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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2013 Apr 23;110(19):E1724–E1733. doi: 10.1073/pnas.1220282110

E4orf4 induces PP2A- and Src-dependent cell death in Drosophila melanogaster and at the same time inhibits classic apoptosis pathways

Antonina Pechkovsky a,b,1, Maoz Lahav a,1, Eliya Bitman a,2, Adi Salzberg b,3, Tamar Kleinberger a,3
PMCID: PMC3651459  PMID: 23613593

Significance

Expression of the adenovirus protein E4orf4 alone in cultured mammalian cells prompts noncanonical apoptosis that is more efficient in oncogene-transformed cells than in normal cells. Here, E4orf4 activity in a whole organism (Drosophila melanogaster) is described, leading to three significant conclusions: (i) E4orf4-induced cell death is an evolutionarily conserved process; (ii) E4orf4 induces a distinctive mode of cell death, differing from well-characterized cell death mechanisms; and (iii) E4orf4 activates cell death but concomitantly inhibits it, thus minimizing damage to normal tissues. The last finding suggests a possible explanation for the differential effect of E4orf4 in normal and cancer cells.

Abstract

The adenovirus E4orf4 protein regulates the progression of viral infection, and when expressed alone in mammalian tissue culture cells it induces protein phosphatase 2A (PP2A)-B55– and Src-dependent cell death, which is more efficient in oncogene-transformed cells than in normal cells. This form of cell death is caspase-independent, although it interacts with classic caspase-dependent apoptosis. PP2A-B55–dependent E4orf4-induced toxicity is highly conserved in evolution from yeast to mammalian cells. In this work we investigated E4orf4-induced cell death in a whole multicellular organism, Drosophila melanogaster. We show that E4orf4 induced low levels of cell killing, caused by both caspase-dependent and -independent mechanisms. Drosophila PP2A-B55 (twins/abnormal anaphase resolution) and Src64B contributed additively to this form of cell death. Our results provide insight into E4orf4-induced cell death, demonstrating that in parallel to activating caspase-dependent apoptosis, E4orf4 also inhibited this form of cell death induced by the proapoptotic genes reaper, head involution defective, and grim. The combination of both induction and inhibition of caspase-dependent cell death resulted in low levels of tissue damage that may explain the inefficient cell killing induced by E4orf4 in normal cells in tissue culture. Furthermore, E4orf4 inhibited JNK-dependent cell killing as well. However, JNK inhibition did not impede E4orf4-induced toxicity and even enhanced it, indicating that E4orf4-induced cell killing is a distinctive form of cell death that differs from both JNK- and Rpr/Hid/Grim-induced forms of cell death.

The adenovirus E4 ORF 4 (E4orf4) protein is a multifunctional viral regulator that contributes to temporal regulation of the progression of viral infection (1–3). When expressed alone, E4orf4 induces p53-independent cell death in transformed cells (4–6). Oncogenic transformation of primary cells in tissue culture sensitizes them to cell killing by E4orf4 (7), indicating that E4orf4 research may have implications for cancer therapy. It has been further reported that E4orf4 induces a caspase-independent, nonclassic apoptotic pathway, which maintains a cross-talk with the classic caspase-dependent pathways (4, 8). We have previously shown that E4orf4 interacts with the heterotrimeric protein phosphatase 2A (PP2A) through a direct association with its regulatory Bα/B55 or B’/B56 subunits (2, 9). The PP2A–E4orf4 interaction mediated by the Bα/B55 subunit is required for induction of cell death (7, 9, 10). E4orf4 also associates with members of the Src kinase family, leading to its Tyr phosphorylation and to deregulation of Src signaling, resulting in enhanced cell death (11). Analysis of E4orf4 mutants further indicated that the interactions of E4orf4 with PP2A and Src had an additive effect on E4orf4-induced cell death (12).

Studies in yeast revealed that E4orf4 induced PP2A-dependent irreversible growth arrest in this organism (13), indicating that at least the PP2A-dependent E4orf4-induced cell death pathway was highly conserved in evolution. This conservation was further confirmed by a yeast genetic screen that revealed an additional component of the E4orf4 cell death network, Ynd1, which associated with E4orf4 and contributed to E4orf4-induced cell killing in mammalian cells as well (14).

Because yeast was expected to uncover only part of the mechanisms underlying E4orf4-induced cell death in mammalian cells, the powerful genetic model fly Drosophila melanogaster was chosen for further studies. Drosophila has served as an excellent model organism for the study of apoptotic processes (15, 16). Most forms of programmed cell death in the fly are caspase-dependent and require the presence of a small genomic region (H99) that contains the reaper (rpr), head involution defective (hid), and grim genes (15, 16). However, under some circumstances, apoptosis can occur in the absence of H99 (16), and recent reports have suggested the existence of caspase-independent programmed cell death in the fly (17–19).

Replication of animal viruses relies on interactions between viral proteins and cellular regulatory factors. These interactions allow the recruitment of host cell mechanisms to optimally support virus replication. Although animal viruses do not infect Drosophila, several regulatory mechanisms are highly conserved between flies and mammalian cells, and Drosophila can therefore serve as a good model system to investigate virus–host cell interactions (reviewed in ref. 20).

In this work we investigated the consequences of adenovirus E4orf4 expression in Drosophila. We found a high degree of conservation in the mechanisms underlying E4orf4-induced cell death in mammalian cells and in the fly and discovered a unique E4orf4 function, namely its ability to inhibit classic apoptosis pathways in addition to its ability to induce cell death. Our results suggest that the fly system can be used to address important aspects of E4orf4 biology.

Results

Generation of Transgenic UAS-E4orf4 Flies.

E4orf4 was cloned into the pUAST vector (21) downstream of the yeast upstream activating sequence (UAS), and a transgenic fly strain carrying the UAS-E4orf4 construct on the third chromosome was generated (E4orf4-1). To obtain fly strains that vary in their E4orf4 expression levels, the original third chromosome transgene was transposed to additional chromosomal locations, and several new strains were obtained. E4orf4 was expressed by crossing the UAS-E4orf4 flies to a variety of fly strains expressing the yeast Gal4 transcription factor from various tissue-specific promoters. The different E4orf4 strains likely expressed varying levels of E4orf4 upon induction as judged by the severity of their resulting phenotypes. To examine the effects of E4orf4 expression in detail, strain E4orf4-17, which generated the strongest E4orf4-induced phenotypes, was chosen for further analysis.

