Synchronized Expression of Two Caspase Family Genes, ice-2 and ice-5, in Hydrogen Peroxide-Induced Cells of the Silkworm, Bombyx mori

Caspase family proteins play important roles in different stages of the apoptotic pathway. To date, however, functions of Bombyx mori L. (Lepidoptera: Bombycidae) caspase family genes are poorly known. This paper focuses on the morphology, mitochondrial membrane potential, and expression profiles of two novel B. mori caspase family genes (ice-2 and ice-5) in 3 µM hydrogen peroxide (H2O2) damaged B. mori cells, which were separated from the ovary of B. mori. In addition, comparisons were made between damage caused by H2O2 and by ultraviolet (UV) irradiation. The results showed that the potential change of the mitochondrial membrane occurred at 0.5 h after H2O2 stimulation, which was sooner than occurred in the UV treated model where the obvious decrease appeared at 6 h after stimulation. In addition, the total change in the potential of the mitochondrial membrane in H2O2 treated B. mori cells was larger than with UV treated cells during the whole process. Analysis of fluorescent quantitative real-time PCR demonstrated that ice-2 and ice-5 might be involved in both H2O2 and UV-induced apoptosis in B. mori cells. Notably, after exposure to H2O2, the expression patterns of ice-5 were remarkably higher than those of ice-2, while the result was the opposite after exposure to UV irradiation. The data indicate that apoptosis induced by H2O2 was directly related to the mitochondrial pathway. The two isoforms of B. mori ice may play different roles in the mitochondrion associated apoptotic pathway in B. mori cells, and the apoptotic pathway in H2O2 induced B. mori cells is different from the UV induced apoptotic pathway.


Introduction
As a member of the caspase (cys-teinylaspartate specific proteinase) family, interleukin -1beta-converting enzyme (ICE) was discovered in mammals and named caspase-1. It is considered the initiator in caspase-dependent apoptosis. ICE was identified as a CED-3-like protein in Caenorhabditis elegans (Yuan et al. 1993). In lepidopteran insects, ice was identified as a pro-death factor in the Heliothis virescens midguts developmental apoptotic process (Parthasarathy and Palli 2007). According to the reported sequences in GenBank, three silkworm ice homologsice, ice-2 and ice-5 -were described (Accession numbers: ice, AY885228; ice-2, DQ360829; and ice-5, DQ360830). In a previous study (Song et al. 2007) ice-2 and ice-5 were cloned with an open reading frame of 852 and 936 base pairs (bps), respectively.
Many agents that induce apoptosis are either oxidants or stimulators of cellular oxidative metabolism (Haddad 2004). H 2 O 2 is a reactive oxygen species. In general, reactive oxygen species are harmful to living organisms because they tend to cause oxidative damage to proteins, nucleic acids, and lipids (Hermes-Lima and Zenteno-Savín 2002). They also can induce various biological processes (Suzuki et al. 1997) and have been proposed as common mediators for apoptosis (Haddad 2004). H 2 O 2 is an oxidant that triggers caspase activation and subsequent apoptosis (Blackstone and Green 1999). Therefore, the oxidative damage model based on H 2 O 2 could be efficient for elucidating the roles of ice-2 and ice-5 in H 2 O 2 induced apoptosis. Kidd (1998) reported that H 2 O 2 -mediated caspase activation was dependent on the release of cytochrome c from mitochondria, suggesting a key role for this peroxide in mitochondrial permeability and leakage. Before the release of cytochrome c from the mitochondria, the mitochondrial membrane potential was lost (Twomey and McCarthy 2005).
This study attempted to characterize the genes of ice-2 and ice-5 in the early phase of H 2 O 2 induced apoptosis and to observe morphological and mitochondrial membrane potential changes in cells of Bombyx mori L. (Lepidoptera: Bombycidae). Meanwhile, time course transcriptional profiles of the two genes were investigated by quantitative realtime PCR. This report will provide new insight into the function of ICEs in insects. Additionally, damage caused by H 2 O 2 and UV irradiation were compared in this paper and may provide insight into the role of insect ICEs during the apoptosis processes.

B. mori cell culture
B. mori ovary-derived cells that were a gift of Dr. Xiangfu Wu (Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology) were cultured in TC-100 insect cell culture medium (Gibco brand, Invitrogen, www.invitrogen.com) supplemented with 10% fetal bovine serum at 27° C. H 2 O 2 was applied to the B. mori cells, which then were plated at a density of 2 106 cells in 6-well plates (Corning, www.corning.com). They were incubated for 3-5 days at 27° C, and then used for further studies.

