Rare and Frequent Promoter Methylation, Respectively, of TSHZ2 and 3 Genes That Are Both Downregulated in Expression in Breast and Prostate Cancers

Neoplastic cells harbor both hypomethylated and hypermethylated regions of DNA. Whereas hypomethylation is found mainly in repeat sequences, regional hypermethylation has been linked to the transcriptional silencing of certain tumor suppressor genes. We attempted to search for candidate genes involved in breast/prostate carcinogenesis, using the criteria that they should be expressed in primary cultures of normal breast/prostate epithelial cells but are frequently downregulated in breast/prostate cancer cell lines and that their promoters are hypermethylated.We identified several dozens of candidates among 194 homeobox and related genes using Systematic Multiplex RT-PCR and among 23,000 known genes and 23,000 other expressed sequences in the human genome by DNA microarray hybridization. An additional examination, by real-time qRT-PCR of clinical specimens of breast cancer, further narrowed the list of the candidates. Among them, the most frequently downregulated genes in tumors were NP_775756 and ZNF537, from the homeobox gene search and the genome-wide search, respectively. To our surprise, we later discovered that these genes belong to the same gene family, the 3-member Teashirt family, bearing the new names of TSHZ2 and TSHZ3. We subsequently determined the methylation status of their gene promoters. The TSHZ3 gene promoter was found to be methylated in all the breast/prostate cancer cell lines and some of the breast cancer clinical specimens analyzed. The TSHZ2 gene promoter, on the other hand, was unmethylated except for the MDA-MB-231 breast cancer cell line. The TSHZ1 gene was always expressed, and its promoter was unmethylated in all cases.TSHZ2 and TSHZ3 genes turned out to be the most interesting candidates for novel tumor suppressor genes. Expression of both genes is downregulated. However, differential promoter methylation suggests the existence of distinctive mechanisms of transcriptional inactivation for these genes

Institutional Turn(s) in Theories of Legal Interpretation

The paper gives an overview of recent doctrines of legal interpretation that can be subsumed under the common name “institutional turn” in theories of legal interpretation. Among the reviewed theoretical positions are the ones from Jeremy Wаldron, Victoria Nourse, Cass Sunstein, Adrian Vermeule, Scott Shapiro. It is concluded that the institutional turn in legal interpretation offers some significant insights into legal interpretation and interesting perspectives on the appropriate interpretative methodologies. The turn, however, does this by eschewing parts of traditional accounts of legal interpretation that are arguably worth preserving in any theory of legal interpretation

Fusion of the Genes EWSR1 and PBX3 in Retroperitoneal Leiomyoma with t(9;22)(q33;q12)

Retroperitoneal leiomyoma is a rare benign smooth muscle tumor almost exclusively found in women and with histopathological features similar to uterine leiomyomas. The pathogenesis of retroperitoneal leiomyoma is unclear and next to nothing is known about the cytogenetics and molecular genetics of the tumor. We present here a retroperitoneal leiomyoma with a t(9;22)(q33;q12) as the sole karyotypic aberration. The translocation resulted in an EWSR1-PBX3 fusion gene in which exon 9 of EWSR1 (nucleotide 1320 accession number NM_013986 version 3) was in-frame fused to exon 5 of PBX3 (nucleotide 824 accession number NM_006195 version 5). The EWSR1-PBX3 fusion transcript codes for a 529 amino acids long chimeric EWSR1-PBX3 protein which contains the N-terminal transactivation part of EWSR1 and the homeodomain of PBX3. The present study, together with our previous finding of a retroperitoneal leiomyoma with t(10;17)(q22;q21) as the sole karyotypic aberration and a KAT6B-KANSL1 fusion gene, indicates that retroperitoneal leiomyomas may be characterized by fusion genes coding for chimeric proteins. However, cytogenetic and molecular heterogeneity exists in these tumors and it is too early to tell how many and which different pathways lead to retroperitoneal leiomyomagenesis

Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential

In the stem cell field there is a lack of non invasive and fast methods to identify stem cell’s metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that the metabolic signature of NPSCs correlates with their differentiation potential, showing that neuronal progenitors and glial progenitors have a different free/bound NADH ratio. Reducing conditions in NPSCs correlates with their neurogenic potential, while oxidative conditions correlate with glial potential. For the first time we show that FLIM NADH metabolic fingerprint provides a novel, and quantitative measure of stem cell potential and a label-free and non-invasive means to identify neuron- or glial- biased progenitors