The tumor suppressor Caliban regulates DNA damage-induced apoptosis through p53-dependent and -independent activity

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Abstract

We previously identified Caliban (Clbn) as the Drosophila homolog of human Serologically defined colon cancer antigen 1 gene and demonstrated that it could function as a tumor suppressor in human non-small-cell lung cancer (NSCLC) cells, although its mode of action was unknown. Herein, we identify roles for Clbn in DNA damage response. We generate clbn knockout flies using homologous recombination and demonstrate that they have a heightened sensitivity to irradiation. We show that normal Clbn function facilitates both p53-dependent and -independent DNA damage-induced apoptosis. Clbn coordinates different apoptosis pathways, showing a two-stage upregulation following DNA damage. Clbn has proapoptotic functions, working with both caspase and the proapoptotic gene Hid. Finally, ecotopic expression of clbn+ in NSCLC cells suppresses tumor formation in athymic nude mice. We conclude that Caliban is a regulator of DNA damage-induced apoptosis, functioning as a tumor suppressor in both p53-dependent and -independent pathways.

Introduction

Cells are continually exposed to a variety of exogenous assaults and intrinsic metabolic by-products that cause DNA damage and/or replication errors. Multiple systems have been established to detect aberrant DNA and delay the cell cycle, allowing time to precisely correct this damage. The disruption of these DNA damage response pathways can lead to severe disease, including cancer.

The tumor suppressor p53 is the most frequently mutated gene in human tumors; expression of the wild-type (wt) TP53 gene in these tumors can cause tumor regression and even complete clearance in vivo.1, 2, 3, 4 When DNA damage occurs, p53 protein is stabilized and activated, this in turn, regulates the expression of downstream genes responsible for cell cycle control and apoptosis.5, 6 p53 regulates the activation of cell cycle inhibitors such as p21 and blocks the G1/S cell cycle transition. This p53-dependent G1/S cell cycle checkpoint is indispensable for genome maintenance.5, 7 The p53 knockout mouse appears morphologically normal, but is highly prone to form spontaneous tumors, mostly because of the cell’s failure to undergo apoptosis. p53-dependent apoptosis is central to the elimination of cells with DNA damage.8, 9

A single p53 ortholog has been identified in Drosophila. Like its mammalian counterpart, overexpression of p53 in Drosophila induces apoptosis and loss of the p53 gene eliminates DNA damage-induced apoptosis.10, 11, 12 However, unlike in mammals, overexpression of Drosophila p53 does not block or delay G1- to S-phase transition.10 Thus, other factors and/or pathways might cooperate with or substitute for p53 to provide the same level of cell cycle progression control in Drosophila as is found in mammals.

The induction of DNA double-strand breaks also activates Ataxia Telangiectasia Mutated (ATM) kinase, which in turn phosphorylates many downstream targets including p53.13 ATM has multiple functions, including DNA damage repair, cell cycle checkpoint and telomere maintenance.14, 15 Both ATM and p53 are bona fide tumor suppressors, as mutants in either gene cause tumors in mice and humans; however, the Drosophila mutants do not seem to increase tumor formation. Drosophila ATM is encoded by the telomere fusion locus (tefu), so named because the primary mutant phenotype is the cytological appearance of telomere fusions.16, 17, 18, 19

We previously identified the Drosophila protein Caliban (Clbn) as the highly conserved ortholog of human Serologically defined colon cancer antigen 1 gene (Sdccag1) and demonstrated that it could act as a nuclear export mediator factor.20 Sdccag1 was initially identified as a protein producing an autologous antibody in some colon cancer patients,21 this implied that Sdccag1 might be mis-expressed in colon cancer cells. Another group showed the accumulation of Sdccag1 in non-small-cell lung cancer (NSCLC) cells and a concomitant G1 cell cycle arrest in response to the anti-cancer drug VT1 (methyl-4-methoxy-3-(3-methyl-2-butanoly) benzoate),22 suggesting that Sdccag1 could act as a tumor suppressor and could be a potential drug target with clinical applications for cancer therapy. We had also demonstrated that fly Clbn could act as a tumor suppressor in human NSCLC cells. The stable integration and ectopic expression of the fly clbn+ gene in two different human adenocarcinoma cell lines, A549 and EKVX, greatly reduced their ability to form colonies on soft agar and invasiveness indices.20 Finally, it was shown that more than half of mismatch repair–deficient lymphoma cells examined in one study had frame shift mutation in the sdccag1 gene.23 While studies have connected Sdccag1/Clbn with cancer, none have addressed a mechanism or pathway for its mode of action.

