Oncogene (2008) 27, 1429–1438 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Gadd45b is a pro-survival factor associated with stress-resistant tumors

A Engelmann1, D Speidel1, GW Bornkamm2, W Deppert1 and C Stocking1

1Heinrich-Pette-Institut, Hamburg, Germany and 2GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Institut fu¨r Klinische Molekularbiologie und Tumorgenetik, Munich, Germany

Tumors that acquire resistance against death stimuli recognized. It is now widely accepted that most cancer constitute a severe problem in the context of cancer therapies work by inducing the intrinsic apoptotic path- therapy. To determine genetic alterations that favor the way; however other mechanisms may also be involved, development of stress-resistant tumors in vivo, we took including activation of senescence, necrosis, mitotic advantage of polyclonal tumors generated after retro- catastrophe or autophagic cell death (Johnstone et al., viral infection of newborn Ek-MYC mice, in which the 2002; Okada and Mak, 2004). Altered expression or retroviral integration acts as a mutagen to enhance tumor mutation of encoding key involved in any progression. Tumor cells were cultivated ex vivo and of these processes can provide cancer cells with both an exposed to c-irradiation prior to their transplantation into intrinsic survival advantage and inherent resistance to syngenic recipients, thereby providing a strong selective therapeutic-induced stress. pressure for pro-survival mutations. Secondary tumors To identify novel genes and thus mechanisms by developing from stress-resistant tumor stem cells were which tumor cells escape stress response, we have deve- analysed for retroviral integration sites to reveal candi- loped an approach in which tumor growth is accelerated date genes whose dysregulation confer survival. In addi- by retroviral insertional mutagenesis, and subclones tion to the encoding the antiapoptotic Bcl-xL , that have acquired stress resistance are selected. This we identified the gadd45b locus to be a novel common approach takes advantage of the fact that retroviral integration site in these tumors, leading to enhanced infection of transgenic mice expressing the human MYC expression. In accord with a thus far undocumented role of oncogene under control of the Igl enhancers, mimicking Gadd45b in tumorigenesis, we showed that NIH3T3 cells human Burkitt’s lymphoma (Kovalchuk et al., 2000), overexpressing Gadd45b form tumors in NOD/SCID mice. leads to the rapid development of polyclonal tumors. Interestingly and differently to other known ‘classical’ Retrovirus infection accelerates tumor progression by antiapoptotic factors, high Gadd45b levels did not protect integration in the vicinity of oncogenes and/or tumor against MYC-, UV- or c-irradiation-induced , but suppressor genes and their subsequent activation or conferred a strong and specific survival advantage to serum inactivation, conferring a selective advantage and withdrawal. subsequent outgrowth of the affected cell (Mikkers Oncogene (2008) 27, 1429–1438; doi:10.1038/sj.onc.1210772; and Berns, 2003). In the approach developed here, the published online 24 September 2007 tumor cells are subsequently subjected to stress factors (ex vivo cultivation and g-radiation) prior to transplan- Keywords: B-cell lymphoma; MYC; p53; Bcl-xL; retroviral tation into recipient mice, thus providing a strong insertional mutagenesis; stress-resistance selective pressure for tumor cells with integrations that confer pro-survival mutations. The strength of this approach was demonstrated by identifying the known antiapoptotic factor Bcl-xL, but also identifying Introduction Gadd45b as a pro-survival factor in this system. Recent studies using gadd45b knockout mice have suggested Despite considerable progress over the past fifty years an antiapoptotic function for Gadd45b (Gupta et al., in the treatment of human malignancies, intrinsic 2005). Extending these studies, we provide evidence that and acquired resistance to chemotherapeutic agents normal levels of Gadd45b may be critical to prevent and radiation remains a severe problem (Bernier et al., apoptosis, but increased levels of Gadd45b contribute 2004; Chabner and Roberts, 2005). Our understanding to tumorigenesis and tumor resistance by a mechanism of mechanisms leading to therapeutic resistance has distinct to classical apoptosis. been expanded considerably during this time, and the crucial role of stress resistance pathways is increasingly Results

