Caveolin-1 is Involved in Regulating the Biological Response of Cells to Nanosecond Pulsed Electric Fields.

Nanosecond pulsed electric fields (nsPEFs) induce changes in the plasma membrane (PM), including PM permeabilization (termed nanoporation), allowing free passage of ions into the cell and, in certain cases, cell death. Recent studies from our laboratory show that the composition of the PM is a critical determinant of PM nanoporation. Thus, we hypothesized that the biological response to nsPEF exposure could be influenced by lipid microdomains, including caveolae, which are specialized invaginations of the PM that are enriched in cholesterol and contain aggregates of important cell signaling proteins, such as caveolin-1 (Cav1). Caveolae play a significant role in cellular signal transduction, including control of calcium influx and cell death by interaction of Cav1 with regulatory signaling proteins. Present results show that depletion of Cav1 increased the influx of calcium, while Cav1 overexpression produced the opposite effect. Additionally, Cav1 is known to bind and sequester important cell signaling proteins within caveolae, rendering the binding partners inactive. Imaging of the PM after nsPEF exposure showed localized depletion of PM Cav1 and results of co-immunoprecipitation studies showed dissociation of two critical Cav1 binding partners (transient receptor potential cation channel subfamily C1 (TRPC1) and inositol trisphosphate receptor (IP3R)) after exposure to nsPEFs. Release of TRPC1 and IP3R from Cav1 would activate downstream signaling cascades, including store-operated calcium entry, which could explain the influx in calcium after nsPEF exposure. Results of the current study establish a significant relationship between Cav1 and the activation of cell signaling pathways in response to nsPEFs.

Receptor- and store-operated mechanisms of calcium entry during the nanosecond electric pulse-induced cellular response.

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP1). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP2) depletion resulting in physiological responses similar to those observed following stimulation of Gq11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd3+, 300 µM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP2 and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD3+ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 µM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP1 uptake compared to Gd3+. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd3+ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP1 and Ca2+ cannot be used solely as markers of nsEP-induced nanoporation.

Nanosecond pulsed electric field induced dose dependent phosphatidylinositol-4,5-bisphosphate signaling and intracellular electro-sensitization.

Previously, it was demonstrated that nanometer-sized pores (nanopores) are formed in outer cellular membranes after exposure to nanosecond electric pulses (nsEPs). We reported that plasma membrane nanoporation affects phospholipids of the cell membrane, culminating in cascading phosphoinositide phosphatidylinositol-4,5-bisphosphate (PIP2) intracellular signaling. In the current study, we show that nsEPs initiated electric field (EF) dose-dependent PIP2 hydrolysis and/or depletion from the plasma membrane. This process was confirmed using fluorescent optical probes of PIP2 hydrolysis: PLCδ-PH-EGFP and GFP-C1-PKCγ-C1a. The 50% maximum response occurs with a single 600ns pulse achieving an effective dose (ED50) of EF~8kV/cm within our model cell system. At 16.2kV/cm, the ED50 for the pulse width was 484ns. Reduction of the pulse width or EF amplitude gradually reduced the observed effect, but twenty 60ns 16.2kV/cm pulses produced an effect similar to a single 600ns pulse of the same amplitude. Propidium iodide (PI) uptake after the nsEP exposure confirmed a strong relationship between EF-induced plasma membrane impact and PIP2 depletion. These results have expanded our current knowledge of nsEPs dependent cell physiological effects, and serve as a basis for model development of new exposure standards, providing novel tools for drug independent stimulation and approaches to differential modulation of key cellular functions.

The biological response of cells to nanosecond pulsed electric fields is dependent on plasma membrane cholesterol.

Previous work from our laboratory demonstrated nanopore formation in cell membranes following exposure to nanosecond pulsed electric fields (nsPEF). We observed differences in sensitivity to nsPEF in both acute membrane injury and 24h lethality across multiple cells lines. Based on these data, we hypothesize that the biological response of cells to nsPEF is dependent on the physical properties of the plasma membrane (PM), including regional cholesterol content. Results presented in this paper show that depletion of membrane cholesterol disrupts the PM and increases the permeability of cells to small molecules, including propidium iodide and calcium occurring after fewer nsPEF. Additionally, cholesterol depletion concurrently decreases the "dose" of nsPEF required to induce lethality. In summary, the results of the current study suggest that the PM cholesterol composition is an important determinant in the cellular response to nsPEF.

Activation of autophagy in response to nanosecond pulsed electric field exposure.

Previous work demonstrated significant changes in cellular membranes following exposure of cells to nanosecond pulsed electric fields (nsPEF), including nanoporation and increases in intracellular calcium concentration. While it is known that nsPEF exposure can cause cell death, how cells repair and survive nsPEF-induced cellular damage is not well understood. In this paper, we investigated whether autophagy is stimulated following nsPEF exposure to repair damaged membranes, proteins, and/or organelles in a pro-survival response. We hypothesized that autophagy is activated to repair nsPEF-induced plasma membrane damage and overwhelming this compensatory mechanism results in cell death. Activation of autophagy and subsequent cell death pathways were assessed measuring toxicity, gene and protein expression of autophagy markers, and by monitoring autophagosome formation and maturation using fluorescent microscopy. Results show that autophagy is activated at subtoxic nsPEF doses, as a compensatory mechanism to repair membrane damage. However, prolonged exposure results in increased cell death and a concomitant decrease in autophagic markers. These results suggest that cells take an active role in membrane repair, through autophagy, following exposure to nsPEF.

