BACKGROUND
programmed cell death, also known as apoptosis, is a normal physiological cell suicide program that is highly conserved among vertebrates and invertebrates
<cit> .
apoptotic cells undergo a series of dramatic and characteristic alterations in cellular morphology, such as dna fragmentation, chromatin condensation, cytoskeleton reorganization, and plasma membrane blebbing
<cit> .
apoptosis plays an important role during development and tissue homeostasis eliminating discarded cells from the organism, including damaged and virus-infected cells.
for this reason, apoptosis acts as a host protection mechanism by which virus-infected cells are removed to limit the proliferation of viruses
<cit> .
thus, to overcome this defense response in cells, baculoviruses carry anti-apoptotic genes to inhibit programmed cell death
<cit> .
spodoptera frugiperda caspase- <dig> , the most studied effector caspase of lepidoptera, is the principal effector caspase of s. frugiperda  <dig>  cells, and is activated by various death stimuli, including baculovirus infection, ultraviolet  irradiation, and over-expression of pro-apoptotic genes
<cit> .
two types of anti-apoptotic genes have been identified in baculoviruses, p <dig>  and inhibitor of apoptosis
<cit> .
the p <dig> gene from autographa californica multicapsid nucleopolyhedrovirus  and p <dig> gene from spodoptera littoralis nucleopolyhedrovirus  are required to prevent apoptosis during the virus infection of s. frugiperda cells, such as sf <dig> and sf21
<cit> .
the op-iap from the orgyia pseudotsugata multicapsid nucleopolyhedrovirus  and sf-iap from the host, s. frugiperda cells, also suppress the apoptosis process
<cit> .
the baculovirus expression vector system  is a powerful tool used for the expression of foreign proteins in numerous insect cells, including sf <dig>  sf <dig> cells and trichoplusi ni cells
<cit> .
therefore, recombinant protein production using a bevs can be used for many pharmaceutical applications
<cit> .
exposure of cells to stressors, including high temperatures or a wide variety of physical and chemical insults, induces the expression of heat-shock proteins  in cells
<cit> .
hsps are molecular chaperones responsible for maintaining cell homeostasis and promoting cell survival
<cit> .
baculovirus infection also serves as a stress factor that can activate both death-inducing and cellular-protective pathways, and the heat-shock response is important for baculovirus replication in insect cells
<cit> .
moreover, the rate-limited expression of endoplasmic reticular  molecular chaperones is strongly associated with the maximal expression of exogenous proteins by bevs
<cit> .
few studies have examined the potential of rna inference  to increase protein production in the bevs
<cit> , however, several studies have demonstrated the efficiency of this approach in both insect cells and larvae
<cit> .
in our previous studies, we used dna vector-based approaches with endogenously expressed double-stranded rna  to silence its target gene, sf-caspase- <dig>  in sf <dig> cells.
therefore, the sf-caspase- <dig> mrna level and plasmid copy number in the sf-caspase-1-repressed stable cells were examined
<cit> .
in addition, sf-caspase-1-repressed stable cells exhibited resistance to apoptosis and enhancement of recombinant protein production
<cit> .
these results were consistent with later findings in t. ni cells
<cit> ; however, the mechanism of recombinant protein augmentation in baculovirus-infected caspase-repressed insect cells was not determined.
therefore, in this present study, we use rnai-mediated sf-caspase-1-repressed stable cells to clarify how resistance to apoptosis could enhance both intracellular  and extracellular  recombination protein production by bevs.
furthermore, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study.
RESULTS
stable transfection plasmid verification by genomic dna pcr and rt-pcr in sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells
stable cells  were established by transfection of the control vector pib or vector pibdscasp , and subsequently selected by bsd
<cit> .
after more than  <dig> passages, stable cell lines were analyzed by genomic dna pcr and rt-pcr to examine the inverted-repeat dna sequence of sf-caspase- <dig> and endogenous expressed sf-caspase- <dig> mrna quantity.
the corresponding primers and pcr cycle number used for the experiments in figure
<dig> are shown in table
<dig>  as shown for the genomic dna pcr performed in figure
3a, we supposed the pibdscasp vectors were stably integrated with sf <dig> cellular genomic dna.
in addition, the data also showed that levels of sf-casp <dig> mrna in sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells were apparently lower than those observed in sf <dig> and sf9/pib cells .
furthermore, control actin mrna was not obviously affected in either of the cell lines.
these results demonstrated that sf-caspase- <dig> mrna was successfully suppressed by sf-caspase- <dig> dsrna in both sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells.
in our previous study, we tested the expression of p <dig>  a substrate inhibitor of sf-caspase- <dig>  in sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells, and found that the level of p <dig> in pibdscasp transfected cells was evidently higher than that found in normal cells
<cit> .
thus, we suggested that sf-caspase- <dig> is suppressed in these sf-caspase-1-repressed cells.
