Chemical Perturbation of Mcl‑1 Pre-mRNA Splicing to Induce Apoptosis in Cancer Cells

The myeloid cell leukemia-1 (<i>MCL1</i>) gene encodes antiapoptotic Mcl-1_L and proapoptotic Mcl-1_S proteins. In cancer, the Mcl-1_L/Mcl-1_S ratio is very high, accounting for the antiapoptotic nature of cancer cells. As such, reducing this ratio can render the cancer cells prone to apoptosis. The Mcl-1_L/Mcl-1_S ratio is determined in the alternative pre-mRNA splicing step that is regulated by splicing factor 3B1 (SF3B1). Here, we report that meayamycin B, a potent inhibitor of SF3B1, reversed the dominant isoform from Mcl-1_L to Mcl-1_S at the mRNA and protein levels. The resulting proapoptotic cellular environment was further exploited; when meayamycin B was combined with Bcl-x_L inhibitor ABT-737, the combination treatment triggered apoptosis in nonsmall cell lung cancer A549 and H1299 cells that were otherwise resistant to ABT-737. These results demonstrate that perturbation of the <i>MCL1</i> splicing with small molecule inhibitors of SF3B1 provides a means to sensitize cancer cells toward Bcl-x_L inhibitors

Development of a Sustainable Enrichment Strategy for Quantification of Mercury Ions in Complex Samples at the Sub-Parts per Billion Level

To limit environmental exposure of mercury species, government bodies restrict emissions of various environmental mercury sources to sub-parts per billion (ppb) levels. Current methods for detection of mercury are time-consuming and expensive and suffer from many drawbacks. Optical methods are in principle less intensive but have not yet been implemented for real-world applications because of a lack of sufficient sensitivity and robustness. We previously reported a fluorometric method for quantifying mercury ions based on the oxymercuration of a vinyl ether with a detection limit of 1 ppb, not meeting the requirement by government bodies. To fill the gap between our previous method and the governments’ restrictions, we have developed a method to enrich complex samples with mercury ions through the use of a recyclable thiol-based resin and the novel chemistry of mercury release. The combination of our previous fluorometric method and the new enrichment chemistry allowed the detection of 0.1 ppb mercury in a complex synthetic sample

A High-Throughput Method To Detect Palladium in Ores

Platinum-group metals are in increasing demand for industrial use. Herein, we present a fluorometric method for palladium detection that can be used to prioritize ore samples on site. The analyses of solid ore samples were sufficiently correlated with that of aqua regia extracts of the same ore samples. Twenty samples could be analyzed in parallel and more samples can be if so desired due to the high throughput nature of the assay method. Although the correlational study was performed after 90 min incubation, 30 min incubation was sufficient to obtain strong fluorescence signals (i.e., 1.5 min per sample). Given the visible nature of the green fluorescence and the procedural simplicity, palladium and coexisting platinum ores are identifiable in the field with the naked eye

Synthesis of Violaceic Acid and Related Compounds through Aryl Triazene

MPC1001 is a potent anticancer natural product that contains a violaceic acid moiety. Herein we report the total synthesis of the natural product violaceic acid and its derivative. In this approach, a triazene-directed Ullman coupling proved to be highly effective. We converted the triazene to a hydroxy group by means of a palladium-catalyzed reaction. Treatment of the triazene with trifluoroacetic acid generated an arenediazonium ion that produced an aryl radical, leading to the protodediazoniation and a tricyclic product

Total Synthesis and Biological Studies of TMC-205 and Analogues as Anticancer Agents and Activators of SV40 Promoter

TMC-205 is a natural fungal metabolite with antiproliferative activity against cancer cell lines. The light- and air-sensitivity prevented in-depth exploitation of this novel indole derivative. Herein, we report the first synthesis of TMC-205. On the basis of its reactivity with reactive oxygen species, we developed air-stable analogues of TMC-205. These analogues are 2–8-fold more cytotoxic than TMC-205 against HCT-116 colon cancer cell line. Importantly, at noncytotoxic dose levels, these analogues activated the transcription of luciferase reporter gene driven by simian virus 40 promoter (SV40). Further, these small molecules also inhibit firefly luciferase, presumably by direct interaction

