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Galectin-1, a mammalian lectin expressed in many tissues, induces death of diverse cell types, including lymphocytes and tumor cells. The galectin-1 T cell death pathway is novel and distinct from other death pathways, including those initiated by Fas and corticosteroids. We have found that galectin-1 binding to human T cell lines triggered rapid translocation of endonuclease G from mitochondria to nuclei. However, endonuclease G nuclear translocation occurred without cytochrome c release from mitochondria, without nuclear translocation of apoptosis inducing factor, and prior to loss of mitochondrial membrane potential. Galectin-1 treatment did not result in caspase activation, nor was death blocked by caspase inhibitors. However, galectin-1 cell death was inhibited by intracellular expression of galectin-3, and galectin-3 expression inhibited the eventual loss of mitochondrial membrane potential. Galectin-1 induced cell death proceeds via a caspase-independent pathway that involves a unique pattern of mitochondrial events, and different galectin family members can coordinately regulate susceptibility to cell death.
Cell death is crucial for proper development of multicellular organisms, for maintenance of immune homeostasis, and for prevention of neoplastic disease. Death in many cell types is a complex process, utilizing multiple death signals and several parallel death pathways (1-3). The complexity of cell death pathways reflects a requirement for tight control of the death process, while redundancy is ensured by overlapping cell death inducers and effectors.
Regulation of cell death is a conserved function of the galectins, a family of fourteen mammalian lectins that are expressed in a wide variety of tissues. Galectins –1, -7, -8, -9 and –12 induce death of various cell types (4–6) including lymphocytes, keratinocytes, eosinophils, adipocytes and tumor cells, while intracellular galectin-3 expression protects T cells and some types of carcinoma cells from death (6–10). We have demonstrated that galectin-1 can induce cell death of T cells and thymocytes, while other groups have shown that galectin-1 can kill B cells, breast cancer cells and prostate cancer cells (11–13). The mechanism of galectin-1 induced T cell death is distinct from that mediated by Fas or glucocorticoids (4,5). However, the galectin-1 death pathway in T cells is still poorly understood. Intriguingly, galectins are an evolutionarily ancient family of molecules, with homologues found in primitive organisms including multicellular fungi and sponges (14). If cell death regulation is a conserved function of galectins in lower organisms, then components of the galectin-1 mediated T cell death pathway may have features common to the simpler cell death mechanisms of organisms such as fungi.
We have found that the galectin-1 death pathway in T cells has several novel features. While galectin-1 mediated T cell death was caspase-independent, galectin-1 binding to T cells results in translocation of endonuclease G (EndoG) (15,16) to the nucleus without release of other mitochondrial death effectors. In addition, intracellular galectin-3 inhibited galectin-1 induced T cell death, indicating that different galectin family members can coordinately regulate cell fate.
Galectin-1 induced cell death has many features such as rapid phosphatidylserine (PS) externalization, membrane blebbing and nuclear DNA fragmentation, which can be mediated by caspase proteases. However, galectin-1 induced T cell death appeared to be caspase-independent.
We examined the effects of caspase inhibitors on galectin-1 induced death of CEM and MOLT-4 T cells. Fas induced death of CEM T cells was used as a positive control, as MOLT-4 cells are resistant to Fas killing (17). Cells were treated with either z-Val-Ala-Asp(OMe)-CH2F (zVAD-fmk), a general caspase inhibitor, or z-Asp-Glu-Val-Asp(OMe)-CH2F (zDEVD-fmk), a downstream caspase inhibitor, for 1 hour prior to the addition of galectin-1, and death assessed by annexin V binding. As shown in Fig. 1A, both zVAD-fmk and zDEVD-fmk significantly inhibited Fas induced CEM cell death. However, 100 μM zVAD-fmk and zDEVD-fmk had no detectable effect on galectin-1 induced death of either CEM or MOLT-4 cells. As phosphatidylserine (PS) externalization can occur independently of caspase activity (2), we also asked whether markers of cell death downstream of PS externalization required caspase activity, by examining the effects of caspase inhibitors on cell loss and membrane permeability to 7-amino-actinomycin D (7AAD). zVAD-fmk and zDEVD-fmk also did not inhibit galectin-1 induced cell loss or uptake of 7AAD, while these inhibitors did block cell loss and 7AAD uptake in Fas induced cell death (Fig. 1B).
