Regulation of melanogenesis in conditionally immortalised mouse melanocytes expressing a temperature-sensitive SV40 large T antigen

The transformation of a normal melanocyte to a malignant melanoma involves a series of poorly understood genotypic and phenotypic alterations. In vitro models of melanoma formation generated by transforming mouse melanocytes with exogenous oncogenes have revealed that this process is frequently accompanied by a loss of pigmentation. The aim of this study was to establish, and to make use of unique cell lines to gain further insight into the mechanism(s) by which oncoproteins alter melanocyte differentiation. Primary cultures of mouse epidermal and dermal melanocytes were infected with a retrovirus carrying a temperature-sensitive mutant SV40 large T antigen. Six immortalised cell lines thus generated were analysed by northern and western blots and by enzymatic assays at the permissive temperature of the oncoprotein. Three epidermal and two dermal melanocyte clones remained pigmented and expressed tyrosinase, TRP-1 and -2 genes and the proteins encoded by them. In addition they expressed the mi gene and the c-kit receptor. In contrast, one dermal melanocyte clone (DMEL-3) gradually depigmented: this was accompanied by enhanced growth and down-regulation of melanocyte-specific gene expression. At the non-permissive temperature of the oncoprotein, proliferation ceased and DMEL-3 cells repigmented with a time-dependent increase in melanocyte-specific gene expression. Moreover, mi gene expression was down-regulated in the DMEL-3 cell line at the permissive temperature and was re-expressed at the non-permissive temperature. These results provided direct evidence for the role of the SV40 large T antigen in melanocyte dedifferentiation and emphasized the pivotal role of Mi in this process. Northern blot analysis of DMEL-3 cells cultured at the permissive and non-permissive temperatures revealed that there were no detectable levels of Pax3 transcripts at either temperature. In addition, Pax3 expression was absent in the highly pigmented DMEL-2 and melan-a cell lines. These results suggest that Pax3 is not required for mi expression and that it is unlikely to be a target of the T antigen-mediated repression of mi. To explore the possibility that other melanocyte markers are also altered as a consequence of alterations in mi expression, the DMEL-3 cells were examined for changes in the α-MSH and c-kit receptors. Melanin synthesis and tyrosinase activity assays showed that alterations in mi expression did not correlate to responsiveness to α-MSH, suggesting that the MSH receptor gene is not regulated by Mi. Furthermore, northern blot analysis showed that DMEL-3 cells did not express c-kit at either the permissive or nonpermissive temperature, suggesting that Mi does not regulate c-kit expression. To address the possible role of RB family members in melanocyte differentiation, it was investigated whether melanocyte differentiation is accompanied by an increase in their mRNAs and protein levels. Northern blot analysis strongly suggested that expression of the RB1, p130 and p107 is not altered when DMEL-3 cells were induced to differentiate at the non-permissive temperature. The results from western blot anaysis were inconclusive and require further investigations. Finally, the pigmented cell lines established in the present study provided a unique opportunity to investigate the stimulatory effect of TPA on melanogenesis because growth curves showed that the cells become TPA-independent. The results showed that stimulation of melanogenesis by TPA in a pigmented melanocyte line, DMEL-2, resulted in an increase in tyrosinase, TRP-1 and TRP-2 proteins and mRNAs. Additionally, TPA increased mi gene expression which suggests that Mi is necessary for the TPA-triggered signalling cascade that induces expression of the tyrosinase gene family. These results disclose, for the first time, a mechanistic link between TPA and the transcriptional induction of pigmentation

The T-box transcription factor, TBX3, is sufficient to promote melanoma formation and invasion

The T-box transcription factor, TBX3, is overexpressed in several cancers and has been proposed as a chemotherapeutic target. Several lines of evidence suggest that TBX3 may be a key contributor to malignant melanoma, a highly aggressive and intractable disease. Using in vitro and in vivo assays we demonstrate here for the first time that overexpressing TBX3 in non-tumourigenic early stage melanoma cells is sufficient to promote tumour formation and invasion. Furthermore, we show that TBX3 may play an important role as a reciprocal switch between substrate dependent cell proliferation and tumour invasion

Gastrointestinal nematode-derived antigens alter colorectal cancer cell proliferation and migration through regulation of cell cycle and epithelial-mesenchymal transition proteins

As the global incidences of colorectal cancer rises, there is a growing importance in understanding the interaction between external factors, such as common infections, on the initiation and progression of this disease. While certain helminth infections have been shown to alter the severity and risk of developing colitis-associated colorectal cancer, whether these parasites can directly affect colorectal cancer progression is unknown. Here, we made use of murine and human colorectal cancer cell lines to demonstrate that exposure to antigens derived from the gastrointestinal nematode Heligmosomoides polygyrus significantly reduced colorectal cancer cell proliferation in vitro. Using a range of approaches, we demonstrate that antigen-dependent reductions in cancer cell proliferation and viability are associated with increased expression of the critical cell cycle regulators p53 and p21. Interestingly, H. polygyrus-derived antigens significantly increased murine colorectal cancer cell migration, which was associated with an increased expression of the adherens junction protein β-catenin, whereas the opposite was true for human colorectal cancer cells. Together, these findings demonstrate that antigens derived from a gastrointestinal nematode can significantly alter colorectal cancer cell behavior. Further in-depth analysis may reveal novel candidates for targeting and treating late-stage cancer