Hydrogen peroxide treatment
Apoptosis was induced in B. mori cells by exposure to different concentrations (0.09 -90 M) of H 2 O 2 , and the median lethal dose (LD 50 ) was calculated. While incubating at the LD 50 H 2 O 2 concentration, B. mori cells were observed microscopically at specified intervals for the appearance of apoptotic bodies, and were collected at regular intervals.

UV irradiation treatment
The cells, with a very thin layer of phosphate buffered saline were irradiated for 20 s under UVA and UVB lamps at different UV doses (50 -5 mJ/cm 2 ). The total dosage was measured by a radiometer (International Light, Inc., www.intl-lighttech.com) fitted with a UV detector. At the LD 50 H 2 O 2 concentration, LD 50 , B. mori cells were observed microscopically at specified intervals for the appearance of apoptotic bodies, and were collected at regular intervals.

MTT assay for cell mortality
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect mortality and was carried out according to Fornelli et al. (2004). Five mg/ml MTT was dissolved in phosphate buffered saline, and 20 l of this stock solution was added to the culture wells. The incubation time with MTT was 3 h at 27° C. The supernatant was removed, and 150 μl of dimethyl sulfoxide was added to each well before reading optical density at 580 nm with fluorescence spectromety (Spectra Max, Gemini EM, Molecular Devices, www.moleculardevices.com). Mortality = 1viability.
For quantitative fluorescence measurement, cells were rinsed once after JC-1 staining and scanned with fluorescence spectrometry at 485-nm excitation and 530 and 590 nm emission, to measure green and red JC-1 fluorescence, respectively. Each well was scanned at 25 areas rectangularly arranged in 5 x 5 pattern with 1-mm intervals and an approximate beam area of 1 mm 2 (bottom scanning).

RNA extraction
Total RNA was extracted from the collected cells using Trizol (Invitrogen) according to the manufacturer's protocol. Contaminated genomic DNA was removed by Rnase-free Dnase I (Promega, www.promega.com). The concentration of the RNA was assessed using the Genspec III spectrophotometer (Hitachi Genetic Systems, www.biospace.com), and the integrity of the RNA was assessed by running 2 μl of RNA on a 1% ethidium bromide/agarose gel. The RNA was stored at -70° C until needed.
Reverse transcription 2 g DNase-treated RNA was reversetranscribed to single stranded cDNA in a 20 μl reaction containing 0.2 μmol/L oligo-dT, 0.5 mmol/L of each dNTP, 5 μl M-MLV 5 reaction buffer, and 200 U M-MLV reverse transcriptase (Promega). The thermal cycling profiles were as follows: 65° C for 5 min, 37° C for 60 min, and 75° C for 5 min. The resultant cDNA was stored at -20° C until needed.
Real-time PCR amplifications were performed to examine the relative expression of ice-2 and ice-5 in treated B. mori cells in the sequence detection system (MX3000P, Stratagene, www.stratagene.com). Duplicates of 0.5 l cDNA from each reverse transcription reaction were used as templates. The reactions were performed in a total volume of 50 μl using SYBR premix EXTaqTM perfect realtime kit (TaKaRa, www.takara-bio.com) as recommended by the manufacturer. The following MX3000P thermocycling program was used: denaturation program (3 min at 95° C), amplification and quantification program repeated 40 times (10 s at 95° C, 30 s at 58° C and 20 s at 72° C with a single fluorescence measurement), and melting curve program (55° C to 95° C with a heating rate of 0.1° C/s).
Relative expression levels of ice-2 and ice-5 were calculated with the comparative Ct (2 -Ct) method. Means and standard errors for each time-point were obtained from the averages of three independent sample sets.

Statistical analysis
Data are presented as the mean ± SD or mean ± SE of results of two or three separate experiments, as specified in the figure legends. Statistical significance was calculated (SPSS11.5, SPSS Inc., www.spss.com) with one-way ANOVA and one-sample T test. The p value lower than 0.05 was considered as significant.

