Molecular and cellular pathogenesis of melanoma initiation and progression

Melanoma is a malignant tumor of melanocytes that can spread to other organs of the body, resulting in severe and/or lethal malignancies. Melanocytes are pigment-producing cells found in the deep layer of the epidermis and are originated from melanocytes stem cells through a cellular process called melanogenesis. Several genes and epigenetic and micro-environmental factors are involved in this process via the regulation and maintenance of the balance between melanocytes stem cells proliferation and their differentiation into melanocytes. Dysregulation of this balance through gain or loss of function of key genes implicated in the control and regulation of cell cycle progression and/or differentiation results in melanoma initiation and progression. This review aims to provide a comprehensive overview about the origin of melanocytes, the oncogenic events involved in melanocytes stem cells transformation, and the mechanisms implicated in the perpetuation of melanoma malignant phenotype.     

Tumor suppressive pathways in the control of neurogenesis

The generation of specialized neural cells in the developing and postnatal central nervous system is a highly regulated process, whereby neural stem cells divide to generate committed neuronal progenitors, which then withdraw from the cell cycle and start to differentiate. Cell cycle checkpoints play a major role in regulating the balance between neural stem cell expansion and differentiation. Loss of tumor suppressors involved in checkpoint control can lead to dramatic alterations of neurogenesis, thus contributing to neoplastic transformation. Here we summarize and critically discuss the existing literature on the role of tumor suppressive pathways and their regulatory networks in the control of neurogenesis and transformation.     

Glial versus melanocyte cell fate choice: Schwann cell precursors as a cellular origin of melanocytes

Melanocytes and Schwann cells are derived from the multipotent population of neural crest cells. Although both cell types were thought to be generated through completely distinct pathways and molecular processes, a recent study has revealed that these different cell types are intimately interconnected far beyond previously postulated limits in that they share a common post-neural crest progenitor, i.e. the Schwann cell precursor. This finding raises interesting questions about the lineage relationships of hitherto unrelated cell types such as melanocytes and Schwann cells, and may provide clinical insights into mechanisms of pigmentation disorders and for cancer involving Schwann cells and melanocytes.     

DNG1, a Dictyostelium homologue of tumor suppressor ING1 regulates differentiation of Dictyostelium cells

dng1 is a Dictyostelium homologue of the mammalian tumor suppressor ING gene. DNG1 protein localizes in the nucleus, and has a highly conserved PHD finger domain found in chromatin-remodeling proteins. Both dng1 disruption and overexpression impaired cell proliferation. In dng1-null cells, the progression of differentiation was delayed in a cell-density-dependent manner, and many tiny aggregates were formed. Exogenously applied cAMP pulses reversed the inhibitory effect caused by dng1 disruption on the aggregation during early development, but formation of tiny aggregates was not restored. dng1-overexpressing cells acquired the ability to undergo chemotaxis to cAMP earlier and exhibited enhanced differentiation. These phenotypes were found to be coupled with altered expressions of early genes such as cAMP receptor 1 (car1) and contact site A (csA). Furthermore, disordered histone modifications were demonstrated in dng1-null cells. These results suggest a regulatory role of dng1 in the transition of cells from growth to differentiation.Received 29 December 2004; received after revision 24 May 2005; accepted 26 May 2005     

MicroRNA miR-196a is a central regulator of HOX-B7 and BMP4 expression in malignant melanoma

Since bone morphogenetic proteins (BMPs) play an important role in melanoma progression, we aimed to determine the molecular mechanisms leading to overexpression of BMP4 in melanoma cells compared to normal melanocytes. With our experimental approach we revealed that loss of expression of a microRNA represents the starting point for a signaling cascade finally resulting in overexpression of BMP4 in melanoma cells. In detail, strongly reduced expression of the microRNA miR-196a in melanoma cells compared to healthy melanocytes leads to enhanced HOX-B7 mRNA and protein levels, which subsequently raise Ets-1 activity by inducing basic fibroblast growth factor (bFGF). Ets-1 finally accounts for induction of BMP4 expression. We were furthermore able to demonstrate that bFGF-mediated induction of migration is achieved via activation of BMP4, thus determining BMP4 as major modulator of migration in melanoma. In summary, our study provides insights into the early steps of melanoma progression and might thereby harbor therapeutic relevance.     

