Regulation of melanin synthesis by the TGF-β family in B16 melanoma cells

Effects of representative members of the transforming growth factor-β (TGF-β) family, TGF-β1, activin A and BMP-2, on melanin content and expression of pigment-producing enzymes were examined in B16 melanoma cells. Treatment with TGF-β1 or activin A but not with BMP-2 significantly decreased melanin content and expression of Tyrosinase and Tyrp-1, suggesting an inhibitory effect of TGF-β1 and activin A on melanin synthesis. TGF-β1 completely inhibited melanin synthesis induced by α-melanin stimulating hormone (α-MSH), whereas activin A only slightly did. As compared with parental B16 cells, the inhibitory effects of TGF-β1 and activin A on melanin content were relative smaller in B16 F10 cells, a subline of B16 cells that contain more pigment. The present study indicates that in addition to TGF-β, activin negatively regulates melanogenesis in the absence of α-MSH, but that the activity in the presence of α-MSH was slightly different between TGF-β and activin.     

Dermis‐derived stem cells: a source of epidermal melanocytes and melanoma?

Human multipotent dermal stem cells (DSCs) have been isolated and propagated from the dermal region of neonatal foreskin. DSCs can self-renew, express the neural crest stem cell markers NGFRp75 and nestin, and are capable of differentiating into a wide variety of cell types including mesenchymal and neuronal lineages and melanocytes, indicative of their neural crest origin. When placed in the context of reconstructed skin, DSCs migrate to the basement membrane zone and differentiate into melanocytes. These findings, combined with the identification of NGFRp75-positive cells in the dermis of human foreskin, which are devoid of hair, suggest that DSCs may be a self-renewing source of extrafollicular epidermal melanocytes. In this review, we discuss the properties of DSCs, the pathways required for melanocyte differentiation, and the value of 3D reconstructed skin to assess the behavior and contribution of DSCs in the naturalized environment of human skin. Potentially, DSCs provide a link to malignant melanoma by being a target of UVA-induced transformation.     

European coding system for tissues and cells: a challenge unmet?

The Comité Européen de Normalisation (European Committee for Standardization, CEN) Workshop on Coding of Information and Traceability of Human Tissues and Cells was established by the Expert Working Group of the Directorate General for Health and Consumer Affairs of the European Commission (DG SANCO) to identify requirements concerning the coding of information and the traceability of human tissues and cells, and propose guidelines and recommendations to permit the implementation of the European Coding system required by the European Tissues and Cells Directive 2004/23/EC (ED). The Workshop included over 70 voluntary participants from tissue, blood and eye banks, national ministries for healthcare, transplant organisations, universities and coding organisations; mainly from Europe with a small number of representatives from professionals in Canada, Australia, USA and Japan. The Workshop commenced in April 2007 and held its final meeting in February 2008. The draft Workshop Agreement went through a public comment phase from 15 December 2007 until 15 January 2008 and the endorsement period ran from 9 April 2008 until 2 May 2008. The endorsed CEN Workshop Agreement (CWA) set out the issues regarding a common coding system, qualitatively assessed what the industry felt was required of a coding system, reviewed coding systems that were put forward as potential European coding systems and established a basic specification for a proposed European coding system for human tissues and cells, based on ISBT 128, and which is compatible with existing systems of donation identification, traceability and nomenclatures, indicating how implementation of that system could be approached. The CWA, and the associated Workshop proposals with recommendations, were finally submitted to the European Commission and to the Committee of Member States that assists its management process under article 29 of the Directive 2004/23/EC on May 25 2008. In 2009 the European Commission initiated an impact assessment on the Workshop proposals and recommendations. In the absence of an agreed pan-European direction various initiatives have continued work using, adopting or adapting their preferred, or existing, methods.     

3′-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides

In this report, we establish that 3-azido-3-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these results may have important implications regarding the potential use of AZT or related glycosylation inhibitors as cancer chemotherapeutics.Abbreviations AZT 3-azido-3-deoxythymidine - Cer ceramide - Crbr cerebroside - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - GD3 disialyl lactosylceramide - GM3 sialyl lactosylceramide - HPTLC high-performance thin layer chromatography - LacCer lactosylceramide - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - PA phosphatidic acid - PBS phosphate-buffered saline - PC phosphatidylcholine - PDMP 1-phenyl-2-decanoylamino-3-morpholino-1-propanol - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SM sphingomyelin     

When do tissues and cells become products? – Regulatory oversight of emerging biological therapies