Correspondence: Dr C Stocking, Molecular Pathology, Heinrich- Mouse model for the identification of tumor-related Pette-Institut, PO Box 201652, D-20206 Hamburg, Germany. E-mail: [email protected] pro-survival genes Received 27 March 2007; revised 11 July 2007; accepted 6 August 2007; To define genetic alterations that contribute to published online 24 September 2007 development of stress-resistant hematopoietic tumors Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1430 in vivo, we extended the method of retroviral insertional show that infection of newborn El-MYC mice with mutagenesis in a mouse model for leukemia by adding Moloney-murine leukemia virus (Mo-MuLV) enhanced an additional selection step. In the first step, we could tumor progression and decreased the median survival

Oncogene Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1431 time of the mice from 100 to 49 days (Figure 1a). Similarly to uninfected controls, the mice succumbed to an aggressive B-cell (B220 þ IgM þ ) or pro-B-cell (B220 þ IgMÀ) neoplasia, characterized by lymphoma and hepatosplenomegaly, as well as high leukocyte counts in the peripheral blood (mean 114 Â 106 cells mlÀ1, n ¼ 44). The accelerated tumor progression can be attributed to additional oncogenic mutations caused by retrovirus integration. To specifically select for tumor subclones in which mutations imparted a pro-survival advantage to stress stimuli, we explanted the tumors, cultivated the tumor cells ex vivo and exposed them to 7 Gy g-radiation, prior to their transplantation into syngenic recipient animals. Such treatments combine several stress stimuli and eliminate the vast majority of tumor cells by apoptosis as indicated by fragmentation of DNA and the presence of active cleaved caspase-3 (Figures 1b and c). Although irradiation clearly provided the major death stimulus, ex vivo cultivation alone was sufficient to provoke a measurable apoptotic response in some tumor cells within a short time, indicating the sensitivity of primary tumor cells to non-genotoxic environmental changes. In concordance with the massive cell death upon irradiation, tumor-initiating cells giving rise to second- ary tumors were detected at a frequency four orders of Figure 2 Secondary tumors arising from irradiated tumors have magnitude lower than that of non-irradiated controls, acquired mutations conferring radiation resistance. (a) Schematic representation of experimental design to test if tumors selected as determined by serial dilution (Figure 1d). Southern after radiation are less sensitive to stress signals. (b) Survival curves blot analysis performed with a Mo-MuLV env probe of mice transplanted with 5 Â 105 secondary tumor cells that were confirmed that the stress treatment induced a strong either irradiated (lightning bolt and solid lines) or untreated selection for specific clones; the viral integration pattern (dashed line). Two sets of secondary tumors were tested: tumors changed significantly from a predominantly diffuse generated from irradiated primary tumors (gray lines) or non- irradiated primary tumors (black lines). Results depicted are a picture in the parental primary tumor (P), indicative of compilation of five experiments with independent primary tumors, a highly polyclonal tumor, to a more distinct band for which three to four mice were used for each experimental pattern in the different, primarily monoclonal, second- condition (total of 68 mice). ary tumors (S1–S9) (Figure 1e). Of note, some bands not prominently appearing in the parental tumor could resistance (secondary tumors derived from irradiated be detected in several of the different independent primary cells), tumor growth was significantly faster and secondary tumors, with specific differences between the with a higher penetrance (95 versus 50%) than that of sets of non-irradiated and irradiated secondary tumors. irradiated tumor cells derived from secondary tumors This finding confirms that both treatments, ex vivo culti- from non-irradiated primary cells (Figure 2b). These vation alone and ex vivo cultivation plus g-irradiation, results demonstrate that tumor cells had been selected provide a distinct selection pressure, selecting for that were significantly less sensitive to irradiation different tumor cell clones. treatment. To determine if spontaneous mutations A second round of radiation and re-transplantation of within the Trp53 gene were responsible for the acquired both irradiated and non-irradiated secondary tumors stress resistance, sequence analysis was performed. Only was performed to verify the selection for stress-resistant 25% of the tumors carried point mutations in the p53 tumors (Figure 2a). Although irradiation still delayed coding region, in agreement with the low incidence of the in vivo growth of tumors cells selected for stress p53 inactivation in Mo-MuLV-induced murine and