E4orf4 Induces Dose-Dependent Phenotypes in Eyes and Wings of Normal Flies.

To explore the phenotypic effects of E4orf4 in flies, the UAS-E4orf4-17 strain was crossed to flies carrying GAL4 driven by the Glass Multimer Reporter (GMR) or engrailed (en) promoters. To examine the dose-dependence of E4orf4-induced phenotypes, crosses were set at varying temperatures, because Gal4-driven expression was reported to rise with increasing temperatures (22). en>E4orf4 third-instar larvae were used for preparation of protein extracts, and the Western blot shown in Fig. 1A confirms that E4orf4 expression levels increased with rising temperatures. Furthermore, E4orf4 expression was higher in males than in females (Fig. 1A). Expression of E4orf4 in the eye under control of GMR-GAL4 (GMR>E4orf4) at 29 °C induced a small and rough eye phenotype, and the severity of this phenotype decreased at lower temperatures (Fig. 1 C–E). GMR>GFP flies served as a control (Fig. 1F). Fig. 1B demonstrates that the E4orf4 effect on eye size was more dramatic in males than in females, probably owing to the higher levels of E4orf4 expression in males.

Fig. 1.

Fig. 1.

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E4orf4 induces dose-dependent phenotypes in the Drosophila eye and wing. (A) Protein extracts were prepared from en>E4orf4 third-instar larvae reared at the indicated temperatures, and Western blot analysis was performed using antibodies to E4orf4 and actin, which served as a loading control. (Left) Protein extracts from male larvae; (Right) extracts from males (m) compared with females (f). (B) Eye sizes of GMR>E4orf4 and GMR>GFP male and female flies reared at the indicated temperatures were measured using the ImageJ program. Sizes of E4orf4-expressing eyes were normalized to eye sizes of GFP-expressing flies. Relative eye sizes are shown for each temperature and each sex. Error bars mark the SE. n = 8–20, P < 0.001 for the differences between E4orf4 and GFP at each temperature. (C–F) Representative eyes of male flies with the indicated genotypes reared at the indicated temperatures. (G) en>E4orf4 flies (males and females) were scored for the following wing defects: ACV, lacking the anterior cross-vein; PCV, lacking the posterior cross-vein; L5, lacking part of the L5 vein; posterior to L5, lacking part of the wing blade posterior to the L5 vein; anterior to L5, lacking a larger portion of the wing blade spanning the L4–L5 intervein domain. Percent of the population containing the various wing defects at each temperature is shown. n = 80–98. (H–K) Representative wings of flies with the indicated genotypes reared at the indicated temperatures. The veins, cross-veins, and alulae are marked on the en>GFP wing (K), and arrows mark the defects in the en>E4orf4 wings (H–J). (L) The number of bristles in the alulae of en>E4orf4 flies were counted at each rearing temperature, and the average number was normalized to the bristle number in control GMR>GFP alulae. The normalized values are shown. Error bars indicate SE. n = 35–58. 18 °C: P = 0.04; 24 °C: P < 10−6; 29 °C: P < 10−6. (M–P) Representative alulae of flies with genotypes as in H–K and reared at the indicated temperatures.

Expression of E4orf4 in the posterior compartment of the wing, using the en-GAL4 driver (en>E4orf4), caused a variety of wing defects, including loss of the anterior cross-vein (ACV), loss of the posterior cross-vein (PCV), partial elimination of the L5 vein, partial elimination of the posterior part of the wing blade, or almost complete elimination of the posterior part of the wing (Fig.1 H–J). The percentage of wing defects at each temperature is summarized in Fig. 1G. The results indicate that increasing the temperature at which the flies were reared resulted in a greater proportion of more severe wing defects caused by E4orf4. We also observed that the wings of en>E4orf4 flies grown at 18 °C (Fig. 1H) were larger than control en>GFP wings (Fig. 1K), which were similar in size at all temperatures. This effect on wing size was not observed at higher temperatures in the presence of higher E4orf4 levels. In addition to causing wing defects, E4orf4 expression reduced the number of bristles in en>E4orf4 alulae in a dose-dependent manner, as demonstrated in Fig. 1 L–O. en>GFP expression at 29 °C did not cause similar effects (Fig. 1P). In summary, the results presented in Fig. 1 indicate that E4orf4 induced dose-dependent defects in eyes and wings.

E4orf4 Induces Caspase-Dependent Apoptosis in Drosophila.

To determine whether E4orf4 effects on eye and wing morphology resulted from induction of apoptosis, eye and wing discs from control GFP- or β-Galactosidase (β-Gal)- and E4orf4-expressing third-instar larvae were stained with antibodies to E4orf4 or the control β-Gal, and to cleaved caspase-3. As seen in Fig. 2 A–F, lacZ expression in the eye disk (GMR>lacZ) was associated with background levels of caspase activation, and expression of E4orf4 (GMR>E4orf4) led to caspase activation in only a small percentage of E4orf4-expressing cells.

Fig. 2.

Fig. 2.

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E4orf4 induces caspase activation in a subpopulation of cells in the Drosophila eye and wing discs. Eye (A–F) and wing (G–R) discs from third-instar larvae (A–L: grown at 29 °C; M–R: grown at 24 °C) with the indicated genotypes [GMR>lacZ (A–C), GMR>E4orf4 (D–F), en>GFP (G–I), en>E4orf4 (J–L), ms1096>lacZ (M–O), and ms1096>E4orf4 (P–R)] were stained with antibodies to β-galactosidase (A, M) or E4orf4 (D, J, P) (Left) or to activated caspase-3 (B, E, H, K, N, Q) (Center). (Right) (C, F, I, L, O, R) Merge of the two channels. Representative images are shown.