UV-mediated Regulation of the anti-senescence factor Tbx2

Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2

p53 requires the stress sensor USF1 to direct appropriate cell fate decision

Genomic instability is a major hallmark of cancer. To maintain genomic integrity, cells are equipped with dedicated sensors to monitor DNA repair or to force damaged cells into death programs. The tumor suppressor p53 is central in this process. Here, we report that the ubiquitous transcription factor Upstream Stimulatory factor 1 (USF1) coordinates p53 function in making proper cell fate decisions. USF1 stabilizes the p53 protein and promotes a transient cell cycle arrest, in the presence of DNA damage. Thus, cell proliferation is maintained inappropriately in Usf1 KO mice and in USF1-deficient melanoma cells challenged by genotoxic stress. We further demonstrate that the loss of USF1 compromises p53 stability by enhancing p53-MDM2 complex formation and MDM2-mediated degradation of p53. In USF1-deficient cells, the level of p53 can be restored by the re-expression of full-length USF1 protein similarly to what is observed using Nutlin-3, a specific inhibitor that prevents p53-MDM2 interaction. Consistent with a new function for USF1, a USF1 truncated protein lacking its DNA-binding and transactivation domains can also restore the induction and activity of p53. These findings establish that p53 function requires the ubiquitous stress sensor USF1 for appropriate cell fate decisions in response to DNA-damage. They underscore the new role of USF1 and give new clues of how p53 loss of function can occur in any cell type. Finally, these findings are of clinical relevance because they provide new therapeutic prospects in stabilizing and reactivating the p53 pathway

Gastrointestinal Dysfunction Criteria in Critically Ill Children: The PODIUM Consensus Conference

CONTEXT Prior criteria to define pediatric multiple organ dysfunction syndrome (MODS) did not include gastrointestinal dysfunction. OBJECTIVES Our objective was to evaluate current evidence and to develop consensus criteria for gastrointestinal dysfunction in critically ill children. DATA SOURCES Electronic searches of PubMed and EMBASE were conducted from January 1992 to January 2020, using medical subject heading terms and text words to define gastrointestinal dysfunction, pediatric critical illness, and outcomes. STUDY SELECTION Studies were included if they evaluated critically ill children with gastrointestinal dysfunction, performance characteristics of assessment/scoring tools to screen for gastrointestinal dysfunction, and assessed outcomes related to mortality, functional status, organ-specific outcomes, or other patient-centered outcomes. Studies of adults or premature infants, animal studies, reviews/commentaries, case series with sample size ≤10, and non-English language studies with inability to determine eligibility criteria were excluded. DATA EXTRACTION Data were abstracted from each eligible study into a standard data extraction form along with risk of bias assessment by a task force member. RESULTS The systematic review supports the following criteria for severe gastrointestinal dysfunction: 1a) bowel perforation, 1b) pneumatosis intestinalis, or 1c) bowel ischemia, present on plain abdominal radiograph, computed tomography (CT) scan, magnetic resonance imaging (MRI), or gross surgical inspection, or 2) rectal sloughing of gut mucosa. LIMITATIONS The validity of the consensus criteria for gastrointestinal dysfunction are limited by the quantity and quality of current evidence. CONCLUSIONS Understanding the role of gastrointestinal dysfunction in the pathophysiology and outcomes of MODS is important in pediatric critical illness

Profiling of patient-specific myocytes identifies altered gene expression in the ophthalmoplegic subphenotype of myasthenia gravis

Background: While extraocular muscles are affected early in myasthenia gravis (MG), but respond to treatment, we observe a high incidence of treatment-resistant ophthalmoplegia (OP-MG) among MG subjects with African genetic ancestry. Previously, using whole exome sequencing, we reported potentially functional variants which associated with OP-MG. The aim of this study was to profile the expression of genes harbouring the OP-MG associated variants using patient-derived subphenotype-specific ‘myocyte’ cultures. Methods From well-characterised MG patients we developed the ‘myocyte’ culture models by transdifferentiating dermal fibroblasts using an adenovirus expressing MyoD. These myocyte cultures were treated with homologous acetylcholine receptor antibody-positive myasthenic sera to induce muscle transcripts in response to an MG stimulus. Gene expression in myocytes derived from OP-MG (n = 10) and control MG subjects (MG without ophthalmoplegia; n = 6) was quantified using a custom qPCR array profiling 93 potentially relevant genes which included the putative OP-MG susceptibility genes and other previously reported genes of interest in MG and experimental autoimmune myasthenia gravis (EAMG). Results OP-MG myocytes compared to control MG myocytes showed altered expression of four OP-MG susceptibility genes (PPP6R2, CANX, FAM136A and FAM69A) as well as several MG and EAMG genes (p  0.78, p < 0.01), but not in control MG samples. OP-MG susceptibility genes and MG-associated genes accounted for the top three significantly correlated gene pairs (r ≥ 0.98, p < 1 × 10− 6) reflecting crosstalk between OP-MG and myasthenia pathways, which was not evident in control MG cells. The genes with altered expression dynamics between the two subphenotypes included those with a known role in gangliosphingolipid biosynthesis, mitochondrial metabolism and the IGF1-signalling pathway. Conclusion Using a surrogate cell culture model our findings suggest that muscle gene expression and co-expression differ between OP-MG and control MG individuals. These findings implicate pathways not previously considered in extraocular muscle involvement in myasthenia gravis and will inform future studies