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses.

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.

Using a portable terahertz spectrometer to measure the optical properties of in vivo human skin.

Terahertz (THz) time-domain spectroscopy systems permit the measurement of a tissue's hydration level. This feature makes THz spectrometers excellent tools for the noninvasive assessment of skin; however, current systems are large, heavy and not ideal for clinical settings. We previously demonstrated that a portable, compact THz spectrometer permitted measurement of porcine skin optical properties that were comparable to those collected with conventional systems. In order to move toward human use of this system, the goal for this study was to measure the absorption coefficient (µa) and index of refraction (n) of human subjects in vivo. Spectra were collected from 0.1 to 2 THz, and measurements were made from skin at three sites: the palm, ventral and dorsal forearm. Additionally, we used a multiprobe adapter system to measure each subject's skin hydration levels, transepidermal water loss, and melanin concentration. Our results suggest that the measured optical properties varied considerably for skin tissues that exhibited dissimilar hydration levels. These data provide a framework for using compact THz spectrometers for clinical applications.

Effects of a TASER® conducted energy weapon on the circulating red-blood-cell population and other factors in Sus scrofa.

In previous studies hematocrit has been consistently increased in an anesthetized animal model after exposures to TASER(®) conducted energy weapons (CEWs). In the present study we analyzed changes in blood cell counts and red blood cell membrane proteins following two 30-s applications of a TASER C2 device (which is designed for civilian use). Hematocrit increased significantly from 33.2 ± 2.4 (mean ± SD) to 42.8 ± 4.6 % immediately after CEW exposure of eleven pigs (Sus scrofa). Red blood cell count increased significantly from 6.10 ± 0.55 × 10(12)/L to 7.45 ± 0.94 × 10(12)/L, and mean corpuscular volume increased significantly from 54.5 ± 2.4 fl to 57.8 ± 2.6 fl. Mean corpuscular hemoglobin concentration decreased significantly from 20.5 ± 0.7 to 18.5 ± 0.6 mM. Thirty protein spots (from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, selected for detailed comparison) exhibited greater densities 30-min post-exposure compared with pre-exposure values. A greater number of echinocytes were observed following CEW exposure. On the basis of these results it appears that, during the strong muscle contractions produced by TASER CEWs, a specific population of red blood cells (RBCs) may be released from the spleen or other reservoirs within the body. The total time of CEW exposure in the present study was relatively long compared with exposures in common law-enforcement scenarios. Despite statistically significant changes in red blood cell counts (and other measures directly related to RBCs), the alterations were short-lived. The transient nature of the changes would be likely to counteract any potentially detrimental effects.

Prevention of metastases with a Mage-b DNA vaccine in a mouse breast tumor model: potential for breast cancer therapy.

Anti-tumor vaccines are a relatively non-toxic alternative to conventional chemotherapeutic strategies to control breast cancer. Immunization with tumor-associated antigens (TAAs) triggers anti-tumor cytotoxic T lymphocytes (CTL), which can limit tumor progression. Here we report on the development and effectiveness of a TAA-based DNA vaccine encoding Mage-b1/2, the mouse homologue of the human MAGE-B1/2. As model system, we used immune competent Balb/c mice with syngeneic non-metastatic (64pT) or metastatic (4TO7cg) breast tumors. First, the presence of Mage-btranscripts in the 64pT and 4TO7cg breast tumors and metastases was demonstrated by RT-PCR, Southern blotting, and DNA sequencing. A DNA-based vaccine was developed from transcripts of one of the 64pT tumors, encoding the complete Mage-b1/2 protein, and subsequently tested for its preventive efficacy in both breast tumor models. Mice were immunized two times intramuscularly with the vaccine (pcDNA3.1-Mage-b1/2-V5), the control vector (pcDNA3.1-V5), or saline. Two weeks after the last immunization, the syngeneic 4TO7cg or 64pT tumor cell lines were injected in a mammary fat pad. Mice were monitored during the next 4 weeks for tumor formation, latency and size, and subsequently sacrificed for analysis. While the Mage-b1/2 vaccine had only a minor effect on the latency and growth of primary tumors, a significant and reproducible reduction in the number of 4TO7cg metastases was observed (vaccine versus control vector, p=0.0329; vaccine versus saline, p=0.0128). The observed protective efficacy of the Mage-b DNA vaccine correlated with high levels of vaccine-induced IFNgamma in spleen and lymph nodes upon re-stimulation in vitro. These results demonstrate the potential of TAA-based DNA vaccines in controlling metastatic disease in breast cancer patients.

Behavior of metastatic and nonmetastatic breast tumors in old mice.

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.