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. Author manuscript; available in PMC: 2008 Apr 11.
Published in final edited form as: J Invest Dermatol. 2006 May 25;126(10):2247–2256. doi: 10.1038/sj.jid.5700381

Activation of Dual Apoptotic Pathways in Human Melanocytes and Protection by Survivin

Tong Liu 1, Diana Biddle 1, Adrianne N Hanks 1, Brook Brouha 2, Hui Yan 1, Ray M Lee 1,3,4, Sancy A Leachman 1,2, Douglas Grossman 1,2,3
PMCID: PMC2292407  NIHMSID: NIHMS44271  PMID: 16728972

Abstract

Apoptosis resistance in melanoma is a primary cause of treatment failure. Apoptotic pathways in melanocytes, from which melanoma arises, are poorly characterized. Human melanocytes were susceptible to apoptosis following exposure to UV radiation (UVB, 24–48 hours), 4- tert-butylphenol (4-TBP, 1–4 hours), and cisplatin (24–48 hours). These responses were associated with Bid cleavage, caspase activation (caspases 3, 8, and 9), mitochondrial depolarization and release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor (AIF), but not endonuclease G. The apoptotic responses and AIF release were caspase-independent, as they were not blocked by zVal-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk). While RNA interference-mediated knockdown of AIF protected melanocytes against apoptosis induced by serum withdrawal, apoptotic responses to UVB, cisplatin, and 4-TBP were not compromised by AIF knockdown, even in the presence of zVAD-fmk. Finally, adenoviral-mediated expression of Survivin, an inhibitor of apoptosis expressed in melanoma but not melanocytes, protected melanocytes against UVB-induced apoptosis. Survivin expression in melanocytes partially blocked caspase activation and release of mitochondrial release of AIF, cytochrome c, and Smac induced by UVB. These data indicate that multiple stimuli can activate both caspase-dependent and caspaseindependent apoptotic pathways in melanocytes, and that endogenous expression of Survivin in melanoma may contribute to apoptosis resistance by multiple mechanisms.

INTRODUCTION

Apoptosis plays a critical role in regulating epidermal development and restraining carcinogenesis. Keratinocytes are highly susceptible to apoptosis and their rapid turnover maintains epidermal homeostasis and eliminates premalignant cells (Raj et al., 2006). The keratinocyte apoptotic program, activated in vivo by UV light and cytokines, includes death receptor signaling (Takahashi et al., 2001), caspase activation (Sitailo et al., 2002), and mitochondrial depolarization (Denning et al., 2002), and leads to caspase-dependent cell death (Grossman et al., 2001a). By contrast, apoptosis in melanocytes is poorly understood and the physiologic signals that induce melanocyte apoptosis are unknown.

Unlike keratinocytes, melanocytes in vivo exhibit slow turnover and limited proliferative capacity (Yaar and Gilchrest, 1991), and physiologic exposure to UVB stimulates melanin production and proliferation rather than apoptosis (Gilchrest et al., 1999). While cyclic involution of the hair follicle during catagen involves massive keratinocyte apoptosis, melanocytes are spared (Botchkareva et al., 2001). Their survival in vivo depends on the expression of Bcl-2, as Bcl-2-deficient mice exhibit progressive hair graying and ultimately become hypopigmented (Veis et al., 1993) as a result of melanocyte apoptosis (Nishimura et al., 2005). In normal nevi, melanocytes are not mitotically active and spontaneous apoptosis cannot be detected (Florell et al., 2002). Nevo-melanocytes demonstrate increased apoptosis resistance compared to isolated melanocytes (Alanko et al., 1999), and in melanoma, apoptosis resistance is associated with metastasis (Glinsky et al., 1997) and is likely responsible for treatment failures (Grossman and Altieri, 2001). In the acquired condition of vitiligo, melanocytes are eliminated by apoptosis resulting from immune attack (Huang et al., 2002). In vitro, melanocyte cell death can be induced by UVB (Zhai et al., 1996), growth factor withdrawal (Halaban et al., 1993), IFN-β (Krasagakis et al., 1991), transforming growth factor-β (Rodeck et al., 1994), and the tyrosine analog 4-tert-butylphenol (4-TBP) (Le Poole et al., 1999). We previously demonstrated that in addition to UVB and 4-TBP, melanocyte apoptosis can be induced in vitro by multiple drugs, but high doses and prolonged incubation are required (Bowen et al., 2003).