Protein-O-mannosyltransferases in virulence and development

Protein-O-mannosyltransferases (Pmt proteins) catalyse the addition of mannose to serine or threonine residues of secretory proteins. This modification was described first for yeast and later for other fungi, mammals, insects and recently also for bacteria. O-mannosylation depends on specific isoforms of the three Pmt1, 2 and 4 subfamilies. In fungi, O-mannosylation determines the structure and integrity of cell walls, as well as cellular differentiation and virulence. O-mannosylation of specific secretory proteins of the human fungal pathogen Candida albicans and of the bacterial pathogen Mycobacterium tuberculosis contributes significantly to virulence. In mammals and insects, Pmt proteins are essential for cellular differentiation and development, while lack of Pmt activity causes Walker-Warburg syndrome (muscular dystrophy) in humans. The susceptibility of human cells to certain viruses may also depend on O-mannosyl chains. This review focuses on the various roles of Pmt proteins in cellular differentiation, development and virulence. Received 6 September 2007; received after revision 3 October 2007; accepted 5 October 2007     

Ras proteins in the control of the cell cycle and cell differentiation

The Ras family of small GTPases includes three closely related proteins: H-, K-, and N-Ras. Ras proteins are involved in the transduction of signals elicited by activated surface receptors, acting as key components by relaying signals downstream through diverse pathways. Mutant, constitutively activated forms of Ras proteins are frequently found in cancer. While constitutive Ras activation induces oncogenic-like transformation in immortalized fibroblasts, it causes growth arrest in primary vertebrate cells. Induction of p53 and cyclin-dependent kinase inhibitors such as p15^INK4b, p16^INK4a, p19^ARF, and p21^WAF1 accounts for this response. Interestingly, while ras has usually been regarded as a transforming oncogene, the analysis of Ras function in most of the cellular systems studied so far indicates that the promotion of differentiation is the most prominent effect of Ras. While in some cell types, particularly muscle, Ras inhibits differentiation, in others such as neuronal, adipocytic, or myeloid cells, Ras induces differentiation, in some cases accompanied by growth arrest. Several possible mechanisms for the pleiotropic effects of Ras in animal cells are discussed. Received 8 March 2000; received after revision 24 May 2000; accepted 24 May 2000     

The positional identity of mouse ES cell-generated neurons is affected by BMP signaling

We investigated the effects of bone morphogenetic proteins (BMPs) in determining the positional identity of neurons generated in vitro from mouse embryonic stem cells (ESCs), an aspect that has been neglected thus far. Classical embryological studies in lower vertebrates indicate that BMPs inhibit the default fate of pluripotent embryonic cells, which is both neural and anterior. Moreover, mammalian ESCs generate neurons more efficiently when cultured in a minimal medium containing BMP inhibitors. In this paper, we show that mouse ESCs produce, secrete, and respond to BMPs during in vitro neural differentiation. After neuralization in a minimal medium, differentiated ESCs show a gene expression profile consistent with a midbrain identity, as evaluated by the analysis of a number of markers of anterior–posterior and dorsoventral identity. We found that BMPs endogenously produced during neural differentiation mainly act by inhibiting the expression of a telencephalic gene profile, which was revealed by the treatment with Noggin or with other BMP inhibitors. To better characterize the effect of BMPs on positional fate, we compared the global gene expression profiles of differentiated ESCs with those of embryonic forebrain, midbrain, and hindbrain. Both Noggin and retinoic acid (RA) support neuronal differentiation of ESCs, but they show different effects on their positional identity: whereas RA supports the typical gene expression profile of hindbrain neurons, Noggin induces a profile characteristic of dorsal telencephalic neurons. Our findings show that endogenously produced BMPs affect the positional identity of the neurons that ESCs spontaneously generate when differentiating in vitro in a minimal medium. The data also support the existence of an intrinsic program of neuronal differentiation with dorsal telencephalic identity. Our method of ESC neuralization allows for fast differentiation of neural cells via the same signals found during in vivo embryonic development and for the acquisition of cortical identity by the inhibition of BMP alone.     

cAMP initiates early phase neuron-like morphology changes and late phase neural differentiation in mesenchymal stem cells

The intracellular second messenger cAMP is frequently used in induction media to induce mesenchymal stem cells (MSCs) into neural lineage cells. To date, an understanding of the role cAMP exerts on MSCs and whether cAMP can induce MSCs into functional neurons is still lacking. We found cAMP initiated neuron-like morphology changes early and neural differentiation much later. The early phase changes in morphology were due to cell shrinkage, which subsequently rendered some cells apoptotic. While the morphology changes occurred prior to the expression of neural markers, it is not required for neural marker expression and the two processes are differentially regulated downstream of cAMP-activated protein kinase A. cAMP enabled MSCs to gain neural marker expressions with neuronal function, such as, calcium rise in response to neuronal activators, dopamine, glutamate, and potassium chloride. However, only some of the cells induced by cAMP responded to the three neuronal activators and further lack the neuronal morphology, suggesting that although cAMP is able to direct MSCs towards neural differentiation, they do not achieve terminal differentiation.     

