Histone deacetylase inhibitors induce invasion of human melanoma cells in vitro via differential regulation of N-cadherin expression and RhoA activity.

BACKGROUND: Histone deacetylase inhibitors (HDACi) exert multiple cytotoxic actions on cancer cells. Currently, different synthetic HDACi are in clinical use or clinical trials; nevertheless, since both pro-invasive and anti-invasive activities have been described, there is some controversy about the effect of HDACi on melanoma cells. METHODS: Matrigel and Collagen invasion assays were performed to evaluate the effect of several HDACi (Butyrate, Trichostatin A, Valproic acid and Vorinostat) on two human melanoma cell line invasion (A375 and HT-144). The expression of N- and E-Cadherin and the activity of the RhoA GTPase were analyzed to elucidate the mechanisms involved in the HDACi activity. RESULTS: HDACi showed a pro-invasive effect on melanoma cells in vitro. This effect was accompanied by an up-regulation of N-cadherin expression and an inhibition of RhoA activity. Moreover, the down-regulation of N-cadherin through blocking antibodies or siRNA abrogated the pro-invasive effect of the HDACi and, additionally, the inhibition of the Rho/ROCK pathway led to an increase of melanoma cell invasion similar to that observed with the HDACi treatments. CONCLUSION: These results suggest a role of N-cadherin and RhoA in HDACi induced invasion and call into question the suitability of some HDACi as antitumor agents for melanoma patients.

Anatomical basis for cell transplantation into mouse seminiferous tubules.

Cell transplantation into the seminiferous tubules is a useful technique for the study of physiological and pathological conditions affecting the testis. However, the precise three-dimensional organization and, particularly, the complex connectivity of the seminiferous network have not yet been thoroughly characterized. To date, the technical approaches to address these issues have included manual dissection under the stereomicroscope, reconstruction of histological serial sections, and injection of contrast dyes, but all of them have yielded only partial information. Here, using an approach based on the microinjection of a self-polymerizing resin followed by chemical digestion of the surrounding soft tissues, we reveal fine details of the seminiferous tubule scaffold and its connections. These replicas of the testis seminiferous network were studied by scanning electron microscopy. The present results not only establish a morphological basis for more precise microinjection into the mouse seminiferous tubules but also enable a more profound investigation of physiological and embryological features of the testis.

Plasma membrane and nuclear envelope integrity during the blebbing stage of apoptosis: a time-lapse study.

BACKGROUND INFORMATION: The execution phase of apoptosis is characterized by extensive blebbing of the plasma membrane, which usually results in secondary lysis in vitro. To analyse the permeability of cellular membranes during this process, we induced apoptosis in human melanoma A375 cells that had been transfected with fluorescently tagged proteins which were targeted to different subcellular locations. RESULTS: The dual treatment of resveratrol and butyrate produced a synergistic induction of apoptosis by blocking different phases of the cell cycle. Changes in the plasma membrane, nuclear envelope and nucleoli were monitored by time-lapse confocal microscopy. Fluorescently labelled proteins were not mis-localized from their original locations in any of the cells undergoing blebbing for several hours. Thus the maintenance of karyophilic and nucleolar proteins within the nucleus during the blebbing stage and the accessibility of vital selective chromatin dyes confirmed a functional preservation of the nuclear compartment until the final necrotic blister. The translocation of phosphatidylserine to the outer leaflet of the plasma membrane was not detected during the blebbing period. CONCLUSION: These results show that the functional integrity of the nuclear envelope and plasma membrane may be conserved until the end of the execution phase of apoptosis.

Hypoxia and pluripotency in embryonic and embryonal carcinoma stem cell biology.

