Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5′-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure

Enhancement in the efficiency of polymerase chain reaction by TiO<SUB>2</SUB> nanoparticles: crucial role of enhanced thermal conductivity

Improvement of the specificity and efficiency of the polymerase chain reaction (PCR) by nanoparticles is an emerging area of research. We observed that TiO2 nanoparticles of ~ 25 nm diameter caused significant enhancement of PCR efficiency for various types of templates (namely plasmid DNA, genomic DNA and complementary DNA). By a series of experiments, the optimal TiO2 concentration was determined to be 0.4 nM, which resulted in up to a seven-fold increase in the amount of PCR product. As much as 50% reduction in overall reaction time (by reduction of the number of cycles and the time periods of cycles) was also achieved by utilizing TiO2 nanoparticles without compromising the PCR yield. Investigations of the mechanism of such PCR enhancement by simulations using the 'Fluent K epsilon turbulent model' provided evidence of faster heat transfer in the presence of TiO2 nanoparticles. Consistent with these findings, TiO2 nanoparticles were observed to augment the denaturation of genomic DNA, indicating more efficient thermal conductivity through the reaction buffer. TiO2 nanoparticle-assisted PCR may be useful for profound reduction of the overall PCR reaction period and for enhanced amplification of DNA amplicons from a variety of samples, including GC-rich templates that are often observed to yield unsatisfactory results

BCR signaling inhibitors differ in their ability to overcome Mcl-1-mediated resistance of CLL B cells to ABT-199

The Bcl-2 antagonist ABT-199 has demonstrated promising clinical activity in patients with CLL. ABT-199 is strongly cytotoxic against unstimulated peripheral blood CLL cellsin vitro, but is much less effective against CLL cells that have received survival signals from the microenvironment. In particular, stimulation of CLL cells with CD40L results in substantial resistance that is mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-xLand Bfl-1. In the present study we investigated whether resistance to ABT-199 can be conferred by B-cell receptor (BCR) stimulation, which is another important survival signal from the leukemic microenvironment. We show that sustained BCR stimulation results in significant ABT-199-resistance, which correlates with induction of the antiapoptotic protein Mcl-1 and less consistently with downregulation of proapoptotic Bmf, Hrk and BimEL A major role for Mcl-1 in conferring ABT-199 resistance is additionally supported by knockdown and enforced expression experiments with primary CLL cells. We further show that SYK, BTK and PI3K\u3b4 inhibitors significantly downregulate Mcl-1, but with different efficacy. Complete Mcl-1 downregulation was consistently achieved only with the SYK inhibitors R406 and GS-9973, whereas the BTK inhibitor ibrutinib and the PI3K\u3b4 inhibitor idelalisib in more than half of the cases had only a partial effect. The greater ability of SYK inhibitors to downregulate Mcl-1 correlated with their greater capacity to block BCR-mediated inactivation of GSK-3, a major negative regulator of Mcl-1. The finding that BCR-signaling inhibitors differ in their ability to target Mcl-1 is relevant for the design of clinical trials combining these agents with ABT-199

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2's hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366's cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.status: publishe