<dig>  pib, sf9/pib cells; dscasp- <dig>  sf9/pibdscasp- <dig> cells; dscasp- <dig>  sf9/pibdscasp- <dig> cells.
er, endoplasmic reticulum.
accumulated seap expression by sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells
seap is a secreted  recombinant protein that can be assayed in culture medium using a standard activity assay.
thus, we used rbacseap to infect the sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells at a multiplicity of infection  of  <dig> - <dig> and analyzed the accumulated seap expression in the culture medium every day post infection .
at 2– <dig> dpi, the accumulated seap expression in rbacseap-infected sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells was obviously higher than that in the control group, sf <dig> and sf9/pib cells, at all mois .
moreover, at an moi of  <dig> and  <dig>  accumulated seap expression in rbacseap-infected sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells was approximately two-fold higher than that in the control group after  <dig> dpi .
moreover, the difference of accumulated seap expression between normal and sf-caspase-1-repressed stable cells increased with moi, such as that at mois of  <dig> and  <dig> .
these data demonstrated that the secreted recombinant protein production in sf-caspase-1-repressed stable cells was higher than that in normal insect cell after baculovirus infection.
luciferase expression in sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells
to estimate different kinds of recombinant protein production in sf-caspase-1-repressed stable cells, we use an intracellular recombinant protein, luciferase, as the assay target.
the rbacluc-infected sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells showed higher luciferase activities than that in control cells at all mois at  <dig> or  <dig> dpi .
however, at all mois, the difference of luciferase expression was not apparent at > <dig> dpi  and might result from the lysis of virus-infected cells during the later phase of infection releasing intracellular proteins
<cit> .
seap expression at each day’s period in sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells
to further investigate the differences of recombinant protein production between the normal and sf-caspase-1-repressed stable cells, specific seap activity at every  <dig> hour period of time in the culture media of rbacseap infected cells was analyzed.
at an moi of  <dig>  and  <dig>  seap activities in culture medium of sf9/pibdscasp- <dig> and sf9/pibdscasp- <dig> cells were higher than that of normal cells at 3– <dig> dpi .
moreover, at an moi of  <dig> and  <dig>  peak seap expression in sf-caspase-1-repressed and normal cells occurred at 2– <dig> dpi .
although during the assay the replacement of culture medium of virus-infected cells may affect not only the energy sources for protein expression but also virus replication in cells, the difference of recombinant protein production between sf-caspase-1-repressed and normal cells may be caused by the diverse cellular states that occur during the middle phase of the infection process.
expression of molecular chaperones in baculovirus-infected sf-caspase-1-repressed stable cells
the heat shock response is important for baculovirus replication in insect cells and the expression of er molecular chaperones is strongly associated with the maximal expression of exogenous proteins in bevs
<cit> .
therefore, we analyzed the mrna expression of some molecular chaperones involved in virus replication, including copper chaperone, cyclophilins, hsps, calreticulin , and protein disulfide isomerase .
the β-actin mrna level in sf <dig> and sf9/pibdscasp- <dig> cells was relatively equivalent at each time point analyzed and decreased at 9– <dig> hours post-infection  .
however, the mrna expression of chaperones, including atx <dig>  cypa, cypb, hsc <dig>  calr, and pdi, exhibited differential expression between baculovirus-infected sf <dig> and sf9/pibdscasp- <dig> cells at 9– <dig> hpi .
most chaperones demonstrated an induction response for virus infection within  <dig> hpi.
furthermore, expression of hsp <dig> was persistent during the infection process and no dramatic difference was observed between normal and sf-caspase-1-repressed cells .
in contrast, expression of hsc <dig> did not show the same stable expression .
these data are consistent with a study performed by nobiron et al.
<cit>  that identified the cause of this effect to be hsc <dig>  a virus-induced member of the hsp <dig> family.