Low oxygen availability (hypoxia) is a hallmark of rapidly proliferating tumors and has been suggested to be a characteristic of the embryonic and adult stem cell niche. The idea of relating cancer to stem cells is increasingly popular due to the identification of specific cancer stem cells sharing the typical plasticity and motility of pluripotent stem cells. Hypoxia plays a critical role in early embryonic development and in tumor progression, participating in processes such as angiogenesis, apoptosis, cell migration, invasion and metastasis. Some of the molecular pathways that have been shown to mediate these hypoxia-induced responses, such as the hypoxia inducible factor (HIF)-1alpha and Notch signaling, appear to be active in both embryonic and neoplastic pluripotent stem cells. Nevertheless, the mechanisms underlying these regulatory processes are not yet fully understood. In this review, we attempt to shed some light on the mechanisms involved in hypoxia-dependent processes related to stem cell features and tumor progression, such as the maintenance of the undifferentiated state, cell proliferation, tumor neovascularization, extra-cellular matrix degradation and motility factor up-regulation. With this purpose in mind, we summarize recent observations in embryonic, adult and cancer stem cells that demonstrate the parallelism existing in their hypoxia responses. Finally, based on the observations of our own laboratory and others, we suggest that the comparative analysis of the response to low oxygen levels of embryonic stem cells and cancer stem cells (such as embryonal carcinoma cells), may throw fresh light on our understanding of the mechanisms underlying hypoxia-induced invasiveness and the resistance to anticancer treatments, thereby stimulating the development of novel therapeutic strategies.

Imprinting of mammalian male gametes is gene specific and does not occur at a single stage of differentiation.

Epigenetic modifications such as DNA methylation and alterations to chromatin structure have been proposed as hallmarks of imprinting in somatic cells after fertilization. In the germ cell line, gene imprinting needs to be reset in order to transmit the correct sex-specific imprinting pattern to the next generation. The precise timing of imprint erasure and re-establishment for many genes remains to be determined and precise molecular mechanisms of genomic imprinting have not yet been fully characterized. Here, we have analysed the methylation state and DNase-I sensitivity of two genes with reciprocal genomic imprinting (U2af1-rs1 and H19 genes) in a male mouse primordial germ cell (PGC) derived cell line (EG-1), isolated post-natal spermatogonia and mature sperm cells. Our results show that establishment of imprinting of the U2af1-rs1 and H19 genes during male germ cell differentiation occurs at different stages of differentiation. Furthermore, the presence of DNase-I hypersensitive sites may constitute a molecular marker to identify alleles and subsequently acquire the appropriate methylation imprint. We propose that this molecular identifier may be present or absent for a specific gene according to the sex of the gamete.

Interleukin-2 induces the proliferation of mouse primordial germ cells in vitro.

Primordial germ cells (PGCs) are the stem cell precursors of the germ line. Several growth factors contribute to enlarging the PGC population by acting as mitogens, survival factors or both. Interleukin-2 (IL-2) has a growth-promoting activity for T and B-lymphocytes, but its role in PGCs had not yet been studied. Here, we show that PGCs isolated from 10.5, 11.5 and 12.5 day postcoitum (dpc) mouse embryos constitutively express the three subunits (alpha, beta and gamma) of the IL-2 receptor (IL-2R). In contrast, IL-2 mRNA was not detected in these cells. However, the addition of recombinant IL-2 to the culture medium increased the number of PGCs in vitro via a mitogenic effect, as indicated by bromodeoxyuridine incorporation assays. Neutralization of the IL-2 receptor using anti-IL-2R subunit antibodies inhibited this IL-2-mediated proliferative effect on PGCs from 11.5 dpc embryos. Together, these data are indicative of a paracrine effect of IL-2 on PGC proliferation. In this regard, we also compared the effect of IL-2 with other compounds such as basic fibroblast growth factor (bFGF), steel factor, leukemia inhibitory factor and forskolin, and found that the degree of proliferation induced by IL-2 was similar to that induced by bFGF and forskolin. These observations support the notion that similar patterns of molecular signaling may underlie the developmental pathways of hematopoietic and germ stem cell precursors.

Polychlorinated biphenyls reduce the kinematics contractile properties of embryonic stem cells-derived cardiomyocytes by disrupting their intracellular Ca2+ dynamics.

