Splicing transitions of the anchoring protein ENH during striated muscle development.

The ENH (PDLIM5) protein acts as a scaffold to tether various functional proteins at subcellular sites via PDZ and three LIM domains. Splicing of the ENH primary transcript generates various products with different repertories of protein interaction modules. Three LIM-containing ENH predominates in neonatal cardiac tissue, whereas LIM-less ENHs are abundant in adult hearts, as well as skeletal muscles. Here we examine the timing of splicing transitions of ENH gene products during postnatal heart development and C2C12 myoblast differentiation. Real-time PCR analysis shows that LIM-containing ENH1 mRNA is gradually decreased during postnatal heart development, whereas transcripts with the short exon 5 appear in the late postnatal period and continues to increase until at least one month after birth. The splicing transition from LIM-containing ENH1 to LIM-less ENHs is also observed during the early period of C2C12 differentiation. This transition correlates with the emergence of ENH transcripts with the short exon 5, as well as the expression of myogenin mRNA. In contrast, the shift from the short exon 5 to the exon 7 occurs in the late differentiation period. The timing of this late event corresponds to the appearance of mRNA for the skeletal myosin heavy chain MYH4. Thus, coordinated and stepwise splicing transitions result in the production of specific ENH transcripts in mature striated muscles.

Increased RLS prevalence in children and adolescents with migraine: a case-control study.

BACKGROUND: Previous studies have reported an increased frequency of restless legs syndrome (RLS) in adult migraine patients. Until now, the frequency of RLS in pediatric patients has not been investigated. We set out to assess the frequency of RLS in children and adolescents with migraine compared to headache-free controls. METHODS: We investigated 111 consecutive patients with a sole diagnosis of migraine with or without aura presenting to the Headache Unit at the Department of Child and Adolescent Psychiatry and 73 headache-free controls for the presence of RLS using a semistructured interview. In addition, we assessed the level of daytime sleepiness by means of the Epworth sleepiness scale (ESS). A second group of headache-free controls was screened for the presence of RLS using an online questionnaire. RESULTS: The frequency of RLS in migraine patients was significantly higher than in controls (22% vs. 5% (p < 0.001) and 8% (p < 0.001)). DISCUSSION: This is the first study suggesting an association between RLS and migraine in the pediatric population. Future studies are needed to determine the extent of sleep disruption in children and adolescents with migraine and comorbid RLS.

Gene expression profiling of rat spermatogonia and Sertoli cells reveals signaling pathways from stem cells to niche and testicular cancer cells to surrounding stroma.

BACKGROUND: Stem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells. RESULTS: The pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance. CONCLUSIONS: Our data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-environment in testicular cancer.

Signal transduction via G protein coupled receptors: a personal outlook.

Intense research continues to address transmembrane signal transduction. Here we recall seven fundamental concepts governing this field. Only signal transduction via G protein coupled receptors (GPCR), is explicitly considered. But the fundamental concepts apply also to other transmembrane receptors such as receptor protein kinases. Although elements of the signal transduction complexes are readily exchangeable, it appears very likely that these complexes are highly organized in situ; how such organization is achieved remains puzzling, and an important question to be answered. Research in signal transduction can continue to explore with 'reductionist' approaches the fine details of individual molecular properties of signaling proteins and sub-cellular events. Attempts of comprehensive description of the biology of signal transduction cannot--at the present time--take into account the whole complexity of the systems involved. Nevertheless, it appears worthwhile to attempt more wholesome approaches, the results of which might turn out to be quite useful in medicine and pharmacology.

Promoter-proximal pausing of RNA polymerase II: an opportunity to regulate gene transcription.

Transcription of eukaryotic genes by RNA polymerase II (pol II) is a complex, highly regulated multiphasic process. Pol II pauses in the proximity of the promoter on a large fraction of transcribed genes. Transcription initiation and elongation of transcripts are under distinct control. Induced gene expression can thus be due to enhanced initiation and/or stimulated elongation. Pausing and resumption of the elongation of transcripts is under the control of transcription elongation factors. Three of them, P-TEFb, DSIF, and NELF have been well characterized as protein complexes with multiple general but also gene specific functions. Elongation factors execute checkpoint functions but serve also as targets for signaling processes which regulate gene expression. Due to the general importance of transcription elongation factors, it is difficult to delineate the mechanisms by which elongation of specific genes is regulated by specific intracellular signals. However, it is clear that the controlled pausing of pol II provides an opportunity to finely control timing and quantity of transcriptional output.

