Apoptosis (2011) 16:249–255 DOI 10.1007/s10495-010-0570-0 ORIGINAL PAPER Rapid cold-hardening blocks cold-induced apoptosis by inhibiting the activation of pro-caspases in the flesh fly Sarcophaga crassipalpis Shu-Xia Yi • Richard E. Lee Jr. Published online: 1 January 2011 Ó Springer Science+Business Media, LLC 2010 Abstract Apoptosis plays important roles in the selective Introduction elimination of sub-lethally damaged cells due to various environmental stresses. The rapid cold-hardening (RCH) The rapid cold-hardening (RCH) response that protects response protects insects from the otherwise lethal conse- insects from non-freezing cold-shock injury was reported quences of injury due to cold-shock. We recently demon- more than two decades ago [1]. RCH occurs very rapidly strated that cold shock induces apoptotic cell death in requiring only minutes to hours for its induction, and is insects and that RCH functions to specifically block cold- elicited in diverse taxa of freezing intolerant insects and shock-induced apoptosis. In the present study we used related arthropods [2, 3]. This acclimatory response func- isolated fat body, midgut, muscle, and Malpighian tubules tions to enhance organismal performance during environ- from adult flesh flies Sarcophaga crassipalpis to test the mental cooling [4, 5]. following hypotheses: (1) cold-induced apoptosis varies At the cellular level, RCH results in changes in the among different tissues and (2) RCH blocks the apoptotic composition of membrane lipids [6–8] and increases in pathway by preventing the activation of pro-caspases. membrane fluidity [9]. This response also requires calcium Cold-shock induced substantial amounts of apoptotic cell for cold-sensing and the induction of cold-hardening [10]. death that matched with tissue damage as determined using Despite the generality of this response, the nature of the vital dyes. RCH treatment significantly reduced apoptotic underlying physiological mechanisms of RCH is not clear. cell death in all tested tissues. Caspase-3 (executioner) The RCH response also blocks cold-induced apoptosis activity was 2–3 times higher in the cold- and heat-shocked [11]. Apoptosis, also called programmed cell death, plays a groups than in control and RCH groups. Likewise, the critical role in the development of multicellular organisms activity of caspase-9 (initiator) showed a similar trend as [12]. Apoptosis is a highly conserved, precisely regulated for caspase-3 in all tissues but midgut. In addition, cold- cascade of biochemical and molecular events for elimi- shock and heat-shock treatments also increased caspase-2 nating aged and sub-lethally damaged cells [13, 14], and activity 2–3 folds in both soluble and membrane fractions can be induced by various stresses, including environ- of fat body and muscle extracts compared to controls. mental, chemical and mechanical damage [11, 15]. Mor- phological changes in the apoptotic cells include blebbing, Keywords Apoptosis Á Caspases Á Cold shock Á RCH Á loss of membrane asymmetry and attachment, cell shrink- Sarcophaga crassipalpis age, chromatin condensation, and chromosomal DNA fragmentation [16]. A vital component in the apoptotic process is a cas- Electronic supplementary material The online version of this article (doi:10.1007/s10495-010-0570-0) contains supplementary cade of caspases (cysteinyl aspartate-specific protease) material, which is available to authorized users. that cleave many cellular substrates [17]. Thus far, 15 caspases have been identified in mammals, 4 in Caeno- & S.-X. Yi ( ) Á R. E. Lee Jr. rhabditis elegans, and 7 in Drosophila melanogaster Department of Zoology, Miami University, Oxford, OH 45056, USA [18]. All caspases are synthesized prior to the induction e-mail: [email protected] of apoptosis and maintained as inactive precursors 123 250 Apoptosis (2011) 16:249–255 (pro-caspases). Induction of apoptosis triggers proteolysis from SAS Institute) with statistical significance set at of pro-caspases to produce two peptides, 17–25 and P \ 0.05. 10–12 kDa, which dimerize to form the active caspases. The initiators (caspase-2, -8, -9, -10) begin the cascade, Detecting apoptosis by TUNEL assay whereas the executioners (caspase-3, -6, -7, -14) are critical for final execution of the cell death program Terminal deoxynucleotidyl transferase-mediated uridine [19, 20]. Activation of pro-caspase-3 is a central step in 50-triphosphate-biotin nick end labeling (TUNEL) assay the execution phase and serves as the convergence point [26, 27] was used to detect the apoptotic cell death on of different apoptotic signaling pathways [21]. Therefore, cryosections of adult tissues, including fat body, muscle, caspase activation is an indicator of apoptosis and can be midgut, and Malpighian tubules. Preparation of cryosec- used to determine whether, and when, apoptosis occurs in tions and TUNEL assay were performed as previously tissues/cells [22]. described by Yi et al. [11] from three groups of flies: the Although the effects of RCH protection against cold- untreated control, the