It had been reported that Drosophila clbn is transcribed in response to ionizing radiation and that this response is controlled by p53.24 This led us to investigate if Clbn functions in the DNA damage response. Here we created Drosophila clbn knockout animals by gene targeting and demonstrated that they are sensitive to irradiation. We show that clbn is upregulated in a two-stage manner after irradiation and that it regulates both p53-dependent and -independent apoptosis. Our results indicate that Clbn functions as a tumor suppressor by having proapoptotic activity and regulating the DNA damage response. We conclude that Clbn may have an important role in maintaining genome integrity.

Results

Generation of clbn knockout flies

To explore the in vivo functions of Clbn, we generated knockout flies by homologous recombination.25 Three independent lines were recovered where the entire clbn protein-coding region was replaced with an enhanced yellow fluorescent protein that is transcribed by the ubiquitously expressing armadillo promoter (Supplementary Figure S1A). All three lines are homozygous viable but show small, reproducible developmental delays and shortened lifespans (data not shown). Molecular analysis by western blot, long-range PCR and microarray analysis confirmed the deletion of the clbn locus (Supplementary Figure S1B and data not shown).

Clbn knockout flies are hypersensitive to irradiation

It was previously shown that clbn transcription is upregulated in response to irradiation, in a p53-dependant manner.24 This suggested to us that Clbn might be involved in the p53-dependant response to DNA damage. To investigate this, we treated wt, p53, clbn and p53 clbn double-mutant third-instar larvae with 500 rad γ-radiation and examined mitotic chromosomes from brains. We found that clbn flies had on average more than twice the number of breaks per nucleus (1.01 (n=413)) compared with wt flies (0.45 (n=393)), a number statistically indistinguishable from p53 flies (1.06 (n=408); Figures 1a and b). Interestingly, the p53 clbn double-mutant flies showed a similar number of breaks to either single mutation alone (1.02 (n=409)). This suggests that Clbn may have an epistatic relationship with p53 in response to DNA damage.

Figure 1
figure1

clbn and p53 knockout flies are equally hypersensitive to irradiation. Larvae were exposed to γ-radiation at 500 rad (a, b) or 4000 rad (c). (a) DAPI (4,6-diamidino-2-phenylindole)-stained unirradiated (top) or irradiated (bottom) larval brain metaphase chromosomes; the latter shows breaks in chromosomes 2 and 3, marked by an arrow and arrowhead, respectively. (b) Average number of breaks per nucleus in larval brains of the indicated genotypes. Data are derived from a minimum of 10 nuclei per larvae and 10 larvae per genotype in three independent experiments. Error bars indicate the standard error of the mean. There is a significant difference between wild type (wt) and each mutant combination tested (*P<0.01), which are statistically indistinguishable from each other. (c) Melanotic masses, indicated by arrows, were induced by irradiation of clbn larvae.

We also noticed that all three clbn fly lines were likely to form melanotic masses following irradiation. Melanotic masses are indicative of an innate immune response and are often associated with over proliferation of hemocytes. To confirm that loss of Clbn contributes to the formation of these masses, we compared clbn1Q (clbn) with a wt line having the identical first and second chromosomes, thus reducing genetic background differences. We place groups of 200 wt or clbn first-instar larvae on plates, treated them with 4000 rad γ-radiation and counted the number of late third-instar larvae with melanotic masses (Figure 1c). In wt third-instar larvae, 1.1±0.4% developed masses (n=14 plates) after irradiation compared with 4.8±1.2% (n=14 plates) of clbn third-instar larvae. clbn larvae develop significantly more masses than wt larvae (P=0.05). While the nature of these masses was not investigated further, our data indicate that Clbn might have roles in preventing irradiation-induced chromosomal damage and over proliferation of hemocytes.