As caspase inhibitors may not completely inhibit all caspase activity, caspase activation was also assayed using the endogenous caspase substrate poly(ADP-ribose)polymerase (PARP), an 116kD protein that can be cleaved by caspases to an 85kD form during apoptosis. The 85kD PARP cleavage fragment appeared in anti-Fas mAb treated cells (Fig 1C lane 4). However, galectin-1 treated CEM and MOLT-4 (Fig 1C lanes 2 and 6) cells did not contain increased amounts of the 85kD PARP cleavage product compared to controls (Fig 1C lanes 1 and 5), although galectin-1 and anti-Fas treatment resulted in equivalent levels of cell loss. We also examined cleavage of DFF40/ICAD, another endogenous caspase substrate (18), after galectin-1 treatment, but detected no DFF40/ICAD cleavage after galectin-1 treatment (data not shown).
Cleavage of zDEVD-7-amino-4-trifluoromethylcoumarin (zDEVD-AFC), a synthetic fluorogenic caspase substrate, was also measured. Extracts of CEM cells treated with anti-Fas mAb cleaved zDEVD-AFC, resulting in the release of free AFC (Figure 1D), and zDEVD-AFC cleavage in Fas-treated cells was inhibited by zDEVD-fmk (data not shown). However, we detected no cleavage of zDEVD-AFC by extracts of galectin-1 treated CEM and MOLT-4 cells (Fig 1D).
Finally, we directly examined activation of several pro-caspases, including 2,3,4,6,8,9 and 10, to active forms, by immunoblotting with a panel of specific antibodies. Although we detected activation of pro-caspases in anti-Fas mAb treated CEM cells, we did not detect activation of any of these pro-caspases in galectin-1 treated CEM and MOLT-4 cells (data not shown). Therefore, galectin-1 induced cell death appears to operate via a caspase-independent mechanism.
As non-caspase proteases have also been implicated in cell death (2, 19–22), we also examined the ability of several other protease inhibitors to inhibit galectin-1 cell death. We observed no reduction in galectin-1 induced PS exposure with calpain inhibitors I and II, serine protease inhibitors TPCK and TLCK, cathepsin inhibitors pepstatin and CA-074 Me, or the proteasome inhibitor lactacystin (data not shown).
Mitochondrial events that occur during different types of cell death include loss of mitochondrial membrane potential (Δψm), release of cytochrome c to the cytosol, and translocation of apoptosis inducing factor (AIF) and EndoG to the nucleus (15, 16, 22–27). Suprisingly, we observed a novel pattern of mitochondrial events during galectin-1 mediated cell death. Galectin-1 treatment induced rapid EndoG translocation from mitochondria to nuclei without cytochrome c release, while loss of Δψm was only detected after EndoG translocation.
We examined the Δψm of CEM cells undergoing galectin-1 and anti-Fas mAb induced death. Figure 2A shows that Δψm was maintained in both galectin-1 and anti-Fas mAb treated cells after 1 hour. However, at this time, significant PS externalization had occurred in galectin-1 treated cells (Fig. 2A). At 6 hours, loss of Δψm was detected in cells undergoing both galectin-1 and Fas induced death. However, at 6 hr, a smaller fraction of galectin-1 treated cells were DiOC6(3)low than were annexin V+. These results indicate that loss of Δψm is not an early step in galectin-1 induced cell death.
While Δψm was maintained during the early stages of galectin-1 death, other mitochondrial events such as release of cytochrome c can trigger apoptosis without loss of Δψm (26). To examine cytochrome c release, CEM cells were treated with galectin-1 or anti-Fas mAb and the translocation of cytochrome c to the cytosol was detected by immunoblotting. Annexin V/PI staining demonstrated 42% cell death of Fas treated cells and 34% cell death of galectin-1 treated cells in these samples. Treatment of cells with anti-Fas mAb resulted in release of cytochrome c to the cytosol. In contrast, we detected no release of cytochrome c in the cytosol of galectin-1 treated cells by 6 hr, although the amount of cell death induced by galectin-1 and Fas were comparable (Fig. 2B). Thus, galectin-1 mediated cell death does not appear to require the release of cytochrome c from the mitochondria to the cytosol.