Time-Insensitive Fluorescent Sensor for Human Serum Albumin and Its Unusual Red Shift

The concentration of human serum albumin (HSA) indicates the health state of individuals and is routinely measured by UV spectroscopy with bromocresol. However, this method tends to overestimate HSA, and more critically, depends highly on the timing, in seconds, of the measurements. Here, we report an analog of 2′,7′-dichlorofluorescein that can be used as a fluorescent sensor to quantify HSA in human sera. The accuracy of this new method proved superior to that of bromocresol when an international standard serum sample was analyzed. This method is more convenient than the bromocresol method because it allows for fluorescence measurements during a >15 min period. Colorimetric analysis was also performed to further investigate the effects of the binding of the sensor to HSA. These spectroscopic studies suggest that absorption and emission changes upon HSA binding may be due to the dehydration of the dye and/or stabilization of the tritylic cation species

PHF5A and U2 snRNP component SF3B1 interact with AAV capsid.

<p><b>(A)</b> HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. <b>(B)</b> Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10⁵) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. <b>(C)</b> AAV2 CMV-Luc vector (3 x 10¹⁰ genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. <b>(D)</b> HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10¹⁰ genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). <b>(E)</b> HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10¹⁰ genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. <b>(F)</b> HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. <b>(G)</b> Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. <b>(H)</b> HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10¹⁰ genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. <b>(I)</b> Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. <b>(J)</b> Control or AAV VP1-over-expressing 293T cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.</p

Screening of the siRNA library for proteasomal pathway genes identifies PHF5A as a factor blocking the transduction by AAV9 vector.

<p><b>(A)</b> Screening of the siRNA library was carried out by reverse transfection of HeLa cells with siRNAs, followed by infection with luciferase-expressing AAV9 vectors (AAV9 CMV-Luc) at a multiplicity of infection (MOI) of 10⁴, and assessment of luciferase expression. Screening of the library identified 12 candidate genes that increased transduction by AAV9 vectors over 10-fold. Further studies were carried out in HeLa cells transfected/transduced with specific siRNAs or shRNA lentivectors for each of the 12 genes to verify the screening candidates. <b>(B)</b> Quantitative real-time RT-PCR was performed to determine the levels of PHF5A transcripts in cells treated with control or PHF5A siRNAs at 48 hours. <b>(C)</b> HeLa cells were transfected with control or PHF5A siRNAs for 24 hours, followed by infection with AAV9 CMV-Luc vectors (MOI 10⁴) for an additional 48 hours. The luciferase assay was performed in order to determine relative luciferase activities in treated cells. <b>(D)</b> Same as C, except that a luciferase-expressing adenoviral vector at an MOI of 3 x 10² or an HIV-1-based lentiviral vector (MOI 0.3) were used to infect siRNA-treated HeLa cells. <b>(E)</b> Lentiviral vector pSIN-PHF5A-Escape with the PHF5A-HA Escape transgene was generated through introduction of three silent mutations in the PHF5A siRNA#1-targeted sequence. Western blotting was performed to verify the expression of the PHF5A-HA-Escape and its resistance to the PHF5A siRNA#1 treatment. Anti-PHF5A antibody was used to detect endogenous and over-expressed PHF5A-HA, while anti-HA antibody detected the HA-tagged PHF5A. <b>(F)</b> HeLa cell lines stably expressing the PHF5A-HA-Escape mutant were generated through lentiviral transduction of the escape mutant, followed by puromycin selection. Upon treatment with the PHF5A siRNA and AAV9 CMV-Luc vector (MOI 10⁴), luciferase expression was determined in control HeLa and PHF5A-HA-Escape-expressing HeLa cells. <b>(B-D, F)</b> Data are shown as averages of three independent experiments with error bars representing standard error of the mean. *p<0.05.</p

Meayamycin B increases AAV vector transduction of clinically relevant cell types.