Upon E4orf4 expression in the wing disk (en>E4orf4) at 29 °C, many E4orf4-expressing cells did not contain activated caspase-3, but a cluster of cells stained with the antibody to cleaved caspase-3 was observed (Fig. 2 J–L). No caspase activation was observed in wing discs of en>GFP flies (Fig. 2 G–I). Furthermore, clusters of caspase-3–stained cells were not observed when E4orf4-expressing flies were reared at 24 °C rather than 29 °C, or when the p35 caspase inhibitor was expressed together with E4orf4 at 29 °C (Fig. S1). Some of the cells expressing an active caspase-3 manifested reduced E4orf4 staining and seemed to invade the anterior part of the disk (Fig. 2L), reminiscent of migrating “undead” cells described previously (23). The cluster of cells with activated caspase-3 may represent dying cells that are being extruded from the wing disk tissue, as can be seen in Fig. S2. Because we observed the appearance of extruded cells undergoing apoptosis when E4orf4 was expressed under the regulation of en-GAL4 in the wing disk but not when a GMR driver was used in the eye disk, we asked whether dead cell extrusion was typical of the wing disk or whether it was specific to en>E4orf4 expression. To address this question, the ms1096 driver, which drives expression in the entire wing blade (24), was used to express E4orf4 at 24 °C. As demonstrated in Fig. 2 M–R and Fig. S3 A and B, caspase activation was observed in a fraction of the cells in ms1096>E4orf4 wing discs but not in ms1096>lacZ discs. Interestingly, expression of lacZ in the control ms1096>lacZ discs was uniform (Fig. 2M and Fig. S3A), whereas in the ms1096>E4orf4 discs many cells with high levels of active caspase-3 exhibited low levels of E4orf4 (Fig. 2 P–R and Fig. S3B), suggesting loss of E4orf4 expression. Colocalization of E4orf4 and active caspase-3 was observed at the margin of the region containing cells with high caspase and low E4orf4 levels (Fig. S3 C and D). This colocalization pattern possibly represents the transition from live cells expressing E4orf4 to dying cells losing E4orf4 expression. Furthermore, optical sectioning and 3D reconstructions of ms1096>E4orf4 discs demonstrated that dying cells containing high levels of active caspase-3, and low E4orf4 levels were extruded from the living tissue (Fig. S3E).

Caspase-Independent Effects Contribute to E4orf4-Induced Phenotypes.

It has been reported that E4orf4-induced cell death in mammalian cells is caspase-independent, although there is a cross-talk between nonclassic apoptosis induced by E4orf4 and classic, caspase-dependent pathways (4, 8). Results shown in Fig. 2 indicated that E4orf4 induced caspase activation in Drosophila eye and wing discs. To determine whether the E4orf4-induced phenotypes described in Fig. 1 resulted exclusively from caspase-dependent apoptosis, or whether caspase-independent mechanisms were involved as well, we examined E4orf4-associated phenotypes in flies expressing caspase inhibitors.

Expression of E4orf4 together with a control GFP transgene under regulation of the GMR driver (GMR>E4orf4,GFP) at 29 °C decreased eye size in males to 48% of control GFP flies (GMR>GFP,GFP) and in females to 68% of control (Fig. 3 B and E, n > 32). Concomitant expression of the caspase inhibitor Drosophila inhibitor-of-apoptosis protein 1 (dIAP1) with E4orf4 (GMR>E4orf4,dIAP1) did not result in complete recovery of normal eye size, reversing the size of E4orf4-induced small eyes to only 75% of control male GMR>GFP,dIAP1 eye size, and to 87% of female control (Fig. 3 C and E, n > 32). These differences in eye sizes were statistically significant (Fig. 3E, P < 10−6). Moreover, dIAP1 expression did not reduce E4orf4-induced eye roughness and did not result in recovery of normal eye shape. In contrast, when dIAP1 was coexpressed with a Rpr transgene (rprH3-13) that acted more efficiently than E4orf4 and eliminated most of the ommatidia in the eye (compare Fig. 3 B and G), eye size was almost completely restored to normal (98% of control GMR>dIAP1,GFP), and no roughness or other morphological abnormalities were observed (Fig. 3 F–J, n > 31). Furthermore, E4orf4 expression under control of the GMR driver did not cause any lethality, whereas Rpr expression under similar conditions resulted in only 20% adult eclosion (n > 350). Coexpression of dIAP1 with Rpr restored survival to 100% of the flies (n > 350), further indicating that dIAP1 was highly efficient in inhibiting cell death induced by Rpr but less efficient in eliminating the more minor effects produced by E4orf4.

Fig. 3.

Fig. 3.

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Caspase inhibitors do not fully rescue E4orf4 phenotypes. Representative eyes of adult male flies with the indicated genotypes are shown in A–D, F–I, K–N, and P–S. Male and female eye sizes were measured and normalized to control GMR>GFP,GFP male or female eye sizes, respectively. The effects of dIAP1 and p35 on the E4orf4-induced small eye phenotype are plotted in E and O, respectively, whereas their effect on Rpr-induced phenotypes is shown in J and T. The flies were reared at 29 °C (A–I) or at 24 °C (K–S). The rpr transgene used here is rprHA3-13. Error bars represent SEs. n = 22–69.

Similar results were obtained with the p35 caspase inhibitor expressed in flies that were reared at 24 °C. When E4orf4 was coexpressed with p35, eye sizes increased but did not revert to the size of control eyes expressing GFP with p35 (Fig. 3 K–O, P < 10−6). Furthermore, eyes expressing E4orf4 and p35 retained the roughness and the more narrowly oval shape typical of eyes expressing E4orf4 with GFP (Fig. 3 K–N). As for the effect of p35 on Rpr-expressing eyes, p35 expressed together with Rpr in females did not increase eye size to the dimensions of GFP+p35-expressing eyes, which were larger by 20% than WT eyes (Fig. 3 P–T). However, in males there was no statistically significant difference between the size of eyes expressing Rpr+p35 and eyes expressing GFP+p35 (Fig. 3T, n > 30, P = 0.74). In addition, p35 eliminated Rpr-induced eye roughness in both males and females. Furthermore, Rpr expression resulted in only 56% adult eclosion, and p35 coexpression led to 100% viability. Expression of a stronger rpr transgene [rpr(X)] under GMR control caused 100% fly lethality at 24 °C, and coexpression with p35 rescued 100% of fly viability, confirming the efficient inhibition of rpr-induced apoptosis by p35.

Thus, the results suggest that E4orf4-induced phenotypes are partially caspase-dependent but that caspase-independent mechanisms also contribute to the E4orf4 effects.

E4orf4 Inhibits Classic Apoptotic Pathways in Drosophila.