Loss of cardiolipin from mitochondria is associated with release of cytochrome c associated with the inner mitochondrial membrane during apoptosis (27). We examined loss of mitochondrial cardiolipin in galectin-1 and Fas-treated cells using the cardiolipin-specific dye 10-N-nonyl acridine orange (NAO); a decrease in mitochondrial cardiolipin results in decreased cell staining with NAO (28). Cardiolipin loss was detected after three hours in Fas-treated cells, while no decrease in NAO staining was detected in galectin-1 treated cells (Fig. 2C), consistent with the lack of cytochrome c release in galectin-1 treated cells seen in Fig. 2B.
DNA degradation has been demonstrated in galectin-1 mediated T cell death by several methods, including TUNEL labeling, detection of cells with subdiploid amounts of DNA, and DNA laddering (4, 5, 29). However, as mentioned above, galectin-1 induced cell death does not appear to involve the caspase-dependent endonuclease DFF40/CAD. EndoG has been identified as a mitochondrial endonuclease responsible for nucleosomal DNA fragmentation (15, 16, 24). In mouse embryonic fibroblast (MEF) cells triggered to die by ultraviolet irradiation or treatment with TNF and cycloheximide, EndoG translocated from mitochondria to nuclei and cleaved nuclear DNA; both EndoG translocation and nuclease activity were caspase-independent (15).
We examined the subcellular location of EndoG at several time points after the initiation of galectin-1 mediated death of CEM T cells (Fig. 3). Strikingly, translocation of EndoG from the mitochondria to the nucleus occurred very rapidly in galectin-1 induced T cell death. Fig. 3A demonstrates the appearance of EndoG in nuclear fractions from galectin-1 treated cells, while there was no increase in another mitochondrial protein, AIF, in these nuclear fractions. By immunofluorescence analysis, nuclear EndoG was detected in a significant fraction of the cells by 1 hr after addition of galectin-1 (Fig. 3C), although no detectable increase in nuclear EndoG occurred at earlier timepoints. By 6 hr after galectin-1 addition, the majority of the cells had translocated EndoG to the nucleus (Fig. 3C). The rapid nuclear translocation of EndoG in galectin-1 treated cells occurred in the absence of significant loss of mitochondrial membrane potential and without cytochrome c release (Fig. 2).
Figure 4 demonstrates the subcellular localization of EndoG in CEM cells after galectin-1 treatment. In control-treated cells, cytochrome c and EndoG remained co-localized in punctate cytoplasmic structures that ring the nucleus, demonstrating the mitochondrial localization of EndoG. However, in CEM cells treated with galectin-1, EndoG was detected in the nucleus (top panel), while cytochrome c remained in punctate structures in the cytoplasm, consistent with results in Fig. 2 demonstrating lack of cytochrome c release in galectin-1 treated cells. In cells that had nuclear fragmentation, a sign of DNA degradation, EndoG was detected in the nuclear fragments (second panel).
As mentioned above, AIF is another mitochondrial protein involved in caspase-independent cell death triggered by a variety of agents, including staurosporine and dexamethasone (30–33). We examined the subcellular localization of AIF after galectin-1 addition to CEM T cells. As shown in Fig. 4, no increase in nuclear translocation of AIF was detected in galectin-1 treated cells compared to cells treated with buffer alone (third and fourth panels), confirming the biochemical results shown in Fig. 3; nuclear staining for AIF was detected in 6% of cells treated with buffer alone, and in 5% of cells treated with galectin-1. In contrast, staurosporine treated CEM cells were examined for AIF nuclear translocation as a positive control; staurosporine treatment resulted in nuclear translocation of AIF in 56% of the CEM cells (data not shown). Thus, nuclear translocation of EndoG occurs early in galectin-1 induced T cell death in the absence of cytochrome c release or AIF nuclear translocation from the mitochondria, indicating that release of various mitochondrial effectors can be uncoupled in different death pathways (34), although the role of EndoG in galectin-1 induced cell death remains unknown.
Galectin-3 is the only known galectin with anti-apoptotic activity. Intracellular galectin-3 expression inhibited death of T cells, myeloid cells, and breast cancer cells induced by a variety of triggers such as Fas, nitric oxide, staurosporine, cisplatin and detachment of adherent cells (6–10, 35–37). However, little is known about the ability of different pro- and anti-death galectins to coordinately regulate cell death. We examined Jurkat T cells transfected with galectin-3 or vector alone for susceptibility to galectin-1. Galectin-3 expression in these cells has been shown to inhibit death induced by anti-Fas or staurosporine (7). We confirmed that, as previously shown (7), the two galectin-3 expressing clones 3.1 and 3.2 produce abundant intracellular galectin-3, while no galectin-3 is detectable on the cell surface (data not shown). Fig. 5A demonstrates that the Jurkat 3.1 and 3.2 cells were resistant to galectin-1 induced cell death, compared to the parental (wt) and control transfected (neo) cells. Thus, intracellular galectin-3 blocked cell death triggered by extracellular galectin-1.