Persistent organic pollutants are a group of chemicals that include polychlorinated biphenyls (PCBs). PCBs exposure during adult life increases incidence and severity of cardiomyopathies, whereas in utero exposure determines congenital heart defects. Being fat-soluble, PCBs are passed to newborns through maternal milk, impairing heart functionality in the adult. It is still unknown how PCBs impair cardiac contraction at cellular/molecular levels. Here, we study the molecular mechanisms by which PCBs cause the observed heart contraction defects, analysing the alterations of Ca2+ toolkit components that regulate contraction. We investigated the effect that Aroclor 1254 (Aroclor), a mixture of PCBs, has on perinatal-like cardiomyocytes derived from mouse embryonic stem cells. Cardiomyocytes, exposed to 1 or 2 µg/ml Aroclor for 24 h, were analyzed for their kinematics contractile properties and intracellular Ca2+ dynamics. We observed that Aroclor impairs cardiomyocytes contractile properties by inhibiting spontaneous Ca2+ oscillations. It disrupts intracellular Ca2+ homeostasis by reducing the sarcoplasmic reticulum Ca2+ content and by inhibiting voltage-gated Ca2+ entry. These findings contribute to the understanding of the molecular underpinnings of PCBs-induced cardiovascular alterations, which are emerging as an additional life-threatening hurdle associated to PCBs pollution. Therefore, PCBs-dependent alteration of intracellular Ca2+ dynamics is the most likely trigger of developmental cardiac functional alteration.

Nucleoporins redistribute inside the nucleus after cell cycle arrest induced by histone deacetylases inhibition.

Nucleoporins are the main components of the nuclear-pore complex (NPC) and were initially considered as mere structural elements embedded in the nuclear envelope, being responsible for nucleocytoplasmic transport. Nevertheless, several recent scientific reports have revealed that some nucleoporins participate in nuclear processes such as transcription, replication, DNA repair and chromosome segregation. Thus, the interaction of NPCs with chromatin could modulate the distribution of chromosome territories relying on the epigenetic state of DNA. In particular, the nuclear basket proteins Tpr and Nup153, and the FG-nucleoporin Nup98 seem to play key roles in all these novel functions. In this work, histone deacetylase inhibitors (HDACi) were used to induce a hyperacetylated state of chromatin and the behavior of the mentioned nucleoporins was studied. Our results show that, after HDACi treatment, Tpr, Nup153 and Nup98 are translocated from the nuclear pore toward the interior of the cell nucleus, accumulating as intranuclear nucleoporin clusters. These transitory structures are highly dynamic, and are mainly present in the population of cells arrested at the G0/G1 phase of the cell cycle. Our results indicate that the redistribution of these nucleoporins from the nuclear envelope to the nuclear interior may be implicated in the early events of cell cycle initialization, particularly during the G1 phase transition.

Purification and Analysis of Male Germ Cells from Adult Mouse Testis.

Isolation of pools of spermatogenic cells at specific developmental stages is essential for the investigations of molecular events controlling critical transitions during spermatogenesis. Large-scale cell purification techniques allow for combined proteomics, genomics, and transcriptomics studies. Herein, we describe a procedure for the purification of meiotic and post-meiotic male germ cells from adult mouse testes. We also describe how the fractionated cell populations could be used for further studies. In our laboratory, these protocols are routinely used to specifically investigate the molecular basis of histone acetylation/acylation-driven epigenetic programming.

Changes in the expression of cyclin dependent kinase inhibitors during differentiation of immortalized fibroblasts into adipocytes.