<p><b>(A)</b> Primary mouse islets were infected with AAV8 CMV-GFP in the presence or absence of 2 nM meayamycin B, and GFP expression was monitored for three days. <b>(B)</b> Primary human islets were treated with AAV2 or AAV9 CMV-Luc vectors for 7 hours and then treated with 0, 2, 5 or 10 nM meayamycin B. Luciferase expression was analyzed 48 hours p.i. <b>(C)</b> Neonatal rat cardiomyocytes were infected with AAV2 CMV-Luc or scAAV9 CMV-GFP vectors and treated with meayamycin B, 3 hours p.i. Luciferase activity was measured 3 days p.i., while GFP expression was monitored at 5 days p.i. <b>(D)</b> Porcine hepatocytes were infected with AAV2 or AAV9 CMV-Luc vectors for 8 hours, virus was then removed and cells were treated with 0, 2, or 20 nM meayamycin B. Cells were harvested 48 hours p.i. for the luciferase assay. In A-D, an MOI of 10⁴ was used.</p

The U2snRNP complex plays the key role in restricting AAV vector transduction.

<p><b>(A)</b> HeLa cells were transfected with control siRNA, or siRNAs targeting PHF5A, histone 4, U2AF1, SF3B1, SF3B2 and SF3B3 for 24 hours, followed by the AAV9 CMV-Luc vector transduction (MOI 10⁴). Relative luciferase expression was determined 48 hours p.i. <b>(B)</b> Same as A, but luciferase-expressing adenoviral vector was used to transduce siRNA-treated cells (MOI of 3 x 10²). <b>(C)</b> Same as A, except that the siRNA-treated cells were transfected with the vector genome plasmid, pAAV CMV-Luc (0.2 μg/well)for 48 hours. Note that this plasmid was used to generate the infectious AAV9 CMV-Luc vector used in (A). <b>(D)</b> HeLa cells were treated with increasing concentrations of U2 snRNP inhibitor, meayamycin B, followed by transduction with the AAV9 CMV-Luc vector (MOI 10⁴). Relative luciferase expression was determined 48 hours p.i. <b>(E)</b> Same as D, except that a prespliceosome/A complex inhibitor, Isoginkgetin, was used. <b>(F)</b> HeLa cells were treated with indicated spliceosome inhibitors for 8 and 24 hours and levels of unspliced and spliced cellular MAPT (microtubule associated protein tau) transcripts were determined by RT-PCR. <b>(G)</b> HeLa cells were treated with 20 nM meayamycin B at various time points before or after AAV9 CMV-Luc vector infection (MOI 10⁴). Relative luciferase expression was determined 48 hours p.i. <b>(H)</b> HeLa cells were infected by AAV9 CMV-GFP (MOI 10³) or scAAV9 CMV-GFP vectors (MOI 6 x 10³), followed by treatment with 20 nM meayamycin B at 8 hours p.i. Flow cytometry analysis was performed to see GFP-positive cell populations at 48 hours p.i. <b>(I)</b> Co-treatment of HeLa cells with SF3B1 siRNA and Meayamycin B. HeLa cells were treated with the siRNAs for 48 hr, followed by infection with AAV9 CMV-Luc (MOI 10⁴). At 9 hours p.i. Meayamycin B (5nM) was added, and cells were harvested for the luciferase assay 48 hours p.i. <b>(J)</b> Co-treatment of HeLa cells with MG-132 and Meayamycin B. 30 min prior to AAV infection cells were treated with MG-132. 9 hours after infection with AAV9 CMV-Luc (MOI 10⁴), cells were treated with meayamycin B, and harvested for luciferase assay 20 hours later. Due to notable toxicity of MG-132, we needed to harvest cells at this early time point. <b>(K)</b> Influence of dual treatment with human adenovirus 5 infection and SF3B1 disruption on AAV vector infection. HeLa cells were treated with control or SF3B1 siRNAs for 24 hours, followed by infection with AAV2 CMV-Luc (MOI 10⁴) or co-infection with AAV2 CMV-Luc and human adenovirus 5 (MOI 3 x 10⁴) for 48 hours. <b>(L)</b> Influence of adenovirus 5 infection on subcellular localization of SF3B1 in HeLa cells. HeLa cells were infected with human adenovirus 5 (MOI 10⁴) for 24 hours, and SF3B1 in control and infected HeLa cells was visualized by anti-SF3B1 antibody (red). Nuclei were counter-stained by DAPI (blue). (A-E, G, I, J and K) Samples were run in triplicate and results are the average of two independent experiments. *p<0.05.</p