The anti-apoptotic effect of galectin-3 has been attributed to the ability of galectin-3 to block mitochondrial events in cell death (7, 8, 10). We examined the mitochondrial membrane potential of Jurkat wt, neo and 3.1 cells treated with galectin-1 for six hours. As shown in Figure 5B, intracellular galectin-3 expression also blocked galectin-1 induced disruption of Δψm. To confirm that the loss of susceptibility to galectin-1 in galectin-3 transfected cells did not result from decreased availability of galectin-1 receptors on the cell surface, we performed binding assays using biotinylated galectin-1 to measure the relative binding of galectin-1 to the cells. As shown in Fig. 5C, expression of galectin-3 did not reduce galectin-1 binding to the surface of the cells, compared with the parental cells or the vector control. In addition, binding to all cell lines was completely abrogated in the presence of 100 mM lactose, demonstrating that all galectin-1 binding we observed was carbohydrate-dependent.
Galectin-1 induced PS exposure on both T cell lines and thymocytes occurs rapidly, with changes in membrane asymmetry detectable after only twenty minutes exposure to galectin-1 (38), implying that galectin-1 induced cell death does not require de novo protein synthesis. To determine the requirement for protein synthesis in galectin-1 cell death, we examined the effect of cycloheximide; as seen in Figure 6, incubation with cycloheximide did not block galectin-1 induced PS externalization. Furthermore, pretreatment with cycloheximide did not inhibit galectin-1 induced cell loss, although this concentration of cycloheximide was effective in inhibiting 98% of dexamethasone induced cell death (data not shown). Thus, de novo protein synthesis is not an absolute requirement for galectin-1 cell death.
Reactive oxygen species can also participate in cell death. We measured peroxides and superoxides after galectin-1 treatment of CEM and MOLT-4 cells, but no increase in reactive oxygen species was detected in galectin-1 treated cells (data not shown). Also, pre-incubation with the anti-oxidants ascorbate and N-acetylcysteine did not protect cells from galectin-1 death, indicating that this pathway does not require generation of reactive oxygen species.
We also examined ceramide generation and calcium flux as possible early events in galectin-1 death. Ceramide generation can initiate caspase-independent cell death (39, 40). However, we did not detect significant ceramide generation during galectin-1 triggered killing, and inhibitors of ceramide synthase and acidic sphingomyelinase did not block galectin-1 cell death (data not shown). Finally, calcium flux has been reported to regulate externalization of PS during apoptosis (41). Addition of galectin-1 to CEM and MOLT-4 cells induced an increase in intracellular calcium; while addition of EGTA blocked the galectin-1 mediated calcium flux, it did not significantly inhibit galectin-1 triggered PS externalization (data not shown). This is consistent with our earlier finding that galectin-1 could induce T cell death in the absence of calcium flux (42). Thus, increases in intracellular ROS, ceramide or calcium do not appear to be essential for galectin-1 cell death.
Galectin-1, an endogenous lectin expressed in many different tissues, can induce cell death of T and B cells, thymocytes, breast cancer cell lines, and prostate cancer cell lines (6). During galectin-1 triggered death, T cells demonstrate PS externalization, chromatin condensation and margination, DNA fragmentation and membrane blebbing. While galectin-1 mediated cell death is distinct from death pathways triggered by Fas or glucocorticoids (4,5,38), the mechanism of galectin-1 mediated T cell death is not well understood. Our studies demonstrate that galectin-1 induced T cell death is caspase-independent, involves rapid nuclear translocation of EndoG from mitochondria without detectable cytochrome c release or AIF translocation and prior to the loss of Δψm, and does not require de novo protein synthesis.