Figure 1 Mouse model to identify pro-survival genes. (a) Survival curves of Moloney-murine leukemia virus (Mo-MuLV) infected (n ¼ 65) and uninfected (n ¼ 47) El-MYC C57Bl/6 mice. Newborn mice infected with Mo-MuLV show an acceleration of disease as compared to uninfected control animals. (b and c) Apoptosis induction in tumor cells upon ex vivo cultivation and irradiation. (b) DNA fragmentation indicative of apoptosis-related nucleases can be observed in all tumors 3 h after irradiation with 7 Gy and to a lesser extent in non-irradiated tumors. (c) Western blot analysis of protein isolated from explanted tumor cells confirmed the presence of cleaved caspase-3, a marker of apoptosis, in tumors 3 h after irradiation and, to a lesser extent, in one non-irradiated tumor. (d) Survival curves of mice transplanted with limiting dilutions of either non-irradiated tumor cells (black lines) or 5 Â 106 g-irradiated tumor cells (gray line). The kinetics of tumor induction allowed the calculation of cells in the tumor mass that were able to form tumors after irradiation. Shown are the results of one tumor, but similar results were obtained with independent tumors (n ¼ 3). (e) Detection of proviral integrations by Southern blot analysis of genomic DNA digested with BglII and hybridized with an env-probe. The arrow marks an endogenous retroviral fragment, also observed in DNA from uninfected lymph nodes. Lightning bolt indicates tumors arising from irradiated cells. P, parental tumor; S1–S9, secondary tumors.

Oncogene Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1432 Table 1 Common integration sites found in secondary tumors Locus Protein Chr Hitsa Mycb Reference

Bmi1 Polycomb complex protein 2 4 + Haupt et al. (1991) and van Lohuizen et al. (1991) Ahi1 Abelson helper integration site 1 10 3 + Poirier et al. (1988) Pim2 Serine/threonine kinase X 3 + van der Lugt et al. (1995) Bcl2l1 Apoptosis regulator Bcl-xL 23 À This article Mcl1 Apoptosis regulator EAT/MCL-1 3 2 + Mikkers et al. (2002) Runx3 Runt transcription factor 3 4 2 + Stewart et al. (2002) Gadd45b Growth arrest and DNA 10 2 À This article damage-inducible protein b

aNumber of independent integrations found in secondary tumors. bPreviously identified as a CIS in MYC-associated tumors.

human lymphopathic tumors (Baxter et al., 1996; Krug et al., 2002).

Integrations into the Bcl2l1 locus as a proof of principle To identify genetic alterations responsible for a pro- survival advantage in stress-resistant tumor cells, we analysed secondary tumors derived from ex vivo culti- vated and irradiated primary tumors for viral integra- tion sites. In total, 76 viral integration sites of 21 secondary tumors from 10 independent tumor sets were determined, and their specific location in the cellular genome was mapped (Supplementary data). Proviral insertions occurring in several, independently derived tumors are classified as common integration sites (CISs) and mark genes contributing to tumorigenicity (and progression), based on the fact that the frequency of finding two independent integrations in the same locus without a selective advantage would be too low to be detected (Mikkers and Berns, 2003). The majority of CISs that we identified in secondary tumors have been described previously in MYC tumor mouse models (Table 1). However, in addition, we found two loci, bcl2l1 and gadd45b, which have not been identified as a CIS in these models, but which were associated with Figure 3 Integrations within the bcl2l1 locus are selectively found stress-treated secondary transplants in our analysis. in secondary tumors derived from irradiated tumor cells and lead to upregulation of Bcl-xL expression. (a) Southern blot analysis of Three independent integrations in two tumor sets BglII digested genomic DNA. Proviral integrations were detected were observed in the bcl2l1 locus, which codes for with a bcl2l1 probe. C, control DNA from uninfected lymph node; Bcl-xL. This is a bona fide antiapoptotic factor, being a P, parental tumor; S1–S6, secondary tumors. Irradiation is member of the Bcl-2 family, which constitutes the indicated by lightning bolt. Asterisks denote rearranged bands in the tumors S4 and S5. (b) Quantitative reverse transcriptase (RT)– master regulator controlling the intrinsic, mito- PCR of bcl211 mRNA levels relative to hprt controls isolated from chondria-mediated pathway of apoptosis (Tsujimoto, the indicated tumors. 2003). Southern blot analysis of DNA isolated from one tumor set showed that integration into the bcl211 that retroviral insertion into the bcl2l1 locus resulted in locus was present in two of three irradiated secondary ca. 10-fold overexpression of Bcl-xL mRNA as com- tumors, while it was absent in all tumors derived from pared to parental tumors (compare tumor P with S3 non-irradiated tumor cells and in the primary tumor and S4 in Figure 3b). However, upregulation was (Figure 3a). Although the secondary tumors were also observed in secondary tumors from the same set, derived from the same primary tumor, the integrations in which no bcl211 integrations were detected (tumor were independent events—occurring approximately S6). This may be due to integration in a region not 1000 bp from another, within the second intron of the detected by the probe, integration and dysregulation of bcl211 gene. A third integration was also detected in a a gene whose product regulates Bcl-xL expression, or secondary tumor arising from an independent primary to an unknown mechanism. Taken together, we found tumor after irradiation. In this case, integration had the Bcl2l1 locus to be a novel CIS characteristic of occurred 300 bp upstream of the first coding exon. radiation-resistant tumors, leading to elevated levels of Quantitative reverse transcriptase (RT)–PCR confirmed Bcl211 expression.