<dig>  dsc <dig>  sf9/pibdscasp- <dig> cells; act, β-actin.
discussion
in this study, our data demonstrated that both intracellular  and extracellular  recombinant protein production in sf-caspase-1-repressed stable cells was higher than that in normal insect cell after baculovirus infection .
although luciferase expression did not correlate with accumulated seap expression, it was apparent that both extracellular and intracellular recombinant protein production in sf-caspase-1-repressed stable cells was higher than for parental cells .
thus, these data indicate that sf-caspase-1-repressed stable cells express a higher amount of recombinant protein in bevs, consistent with previous studies in s. frugiperda cells by our group
<cit>  and in t. ni cells by bentley’s group
<cit> .
hence, we can suggest that the apoptotic repressed insect cells have greater recombinant protein production when infected with recombinant baculovirus, providing an effective production tool for bevs.
besides, results also indicated that the difference of recombinant protein production between sf-caspase-1-repressed and normal cells may be caused by the diverse cellular states that occur during the middle phase of the infection process .
molecular chaperones are important and highly associated with the state of cells.
the results of molecular chaperone mrna levels in baculovirus-infected normal and sf-caspase-1-repressed stable cells demonstrated that these cells were appeared in different states during the infection progression .
therefore, we assumed that this is a key explanation for baculovirus-infected sf-caspase-1-repressed stable cells have a higher recombinant protein production than that in normal cells.
the persistent expression of molecular chaperones in baculovirus-infected sf-caspase-1-repressed stable cells resulted in a higher recombinant protein production than that in normal cells, which can be suggested by some earlier studies that focused on the co-expression of molecular chaperones, bip , calreticulin and calnexin in bevs to improve the recombinant production and secretion from cells and larvas
<cit> .
therefore, we also suppose that expression of molecular chaperon in combined with rnai technique may have opportunity to further improve bevs.
during the baculovirus infection process, the condition of infected insect cells often represents key factors that affect both baculovirus replication and recombinant production.
therefore, there appears to be a distinct difference of the baculovirus infection process that occurs in normal and sf-caspase-1-repressed cells.
we proposed that the sf-caspase-1-repressed stable cells have a different status and this response improves the ability of infected cells to express a higher amount of recombinant proteins.
CONCLUSIONS
in summary, the differential expression of molecular chaperones in baculovirus-infected sf-caspase-1-repressed stable cells affects the production of recombinant protein when compare with normal cells.
therefore, the current study identified critical virus-cell interactions that are likely to improve the development of bevs in future studies.

 programmed cell death, also known as apoptosis, is a normal physiological cell suicide program that is highly conserved among vertebrates and invertebrates. apoptotic cells undergo a series of dramatic and characteristic alterations in cellular morphology, such as dna fragmentation, chromatin condensation, cytoskeleton reorganization, and plasma membrane blebbing. apoptosis plays an important role during development and tissue homeostasis eliminating discarded cells from the organism, including damaged and virus-infected cells. for this reason, apoptosis acts as a host protection mechanism by which virus-infected cells are removed to limit the proliferation of viruses. thus, to overcome this defense response in cells, baculoviruses carry anti-apoptotic genes to inhibit programmed cell death. spodoptera frugiperda caspase-, the most studied effector caspase of lepidoptera, is the principal effector caspase of s. frugiperda cells, and is activated by various death stimuli, including baculovirus infection, ultraviolet irradiation, and over-expression of pro-apoptotic genes. two types of anti-apoptotic genes have been identified in baculoviruses, p and inhibitor of apoptosis. the p gene from autographa californica multicapsid nucleopolyhedrovirus and p gene from spodoptera littoralis nucleopolyhedrovirus are required to prevent apoptosis during the virus infection of s. frugiperda cells, such as sf and sf21. the op-iap from the orgyia pseudotsugata multicapsid nucleopolyhedrovirus and sf-iap from the host, s. frugiperda cells, also suppress the apoptosis process. the baculovirus expression vector system is a powerful tool used for the expression of foreign proteins in numerous insect cells, including sf sf cells and trichoplusi ni cells. therefore, recombinant protein production using a bevs can be used for many pharmaceutical applications. exposure of cells to stressors, including high temperatures or a wide variety of physical and chemical insults, induces the expression of heat-shock proteins in cells. hsps are molecular chaperones responsible for maintaining cell homeostasis and promoting cell survival. baculovirus infection also serves as a stress factor that can activate both death-inducing and cellular-protective pathways, and the heat-shock response is important for baculovirus replication in insect cells. moreover, the rate-limited expression of endoplasmic reticular molecular chaperones is strongly associated with the maximal expression of exogenous proteins by bevs. few studies have examined the potential