Supplementary Tables from A Mouse Skin Multistage Carcinogenesis Model Reflects the Aberrant DNA Methylation Patterns of Human Tumors

Melanin and melanoma tumors are two fields of increasing interest in biomedical research. Melanins are ubiquitous biopigments with adaptive value and multiple functions, and occur in the malignant melanoma. Although several chemical structures have been proposed for eumelanin, molecular modeling and orbitals indicate that a planar or spiral benzoquinone-porphycene polymer would be the model that better explains the broad-band light and ultrasound absorption, electric conductivity, and graphite-like organization shown by X-ray crystallography and electron microscopy. Lysosomes and melanosomes are selectively labeled by vital probes, and melanin also binds to metal cations, colorants, and drugs, with important consequences in pharmacology, pathology, and melanoma therapy. In addition to traditional and recent oncologic treatments, photodynamic, photothermal, and ultrasound protocols represent novel modalities for melanoma therapy. Since eumelanin is practically the ideal photothermal and ultrasound sensitizer, the vibrational decay from photo-excited electrons after NIR irradiation, or the electrochemical production of ROS and radicals after ultrasound absorption, induce an efficient heating or oxidative response, resulting in the damage and death of tumor cells. This allows repetitive treatments due to the remaining melanin contained in tumoral melanophages. Given that evolution and prognosis of the advanced melanoma is still a concern, new biophysical procedures based on melanin properties can now be developed and applied.

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.

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.

This book chapter is drafted for biologists with experimental experiences in ROS biology but being newcomers in the field of modeling. We start with a general introduction about computational modeling in biology and an overview of software tools suitable for beginners. This chapter encompasses an introduction to computational models with special focus on simulation of ROS dynamics. A step-by-step tutorial follows providing guidance for all relevant model development processes. This course of action gives a comprehensible way to understand the benefits of computational models and to gain the necessary knowledge to build own small equation-based models. Small models can be created without any special programming expertise or in-depth technical and mathematical knowledge. Afterward in the final section, a short overview of pitfalls, challenges, and limitations is provided, combined with suggestions for further reading to improve and expand modeling skills of biologists.

The skin is the largest organ of the human body and constitutes the most external and essential defensive barrier against pathogens and physical-chemical damage involving thermal deregulation, dehydration, chemical attack and ultraviolet radiation. To deal with these critical challenges, the skin exhibits a high self-renewal potential driven by dynamic stem cell niches. However, the cutaneous homeostatic and regenerative potential is at present critically impaired by the increasing accumulation of several air pollutants, directly implicated in a plethora of dermatologic conditions, including premature skin aging, altered pigmentation and cancer. In this scenario, innovative strategies are required to tackle the effects of severe air pollution on skin function. Here we present an aqueous extract of the polyextremophile Gramineae native to Antarctica, Deschampsia antarctica (EDA). This organism is able to live under extreme conditions showing highly efficient protection against severe environmental aggression. We have used a human skin organ culture (HSOC) model to investigate the potential of EDA as a protective agent against the action of common and highly toxic pollutants. In particular, we have evaluated the biological effects (changes in morphological parameters and expression/localization of proliferation, cell death, toxic response/inflammation markers) of a prolonged (7 days) exposure to high doses of arsenic, cadmium, chrome and dioxins in the presence or absence of EDA. Our results clearly indicate that EDA exhibits a strong protective function against chemical pollutants in the HSOC model, suggesting that this natural extract might be effectively used in vivo to protect human skin routinely in different daily conditions.

The impact of the interaction of all combined environmental agents to which an individual is exposed during his/her lifetime, as well as how his/her organism responds to these influences, defines health, aging, and disease. The systematic, integrative characterization of the different elements making up the “exposome” is thus necessary to identify and exploit the potential of compounds capable of conferring protection with minimal side effects. Extracts from the natural world, containing synergistic combinations of compounds with antioxidant and protective properties, have long been used in traditional medicine. Modern science has the opportunity to leverage these substances honed by evolution and use them safely and reliably, with a profound mechanistic knowledge and guaranteeing standardization and absence of toxicity. Here, we discuss our current knowledge regarding the potential of a soluble extract of the hair grass Deschampsia antarctica (as its standardized commercial preparation Edafence®) to counteract the skin exposome and its impact on skin aging and disease.

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.

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.

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/β-catenin signaling targets (CCDN1, AXIN2) and with the expression of stem cell markers (CK15, CD34) and growth factors implicated in the telogen/anagen transition (TGFβ2, 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.

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, Gsk3β) 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.

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/β-catenin signalling, while regression and resting phases are mainly regulated by Tgf-β/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-β/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 β-catenin binds to the Eng promoter depending on Bmp signalling. Moreover, we show that β-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/β-catenin and Bmp/Smad signals.

The members of the Src family of nonreceptor tyrosine kinases (SFKs) are implicated in multiple signaling processes that regulate key cellular functions, including proliferation, migration, differentiation, and survival. SFKs are activated by a large number of receptors for growth factors, cytokines, steroid hormones, G protein-coupled receptors, and also by adhesion proteins and other signaling partners. Through their common modular kinase an adapter protein domains, SFKs critically contribute to diversify different signal inputs, weaving a complex and dynamic network of cellular responses. Not surprisingly, SFKs are involved in embryo development and in the maintenance of different adult tissues and organs. Conversely, dysfunction of SFKs is associated with different pathologies, including cancer. Despite the continuous research in the field, several aspects of SFKs regulation and function are still not well defined and new roles for these proteins are steadily reported. The aim of this review is to provide an update on the major regulatory mechanisms of SFKs activity, including the emerging redox-dependent pathway. We have also focused on the functional implications of SFKs in Prolactin signaling and breast cancer development, two increasingly important aspects of SFKs biology. Finally, we briefly revisited the role of SFKs during embryo development and provide insights on the involvement of these proteins in the regulation of embryonic, somatic, and breast cancer stem cells.

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.

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.

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.

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.

Homeostasis, whose regulation at the molecular level is still poorly understood, is intimately related to the functions of epidermal stem cells. Five research groups have been brought together to work on new in vitro and in vivo skin models through the SkinModel-CM program, under the auspices of the Spanish Autonomous Community of Madrid. This project aims to analyze the functions of DNA methyltransferase 1, endoglin, and podoplanin in epidermal stem cell activity, homeostasis, and skin cancer. These new models include 3-dimensional organotypic cultures, immunodeficient skin-humanized mice, and genetically modified mice. Another aim of the program is to use skin-humanized mice to model dermatoses such as Gorlin syndrome and xeroderma pigmentosum in order to optimize new protocols for photodynamic therapy. (C) 2013 Elsevier Espana, S.L.U. and AEDV. All rights reserved.