Oncogene Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1433 upregulation (see Discussion). Taken together, we found retroviral integrations into the gadd45b locus in two independent sets of tumors thereby clearly identifying this gene as a CIS. Significantly, integration and the resulting upregulation of Gadd45b expression were exclusively observed in secondary tumors, again indica- tive of a selection advantage that this integration confers to tumor cells that have been stressed by ex vivo cultivation with or without subsequent g-irradiation.

Gadd45b is an oncogenic pro-survival factor The finding that tumor cells with elevated Gadd45b levels are enriched upon stress treatment prompted us to ask whether high intracellular concentrations of this factor actively support tumorigenesis. To study the consequence of high Gadd45b expression, we stably expressed Gadd45b in NIH3T3 cells using a retroviral vector co-expressing enhanced green fluorescent protein (eGFP). The transduced cell population was enriched (75–95%) by flow cytometric sorting of GFP positive cells. Expression of Gadd45b and GFP in these cells was verified by western blot analysis (data not shown). Subcutaneous injection of 1 Â 106 Gadd45b expressing NIH3T3 cells reproducibly induced tumors in NOD/ SCID mice (n ¼ 10), which were clearly visible 15 days post injection (mean weight 0.5970.32 g; range 0.25– 1.28 g). At the same time and up to the end of the Figure 4 Integrations within the gadd45b locus are found in observation period (21 days post injection), animals that secondary tumors and lead to upregulation of gadd45b expression. had received parental- (n ¼ 9) or vector-transduced (a and b) Southern blot analysis of HindIII (a)orBglII (b) digested (n ¼ 9) NIH3T3 cells did not show any tumors. Over- genomic DNA hybridized to a locus-specific probe. C, control DNA from uninfected lymph node; P1 and P2, parental tumors; expression of Gadd45b thus transforms NIH3T3 mouse S1–S21, secondary tumors. Lightning bolts indicate tumors arising fibroblasts and supports tumorigenesis. Moreover, these from irradiated cells. Arrows denote rearranged bands. (c) North- findings indicate that Gadd45b has an oncogenic ern blot analysis of RNA isolated from tumors and hybridized with activity, which is independent of deregulated MYC a gadd45b probe. expression. We speculated that the pro-tumorigenic potential of Gadd45b is due to inhibition of stress-induced cell death The gadd45b locus is a novel CIS in a way similar to Bcl-xL. To address this, we analysed From integrations in two independent tumor sets, we the impact of Gadd45b overexpression in several identified the gadd45b gene locus to be a novel CIS. systems. DNA damage, as induced by high-dose Gadd45b is a member of the growth arrest and DNA irradiation, leads to apoptotic cell death by engaging damage-inducible family that is upregulated by the intrinsic mitochondria-mediated pathway. This can various stress stimuli (Liebermann and Hoffman, 2002). be measured via western blot analysis monitoring the In one tumor set, several secondary tumors (four of five) presence of the cleaved and thereby active form of from an irradiated primary tumor contained an caspase-3 or assessing DNA fragmentation (% sub-G1) integration 1.5 kb downstream of the gadd45b gene, by fluorescence-activated cell sorting (FACS) analysis. which was not observed in five of seven secondary NIH3T3 fibroblasts exposed to two different doses of tumors that had developed from non-irradiated cells UV irradiation show the characteristics of apoptosis to (Figure 4a). No integration in this locus was detectable varying degrees (Figures 5a and b). As expected, in the parental tumor. In a second independent tumor overexpression of Bcl-xL completely blocked apoptosis. set, integration was detected 6.4 kb downstream of the In contrast, Gadd45b overexpressing NIH3T3 cells gadd45b gene. This integration was detectable in three of underwent apoptosis upon UV treatment to the same four tumors derived from irradiated tumor cells, but not extent as parental- or vector-transduced cells (Figures 5a in the parental primary tumor (Figure 4b). Northern and b). Similar results were obtained when murine blot and quantitative RT–PCR analyses showed that hematopoietic Ba/F3 cells were subject to 6 Gy g- expression in tumors performing the integration was signi- irradiation (Figure 5b). We conclude that elevated ficantly elevated, with the exception of S19 (Figure 4c and Gadd45b levels do not protect against DNA damage- data not shown). Also two tumors (S6, S7) without inte- induced apoptosis mediated by the intrinsic pathway. gration in this specific locus showed elevated gadd45b Another mechanism to induce cell death that might be expression, suggesting alternative mechanisms for relevant for our model system is activation of apoptosis