EndoG is a mitochondrial protein that is an important apoptogenic endonuclease (15, 16). During MEF cell death induced by UV radiation or by treatment with TNF plus cycloheximide, EndoG translocates from mitochondria to the nucleus and cleaves nuclear DNA in a caspase-independent manner (15). As shown in Figs. 1, ,33 and and4,4, galectin-1 mediated cell death involved rapid EndoG translocation in the absence of caspase activation. In cells treated with galectin-1, EndoG translocation occurred in most cells prior to the loss of Δψm and without detectable release of cytochrome c or AIF (Fig. 2, ,4).4). This is in contrast to MEF cell death induced by UV radiation, and TNF, and Fas mediated death of lymphoid cells, in which both EndoG and cytochrome c are released from mitochondria (15, 43). While cytochrome c release can occur without disruption of Δψm or mitochondrial function (26, 44), the absence of detectable cytochrome c release during EndoG translocation is a novel feature of galectin-1 T cell death. As mitochondrial remodeling has been shown to be important for cytochrome c release, our results suggest that galectin-1 binding does not result in the same mitochondrial structural alterations that are caused by other apoptotic triggers (45), as indicated by the lack of cytochrome c release observed in galectin-1 treated cells (Fig. 2B).
While several studies have shown concomitant release of EndoG and AIF during cell death (33, 34, 46), EndoG alone was sufficient for DNA degradation in MEF cells (15). Thus, the lack of AIF release that we observed may indicate that EndoG participates alone in galectin-1 mediated cell death, or may indicate that other factors cooperate with EndoG in the galectin-1 death pathway, although the precise role for EndoG in galectin-1 cell death has not been determined. Though the exact mechanisms regulating release and translocation of specific mitochondrial proteins during cell death are not completely understood, release appears to be regulated and to be protein specific, rather than the result of general mitochondrial destruction; proposed models include a hierarchical model of mitochondrial protein release with different thresholds for release of specific proteins, as well as selective channels for different mitochondrial effectors (25, 33, 46–48). For example, in MEF cells treated with UV radiation or TNF, translocation of EndoG did not result in release of mitochondrial hsp70 (15). The initiator(s) of EndoG release from mitochondria during galectin-1 induced death are not known; while truncated Bid (tBid) can trigger EndoG release from mitochondria in Fas-induced liver cell death (43), generation of tBid results from caspase cleavage, and tBid also releases cytochrome c (43, 44, 49, 50), features that we did not observe in galectin-1 treated cells. The roles of tBid and other Bcl family members in galectin-1 death remain to be elucidated.
Several members of the galectin family can positively regulate cell death, while the only anti-apoptotic galectin is galectin-3. As mentioned above, intracellular galectin-3 can block cell death induced by a variety of stimuli, including staurosporine, nitric oxide, cisplatin, Fas ligation, and death triggered by loss of adhesion (7,9,10,37). We found that galectin-3 expression in Jurkat T cells also blocked galectin-1 induced cell death. Galectin-3 expression in these clones was entirely intracellular, as no galectin-3 was detected on the cell surface. In addition, galectin-1 bound to all the Jurkat cell lines in a carbohydrate dependent manner, as detected by flow cytometry, regardless of galectin-3 expression status (Fig. 5C). Therefore, the protective effect of galectin-3 was not due to simple competitive inhibition with galectin-1 binding for cell surface carbohydrate ligands. In our study, intracellular expression of galectin-3 inhibited the eventual loss of mitochondrial membrane potential in galectin-1 treated cells. While intracellular galectin-3 has been shown to prevent release of cytochrome c in breast cancer cells treated with cisplatin or staurosporine (10), this specific event is likely not contributing to resistance to galectin-1 death, as we detected no release of cytochrome c in galectin-1 treated T cells (Fig. 2). However, galectin-3 expression may directly stabilize mitochondria and prevent other mitochondrial changes in galectin-1 death. Alternatively, galectin-3 can bind to anti-apoptotic Bcl-2 (7,8), and may antagonize the decrease in Bcl-2 that has been observed in galectin-1 induced T cell death (51).
We have previously shown that galectin-1 induced T cell death proceeds rapidly, suggesting that de novo protein synthesis was not required for death (38). This was confirmed in the present study, as cycloheximide treatment did not inhibit galectin-1 induced T cell death; similarly, protein synthesis was not required for EndoG translocation or death of MEF cells (15). In contrast, Rabinovich and co-workers found activation of the AP-1 transcription factor during galectin-1 apoptosis in rat T cells (29), and also described caspase activation triggered by galectin-1 binding (51). It is possible that galectin-1 may be activating different death pathways in different cell types. Alternatively, galectin-1 may activate more than one cell death mechanism, similar to the dual death mechanisms activated by Fas (3). Of note, galectin-9 has been shown to trigger death of various cell types via a caspase-1 dependent pathway (52). In addition, Cummings and co-workers have determined that galectin-1 binding to some cell types does not result in DNA cleavage, but that the galectin-1 induced PS exposure on the cell membrane is sufficient for phagocytosis of the cells by macrophages (53). Thus, galectin-1 binding may result in different endpoints in different cell types.