cold-shocked, and the RCH, using shock injury are well known at the organismal level for the in situ cell death detection kit, TMR Red (Roche, many species, the underlying molecular mechanisms con- Indianapolis, IN). Nuclear DNA was stained with 40,6- ferring increased cold tolerance remain poorly understood. diamidino-2-phenylindoleÁ2HCl (DAPI) (Pierce Biotech- Previously, we demonstrated that both high and low tem- nology, Rockford, IL) solution. Then the slides were perature stimuli including heat shock, cold shock, and covered with cover slips and Vectashield (Vector Labo- freezing induced apoptotic cell death in D. melanogaster ratories, Burlingame, CA). Apoptotic cells were stained [11]. We also discovered that cold-shock-induced apopto- red by TUNEL reagent, while all nuclei were stained sis was prevented by RCH treatment by suppressing levels blue by DAPI when observed with a fluorescence of caspase-like proteins and elevating anti-apoptotic pro- microscope [11]. Cell apoptosis was quantified by com- tein, Bcl-2. In this study, we used isolated tissues (fat body, paring the ratio of red to blue cells using ANOVA midgut, muscle, and Malpighian tubule) from adults of Bonferroni–Dunn test (Statview, SAS Institute) with the flesh fly Sarcophaga crassipalpis to determine whether: statistical significance set at P \ 0.05. Each mean value (1) cold-induced apoptosis varies among different tissues is based on the counts of eight fields of 50–200 cells for and (2) RCH blocks the apoptotic pathway by preventing each of three replicates. the activation of pro-caspases. DNA isolation and gel electrophoresis Materials and methods DNA was isolated as described by Yi et al. [11] from fat Insect culture and assessment of cell viability body and midgut tissues of three groups of flies: control, cold-shocked and RCH. The isolated DNA was washed The flesh flies, S. crassipalpis (Diptera, Sarcophagidae), with 70% ethanol, air dried, and stored at -80°C. Before were reared as described by Lee and Denlinger [23]. All electrophoresis, the DNA was dissolved in TE buffer experiments used 5- to 8-day-old adult flies. To determine (10 mM Tris, 1 mM EDTA, pH 7.4) and its concentration the effect of RCH on cold tolerance of tissues and cells, was measured on a Nanodrop automatic system. DNA adults were divided into three groups: control (untreated at fragmentation was evaluated by electrophoresis on 2% 23°C), cold-shocked (directly exposed to -8°C for 2 h), agarose gel containing ethidium bromide in TAE buffer and RCH (exposed to 0°C for 2 h prior to a 2 h exposure to (40 mM Tris, 20 mM acetate, 2 mM EDTA, pH 8.1) and -8°C). 2 h after each treatment, fat body, midgut, and visualized under ultraviolet light [28]. Fifteen micrograms Malpighian tubules were dissected from adults in Coast’s of DNA samples were loaded onto each lane of the gel solution. Following dissection, cell viability of each tissue using 100 bp Ladder (Fisher, Fair Lawn, NJ) as standard was assessed with the LIVE/DEAD sperm viability kit markers. (Molecular Probes, Eugene, OR) as described by Yi and Lee [24, 25]. Green or yellow green fluorescence indicates Caspase activity assays live cells with intact cell membranes, while dead cells fluoresced red or orange-red. Percent cell survival of each Enzymatic activities of three caspases: caspase-2, caspase- tissue from individual flies was based on the mean value 3, and caspase-9, were determined, respectively, using the of three counts of 100 cells. Three to five individuals ICH-1/Caspase-2 colorimetric protease assay kit, Caspase- (n = 3–5) were used in each treatment group. Means were 3 colorimetric activity assay kit (DEVD), and Caspase-9 compared using ANOVA Bonferroni–Dunn test (Statview colorimetric activity assay kit (Chemicon International, 123 Apoptosis (2011) 16:249–255 251 Temecula, CA) as per the manufacturer’s instructions. In these assays, caspase-2 proteases in both soluble and membrane-bound protein samples recognized the sequence of VDVAD (Val-Asp-Val-Ala-Asp) and specifically cleaved at the C-terminal side of the aspartate residue. The assay is based on spectrophotometric detection (405 nm) of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate VDVAD-pNA. Comparing the absorbance of pNA from an apoptotic sample with an untreated control allows determination of the increase in caspase-2 activity. Similarly, caspase-3 protease in the samples recognized the sequence DEVD (Asp-Glu- Val-Asp) and cleaved pNA from the labeled substrate DEVD-pNA; while caspase-9 protease recognized the Fig. 1 Cell death detected in the fat body, midgut, and Malpighian sequence LEHD (Leu-Glu-His-Asp) and cleaved the tubules (MT) from control, RCH, and cold-shock treated adults by labeled substrate LEHD-pNA to release free pNA. In each Live/Dead viability kit. A significant difference (ANOVA Bonfer- case, the protein samples were diluted to a protein con- roni–Dunn test, P \ 0.05) between treatment groups in each tissue is centration of 100–200 lg in a volume of 90 ll with the indicated by different letters provided assay buffer.