It has been previously observed that inhibition of apoptosis in the wing disk by the p35 caspase inhibitor resulted in the generation of so-called “undead” cells (25–27). These cells exhibit most apoptosis markers, and they round up and can invade neighbor compartments, although they do not gain the identity of the invaded compartment (23). The appearance of a cluster of cells containing active caspase-3 and partially invading the anterior part of the wing disk upon en>E4orf4 expression (Fig. 2) was reminiscent of such clusters of “undead” cells and led us to examine whether E4orf4 had an antiapoptotic function. To test this idea we coexpressed each of the three strong Drosophila proapoptotic genes rpr, hid, and grim either with E4orf4 or with GFP as a control. When hid, grim, and two rpr transgenes were expressed together with GFP under the regulation of GMR-GAL4 at 18 °C, only 69% of GMR>GFP,hid pupae, 32.5% of GMR>GFP,rpr(X) pupae, 0.5% of GMR>GFP,rpr(2) pupae, and 0.01% of GMR>GFP,grim pupae eclosed, whereas 100% of the flies survived E4orf4 and GFP expression (Fig. 4A, n > 300 pupae for each sample). When E4orf4 was expressed together with the proapoptotic genes, 95% of GMR>E4orf4,hid pupae, 78% of GMR>E4orf4,rpr(X), 27.4% of GMR>E4orf4,rpr(2), and 78% of GMR>E4orf4,grim pupae eclosed (Fig. 4A, n > 300). Measuring eye sizes of surviving females revealed that E4orf4 expression significantly increased eye size in all these genetic backgrounds (Fig. 4 B–L, P < 0.01). These results indicate that E4orf4 counteracts classic apoptotic pathways in normal flies.

Fig. 4.

Fig. 4.

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E4orf4 inhibits Rpr-, HID-, and Grim-induced apoptosis. (A) Flies expressing GFP, Rpr(X), Rpr(2), HID, or Grim together with GFP or E4orf4 under GMR control were reared at 18 °C, and the percent of adult eclosion was calculated for each genotype. n > 300 for each sample, and error bars represent SEs. *P < 0.01 for all GFP-E4orf4 pairs except in combination with UAS-GFP (NS, nonsignificant). (B–F) Representative eyes of female adults with the indicated genotypes, not expressing E4orf4. (G) Sizes of female eyes with the indicated genotypes were determined and normalized to eye sizes of control GMR>GFP,GFP females. The average normalized value is shown in the graph. n > 20, except for UAS-rpr(2), n = 4, and UAS-grim, n = 2, owing to lethality. Error bars represent SEs. *P < 0.01 for all pairs. (H–L) Representative female eyes with the indicated genotypes, expressing E4orf4, are shown.

E4orf4 Inhibits JNK-Dependent Apoptosis in Drosophila.

Stress signals in Drosophila activate the proapoptotic genes rpr and hid through the JNK pathway and Drosophila p53 (dp53). JNK and dp53 also function downstream of proapoptotic genes and the Dronc caspase, thus establishing a feedback loop that amplifies the apoptotic stimulus (27–29). Because E4orf4 inhibited Rpr- and Hid-induced apoptosis (Fig. 4), we inquired whether it also inhibited JNK signaling. Eiger, the Drosophila tumor necrosis factor ortholog, has been shown to induce cell death by triggering JNK signaling (17, 19). We first tested therefore the E4orf4 effects on Eiger-induced apoptosis. Overexpression of eiger under the regulation of GMR-GAL4 (GMR>GFP,eiger) resulted in reduced adult eclosion, from 81% of adults at 18 °C, through 49% at 24 °C to 38% at 29 °C. However, coexpression of E4orf4 rescued the flies, and 99% of adults emerged at 18 °C, 100% emerged at 24 °C, and 94% at 29 °C (Fig. 5 A–C, n > 400, P < 0.05). When the eyes of adult flies reared at 18 °C were examined, we found that eye sizes of GMR>GFP,eiger flies were dramatically reduced to 13% of GMR>GFP,GFP eyes in females and to 26% in males (Fig. 5 D, F, and I, n > 25). In contrast, coexpression of E4orf4 with Eiger (GMR>E4orf4,eiger) significantly increased eye sizes to 39% of GMR>GFP,eiger control in females and 53% in males (Fig. 5 D, I, and K, n > 30, P < 0.001). Thus, E4orf4 seems to inhibit Eiger-induced cell death.

Fig. 5.

Fig. 5.

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E4orf4 inhibits JNK-induced cell death. (A–C) Flies expressing GFP, Eiger, or HepCA together with GFP or E4orf4 under GMR regulation were reared at 18 °C (A), 24 °C (B), or 29 °C (C), and the percent of adult eclosion was calculated for each group. n > 400, error bars represent SEs. All nonsignificant changes (P > 0.05) are marked as NS. *P < 0.001, **P < 0.05. (E–H and J–M) Representative male eyes with the indicated genotypes, reared at 18 °C, expressing GFP (E–H) or E4orf4 (J–M). Two different fly strains containing HepCA [hepCA(1) and hepCA(2)] were used. (D and I) Eye sizes of each genotype shown in E–H and J–M were normalized to control GMR>GFP,GFP eye sizes, and the average normalized value is shown in the graph. n > 30, except for male UAS-HepCA(1): n = 6. *P < 0.001 for all pairs. Error bars represent SEs. D, female eyes; I, male eyes.

To investigate which part of the JNK pathway is inhibited by E4orf4 we activated JNK signaling downstream of Eiger by expressing a constitutively active form of the JNK kinase Hemipterous (HepCA) and tested the effect of GFP or E4orf4 on HepCA-induced cell death. As demonstrated in Fig. 5 A and B, GMR>GFP,HepCA(1) expression decreased eclosion rates to 67% at 18 °C and to 0.75% at 24 °C. When E4orf4 was coexpressed with HepCA, the eclosion rate at 18 °C was not significantly altered, but frequency of eclosion was dramatically increased at 24 °C to 89% (n > 400, P < 0.001). Determination of eye sizes in viable adults reared at 18 °C, using two different HepCA transgenic strains, revealed that GMR>GFP,HepCA(1) eye sizes were reduced to 17% of GMR>GFP,GFP eye sizes in females and to 18% of control in males, and GMR>GFP,HepCA(2) eye sizes were reduced to 19% of control in both females and males (Fig. 5 D and G–I, n > 30 except for male GMR>GFP,HepCA(1): n = 6). When E4orf4 was coexpressed with HepCA(1) it increased eye size in females to 25% of control, and in males to 31% (Fig. 5 D, I, and L, n > 30). When expressed with HepCA(2), E4orf4 increased eye size to 23% of control in females and 31% in males (Fig. 5 D, I, and M, n > 30). All E4orf4 effects on eye size were statistically significant (P < 0.001). Thus, when expressed at sufficient levels, E4orf4 inhibited HepCA-induced cell death, indicating that it interacted with the JNK pathway downstream of Hep. The results presented in Fig. 5 are consistent with inhibition of JNK signaling by the viral protein.

