Gene-Specific Intron Retention Serves as Molecular Signature that Distinguishes Melanoma from Non-Melanoma Cancer Cells in Greek Patients.

BACKGROUND: Skin cancer represents the most common human malignancy, and it includes BCC, SCC, and melanoma. Since melanoma is one of the most aggressive types of cancer, we have herein attempted to develop a gene-specific intron retention signature that can distinguish BCC and SCC from melanoma biopsy tumors. METHODS: Intron retention events were examined through RT-sqPCR protocols, using total RNA preparations derived from BCC, SCC, and melanoma Greek biopsy specimens. Intron-hosted miRNA species and their target transcripts were predicted via the miRbase and miRDB bioinformatics platforms, respectively. Ιntronic ORFs were recognized through the ORF Finder application. Generation and visualization of protein interactomes were achieved by the IntAct and Cytoscape softwares, while tertiary protein structures were produced by using the I-TASSER online server. RESULTS: c-MYC and Sestrin-1 genes proved to undergo intron retention specifically in melanoma. Interaction maps of proteins encoded by genes being potentially targeted by retained intron-accommodated miRNAs were generated and SRPX2 was additionally delivered to our melanoma-specific signature. Novel ORFs were identified in MCT4 and Sestrin-1 introns, with potentially critical roles in melanoma development. CONCLUSIONS: The property of c-MYC, Sestrin-1, and SRPX2 genes to retain specific introns could be clinically used to molecularly differentiate non-melanoma from melanoma tumors.

The resurgence of Hormone-Sensitive Lipase (HSL) in mammalian lipolysis.

The ability to store energy in the form of energy-dense triacylglycerol and to mobilize these stores rapidly during periods of low carbohydrate availability or throughout the strong metabolic demand is a highly conserved process, absolutely essential for survival. In the industrialized world the regulation of this pathway is viewed as an important therapeutic target for disease prevention. Adipose tissue lipolysis is a catabolic process leading to the breakdown of triacylglycerols stored in fat cells, and release of fatty acids and glycerol. Mobilization of adipose tissue fat is mediated by the MGL, HSL and ATGL, similarly functioning enzymes. ATGL initiates lipolysis followed by the actions of HSL on diacylglycerol, and MGL on monoacylglycerol. HSL is regulated by reversible phosphorylation on five critical residues. Phosphorylation alone, however, is not enough to activate HSL. Probably, conformational alterations and a translocation from the cytoplasm to lipid droplets are also involved. In accordance, Perilipin functions as a master regulator of lipolysis, protecting or exposing the triacylglycerol core of a lipid droplet to lipases. The prototype processes of hormonal lipolytic control are the ß-adrenergic stimulation and suppression by insulin, both of which affect cytoplasmic cyclic AMP levels. Lipolysis in adipocytes is an important process in the management of body energy reserves. Its deregulation may contribute to the symptoms of type 2 diabetes mellitus and other pathological situations. We, herein, discuss the metabolic regulation and function of lipases mediating mammalian lipolysis with a focus on HSL, quoting newly identified members of the lipolytic proteome.

Cloning and functional characterization of the ovine malic enzyme promoter.

While in human and rodents lipogenesis occurs predominantly in the liver, adipose tissue is the major site in ruminants. Here we report the nucleotide sequence of the 5'-flanking region of the ovine malic enzyme gene (ME1). The ME1 promoter is located within a GC-rich region fulfilling the criteria of CpG islands and lacks a TATA-box. Deletion analysis identified a region (-231/-170) that suppressed promoter activity in luciferase assays in HepG2 hepatoma cells but not in 3T3-L1 adipocytes. This region contains a putative triiodothyronine response element (T3RE) that differs from the human ME1 T3RE by two nucleotides. When the human ME1 T3RE was introduced into the ovine ME1 promoter context, transcriptional activity was increased in the hepatic cell lines HepG2 and H4IIE but not in differentiated 3T3-L1 cells. Our results suggest that the sequence of the T3RE in the ME1 promoter determines differences in the tissue/species activity of malic enzyme in ruminants and human.

Grade-dependent effects on cell cycle progression and apoptosis in response to doxorubicin in human bladder cancer cell lines.