Clbn regulates both p53-dependent and -independent apoptosis

As clbn mutant flies are hypersensitive to irradiation, we wished to determine whether this effect was due to defects in cell death, which can be induced by DNA damage. Previous reports had shown that DNA damage induces cell death in Drosophila, as in mammals, by both p53-dependent and -independent mechanisms. p53-dependent cell death occurs rapidly after DNA damage, while p53-independent cell death is delayed.12, 26, 27 We treated third-instar larvae with 4,000 rad γ-radiation, then dissected and stained wing discs with acridine orange, a marker for dead cells, at 4, 8, 16 or 24 h after treatment. In the absence of irradiation, dead cells are rarely seen in wing discs from wt, p53, clbn and p53 clbn flies. Representative irradiated discs are shown (Supplementary Figure S2A). These data were quantified and an index of cell death over the time course after irradiation is shown (Supplementary Figure S2B). Wt wing discs show two clear phases of cell death, with the first visible after 4 h of irradiation and appearing to plateau around 8 h and the second visible as the change in the index seen from 16 h until the final 24 h time point. In contrast, p53 discs do not show the early induction of cell death, consistent with a previous report,26 but do show an elevation during the second phase, between 16 and 24 h (Supplementary Figure S2B). clbn discs show the early induction of cell death, although to a significantly lower level than wt discs (P=0.026 at 8 h).

The second phase of cell death in clbn discs appears to be largely blocked, as there is only a small increase in the cell death index from 8 through 24 h post-irradiation. Clearly Clbn is required for the second p53-independent phase of cell death and may also contribute to the first p53-dependent phase. The p53 clbn double-mutant discs appear to have a complete block to irradiation-induced cell death (Supplementary Figure S2B).

As acridine orange stains all dead cells, not just those undergoing apoptosis, we next looked more directly at apoptosis. We used the antibody recognizing the cleaved caspase-3 (C3), a universal marker for apoptosis in Drosophila. The flies were treated as previously described. Clearly early stage p53-dependent apoptosis, visible at 4 and 8 h, is greatly reduced after irradiation of clbn flies (Figures 2a and b). Late stage p53-independent apoptosis is also largely reduced in clbn flies. Moreover, apoptosis is almost completely eliminated in p53 clbn double mutants throughout the whole process. We suggest that Clbn facilitates both p53-dependent and -independent apoptosis.

Figure 2
figure2

Clbn regulates p53-dependent and -independent apoptosis. Time course of irradiation-induced apoptosis in wild-type (wt) and mutant imaginal wing discs was monitored by anti-caspase 3 (C3) immunostaining. Third-instar larvae were exposed to 4000 rad γ-radiation and then wing discs were dissected and stained with C3 antibody at the indicated times following treatment. (a) Fluorescent photomicrographs of representative wing discs. (b) Apoptotic index (anti-C3 index) was quantified as C3 staining area in whole disc at the indicated time points, it is the total isosurface volume of positive region divided by isosurface of the entire disc. The data were derived from the average of at least five discs for each time point and three independent experiments. Error bar represents the standard error of mean.

A two-phase elevation of clbn transcription in response to irradiation

Irradiation-induced apoptosis occurs in two phases with an early rapid response, which is p53-dependent, and a delayed response, which is p53-independent.26 Mutant clbn appears to reduce both of these phases. As a first step in determining the mechanism of Clbn regulation of apoptosis, we irradiated third-instar larvae and determined the levels of clbn transcripts, using reverse transcription–quantitative PCR, at different time points after irradiation. In wt flies, we see two peaks of elevated clbn mRNA, at 1.5 and 16 h post irradiation; the levels increased by 2.9- and 2.8-fold, respectively (Figure 3a). The level of increased transcript we see at 1.5 h post irradiation is similar to a 2.6-fold increase reported previously.24

Figure 3
figure3

A two-phase induction of clbn transcription in response to irradiation. Third-instar larvae were irradiated as in the previous experiments, and relative levels of clbn RNA were measured by reverse transcription–quantitative PCR after the indicated times. Data represent the average from triplicate samples in three independent experiments. Error bar represents the standard error of mean. Values were normalized to rp49 RNA levels. (a) Relative levels of clbn RNA in wt flies. (b) Relative levels of clbn RNA in p53 flies.

