Zinc finger transcription factor ecotropic viral integration site 1 is induced by all-trans retinoic acid (ATRA) and acts as a dual modulator of the ATRA response

Ecotropic viral integration site 1 (EVI1) plays important roles in leukaemia and development, and its expression is temporally and spatially highly restricted during the latter process. Nevertheless, the only physiological agent that to date has been shown to regulate transcription of this gene in mammalian cells is all-trans retinoic acid. Here we describe the identification of a retinoic acid response element that was located in the most distal of several alternative first exons of the human EVI1 gene and was constitutively bound by canonical retinoid receptors in NTERA-2 teratocarcinoma cells. Furthermore, it was the target of negative feedback by EVI1 on the induction of its own promoter by retinoic acid. This process required a previously described transcription repression domain of EVI1. Extending its role as a modulator of the retinoic acid response, EVI1 had the opposite effect on the RARβ retinoic acid response element, whose induction by all-trans retinoic acid it enhanced through a mechanism that involved almost all of its known functional domains. Augmentation of the retinoic acid response by EVI1 was also observed for the endogenous RARβ gene. Thus, we have established EVI1 as a novel type of modulator of the retinoic acid response, which can both enhance and repress induction by this agent in a promoter-specific manner

Analysis of genetic copy number changes in cervical disease progression

<p>Abstract</p> <p>Background</p> <p>Cervical dysplasia and tumorigenesis have been linked with numerous chromosomal aberrations. The goal of this study was to evaluate 35 genomic regions associated with cervical disease and to select those which were found to have the highest frequency of aberration for use as probes in fluorescent in-situ hybridization.</p> <p>Methods</p> <p>The frequency of gains and losses using fluorescence in-situ hybridization were assessed in these 35 regions on 30 paraffin-embedded cervical biopsy specimens. Based on this assessment, 6 candidate fluorescently labeled probes (8q24, Xp22, 20q13, 3p14, 3q26, CEP15) were selected for additional testing on a set of 106 cervical biopsy specimens diagnosed as Normal, CIN1, CIN2, CIN3, and SCC. The data were analyzed on the basis of signal mean, % change of signal mean between histological categories, and % positivity.</p> <p>Results</p> <p>The study revealed that the chromosomal regions with the highest frequency of copy number gains and highest combined sensitivity and specificity in high-grade cervical disease were 8q24 and 3q26. The cytological application of these two probes was then evaluated on 118 ThinPrep™ samples diagnosed as Normal, ASCUS, LSIL, HSIL and Cancer to determine utility as a tool for less invasive screening. Using gains of either 8q24 or 3q26 as a positivity criterion yielded specificity (Normal +LSIL+ASCUS) of 81.0% and sensitivity (HSIL+Cancer) of 92.3% based on a threshold of 4 positive cells.</p> <p>Conclusions</p> <p>The application of a FISH assay comprised of chromosomal probes 8q24 and 3q26 to cervical cytology specimens confirms the positive correlation between increasing dysplasia and copy gains and shows promise as a marker in cervical disease progression.</p

The Oncoprotein EVI1 and the DNA Methyltransferase Dnmt3 Co-Operate in Binding and De Novo Methylation of Target DNA

EVI1 has pleiotropic functions during murine embryogenesis and its targeted disruption leads to prenatal death by severely affecting the development of virtually all embryonic organs. However, its functions in adult tissues are still unclear. When inappropriately expressed, EVI1 becomes one of the most aggressive oncogenes associated with human hematopoietic and solid cancers. The mechanisms by which EVI1 transforms normal cells are unknown, but we showed recently that EVI1 indirectly upregulates self-renewal and cell-cycling genes by inappropriate methylation of CpG dinucleotides in the regulatory regions of microRNA-124-3 (miR-124-3), leading to the repression of this small gene that controls normal differentiation and cell cycling of somatic cells. We used the regulatory regions of miR-124-3 as a read-out system to investigate how EVI1 induces de novo methylation of DNA. Here we show that EVI1 physically interacts with DNA methyltransferases 3a and 3b (Dnmt3a/b), which are the only de novo DNA methyltransferases identified to date in mouse and man, and that it forms an enzymatically active protein complex that induces de novo DNA methylation in vitro. This protein complex targets and binds to a precise region of miR-124-3 that is necessary for repression of a reporter gene by EVI1. Based on our findings, we propose that in cooperation with Dnmt3a/b EVI1 regulates the methylation of DNA as a sequence-specific mediator of de novo DNA methylation and that inappropriate EVI1 expression contributes to carcinogenesis through improper DNA methylation