Galectin-1 induced cell death may utilize a death mechanism that has been conserved during evolution. Galectins are evolutionarily ancient molecules, with homologs found in primitive organisms including multicellular fungi, sponges, and C. elegans (14). The galactose-containing ligands preferentially recognized by galectins are present on cell surface glycoconjugates of these primitive organisms (54). Mitochondrial EndoG release participates in apoptosis in C. elegans, with no requirement for cytochrome c release (16, 33). Intriguingly, aspects of galectin-1 induced cell death, such as caspase- and cytochrome c-independence, are reminiscent of cell death pathways of organisms such as yeast and Dictyostelium (55, 56).
Understanding the unique pathway of galectin-1 induced cell death is critical for development of new approaches to regulating cell survival. The potential of galectin-1 in modulating immune responses in T cell-dependent autoimmune disorders and in cancer has been demonstrated in animal models (6, 57). In addition, galectin-1 may also be a useful anti-neoplastic agent, by killing cells that have escaped other apoptotic triggers (12, 13). That the galectin-1 death pathway is caspase-independent and appears to be distinct from other death pathways suggests that galectin-1 can synergize with other apoptotic agents, as has been shown with T cell receptor engagement or dexamethasone treatment (5, 58). Further elucidation of the galectin-1 death mechanism will facilitate identification of target cells susceptible to this type of death, and the design of agents to therapeutically manipulate this novel death pathway.
MOLT-4 human T lymphoblastoid cells were purchased from ATCC (Rockville, MD). CEM T cells were the gift of Dr. Blair Ardman, New England Medical Center, Boston, MA. Jurkat T cells were obtained and galectin-3 expressed and purified as previously described (7). Reagents were from the indicated suppliers: cycloheximide (ICN, Costa Mesa, CA), z-Val-Ala-Asp(OMe)-CH2F (zVAD-fmk) and z-Asp-Glu-Val-Asp(OMe)-CH2F (zDEVD-fmk) (Enzyme Systems Products, Livermore, CA), zDEVD-7-amino-4-trifluoromethylcoumarin (zDEVD-AFC) (Kamiya, Seattle, WA), DiOC6(3), 10-N-nonyl acridine orange (NAO) and Prolong Antifade Mounting Media (Molecular Probes, Eugene, OR), annexin V-FITC Apoptosis kit (R&D Systems, Minneapolis, MN), pepstatin (Roche, Indianapolis, IN), CA-074 Me (Calbiochem, San Diego, CA). Recombinant galectin-1 was synthesized as previously described (4). Antibodies were from the following sources: mouse anti-human Fas, clone CH11 and mouse anti-human histone H1, clone AE-4 (Upstate Biotechnology, Lake Placid, NY); anti-poly(ADP-ribose)polymerase (PARP), clone C2.10 (Enzyme Systems Products, Livermore, CA); anti-cytochrome c, clone 7H8.2C12 (Pharmingen, San Diego, CA); anti-apoptosis inducing factor (AIF) (ProSci Inc., Poway, CA); FITC conjugated goat-anti rabbit serum (Jackson Laboratories, West Grove, PA); Texas Red conjugated goat anti-mouse IgG (Southern Biotech Associates, Birmingham, AL). Rabbit anti-EndoG was produced as in (15).
Galectin-1 death assays were performed as described (4) with indicated times. For Fas death assays, 2 x 106 cells in 400 μl final volume were treated with 1μg/ml anti-Fas mAb and incubated at 37ºC for indicated times. Analysis was performed on a BectonDickinson FACScan flow cytometer using CellQuest software. Cell loss was determined by forward v. side scatter gating, as described (5). Cells were analyzed for annexin V binding and uptake of propidium iodide (PI) or 7-amino-actinomycin D (7AAD). Percent viable cells was calculated as % viable cells = [100 x (# annexin V−, PI− cells)/(total number of cells)], and percent cell death was calculated as: % cell death = 100 x [1 - % viable cells (galectin-1 or Fas) / % viable (control)].