The mechanisms implicated in the differentiation of fibroblastic precursors into adipocytes can be analyzed in vitro using cell models, such as the 3T3-L1 cell line. Since cell differentiation involves an exit from the cell cycle, it is likely that molecules that inhibit proliferation participate in the control of adipogenesis. This study was aimed at determining the role, if any, of several cyclin-dependent kinase (CDK)-inhibitors and the transcription factor C/EBPα in the process of adipocyte differentitation. We analyzed by Western blot the expression of distinct cyclin-dependent kinase (CDK)-inhibitors and C/EBPα during various stages of differentiation of 3T3-L1 cells to adipocytes. We observed specific changes in the expression of CDK inhibitors and C/EBPα, during the various phases of adipogenesis. Levels of p15INK4B were maximal in confluent cells prior to the induction of differentiation and minimal in differentiated cells. Maximal levels of p16INK4A were detected following 48 h of differentiation treatment. Highest levels of p18INK4C were measured during the phase of cell confluence prior to treatment and in differentiated cells. p21CIP1 was expressed during the exponential growth phase, during exit from clonal expansion, and in differentiated cells, while p27KIP1 was found above all in confluent and differentiated cells. The present results support the participation of CDK-inhibitors in the process of in vitro adipogenesis. Specifically, the proteins p18INK4C, p21CIP1 and p27KIP1 seem to play an outstanding role in the maintenance of the differentiated state of adipocytes. Understanding the molecular mechanisms involved in adipocyte differentiation will presumably facilitate the design of new drugs aimed at novel therapeutic targets.

Germinal tumor invasion and the role of the testicular stroma.

Testicular germ cell tumors (TGCTs) are the most frequent neoplasia among young people and their incidence has grown very quickly during recent decades in North America and Europe. Many studies have been carried out in order to elucidate the factors involved in the appearance and progression of these tumors. Little is known about the role of cancer cell-stroma crosstalk in TGCT invasive processes. Here, we review several factors which may be implicated in germ cell tumor progression, such as matrix metalloproteinases, insulin-like growth factor, transforming growth factor beta, the cadherin/catenin complex and integrins. Paradoxically, some of these molecules are also involved in the regulation of normal testicular function. Finally, we discuss prospects for future research on the role of the stroma in the progression and differentiation of male germ cell tumors.

Vasculogenesis and angiogenesis in nonseminomatous testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) comprise the vast majority of all testicular malignancies and are the most common type of cancer among young male adults. The nonseminomatous variant of TGCTs is characterized by the presence of embryonic and extraembryonic tissues together with a population of pluripotent cancer stem cells, the so-called embryonal carcinoma. One of the main causes of the resistance of these tumors to therapy is their ability to invade adjacent tissues and metastasize into distant sites of the body. Both of these tumor processes are highly favored by the neovascularization of the malignant tissue. New vessels can be generated by means of angiogenesis or vasculogenesis, and both have been observed to occur during tumor vascularization. Nevertheless, the precise contribution of each process to the neoplastic vascular bed of TGCTs remains unknown. In addition, another process known as tumor-derived vasculogenesis, in which malignant cells give rise to endothelial cells, has also been reported to occur in a number of tumor types, including experimental TGCTs. The participation and cross talk of these 3 processes in tumor vascularization is of particular interest, given the embryonic origin of teratocarcinomas. Thus, in the present review, we discuss the importance of all 3 vascularization processes in the growth, invasion, and metastasis of testicular teratocarcinomas and summarize the current state of knowledge of the TGCT microenvironment and its relationship with vascularization. Finally, we discuss the importance of vascularization as a therapeutic target for this type of malignancy.

Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells.

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca(2+)-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

The spermatogonial stem cell niche in testicular germ cell tumors.

Spermatogonial stem cells (SSCs) are pluripotent elements found in the adult seminiferous epithelium between Sertoli cells and a basal lamina which covers the multilayered external wall of peritubular myoid cells. The microenvironment of this pluripotent stem cell niche creates the complex and dynamic system that is necessary for the initiation of spermatogenesis, but this system also contains factors which can potentially collaborate in the progression of testicular germ cell tumors (TGCTs). In this review, we summarize our current knowledge about some important structural and molecular features related to the SSC niche, including growth factors, adhesion molecules, extracellular matrix, mechanical stress and vascularization. We discuss their possible collaborative effects on the generation and progression of TGCTs, which are a type of cancer representing the most frequent neoplasia among young men and whose incidence has grown very quickly during the past decades in North America and Europe. In this regard, a better understanding of the pluripotent stem cell niche where these malignancies arise will provide further insights into the origin of TGCTs and the mechanisms underlying their growth and invasion of adjacent and distant tissues.