Although therapeutics derived from biological sources have been subjected to regulatory oversight for some time, the products used in transplantation procedures have historically been exempt from this oversight. These products have been viewed as being part of medical practice rather than as the result of mainstream pharmaceutical manufacture. Furthermore, their unique source makes them difficult to assess in traditional regulatory systems based on the␣tenets of pharmaceutical quality control. With the␣increasing use of transplantation therapies to both replace dysfunctional organs and to influence genetic and metabolic processes, public health concerns on these therapies have increased. In addition, it is recognized that therapeutic claims for some of these interventions need to be properly assessed. These considerations have led the established regulatory agencies of the developed world to develop new regulatory paradigms for the products of transplantation practice. While a number of concerns have driven these developments, the minimization of infectious disease risk remains the paramount driver for introducing these regulatory systems. More than the regulation of medicines and medical devices manufactured in traditional pharmaceutical modes, the regulation of cell and tissue products is intimately linked to areas of public health policy and funding. This places regulators in a challenging position as they attempt to reconcile their roles as independent assessors with the needs of the overall public health framework. This is particularly difficult when considering measures which may affect access to life saving therapies. Regulators have recognized the need to assess these therapies through systems which incorporate consideration of risk-benefit ratios and include mechanisms for transparent and accountable release of products when full compliance to traditional concepts of manufacturing practice is not possible.     

Real-time PCR: what relevance to plant studies?

The appearance of genetically modified organisms on the food market a few years ago, and the demand for more precise and reliable techniques to detect foreign (transgenic or pathogenic) DNA in edible plants, have been the driving force for the introduction of real-time PCR techniques in plant research. This was followed by numerous fundamental research applications aiming to study the expression profiles of endogenous genes and multigene families. Since then, the interest in this technique in the plant scientist community has increased exponentially. This review describes the technical features of quantitative real-time PCR that are especially relevant to plant research, and summarizes its present and future applications.     

Dilation of the endoplasmic reticulum in beta cells due to molecular overcrowding?: Kinetic simulations of extension limits and consequences on proinsulin synthesis

Insulin regulates the energy homeostasis of the human body. This is synthesized in the endoplasmic reticulum (ER) of pancreatic beta cells from proinsulin. Chronic hyperglycemia increases considerably the proinsulin secretion, overcrowding the ER. Recent experimental evidence demonstrates that such states favor the proinsulin denaturation. The biophysical mechanism of this cellular dysfunction remains largely unknown. We use basic molecular principles and numerical simulations of time-dependent crowding conditions in the ER to show that crowding effects enhance the propensity of proinsulin molecules to (mis)fold in compressed, nonnative structures. Present results suggest: i) misfolding events and toxic accumulations increase dramatically if the proinsulin load exceeds 50% of the available space and ii) insufficient lag time for the relaxation of the ER between consecutive proinsulin uploads can cause irreversible alterations of folding capabilities. Present study may prove useful in generating new testable statements on circumstances leading to the development of diabetes.     

Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17beta given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [(14)C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17beta has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [(14)C]lysine into stromal nuclear proteins, but changes after oestradiol-17beta treatment were similar to those seen in epithelium with oestradiol-17beta alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17beta. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17beta alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-(3)H]oestradiol-17beta by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.     

Evaluation of 3,4-dihydroquinazoline-2(1H)-thiones as inhibitors of α-MSH-induced melanin production in melanoma B16 cells

Novel 3,4-dihydroquinazoline-2(1H)-thiones (QNTs) 1 were found to be potent inhibitors of α-MSH-induced melanin production. The effect of QNTs to inhibit melanin formation in B16 melanoma cells was screened in the presence of α-MSH. In defining the mechanism of activity, the effects on tyrosinase activity, on tyrosinase synthesis and on the depigmentation of melanin were evaluated. QNTs did not affect the catalytic activity of tyrosinase, but rather acted as an inhibitor of tyrosinase synthesis.     

Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?

Mesenchymal stromal cells(MSCs) are currently being investigated for use in a wide variety of clinical applications. For most of these applications, systemic delivery of the cells is preferred. However, this requires the homing and migration of MSCs to a target tissue. Although MSC hominghas been described, this process does not appear to be highly efficacious because only a few cells reach the target tissue and remain there after systemic administration. This has been ascribed to low expression levels of homing molecules, the loss of expression of such molecules during expansion, and the heterogeneity of MSCs in cultures and MSC culture protocols. To overcome these limitations, different methods to improve the homing capacity of MSCs have been examined. Here, we review the current understanding of MSC homing, with a particular focus on homing to bone marrow. In addition, we summarize the strategies that have been developed to improve this process. A better understanding of MSC biology, MSC migration and homing mechanisms will allow us to prepare MSCs with optimal homing capacities. The efficacy of therapeutic applications is dependent on efficient delivery of the cells and can, therefore, only benefit from better insights into the homing mechanisms.     