Negative elongation factor NELF controls transcription of immediate early genes in a stimulus-specific manner.

The transcription rate of immediate early genes (IEGs) is controlled directly by transcription elongation factors at the transcription elongation step. Negative elongation factor (NELF) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) stall RNA polymerase II (pol II) soon after transcription initiation. Upon induction of IEG transcription, DSIF is converted into an accelerator for pol II elongation. To address whether and how NELF as well as DSIF controls overall IEG transcription, its expression was reduced using stable RNA interference in GH4C1 cells. NELF knock-down reduced thyrotropin-releasing hormone (TRH)-induced transcription of the IEGs c-fos, MKP-1, and junB. In contrast, epidermal growth factor (EGF)-induced transcription of these IEGs was unaltered or even slightly increased by NELF knock-down. Thus, stable knock-down of NELF affects IEG transcription stimulation-specifically. Conversely, DSIF knock-down reduced both TRH- and EGF-induced transcription of the three IEGs. Interestingly, TRH-induced activation of the MAP kinase pathway, a pathway essential for transcription of the three IEGs, was down-regulated by NELF knock-down. Thus, stable knock-down of NELF, by modulating intracellular signaling pathways, caused stimulation-specific loss of IEG transcription. These observations indicate that NELF controls overall IEG transcription via multiple mechanisms both directly and indirectly.

Dedifferentiation of human articular chondrocytes is associated with alterations in expression patterns of GDF-5 and its receptors.

Human articular chondrocytes are expanded in monolayer culture in order to obtain sufficient cells for matrix-associated cartilage transplantation. During this proliferation process, the cells change their shape as well as their expression profile. These changes resemble those that occur during embryogenesis, when the limb anlagen form the interzone that later develops the joint cleft. We analysed the expression profile of genes that are reportedly important for these changes during embryogenesis within the dedifferentiation process of adult articular chondrocytes. We found GDF-5, BMPR-Ib and connexin 43 up-regulated, as well as a down-regulation of BMPR-Ia and noggin. Connexin 32 could not be detected in either native cartilage or in dedifferentiated cells. The newly synthesized proteins were detected by immunofluorescence. There is evidence from our results that dedifferentiated chondrocytes resemble the cells from the interzone in developing synovial joints.

The FoxO/Bcl-6/cyclin D2 pathway mediates metabolic and growth factor stimulation of proliferation in Min6 pancreatic beta-cells.

Lack of nutrients and growth factors activates FoxO transcription factors in pancreatic beta-cells, whereas PI3K/Akt-dependent inactivation of FoxO favors proliferation. To address the link between FoxO and cell cycle control, we deprived Min6 cells of serum and glucose which activated FoxO and inhibited proliferation. Concomitantly, expression of the transcriptional repressor Bcl-6 was stimulated, whereas cyclin D2 was lowered. Gain of function approaches indicated that FoxO activation was sufficient to activate bcl-6 transcription, while Bcl-6 repressed cyclin D2 transcription and proliferation. Thus, in pancreatic beta-cells, the FoxO/Bcl6/cyclin D2 pathway connects nutrient and growth factor status to cell cycle control, and may therefore be considered for its therapeutic potential in diabetes.

Alterations in CD44 isoforms and HAS expression in human articular chondrocytes during the de- and re-differentiation processes.

The purpose of this study was to investigate the expression of different CD44 and hyaluronan synthase isoforms in cartilage, their alterations during the chondrocyte dedifferentiation process in monolayer culture and during the redifferentiation process on 3D scaffolds. Chondrocytes isolated from human articular cartilage were cultured as a monolayer for up to 36 days and were seeded on two different 3D scaffolds (HYAFF 11 and Bio-Gide). Expression levels of CD44s, CD44-lt, CD44-st, HAS1, HAS2, HAS3 and UDPGD were determined by real-time RT-PCR at different time points. At the protein level CD44 and CD90 were analyzed by flow cytometry. HAS2 was found to be the predominantly expressed hyaluronan synthase in chondrocytes and was not subjected to any regulation during the dedifferentiation process. CD44s, CD44-lt, CD44-st and UDPGD, however, were upregulated immediately after cell isolation. In addition, a high cell density was found to significantly increase CD44-st and CD44-lt expression. Redifferentiation on 3D scaffolds reversed the increase of the CD44 expression. Our data point out that CD44 expression does not correlate with matrix assembly in chondrocytes and that CD44 has a regulatory function in chondrocytes, not necessarily on differentiation, but probably on proliferation.