Cell surface antigen profiling using a novel type of antibody array immobilised to plasma ion-implanted polycarbonate

To identify and sort out subpopulations of cells from more complex and heterogeneous assemblies of cells is important for many biomedical applications, and the development of cost- and labour-efficient techniques to accomplish this is warranted. In this report, we have developed a novel array-based platform to discriminate cellular populations based on differences in cell surface antigen expressions. These cell capture microarrays were produced through covalent immobilisation of CD antibodies to plasma ion immersion implantation-treated polycarbonate (PIII-PC), which offers the advantage of a transparent matrix, allowing direct light microscopy visualisation of captured cells. The functionality of the PIII-PC array was validated using several cell types, resulting in unique surface antigen expression profiles. PIII-PC results were compatible with flow cytometry, nitrocellulose cell capture arrays and immunofluorescent staining, indicating that the technique is robust. We report on the use of this PIII-PC cluster of differentiation (CD) antibody array to gain new insights into neural differentiation of mouse embryonic stem (ES) cells and into the consequences of genetic targeting of the Notch signalling pathway, a key signalling mechanism for most cellular differentiation processes. Specifically, we identify CD98 as a novel marker for neural precursors and polarised expression of CD9 in the apical domain of ES cell-derived neural rosettes. We further identify expression of CD9 in hitherto uncharacterised non-neural cells and enrichment of CD49e- and CD117-positive cells in Notch signalling-deficient ES cell differentiations. In conclusion, this work demonstrates that covalent immobilisation of antibody arrays to the PIII-PC surface provides faithful cell surface antigen data in a cost- and labour-efficient manner. This may be used to facilitate high throughput identification and standardisation of more precise marker profiles during stem cell differentiation and in various genetic and disease contexts.     

Regulation of G1 phase progression by growth factors and the extracellular matrix

Cell cycle progression is regulated by both intracellular and extracellular control mechanisms. Intracellular controls ensure that cell cycle progression is stopped in response to irregularities such as DNA damage or faulty spindle assembly, whereas extracellular factors may determine cell fate such as differentiation, proliferation or programmed cell death (apoptosis). When extracellular factors bind to receptors at the outside of the cell, signal transduction cascades are activated inside the cell that eventually lead to cellular responses. We have shown previously that MAP kinase (MAPK), one of the proteins involved in several signal transduction processes, is phosphorylated early after mitosis and translocates to the nucleus around the restriction point. The activation of MAPK is independent of cell attachment, but does require the presence of growth factors. Moreover, it appears that in Chinese hamster ovary cells, a transformed cell line, growth factors must be present early in the G1 phase for a nuclear translocation of MAPK and subsequent DNA replication to occur. When growth factors are withdrawn from the medium immediately after mitosis, MAPK is not phosphorylated, cell cycle progression is stopped and cells appear to enter a quiescent state, which may lead to apoptosis. Furthermore, in addition to this growth-factor-regulated decision point in early G1 phase, another growth-factor-sensitive period can be distinguished at the end of the G1 phase. This period is suggested to correlate with the classical restriction point (R) and may be related to cell differentiation.     

Increased apoptosis in differentiating p27-deficient mouse embryonic stem cells

In mouse embryonic stem (mES) cells, the expression of p27 is elevated when differentiation is induced. Using mES cells lacking p27 we tested the importance of p27 for the regulation of three critical cellular processes: proliferation, differentiation, and apoptosis. Although cell cycle distribution, DNA synthesis, and the activity of key G1/S-regulating cyclin-dependent kinases remained unaltered in p27-deficient ES cells during retinoic acid-induced differentiation, the amounts of cyclin D2 and D3 in such cells were much lower compared with normal mES cells. The onset of differentiation induces apoptosis in p27-deficient cells, the extent of which can be reduced by artificially increasing the level of cyclin D3. We suggest that the role of p27 in at least some differentiation pathways of mES cells is to prevent apoptosis, and that it is not involved in slowing cell cycle progression. We also propose that the pro-survival function of p27 is realized via regulation of metabolism of D-type cyclin(s).Received 25 February 2004; received after revision 5 April 2004; accepted 15 April 2004     

Intracellular injection of cAMP and cGMP into snail neurones induces an increase in Na+-conductance

Summary Injection of cAMP and more rarely cGMP into the neurones of the snailHelix pomatia induces an increase in membrane conductance, membrane depolarization and excitation. The effect is theophylline-dependent and has a reversal potential near — 10 mV.