In the previous study, we treated HSV-1-infected cells with a calcium ionophore, ionomycin, 18 h post infection (p.i.) in order to detect its enhancing effect on the release of HSV-120. In this condition, most cells attached to the plate and were releasing progeny viruses into culture medium, although further incubation gradually increased the number of detached cells. In the similar condition, we examined the effect of H₂O₂ on the release of HSV-1. When FI cells were infected with HSV-1 at a multiplicity of infection (MOI) of 2 plaque forming units (PFU)/cell, cultured for 18 h and treated with H₂O₂ at concentrations ranging from 0.1 to 5 mM for 2 h, cell-free virus was increased at 0.5, 1 and 5 mM; the increase at 1 and 5 mM was significant as compared with the untreated control (Fig. 1A). In contrast, the amount of cell-associated virus was not significantly changed (Fig. 1B). In the absence of H₂O₂, mean virus titers in cell-free and cell-associated fractions were 4.6 × 10⁶ and 1.1 × 10⁸ PFU/ml. After treatment with 1 mM H₂O₂ for 2 h, mean virus titers in these fractions were 2.6 × 10⁷ and 1.1 × 10⁸ PFU/ml, respectively. A six-fold increase as compared with the untreated control was observed in the cell-free fraction, but no increase was observed in the cell-associated fraction. The proportions of cell-free virus in the total amount of virus in the presence or absence of H₂O₂ were 22% and 4%, respectively, indicating that H₂O₂ markedly increased cell-free virus in the cultures.

It has been shown that H₂O₂ caused a sustained and uncontrolled rise in [Ca²⁺]i that preceded substantial cell injury or irreversible cell death 8. Whether H₂O₂ could affect the [Ca²⁺]i was examined at concentrations to enhance the virus release. FI cells were infected with HSV-1 at an MOI of 2 PFU/cell and cultured for 18 h. The mean level of [Ca²⁺]i in HSV-1-infected cells was approximately 200 nM. When the infected cells were treated with 1 mM H₂O₂, a significant rise in [Ca²⁺]i beginning approximately 30 sec after the exposure to H₂O₂ was observed. Subsequently, there was a secondary rise in [Ca²⁺]i, that appeared within 40 sec; a maximal level (460 nM) was attained in 6 min (Fig. 2A).

To determine the effect of calcium chelators, infected cells were treated with an extracellular calcium chelating agent, glycol-bis (beta-aminoethyl ether)-N',N',N',N'-tetraacetic acid (EGTA), for 20 min until 18 h p.i., and then H₂O₂ treatment was initiated. EGTA did not inhibit the immediate rise in [Ca²⁺]i significantly, but suppressed the secondary rise at a low level (Fig. 2B). When HSV-1-infected cells were exposed to an intercellular Ca²⁺ chelator, 1,2-bis (2-aminophenoxy)ethane- N',N',N',N'-tetraacetic acid (BAPTA) or quin-2, for 20 min prior to the H₂O₂ treatment, both the initial and secondary rises in [Ca²⁺]i were suppressed. Although the secondary rise was suppressed by this treatment, the level of [Ca²⁺]i gradually increased to 300–350 nM in 8 min (Fig. 2C and 2D).

The effect of Ca²⁺ depletion on the release of HSV-1 was examined. Eighteen hours after infection, cells were pretreated with 10 mM EGTA for 20 min to deplete extracellular Ca²⁺. Thereafter, treatment with 1 mM H₂O₂ for 2 h was initiated. In this condition, the amount of cell-free virus was 150% of that in the untreated control, whereas it was increased to 450% of the control value by the treatment with H₂O₂ (Fig. 3A). The amounts of cell-free virus in the presence of 50 μM BAPTA and 50 μM quin-2 were 250 % and 230 % of the control, respectively, indicating that the H₂O₂-mediated increase was diminished by BAPTA and quin-2. The amount of cell-associated virus in the cultures was not significantly altered by H₂O₂ in combination with EGTA, BAPTA or quin-2 (Fig. 3B). When HSV-1-infected cells were treated with EGTA, BAPTA or quin-2 only, the amount of cell-free virus was unchanged as compared with that in the untreated control (data not shown).