Gene-specific recruitment of positive and negative elongation factors during stimulated transcription of the MKP-1 gene in neuroendocrine cells.

MAP kinase phosphatase-1 (MKP-1) controls nuclear MAP kinase activity with important consequences on cell growth or apoptosis. MKP-1 transcription is initiated constitutively but elongation is blocked within exon 1. It is unclear how induction of MKP-1 is controlled. Here, we report that the transcriptional elongation factors P-TEFb, DSIF and NELF regulate MKP-1 transcription in the pituitary GH4C1 cell line. Prior to stimulation, DSIF, NELF and RNA polymerase II (pol II) associate with the promoter-proximal region of the MKP-1 gene upstream of the elongation block site. Thyrotropin-releasing hormone (TRH) leads to recruitment of P-TEFb along the whole gene and a marked increase of DSIF and pol II downstream of the elongation block site, whereas NELF remains confined to the promoter-proximal region. 5,6-Dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) an inhibitor of P-TEFb eliminated TRH stimulation of MKP-1 transcription. DRB specifically inhibited TRH-induced recruitment of DSIF and P-TEFb to the MKP-1 gene. Furthermore, DRB treatment eliminated TRH-induced progression along the MKP-1 gene of pol II phosphorylated on Ser-2 of its CTD. These results indicate that P-TEFb is essential for gene-specific stimulated transcriptional elongation in mammalian cells via mechanisms which involve the activation of the DSIF-NELF complex and Ser-2 phosphorylation of pol II.

Enigma homolog 1 scaffolds protein kinase D1 to regulate the activity of the cardiac L-type voltage-gated calcium channel.

AIMS: In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase Cepsilon, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, alpha1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel. METHODS AND RESULTS: PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiomyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with alpha1C in cardiomyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to alpha1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the alpha-adrenergic-induced increase of L-type calcium currents. CONCLUSION: We found a new binding partner, ENH1, and a new target, alpha1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to alpha1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca(2+) channels.

Sequential actions of ERK1/2 on the AP-1 transcription factor allow temporal integration of metabolic signals in pancreatic beta cells.

The AP-1 transcription factor composed of fos and jun gene products mediates transcriptional responses to hormonal and metabolic stimulations of pancreatic beta cells. Here, we investigated the mechanisms that dynamically control expression of AP-1 subunit proteins. In MIN6 cells, glucose and GLP-1 raised c-FOS protein with biphasic kinetics, an initial peak being followed by a plateau that persisted as long as stimuli were maintained. ERK1/2 activation paralleled c-FOS expression. Whereas initial induction of c-FOS protein required ERK1/2-dependent activation of c-fos transcription and de novo protein synthesis, persistent accumulation of c-FOS under sustained stimulation did not. Indeed, dependent on ERK1/2 activation, c-FOS accumulated in its hyperphosphorylated form protected from degradation through the proteasome pathway. The implication of ERK1/2 in the accumulation of c-FOS protein was confirmed in rat primary beta cells, and the functional consequences of this mechanism were demonstrated with DNA-binding and reporter assays. Altogether these findings reveal a sequential regulation of AP-1 by ERK1/2, which initially increases transcription of c-fos and, if stimulation persists, stabilizes freshly synthesized c-FOS protein to efficiently activate the transcription of AP-1-regulated genes. This ERK1/2-AP-1 module can function as a temporal integrator converting metabolic stimuli of different durations into differential transcriptional outputs.

Scaffold-dependent differentiation of human articular chondrocytes.