There has been limited investigation of apoptotic pathways and regulators in melanocytes, or the role of caspase activation in these various responses. Zhang et al. (2000) reported that melanocytes are resistant to death receptor signaling by tumor necrosis factor-α, Fas ligand, and tumor necrosis factor-related apoptosis-inducing factor, which they attributed to lack of the expression of tumor necrosis factor-related apoptosis-inducing factor R1–R4 receptors. Zhai et al. (1996) demonstrated that nerve growth factor protects melanocytes from UVB-induced apoptosis, and is associated with the upregulation of Bcl-2. Kim et al. (2000) found upregulation and redistribution of Bax in UVB-irradiated melanocytes. Kadekaro et al. (2005) reported that α-melanocortin protects melanocytes from UVB-induced apoptosis through the inositol triphosphate kinase-Akt pathway and by increasing phosphorylation and expression of the micro-phthalmia-related transcription factor, known to upregulate Bcl-2 expression (McGill et al., 2002). The resistance to death receptor activation and potential regulation by Bcl-2 and Bax (Bivik et al., 2005) suggests that melanocytes primarily employ intrinsic (mitochondrial) rather than extrinsic (caspase-8-dependent) apoptotic pathways. Consistent with this notion, our survey of apoptotic regulatory proteins in melanocytes found high expression of the primary regulators of the intrinsic pathway, namely inhibitor of apoptosis (IAP) and Bcl-2 family proteins, and the absence of the extrinsic pathway regulator FLICE inhibitor protein (Bowen et al., 2003). The only known apoptotic regulator expressed in melanoma and nevi but not melanocytes appears to be the inhibitor of apoptosis protein Survivin (Bowen et al., 2003). Survivin may confer apoptotic protection in cancer cells by multiple mechanisms, including caspase inhibition similar to other inhibitor of apoptosis (Altieri, 2003). In a recent study, we found that in addition to caspase activation, Survivin inhibition in melanoma cells triggered translocation of mitochondrial apoptosis-inducing factor (AIF) (Liu et al., 2004), previously characterized as the primary mediator of caspase-independent apoptosis (Susin et al., 1999).

In the present study, we demonstrate that in human melanocytes multiple stimuli can trigger both caspase-dependent and caspase-independent apoptosis associated with caspase activation and mitochondrial AIF translocation, respectively. Forced expression of Survivin in melanocytes confers apoptotic protection, suggesting its potential contribution to apoptosis resistance in nevi and melanoma.

RESULTS

Kinetics of melanocyte apoptosis

In previous studies, human melanocytes were stimulated with UVB and a panel of drugs to determine optimal conditions of apoptosis induction for each agent (Bowen et al., 2003). Examination of the melanocyte cultures by light microscopy revealed a difference between these stimuli that was not appreciated in our initial examination of late-stage apoptosis. We noted that 4-TBP had a dramatic and rapid effect, inducing cell rounding and detachment within 1 hour of exposure, whereas cells treated with UVB, cisplatin, or staurosporine did not become detached until 24–48 hours following exposure (not shown). This difference in kinetics of cell detachment induced by 4-TBP compared to other agents was recapitulated in our analysis of phosphatidylserine externalization as an indicator of early-stage apoptosis. Cells treated with 4-TBP uniformly exhibited Annexin binding at 1 hour, with increased binding at 4 hours (Figure 1). A subpopulation of cells treated with UVB and cisplatin was Annexin+ at 24 hours, with most of the remaining cells becoming positive by 48 hours (Figure 1). The kinetics of these responses are also shown (Figure 1, right panels). These experiments established relevant time frames for further investigation of melanocyte apoptotic pathways.

Figure 1. Kinetics of melanocyte apoptosis.

Figure 1

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Melanocytes were untreated (control, filled histogram) or exposed to 1,200 J/m2 UVB, 0.9mM 4-TBP, or 90 μM cisplatin (CP) for the indicated times. Cells were stained with Annexin-PE and analyzed by flow cytometry. Shift to the right (left panels) indicates increased red (FL2) fluorescence and Annexin positivity. Kinetics of phosphatidylserine externalization are also shown (right panels).

We observed concomitant activation of the effector caspase-3 in response to all three stimuli, as indicated by a decrease in the 32-kDa precursor and appearance of 20- and 17-kDa cleavage fragments. Caspase-3 cleavage fragments were evident by 24–48 hours in both UVB- and cisplatin-treated cells, and at 1 hour in 4-TBP-treated cells (Figure 2a). We next evaluated upstream caspases representing extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways. Activation of the initiator caspase-8, reflected by a decreased level of the 55-kDa precursor and appearance of the 35-kDa cleavage fragment, was not prominent in cells treated with 4-TBP or cisplatin, but was evident by 48 hours in UVB-treated cells (Figure 2a). Although the 35-kDa fragment was not seen in response to 4-TBP or cisplatin, slight reduction of the 55-kDa precursor suggests some level of caspase-8 activation occurs in response to these agents. Indeed, all three agents resulted in the cleavage of Bid, known to be mediated by activated caspase-8 (Green, 1998), as indicated by disappearance of the 23-kDa precursor (Figure 2a). Activation of the initiator caspase-9, as indicated by a decrease in the 45-kDa precursor and appearance of the 37-kDa cleavage fragment, was prominently seen in both UVB- and cisplatin-treated cells at 24–48 hours and in 4-TBP-treated cells at 1–4 hours (Figure 2a). The ability of all three agents to induce Bid cleavage suggested, as noted above, that caspase-8 was activated but in our hands Western blotting is a relatively insensitive technique for detecting caspase-8 activation. We therefore employed a more sensitive enzymatic assay to confirm activation of caspase-8 by directly assaying cell lysates using specific fluorogenic caspase substrates. As shown in Figure 2b, activated caspase-3 and -8 were readily detected following treatment with each apoptotic stimulus. Thus, all three agents were capable of activating upstream caspases and caspases representing both extrinsic and intrinsic apoptotic pathways.

Figure 2. Caspase activation following apoptotic stimulation in melanocytes.

