Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells

Smac/DIABLO was recently identified as a protein released from mitochondria in response to apoptotic stimuli which promotes apoptosis by antagonizing inhibitors of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis. However, little is known about the role of Smac/DIABLO in hydrogen peroxide (H(2)O(2))-induced apoptosis of C2C12 myogenic cells. In this study, Hoechst 33258 staining was used to examine cell morphological changes and to quantitate apoptotic nuclei. DNA fragmentation was observed by agarose gel electrophoresis. Intracellular translocation of Smac/DIABLO from mitochondria to the cytoplasm was observed by Western blotting. Activities of caspase-3 and caspase-9 were assayed by colorimetry and Western blotting. Full-length Smac/DIABLO cDNA and antisense phosphorothioate oligonucleotides against Smac/DIABLO were transiently transfected into C2C12 myogenic cells and Smac/DIABLO protein levels were analyzed by Western blotting. The results showed that: (1) H(2)O(2) (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondria to cytoplasm 1 h after treatment, activation of caspase-3 and caspase-9 4 h after treatment, and specific morphological changes of apoptosis 24 h after treatment; (2) overexpression of Smac/DIABLO in C2C12 cells significantly enhanced H(2)O(2)-induced apoptosis and the activation of caspase-3 and caspase-9 (P<0.05). (3) Antisense phosphorothioate oligonucleotides against Smac/DIABLO markedly inhibited de novo synthesis of Smac/DIABLO and this effect was accompanied by decreased apoptosis and activation of caspase-3 and caspase-9 induced by H(2)O(2) (P<0.05). These data demonstrate that H(2)O(2) could result in apoptosis of C2C12 myogenic cells, and that release of Smac/DIABLO from mitochondria to cytoplasm and the subsequent activation of caspase-9 and caspase-3 played important roles in H(2)O(2)-induced apoptosis in C2C12 myogenic cells.     

Endogenously released Smac is insufficient to mediate cell death of human lung carcinoma in response to etoposide

Cytotoxic agents eliminate tumor cells via different mechanisms including apoptosis, although this process is not equally efficient in all kinds of cancer cells. Thus, small cell lung carcinomas (SCLCs) are more sensitive than non-small cell lung carcinomas (NSCLCs) to therapy-induced killing. During apoptosis, several apoptogenic proteins release from the mitochondria. Among these proteins is Smac/DIABLO. Overexpression of Smac effectively potentiates apoptosis by neutralizing the caspase-inhibitory function of the inhibitors of apoptosis proteins (IAPs). However, the physiological relevance of endogenously released Smac in the promotion of malignant cell death is still unclear. Analysis of a panel of human lung cancer cell lines revealed that there is no altered Smac expression in NSCLC and SCLC that might initially impair the drug-induced cell death. Upon engagement of the mitochondrial pathway of apoptosis, etoposide provoked cytosolic accumulation of Smac along with cytochrome c and loss of the mitochondrial membrane potential. Most of these events as well as nuclear apoptotic changes required caspase activation in SCLC, but not in NSCLC. Unexpectedly, pan-caspase inhibition had no effect on Smac release. Co-treatment of SCLC with the IAP-binding peptide Smac-N7 enhanced etoposide-induced apoptosis in a concentration-dependent manner, whereas Smac downregulation by small interfering RNA (siRNA) did not influence caspase-3/-7 activities, nuclear morphological changes, DNA fragmentation, and plasma membrane integrity. Release of cytochrome c and mitochondrial protease Omi/HtrA2 is still detectable at these conditions. These data suggest that Smac deficiency may be compensated for by action of redundant determinants to kill cancer cells. Thus, translocation of endogenous Smac into cytosol does not play a critical role in cell death of human lung carcinoma after etoposide treatment.     

Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death

Inhibitor of apoptosis protein (IAP)-binding proteins such as Grim, Reaper and HID have been shown to exert a critical role in regulating caspase activity in species such as D. Melanogaster. However, a comparable role for the mammalian homologue of second mitochondrial-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) has yet to be clearly established in vivo. Despite tremendous interest in recent years in the use of so-called Smac mimetics to enhance chemotherapeutic potency, our understanding of the true physiologic nature of Smac/DIABLO in regulating programmed cell death (PCD) remains elusive. In order to critically evaluate the role of Smac/DIABLO in regulating mammalian PCD, deficiency of caspase-3 was used as a sensitizing mutation in order to reduce aggregate levels of executioner caspase activity. We observe that combinatorial deletion of Diablo and Casp3, but neither alone, results in perinatal lethality in mice. Consistent with this, examination of both intrinsic and extrinsic forms of PCD in lines of murine embryonic fibroblasts demonstrate that loss of Smac/DIABLO alters both caspase-dependent and caspase-independent intrinsic PCD. Comparative small interfering RNA inhibition studies of X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis (cIAP)-1, cIAP-2, caspase-6 and -7 in both wild-type and Casp3/Diablo DKO mouse embryonic fibroblast lineages, supports a model in which Smac/DIABLO acts to enhance the early phase executioner caspase activity through the modulation of inhibitory interactions between specific IAP family members and executioner caspases-3 and -7.     

Smac/DIABLO is required for effector caspase activation during apoptosis in human cells

Mitochondria play a pivotal role during stress-induced apoptosis as several proapoptotic proteins are released to the cytosol to activate caspases. Smac/DIABLO is one of the proapoptotic proteins released from the mitochondria and has been shown to inactivate IAPs. However, gene knockout studies in mice revealed a redundant role for Smac during development and cell death. By applying RNA interference-mediated loss of function approach, we demonstrate that Smac/DIABLO is required for the activation of effector but not initiator caspases during stress and receptor-mediated cell death in HeLa cells. Cells with reduced Smac resist apoptosis and retained clonogenicity. Our results suggest an obligatory role for Smac/DIABLO in these tumor cells during several pathways of apoptosis induction.     

TRAF3 interacts with Smac/DIABLO and enhances the proapoptotic effect of Smac/DIABLO in cytoplasm

Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding proteinwith low PI) is a 29 kDa mitochondrial precursor protein,which is proteolytically processed in mitochondriainto a 23 kDa mature protein.It is released from the mitochondrial intermembrane space to cytosol after anapoptotic trigger.Smac/DIABLO acts as a dimer and it contributes to caspase activation by sequestering theinhibitor of apoptosis proteins (IAPs).In order to further investigate the mechanism of Smac/DIABLOaction,we used the mature form of Smac/DIABLO as a bait and screened proteins that interact with matureSmac/DIABLO in human liver cDNA library using the yeast two-hybrid system.Forty-two colonies wereobtained after 5.8x 10~6 colonies were screened by nutrition limitation and X-galactosidase assay.After DNAsequence analysis and homology retrieval,one of the candidate proteins was identified as TRAF domain ofthe TNF receptor associated factor 3 (TRAF3).The interaction site between TRAF3 and Smac/DIABLOwas identified by β-galactosidase test. The interaction between TRAF3 and Smac/DIABLO via TRAF domainwas identified in vivo by co-immunoprecipitation in HepG2 cells,and the direct interaction between TRAF3and Smac/DIABLO in vitro was identified by GST-pull down assay.Co-expression of TRAF3 and matureSmac/DIABLO in 293 cells could enhance the Smac/DIABLO-mediated apoptosis.These results suggestedthat TRAF3 interacted with Smac/DIABLO via TRAF domain,leading to an increased proapoptotic effectof Smac/DIABLO in cytoplasm.     

Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2.

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV- or gamma-irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpressing cells. Thus, in addition to modulating apoptosis-associated mitochondrial cytochrome c release, Bcl-2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress-associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase-catalysed event that occurs downstream of cytochrome c release.     