Doxorubicin is an important component of combination therapy for muscle-invasive urinary bladder cancer. Treatment with this topoisomerase II poison is able to interfere with cell cycle progression and lead to cancer cell death. Using FACS analysis, Western immunoblotting and semi-quantitative RT-PCR, we studied the effects of doxorubicin on cell cycle progression and apoptosis, and also explored the possibility of using groups of genes as biomarkers of prognosis and/or response to doxorubicin treatment in human urinary bladder cancer cells. Doxorubicin induced dose-dependent G2/M and/or G1/S cell cycle arrest, followed by grade- and dose-dependent reduction in the amount of the cytosolic trimeric form of FasL, activation of Caspase-8, Caspase-9, Caspase-3, cleavage of PARP, Lamin A/C, Bcl-XL/S and interestingly Hsp90, and finally cell death. Data presented here also suggest the use of the expression patterns of Cyclin-E2, Cyclin-F, p63, p73, FasL, TRAIL, Tweak, Tweak-R, XAF-1, OPG and Bok genes for identification of the differentiation grade, and Cyclin-B2, GADD45A, p73, FasL, Bik, Bim, TRAIL, Fas, Tweak-R, XAF-1, Bcl-2, Survivin, OPG, DcR2 and Bcl-XL genes for the detection of response to doxorubicin in human bladder cancer cells.

Cloning and functional characterization of the 5' regulatory region of ovine Hormone Sensitive Lipase (HSL) gene.

Hormone Sensitive Lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signaling cascade reactions. HSL constitutes the critical enzyme in the modulation of lipid stores and the only component being subjected to hormonal control in terms of the recently identified Adipose Triglyceride Lipase (ATGL). In order to acquire detailed knowledge with regard to the mechanisms regulating ovine HSL (ovHSL) gene transcription activity, we initially isolated and cloned the 5' proximal and distal promoter regions through a genome walking approach, with the utilization of the already characterized ovHSL cDNAs. As evinced by BLAST analysis and a multiple alignment procedure, the isolated genomic fragment of 2.744 kb appeared to contain the already specified 5'-untranslated region (5'-UTR), which was interrupted by a relatively large intron of 1.448 kb. Regarding the upstream remaining part of 1.224 kb, it was demonstrated to represent a TATA-less promoter area, harboring several cis-regulatory elements that could be putatively recognized by relatively more general transcription factors, mainly including Stimulating protein 1 (Sp1), CCAAT-box Binding Factors (CBFs), Activator Protein 2 (AP2) and Glucocorticoid Receptor (GR), as well as other cis-acting regions denominated as Insulin Response Element (IRE), Glucose Response Element (GRE), Fat Specific Element (FSE) and cAMP Response Element (CRE), which could likely function in a nourishment (i.e. glucose)-/hormone-dependent fashion. When different genomic fragments were directionally (5' to 3') cloned into a suitable reporter vector upstream of a promoter-less luciferase gene and transiently transfected into 3T3-L1 (mouse fibroblasts) as well as T24 (human bladder cancer) cell lines, strong promoter activities were unambiguously detected, with the -140/+18 nucleotide sequence bearing the highest transcriptional response, thus indicating that the 1.224 kb 5' flanking region, isolated by genome walking, veritably contains the ovHSL gene promoter. Of particular significance are the observations that the functional promoter fragments could trigger the transcriptional activity of luciferase gene only under high concentration of glucose conditions in both cell lines.

Cloning and functional characterization of the ovine Hormone Sensitive Lipase (HSL) full-length cDNAs: an integrated approach.

Hormone Sensitive Lipase (HSL) is a highly regulated enzyme that mediates lipolysis in adipocytes. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signalling cascade reactions. Since HSL constitutes the key enzyme in the regulation of lipid stores and the only enzyme being subjected to hormonal regulation [in terms of the recently identified Adipose Triglyceride Lipase (ATGL)], the ovine Hormone Sensitive Lipase (ovHSL) full-length cDNA clones were isolated, using a Polymerase Chain Reaction-based (PCR) strategy. The two isolated isoforms ovHSL-A and ovHSL-B contain two highly homologous Open Reading Frame (ORF) regions of 2.089 Kb and 2.086 Kb, respectively, the latter having been missed the 688th triplet coding for glutamine (DeltaQ(688)). The putative 695 and 694 amino acid respective sequences bear strong homologies with other HSL protein family members. Southern blotting analysis revealed that HSL is represented as a single copy gene in the ovine genome, while Reverse Transcription-PCR (RT-PCR) approaches unambiguously dictated its variable transcriptional expression profile in the different tissues examined. Interestingly, as undoubtedly corroborated by both RT-PCR and Western blotting analysis, ovHSL gene expression is notably enhanced in the adipose tissue during the fasting period, when lipolysis is highly increased in ruminant species. Based on the crystal structure of an Archaeoglobus fulgidus enzyme, a three-dimensional (3D) molecular model of the ovHSL putative catalytic domain was constructed, thus providing an inchoative insight into understanding the enzymatic activity and functional regulation mechanisms of the ruminant HSL gene product(s).