As our previous experiments suggested that Clbn might act through both p53-dependent and -independent mechanisms (Figure 2 and Supplementary Figure S2), we repeated the above experiment in a p53 mutant background. Most or all of the elevated level of clbn mRNA is abrogated 1.5 h after irradiation of p53 mutant larvae; however, we still see an increase 12–16 h post irradiation (Figure 3b). This suggests that the two-phased regulation of clbn in response to irradiation-induced DNA damage occurs at the level of transcription with the early phase being p53-dependent and the later phase not requiring p53.

Clbn works with Hid to regulate apoptosis

The proapoptotic gene family Reaper-Hid-Grim (RHG) is required for both p53-dependent and -independent apoptosis in Drosophila.28, 29 As Clbn showed similar characteristics to the RHG family in response to DNA damage-induced apoptosis, we wished to examine the interactions between Clbn and Hid, a major proapoptotic gene responsible for p53-induced apoptosis and p53-independent apoptosis in Drosophila.30, 31, 32 Third-instar larvae of the indicated genotypes were irradiated as before, and the wing imaginal discs were stained with anti-caspase 3 (Figure 4a). The levels of staining were quantified and the average caspase-3 index is shown (Figure 4b). Again wt discs show a two-phase induction of apoptosis, the first detectable at 4 h and the second visible as an increase in caspase-3 staining from 16–24 h. Significantly, both clbn and hid mutant discs show a similar reduction in caspase-3 staining 4 h after irradiation (P<0.001 for both). The second phase of caspase-3 induction, from 16–24 h, approximately parallels the second phase of induction seen in wt discs, although the overall levels are significantly reduced (P<0.001). Finally, the caspase-3 staining in hid clbn double-mutant discs is indistinguishable from either mutant alone (P=0.983), suggesting that there is a requirement for both Hid and Clbn in apoptosis and that they might be working through the same pathway (Figure 4b).

Figure 4
figure4

Clbn regulation of apoptosis is epistatic with Hid. Time course of irradiation-induced apoptosis is shown. Larvae and discs of the indicated genotypes were treated as in Figure 2. (a) Fluorescent photomicrographs of representative wing discs. (b) Anti-C3 immunostaining was quantified as in Figure 2.

As Clbn and Hid may work together to regulate DNA damage-induced apoptosis, we wished to investigate their relationship. We induced the overexpression of hid in clbn mutant, or overexpression of clbn in hid mutant by GMR driver, stained with anti-caspase 3 to detect apoptosis. While overexpression of hid or clbn induced apoptosis in flies eye, they showed differently at mutant background (Figures 5a and c). Overexpression of hid showed similar level of apoptosis in wt and clbn mutant (Figures 5c and d), whereas clbn overexpression in hid allele could not induce apoptosis (Figures 5a and b), suggesting Hid might work downstream of Clbn to regulate apoptosis.

Figure 5
figure5

Hid is essential for Clbn regulated apoptosis. (a) Overexpression of clbn induces apoptosis in wt, not in hid mutant. Imaginal eye discs from third-instar larvae of the indicated genotypes stained with the apoptotic marker anti-caspase 3. (b) Quantification of a as in Figure 2. The data were derived from the average of at least 10 discs. Error bar represents the standard error of mean. (c) Overexpression of hid induces apoptosis in wt and clbn mutant. (d) Quantification of c.

Clbn induces apoptosis through regulating expression of caspase

clbn mutants showed a similar defect in DNA damage-induced apoptosis to hid mutants, suggesting Clbn could be a bridge to mediate apoptotic signals from the transcriptional factor p53 to proapoptotic genes and/or caspases. To determine whether Clbn has this proapoptotic function, we generated transgenic flies that could express wt clbn+ under the control of the Gal4–UAS system.33 We expressed the clbn+ gene in the developing fly eye using a synthetic GMR promoter to drive Gal4 expression. Expression of a single copy of UAS-clbn+ caused a mild rough-eye phenotype, which is made more extreme when two copies of UAS-clbn+ are present in the fly (Figure 6a). Scanning electron microscopes of these eyes showed the disruption of ommatidia caused by ectopic clbn expression (Figures 6b and c). As this suggests that Clbn has a proapoptotic function, we next examined whether Clbn-induced apoptosis acts through regulated expression of Drosophila caspases. We observed very little C3 immunostaining in eye discs from either GMR-Gal4/+ or UAS-clbn+/+ flies; however, a large amount of C3-positive cells is observed when clbn is overexpressed in eye discs (Figure 6d). We suggest that Clbn regulates apoptosis through induced expression of caspases.

