Epigenetic inactivation of the p53-induced long noncoding RNA TP53 target 1 in human cancer.

Long noncoding RNAs (lncRNAs) are important regulators of cellular homeostasis. However, their contribution to the cancer phenotype still needs to be established. Herein, we have identified a p53-induced lncRNA, TP53TG1, that undergoes cancer-specific promoter hypermethylation-associated silencing. In vitro and in vivo assays identify a tumor-suppressor activity for TP53TG1 and a role in the p53 response to DNA damage. Importantly, we show that TP53TG1 binds to the multifaceted DNA/RNA binding protein YBX1 to prevent its nuclear localization and thus the YBX1-mediated activation of oncogenes. TP53TG1 epigenetic inactivation in cancer cells releases the transcriptional repression of YBX1-targeted growth-promoting genes and creates a chemoresistant tumor. TP53TG1 hypermethylation in primary tumors is shown to be associated with poor outcome. The epigenetic loss of TP53TG1 therefore represents an altered event in an lncRNA that is linked to classical tumoral pathways, such as p53 signaling, but is also connected to regulatory networks of the cancer cell.

Sustained Human Hair Follicle Growth Ex Vivo in a Glycosaminoglycan Hydrogel Matrix.

Glycosaminoglycans (GAGs) and associated proteoglycans have important functions in homeostatic maintenance and regenerative processes (e.g., wound repair) of the skin. However, little is known about the role of these molecules in the regulation of the hair follicle cycle. Here we report that growing human hair follicles ex vivo in a defined GAG hydrogel mimicking the dermal matrix strongly promotes sustained cell survival and maintenance of a highly proliferative phenotype in the hair bulb and suprabulbar regions. This significant effect is associated with the activation of WNT/ß-catenin signaling targets (CCDN1, AXIN2) and with the expression of stem cell markers (CK15, CD34) and growth factors implicated in the telogen/anagen transition (TGFß2, FGF10). As a whole, these results point to the dermal GAG matrix as an important component in the regulation of the human hair follicle growth cycle, and to GAG-based hydrogels as potentially relevant modulators of this process both in vitro and in vivo.

Switching on a transient endogenous ROS production in mammalian cells and tissues.

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide. We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems.

In situ production of ROS in the skin by photodynamic therapy as a powerful tool in clinical dermatology.

Photodynamic therapy (PDT) is a clinical modality of photochemotherapy based on the accumulation of a photosensitizer in target cells and subsequent irradiation of the tissue with light of adequate wavelength promoting reactive oxygen species (ROS) formation and cell death. PDT is used in several medical specialties as an organ-specific therapy for different entities. In this review we focus on the current dermatological procedure of PDT. In the most widely used PDT protocol in dermatology, ROS production occurs by accumulation of the endogenous photosensitizer protoporphyrin IX after treatment with the metabolic precursors 5-methylaminolevulinic acid (MAL) or 5-aminolevulinic acid (ALA). To date, current approved dermatological indications of PDT include actinic keratoses (AK), basal cell carcinoma (BCC) and in situ squamous cell carcinoma (SCC) also known as Bowen disease (BD). With regards to AKs, PDT can also treat the cancerization field carrying an oncogenic risk. In addition, an increasing number of pathologies, such as other skin cancers, infectious, inflammatory or pilosebaceous diseases are being considered as potentially treatable entities with PDT. Besides the known therapeutic properties of PDT, there is a modality used for skin rejuvenation and aesthetic purposes defined as photodynamic photorejuvenation. This technique enables the remodelling of collagen, which in turn prevents and treats photoaging stygmata. Finally we explore a new potential treatment field for PDT determined by the activation of follicular bulge stem cells caused by in situ ROS formation.

Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer.

CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.

Regulation of SNAIL1 and E-cadherin function by DNMT1 in a DNA methylation-independent context.

Mammalian DNA methyltransferase 1 (DNMT1) is essential for maintaining DNA methylation patterns after cell division. Disruption of DNMT1 catalytic activity results in whole genome cytosine demethylation of CpG dinucleotides, promoting severe dysfunctions in somatic cells and during embryonic development. While these observations indicate that DNMT1-dependent DNA methylation is required for proper cell function, the possibility that DNMT1 has a role independent of its catalytic activity is a matter of controversy. Here, we provide evidence that DNMT1 can support cell functions that do not require the C-terminal catalytic domain. We report that PCNA and DMAP1 domains in the N-terminal region of DNMT1 are sufficient to modulate E-cadherin expression in the absence of noticeable changes in DNA methylation patterns in the gene promoters involved. Changes in E-cadherin expression are directly associated with regulation of ß-catenin-dependent transcription. Present evidence suggests that the DNMT1 acts on E-cadherin expression through its direct interaction with the E-cadherin transcriptional repressor SNAIL1.

DNA methylation and the functional organization of the nuclear compartment.