Figure 2

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(a) Melanocytes were treated with 1,200 J/m2 UVB, 0.9mM 4-TBP, or 90 μM cisplatin (CP) for the indicated times. Cell lysates (100 μg protein) were then subjected to Western blotting for caspase-3, caspase-8, Bid, and caspase-9 as indicated. Blotting for actin served as a loading control. Markers indicate caspase-3 precursor (32 kDa) and cleavage fragments (20, 17 kDa), caspase-8 precursor (55 kDa) and cleavage fragment (35 kDa, asterisk), Bid 23 kDa precursor, and caspase-9 precursor (45 kDa) and cleavage fragment (37 kDa). Cleaved Bid fragment (tBid) is unstable and was not visualized. (b) Melanocytes were untreated (control) or treated with 1,200 J/m2 UVB (24 hours), 0.9mM 4-TBP (30minutes), or 90 μM cisplatin (CP, 24 hours) as indicated. Cell lysates were assayed for activated caspase activity using fluorogenic substrates for activated caspase-3 (open bars) or activated caspase-8 (shaded bars). Induction of significant activity for each treatment compared to control is indicated by asterisks (*P<0.001; **P<0.0001).

Mitochondria may either initiate or amplify caspase-dependent apoptosis, depending on the experimental context (Arnoult et al., 2002). We thus examined the kinetics of mitochondrial depolarization in melanocytes treated with UVB, 4-TBP, and cisplatin by monitoring changes in mitochondrial transmembrane potential. As shown in Figure 3, responses to all three agents were characterized by a time-dependent left-shift in JC-1 fluorescence, corresponding to loss in transmembrane potential. Mitochondrial depolarization proceeded gradually over 24–48 hours in UVB- and cisplatin-treated cells and was complete by 1 hour in cells treated with 4-TBP (Figure 3). The kinetics of these responses are also shown (Figure 3, right panels). We next investigated whether this mitochondrial depolarization was temporally associated with the release of proapoptotic factors. Melanocytes were treated with UVB, 4-TBP, and cisplatin, and then fractionated into mitochondrial and cytosolic components for Western blotting. Cytochrome c, Smac/DIABLO, and AIF were released into the cytosol in response to all three apoptotic stimuli in the same time frame as mitochondrial depolarization (Figure 4a). In contrast, none of these stimuli resulted in release of endonuclease G (EndoG) (Figure 4a), a mitochondrial protein that like AIF (Susin et al., 1999) can translocate to the nucleus and mediate caspase-independent apoptosis (Li et al., 2001). By fluorescence microscopy, we confirmed that AIF was translocated to the nucleus in apoptotic melanocytes treated with UVB, 4-TBP, and cisplatin (Figure 4b), whereas neither cytochrome c (Figure 4c) nor EndoG (Figure 4d) exhibited nuclear translocation under similar conditions.

Figure 3. Mitochondrial depolarization.

Figure 3

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Melanocytes were untreated (control, filled histogram) or treated with 1,200 J/m2 UVB, 0.9mM 4-TBP, or 90 μM cisplatin (CP), and then stained with JC-1 at the indicated times (dotted and solid line histograms) and analyzed by flow cytometry. Shift to the left indicates decreased red (FL2) fluorescence (left panels). Kinetics of loss of mitochondrial transmembrane potential (MTP) are also shown (right panels). All values are normalized to mean JC-1 fluorescence in untreated cells.

Figure 4. Mitochondrial content release in apoptotic melanocytes.

Figure 4

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(a) Melanocytes were treated with 1,200 J/m2 UVB, 0.9mM 4-TBP, or 90 μM cisplatin (CP) for the indicated times. Cells were then fractionated into mitochondrial (M) and cytosolic (C) components, and electrophoresed and blotted for cytochrome c, Smac/DIABLO, AIF, and EndoG as indicated. Blots for voltage-dependent anion channel and actin confirm equivalent loading among mitochondrial and cytosolic lysates, respectively. Amount loaded per lane of mitochondrial and cytosolic lysates was 20 and 50 μg, respectively. (b) Melanocytes were untreated (control), or treated with 1,200 J/m2 UVB (24 hours), 0.9mM 4-TBP (1 hour), or 90 μM cisplatin (CP, 24 hours) as indicated. Cells were then stained with anti-AIF (red) and 4,6-diamidino-2-phenylindole (blue), and images were overlaid as shown. Purple color indicates co-localization of AIF and nucleus. (c) Melanocytes were prepared as in (b), and stained with anti-cytochrome c (red) and 4,6-diamidino-2-phenylindole (blue), and images were overlaid as shown. Similar blue color of nuclei in 4,6-diamidino-2-phenylindole and overlay panels indicates the absence of cytochrome c in the nucleus. (d) Melanocytes were prepared as in (b), and stained with anti-EndoG (red) and 4,6-diamidino-2-phenylindole (blue), and images were overlaid as shown. Similar blue color of nuclei in 4,6-diamidino-2-phenylindole and overlay panels indicates the absence of EndoG in the nucleus.

Caspase-dependent and -independent apoptosis

Given the capacity of these apoptotic stimuli to induce AIF translocation, we asked whether the apoptotic responses were caspase-independent. Melanocyte responses to UVB, 4-TBP, and cisplatin were assessed in the presence of zVal-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk), a pancaspase inhibitor. In response to all these agents, apoptosis proceeded despite the addition of zVAD-fmk (Figure 5a). To confirm that induced terminal caspase activation was blocked by zVAD-fmk under these experimental conditions, caspase activity was measured directly in lysates prepared from melanocytes treated at various time points in the absence or presence of zVAD-fmk. Addition of zVAD-fmk was sufficient to block the generation of terminal caspase-3 activity in cells treated with UVB and cisplatin (Figure 5b) and 4-TBP (not shown). Next, we examined whether AIF translocation was independent of caspase activation by stimulating cells with UVB and 4-TBP in the presence of zVAD-fmk. As shown in Figure 5c, AIF release in UVB-, 4-TBP-, and cisplatin-treated melanocytes was not inhibited by the addition of zVAD-fmk, indicating its lack of dependence on caspase activation. Taken together, these findings suggest that apoptotic stimulation in melanocytes independently activates both a caspase cascade and a mitochondrial response that includes AIF translocation.