Above the borderland between normal and neoplastic development.

This dialogue between the Editor-in-Chief of the Int. J. Dev. Biol. and a leading figure in human pathology and mammalian embryology highlights the close links between the biological interpretation of neoplasia and differentiation processes which normally occur during development, particularly in the case of teratomas. In addition, it emphasizes how a capacity for work, a firm will to progress, and enthusiasm for science and medical practice can overcome the not insignificant obstacles with which one meets during a life of scientific, academic and clinical dedication.

Embryonic stem cell transplantation into seminiferous tubules: a model for the study of invasive germ cell tumors of the testis.

Over the last 15 years, cell transplantation into seminiferous tubules has become a valuable tool to study germinal cell biology and related matters. This is particularly so, because the blood-testis permeability barrier establishes a sealed compartment which protect against certain influences such as immunological rejection. In the light of the functional and genetic similarities between carcinoma in situ (CIS) of the testis and embryonic stem (ES) cells, our laboratory has developed a tumor assay to study cancer invasion processes in testicular germ cell tumors (TGCT) based on the transplantation of ES cells into the seminiferous tubules. Here, we describe this new tumor assay and provide additional information regarding the transplantation techniques used and their application for the study of TGCTs. Finally, we discuss the practical implications of our experimental approach and its potential application for the understanding of TGCT invasive processes and the development of new antineoplastic strategies.

The role of microenvironment in testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) are the most frequent malignancies in adolescents and young adults. The incidence of TGCTs has doubled over the last few decades and the mechanisms underlying their pervasive growth are still poorly understood. Among them, seminomatous and non-seminomatous tumors have carcinoma in situ of the testis (CIS) as a common precursor lesion. It is currently accepted that the acquisition of genetic alterations and/or exposure to environmental factors are involved in the transition from CIS to invasive tumors. Nevertheless, although several TGCT-associated genetic aberrations have been identified, the mechanisms mediating their effects on TGCT development are still largely unknown. The aim of this review is to analyze the potential role of testicular microenvironmental factors, such as hypoxia and stroma cell-derived factors, in the acquisition by TGCT cells of an aggressive phenotype and the importance of these factors as potential therapeutic targets.

A small great history of the sister Societies of Developmental Biology in Spain and Portugal.

We revise the historical evolution of the societies devoted to Developmental Biology from the early activities of the Institut International dEmbryologie (IIE), founded in 1911, with particular emphasis on the more recent constitution of the Spanish Sociedad Española de Biología del Desarrollo (SEBD), founded in 1994, and the Portuguese Sociedade Portuguesa de Biologia do Desenvolvimento (SPBD), founded in 2006. We also describe the role played by The International Journal of Developmental Biology (IJDB) in the constitution of the SEBD and its projection and support to international Developmental Biology societies and individual researchers in the world, according to its mission to be a non-for-profit publication for scientists, by scientists.

Early bases of modern embryology in Spain: microscopical anatomy and the introduction of cell theory and histology in their scientific and social European context.

We present a survey of the introduction and evolution of microscopy techniques in Spain, and the concepts and lines of research developed around this instrument, particularly in the field of Biomedical research. We cover in our article the long period from the XVII Century to the arrival of the great figure of Santiago Ramon y Cajal (1853-1934). We particularly want to mention many of the previously neglected pioneers who certainly paved the route for his discoveries and, we believe that without them, he would never have arrived to his important position in the annals of Biology and Medicine. The historical, scientific and social framework of that period also helped the approach to important biological concepts such as the cell and tissue, which are previous and essential ideas for a correct understanding of Development.