Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells.

The ability of anti-heat shock protein 90 (Hsp90) drugs to attenuate NF-κB-mediated transcription is the major basis for their anti-inflammatory properties. While the molecular mechanisms underlying this effect are not clear, they appear to be distinct in human endothelial cells. We now show for the first time that type 2 sirtuin (Sirt-2) histone deacetylase binds human NF-κB target gene promoter and prevents the recruitment of NF-κB proteins and subsequent assembly of RNA polymerase II complex in human lung microvascular endothelial cells. Hsp90 inhibitors stabilize the Sirt-2/promoter interaction and impose a "transcriptional block," which is reversed by either inhibition or downregulation of Sirt-2 protein expression. Furthermore, this process is independent of NF-κB (p65) Lysine 310 deacetylation, suggesting that it is distinct from known Sirt-2-dependent mechanisms. We demonstrate that Sirt-2 is recruited to NF-κB target gene promoter via interaction with core histones. Upon inflammatory challenge, chromatin remodeling and core histone H3 displacement from the promoter region removes Sirt-2 and allows NF-κB/coactivator recruitment essential for RNA Pol II-dependent mRNA induction. This novel mechanism may have important implications in pulmonary inflammation.

Facilitation of electroporative drug uptake and cell killing by electrosensitization.

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre-treated with 100-µs EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100-µs EP into two trains of four EP each (with 5-min. interval) decreased the LD(50) 1.5-2 times. Sensitization profoundly enhanced the electroporation-assisted uptake of bleomycin, a cell-impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split-dose EP exposure increased bleomycin-mediated lethality to the same extent as a 10-fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split-dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization.

Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells.

BACKGROUND: Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-µs EP. METHODS: Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry. RESULTS: 10-ns EP caused apoptotic or necrotic death within 2-20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S=alphaD((-K)), where coefficients K and alpha determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas alpha was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only. CONCLUSIONS: 1.8- and 9-µs EP cause cell death efficiently and indiscriminately (LD50 1-3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD50 50-80 J/g for Jurkat and 400-500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms. GENERAL SIGNIFICANCE: Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.

Diagnosis of Ovarian Cancer Using Decision Tree Classification of Mass Spectral Data.

Recent reports from our laboratory and others support the SELDI ProteinChip technology as a potential clinical diagnostic tool when combined with $n$ -dimensional analyses algorithms. The objective of this study was to determine if the commercially available classification algorithm biomarker patterns software (BPS), which is based on a classification and regression tree (CART), would be effective in discriminating ovarian cancer from benign diseases and healthy controls. Serum protein mass spectrum profiles from 139 patients with either ovarian cancer, benign pelvic diseases, or healthy women were analyzed using the BPS software. A decision tree, using five protein peaks resulted in an accuracy of 81.5% in the cross-validation analysis and 80%in a blinded set of samples in differentiating the ovarian cancer from the control groups. The potential, advantages, and drawbacks of the BPS system as a bioinformatic tool for the analysis of the SELDI high-dimensional proteomic data are discussed.

Two modes of cell death caused by exposure to nanosecond pulsed electric field.

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity ("nanoelectroporation"), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1-2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6-24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.

Electroporation-induced electrosensitization.

BACKGROUND: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. METHODOLOGY/PRINCIPAL FINDINGS: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold. CONCLUSIONS/SIGNIFICANCE: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency.

In vitro estrogen receptor binding of PCBs: measured activity and detection of hydroxylated metabolites in a recombinant yeast assay.

The estrogenic activities of 17beta-estradiol, biphenyl, chlorinated biphenyls, and Aroclor mixtures 1221, 1242, and 1248 were measured with a modified recombinant yeast estrogen assay (i.e., a Saccharomyces cerevisiae-based lac-Z (beta-galactosidase) reporter assay). Modifications of the assay included the use of glass vials instead of plastic microtiter plates and the addition of the medium and yeast before the test substrate. 14C-labeled compounds were used to follow improvements in the assay procedures. 14C-17beta-estradiol recovery from plastic microtiter plates and glass vials using the standard or the modified procedure was approximately 89%. However, 14C-4-CB (4-chlorobiphenyl) recovery was considerably less, ranging from 3% in plastic microtiter plates using the standard procedure to 26% in vials using the modified procedure. These results suggest that the toxicity of strongly hydrophobic chemicals may be underestimated. Using the modified yeast estrogen assay, full agonist activity was observed for 4-CB, 2,4,6-CB, and 2,5-CB while each of the Aroclor mixtures were only partial agonists. The equivalent EC50 values in ppm were in environmentally relevant concentrations for biphenyl (19 ppm), 4-CB (4.5 ppm), 2,5-CB (21 ppm), 2,4,6-CB (0.8 ppm), Aroclor 1221 (2.9 ppm), Aroclor 1242 (0.65 ppm), and Aroclor 1248 (2.3 ppm). Estrogen receptor binding for the individual PCB congeners was 25- to 650-fold less than the reported estrogen binding for the corresponding hydroxylated PCB metabolite. Gas chromatographic/mass spectrometric analysis of yeast extracts indicated that S. cerevisiae hydroxylated the individual PCB congeners in the ppb range. With the exception of biphenyl, the concentration of hydroxylated metabolites obtained from incubation of S. cerevisiae with PCB congeners was consistent with the concentration necessary to elicit a positive estrogen receptor-binding response. This work provides evidence that S. cerevisiae are capable of metabolic transformation of PCBs and that estrogen receptor binding of PCBs is mediated through the hydroxylated metabolite rather than through the direct interaction of the PCB congeners with the estrogen receptor.

Pharmacoproteomic analysis of prechemotherapy and postchemotherapy plasma samples from patients receiving neoadjuvant or adjuvant chemotherapy for breast carcinoma.

BACKGROUND: In this study, proteomic changes were examined in response to paclitaxel chemotherapy or 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC) chemotherapy in plasma from patients with Stage I-III breast carcinoma. The authors also compared the plasma profiles of patients with cancer with the plasma profiles of healthy women to identify breast carcinoma-associated protein markers. METHODS: Sixty-nine patients and 15 healthy volunteers participated in the study. Plasma was sampled on Day 0 before chemotherapy and on Day 3 posttreatment in the 69 patients or 3 days apart in the 15 healthy women. Twenty-nine patients received preoperative chemotherapy, and 40 received postoperative chemotherapy. Surface-enhanced laser desorption/ionization mass spectrometry was used to generate protein mass profiles. RESULTS: Few changes were observed in plasma during treatment. Only 1 protein peak was identified (mass/charge ratio [m/z], 2790) that was induced by paclitaxel and, to a lesser extent, by FAC chemotherapy. This proteomic response was detectable in 80% of patients who were treated preoperatively but also was present with lesser intensity in approximately 40% of patients treated postoperatively. There was no clear correlation between induction of m/z 2790 during a single course of treatment and final tumor response to preoperative chemotherapy. Five other peaks also were identified that discriminated between plasma from patients with breast carcinoma and plasma from normal women. These same peaks also were detectable in a subset of patients who already had undergone surgery to remove their tumors. CONCLUSIONS: A single chemotherapy-inducible SELDI-MS peak and five other peaks that distinguished plasma obtained from patients with breast carcinoma from plasma obtained from normal, healthy women were identified. The (as yet unsequenced) proteins represented by these peaks are candidate markers of micrometastatic disease after surgery.