Figure 6
figure6

Overexpression of wild-type clbn+ induces caspase-dependent apoptosis. Fly eyes from the genotypes UAS-clbn/+ (clbn/+), GMR-Gal4/+ (GMR/+), GMR-Gal4>UAS-clbn/+ (GMR>clbn/+) and GMR-Gal4>UAS-clbn/UAS-clbn (GMR>clbn/clbn). (a) Light micrographs of fly eyes of the indicated genotypes. (b, c) Scanning electron micrographs of fly eyes of the indicated genotypes with the scale bars being 100 μm (b) or 20 μm (c). (d) Overexpression of clbn induces caspase-dependent apoptosis. Imaginal eye discs from third-instar larvae of the indicated genotypes stained with the apoptotic marker anti-caspase 3 (anti-C3).

It is known that antiapoptotic protein p35 from baculovirus suppresses the expression of effector caspases Drice and DCP-1,34, 35, 36 as Clbn induces expression of caspases (Figure 6d), next we induced overexpression of clbn and p35 at the same time by GMR driver. Overexpression of p35 rescues the eye phenotype induced by overexpression of clbn+ (Supplementary Figure S3) and completely abolished apoptosis in eye discs induced by clbn+ expression (Supplementary Figure S4), which provide more evidence that Clbn function at apoptosis is through caspases in Drosophila.

Synergistic spontaneous tumor formation in tefu/atm mutants combined with p53 or clbn

The major defect of tefu/dATM mutation is telomeres association, which leads to p53-dependent and -independent apoptosis;37 we generated the double-mutant flies tefu p53 and tefu clbn, to investigate whether ATM might have a role in p53 and/or Clbn tumor formation. It was readily apparent that third-instar larvae from these stocks formed spontaneous melanotic tumors. We quantified tumors by scoring wt, single and double-mutant third-instar larvae (Table 1). While wt, p53 and clbn larvae do not form spontaneous melanotic tumors, tefu third-instar larvae showed a low level of tumor formation (Table 1, 1.2%). In contrast, combining either p53 or clbn with tefu, produced high rates of tumor formation in both double-mutant lines, 36.3% (n=956) and 29.5% (n=811), respectively (Table 1). There is a clear synergistic interaction between ATM and either p53 or Clbn.

Table 1 Spontaneous tumors in flies

Clbn+ expression in NSCLC cells suppress tumor formation in mice

We previously showed that Clbn could act as a nuclear export mediator. We used an enhanced yellow fluorescent protein fused to a Clbn target sequence, pEYFP-HDA, to demonstrate that Clbn+ function is required to translocate pEYFP-HDA from the nucleus to the cytoplasm of both fly and human cells. The nuclear export of pEYFP-HDA was then used to assay human Clbn+ function. While normal human cells and human colon cancer cell lines were able to export our reporter gene, none of five human NSCLC cell lines were able to export the reporter gene. Stable integration and expression of fly clbn+ restored the export function to these cells. This expression also reduced their ability to form colonies on soft agar and invasiveness, but did not affect other growth parameters.20

Here we compared the ability of the human lung adenocarcinoma cell line A549 and two of its derivatives, A549-Clbn-M and A549-Clbn-S, which ectopically express a Myc-tagged and untagged wt fly clbn+ gene, respectively, to form tumors when injected into athymic nude mice. Subcutaneous injections of the NSCLC line A549 into mice produced fast-growing tumors that obtained a large size (85 mm2). In contrast, two different A549 lines, ubiquitously expressing fly clbn+, formed slower growing and smaller tumors (Figures 7A and B). Of particular note, the untagged A549-Clbn-S cells completely abrogated tumor formation in five of eleven mice injected from two separate experiments. Fly Clbn appears to be able to function as a tumor suppressor in human lung cancer cells.

Figure 7
figure7

Ecotopic expression of clbn suppresses tumor formation in athymic nude mice. Nude mice were injected with three different human A549 non-small-cell lung cancer cell lines, the parent line or two lines expressing the wild-type Drosophila clbn+ gene. (A) [a] Parent A549 cells, [b] A549-Clbn-M cells, [c] A549-Clbn-S cells. (B) Average tumor size in the mice injected with the cells used in A on the indicated days after injection. Smaller tumors were formed when athymic nude mice were injected with clbn stably transformed human A549 cells.