Oncogene Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1434 as a direct consequence of MYC overexpression. Similar To test whether overexpression of Gadd45b protects to DNA damage, deregulated MYC expression can also against MYC induced cell death, we took advantage induce apoptosis via the intrinsic pathway, but the of a murine hematopoietic cell line (ERP 15–41) that signaling upstream of the mitochondria is different. expresses a fusion protein coupling MYC to the

Oncogene Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1435 estrogen receptor (MYC-ER) (Blyth et al., 2000). Upon Discussion addition of 4-hydroxytamoxifen (4OHT), MYC-ER becomes activated and induces apoptosis as evidenced The goal of this study was to define molecular by DNA fragmentation and a high percentage of cells mechanisms by which hematopoietic tumors escape positive for propidium iodide (PI) staining (Figure 5c). Overexpression of Bcl-xL also blocked MYC-induced cell death in these cells. No protective effect upon MYC activation, however, was observed in Gadd45b over- expressing cells, which behaved as the control cells. Thus, elevated Gadd45b levels do not confer a pro-survival effect by means of inhibiting the intrinsic pathway of apoptosis induced by DNA damage or activation of MYC. Since integration in the gadd45b locus and elevated expression of its gene product was also observed in some secondary tumors that were not irradiated (see Figure 4), we reasoned that Gadd45b might help the tumor cells to better survive non-genotoxic stress as a result of the ex vivo cultivation. To mimic this particular type of stress in a cell culture setting, we cultivated the differently transduced NIH3T3 fibroblasts in medium without serum. Serum withdrawal efficiently led to cell death of the vector-transduced control cells as evidenced by PI staining (Figure 6a). Interestingly, overexpression of Bcl-xL provided only an initial, slightly protective effect against this stress stimulus, but could not prevent cell death. In striking contrast, Gadd45b expression prevented cell death under these conditions and main- tained cell viability and growth (Figure 6b). As it has been reported that serum deprivation induces classical caspase-dependent apoptosis (Paddenberg et al., 2001), we analysed the presence of cleaved and thus active caspase-3 in these cells (Figure 6c). Cleaved caspase-3 was detectable in all serum-starved NIH3T3 cells, but, consistent with the ability of Bcl-xL but not Gadd45b to block the caspase pathway, levels were significantly reduced in cells expressing Bcl-xL (Figure 6c). However, since only a small proportion of Gadd45b overexpressing cells died upon serum with- Figure 6 Overexpression of Gadd45b protects NIH3T3 cells from cell death upon serum withdrawal. Transduced and sorted NIH3T3 drawal, and Bcl-xL overexpressing cells died despite cells (same cells as in Figure 5) were cultivated with 10% or no suppression of caspase-3 activation, we conclude that serum. (a) To determine cell viability, cells were harvested at the the major death pathway in NIH3T3 cells induced by indicated time, stained with propidium iodide (PI) and analysed by serum withdrawal differs from known caspase-induced flow cytometry. The percentage of dead cells upon serum withdrawal apoptosis. This conclusion is supported by other studies was calculated as % PI positive cells (0% serum)À% PI positive cells (10% serum). Figure shows the result and s.d. of three independent investigating the death pathway initiated by serum experiments. (b) To determine cell proliferation rates, the number withdrawal (Simm et al., 1997; Kues et al., 2002; Leicht of viable cells was determined by Trypan blue exclusion at the et al., 2003). Gadd45b overexpression clearly protects indicated times. Figure shows the result and s.d. of two independent against this yet to be identified death program. In experiments. (c) Western blot analysis of NIH3T3 fibroblasts transduced with the retroviral vectors and subjected to serum summary, our data show that elevated levels of withdrawal for 30 h. Proteins were detected using specific antibodies Gadd45b confer a potent and very specific protection as indicated. The presence of cleaved caspase-3, the active form of the against stress-induced cell death. executioner caspase, was used as a marker for apoptosis.