Solid phase synthesis of Smac/DIABLO-derived peptides using a ‘Safety-Catch’ resin: Identification of potent XIAP BIR3 antagonists

The N-terminal sequence of the Smac/DIABLO protein is known to be involved in binding to the BIR3 domain of the anti-apoptotic proteins IAPs, antagonizing their action. Short peptides and peptide mimetics based on the first 4-residues of Smac/DIABLO have been demonstrated to re-sensitize resistant cancer cells, over-expressing IAPs, to apoptosis. Based on the well-defined structural basis for this interaction, a small focused library of C-terminal capped Smac/DIABLO-derived peptides was designed in silico using docking to the XIAP BIR3 domain. The top-ranked computational hits were conveniently synthesized employing Solid Phase Synthesis (SPS) on an alkane sulfonamide ‘Safety-Catch’ resin. This novel approach afforded the rapid synthesis of the target peptide library with high flexibility for the introduction of various C-terminal amide-capping groups. The library members were obtained in high yield (>65%) and purity (>85%), upon nucleophilic release from the activated resin by treatment with various amine nucleophiles. In vitro caspase-9 activity reconstitution assays of the peptides in the presence of the recombinant BIR3-domain of human XIAP (500 nM) revealed N-methylalanyl-tertiarybutylglycinyl-4-(R)-phenoxyprolyl-N-biphenylmethyl carboxamide (11a) to be the most potent XIAP BIR3 antagonist of the series synthesized inducing 93% recovery of caspase-9 activity, when used at 1 μM concentration. Compound (11a) also demonstrated moderate cytotoxicity against the breast cancer cell lines MDA-MB-231 and MCF-7, compared to the Smac/DIABLO-derived wild-type peptide sequences that were totally inactive in the same cell lines.     

A high-throughput screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay

Resistance to apoptosis is afforded by inhibitor of apoptosis proteins (IAPs) which bind to and inhibit the caspases responsible for cleavage of substrates leading to apoptotic cell death. Smac (or DIABLO), a proapoptotic protein released from the mitochondrial intermembrane space into the cytosol, promotes apoptosis by binding to IAPs, thus reversing their inhibitory effects on caspases. We have developed a high-throughput fluorescence polarization assay utilizing a fluorescein-labeled peptide similar to the "IAP binding" domain of Smac N terminus complexed with the BIR3 domain of X-linked IAP (XIAP) to identify small-molecule mimics of the action of Smac. The IC(50)s of peptides and a tetrapeptidomimetic homologous to the N terminus of Smac demonstrated the specificity and utility of this assay. We have screened the National Cancer Institute "Training Set" of 230 compounds, with well-defined biological actions, and the "Diversity Set" of 2000 chemically diverse structures for compounds which significantly reduced fluorescence polarization. Highly fluorescing or fluorescence-quenching compounds (false positives) were distinguished from those which interfered with Smac peptide binding to the XIAP-BIR3 in a dose-dependent manner (true positives). This robust assay offers potential for high-throughput screening discovery of novel compounds simulating the action of Smac/DIABLO.     

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17β-estradiol at estrus

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17β-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17β-estradiol treatment. Expression of NF-κB and IκB proteins, and IκB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus.     

Relative timing of redistribution of cytochrome c and Smac/DIABLO from mitochondria during apoptosis assessed by double immunocytochemistry on mammalian cells

Redistribution of cytochrome c and Smac/DIABLO from mitochondria occurs during apoptosis, although the relative timing of their release is not well characterized. Double immunocytochemistry was utilized here to study quantitatively the patterns of release of cytochrome c and Smac/DIABLO from mitochondria in single cells. Human osteosarcoma cells and murine embryonic cortical neurons were analyzed during apoptosis induced by staurosporine. In osteosarcoma cells treated with staurosporine for 24 h, a substantial proportion of cells (36%) released cytochrome c from the mitochondria before Smac/DIABLO. In contrast, these proteins were released mostly concordantly in neurons; only a minority of cells (< or = 15%) released cytochrome c without Smac/DIABLO (or vice versa) from mitochondria. Patterns of release in either cell type were unaltered by addition of the caspase inhibitor, zVAD-fmk. The double immunocytochemistry procedure facilitated clear definition of the temporal release of cytochrome c and Smac/DIABLO from mitochondria in intact apoptotic cells, enabling us to demonstrate for the first time that their mutual redistribution during apoptosis varies between different cell types.     

Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway

Smac/DIABLO is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes cytochrome c-dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs). We provide evidence that Smac/DIABLO functions at the levels of both the Apaf-1-caspase-9 apoptosome and effector caspases. The N terminus of Smac/DIABLO is absolutely required for its ability to interact with the baculovirus IAP repeat (BIR3) of XIAP and to promote cytochrome c-dependent caspase activation. However, it is less critical for its ability to interact with BIR1/BIR2 of XIAP and to promote the activity of the effector caspases. Consistent with the ability of Smac/DIABLO to function at the level of the effector caspases, expression of a cytosolic Smac/DIABLO in Type II cells allowed TRAIL to bypass Bcl-xL inhibition of death receptor-induced apoptosis. Combined, these data suggest that Smac/DIABLO plays a critical role in neutralizing IAP inhibition of the effector caspases in the death receptor pathway of Type II cells.     

Smac/DIABLO is not released from mitochondria during apoptotic signalling in cells deficient in cytochrome c

We have characterised the apoptotic defects in cells null for cytochrome c (cyt c-/-). Such cells treated with staurosporine (STS) exhibited translocation to the mitochondria and activation of the proapoptotic signalling molecule Bax but failed to release Smac/DIABLO from these organelles, judged by both confocal microscopy and Western blotting. While reference cells expressing cytochrome c released both it and Smac/DIABLO under a variety of conditions of apoptotic induction, we have never observed release of Smac/DIABLO from cyt c-/- cells. We eliminate the possibility that proteasomal degradation of cytosolically localised Smac/DIABLO is responsible for our failure to visualise the protein outside the mitochondria. Our findings indicate an unanticipated nexus between release of cytochrome c and Smac/DIABLO from mitochondria, previously thought to be a more or less synchronised event early in apoptosis. We suggest that the failure of cyt c-/- cells to release Smac/DIABLO after recruitment of Bax to mitochondria represents an extreme manifestation of some inherent difference in the regulation of release of these two proteins from mitochondria.     

Mitochondrial fission leads to Smac/DIABLO release quenched by ARC

Apoptosis plays a critical role for the development of a variety of cardiac diseases. Cardiomyocytes are enriched in mitochondria, while mitochondrial fission can regulate apoptosis. The molecular mechanism governing cardiomyocyte apoptosis remain to be fully elucidated. Our results showed that Smac/DIABLO is necessary for apoptosis in cardiomyocytes, and it is released from mitochondria into cytosol in response to apoptotic stimulation. Smac/DIABLO release is a consequence of mitochondrial fission mediated by dynamin-related protein-1 (Drp1). Upon release Smac/DIABLO binds to X-linked inhibitor of apoptosis protein (XIAP), resulting in the activation of caspase-9 and caspase-3. Their activation is a prerequisite for the initiation of apoptosis because the administration of z-LEHD-fmk and z-DQMD-fmk, two relatively specific inhibitors for caspase-9, and caspase-3, respectively, could significantly attenuate apoptosis. Smac/DIABLO release could not be blocked by these caspase inhibitors, indicating that it is an event upstream of caspase activation. ARC (apoptosis repressor with caspase recruitment domain), an abundantly expressed apoptotic repressor in cardiomyocytes, could inhibit mitochondrial fission and Smac/DIABLO release. Our data reveal that Smac/DIABLO is a target of ARC in counteracting apoptosis.     

Livin promotes Smac/DIABLO degradation by ubiquitin-proteasome pathway

Livin, a member of the inhibitor of apoptosis protein (IAP) family, encodes a protein containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. It has been reported that Livin directly interacts with caspase-3 and -7 in vitro and caspase-9 in vivo via its BIR domain and is negatively regulated by Smac/DIABLO. Nonetheless, the detailed mechanism underlying its antiapoptotic function has not yet been fully characterized. In this report, we provide, for the first time, the evidence that Livin can act as an E3 ubiquitin ligase for targeting the degradation of Smac/DIABLO. Both BIR domain and RING finger domain of Livin are required for this degradation in vitro and in vivo. We also demonstrate that Livin is an unstable protein with a half-life of less than 4 h in living cells. The RING domain of Livin promotes its auto-ubiquitination, whereas the BIR domain is likely to display degradation-inhibitory activity. Mutation in the Livin BIR domain greatly enhances its instability and nullifies its binding to Smac/DIABLO, resulting in a reduced antiapoptosis inhibition. Our findings provide a novel function of Livin: it exhibits E3 ubiquitin ligase activity to degrade the pivotal apoptotic regulator Smac/DIABLO through the ubiquitin-proteasome pathway.     

Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential

Background

We have previously shown that prostate cancer LNCaP cells are resistant to TRAIL, and downregulation of PI-3K/Akt pathway by molecular and pharmacological means sensitizes cells to undergo apoptosis by TRAIL and curcumin. The purpose of this study was to examine the molecular mechanisms by which resveratrol sensitized TRAIL-resistant LNCaP cells.

Results

Resveratrol inhibited growth and induced apoptosis in androgen-dependent LNCaP cells, but had no effect on normal human prostate epithelial cells. Resveratrol upregulated the expression of Bax, Bak, PUMA, Noxa, Bim, TRAIL-R1/DR4 and TRAIL-R2/DR5, and downregulated the expression of Bcl-2, Bcl-X_L, survivin and XIAP. Treatment of LNCaP cells with resveratrol resulted in generation of reactive oxygen species, translocation of Bax and p53 to mitochondria, subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, AIF, Smac/DIABLO and Omi/HtrA2), activation of caspase-3 and caspase-9 and induction of apoptosis. The ability of resveratrol to sensitize TRAIL-resistant LNCaP cells was inhibited by dominant negative FADD, caspase-8 siRNA or N-acetyl cysteine. Smac siRNA inhibited resveratrol-induced apoptosis, whereas Smac N7 peptide induced apoptosis and enhanced the effectiveness of resveratrol.

Conclusion

Resveratrol either alone or in combination with TRAIL or Smac can be used for the prevention and/or treatment of human prostate cancer.     

Real-time single cell analysis of Smac/DIABLO release during apoptosis

We examined the temporal and causal relationship between Smac/DIABLO release, cytochrome c (cyt-c) release, and caspase activation at the single cell level during apoptosis. Cells treated with the broad-spectrum caspase inhibitor z-VAD-fmk, caspase-3 (Casp-3)-deficient MCF-7 cells, as well as Bax-deficient DU-145 cells released Smac/DIABLO and cyt-c in response to proapoptotic agents. Real-time confocal imaging of MCF-7 cells stably expressing Smac/DIABLO-yellow fluorescent protein (YFP) revealed that the average duration of Smac/DIABLO-YFP release was greater than that of cyt-c-green fluorescent protein (GFP). However, there was no significant difference in the time to the onset of release, and both cyt-c-GFP and Smac/DIABLO-YFP release coincided with mitochondrial membrane potential depolarization. We also observed no significant differences in the Smac/DIABLO-YFP release kinetics when z-VAD-fmk-sensitive caspases were inhibited or Casp-3 was reintroduced. Simultaneous measurement of DEVDase activation and Smac/DIABLO-YFP release demonstrated that DEVDase activation occurred within 10 min of release, even in the absence of Casp-3.     

Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism

We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death. This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac. Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition.     

Novel link between E2F1 and Smac/DIABLO: proapoptotic Smac/DIABLO is transcriptionally upregulated by E2F1

Deregulated expression of E2F1 not only promotes S-phase entry but also induces apoptosis. Although it has been well documented that E2F1 is able to induce p53-dependent apoptosis via raising ARF activity, the mechanism by which E2F induces p53-independent apoptosis remains unclear. Here we report that E2F1 can directly bind to and activate the promoter of Smac/DIABLO, a mitochondrial proapoptotic gene, through the E2F1-binding sites BS2 (-542 approximately -535 bp) and BS3 (-200 approximately -193 bp). BS2 and BS3 appear to be utilized in combination rather than singly by E2F1 in activation of Smac/DIABLO. Activation of BS2 and BS3 are E2F1-specific, since neither E2F2 nor E2F3 is able to activate BS2 or BS3. Using the H1299 ER-E2F1 cell line where E2F1 activity can be conditionally induced, E2F1 has been shown to upregulate the Smac/DIABLO expression at both mRNA and protein levels upon 4-hydroxytamoxifen treatment, resulting in an enhanced mitochondria-mediated apoptosis. Reversely, reducing the Smac/DIABLO expression by RNA interference significantly diminishes apoptosis induced by E2F1. These results may suggest a novel mechanism by which E2F1 promotes p53-independent apoptosis through directly regulating its downstream mitochondrial apoptosis-inducing factors, such as Smac/DIABLO.