of rna inference to increase protein production in the bevs, however, several studies have demonstrated the efficiency of this approach in both insect cells and larvae. in our previous studies, we used dna vector-based approaches with endogenously expressed double-stranded rna to silence its target gene, sf-caspase- in sf cells. therefore, the sf-caspase- mrna level and plasmid copy number in the sf-caspase-1-repressed stable cells were examined. in addition, sf-caspase-1-repressed stable cells exhibited resistance to apoptosis and enhancement of recombinant protein production. these results were consistent with later findings in t. ni cells; however, the mechanism of recombinant protein augmentation in baculovirus-infected caspase-repressed insect cells was not determined. therefore, in this present study, we use rnai-mediated sf-caspase-1-repressed stable cells to clarify how resistance to apoptosis could enhance both intracellular and extracellular recombination protein production by bevs. furthermore, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study.  stable transfection plasmid verification by genomic dna pcr and rt-pcr in sf9/pibdscasp- and sf9/pibdscasp- cells stable cells were established by transfection of the control vector pib or vector pibdscasp, and subsequently selected by bsd. after more than passages, stable cell lines were analyzed by genomic dna pcr and rt-pcr to examine the inverted-repeat dna sequence of sf-caspase- and endogenous expressed sf-caspase- mrna quantity. the corresponding primers and pcr cycle number used for the experiments in figure are shown in table as shown for the genomic dna pcr performed in figure 3a, we supposed the pibdscasp vectors were stably integrated with sf cellular genomic dna. in addition, the data also showed that levels of sf-casp mrna in sf9/pibdscasp- and sf9/pibdscasp- cells were apparently lower than those observed in sf and sf9/pib cells. furthermore, control actin mrna was not obviously affected in either of the cell lines. these results demonstrated that sf-caspase- mrna was successfully suppressed by sf-caspase- dsrna in both sf9/pibdscasp- and sf9/pibdscasp- cells. in our previous study, we tested the expression of p a substrate inhibitor of sf-caspase- in sf9/pibdscasp- and sf9/pibdscasp- cells, and found that the level of p in pibdscasp transfected cells was evidently higher than that found in normal cells. thus, we suggested that sf-caspase- is suppressed in these sf-caspase-1-repressed cells. pib, sf9/pib cells; dscasp- sf9/pibdscasp- cells; dscasp- sf9/pibdscasp- cells. er, endoplasmic reticulum. accumulated seap expression by sf9/pibdscasp- and sf9/pibdscasp- cells seap is a secreted recombinant protein that can be assayed in culture medium using a standard activity assay. thus, we used rbacseap to infect the sf9/pibdscasp- and sf9/pibdscasp- cells at a multiplicity of infection of - and analyzed the accumulated seap expression in the culture medium every day post infection. at 2– dpi, the accumulated seap expression in rbacseap-infected sf9/pibdscasp- and sf9/pibdscasp- cells was obviously higher than that in the control group, sf and sf9/pib cells, at all mois. moreover, at an moi of and accumulated seap expression in rbacseap-infected sf9/pibdscasp- and sf9/pibdscasp- cells was approximately two-fold higher than that in the control group after dpi. moreover, the difference of accumulated seap expression between normal and sf-caspase-1-repressed stable cells increased with moi, such as that at mois of and. these data demonstrated that the secreted recombinant protein production in sf-caspase-1-repressed stable cells was higher than that in normal insect cell after baculovirus infection. luciferase expression in sf9/pibdscasp- and sf9/pibdscasp- cells to estimate different kinds of recombinant protein production in sf-caspase-1-repressed stable cells, we use an intracellular recombinant protein, luciferase, as the assay target. the rbacluc-infected sf9/pibdscasp- and sf9/pibdscasp- cells showed higher luciferase activities than that in control cells at all mois at or dpi. however, at all mois, the difference of luciferase expression was not apparent at > dpi and might result from the lysis of virus-infected cells during the later phase of infection releasing intracellular proteins. seap expression at each day’s period in sf9/pibdscasp- and sf9/pibdscasp- cells to further investigate the differences of recombinant protein production between the normal and sf-caspase-1-repressed stable cells, specific seap activity at every hour period of time in the culture media of rbacseap infected cells was analyzed. at an moi of and seap activities in culture medium of sf9/pibdscasp- and sf9/pibdscasp- cells were higher than that of normal cells at 3– dpi. moreover, at an moi of and peak seap expression in sf-caspase-1-repressed and normal cells occurred at 2– dpi. although during the assay the replacement of culture medium of virus-infected cells may affect not only the energy sources for protein expression but also virus replication in cells, the difference of recombinant protein production between sf-caspase-1-repressed and normal cells may be caused by the diverse cellular states that occur during the middle phase of the infection process. expression of molecular chaperones in baculovirus-infected sf-caspase-1-repressed stable cells the heat shock response is important for baculovirus replication in insect cells and the expression of er molecular chaperones is strongly associated with the maximal expression of exogenous proteins in bevs. therefore, we analyzed the mrna expression of some molecular chaperones involved in virus