Molecular mechanisms underpinning sarcomas and implications for current and future therapy

Sarcomas are complex mesenchymal neoplasms with a poor prognosis. Their clinical management is highly challenging due to their heterogeneity and insensitivity to current treatments. Although there have been advances in understanding specific genomic alterations and genetic mutations driving sarcomagenesis, the underlying molecular mechanisms, which are likely to be unique for each sarcoma subtype, are not fully understood. This is in part due to a lack of consensus on the cells of origin, but there is now mounting evidence that they originate from mesenchymal stromal/stem cells (MSCs). To identify novel treatment strategies for sarcomas, research in recent years has adopted a mechanism-based search for molecular markers for targeted therapy which has included recapitulating sarcomagenesis using in vitro and in vivo MSC models. This review provides a comprehensive up to date overview of the molecular mechanisms that underpin sarcomagenesis, the contribution of MSCs to modelling sarcomagenesis in vivo, as well as novel topics such as the role of epithelial-to-mesenchymal-transition (EMT)/mesenchymal-to-epithelial-transition (MET) plasticity, exosomes, and microRNAs in sarcomagenesis. It also reviews current therapeutic options including ongoing preclinical and clinical studies for targeted sarcoma therapy and discusses new therapeutic avenues such as targeting recently identified molecular pathways and key transcription factors.The authors were supported by grants from the South Africa Medical Research Council (SAMRC), the National Research Foundation (NRF), Cancer Association of South Africa (CANSA) and the Universities of Cape Town and Pretoria.The South Africa Medical Research Council (SAMRC), the National Research Foundation (NRF), Cancer Association of South Africa (CANSA) and the Universities of Cape Town and Pretoria.http://www.nature.com/sigtrans/Immunolog

Intensity Inhomogeneity Correction of SD-OCT Data Using Macular Flatspace

Images of the retina acquired using optical coherence tomography (OCT) often suffer from intensity inhomogeneity problems that degrade both the quality of the images and the performance of automated algorithms utilized to measure structural changes. This intensity variation has many causes, including off-axis acquisition, signal attenuation, multi-frame averaging, and vignetting, making it difficult to correct the data in a fundamental way. This paper presents a method for inhomogeneity correction by acting to reduce the variability of intensities within each layer. In particular, the N3 algorithm, which is popular in neuroimage analysis, is adapted to work for OCT data. N3 works by sharpening the intensity histogram, which reduces the variation of intensities within different classes. To apply it here, the data are first converted to a standardized space called macular flat space (MFS). MFS allows the intensities within each layer to be more easily normalized by removing the natural curvature of the retina. N3 is then run on the MFS data using a modified smoothing model, which improves the efficiency of the original algorithm. We show that our method more accurately corrects gain fields on synthetic OCT data when compared to running N3 on non-flattened data. It also reduces the overall variability of the intensities within each layer, without sacrificing contrast between layers, and improves the performance of registration between OCT images

The T-box transcription factor TBX3 drives proliferation by direct repression of the p21WAF1 cyclin-dependent kinase inhibitor

Background: TBX3, a member of the T-box family of transcription factors, is essential in development and has emerged as an important player in the oncogenic process. TBX3 is overexpressed in several cancers and has been shown to contribute directly to tumour formation, migration and invasion. However, little is known about the molecular basis for its role in development and oncogenesis because there is a paucity of information regarding its target genes. The cyclin-dependent kinase inhibitor p21WAF1 plays a pivotal role in a myriad of processes including cell cycle arrest, senescence and apoptosis and here we provide a detailed mechanism to show that it is a direct and biologically relevant target of TBX3. Results: Using a combination of luciferase reporter gene assays and in vitro and in vivo binding assays we show that TBX3 directly represses the p21WAF1 promoter by binding a T-element close to its initiator. Furthermore, we show that the TBX3 DNA binding domain is required for the transcriptional repression of p21WAF1 and that pseudo-phosphorylation of a serine proline motif (S190) located within this domain may play an important role in regulating this ability. Importantly, we demonstrate using knockdown and overexpression experiments that p21WAF1 repression by TBX3 is biologically significant and required for TBX3-induced cell proliferation of chondrosarcoma cells. Conclusions: Results from this study provide a detailed mechanism of how TBX3 transcriptionally represses p21WAF1 which adds to our understanding of how it may contribute to oncogenesis