Recently, periostin (POSTN), 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 POSTN 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. RESULTS: Periostin expression levels are highly variable in both normal tissues and tumors and strong POSTN overexpression is mostly detected in tumors from pancreas and liver. POSTN is not expressed in blood cancers. In melanoma samples, average periostin expression is not increased in primary tumors whereas POSTN 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 COL6A3 stromal marker mRNA in all cell lines, significant POSTN 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 POSTN and 2) melanoma cells from distinct metastases of the same patient were associated with very different levels of periostin expression. CONCLUSION: 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 POSTN expression levels with increased primary tumor thickness and metastatic process development.

HIPK2 inhibits both MDM2 gene and protein by, respectively, p53-dependent and independent regulations.

We address here the involvement of the homeodomain-interacting protein kinase 2 (HIPK2)/p53 complex on MDM2 regulation following apoptotic DNA damage. Our results provide a plausible transcriptional (p53-dependent) and posttranscriptional (p53-independent) double mechanism by which HIPK2 accomplishes MDM2 downmodulation. First, in wtp53-carrying cells HIPK2-dependent p53Ser46 phosphorylation selectively inhibits MDM2 at transcriptional level. Secondly, HIPK2 interacts with MDM2 in vitro and in vivo and promotes MDM2 nuclear export and proteasomal degradation, in p53-null cellular context. This p53-independent effect is likely mediated by HIPK2 catalytic activity and we found that HIPK2 phosphorylates MDM2 in vitro. In response to DNA damage, depletion of HIPK2 by RNA-interference abolishes MDM2 protein degradation. We propose that HIPK2 contributes to drug-induced modulation of MDM2 activity at transcriptional (through p53Ser46 phosphorylation) and posttranscriptional (through p53-independent subcellular re-localization and proteasomal degradation) levels.