The effect of H₂O₂ on cell viability was examined by trypan blue exclusion. In mock-infected FI cells, the proportion of trypan blue-positive dead cells was 8%. After treatment with 1 mM H₂O₂ for 2 h, 28% of cells were positive trypan blue (Fig. 4A). When cells were infected with HSV-1 at an MOI of 2 PFU/cell and cultured for 20 h, 29% of cells were stained. After the treatment with 1 mM H₂O₂ from 18 to 20 h p.i., the proportion of dead cells was increased to 56% (Fig. 4B). The only detectable morphological change of H₂O₂-treated cells was enlargement of intercellular space due to cell rounding, irrespective of HSV-1 infection.

To determine the effect of Ca²⁺ chelators, HSV-1-infected cells were pretreated with 10 mM EGTA, 50 μM BAPTA or 50 μM quin-2 for 20 min and then treated with 1 mM H₂O₂ for 2 h. In the presence of EGTA, BAPTA and quin-2, the proportions of dead cells in H₂O₂-treated cultures were 38%, 34% and 36%, respectively, indicating that Ca²⁺ chelators reversed the effect of H₂O₂ (Fig. 5). When HSV-1-infected cells were treated with EGTA, BAPTA or quin-2 only, there were no changes in the proportion of dead cells (data not shown).

A number of studies have shown that H₂O₂ induced apoptosis with DNA fragmentation 891011. To clarify this issue, DNA was labeled by propidium iodide (PI) and subjected to flow cytometric analysis. In mock-infected cells treated with 1 mM H₂O₂ for 2 h, there were no apparent changes in the pattern of the cell cycle as compared with the untreated control (Fig. 6A and 6B). However, after treatment for 24 h, a sub-G1 peak appeared (Fig. 6C), indicating the induction of DNA fragmentation. When FI cells were infected with HSV-1 at an MOI of 2 PFU/cell and cultured for 18 h, the profile of DNA content was different from that of mock-infected cells. A broad peak was observed at the position of G₀/G₁ and the population of G₂/M phase was decreased (Fig. 6D), indicating the disturbance of cell cycle due to HSV-1 infection. Even if infected cells were treated with 1 mM H₂O₂ for 2 h or 24 h, a specific sub-G1 peak was not demonstrated (Fig. 6E and 6F)

When HSV-1-infected cells were treated with 1 mM H₂O₂ from 18 to 20 h after infection and subjected to Hoechst staining and annexin V staining, increase of apoptotic cells was not demonstrated (data not shown)

To gain further insight into the alterations caused by H₂O₂, electron microscopy was used. The cultures were fixed in situ and sections parallel to the dish surface were prepared. HSV-1-infected cells had large vesicular nuclei with dispersed chromatin. In the portion where cell-to-cell interaction was tight, a large number of viral particles were pooled in a narrow intercellular space (Fig. 7A and 7B). When HSV-1-infected cells were treated with 1 mM H₂O₂ from 18 to 20 h p.i., ruffling of the nuclear membrane and clustering of condensed chromatin at the nuclear periphery were observed, but the nuclear and cytoplasmic density was apparently unaltered. Cell shrinkage observed in apoptotic cells was not demonstrated. Generally, cell-to-cell junctions were enlarged, and as a consequence, viral particles pooled in the space were lost (Fig. 7C). Although the integrity of most of the plasma membrane was preserved, there were bubble-like structures that arose from the cell membrane (Fig. 7E). Occasionally, rapture of vacuoles containing organelles was observed on the cell surface (Fig. 7D). A focal defect of the plasma membrane was observed adjacent to transport vesicles containing viral particles at cell periphery (Fig. 7F and 7G).