Figure 5. Melanocyte apoptotic responses are caspase-independent.

Figure 5

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(a) Melanocytes were treated with 1,200 J/m2 UVB (48 hours), 0.9mM 4-TBP (24 hours), or 90 μM cisplatin (CP, 48 hours) in the absence (solid bars) or presence (open bars) of 20 μM zVAD-fmk. Percent apoptotic cells was then determined from the sub-G1 fraction obtained by flow cytometry of propidium iodide-stained cells. Error bars reflect SEM from three independent experiments. P-values for responses in the presence and absence of zVAD-fmk were not significant (UVB, P=0.59; 4-TBP, P=0.25; CP, P=0.82). (b) Caspase-3 activity in apoptotic melanocytes is blocked by zVAD-fmk. Melanocytes were untreated (solid bars) or treated with 1,200 J/m2 UVB (left panel) or 90 μM cisplatin (CP, right panel) in the absence (shaded bars) or presence of 20 μM zVAD-fmk (open bars) for 24 or 48 hours as indicated. Whole-cell lysates prepared at each time point were assayed for caspase-3 activity. Error bars reflect SEM from three measurements. Significant P-values for caspase inhibition indicated by asterisks (*P=0.03; **P=0.003; ***P<0.001). (c) Caspase inhibition does not block AIF release. Melanocytes were treated with 1,200 J/m2 UVB, 0.9mM 4-TBP, or 90 μM cisplatin (CP) as indicated in the absence or presence of 20 μM zVAD-fmk. Mitochondrial (M, 20 μg) and cytosolic (C, 50 μg) lysates were electrophoresed and blotted for AIF and actin.

Finally, we attempted to assess the contribution of the caspase-independent component of the melanocyte apoptotic response by knocking down AIF. We screened small interfering RNAs (siRNAs) directed against AIF, and were able to achieve approximately 70% reduction of AIF levels compared to untransfected cells or cells transfected with control siRNA (Figure 6a). Under these conditions of AIF knockdown, apoptotic responses to UVB were not appreciably affected compared to untransfected cells (Figure 6b). In addition, apoptotic responses of AIF-depleted cells were not significantly altered in the presence of zVAD-fmk (Figure 6b). Similar results were obtained for responses to cisplatin (Figure 6c) and 4-TBP (not shown). We considered the possibility that the residual AIF was sufficient to mediate caspase-independent apoptosis; however, our experimental conditions of AIF knockdown in the presence of zVAD-fmk were sufficient to protect melanocytes against apoptosis induced by serum withdrawal (Figure 6d) – an apoptotic response shown in other cell types to be dependent on AIF (Joza et al., 2001). Thus, the susceptibility of AIF-depleted melanocytes to UVB- and drug-induced apoptosis in the presence of zVAD-fmk raises the possibility that an additional mediator of caspase-independent apoptosis distinct from AIF (and EndoG) may be involved in these responses.

Figure 6. AIF knockdown does not protect against apoptosis.

Figure 6

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(a) Knockdown of AIF by RNAi. Melanocytes were unmanipulated (−) or transfected with control siRNA (Con) or siRNA targeting AIF (AIF). After 48 hours, whole-cell lysates were blotted for AIF and actin. Numbers below AIF blot indicate relative densitometry readings, with untransfected cells set at 1. (b) Unmanipulated melanocytes (no RNAi) or melanocytes transfected with control siRNA (Con-RNAi) or siRNA targeting AIF (AIF-RNAi) were untreated, or treated with UVB (48 hours) in the absence or presence of zVAD-fmk as indicated. Apoptotic cells were assessed by flow cytometry as in Figure 5a. In each histogram, the bar indicates the percentage of apoptotic cells (sub-G1 fraction). Experiment is representative of three performed in which significant AIF knockdown was achieved. (c) Melanocytes transfected with Con-RNAi or AIF-RNAi were untreated, or treated with cisplatin (CP, 48 hours) in the absence or presence of zVAD-fmk as indicated. Apoptotic cells were assessed as in (b). Experiment is representative of two performed in which significant AIF knockdown was achieved. (d) Melanocytes were transfected with Con-RNAi or AIF-RNAi, then 8 hours later were incubated in normal medium or medium without serum (ser. WD) in the absence or presence of zVAD-fmk as indicated. After 48 hours, zVAD-fmk was re-added to appropriate wells. Apoptotic cells were assessed as in (b) 96 hours after transfection. Experiment shown is representative of two performed in which significant AIF knockdown was achieved.