Discussion

Genome instability is one of the hallmarks of most human cancers. Tumorigenesis is normally blocked by the expression of a number of tumor suppressor genes, which function in a variety of cellular functions, including cell cycle arrest, DNA damage response, apoptosis, DNA replication, proliferation, differentiation and angiogenesis.38 Genetically mutated or dysfunctional tumor suppressor genes disrupt molecular signaling pathways to initiate or promote tumor progression. The identification and characterization of these tumor suppressor genes provide the knowledge needed to develop new tools to treat cancer.

We previously identified Drosophila Clbn as a nuclear export mediator factor, suggested that it might function as a tumor suppressor, and demonstrated that is the homolog of mammalian Sdccag1.20 While clbn was shown to be upregulated in a p53-dependent manner after irradiation,24 its role in DNA damage response and tumor suppression was obscure. Here, we report the generation of knockout clbn flies and use them to investigate its functions in DNA damage response. Our results show that loss-of-function clbn flies are hypersensitive to irradiation. This can be seen as an increase in the number of visible larval chromosomal breaks in mitotic brains, thus indicating that Clbn could be involved in a DNA damage response. This hypothesis was reinforced upon showing an epistatic relationship between clbn and p53 for the formation of chromosomal breaks.

Apoptosis regulation is one central mechanism for tumor suppression, with p53 having a critical role in the early stage response following DNA damage. Because the transcription of clbn is regulated in part by p53,24 we investigated the relationship between Clbn and p53 in regulating DNA damage-induced apoptosis. Third-instar larvae imaginal discs were examined at different times following irradiation, using the apoptosis marker cleaved caspase 3. We found that loss of Clbn reduces the number of apoptotic cells 4 h after irradiation by half while loss of p53 eliminates most or all cell death at this early stage. A second stage of cell death is visible between 16 and 24 h post-irradiation in wt wing discs. This second stage of cell death is clearly visible in p53 loss-of-function mutations, but is eliminated in the p53 clbn double mutants. On the basis of these observations, we conclude that Clbn is necessary for both the full expression of early stage p53-dependent apoptosis and the later p53-independent stage. While p53 regulates clbn transcription at DNA damage-induced early stage apoptosis, who is regulating clbn at p53-independent apoptosis is unknown. Recent report suggests that dE2F1 is required for irradiation-induced p53-independent apoptosis, and it is necessary for transcriptional activation of hid.39 Other report finds that JNK inhibitor puc blocks Hid induction by irradiation in p53 mutant cells, and reduces p53-independent apoptosis.30 The p53-independent apoptosis might be triggered by DNA damage signal, as it is irradiation dosage dependent,26 and can eliminate aneuploid cells.30 Whether Clbn involved p53-independent apoptosis is regulated as Hid by dE2F1 or JNK is unknown yet, which needs further investigation.

Clbn displays proapoptotic function as mutations in it or the proapoptotic protein Hid show similar reductions to each other in the amount of cleaved caspase-3 induced after irradiation. The hid clbn double-mutant larvae are indistinguishable from either single mutant alone, suggesting that Clbn and Hid are working through the same pathway.

We also observed a two-stage transcriptional upregulation of clbn after irradiation. The timing of this transcription at 1.5 and 16 h after irradiation precedes the Clbn-dependent induction of apoptosis, which our mutant analysis detects at 4 and 24 h. Thus, the regulation of Clbn’s proapoptotic function is at the level of clbn transcription, with the early stage induction requiring p53 and the later stage being p53 independent.

Knockout clbn flies have a reduced ability to undergo apoptosis, suggesting that Clbn is necessary for normal proapoptotic functions in flies. We next demonstrated that Clbn is sufficient to induce apoptosis by ectopically expressing it in the Drosophila eye. This expression resulted in a rough-eye phenotype, which became more extreme upon expressing higher levels of Clbn. Similar results were obtained by others ectopically expressing p53 in the eye.10 This phenotype most likely results from increased apoptosis as the levels of cleaved caspase 3 increase in parallel with the severity of the mutant eye phenotype.