Evaluation of plasma and tissue estrogen suppression with third-generation aromatase inhibitors: Of relevance to clinical understanding?

Development of aromatase inhibition and aromatase inhibitors as a therapeutic strategy was initiated through two different pathways. The one pathway went through systematic exploration of aromatase substrate analogues for enzyme inhibitions, subsequently leading to the development of steroidal agents for clinical use. The second involved clinical observation with an unsuccessful anti-epileptic compound named aminoglutethimide, attempting to achieve a “medical adrenalectomy”. Endocrine studies on patients treated with aminoglutethimide lead to direct assessment of in vivo aromatase inhibition in patients on treatment, thus identifying a novel therapeutic strategy. As such, both research programs represent different examples of pioneering translational work leading towards a successful therapeutic strategy. Subsequent studies with respect to total aromatase inhibition have led to successful development of more potent strategies. Most importantly, these studies have revealed a correlation between aromatase inhibition and clinical outcome. Ongoing studies exploring tissue estrogen levels as well as gene expression profiles on therapy may further improve this important therapeutic area.     

Metabolic depression: a response of cancer cells to hypoxia?

Hypoxic tumours have the worst prognosis because they are the most aggressive and the most likely to metastasize. This may be because these aggressive cancers have a hypoxic core which generates signals that activate angiogenesis which enables the supply of nutrients and oxygen to a rapidly growing outer oxidative shell. The hypoxic core is a crucial element of this hypothesis, as is the fact that the cells in the hypoxic core are inherently adapted to survive hypoxia. We reasoned therefore that cancer cells exposed to hypoxia/anoxia should show the hallmarks of adaptation to hypoxia/anoxia, i.e. a down-regulation of protein synthesis and a reverse Pasteur effect. We tested this hypothesis in transformed (MCF-7) and normal (HME) human mammary epithelial cells, by exposing both cell types to a range of oxygen concentrations, including anoxia. We find that indeed protein synthesis is down-regulated in the MCF-7, but not in the HME cells in response to anoxia. The data on glycolysis are not as clear-cut, but in the light of similar previous measurements on hypoxia-tolerant animals, is still consistent with the hypothesis.     

Expression of NK-associated receptors on cytotoxic T cells from melanoma patients: a two-edged sword?

The coexistence of tumor progression with a tumor-specific immune response constitutes a major paradox of tumor immunity. During the last decade, the presence of cytotoxic T lymphocytes (CTLs) recognising melanoma-associated antigens has been unequivocally demonstrated in numerous different in vivo and in vitro models. However, most often these melanoma-specific T lymphocytes do not control tumor growth. Several mechanisms that involve changes in melanoma phenotype and/or in T-cell differentiation and function could explain the inability of the immune response to control melanoma. In the last few years it has been demonstrated that cellular cytotoxicity is the result of a balance between activating signals triggered by the TCR and costimulatory molecules and inhibitory signals triggered by inhibitory receptors expressed by the CTL. Because the final outcome of the immune response against melanoma depends on the balance between activating and inhibitory signals, the expression de novo on melanoma cells of ligands for inhibitory NKRs and the down-regulation of costimulatory molecules may favor the escape of tumor cells from immunosurveillance. In this paper we review how altered expression of molecules required for T-cell costimulation could result in impaired lysis of melanoma. The modulation of antimelanoma T-cell responses by a group of receptors originally described on NK cells (NK-associated receptors) but which are now known also to be expressed on a subset of cytolytic effector cells is reviewed. We hypothesize that the expression of ligands for NKRs on melanoma cells may contribute to T-cell-mediated immune responses against melanoma either enhancing or inhibiting activation and differentiation to effector cells. Blocking inhibitory receptors or increasing activating receptors could result in new strategies to improve T-cell-mediated rejection of melanoma.     

Rather rule than exception? How to evaluate the relevance of dual protein targeting to mitochondria and chloroplasts

Dual targeting of a nuclearly encoded protein into two different cell organelles is an exceptional event in eukaryotic cells. Yet, the frequency of such dual targeting is remarkably high in case of mitochondria and chloroplasts, the two endosymbiotic organelles of plant cells. In most instances, it is mediated by “ambiguous” transit peptides, which recognize both organelles as the target. A number of different approaches including in silico, in organello as well as both transient and stable in vivo assays are established to determine the targeting specificity of such transit peptides. In this review, we will describe and compare these approaches and discuss the potential role of this unusual targeting process. Furthermore, we will present a hypothetical scenario how dual targeting might have arisen during evolution.