Survivin confers apoptotic protection

The expression of Survivin in melanoma but not normal melanocytes, and its capacity to protect against melanoma cell apoptosis (Grossman et al., 1999, 2001b), suggested that the acquisition of Survivin expression in melanocytes may contribute to apoptosis resistance in nevi and melanoma. We thus investigated whether forced expression of Survivin in melanocytes could protect against these apoptotic pathways. Melanocytes, which did not express Survivin, expressed high levels of wild-type and mutant Survivin protein following infection with adenoviruses expressing wild-type Survivin (pAd-Surv) or a dominant-negative Survivin mutant (pAd-T34A) (Figure 7a). Melanocytes were infected with one of these adenoviruses or a control adenovirus (pAd- green fluorescence protein (GFP)), treated with UVB, and then apoptosis was assessed by flow cytometry. Whereas UVB treatment resulted in marked apoptosis in melanocytes infected with either pAd-GFP or pAd-T34A, there was not a significant difference between untreated and UVB-treated pAd-Surv-infected cells (P=0.39) indicating Survivin-mediated protection against UVB-induced apoptosis (Figure 7b).

Figure 7. Forced Survivin expression in melanocytes and apoptotic protection.

Figure 7

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(a) Melanocytes were uninfected or infected for 48 hours with GFP-adenovirus (pAd-GFP), virus expressing wild-type human Survivin (pAd-Surv), or virus expressing Survivin-T34A mutant (pAd-T34A). Cells infected with pAd-Surv, seen by fluorescence microscopy (left panel). Whole-cell lysates prepared from infected cells were subjected to Western blotting using antibodies to Survivin or actin as indicated (right panel). The polyclonal Survivin antibody recognizes both wild-type and mutant Survivin. (b) Melanocytes were infected with the indicated GFP-expressing adenoviruses for 24 hours, then sham-irradiated (untreated, open bars) or exposed to 1,200 J/m2 UVB (shaded bars). Cells were stained with Annexin-PE 24 hours later, and then analyzed by two-color flow cytometry. To normalize for different rates of adenoviral infection and restrict the analysis to infected cells, the percent of GFP+ cells that were Annexin+ was calculated for each group. Shown are the percent of GFP+ cells that were Annexin+ from each group. Error bars indicate SEM from two independent experiments. Asterisks indicate induction of apoptosis in UVB-treated compared to untreated cells (*P=0.095; **P=0.04). Apoptosis induction in UVB-treated versus untreated pAd-Surv-infected cells was not significant (P=0.39).

The ability of exogenous Survivin to protect against melanocyte apoptosis suggests it may inhibit both caspase-dependent and caspase-independent apoptotic pathways. Next, we further investigated protection against apoptosis by Survivin at a mechanistic level by examining its effect on potential mediators of these two pathways. First, melanocytes from multiple donors were infected with pAd-GFP or pAd-Surv, and then assayed for terminal caspase activation following UVB treatment. While control and pAd-GFP-infected cells exhibited a significant increase in caspase activity in response to UVB, pAd-Surv-infected cells revealed only minimal UVB-induced caspase activation (Figure 8a). The induction of caspase activation in pAd-Surv-infected cells was significantly reduced from that in uninfected (P=0.007) and pAd-GFP-infected (P=0.003) cells following UVB treatment. Finally, we examined mitochondrial translocation of proapoptotic mediators in Survivin-expressing melanocytes following UVB treatment. While translocation of AIF from mitochondria to cytosol was similarly observed in control and pAd-GFP-infected cells following UVB, cytosolic AIF levels were reduced by greater than 50% in cells pre-infected with pAd-Surv (Figure 8b). Mitochondrial translocation of cytochrome c and Smac was similarly reduced in cells infected with pAd-Surv compared to those infected with pAd-GFP (Figure 8b). These results indicate that the capacity of Survivin to protect against melanocyte apoptosis likely relates to its previously described capacities to inhibit caspases (O’Connor et al., 2000) as well as suppress translocation of mitochondrial AIF (Liu et al., 2004), cytochrome c, and Smac.

Figure 8. Survivin expression in melanocytes reduced caspase activation and AIF release.

Figure 8

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(a) Melanocytes were uninfected (control) or infected for 48 hours with pAd-GFP or pAd-Surv as indicated. Cells were then untreated (open bars) or treated with 1,200 J/m2 UVB (shaded bars), and 24 hours later, whole-cell lysates were prepared and assayed for caspase-3 activity as in Figure 5b. Asterisk indicates that the level of UVB-induced caspase activation in pAd-Surv-infected cells is reduced from that in uninfected (P=0.007) and pAd-GFP-infected (P=0.003) cells. Experiment shown is representative of four out of five performed. (b) Melanocytes were uninfected (control) or infected for 48 hours with pAd-GFP or pAd-Surv as indicated. Cells were then untreated or treated with 1,200 J/m2 UVB as indicated, and 24 hours later were fractionated into mitochondrial (M) and cytosolic (C) components and subjected to Western blotting as in Figure 4a. Note that there is some translocation of mitochondrial factors resulting from adenoviral infection, so the relevant comparisons are between pAd-GFP- and pAd-Surv-infected cells. Densitometry values are indicated for cytosolic lanes comparing pAd-GFP (arbitrarily set at 1) and pAd-Surv-infected cells treated with UVB. Experiment shown is representative of two performed.