Sequence analysis of ice-2 and ice-5
Sequence analysis suggested that B. mori ice-2 and ice-5 resemble human caspase-3, which plays a role as an effector and depends on the release of cytochrome c from the mitochondrion. Interestingly, expression of the ice isoform was not detected in the previous study, since no copies of ice were found in dozens of sequenced clones in UVtreated B. mori cell line (Song et al. 2007). Moreover, the isoforms, ice-2 and ice-5, were transcribed from the same gene but spliced differently under UV irradiation, and they both have a QACRG active site that belongs to the caspase family (Song et al. 2007). Sequence analysis revealed that ice-2 had seven exons, while ice-5 had eight. The difference between the two genes was that ice-5 contained an extra exon with 84 bp, and the 28 amino acids are unique to ice-5 (Figure 1).

LD 50 values for H 2 O 2 and UV irradiation that induce cell apoptosis
Apoptosis was induced in B. mori cells by exposure to different concentrations (0.09 -90 M) of H 2 O 2 , and the LD 50 value was calculated using the MTT assay. The same test was repeated with UV irradiation. Table1 shows that the best concentration of H 2 O 2 was 3 M because mortality (49.074%) of 3 Mtreated B. mori cells was nearest to LD 50 . The best dose of UV irradiation was 20 mJ/cm 2 ,  with a mortality rate of 45.961%, which was the nearest to LD 50 .

Morphological change of cells after H 2 O 2 stimulation
Using a microscope, B. mori cells were observed after H 2 O 2 stimulation at regular intervals from 0 to 12 h. As time passed, the morphology of the cells changed. However, in the first 4 h after stimulation, there were a few cells that had different morphology from the normal cells (Figure 2). Then some cell membranes wrinkled and the cells became smaller than normal cells by 5 h after stimulation. By 6 h after stimulation, wrinkling was more obvious. Bubble-like bodies appeared around wrinkled cells at 9 h post-stimulation. Vesicles formed in cell membranes, and apoptotic bodies were observed from the 10 h to 12 h phase.
Change in mitochondrial membrane potentials B. mori cells were acutely exposed to 3 M H 2 O 2 and were tested at different times using the JC-1 assay. The results showed that during  . Expression profiles of ice-2 and ice-5 in Bombyx mori cells after H2O2 stimulation. Real-time PCR analyses were performed using total RNA from cells that were collected at regular intervals from 0 h to 6 h after H2O2 stimulation. The relative ice-2 (F = 255.187; df = 7, 16; p = 0.0001) and ice-5 (F = 102.894; df = 7, 16; p = 0.0001) expression levels as calculated by 2 Ct are shown for each group, and the bar charts (mean ± SE) represent three independent experiments with three replications. High quality figures are available online. the first 5 h, the 590:530 fluorescence ratio of JC-1 dye declined sharper than that during the following 7 h, and the change could be omitted compared to the later change ( Table  2). The red-green JC-1 fluorescence ratio started to decrease at 0.5 h after H 2 O 2 stimulation. After dramatically declining, the red-green JC-1 fluorescence ratio tailed off steadily from 6 h to 12 h after-stimulation.

Expression profiles of the ice-2 and ice-5 genes
The relative expression of mRNA of ice-2 and ice-5 of H 2 O 2 stimulated B. mori cells was analyzed by quantitative real-time PCR. The ice-2 gene was highly expressed at two time points, 0.5 and 5 h after H 2 O 2 stimulation, while the expression level of ice-5 peaked at 0.5, 3, and 5 h after H 2 O 2 stimulation ( Figure  3). In other times, however, very low levels of both ice-2 and ice-5 mRNAs were detected. The mRNA level of ice-5 was higher than that of ice-2 at the majority of time stages from 0 to 6 h, except for the 5 h time point.

Comparisons between damage from H 2 O 2 and UV irradiation
Although at 5 h post stimulation, the images of dying B. mori cells treated with H 2 O 2 were distinct from UV treated cells, they both had similar appearances at 12 h (Figure 4). Apoptotic bodies could be found easily under a microscope at 200x magnification. Moreover H 2 O 2 treated cells formed membrane vesicles at 9 h, while UV treated cells started to vesicluate at 5 h, when the response of the cells to the stimuli was first detected. Additionally, throughout the process, the change in the fluorescence ratio of H 2 O 2 treated cells (10.413) was more obvious than that of the UV treated cells (4.938) ( Table 2). In H 2 O 2 treated cells, the fluorescence ratio declined at 0.5 h, but it declined at 6 h in UV treated cells (Table 2).