In addition to a direct role for Clbn in the Drosophila DNA damage response, we show three lines of evidence that Clbn functions as a bona fide tumor suppressor. First, irradiated knockout clbn larvae developed twice the number of melanotic masses as wt larvae. Second, 29.5% of tefu clbn larvae formed spontaneous melanotic tumors compared with 0% and 1.2% of clbn and tefu larvae, respectively. Third, ectopic expression of the wt fly clbn+ gene in human NSCLC cells suppressed tumor formation when injected into athymic nude mice, both slowing the growth of tumors and reducing their size.

On the basis of our findings we propose the following model (Figure 8). DNA damage activates p53 and Clbn. Activation of Clbn is at the transcription level, as we demonstrate two waves of clbn transcription following irradiation, the early being p53 dependent and the latter being p53 independent.

Figure 8
figure8

Clbn functions at DNA damage-induced apoptosis to suppress tumor formation. Upon DNA damage, clbn is transcriptionally upregulated by p53-dependent and -independent pathways. Upregulated Clbn performs proapoptotic function through caspases and a role epistatic with Hid, a critical proapoptotic gene. Clbn is essential to eliminate tefu/dATM mutation caused by damaged cells through apoptosis.

While Clbn and p53 proteins might interact directly, a recent report failed to find such a protein interaction.40 We favor the hypothesis that p53 regulates clbn+ transcription either directly or indirectly. We note several putative p53-binding sites near clbn and are currently investigating if they are required for p53-dependent activation of clbn. We have shown Clbn is a bipartite nuclear exporting protein, is there any link between its nuclear exporting function and apoptosis function? A recent paper by Long et al.41 finds that MeCP2 (methyl CpG binding protein 2) interacts with Sdccag1, and suggested that Sdccag1 may export MeCP2 to regulate its functions. Partial silencing of MeCP2 in human mesenchymal stem cells suppresses apoptosis42 and MeCP2 may link with increased senescence.43 Others suggest MeCP2 and p53 may have feedback regulation.44 Our results show that Clbn has a physical interaction with a transcriptional repressor of p53 (our unpublished data). Clbn may export transcription suppressors from nucleus upon DNA damage, releases suppression to p53, which needs more study.

As p53 is mutated in more than half of tumors, the p53-independent regulation of apoptosis may provide an additional avenue for developing molecular targets for preventing tumor formation. In this study, we identify Clbn as a critical player in p53-independent apoptosis and believe it represents a potential target for therapeutic applications.

Materials and methods

Drosophila melanogaster genetics

All flies were maintained at 25 °C on standard cornmeal-sucrose-yeast agar medium. w1118 was used as the wt strain. The following mutant genotypes were used: p535A-1-4; p5311-1B-1; w1; GMR-hid were from Bloomington Drosophila Stock Center; hid05014 (gift from Hermann Steller and Laura Johnston); UAS-p35(gift from Laura Johnston and Tin Tin Su); GMR-gal4/+; w1/hid05014 (hid); p535A-1-4 clbn1Q (p53 clbn); p5311-1B-1 clbn1Q; w1 clbn1Q/hid05014 clbn1Q (hid clbn); UAS-clbn; GMR> UAS-clbn/+; GMR>UAS-clbn/UAS-clbn.

Larvae irradiation

Either first-instar larvae or active crawling third-instar larvae were collected and irradiated with a cobalt-60 γ-ray source (Beijing Institute of Radiation Medicine).

Mitotic chromosomes spreading

Mitotic chromosomes spreading experiment was performed as described.45 Larvae were irradiated with 500 rad γ-ray radiation. After recovered at 25 °C for 1.5 h, larval brains were dissected in 1 × phosphate-buffered saline, incubated in hypotonic solution (0.5% sodium citrate·2 H2O) for 10 min, fixed in freshly prepared acetic acid/methanol/H2O (11:11:2) mixture for a few seconds. Fixed brains were treated in 45% acetic acid for 1–2 min, followed by squashing. Slides were frozen in liquid nitrogen, dehydrated in absolute ethanol at −20 °C. Air dried slides and stained in mounting medium with DIAP (Vectashield, Burlingame, CA, USA). Images were taken using UltraVIEW VoX 3D live cell imaging system (PerkinElmer, Waltham, MA, USA).