Protein Kinase Cδ Stimulates Proteasome-Dependent Degradation of C/EBPα during Apoptosis Induction of Leukemic Cells

BACKGROUND:The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction. METHODOLOGY/PRINCIPAL FINDINGS:Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells. CONCLUSIONS/SIGNIFICANCE:These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis

A Molecular Signature of Proteinuria in Glomerulonephritis

Proteinuria is the most important predictor of outcome in glomerulonephritis and experimental data suggest that the tubular cell response to proteinuria is an important determinant of progressive fibrosis in the kidney. However, it is unclear whether proteinuria is a marker of disease severity or has a direct effect on tubular cells in the kidneys of patients with glomerulonephritis. Accordingly we studied an in vitro model of proteinuria, and identified 231 “albumin-regulated genes” differentially expressed by primary human kidney tubular epithelial cells exposed to albumin. We translated these findings to human disease by studying mRNA levels of these genes in the tubulo-interstitial compartment of kidney biopsies from patients with IgA nephropathy using microarrays. Biopsies from patients with IgAN (n = 25) could be distinguished from those of control subjects (n = 6) based solely upon the expression of these 231 “albumin-regulated genes.” The expression of an 11-transcript subset related to the degree of proteinuria, and this 11-mRNA subset was also sufficient to distinguish biopsies of subjects with IgAN from control biopsies. We tested if these findings could be extrapolated to other proteinuric diseases beyond IgAN and found that all forms of primary glomerulonephritis (n = 33) can be distinguished from controls (n = 21) based solely on the expression levels of these 11 genes derived from our in vitro proteinuria model. Pathway analysis suggests common regulatory elements shared by these 11 transcripts. In conclusion, we have identified an albumin-regulated 11-gene signature shared between all forms of primary glomerulonephritis. Our findings support the hypothesis that albuminuria may directly promote injury in the tubulo-interstitial compartment of the kidney in patients with glomerulonephritis

Cracking the BAFF code.

The tumour necrosis factor (TNF) family members B cell activating factor (BAFF) and APRIL (a proliferation-inducing ligand) are crucial survival factors for peripheral B cells. An excess of BAFF leads to the development of autoimmune disorders in animal models, and high levels of BAFF have been detected in the serum of patients with various autoimmune conditions. In this Review, we consider the possibility that in mice autoimmunity induced by BAFF is linked to T cell-independent B cell activation rather than to a severe breakdown of B cell tolerance. We also outline the mechanisms of BAFF signalling, the impact of ligand oligomerization on receptor activation and the progress of BAFF-depleting agents in the clinical setting

Науково-практичний коментар Закону України «Про запобігання корупції» [станом на 1 лип. 2018 р.]

Науково-практичний коментар Закону України «Про запобігання корупції»: станом на 1 лип. 2018 р. / [А. В. Андрєєв, І. Л. Антипова, С. В. Банах та ін.], за заг. ред. Журавльова Д. В. – Київ: Видав. дім «Професіонал», 2018. – 512 с.Коментар розрахований на осіб, уповноважених на виконання функцій держави або місцевого самоврядування та прирівняних до них осіб, які є суб’єктами, на яких поширюється дія Закону України «Про запобігання корупції», на уповноважених осіб, відповідальних за реалізацію антикорупційних програм, суддів, прокурорів, слідчих, детективів, адвокатів, а також на студентів вищих учбових закладів, які готують фахівців в галузі права

The Protein Kinase Cδ Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint*

Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G2/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G2/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr15), a critical event in the G2/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G2/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G2/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G2/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G2/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G2/M cell cycle checkpoint may be a critical component of its tumor suppressor function