Figure 5 Bcl-xL but not Gadd45b protects against DNA damage and MYC-induced apoptosis. (a) Western blot analysis of NIH3T3 fibroblasts transduced with the retroviral vectors and either mock-treated or subjected to UV irradiation at the indicated dose. Cells were harvested 42 h after irradiation. Proteins were detected using specific antibodies as indicated. The presence of cleaved caspase-3, the active form of the executioner caspase, was used as a marker for apoptosis. (b) Fluorescence-activated cell sorting (FACS) analysis of NIH3T3 cells, subjected to 50 J mÀ2 UV-irradiation, and Ba/F3 cells, subjected to 6 Gy g-irradiation. Cells were fixed in ethanol and stained with propidium iodide (PI) 48 h after treatment. The percentage of sub-G1 fragments is a measure of apoptosis. (c) FACS analysis of transduced and sorted ERP15-41 cells after MYC-ER activation. Transduced ERP15-41 cells were treated with 4-hydroxytamoxifen (4OHT) to activate MYC-ER or with ethanol as a control. Cells were harvested at the indicated time points. To measure apoptosis or viability, ethanol-fixed (left) or non-fixed (right) cells, respectively, were stained with PI. Viability was calculated as % PI positive cells (4OHT treated)À% PI positive cells (ethanol treated). Figure shows the result and s.d. of two independent experiments.