Matrix-associated autologous chondrocyte transplantation (MACT) is a tissue-engineered approach for the treatment of cartilage defects and combines autologous chondrocytes seeded on biomaterials. The objective of the study is the analysis of growth and differentiation behaviour of human articular chondrocytes grown on three different matrices used for MACT. Human articular chondrocytes were kept in monolayer culture for 42 days and then seeded on matrices consisting of either collagen type I/III, hyaluronan, or gelatine. During the culture time of 4 weeks the constructs were analyzed weekly. Morphological criteria were studied by scanning and transmission electron microscopy. The expression of the main type collagens was analyzed by real-time PCR. The collagen type I/III matrix supported a differentiation that closely resembled the tissue organisation of native cartilage, but cell number and type II collagen synthesis were low and differentiation occurred rather late in the cultivation period. The hyaluronan matrix and the gelatine-based matrix supported a rather rapid differentiation, with a high number of cells and a relatively high amount of type II collagen, but there was no spatial assembly that mimicked native cartilage. These facts indicate that the nature of the matrix is of great influence in the differentiation behaviour of dedifferentiated chondrocytes.

NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation.

NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24 h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas apocynin and gliotoxin did not (IC(50)>100 muM). The pattern of NOX4-dependent ROS generation was unique: (i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and (ii) the type of ROS released from NOX4-expressing cells was H(2)O(2), whereas superoxide (O(2)(-)) was almost undetectable. Probes that allow detection of intracellular O(2)(-) generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O(2)(-) within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as an inducible NOX isoform.

Mechanisms of transcriptional regulation underlying temporal integration of signals.

How cells convert the duration of signals into differential adaptation of gene expression is a poorly understood issue. Signal-induced immediate-early gene (IEG) expression couples early signals to late expression of downstream genes. Here we study how kinetic features of the IEG- system allow temporal integration of stimuli in a pancreatic beta cell model of metabolic stimulation. Gene expression profiling revealed that beta cells produce drastically different transcriptional outputs in response to different stimuli durations. Noteworthy, most genes (87%) regulated by a sustained stimulation (4 h) were not regulated by a transient stimulation (1 h followed by 3 h without stimulus). We analyzed the induction kinetics of several previously identified IEGs and . IEG expression persisted as long as stimulation was maintained, but was rapidly lost upon stimuli removal, abolishing the delayed induction. The molecular mechanisms coupling the duration of stimuli to quantitative transcription were demonstrated for the AP-1 transcription factor. In conclusion, we propose that the network composed of IEGs and their dynamically functions to convert signal inputs of different durations into quantitative differences in global transcriptional adaptation. These findings provide a novel and more comprehensive view of dynamic gene regulation.

Transcriptional response of pancreatic beta cells to metabolic stimulation: large scale identification of immediate-early and secondary response genes.

BACKGROUND: Physiological long term adaptation of pancreatic beta cells is driven by stimuli such as glucose and incretin hormones acting via cAMP (e.g. GLP-1) and involves regulated gene expression. Several rapidly inducible immediate-early genes (IEGs) have been identified in beta cells. Many of these IEGs code for transcription factors and have the potential to control the transcription of downstream target genes likely involved in long term cellular adaptation. The identity of these target genes has not been determined, and the sequence of events occurring during beta cell adaptation is still unclear. RESULTS: We have developed a microarray-based strategy for the systematic search of targets. In Min6 insulin-secreting cells, we identified 592 targets and 1278 IEGs responding to a co-stimulation with glucose and cAMP. Both IEGs and targets were involved in a large panel of functions, including those important to beta cell physiology (metabolism, secretion). Nearly 200 IEGs were involved in signaling and transcriptional regulation. To find specific examples of the regulatory link between IEGs and targets, target promoter sequences were analyzed in silico. Statistically significant over-representation of AP-1 response elements notably suggested an important role for this transcription factor, which was experimentally verified. Indeed, cell stimulation altered expression of IEG-encoded components of the AP-1 complex, activating AP-1-dependent transcription. Loss and gain-of-function experiments furthermore allowed to validate a new AP-1 regulated gene (sulfiredoxin) among the targets. AP-1 and sulfiredoxin are sequentially induced also in primary cells from rat islets of Langerhans. CONCLUSION: By identifying IEGs and their downstream targets, this study brings a comprehensive description of the transcriptional response occurring after beta cell stimulation, as well as new mechanistic insights concerning the AP-1 transcription factor.

NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation.