Inhibition of the JNK Pathway Enhanced E4orf4-Induced Cell Death.

The finding that E4orf4 inhibited the JNK pathway suggested that JNK inhibition may contribute to E4orf4-induced phenotypes in Drosophila. We set out therefore to determine whether inhibition of JNK cooperated with E4orf4 to generate a small eye phenotype. Inhibition of the JNK pathway was achieved in two ways: reduced Eiger levels in eiger heterozygous flies and overexpression of Puckered (Puc), a JNK phosphatase (30). When the effect of GMR>E4orf4 on eye size was compared in WT and eiger heterozygous backgrounds at 3 different temperatures, we found that reduction of eye size by E4orf4 was significantly more efficient in the eiger heterozygous background for both male and female flies at 24 °C and 29 °C (Fig. 6A, *P < 0.00001). The same effect was observed in females at 18 °C, but no change in E4orf4 effect on eye size was observed for male flies at this temperature (Fig. 6A). When E4orf4 was expressed together with Puc under GMR regulation, eye sizes in both females and males were reduced more significantly than when E4orf4 was expressed together with control GFP, at all temperatures (Fig. 6B, P < 0.05). Puc expression in the absence of E4orf4 did not affect eye size. Thus, JNK inhibition enhanced the E4orf4-induced small eye phenotype, suggesting that JNK inhibition contributed to induction of cell death by E4orf4 in normal Drosophila tissues, and that E4orf4 did not induce cell death through the JNK pathway.

Fig. 6.

Fig. 6.

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JNK inhibition enhances E4orf4-induced cell death. (A) Eye sizes of WT (Canton-S) or eiger−/+ flies containing GMR>GFP or GMR>E4orf4 were measured. GMR>GFP eye sizes in each genetic background, temperature, and sex were defined as 100%, and the graph shows the relative GMR>E4orf4 eye sizes. n > 14, *P < 0.00001. NS, P > 0.05. Error bars represent SEs. (B) Eye sizes of each of the indicated fly groups were measured. The average size of control eyes (GMR>GFP,GFP or GMR>GFP,puc) for each temperature and sex was defined as 100%. Eye sizes of GMR>E4orf4,GFP and GMR>E4orf4,puc were normalized to control GMR>GFP,GFP or GMR>GFP,puc eye sizes, respectively. The average normalized value is shown in the graph. n > 10, *P < 0.00001, **P < 0.05. Error bars represent SEs.

PP2A and Src Kinases Contribute Additively to E4orf4-Induced Cell Death in Drosophila.

It was previously shown in mammalian tissue culture cells that interactions with PP2A (through its regulatory B55 subunit) and with Src kinases contributed to E4orf4-induced cell death (7, 9–11). To examine whether these major E4orf4 partners contributed to E4orf4-induced cell death in the fly, we carried out a series of experiments investigating the effects of WT E4orf4 in a genetic background of reduced PP2A-B55 or Src expression, and the effects of E4orf4 mutants lacking the ability to bind PP2A and/or Src.

The PP2A-B55 subunit is encoded in Drosophila by the twins (tws)/abnormal anaphase resolution (aar) gene, and various alleles of this gene have been described. Two alleles were used here, tws60 (31) and aar1 (32). To investigate whether tws/aar was required for E4orf4-induced cell death in the fly, the UAS-E4orf4-17 insertion was recombined to the aar1 chromosome and the aar1,UAS-E4orf4-17 flies were crossed to en-GAL4 or GMR-GAL4 flies carrying the tws60 mutation. To determine the levels of tws/aar expression in transheterozygotes (tws60/aar1) and in the heterozygous mutants, Western blot analysis was carried out using protein extracts from third-instar larvae of the appropriate genotypes. As seen in Fig. 7A, no PP2A-B55 subunit protein was detected in tws60/aar1 larvae, and lower levels of this protein were detected in the heterozygous tws60/+ in comparison with the WT larvae. We next examined the effect of E4orf4 on eye size in the various genetic backgrounds. In this experiment, the transheterozygous flies, which cannot eclose, were dissected out from the pupal case so their eyes could be visualized. As seen in Fig. 7B, GMR>E4orf4 expression in a WT background at 29 °C reduced eye sizes to 61% of control GMR>GFP eyes in females and to 52% in males (n > 9). Heterozygous flies expressing E4orf4 (GMR>E4orf4 in tws60/+ or aar1/+ backgrounds) had significantly larger eyes, which were 100% of WT eyes in tws60/+ females and 82% in males, and 85% of WT eyes in aar1/+ females and 74% in males (n > 9, P < 0.017). GMR>E4orf4 expression in the tws60/aar1 transheterozygotes did not reduce eye size compared with WT eyes (nfemales = 7, nmales = 2). Quantitation of eye sizes of the various fly strains revealed that the differences between the effects of E4orf4 in a WT background and in the heterozygotes or the transheterozygotes were statistically significant (Fig. 7B, P < 0.045).

Fig. 7.

Fig. 7.