Discussion
As previously reported, the decrease of mitochondrial membrane potential started at the very beginning of the treatment and preceded the morphological change of the cells. This implies that apoptosis induced by H 2 O 2 might relate to the intrinsic apoptotic pathway via effects on the mitochondria. The peak levels of ice-2 and ice-5 were reached when the cellular morphology was still unchanged but the mitochondrial membrane potential had already changed considerably (Figures 2 and 3, Table 2), suggesting that the activation of B. mori ice-2 and ice-5 might be related to the release of cytochrome c from the mitochondria. Later, at 5 h after stimulation, changes in all the data were obvious. First, cell membranes were triggered to wrinkle, and cells became smaller than the ordinary cells. At the same time, the mitochondrial membrane potential steadily declined, beyond the dramatic decrease during the first 5 h. There was also another increase in the expression of ice-2 and ice-5. In Spodoptera frugiperda cells, oxidant treatments resulted in the release of cytochrome c followed by the activation of caspase-3 (Sahdev et al. 2003). Therefore, B. mori ICEs might be regulated by H 2 O 2 , and related to the dysfunction of mitochondria. ice-2 and ice-5 may also be initiators associated with mitochondria initially, and then be effectors following the dysfunction of mitochondria in H 2 O 2 induced apoptosis.
The fact that the genes of ice-2 and ice-5 were different from each other by just one exon implied that different mRNAs are present. This is likely related to the different patterns in their expression profiles. From 0 to 0.5 h after exposure to H 2 O 2 , while the level of ice-2 increased from low to high, the level of ice-5 increased from being undetectable to the highest level ( Figure 3). Then, after expressing stable levels for a while, ice-2 rose to its highest level, and ice-5 reached its second peak, suggesting that ice-5 may play a more active role in the early phase of H 2 O 2induced apoptosis than ice-2, and that they may have complementary functions. ice-2 and ice-5 might induce their own expression of in the later phases of apoptosis.
Based on the expression profiles, the levels of both ice-2 and ice-5 decreased significantly at 1 h after H 2 O 2 stimulation, and the level of ice-2 remained low from 1 to 4 h after H 2 O 2 stimulation. In contrast, the level of ice-5 fluctuated from low to a medium during this period. This was quite different from the profile of UV induced apoptosis ( Figure 5). During UV induced apoptosis, from 1 to 4 h post treatment, ice-5 was almost undetectable. This difference may have resulted in the changing morphology of B. mori cells at 5 h after stimulation. The unique expression patterns of ice-2 and ice-5 suggest that the single exon difference between them may be the reason for the unique role of ice-5 in the apoptotic pathway.
In addition, the total reduction in fluorescence ratio of H 2 O 2 treated cells is about 3 times more than the reduction in fluorescence ratio of UV treated cells. This suggests that H 2 O 2induced damage led to a more serious loss in the potential of the mitochondrial membrane (Table 2). This may have happened because UV irradiation damage to cells is only partly due to oxidative damage causing mitochondrion dysfunction (Kannan and Jain 2000). When the UV irradiation causes DNA mutation, DNA repair mechanisms might function to restore some mutations, so that both ice-2 and ice-5 were less active in UV stimulated cells.
In conclusion, ice-2 and ice-5 synchronal expression profiles indicate that activation of ice-2 and ice-5 may be related to mitochondrial dysfunction after H 2 O 2 -induced damage and that ice-2 and ice-5 might cooperate in the early phases of both H 2 O 2 and UV induced apoptosis in a B. mori cell line. The comparison between relative expression profiles of H 2 O 2 and UV-induced apoptosis suggests that the absence in ice-2 of an 84bp exon that exists in ice-5 might be the reason for lower activity of ice-2 than of ice-5 in the H 2 O 2 induced apoptosis pathway. Because UV irradiation not only induces the generation of OH and H 2 O 2 (Kannan and Jain 2000), but also can cause mutation of DNA, UV induced apoptosis is more complex than H 2 O 2 -induced apoptosis. This phenomenon would occur uniquely in UV irradiationinduced apoptosis and is a topic for further study.  (Wang et al. 2008). Real-time PCR analyses were performed using total RNA from cells that were collected at regular intervals from 0 h to 6 h after H2O2 stimulation. The relative ice-2 and ice-5 expression levels as calculated by 2 Ct are shown for each group, and the bar charts (mean ± SE) represent three independent experiments with three replications. High quality figures are available online.