Oncogene Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1436 stress-induced cell death. We established a powerful stimulation with TNFa or Fas ligand) (De Smaele et al., mouse model using retroviral insertional mutagenesis to 2001; Zazzeroni et al., 2003) or have inferred an identify genetic lesions that can abrogate cell death in antiapoptotic function by evaluating genotoxic-stress- lymphoid cells after induction of stress. One locus induced apoptosis in gadd45bÀ/À bone marrow cells identified in this manner was mapped to the Bcl2l1 gene. (Gupta et al., 2005, 2006). In accordance with our data, This locus has been identified as a CIS in other mouse other work demonstrated that Gadd45b does not have models (Packham et al., 1998; Hwang et al., 2002), but an influence on apoptosis (Zerbini et al., 2004). has not been identified as a CIS for MYC mouse models. Although high levels of Gadd45b were unable to The finding that Bcl-xL expressing cells are protected protect cells from radiation or MYC-induced apoptosis, against apoptosis induced by irradiation was not we were able to show that Gadd45b overexpression surprising. The protein belonging to the Bcl-2 family is protected fibroblasts from cell death after serum with- a well-known antiapoptotic molecule, which blocks drawal, showing that Gadd45b expression inhibits cell apoptosis at the level of the mitochondria and inhibits death under certain circumstances. Importantly, the release of apoptogenic molecules from the inter- Gadd45b interferes with a cell death-inducing pathway membrane space of the organelle (Tsujimoto, 2003). Our that is different from the ‘classical’ caspase-3 driven findings are in accord with studies showing that elevated apoptosis, which can be suppressed by Bcl-xL, indicating Bcl-xL levels correlate with therapy resistance of tumor that the intrinsic pathway of apoptosis plays only a cells from hematologic and solid tumors. The identifica- minor role upon serum withdrawal, and another, yet to tion of the bcl2l1 locus as a CIS in secondary tumors be determined signaling cascade is the driving force to after irradiation thus provides a satisfying ‘proof-of- eliminate the cells under these conditions. Recent principle’ to our approach. evidence suggests that besides apoptosis and senescence, The second CIS identified in this study was mapped to therapeutic regimens can eliminate tumor cells by the gadd45b locus. This locus is a new retroviral CIS, alternative pathways (Okada and Mak, 2004). For since integrations in this locus have, to our knowledge, example, it has been shown that cells protected from never been found in any mouse model. Integrations were apoptosis by Bax and Bak knockout undergo necrosis or primarily but not exclusively found in secondary tumors autophagic cell death in response to otherwise apoptotic derived from irradiated primary tumor cells, leading to stimuli (Lindsten et al., 2003; Shimizu et al., 2004). It an approximately fivefold increase in expression levels. has also been suggested that the antitumor effect of High expression of gadd45b was also observed in two mTOR inhibitors is mediated by their ability to induce secondary tumors that did not have integrations within autophagy, although it is unknown to what extent the the gadd45b locus. Owing to the extremely high levels autophagy-inducing effect of the mTOR inhibitors is of expression in these tumors, the integration sites involved in their antineoplastic activity (Kroemer and of these two tumors were also cloned and sequenced. Jaattela, 2005). Interestingly, this analysis revealed integration in the Many synergistic mechanisms may contribute to the tpl2 locus. Tpl2 is an activator of the transcription elimination of tumor cells in the course of therapy and it factor NF-kB (Das et al., 2005), which is a well-known is likely that inhibition of any one such mechanism activator of gadd45b (De Smaele et al., results in escape of cell death. Our work shows for the 2001). Thus the induction of gadd45b expression by first time a novel role for Gadd45b in the emergence of NF-kB could account for the high expression levels. In stress-resistant clones. The specificity of the Gadd45b- summary, there appears to be a high selective advantage mediated effect and its difference to Bcl-xL support the for upregulation of gadd45b in the development of hypothesis that different mechanisms act in concert to tumors after stress, from either in vitro cultivation or protect tumor cells from stress signals. Understanding irradiation. these alternative cell death pathways will be important One explanation for this is that Gadd45b acts as an to develop alternative therapies to eliminate apoptosis- oncogene during tumor progression, either alone or in resistant tumors. combination with existing mutations. An oncogenic function was indeed demonstrated by assaying tumor development of fibroblasts engineered to overexpress Materials and methods Gadd45b in NOD/SCID mice. To determine whether it is also an antiapoptotic protein, the cells were also Mice, infections and tumor formation subjected to different apoptotic-inducing stimuli. Using Mice carrying the human MYC oncogene under the control of two different cell lines, we could show that Bcl-xL but the Igl regulatory elements were crossed with wild-type not Gadd45b overexpression protects cells from irradia- C57BL/6 mice, and newborns were infected intraperitoneally tion-induced apoptosis. Those findings were somewhat within 24 h after birth with 0.05 ml Mo-MuLV-containing 6 surprising, because, although Gadd45b was originally supernatant with a virus titer of 1.6 Â 10 infectivity units per described as a pro-apoptotic protein in response to milliliter. Non-transgenic littermates served as control in these experiments. Infected mice were monitored for lymphoma TGFb (Selvakumaran et al., 1994; Yoo et al., 2003), development, and killed when moribund. Cells dispersed from more recent results have revealed antiapoptotic func- lymph nodes were transplanted (5 Â 106 cells or 5 Â 105 for the tions for gadd45b. Importantly, however, these studies first and second transplantations, respectively, or after serial have only demonstrated an antiapoptotic function for dilutions in RPMI1640) intraperitoneally into syngenic Gadd45b in the extrinsic apoptotic pathway (induced by C57BL/6 recipients. To determine the tumorigenic potential