Symmetrical methylation of cytosine residues at CpG dinucleotides of the DNA molecule is a central epigenetic and heritable hallmark of the genome. This epigenetic modification of DNA is directly associated with a closed molecular conformation of the chromatin fibre which is, in turn, intrinsically linked to an inactive transcriptional status. Thus, DNA methylation is a major determinant of the functional outcome of the nucleus. Equally important, DNA methylation is also involved in the large-scale maintenance of the nuclear architecture, which is required for proper nuclear function. Densely DNA methylated regions tend to occupy large and discrete regions of the genome and can act as referential structural blocks for building up the whole functional organization of the nucleus. In this context, interpreting the three-dimensional pattern of DNA methylation is crucial to our understanding of the dynamic biology of genomes.

A Photodynamic Tool to Promote a Sustained, ROS-Dependent Growth of Human Hair Follicles in Ex Vivo Culture.

Reactive oxygen species (ROS) may severely affect the biochemical viability of most cells. However, ROS may act also as key second messengers regulating important physiological functions in eukaryotic organisms. Of special interest is the potential role of ROS in the regulation of stem cell function and tissue homeostasis and regeneration in adult mammalian tissues. In this context, the hair follicle constitutes an excellent experimental model to study this aspect of ROS biology.Here we present a robust protocol to promote a sustained growth of ex vivo cultured human hair follicles based on the induction of a transient/modulable production of nonlethal endogenous ROS levels in the tissue through a protoporphyrin IX-dependent photodynamic procedure. The light-switchable ROS production activates hair follicle stem cell niches, induces cell proliferation, and maintains the growth/anagen phase for long time. This approach constitutes a complementary experimental tool to study the physiological roles of ROS in human tissues.

The deleterious effects induced by an acute exposure of human skin to common air pollutants are prevented by extracts of Deschampsia antarctica.

The homeostatic and regenerative potential of the skin is critically impaired by an increasing accumulation of air pollutants in human ecosystems. These toxic compounds are frequently implicated in pathological processes such as premature cutaneous ageing, altered pigmentation and cancer. In this scenario, innovative strategies are required to tackle the effects of severe air pollution on skin function. Here we have used a Human Skin Organotypic Culture (HSOC) model to characterize the deleterious effects of an acute topic exposure of human skin to moderately high concentrations of common ambient pollutants, including As, Cd, Cr, dioxins and tobacco smoke. All these toxic compunds inflict severe damage in the tissue, activating the AHR-mediated response to xenobiotics. We have further evaluated the potential of an aqueous leaf extract of the polyextremophile plant Deschampsia antarctica (Edafence) to protect human skin against the acute exposure to toxic pollutants. Our results indicate that pre-treatment of HSOC samples with this aqueous extract conuterbalances the deleterious effects of the exposure to toxic comunds and triggers the activation of key genes invoved in the redox system and in the pro-inflammatory/wound healing response in the skin, suggesting that this natural compound might be effectively used in vivo to protect human skin routinely in different daily conditions.

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate.

Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 µg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.

Oncogenic H-Ras and PI3K signaling can inhibit E-cadherin-dependent apoptosis and promote cell survival after photodynamic therapy in mouse keratinocytes.

Maintenance of E-cadherin mediated cell-cell contacts is often required for the survival of epithelial cells and tissues. Here we report that oncogenic activation of H-Ras in murine keratinocytes can prevent cell death induced by immunological disruption of E-cadherin adhesion. A similar situation was observed in cells showing constitutive activation of the p110 alpha catalytic subunit of class IA PI3K. This protective effect is associated with beta-catenin-dependent transcription and with activation of survival factor Akt/PKB. In addition, we induced cell death by employing photodynamic therapy, using Zn-phthalocyanine as a photosensitizer that targets E-cadherin adhesion complexes. We have found that cell death based on this photodynamic action is also bypassed in cells showing constitutive activation of H-Ras and p110 alpha. Taken together, these results indicate that H-Ras/PI3K/Akt signaling plays a key role in cell survival mediated by E-cadherin cell-cell contacts.

Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmt1) deficient cells.

The nucleolus is the site of ribosome synthesis in the nucleus, whose integrity is essential. Epigenetic mechanisms are thought to regulate the activity of the ribosomal RNA (rRNA) gene copies, which are part of the nucleolus. Here we show that human cells lacking DNA methyltransferase 1 (Dnmt1), but not Dnmt33b, have a loss of DNA methylation and an increase in the acetylation level of lysine 16 histone H4 at the rRNA genes. Interestingly, we observed that SirT1, a NAD+-dependent histone deacetylase with a preference for lysine 16 H4, interacts with Dnmt1; and SirT1 recruitment to the rRNA genes is abrogated in Dnmt1 knockout cells. The DNA methylation and chromatin changes at ribosomal DNA observed are associated with a structurally disorganized nucleolus, which is fragmented into small nuclear masses. Prominent nucleolar proteins, such as Fibrillarin and Ki-67, and the rRNA genes are scattered throughout the nucleus in Dnmt1 deficient cells. These findings suggest a role for Dnmt1 as an epigenetic caretaker for the maintenance of nucleolar structure.