Binding of biotinylated galectin-1 was determined by flow cytometric analysis exactly as described in (59), in the presence or absence of 100 mM lactose to demonstrate carbohydrate-specific binding.
For PARP analysis, 50 μg of total cell extract was separated by 12% SDS-PAGE and immunoblotting was performed according to the manufacturer’s protocol. For cytochrome c and EndoG analysis, mitochondria were extracted from whole cell lysates using the ApoAlert Cell Fractionation kit (BD Biosciences, Palo Alto, CA), according to the manufacturer’s directions. Briefly, cells were homogenized in ice cold mitochondrial fractionation buffer using a glass Dounce homogenizer and Teflon pestle. Cell homogenates were centrifuged at 750xg for 10 minutes to remove cell nuclei. The mitochondrial fraction was obtained by centrifugation at 10,000xg for 25 minutes at 4ºC and the supernatant was used as cellular cytosolic extract. 30 μg of each fraction were separated on 15 % SDS-PAGE and immunoblotting performed according to the manufacturer’s protocol. Antibodies to cytochrome c and the mitochondrial control cytochrome oxidase 4 (COX4) were included in the kit. Blots were visualized using Enhanced Chemiluminescence (ECL) (Amersham, Arlington Heights, IL).
zDEVD-AFC cleavage assay was performed using the manufacturer’s protocol. Briefly, 5 x 106 cells were treated with galectin-1, buffer control, anti-Fas mAb or media for 3 hours at 37ºC. Cells were washed with PBS and extracted with 0.2 ml lysis buffer (1 mM PMSF, 5 mM DTT, 25 mM HEPES pH 7.5, 0.1% Triton-X100, 10% glycerol), and cell extracts were added to assay buffer (50 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS), 10 mM DTT, and 100 μM zDEVD-AFC, and incubated at 37ºC for 1 hour. For zDEVD-fmk controls, 50 μM zDEVD-fmk was added to this cocktail and pre-incubated for 1 hour at 37ºC before addition of zDEVD-AFC. Analysis was done on a Cytofluor plate reader (ex=395, em=530).
After incubation with the indicated agent, 2 x 105 cells were incubated with 40nM DiOC6(3) to measure mitochondrial membrane potential, or 40 nM 10-N-nonyl acridine orange (NAO) (28) to measure mitochondrial cardiolipin, and propidium iodide for 15 minutes at room temp prior to analysis by flow cytometry. Loss of mitochondrial membrane potential or of mitochondrial cardiolipin was indicated by decreased cellular staining with the respective dyes.
CEM cells were treated with galectin-1 or buffer control as above and aliquots were removed for flow cytometry analysis. Cells were washed in cold PBS, fixed in 1 ml 2% paraformaldehyde, PBS for 30 minutes on ice, and quenched with 3 ml 0.2M glycine, PBS. Pelleted cells were permeabilized with 0.1% TritonX-100, washed with PBS, and blocked with 100 μl of 2% goat serum, 4 mg/ml BSA, PBS overnight at 4°C. Cells were incubated with rabbit anti-human EndoG antiserum (1:250) or rabbit anti-human AIF (1:100), and mouse anti-human cytochrome c mAb (1:100) in 2% goat serum, BSA, PBS, overnight at 4ºC. After washing with 1%BSA/PBS, cells were incubated with FITC conjugated goat anti-rabbit mAb (1:100), and Texas Red conjugated goat anti-mouse mAb (1:100) for 1 hour at 4ºC. Alternatively, cells were stained with anti-EndoG or anti-AIF, and with propidium iodide (PI) (2.5 μg/ml) during addition of secondary antibody. Cells were washed with PBS, mounted on slides with Antifade mounting media and dried at RT overnight in the dark. Analysis was done on a Fluoview laser scanning confocal microscope, and images processed with Fluoview imaging analysis software (Olympus America, Inc, Melville, NY). To quantify labeled nuclei, at least 50 cells in five different fields were examined.
The authors thank Myles Cabot and Brett Premack, and the staff at the Jonsson Comprehensive Cancer Center Flow Cytometry Core.
1This work was supported in part by NIH training grants GM08042 (to H.P.H. and J.D.H.) and AI52031 (to J.D.H), NIH GM63281 (to L.G.B.), NIH GM57158 and I-1412 from the Welch Foundation (to X.W.) and NIH AI20958 (to F.-T.L. ).