DISCUSSION

This study provides an initial characterization of the apoptotic program in melanocytes. We found that diverse apoptotic stimuli activate dual apoptotic pathways: one mediated by caspases, and another that is caspase-independent and associated with nuclear translocation of mitochondrial AIF. The expression in melanocytes of many inhibitors of apoptosis (Bowen et al., 2003), and their relative resistance to common apoptotic stimuli (Bowen et al., 2003), is consistent with their specialized function and longevity in the skin (Vancoillie et al., 1999). However, given the extreme hazard that transformation of these cells represents in the development of melanoma, perhaps it is not surprising that normal melanocytes are endowed with potentially redundant apoptotic pathways. Forced expression of Survivin in melanocytes conferred protection against both pathways.

Melanocytes were capable of generating a caspase cascade in response to all the stimuli tested, consistently revealing prominent activation of terminal caspase-3 and initiator caspase-9, with minimal activation of initiator caspase-8. The activation of terminal caspase-3 may originate both from upstream caspase activation as well as mitochondrial release of factors known to enhance caspase-9 activation (cytochrome c) and relieve caspase inhibition (Smac/DIABLO) (Reed, 2000). Our analysis of the kinetics of apoptotic events indicated that caspase activation and mitochondrial depolarization with content release occurred concomitantly in response to a given stimulus, although these responses to 4-TBP were much more rapid than those induced by UVB or cisplatin. This tendency for greater activation of intrinsic (caspase-9) versus extrinsic (caspase-8) pathway-associated caspases is consistent with previous reports implicating Bcl-2 family proteins in the regulation of melanocyte apoptosis in vitro (Zhai et al., 1996; Kim et al., 2000) and in vivo (Nishimura et al., 2005), and the lack of death receptor signaling in melanocytes (Zhang et al., 2000). Using a concentration of zVAD-fmk sufficient to neutralize completely this caspase activity, we found that the addition of zVAD-fmk did not prevent or reduce the apoptosis induced by multiple stimuli, indicating that caspase activation was not required for apoptosis induction. The resistance of these apoptotic responses to zVAD-fmk was consistent with the observed mitochondrial release and nuclear translocation of AIF, previously characterized as the primary mediator of caspase-independent apoptosis (Susin et al., 1999).

Several lines of evidence initially supported the notion that caspases are not required for AIF-mediated (caspase-independent) apoptosis. These included experiments demonstrating insensitivity to chemical caspase inhibitors such as zVAD-fmk (Susin et al., 1999; Cregan et al., 2002; Yu et al., 2002), and translocation and apoptosis induction following microinjection of AIF into caspase-deficient cells (Susin et al., 2000). However, a report by Zamzami et al. (2000) of AIF release from purified mitochondria incubated with activated caspases suggested the possibility that AIF release could actually be triggered by caspase activation. In addition, subsequent reports by Arnoult et al. (2002, 2003) examining Bax-overexpressing 293 cells and HeLa cells treated with staurosporine, actinomycin D, and hydrogen peroxide found that AIF release could be blocked by zVAD-fmk and occurred downstream of cytochrome c release. These studies, taken together with one in Caenorhabditis elegans demonstrating that WAH-1 (AIF) translocation stimulated by the proapoptotic factor EGL-1 required the caspase homolog CED-3 (Wang et al., 2002), suggested the possibility in some cells of a retrograde pathway in which AIF release depends upon caspase activation. Although such a phenomenon may occur in certain cell types exposed to particular apoptotic stimuli, our present finding that AIF release occurred in the presence of zVAD-fmk suggest that in melanocytes caspase activation is not required for AIF release. Rather, various apoptotic stimuli appear to elicit simultaneously distinct caspase-dependent and caspase-independent apoptotic pathways in melanocytes.

In C. elegans, the AIF homolog (WAH-1) associates with EndoG (CPS-6) and both are required to mediate DNA degradation (Wang et al., 2002). Since we did not observe co-translocation of EndoG with AIF from mitochondria in apoptotic melanocytes, AIF appeared to be the sole mediator of caspase-independent apoptosis in these cells. We attempted to dissociate the caspase-dependent component of the apoptotic response by AIF knockdown, which was problematic given the resistance of normal melanocytes to lipid-based transfection (T Liu and D Grossman, unpublished observation). Gene suppression in normal human melanocytes using siRNA or antisense is impaired by the poor transfectability of these cells. Nevertheless, we were able to achieve 70% knockdown of AIF, but found that this level of AIF depletion did not significantly affect melanocyte apoptotic responses even in the presence of zVAD-fmk. Our experimental conditions for AIF knockdown coupled with zVAD-fmk were, however, sufficient to protect melanocytes against apoptosis induced by serum withdrawal. This apoptotic response has been shown in other cell types to be dependent on AIF (Joza et al., 2001). Thus, although it is possible that the residual AIF was sufficient to mediate caspase-independent apoptosis, our results suggest the intriguing possibility that an alternate factor distinct from AIF may be involved in these melanocyte apoptotic responses.