Hedgehog signalling as a target in cancer stem cells.

Hedgehog (Hh) is one of the most important signalling pathways. Together with the Wnt, TGF-Beta/BMP and Notch pathways, it is involved in both embryonic development and adult tissue homeostasis. This is because Hh plays a central role in the proliferative control and differentiation of both embryonic stem cells and adult stem cells. In this way, an alteration in the Hh pathway, either by misexpression of components of that pathway or by changes in the expression of other cellular components that interfere with the Hh signalling system, may trigger the development of several types of cancer. This occurs because normal stem cells or their intermediaries toward differentiated mature cells are not part of the normal proliferative/ differentiation balance and begin to expand without control, triggering the generation of the so-called cancer stem cells. In this review, we will focus on the molecular aspects and the role of Hh signalling in normal tissues and in tumour development.

Publisher Correction: Intrinsic activation of cell growth and differentiation in ex vivo cultured human hair follicles by a transient endogenous production of ROS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intrinsic activation of cell growth and differentiation in ex vivo cultured human hair follicles by a transient endogenous production of ROS.

The emerging variety of signalling roles for ROS in eukaryotic cells and tissues is currently a matter of intense research. Here we make use of ex vivo cultured single human hair follicles as an experimental model to demonstrate that a transient production of non-lethal endogenous ROS levels in these mini-organs promotes efficiently the entry into the growth phase (anagen). The stimulatory process implicates the specific activation of the hair follicle stem cell niche, encompassing the induction of stem cell differentiation markers (Ck15), overall cell proliferation and sustained growth of the tissue associated with expression of gen targets (Ccnd1) concomitant with the inhibition of Wnt signaling antagonists and repressors (Dkk1, Gsk3ß) of Wnt signaling. As a whole, this observation indicates that, once activated, ROS signalling is an intrinsic mechanism regulating the hair follicle stem cell niche independently of any external signal.

A role for the Tgf-ß/Bmp co-receptor Endoglin in the molecular oscillator that regulates the hair follicle cycle.

The hair follicle is a biological oscillator that alternates growth, regression, and rest phases driven by the sequential activation of the proliferation/differentiation programs of resident stem cell populations. The activation of hair follicle stem cell niches and subsequent entry into the growing phase is mainly regulated by Wnt/ß-catenin signalling, while regression and resting phases are mainly regulated by Tgf-ß/Bmp/Smad activity. A major question still unresolved is the nature of the molecular switch that dictates the coordinated transition between both signalling pathways. Here we have focused on the role of Endoglin (Eng), a key co-receptor for members of the Tgf-ß/Bmp family of growth factors. Using an Eng haploinsufficient mouse model, we report that Eng is required to maintain a correct follicle cycling pattern and for an adequate stimulation of hair follicle stem cell niches. We further report that ß-catenin binds to the Eng promoter depending on Bmp signalling. Moreover, we show that ß-catenin interacts with Smad4 in a Bmp/Eng-dependent context and both proteins act synergistically to activate Eng promoter transcription. These observations point to the existence of a growth/rest switching mechanism in the hair follicle that is based on an Eng-dependent feedback cross-talk between Wnt/ß-catenin and Bmp/Smad signals.

Inactivation of the lamin A/C gene by CpG island promoter hypermethylation in hematologic malignancies, and its association with poor survival in nodal diffuse large B-cell lymphoma.

PURPOSE: Lamins support the nuclear envelope and provide anchorage sites for chromatin, but they are also involved in DNA synthesis, transcription, and apoptosis. Although the lack of expression of A-type lamins in lymphoma and leukemia has been reported, the mechanism was unknown. We investigated the possible role of CpG island hypermethylation in lamin A/C silencing and its prognostic relevance. PATIENTS AND METHODS: The promoter CpG island methylation status of the lamin A/C gene, encoding the A-type lamins, was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction in human cancer cell lines (n = 74; from 17 tumor types), and primary leukemias (n = 60) and lymphomas (n = 80). Lamin A/C expression was determined by reverse-transcription polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence. RESULTS: seven (50%) of 14 leukemia- and five (42%) of 13 lymphoma cell lines. The presence of hypermethylation was associated with the loss of gene expression while a demethylating agent restored expression. In primary malignancies, lamin A/C hypermethylation was present in 18% (nine of 50) of acute lymphoblastic leukemias and 34% (14 of 41) of nodal diffuse large B-cell lymphomas. The presence of lamin A/C hypermethylation in nodal diffuse large B-cell lymphomas correlated strongly with a decrease in failure-free survival (Kaplan-Meier, P = .0001) and overall survival (Kaplan-Meier, P = .0005). CONCLUSION: Epigenetic silencing of the lamin A/C gene by CpG island promoter hypermethylation is responsible for the loss of expression of A-type lamins in leukemias and lymphomas. The finding that lamin A/C hypermethylation is associated with poor outcome in diffuse large B-cell lymphomas suggests important clinical implications.