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dPP2A-B55 and dSrc64B contribute additively to E4orf4-induced cell death. (A) Protein extracts were prepared from WT, tws60/+, and tws60/aar1 third-instar larvae, and Western blot analysis was performed using antibodies to dPP2A-B55 and to actin, which served as a loading control. Asterisks mark nonspecific bands. (B) Sizes of male or female eyes with the indicated genotypes and expressing GMR>GFP or GMR>E4orf4 were measured. The average eye size of WT male or female flies expressing GFP was defined as 100%, and relative eye size of the various genotypes is plotted. The genotypes examined here included WT, tws60/+ and aar1/+ heterozygotes, and tws60/aar1 transheterozygotes. n > 9, except for the transheterozygotes, for which: nfemales = 7 and nmales = 2. Error bars represent the SE. P values for the differences between eye sizes of WT and mutant flies expressing E4orf4 were less than 0.045. (C) The frequency of wing defects (described in Fig. 1) in flies with the indicated genotypes is plotted. n = 23–85. (D) E4orf4 was expressed under en regulation in WT flies (WT B55) (n = 26), in tws60/+ or aar1/+ heterozygous flies (n = 33), or together with an interfering RNA to PP2A-B55 (B55-IR) in WT flies (n = 23). Wing discs from third-instar larvae were stained with antibodies to E4orf4 and active caspase-3, and the percentage of discs containing a cluster of cells with active caspase-3 (as seen in Fig. 2K) is shown. (E) GFP or E4orf4 were expressed in male eyes at 29 °C together with GFP or with interfering RNAs (IR) to dSrc64B or PP2A-B55, using the GMR driver. Eye sizes were measured, and the average size of eyes expressing GFP without E4orf4 (GMR>GFP,GFP) was defined as 100%. Relative eye size is shown. Error bars represent the SE. n > 40, except for GMR>E4orf4,B55-IR: n = 14. (F) Transgenic flies containing a pUAST-AttB vector encoding a control RFP protein, WT E4orf4 (WT), or E4orf4 mutants, which do not bind PP2A-B55 (B55−), or Src (Src−), or both (B55−,Src−) were crossed to flies containing en>GAL4 and grown at 29 °C. The frequency of loss of the ACV (marked in Inset with an arrow) in each type of transgenic fly is plotted. n = 23–61. (Inset) Wings containing control RFP and WT E4orf4 transgenes. (G) Protein extracts were prepared from third-instar larvae harboring WT E4orf4, E4orf4 mutants, or RFP, described in F. A Western blot was stained with antibodies to E4orf4 and actin, which served as a loading control.

The contribution of tws/aar to E4orf4-induced effects was further examined in adult wings. As described in Fig. 1, E4orf4 induced dose-dependent phenotypes in the adult wing. We used growth conditions (29 °C) in which en>E4orf4 expression caused a strong effect (i.e., loss of the posterior part of the wing in WT flies). Under these conditions, the effect of E4orf4 on adult wings was examined in heterozygous tws60/+ or aar1/+ flies. As seen in Fig. 7C, 100% of E4orf4-expressing flies with a WT genetic background suffered loss of the posterior part of the wing; however, only 8% of aar1/+ or tws60/+ heterozygotes expressing E4orf4 exhibited this phenotype, and most of them manifested only the more minor wing defects. Eleven percent of tws60/+ and 25% of aar1/+ heterozygotes expressing E4orf4 had normal looking wings, as did 100% of WT flies expressing en>GFP, or aar1 heterozygotes (n > 23). Transheterozygotes were not examined in this experiment owing to difficulties in visualization of pupal wings.

To confirm that reversal of the E4orf4-induced wing defects by the tws60 or aar1 mutations resulted from inhibition of E4orf4-induced cell death, we examined the appearance of the cluster of cells containing active caspase-3 (Fig. 2) in wing discs of flies with various genotypes (Fig. 7D). Expression of en>E4orf4 at 29 °C was associated with accumulation of a cluster of caspase-stained cells in 100% of WT wing discs. When E4orf4 was expressed in tws60 or aar1 heterozygotes, only 30% of wing discs manifested a caspase-stained cluster of cells. Similarly, only 34% of wing discs expressing a PP2A-B55–specific RNAi transgene accumulated a cluster of dead cells (Fig. 7D, n > 23).

Taken together, the effects of tws/aar mutations on E4orf4-induced phenotypes indicate that the PP2A-B55 subunit contributes to E4orf4-induced cell death in the fly, similarly to the findings in mammalian tissue culture cells.

To examine whether the involvement of Src family kinases in E4orf4-induced cell death was also conserved in flies, control GFP or an RNAi transgene targeting the Drosophila Src ortholog dSrc64B were expressed in male eyes under GMR regulation at 29 °C together with E4orf4 or GFP. As seen in Fig. 7E, GMR>GFP,E4orf4 expression in male eyes produced the typical small eye phenotype with an average eye size that was 42% of control GMR>GFP,GFP eyes. Knockdown of dSrc64B increased E4orf4-expressing eye sizes to 63% of GFP-expressing eyes, and knockdown of PP2A-B55 increased eye sizes to 79% of GFP control (n > 14). These results indicate that dSrc64B as well as PP2A-B55 contributed to E4orf4-induced cell death in Drosophila.

To further confirm the contribution of both PP2A-B55 and Src to E4orf4-induced cell death in the fly, we cloned control RFP, WT E4orf4, and E4orf4 mutants unable to bind either the PP2A-B55 subunit (B55−: R81F84A (10), or Src proteins (Src−: R73/74/75A (7, 12), or both (B55−,Src−: R81F84A+R73/74/75A) into a pUAST-AttB vector that can integrate into the Drosophila genome at a specific landing site, using Phi C31 integrase technology (33). The specific integration facilitates similar expression levels of the various mutants by eliminating position effects. Transgenic flies containing the constructs at the attP40 chromosomal site have been generated. WT E4orf4 expressed at this site using the en-GAL4 driver caused only a minor phenotype in the wing, namely loss of the ACV (Fig. 7F, Inset). As seen in Fig. 7F, 100% of WT E4orf4 flies, 48% of Src− flies, 26% of B55− flies, 0.9% of the B55−,Src− flies, and none of the control RFP flies lost the ACV (n > 23). The reduced ability of the E4orf4 mutants to induce loss of the ACV was not caused by a decrease in their expression levels (Fig. 7G). Thus, these results substantiate the results obtained with the tws/aar mutant flies and with dSrc64B and dPP2A-B55 RNAi, and demonstrate that both PP2A-B55 and Src kinases contribute additively to E4orf4-induced cell death in the fly.

Discussion

In this report we present results of an investigation into E4orf4 function in a whole multicellular organism, D. melanogaster. We found several common features of E4orf4-induced cell death in flies and mammals in addition to some differences, and the results provided insight into the mechanisms underlying E4orf4-induced cell death in normal tissues.

The E4orf4 protein has been reported to induce nonclassic apoptosis in transformed mammalian cells in tissue culture (4–7) and irreversible growth arrest in yeast (13, 34). These E4orf4-induced events required the PP2A-B55 subunit in both mammalian cells and yeast (7, 10, 13, 34, 35), underscoring the high degree of evolutionary conservation of the underlying mechanisms. In addition, an interaction with Src family kinases was shown to contribute to E4orf4-induced cell death in mammalian cells in tissue culture (11). In the present study we show that E4orf4-induced cell death in Drosophila required both the PP2A-B55 subunit (tws/aar) and the fly Src family member dSrc64B and that both these E4orf4 partners contributed additively to the generation of E4orf4-induced phenotypes (Fig. 7), just as they have been reported to do in mammalian cells (12). Thus, E4orf4-induced cell death in the fly is a specific process, which relies on evolutionarily conserved mechanisms.