Oral squamous cell carcinoma FI cells 37 were used as an epithelial cell line throughout the experiments. FI cells were grown in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and supplemented with a penicillin-streptomycin antibiotic mixture. The stock of HSV-1 strain KOS was grown and infectivity was determined by plaque assay in Vero cells.

To measure the amounts of cell-free virus, FI cells were infected with HSV-1 at an MOI of 2 PFU/cell. Thereafter, the infected cells were cultured for 18 h and then treated with H₂O₂. The culture plates were centrifuged at 400 × g for 5 min and the supernatant was harvested as a cell-free fraction and stored at -80°C until use. An equal volume of medium was added to each culture plate. For the measurement of cell-associated virus in a culture, the cells were subjected to two cycles of freezing and thawing. They were then centrifuged and the supernatant was harvested as a cell-associated fraction and stored at -80°C. The viral titer in each fraction was measured by assaying the formation of plaques in Vero cell monolayers and means of three determinations were obtained. Results were compared to those for the untreated controls and a percentage value was calculated. Differences of means were analyzed with the unpaired t-test.

[Ca²⁺]i was measured using the fluorescent Ca²⁺ indicator fura-2, which was incorporated intracellularly as its acetoxymethyl ester (fura-2/AM; Calbiochem, Cambridge, MA, USA). Cells were grown on glass-based plastic dishes and incubated with 4 μM fura-2/AM in DMEM for 30 min at 37°C. Cells were then washed in modified Hank's solution (Sigma) containing 137 mM NaCl, 3.5 mM KCl, 0.44 mM KH₂PO₄, 25 mM NaHCO₃, 0.33 mM Na₂HPO₄ and 0.5 mM CaCl₂ for a further 20 min at room temperature. To deplete extracellular Ca²⁺, cells were treated with 10 mM EGTA (Calbiochem) for 10 min prior to the H₂O₂ treatment. For buffering [Ca²⁺]i, cells were pretreated with 50 μM of the acetoxymethyl ester of BAPTA (BAPTA/AM; Calbiochem) or 50 μM of the acetoxymethyl ester of quin-2 (quin-2/AM; Calbiochem) for 10 min. After the addition of H₂O₂, [Ca²⁺]i was measured in individually identified fura-2-loaded cells using alternating excitation wavelengths (340 and 380 nm) with an AQUACOSMOS ratio imaging application software (HAMAMATSU Photonics, Hamamatsu, Japan) and an inverted epifluorescence microscope (DIAPHOT 300, Nikon). In order to evaluate its ability to quantify [Ca²⁺]i, the instrument was tested on Ca²⁺ buffer solutions (Molecular Probes) with known values of [Ca²⁺]i, using fura-2/AM 38; 7 cells were monitored for each experiment.

Cell viability was determined by trypan blue dye exclusion analysis. Cells dissociated by the EDTA-trypsin solution were mixed with an equal volume of phosphate-buffered saline containing 0.24% trypan blue and observed with a microscope. We counted the numbers of stained and unstained cells. Results were compared to those for the untreated controls and a percentage value was calculated. Differences of means were analyzed with unpaired t-test.

FI cells were dissociated in the EDTA-trypsin solution. Isolated cells were added to ice-cold 70% ethanol and then incubated at -20°C for 4 h. Thereafter, cells were centrifuged and incubated with phosphate-citrate buffer for 30 min at room temperature. They were again centrifuged, incubated with 10 μg/ml PI and 10 μg/ml RNase A for 20 min at room temperature, and then analyzed with a Becton Dickinson FACSort (Becton Dickinson, San Jose, CA).

Cells grown on plastic dishes were fixed in 2% glutaraldehyde (TAAB, Berkshire, England) for 2 h, washed with sodium cacodylate buffer and then postfixed in 1% osmium tetroxide (TAAB) for 2 h. Thereafter, cells were dehydrated in a graded series of ethanol and flat embedded in epoxy resin. Sections were cut parallel to the surface of the dishes. They were then stained with 4% uranyl acetate and 0.1% lead citrate (TAAB) and examined with a HITACHI H-7500 electron microscope.