Immunostaining

Immunostaining was performed as described.46 Imaginal wing discs were dissected in 1 × phosphate-buffered saline, fixed in fixative solution (0.1 M PIPES, pH 6.9, 1 mM EGTA, pH 6.9, 1.0% Triton X-100, 1 mM MgSO4, 1% formaldehyde) for 23 min, blocked for 2 h at 4 °C . Samples were incubated with rabbit anti-cleaved caspase-3 (1:200, Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C and detected with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (1:500, BETHYL, Montgomery, TX, USA).

Quantitative analysis of immunostaining

A confocal z-series of image was taken at intervals of 0.75 μm with × 20 objective for discs and × 10 for brains. The overlapped z-series images were generated by Volocity 3D image analysis software (version 5.0, PerkinElmer). For acridine orange staining, the images were taken at a single layer of wing discs. The confocal images were quantified as follows. The isosurfaces of stained positive region were created by Image J software (Version 1.43u, http://rsb.info.nih.gov/ij) with a high-intensity threshold at 40 for anti-caspase 3 staining, 75 for acridine orange staining. The isosurface of the entire disc was created with a low-intensity threshold of 10. The staining index is the total isosurfaces volume of stained positive region divided by isosurface of the entire disc. Images were taken from at least five wing discs or eye discs for each experiment. A total of three independent experiments were analyzed.

Scan electronic microscope

Flies were dehydrated in 75, 85, 95 and 100% (two times) ethanol for 15–20 min each, and then dried with a CPD-030 Baltec critical point dryer (Balzers, Liechtenstein). Samples were observed in low-vacuum using Quanta 200 field emission scanning electron microscope (FEI, Hillsboro, OR, USA).

RNA extraction and reverse transcription–quantitative PCR

Total RNA was extracted from third-instar larvae with RNAqueous Kit (Ambion, Grand Island, NY, USA), and reverse transcribed with oligo-dT and MMLV (Promega, Madison, WI, USA). The clbn transcript was quantified by quantitative PCR with iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and normalized to expression of rp49. Real-time quantitative PCR primer pairs were designed with Primer3 and tested by electrophoresis to verify correctly sized products. The following primer pairs were used: rp49-58F, 5′-IndexTermTACAGGCCCAAGATCGTGAAG-3′; rp49-175R, 5′-IndexTermGACGCACTCTGTTGTCGATACC-3′; clbn-1503F, 5′-IndexTermGCGCAAGACGCAGCAGACG-3′; clbn-1645R, 5′-IndexTermTCTGCTGGGCATCTCTTCCTCC-3′. Reactions were prepared with iQ SYBR Green Supermix (Bio-Rad), and quantified with comparative CT method. Relative expression change was calculated from three independent experiments.

Tumor formation in athymic nude mice

Tumor formation was monitored following the subcutaneous inject of 5 × 106 cells per mouse of one of three cell lines, the parental human lung adenocarcinoma line, A549, and two stably transformed derivatives, A549-Clbn-M and A549-Clbn-S.20 The derivatives constitutively express the full-length Drosophila clbn gene either fused to a Myc-His epitope tag at its carboxy terminus (A549-Cbn-M) or terminating at the normal translation stop site of clbn (A549-Clbn-S).

Statistical analysis

Statistic significance (P-value) was determined by two-way analysis of variance and Student–Newman–Keuls test (SNK-q test).

References

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Acknowledgements

We thank Drs Hermann Steller, Laura Johnston and Tin Tin Su; the Bloomington Stock Center for fly stocks; Dr Dong Han at National Center for Nanoscience and Technology for help with the scanning electron microscope; and the members of the Bi laboratory for advice and discussions. We also thank Dr Mark Mortin for generating the knockout clbn flies and for critically reading this manuscript. We are grateful for comments on this manuscript from Tehyen Chu and Brent McCright. This work was supported by grants from National Basic Research Program of China (973 Program grant no. 2010CB934004), National Natural Science Foundation of China (grant no. 30871388) and CAS Knowledge Innovation Program to XB and by the Food and Drug Administration, Center for Biologics Evaluation and Research.

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Correspondence to X Bi.

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Supplementary Information accompanies the paper on the Oncogene website

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Keywords

  • Apoptosis
  • Caliban
  • p53
  • tumor suppressor

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