Two additional features of the evolutionary conservation of the E4orf4 cell death network emerging from this work include its dose dependence (Fig. 1 and ref. 36) and the contribution of both caspase-dependent and independent mechanisms. It was previously shown in mammalian cells that E4orf4 induced caspase-independent apoptosis, but a cross-talk existed between this pathway and caspase-dependent signaling (4, 8). We show here that E4orf4 induced classic, caspase-dependent apoptosis in the Drosophila eye and wing discs (Figs. 2 and 3) but that caspase-independent pathways contributed to E4orf4-associated phenotypes as well (Fig. 3). However, caspase inhibition seems to have a more major effect on E4orf4-associated phenotypes in flies compared with mammalian cells (Fig. 3) (8). In Drosophila, caspases are constitutively active and dIAPs inhibit them, resulting in apoptosis being the default pathway. In contrast, in mammalian cells caspases are inactive and have to be activated by apoptotic stimuli (37). This fundamental difference may explain the different “readout” of E4orf4 expression in the two organisms. Whereas the immediate E4orf4 partners, including PP2A and Src, are targeted similarly in mammalian cells and in flies, the cross-talk between the upstream caspase-independent part of the pathway and downstream classic apoptotic pathways may be more pronounced in the fly. Notwithstanding this caveat, important parts of the E4orf4-induced pathway seem to be highly conserved and can therefore be studied in Drosophila.

In addition to the similar features of E4orf4-induced cell death in mammalian cells and in flies, one difference was observed, namely the inhibition of JNK signaling by E4orf4 in normal fly tissues demonstrated here (Fig. 5), compared with the previously reported E4orf4-induced JNK activation in transformed mammalian cells in tissue culture (12, 38). Furthermore, JNK inhibition was reported to impede E4orf4-induced cell death in some mammalian tissue culture cells (38), whereas our results demonstrated that inhibition of the JNK pathway did not reduce E4orf4-induced cell death in Drosophila and even enhanced it (Fig. 6). It has been previously demonstrated that JNK signaling in Drosophila switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras (39). It has also been reported that JNK signaling in mammalian cells is activated in many tumor types, but in contrast, JNK also functions as a negative regulator of tumor development in Ras/p53-transformed fibroblasts and induces apoptosis (40–42). Thus, the role of JNK signaling seems to be highly dependent on cellular context and on the nature of the stimulus, and the context-induced differences in JNK signaling may also impact the interaction of E4orf4 with the JNK pathway. Future studies will have to determine whether JNK inhibition or activation by E4orf4 depend on the tumorigenic state of the cells, on the cell environment (monolayer or tissue), or on the type of organism studied.

It has been previously reported that JNK signaling induced caspase-independent cell death in the eye, which relied on metabolic energy production pathways (17–19). However, this mode of nonapoptotic cell death must be distinct from the E4orf4-induced caspase-independent cell death, which is not impeded by JNK inhibition (Fig. 6). The findings that E4orf4 inhibited JNK-induced cell death (Fig. 5) and that JNK inhibition cooperated with E4orf4 to induce its own unique mode of cell death (Fig. 6) are intriguing, and two explanations could be considered. It is possible that one or more effector molecules are required for both JNK- and E4orf4-induced cell death, and JNK and E4orf4 compete for them. It should be noted in this context that both E4orf4 and JNK potentially share common targets: both were shown to inhibit JunB expression, although by different mechanisms (3, 43). Alternatively, inhibition of a JNK function other than cell death induction may contribute to E4orf4-induced cell death. This possibility is reminiscent of findings reported previously, which demonstrated that only the Raf/MAPK effector branch of Ras1 signaling but not other effector branches involving PI3 kinase or Ral-GDS were required for Hid-induced apoptosis (44).

Another question raised by our results is whether inhibition of Rpr, Hid, and Grim and inhibition of JNK by E4orf4 occur independently or through only one of these targets. The finding that inhibition of JNK signaling by E4orf4 seemed to occur downstream of the JNK kinase Hep (Fig. 5) raises the possibility that it may not result from inhibition of Rpr or Hid alone (Fig. 4), because it has been reported that the caspase inhibitor dIAP1 did not inhibit HepCA signaling (45). Furthermore, E4orf4 very effectively inhibited Grim-induced cell death, whereas Grim inhibition was reported not to affect JNK-induced cell death (46), suggesting that JNK inhibition by E4orf4 was not mediated through Grim. On the other hand, inhibition of JNK should not affect expression of exogenous UAS-rpr or hid used in our experiments (Fig. 4), because only endogenous rpr or hid transcription is activated by JNK (47). Thus, it is possible that E4orf4 inhibits JNK and Rpr/Hid/Grim by two separate mechanisms. It is also not known yet whether JNK inhibition by E4orf4 affects both caspase-dependent and -independent mechanisms of JNK-induced cell death. However, the results presented here clearly indicate that in normal Drosophila tissues, E4orf4 activates both caspase-dependent and -independent cell death (Figs. 2 and 3) and at the same time inhibits cell death (Figs. 4 and 5). The sum of these E4orf4 activities, presented in the model described in Fig. 8, resulted in a modest effect on tissue morphology and no effect on fly survival (Figs. 1 and 3–5). The weak pro-cell death activity of E4orf4 in normal Drosophila tissues is consistent with results in mammalian tissue culture cells, which indicated that E4orf4 induced cell death inefficiently in primary cells (7). E4orf4 pro-cell death activity was enhanced in transformed mammalian cells (7), and future studies will determine whether cancer cells in a whole organism, such as Drosophila, will also be more sensitive to the unique mode of cell death induced by E4orf4.

Fig. 8.

Fig. 8.

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Model of E4orf4-induced cell death in normal Drosophila tissues. E4orf4 in collaboration with PP2A and Src induces caspase-dependent and -independent types of cell death. At the same time it also inhibits rpr, hid, and grim (RHD)-induced apoptosis, although its direct target in this pathway is not currently known. E4orf4 also inhibits JNK-induced cell death downstream of Hep. Inhibition of JNK signaling enhances E4orf4-induced cell death. These features of cell killing by E4orf4 indicate that it is a unique form of cell death, distinct from RHD- or JNK-induced death signaling. As a result of combined activation and inhibition of cell death, E4orf4 induces minor damage to normal tissues. Discontinuous arrows represent a weak effect.