Finally, these studies provide a rationale for the expression of Survivin in melanoma. Survivin is notable among the many known regulators of apoptosis for its selective expression in cancer (Velculescu et al., 1999), and appears to be the only one expressed in melanoma and nevi but not normal melanocytes (Grossman et al., 1999; Bowen et al., 2003; Florell et al., 2005). Survivin expression is a critical viability factor in melanoma cells, as its inhibition results in spontaneous apoptosis (Grossman et al., 1999, 2001b). Through disabling apoptotic responsiveness in melanocytes, Survivin expression may thereby promote nevus development and transformation to melanoma. In an attempt to understand its function in melanoma cells, we recently examined the apoptotic events following expression of an inducible Survivin antagonist. Apoptosis induced by Survivin targeting was associated with caspase activation but resistant to zVAD-fmk, and AIF translocation appeared to precede caspase activation following Survivin inhibition (Liu et al., 2004). These findings suggested to us that acquisition of Survivin expression in melanoma cells may confer protection against both caspase-dependent and caspase-independent apoptotic pathways, and that the latter may involve suppression of AIF release. The AIF pathway can be viewed as an accessory apoptotic pathway enabling cells to remain apoptosis-competent should caspase pathways fail – as known to occur in melanoma (Grossman and Altieri, 2001) and other cancers (Schimmer et al., 2003). Malignant cells thwart this “back-up” function through the expression of Survivin. On the other hand, melanocytes, which do not express Survivin (Grossman et al., 1999; Bowen et al., 2003), were shown here to be highly susceptible to caspase-independent apoptosis. We observed that forced expression of Survivin in melanocytes conferred apoptotic protection and was associated with reduced caspase activation and AIF translocation, supporting the notion that it may protect both against caspase-dependent and caspase-independent apoptosis as previously suggested in other cell types (Shankar et al., 2001; Liu et al., 2004). The protective capacity of Survivin, despite its inability to suppress completely caspase activation and AIF release, is consistent with the possibility of additional antiapoptotic functions associated with its expression as suggested recently (Fortugno et al., 2003).

MATERIALS AND METHODS

Drugs and chemicals

4-TBP (Aldrich, Milwaukee, WI) was prepared monthly in 70% ethanol and stored at −20°C. Cisplatin (Sigma Chemical Co, St Louis, MO) was dissolved in dimethyl formamide and stored at 4°C. The pancaspase inhibitor zVAD-fmk was obtained from Enzyme Systems Products (Livermore, CA), solubilized in DMSO, and stored at −20°C.

Antibodies

Purified rabbit polyclonal antibody recognizing Survivin has been described previously (Grossman et al., 1999) and is commercially available from NOVUS Biologicals (Littleton, CO). Rabbit polyclonal antibodies against precursor and cleavage fragments of caspase-3 (sc-7148), caspase-8 (sc-7890), and caspase-9 (sc-7885) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody against cytochrome c (sc-7159), Smac/DIABLO, and voltage-dependent anion channel were from Santa Cruz, Imgenex (San Diego, CA), and Affinity BioReagents Inc. (Golden, CO), respectively. Goat polyclonal antibodies against Bid (sc-6538) and AIF (sc-9416) were also from Santa Cruz. Mouse mAb against β-actin (clone AC-74) was obtained from Sigma. Rabbit polyclonal antibody against EndoG was obtained from ProSci Inc. (Poway, CA).

Cells

Normal human melanocytes were obtained from discarded foreskins, in accordance with the Declaration of Helsinki Principles, and as approved by the Institutional Review Board at the University of Utah (no. 8476). Melanocytes were isolated and propagated in Ham’s F10 containing 7.5% fetal bovine serum, 0.1mM 3-isobutyl-1-methylxanthine (Sigma), 1 μM sodium orthovanadate (Sigma), 2.5 nM cholera toxin (Sigma), 50 ng/ml phorbol 12-myristate 13-acetate (Sigma) and antibiotics as described previously (Bowen et al., 2003). Second and third passage melanocytes were used for all experiments.

Apoptosis induction and detection

Cells were exposed to UVB or drugs and recovered by trypsinization (including non-adherents) as described previously (Bowen et al., 2003). For serum withdrawal experiments, melanocytes were plated in normal medium the day before, then washed and incubated with medium lacking serum but containing other components. Late-stage apoptosis was assessed by total cellular DNA content using propidium iodide and flow cytometry as described previously (Grossman et al., 1999). Activity of caspase-3 and -8 in cell lysates was measured using the fluorogenic substrates Ac-DEVD-MCA and Ac-IETD-MCA, respectively, according to the manufacturer’s instructions (Peptides International Inc., Louisville, KY). Early-stage apoptosis was assessed by phosphatidylserine staining using an Annexin V-PE kit (Santa Cruz), and mitochondrial depolarization was assessed by JC-1 (Molecular Probes, Eugene, OR) fluorescence, both as described previously (Liu et al., 2004).

Mitochondrial isolation

Trypsinized melanocytes were pooled from multiple dishes (200–300cm2), washed in cold phosphate-buffered saline, and resuspended in mitochondrial isolation buffer (0.35 ml) consisting of 0.3M mannitol (Sigma), 70mM sucrose, 1mM EGTA, and 10mM HEPES (Calbiochem, La Jolla, CA). This solution was drawn into a 1cm3 insulin syringe and forcefully passed through a 0.5-inch 28-G needle 20 times and then transferred to a 1.5 ml microfuge tube. After centrifugation (1,000 ×g) at 4°C for 10 minutes, the supernatant was collected and the pellet containing undisrupted cells and nuclei was discarded. This supernatant was spun again (14,000 ×g) at 4°C for 15 minutes. The resulting supernatant (cytosolic fraction) was collected, and the pellet was washed once in mitochondrial isolation buffer and then resuspended in 80 μl mitochondrial isolation buffer (mitochondrial fraction). Protein content in the fractions was determined using the Protein Assay reagent (Bio-Rad Laboratories, Hercules, CA). Fractions were diluted in SDS-containing sample buffer, aliquotted, and stored at −20°C.