NOX5 is a ROS-generating NADPH oxidase which contains an N-terminal EF-hand region and can be activated by cytosolic Ca(2+) elevations. However the C-terminal region of NOX5 also contains putative phosphorylation sites. In this study we used HEK cells stably expressing NOX5 to analyze the size and subcellular localization of the NOX5 protein, its mechanisms of activation, and the characteristics of the ROS released. We demonstrate that NOX5 can be activated both by the protein kinase C activating phorbol esther PMA and by the Ca(2+) ionophore ionomycin. The PMA- but not the ionomycin-dependent activation can be inhibited by protein kinase C inhibitors. NOX5 activity is inhibited by submicromolar concentrations of diphenyl iodonium (DPI), but not by apocynin. Western blot analysis showed a lower ( approximately 70 kDa) than expected (82 kDa) molecular mass. Two arguments suggest that NOX5 is at least partially expressed on the plasma membrane: (i) the membrane-impermeant superoxide was readily detected by extracellular probes, and (ii) immunofluorescent labeling of NOX5 detected a fraction of the NOX5 protein at the plasma membrane. In summary, we demonstrate that NOX5 can be found intracellularly and at the cell surface. We also describe that it can be activated through protein kinase C, in addition to its Ca(2+) activation.

The emerging role of FOXO transcription factors in pancreatic beta cells.

FOXO transcription factors critically control fundamental cellular processes, including metabolism, cell differentiation, cell cycle arrest, DNA repair, and other reactions to cellular stress. FOXO factors sense the balance between stimuli promoting growth and differentiation versus stress stimuli signaling damage. Integrated through the FOXO system, these divergent stimuli decide on cell fate, a choice between proliferation, differentiation, or apoptosis. In pancreatic beta cells, most recent evidence highlights complex FOXO-dependent responses to glucose, insulin, or other growth factors, which include regulatory feedback. In the short term, FOXO-dependent mechanisms help beta cells to accomplish their endocrine function, and may increase their resistance to oxidative stress due to transient hyperglycemia. In the long term, FOXO-dependent responses lead to the adaptation of beta cell mass, conditioning the future ability of the organism to produce insulin and cope with changes in fuel abundance. FOXO emerges as a key factor for the maintenance of a functional endocrine pancreas and represents an interesting element in the development of therapeutic approaches to treat diabetes. This review on the role of FOXO transcription factors in pancreatic beta cells has three parts. In Part I, FOXO transcription factors will be presented in general: structure, molecular mechanisms of regulation, cellular functions, and physiological roles. Part II will focus on specific data about FOXO factors in pancreatic beta cells. Lastly in Part III, it will be attempted to combine general and beta cell-specific knowledge with the aim to envisage globally the role of FOXO factors in beta cell-linked physiology and disease.

Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription.

The melanocyte-specific isoform of the microphthalmia transcription factor affects the phenotype of human melanoma.

The microphthalmia transcription factor MITF plays a pivotal role in the development and differentiation of melanocytes. The purpose of this work was to investigate the expression and function of the melanocyte-specific isoform MITF-M in human melanoma. We found that MITF-M is repressed in 8 of 14 established melanoma cell lines tested. Transfection of MITF-M into a melanoma cell line (518A2) lacking the M-isoform and into a permanent cell line established from normal melanocytes (NMel-II) resulted in slower tumor growth in a severe combined immunodeficient-mouse xenotransplantation model. The growth difference between vector control-transfected tumors derived from the NMel-II cell line (mean tumor weight +/- SD, 3.2 g +/- 1.13) and MITF-M (+) transfectants (mean tumor weight +/- SD, 1.1 g +/- 0.49) was significant (P = 0.018). The mean tumor weight of control-transfected 518A2 tumors was 0.99 g +/- 0.22 and of MITF-M (+) transfectants, 0.69 g +/- 0.32. The difference in growth between 518A2 controls and the MITF-M (+) transfectants was clear, however it did not reach statistical significance (P = 0.08). In addition to the growth-inhibitory effects, MITF-M expression led to a change in the histopathological appearance of tumors from epitheloid toward a spindle-cell type in vivo. These results indicate a role for the MITF-M isoform in the in vivo growth control and the phenotype of human melanoma. In conclusion, MITF-M may qualify as a marker capable of identifying subgroups of melanoma patients with different tumor biology and prognosis.