The findings obtained in the Drosophila system, not only provide insight into E4orf4-induced cell death but may also be relevant to the physiological function of E4orf4 during viral infection. A previous report (3) showed that an adenovirus mutant lacking E4orf4 was much more cytotoxic than the WT virus upon infection of rat fibroblasts. These results are consistent with the results presented here demonstrating that E4orf4 is capable of inhibiting apoptosis in normal Drosophila tissues. The protective role of E4orf4 may contribute to the prevention of premature cell death induced by the activities of early viral proteins and could be important in natural infections, in which adenoviruses can maintain long-term association with their infected hosts.

Materials and Methods

Fly Stocks and Culture.

The following strains are described in FlyBase (http://flybase.org/): Canton-S (used as WT); GAL4 drivers and UAS strains: GMR-GAL4, ms1096-GAL4, en-GAL4, UAS-lacZ, UAS-GFP, UAS-rpr [on the X chromosome: rpr(X)], UAS-hep.CA [Hep-CA(1)] and UAS-hep.Act [Hep-CA(2)], P{GD12263}v35252 (UAS-Src64B-IR), p{GD10742}v34340 (UAS-PP2A B55-IR). The following strains were also used: UAS-Rpr-HA3-13 (rpr) and UAS-Rpr-HA3-2b [rpr(2)] were provided by H. Steller (Rockefeller University, New York), FRT82B, tws60/TM6B was provided by T. Uemura (Kyoto University, Kyoto, Japan), UAS-p-p35/TM6B and UAS-myc-dIAP1/TM6B were provided by E. Arama (The Weizmann Institute of Science, Rehovot, Israel), aar1/TM6B was provided by D. Glover (University of Cambridge, Cambridge, UK), UAS-hid and UAS-puc were provided by O. Gerlitz (The Hebrew University, Jerusalem, Israel), UAS-Grim was provided by A. Orian (Technion, Haifa, Israel), UAS-eiger and a loss-of-function allele of eiger− were provided by M. Miura (University of Tokyo, Tokyo, Japan). The UAS-E4orf4-17, UAS-RFP-attP40, UAS-E4orf4-attP40, UAS-E4orf4R81F84A-attP40 UAS-E4orf4R73/74/75A-attP40, and UAS-E4orf4R81F84A+R73/74/75A-attP40 strains were generated in this work by germ-line transformation. Injections to the attP40 strain were done by Genetic Services.

The following strains were generated by recombination: UAS-p35,UAS-E4orf4-17.22/TM6B, FRT82B,aar1,UAS-E4orf4-17/TM6B, FRT82B,tws60,UAS-E4orf4-17/TM6B, and FRT82B,UAS-E4orf4-17. The following strains were generated by mating the appropriate strains: GMR-GAL4;FRT82B,UAS-E4orf4-17, GMR-GAL4;UAS-GFP, GMR-GAL4:FRT82B,tws60/TM6B, en-GAL4;FRT82B,tws60/TM6B, GMR-GAL4/CyO;UAS-p-p35/TM6B, and GMR-GAL4;UAS-myc-dIAP1/TM6B. Fly stocks were cultured on standard cornmeal/yeast media. The flies in all samples were grown under equal conditions: each cross was started from 10 virgin females and 7 males. Parents were allowed to mate for 4 d and were then discarded.

Adult Eclosion Test.

The number of pupae and the number of eclosed adults were counted in each vial for up to 3 wk after pupa formation. Eclosed adults were removed daily. The ratio between eclosed adults and the total number of pupae was calculated and presented as eclosion rate.

Eye Size Measurement and Wing Imaging.

For eye size measurements, adult flies were anesthetized with CO2. Equal numbers of left and right eyes were measured (n > 20 unless otherwise stated) for each genotype, sex, and temperature. The eyes were photographed using AxioCam MRc and Zeiss Discovery V8 binocular. If the depth of field was not sufficient, a series of images with different foci was taken, and the images were stacked using the Helicon Focus software. Resulting images were measured using the ImageJ program. Eye size for each genotype, sex, and temperature was normalized to the appropriate control. For representative pictures, eyes, and wings were photographed using a high-resolution Olympus DP70 camera and a Zeiss Axioscop 2 microscope.

Western Blots.

Whole-cell extracts were prepared from third-instar larvae in lysis buffer [50 mM Tris·HCl (pH 7.4), 250 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.5% Nonidet P-40, and a 1/10 volume of Complete protease inhibitor mixture (Roche)]. Proteins were analyzed by Western blots using antibodies to E4orf4 (6), Actin (MP Biomedicals), or Tws/Aar (31).

Staining Imaginal Discs.

Larval tissues were stained following standard procedures. Primary antibodies used: rabbit polyclonal anti-E4orf4 (1:500); mouse monoclonal anti-E4orf4 (#16, 1:100) (6); rabbit anti-cleaved caspase-3 (Cell Signaling, 1:100); mouse monoclonal anti-beta-Gal (Promega, 1:1,000). Secondary fluorescent antibodies for fluorescent staining were Cy2, Cy3, or Cy5-conjugated anti-rabbit/mouse (1:50) (Jackson Laboratory). TRITC-labeled Phalloidin (Sigma) was used at a 1:500 dilution. Samples were mounted with a DakoCytomation mounting medium (Dako).

Data Analysis.

The statistical significance of differences in eclosion rates or eye sizes between the various fly groups was calculated by an unpaired t test.

Supplementary Material

Supporting Information
supp_110_19_E1724__index.html (7KB, html)

Acknowledgments

We thank M. Mahroum and A. Livne for their help in generation of the E4orf4-17 transgenic strain; R. Sharf for help with Western blots; and the Bloomington Stock Center, the Vienna Drosophila RNAi Center, and the following scientists for generous gifts of fly strains and antibodies: T. Uemura, D. Glover, O. Gerlitz, A. Orian, M. Miura, H. Steller, and E. Arama. This research was supported in part by Israel Science Foundation Grant 399/11, by the Deutsche Forschungsgemeinschaft within the framework of The German-Israeli Project Cooperation, by the Israel Cancer Association through the Goldberg Dina Fund, by the Israel Cancer Research Fund, and by The Rappaport Faculty of Medicine and Research Institute, Technion–Israel Institute of Technology.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1220282110/-/DCSupplemental.

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