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Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells ¹

Eastern Virginia Medical School, Center for Pediatric Research, Norfolk, Virginia, USA; and
^* Old Dominion University, Physical Electronics Research Institute, Hampton Boulevard, Norfolk, Virginia, USA

²Correspondence: Center for Pediatric Research, 855 W. Brambleton Ave., Norfolk, VA 23510, USA. E-mail: sbeebe{at}chkd.com

SPECIFIC AIMS

These studies were designed to determine the effects of high-intensity nanosecond pulsed electric fields (nsPEF) on cell structure and function. Because cells were never exposed to NsPEF during evolution, it is of interest to determine what signal transduction mechanisms and pathways are recruited by cells in response to nsPEF.

PRINCIPAL FINDINGS

1. As the pulse duration decreases, electroporation of the plasma membrane decreases (Fig. 1) and poration of intracellular membranes increases

View larger version (23K): [in this window] [in a new window]   Figure 1. Decreasing the pulse duration in the nanosecond range results in less plasma membrane poration: differences between cell types. HL-60 cells, and Jurkat cells were exposed in the presence of EthD-1 to 5 repetitive pulses (1–2 s intervals) for 10 ns (150 kV/cm), 60 ns (60 kV/cm), and 300 ns (26 kV/cm). The energy density was 1.7 J/cc/pulse in all cases. Cells were analyzed by flow cytometry. This shows the geometric mean EthD-1 fluorescence ±SE in 6–8 experiments for the cell types indicated.

2. NsPEF effects on the plasma membrane are independent of the energy density (Fig. 1)

3. Cell plasma membrane electroporation responses to nsPEF are different
Jurkat cells are more readily porated than HL60 cells (Fig. 1) and 3T3-L1 cells are not porated at all in response to nsPEF.

4. When the electric fields are sufficiently high, nsPEF induce apoptosis in human and mouse cells
Phosphatidylserine is externalized on the plasma membrane of intact cells, as indicated by the presence of annexin-V-FITC and the absence of ethidium homodimer fluorescence; caspase activity toward DEVD-afc is increased and the binding of a cell-permeable, fluorescent, irreversible inhibitor of active caspases (FITC-VAD-fmk) is increased in intact cells; Cytochrome c is released into the cytoplasm; and forward light scatter is decreased, consistent with cell shrinkage.

5. The caspase inhibitor z-VAD-fmk partially attenuated nsPEF-induced annexin-V-FITC binding, but had no significant effect on the nsPEF-induced decrease in forward light scatter or the increase in side light scatter

6. As the pulse duration of nsPEF is decreased (and effects on the plasma membrane decrease and those on intracellular membranes increase), caspase activation proceeds more slowly

7. When plasma membrane electroporation does occur in response to nsPEF, apoptosis markers appear more rapidly

8. NsPEF-induced apoptosis can occur in the absence of plasma membrane electroporation (Fig. 2)

View larger version (17K): [in this window] [in a new window]   Figure 2. In parallel experiments Jurkat cells were exposed to a single 10 nsPEF at various electric fields as indicated or analyzed without a pulse (control). Cells were either treated with nsPEF in the presence of EthD-1 and analyzed 3–5 minutes after treatment by flow cytometry (left panel) or treated with nsPEF in the absence of EthD-1, incubated with annexin-V-FITC as described in materials and methods, and analyzed by flow cytometry (right panel). Nearly identical results were observed in 4–5 experiments. HL60 cells responded similarly (not shown).

CONCLUSIONS AND SIGNIFICANCE

The application of nsPEF is a new, emerging technology derived from pulse power physics and developed originally for military applications. Recently this technology was found to be effective for bacterial decontamination, but effects on mammalian cells have not been investigated thoroughly until now.

As indicated elsewhere and further detailed in this report, the effects of nsPEF are distinct from classical plasma membrane electroporation. An electrical model for biological cells predicts increasing transparency of the outer membrane for electromagnetic waves with frequencies greater than the ß frequency. This is equivalent to the statement that for pulses with durations shorter than the charging time of the plasma membrane, there is an increasing probability for electric field interactions to occur with intracellular structures and a decreasing probability for modification of the plasma membrane. We recently presented the first experimental evidence that supported this hypothesis by showing that intracellular granules in human blood eosinophils were breached while the plasma membranes remained intact. The studies reported here further support this hypothesis.

In the studies reported here, we determine more specifically the effects of nsPEF on mammalian cell structure and function. Using flow cytometry, enzymatic caspase assays, and immunoblot analysis, we show that as the pulse duration decreases, effects on the plasma membrane decrease, and apoptosis is induced rapidly, at least in part through mitochondrial- and caspase-dependent mechanisms. The temporal progress of apoptosis is dependent on the pulse duration, with apoptosis proceeding more rapidly with longer pulses (300>60>10 ns). This suggests that as the pulse duration decreases there is a continuum of effects on apoptosis signaling mechanisms from the outside of the cell to the cell interior. In addition to this or alternatively, apoptosis signals originating from the plasma membrane are more robust than apoptosis signals originating from the cell interior.

We show that nsPEF-induced apoptosis is independent of plasma membrane electroporation and thermal changes and occurs by recruiting intracellular and plasma membrane apoptosis signaling mechanisms that are used by other apoptotic stimuli. This latter point is relevant because, unlike other apoptotic stimuli such as Fas, serum withdrawal, UV radiation, cytotoxic stimuli, and other stresses, cells have never been exposed to nsPEF until now.

When plasma membrane electroporation does occur in response to nsPEF (when the pulses are relatively long and/or the electric fields are relatively high), the postpulse responses to ethidium homodimer uptake suggest that putative plasma membrane pore number is lower, the pore size smaller, and/or pore resealing rates are more rapid. Further more, the structure and/or function of the pores appear to be different for nsPEF and classical electroporation conditions. Finally, in contrast to nsPEF, cells survive classical electroporation pulses.

As indicated in Fig. 3 ,potential intracellular targets for nsPEF include the mitochondria, the nucleus, and the plasma membrane. Other organelles may be involved in cell responses to nsPEF including the endoplasmic reticulum and Golgi complex (not illustrated). It is possible that depending on the nsPEF duration and/or electric field intensity, these structures may be affected differentially. The release of cytochrome c from the mitochondria into the cytoplasm suggests that the mitochondria are intracellular targets for nsPEF. However, it is not clear whether they are primary or secondary responders. Although Jurkat and HL60 cells exhibit mutated p53, it is possible that nuclear responses through p53 pathways are still possible. It is likely under some of the conditions tested here that there are multiple targets. Further studies are required to determine the mechanism and signaling pathways used by the cell in response to nsPEF, to define thresholds for apoptosis induction, and to determine the pulse characteristics that lead to cell responses.

View larger version (46K): [in this window] [in a new window]   Figure 3. Schematic diagram.

The results suggest that with decreasing pulse durations, nsPEF modulate cell signaling from the plasma membrane to intracellular structures and functions. NsPEF technology provides a unique, energy-independent tool to modulate intracellular structures and functions that can delete aberrant cells by apoptosis. Further analysis of nsPEF-induced effects on cells and tissues may reveal additional applications for this technology in basic cell biology and medicine.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0859fje; doi: 10.1096/fj.02-0859fje

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