Western blotting

Cell lysates were prepared, electrophoresed, transferred to PVDF membranes, and blocked with nonfat milk as described previously (Grossman et al., 1999). Blots were incubated with primary antibodies against Survivin (0.5 μg/ml), caspase-3 (1:400), caspase-8 (1:200), caspase-9 (1:200), Bid (1:200), cytochrome c (1:400), Smac/DIABLO (1 μg/ml), voltage-dependent anion channel (1:750), β-actin (1:30,000), AIF (1:250), or EndoG (1 μg/ml) for 1–2 hours at room temperature, then with species appropriate horseradish peroxidase-conjugated Ig (New England Nuclear, Boston, MA). Bands were visualized by enhanced chemiluminescence (Perkin-Elmer Life Sciences Inc., Boston, MA) and autoradiography.

Fluorescence microscopy

Cells were plated on coverslips, and following exposure to apoptotic stimuli, were stained with anti-AIF antibody (Santa Cruz) as described previously (Liu et al., 2004). Staining for cytochrome c (1:200) and EndoG (15 μg/ml) was performed for 1 hour at 37°C, then after washing in phosphate-buffered saline, cells were stained with Alexa Fluor-conjugated goat-anti-rabbit IgG (1:200, Molecular Probes) for 1 hour at 37°C. Coverslips were adhered to glass slides using a mounting solution containing 4,6-diamidino-2-phenylindole (Sigma) as described (Liu et al., 2004). Cells were visualized using a Olympus IX70 fluorescence microscope, and images were obtained with an Olympus Microfire camera and overlaid using Pictureframe software (Optronics, Goleta, CA). Images were cropped, merged into panels, and colors for each panel were simultaneously optimized in Photoshop.

RNA interference

The RNA oligos targeting nucleotides 151–171 in human AIF, 5′-CUUGUUCCAGCGAUGGCAUtt-3′ (sense) and 5′-AUGCCAUCGCUGGAACAAGtt-3′ (antisense), and control oligos 5′-AGACAGAA GACAGAUAGGCtt-3′ (sense) and 5′-GCCUAUCUGUCUUCUGUCUtt-3′ (antisense), were synthesized by our core facility. Each pair of oligos was solubilized in RNA interference (RNAi) suspension buffer (Qiagen, Valencia, CA) and mixed at a final concentration of 20 μM, heated at 90°C for 1 minute, incubated at 37°C for 1 hour to anneal, and then stored at −20°C. Oligos targeting other sequences in AIF were also prepared, but found to be less effective in preliminary experiments. We tested multiple additional parameters, including cell density, siRNA and lipid concentration, and incubation times. In our optimized protocol, 2 × 105 cells were plated in 2ml medium without antibiotics per well in six-well plates 1 day prior to transfection. For each transfection, 2 μl of stock siRNA duplex was added to 150 μl of OptiMEM medium (Life Technologies, Rockville, MD) and 7 μl of Lipofectamine2000 (Life Technologies) was added to 150 μl of OptiMEM. After 5 minutes, the diluted siRNA was combined with the diluted Lipofectamine2000 and the mixture was incubated for 20 minutes at room temperature to allow complexes to form. Complexes (309 μl) were added to cells (70–80% confluency) in 1.5 ml antibiotic-free medium per well, and plates were incubated at 37°C in 5% CO2 incubator for 48 hours. For RNAi experiments involving serum withdrawal, serum was removed from the medium 8 hours following transfection.

Adenoviral infection

The recombinant replication-defective GFP-expressing pAd adenovirus (control, pAd-GFP) expressing human wild-type Survivin (pAd-Surv) and mutant Survivin-T34A (pAd-T34A) were kindly provided by Dario Altieri (University of Massachusetts) and are described elsewhere (Mesri et al., 2001). These viruses were propagated in 293 cells, purified, and titered as described (Mesri et al., 2001). Aliquots were stored at −80°C. Melanocytes seeded at 70% confluency into 35mm dishes were infected by the addition of virus to the culture medium. After 8 hours, the medium was aspirated and replaced with fresh medium. Since all the viruses express GFP, infection could be monitored by fluorescence microscopy of GFP-expressing cells. For each experiment, each virus was used at an inoculation dose that resulted in comparable percentages of GFP-expressing cells after 24 (40–50%) and 48 hours (80–90%).

Statistical analysis

Data derived from multiple determinations were subjected to unpaired t-tests using Welch’s correction (Prism 3.0, Graphpad software, San Diego, CA). Data from some experiments were subjected to two-way analysis of variance analysis using Statistica 6.0 (StatSoft Inc., Tulsa, OK) to assess the significance of differences between groups.

Acknowledgments

We thank Dr Dario Altieri for the GFP- and Survivin-expressing adenoviruses, Don Gentry in our core facility for help with fluorescence microscopy, and Ken Boucher for discussions on statistical analysis. This work was supported by NIH Grant AR050102 (DG), and the Huntsman Cancer Foundation (DG, RML, SAL).

Abbreviations

AIF

apoptosis-inducing factor

EndoG

endonuclease G

GFP

green fluorescence protein

4-TBP

4-tert-butylphenol

RNAi

RNA interference

siRNA

small interfering RNAs

zVAD-fmk

zVal-Ala- Asp(OMe)-fluoromethyl ketone

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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