Oncogene Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1437 of Gadd45b,1Â 106 transduced NIH3T3 cells in 100 ml PBS Production of retroviral supernatant and infection of cells were injected subcutaneously into NOD/SCID mice. The coding regions of bcl2l1 and gadd45b were amplified via PCR (primers and conditions available upon request) and Southern blot analysis of genomic DNA and isolation of cloned into an FMEV-based retroviral vector (Schwieger et al., integration sites 2002) to generate pSF91-Bcl-xL (R1116) and pSF91-Gadd45b High-molecular-weight genomic DNA was extracted from (R1117). Retroviral pseudo-types were produced as described frozen tissue samples and subjected to Southern blot analysis (Schwieger et al., 2002). Transduced cells were sorted for by standard methods. For the analysis of apoptosis, high- GFP-expressing cells with a FACS Aria (BD Biosciences, molecular-weight DNA was extracted from 1 Â 107 tumor cells Heidelberg, Germany). cultured for 3 h, as described (Heinrichs and Deppert, 2003). Retroviral integration sites were isolated using a modified Western blot analysis protocol of a previously described method (Schmidt et al., Cells were washed with ice-cold PBS, harvested and stored at 2001). Details are available upon request. Integrations were À70 1C. Cells were lysed in 50 mM HEPES, pH 7.5, 150 mM mapped to the murine genome using the BLAT and BLAST NaCl, 0.1% NP40, supplemented with a proteinase inhibitor programs of the UCSC (http://genome.ucsc.edu/) and En- cocktail (complete mini, Roche). Sixty micrograms of lysate sembl (http://www.ensembl.org/) databases, respectively, and was separated on 13% SDS–PAGE gels. For western blotting compared to known CISs in the RTCGD databank (http:// and ECL we used standard protocols. Antibodies against rtcgd.abcc.ncifcrf.gov/mm7/index.html). the following antigens were used, that is, cleaved caspase-3 (9661; Cell Signalling, Danvers, MA, USA), tubulin (CP06; RNA analysis Oncogene/Calbiochem, San Diego, CA, USA), Bcl-xL (sc8392; Total RNA was isolated from fresh or frozen tissue lysed in Santa Cruz Biotechnology, Santa Cruz, CA, USA) and guanidine isothiocyanate and pelleted through a CsC1 cushion Gadd45b (BC100; Novus Biologicals, Littleton, CO, USA). using the standard protocols. Northern blot analysis was performed using standard methods. To quantify expression levels, cDNA was produced with AMV reverse transcriptase (Promega, Mannheim, Germany) and subjected to real-time Abbreviations PCR using the LightCycler FastStart DNA Master SYBR Green I Kit (Roche, Penzberg, Germany). Conditions and 4OHT, 4-hydroxytamoxifen; CIS, common integration site; primers are available upon request. Relative quantification of eGFP, enhanced green fluorescent protein; FACS, fluores- real-time PCR products was performed using the calibrator- cence-activated cell sorting; Mo-MuLV, Moloney-murine normalized method. Two independent experiments were leukemia virus; PI, propidium iodide; RT, reverse transcriptase. performed in duplicates for each sample.

Cell culture and assays Acknowledgements Cell lines were cultured under standard conditions in either DMEM or RPMI1640 media supplemented with 10% FCS We thank Karen Blyth and Ewan Cameron for conditional and 4 mM glutamine, or in the case of Ba/F3 cells with MYC overexpressing cell lines, Arne Du¨ sedau for providing interleukin-3. Induction of MYC was achieved by adding FACS sorter support and other members of the Stocking 250 nM 4-OHT (Sigma). Cells were subjected to UV irradiation laboratory for helpful discussions. This work was part of the in a UV-crosslinker (1800, Stratagene, La Jolla, CA, USA). doctoral thesis of A Engelmann, Department of Biology, g-irradiation was performed using a Cs137 source. Viable cells University of Hamburg, Hamburg, Germany and was were determined by PI exclusion and counted by FACS supported by a grant of the Deutsche Krebshilfe. The analysis. To determine sub-G1 cell fraction, cells were fixed in Heinrich-Pette-Institut is supported by the Freie und Hanses- 80% ethanol (À20 1C), stained with PI and analysed by FACS tadt Hamburg and the German Ministry of Health and Social as described (Speidel et al., 2006). Safety.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene