Susan L. Woods

A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma

So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families. Mutations in CDKN2A account for approximately 40% of familial cases, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds. Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma. We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF). Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant. Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case–control sample. Likewise, it was similarly associated in an independent case–control sample from the United Kingdom. In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both. The variant allele was also associated with increased naevus count and non-blue eye colour. Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.

miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN amplified neuroblastoma

Inactivation of the p53 tumor suppressor pathway allows cell survival in times of stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as neuroblastoma maintain wild-type human TP53 and mouse Trp53 (referred to collectively as p53 herein). Here we describe a miRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3′ untranslated region (UTR). miR-380-5p is highly expressed in mouse embryonic stem cells and neuroblastomas, and high expression correlates with poor outcome in neuroblastomas with neuroblastoma derived v-myc myelocytomatosis viral-related oncogene (MYCN) amplification. miR-380 overexpression cooperates with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice. Conversely, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of p53, and extensive apoptotic cell death. In vivo delivery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma.

Melanoma cell invasiveness is regulated by miR-211 suppression of the BRN2 transcription factor

To identify microRNAs potentially involved in melanomagenesis, we compared microRNA expression profiles between melanoma cell lines and cultured melanocytes. The most differentially expressed microRNA between the normal and tumor cell lines was miR‐211. We focused on this pigment‐cell‐enriched miRNA as it is derived from the microphthalmia‐associated transcription factor (MITF)‐regulated gene, TRPM1 (melastatin). We find that miR‐211 expression is greatly decreased in melanoma cells and melanoblasts compared to melanocytes. Bioinformatic analysis identified a large number of potential targets of miR‐211, including POU3F2 (BRN2). Inhibition of miR‐211 in normal melanocytes resulted in increased BRN2 protein, indicating that endogenous miR‐211 represses BRN2 in differentiated cells. Over‐expression of miR‐211 in melanoma cell lines changed the invasive potential of the cells in vitro through directly targeting BRN2 translation. We propose a model for the apparent non‐overlapping expression levels of BRN2 and MITF in melanoma, mediated by miR‐211 expression.

Differential Activities of Murine Single Minded 1 (SIM1) and SIM2 on a Hypoxic Response Element CROSS-TALK BETWEEN BASIC HELIX-LOOP-HELIX/Per-Arnt-Sim HOMOLOGY TRANSCRIPTION FACTORS

The basic helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) protein family comprises a group of transcriptional regulators that often respond to a variety of developmental and environmental stimuli. Two murine members of this family, Single Minded 1 (SIM1) and Single Minded 2 (SIM2), are essential for postnatal survival but differ from other prototypical family members such as the dioxin receptor (DR) and hypoxia-inducible factors, in that they behave as transcriptional repressors in mammalian one-hybrid experiments and have yet to be ascribed a regulating signal. In cell lines engineered to stably express SIM1 and SIM2, we show that both are nuclear proteins that constitutively complex with the general bHLH/PAS partner factor, ARNT. We report that the murine SIM factors, in combination with ARNT, attenuate transcription from the hypoxia-inducible erythropoietin (EPO) enhancer during hypoxia. Such cross-talk between coexpressed bHLH/PAS factors can occur through competition for ARNT, which we find evident in SIM repression of DR-induced transcription from a xenobiotic response element reporter gene. However, SIM1/ARNT, but not SIM2/ARNT, can activate transcription from the EPOenhancer at normoxia, implying that the SIM proteins have the ability to bind hypoxia response elements and affect either activation or repression of transcription. This notion is supported by co-immunoprecipitation of EPO enhancer sequences with the SIM2 protein. SIM protein levels decrease with hypoxia treatment in our stable cell lines, although levels of the transcripts encoding SIM1 and SIM2 and the approximately 2-h half-lives of each protein are unchanged during hypoxia. Inhibition of protein synthesis, known to occur in cells during hypoxic stress in order to decrease ATP utilization, appears to account for the fall in SIM levels. Our data suggest the existence of a hypoxic switch mechanism in cells that coexpress hypoxia-inducible factor and SIM proteins, where up-regulation and activation of hypoxia-inducible factor-1α is concomitant with attenuation of SIM activities.

NRAS and BRAF Mutations in Cutaneous Melanoma and the Association with MC1R Genotype: Findings from Spanish and Austrian Populations

There is increasing epidemiologic and molecular evidence that cutaneous melanomas arise through multiple causal pathways. To further define the pathways to melanoma, we explored the relationship between germline and somatic mutations in a series of melanomas collected from 134 Spanish and 241 Austrian patients. Tumor samples were analyzed for melanocortin-1 receptor (MC1R) variants and mutations in the BRAF and NRAS genes. Detailed clinical data were systematically collected from patients. We found that NRAS-mutant melanomas were significantly more likely from older patients and BRAF-mutant melanomas were more frequent in melanomas from the trunk. We observed a nonsignificant association between germline MC1R status and somatic BRAF mutations in melanomas from trunk sites (odds ratio (OR) 1.8 (0.8-4.1), P=0.1), whereas we observed a significant inverse association between MC1R and BRAF for melanomas of the head and neck (OR 0.3 (0.1-0.8), P=0.02). This trend was observed in both the Spanish and Austrian populations.

The Graduate Record Examination: Help or Hindrance in Nursing Graduate School Admissions?

Do graduate record examination (GRE) scores serve as strong predictors of student success in graduate school in nursing, and if so, is the extent to which they may indicate success outweighed by their perceived role as a barrier to application to graduate school in nursing? Academic ability, defined as cumulative grade point average (GPA), was used as the outcome indicator for success in graduate school and was compared with admission GRE scores for 217 students admitted to graduate programs at the University of Washington School of Nursing over a 1-year period. The GRE presented a large barrier to application that far outweighed the limited benefit of predicting 5% to 8% of explained variance in GPA.

The bHLH/Per-Arnt-Sim transcription factor SIM2 regulates muscle transcript myomesin2 via a novel, non-canonical E-box sequence

Despite a growing number of descriptive studies that show Single-minded 2 (Sim2) is not only essential for murine survival, but also upregulated in colon, prostate and pancreatic tumours, there is a lack of direct target genes identified for this basic helix–loop–helix/PAS transcription factor. We have performed a set of microarray experiments aimed at identifying genes that are differentially regulated by SIM2, and successfully verified that the Myomesin2 (Myom2) gene is SIM2-responsive. Although SIM2 has been reported to be a transcription repressor, we find that SIM2 induces transcription of Myom2 and activates the Myom2 promoter sequence when co-expressed with the heterodimeric partner protein, ARNT1, in human embryonic kidney cells. Truncation and mutation of the Myom2 promoter sequence, combined with chromatin immunoprecipitation studies in cells, has lead to the delineation of a non-canonical E-box sequence 5′-AACGTG-3′ that is bound by SIM2/ARNT1 heterodimers. Interestingly, in immortalized human myoblasts knock down of Sim2 results in increased levels of Myom2 RNA, suggesting that SIM2 is acting as a repressor in these cells and so its activity is likely to be highly context dependent. This is the first report of a direct SIM2/ARNT1 target gene with accompanying analysis of a functional response element.

A new transgenic mouse line for tetracycline inducible transgene expression in mature melanocytes and the melanocyte stem cells using the Dopachrome tautomerase promoter

We have generated a novel transgenic mouse to direct inducible and reversible transgene expression in the melanocytic compartment. The Dopachrome tautomerase (Dct) control sequences we used are active early in the development of melanocytes and so this system was designed to enable the manipulation of transgene expression during development in utero and in the melanocyte stem cells as well as mature melanocytes. We observed inducible lacZ and GFP reporter transgene activity specifically in melanocytes and melanocyte stem cells in mouse skin. This mouse model will be a useful tool for the pigment cell community to investigate the contribution of candidate genes to normal melanocyte and/or melanoma development in vivo. Deregulated expression of the proto-oncogene MYC has been observed in melanoma, however whether MYC is involved in tumorigenesis in pigment cells has yet to be directly investigated in vivo. We have used our system to over-express MYC in the melanocytic compartment and show for the first time that increased MYC expression can indeed promote melanocytic tumor formation.

Cardiac Output Determination

Critical care nurses frequently are involved in obtaining cardiac output measurements and in using these data to assess and to plan therapy. This article reviews the physiologic determinants of cardiac output and the clinical factors that influence these determinants. Principles and techniques of common methods of cardiac output measurement are discussed. A thorough presentation of guidelines for troubleshooting problems with thermodilution cardiac output measurement is provided in a table. Nursing management issues are discussed using relevant nursing research. Future considerations in cardiac output measurement are discussed, and suggestions of an ideal cardiac output system are provided.

Oncogenic BRAF mutation induces DNA methylation changes in a murine model for human serrated colorectal neoplasia

ABSTRACT Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.