replication, including copper chaperone, cyclophilins, hsps, calreticulin, and protein disulfide isomerase. the β-actin mrna level in sf and sf9/pibdscasp- cells was relatively equivalent at each time point analyzed and decreased at 9– hours post-infection. however, the mrna expression of chaperones, including atx cypa, cypb, hsc calr, and pdi, exhibited differential expression between baculovirus-infected sf and sf9/pibdscasp- cells at 9– hpi. most chaperones demonstrated an induction response for virus infection within hpi. furthermore, expression of hsp was persistent during the infection process and no dramatic difference was observed between normal and sf-caspase-1-repressed cells. in contrast, expression of hsc did not show the same stable expression. these data are consistent with a study performed by nobiron et al. that identified the cause of this effect to be hsc a virus-induced member of the hsp family. dsc sf9/pibdscasp- cells; act, β-actin. discussion in this study, our data demonstrated that both intracellular and extracellular recombinant protein production in sf-caspase-1-repressed stable cells was higher than that in normal insect cell after baculovirus infection. although luciferase expression did not correlate with accumulated seap expression, it was apparent that both extracellular and intracellular recombinant protein production in sf-caspase-1-repressed stable cells was higher than for parental cells. thus, these data indicate that sf-caspase-1-repressed stable cells express a higher amount of recombinant protein in bevs, consistent with previous studies in s. frugiperda cells by our group and in t. ni cells by bentley’s group. hence, we can suggest that the apoptotic repressed insect cells have greater recombinant protein production when infected with recombinant baculovirus, providing an effective production tool for bevs. besides, results also indicated that the difference of recombinant protein production between sf-caspase-1-repressed and normal cells may be caused by the diverse cellular states that occur during the middle phase of the infection process. molecular chaperones are important and highly associated with the state of cells. the results of molecular chaperone mrna levels in baculovirus-infected normal and sf-caspase-1-repressed stable cells demonstrated that these cells were appeared in different states during the infection progression. therefore, we assumed that this is a key explanation for baculovirus-infected sf-caspase-1-repressed stable cells have a higher recombinant protein production than that in normal cells. the persistent expression of molecular chaperones in baculovirus-infected sf-caspase-1-repressed stable cells resulted in a higher recombinant protein production than that in normal cells, which can be suggested by some earlier studies that focused on the co-expression of molecular chaperones, bip, calreticulin and calnexin in bevs to improve the recombinant production and secretion from cells and larvas. therefore, we also suppose that expression of molecular chaperon in combined with rnai technique may have opportunity to further improve bevs. during the baculovirus infection process, the condition of infected insect cells often represents key factors that affect both baculovirus replication and recombinant production. therefore, there appears to be a distinct difference of the baculovirus infection process that occurs in normal and sf-caspase-1-repressed cells. we proposed that the sf-caspase-1-repressed stable cells have a different status and this response improves the ability of infected cells to express a higher amount of recombinant proteins.  in summary, the differential expression of molecular chaperones in baculovirus-infected sf-caspase-1-repressed stable cells affects the production of recombinant protein when compare with normal cells. therefore, the current study identified critical virus-cell interactions that are likely to improve the development of bevs in future studies. 
since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in bevs, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study. there are few studies that have examined the potential of rna inference to increase protein production in the baculovirus expression vector system. however, the mechanism of recombinant protein augmentation in baculovirus-infected caspase-repressed insect cells has not been elucidated. spodoptera frugiperda -caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. in the current study, we utilized rnai-mediated sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular and extracellular recombinant protein production in bevs. the data indicated that the retention of expression of molecular chaperones in baculovirus-infected sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.
 there are few studies that have examined the potential of rna inference to increase protein production in the baculovirus expression vector system. spodoptera frugiperda -caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. however, the mechanism of recombinant protein augmentation in baculovirus-infected caspase-repressed insect cells has not been elucidated.  in the current study, we utilized rnai-mediated sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular and extracellular recombinant protein production in bevs. since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in bevs, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study.  the data indicated that the retention of expression of molecular chaperones in baculovirus-infected sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation. apoptosisbaculoviruschaperonerna interferencesf-caspase-1 