Impaired DNA damage checkpoint response in MIF-deficient mice
2007; Springer Nature; Volume: 26; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7601564
ISSN1460-2075
AutoresAlice Nemajerová, Patricio Mena, Günter Fingerle‐Rowson, Ute M. Moll, Oleksi Petrenko,
Tópico(s)Nuclear Receptors and Signaling
ResumoArticle8 February 2007free access Impaired DNA damage checkpoint response in MIF-deficient mice Alice Nemajerova Alice Nemajerova Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Patricio Mena Patricio Mena Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Gunter Fingerle-Rowson Gunter Fingerle-Rowson University Hospital Cologne, Medical Clinic I, Hematology and Oncology, Cologne, Germany Search for more papers by this author Ute M Moll Ute M Moll Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Oleksi Petrenko Corresponding Author Oleksi Petrenko Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Alice Nemajerova Alice Nemajerova Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Patricio Mena Patricio Mena Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Gunter Fingerle-Rowson Gunter Fingerle-Rowson University Hospital Cologne, Medical Clinic I, Hematology and Oncology, Cologne, Germany Search for more papers by this author Ute M Moll Ute M Moll Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Oleksi Petrenko Corresponding Author Oleksi Petrenko Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Author Information Alice Nemajerova1, Patricio Mena1, Gunter Fingerle-Rowson2, Ute M Moll1 and Oleksi Petrenko 1 1Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA 2University Hospital Cologne, Medical Clinic I, Hematology and Oncology, Cologne, Germany *Corresponding author. Department of Pathology, State University of New York at Stony Brook, BST L9, Stony Brook, NY 11794, USA. Tel.: +1 631 444 3520; Fax: +1 631 444 3424; E-mail: [email protected] The EMBO Journal (2007)26:987-997https://doi.org/10.1038/sj.emboj.7601564 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Recent studies demonstrated that proinflammatory migration inhibitory factor(MIF) blocks p53-dependent apoptosis and interferes with the tumor suppressor activity of p53. To explore the mechanism underlying this MIF-p53 relationship, we studied spontaneous tumorigenesis in genetically matched p53−/− and MIF−/−p53−/− mice. We show that the loss of MIF expression aggravates the tumor-prone phenotype of p53−/− mice and predisposes them to a broader tumor spectrum, including B-cell lymphomas and carcinomas. Impaired DNA damage response is at the root of tumor predisposition of MIF−/−p53−/− mice. We provide evidence that MIF plays a role in regulating the activity of Cul1-containing SCF ubiquitin ligases. The loss of MIF expression uncouples Chk1/Chk2-responsive DNA damage checkpoints from SCF-dependent degradation of key cell-cycle regulators such as Cdc25A, E2F1 and DP1, creating conditions for the genetic instability of cells. These MIF effects depend on its association with the Jab1/CSN5 subunit of the COP9/CSN signalosome. Given that CSN plays a central role in the assembly of SCF complexes in vivo, regulation of Jab1/CSN5 by MIF is required to sustain optimal composition and function of the SCF complex. Introduction Macrophage migration inhibitory factor (MIF) is a ubiquitously expressed, predominantly cytoplasmic protein that has been implicated in the regulation of cell growth and development (Metz and Bucala, 2000). In addition, MIF has been recognized as a critical component of the immune system that regulates the proinflammatory properties of immune cells (Calandra and Roger, 2003). Previous work implicated MIF in the pathogenesis of acute and chronic inflammation such as wound repair, allograft rejection, chronic colitis and septic shock (Metz and Bucala, 2000; Calandra and Roger, 2003). However, the process by which MIF may exert its diverse biological effects is not completely understood. Intracellularly, MIF associates with the Jab1/CSN5 subunit of the COP9 signalosome (Kleemann et al, 2000), a highly conserved protein complex implicated in ubiquitin-mediated protein degradation (Cope and Deshaies, 2003; Wolf et al, 2003). Recently, Jab1/CSN5 was shown to possess a metalloprotease activity that removes the post-translational modification of Nedd8 from cullin, the enzymatic component of the SCF ubiquitin E3 ligase complex (Cope and Deshaies, 2003). Given that Nedd8 conjugation of cullins promotes their ubiquitin ligase activity in vivo, the involvement of Jab1/CSN5 in a number of cellular and developmental processes has been attributed to its control over ubiquitin- and proteasome-dependent proteolysis (Cope and Deshaies, 2003; Wolf et al, 2003). It is believed that the functions of MIF in cell-cycle regulation encompass its binding to Jab1/CSN5 (Kleemann et al, 2000; Calandra and Roger, 2003). In addition to its role in inflammatory and autoimmune diseases, MIF has been implicated in promoting tumorigenesis. Thus, MIF overexpression has been observed in various human cancer tissues, including colorectal, breast, lung and prostate cancer (Mitchell, 2004). Genetic studies demonstrated that MIF promotes B-cell lymphomagenesis and intestinal tumorigenesis in mice (Talos et al, 2005; Wilson et al, 2005). Conversely, elimination of MIF expression in several animal models impaired tumor growth (Fingerle-Rowson et al, 2003; Fan et al, 2005). In agreement with this, we recently showed that loss of MIF expression coincides with the induction of a p53-dependent proliferative block, which profoundly affects normal B-cell development (Talos et al, 2005). Moreover, inhibited S-phase progression and subsequent differentiation block are at the root of the predisposition of MIF-deficient B cells to undergo spontaneous p53-dependent apoptosis (Talos et al, 2005). Accordingly, almost all lymphomas that arise in MIF-deficient Eμ-Myc mice can be accounted for by mutations within the ARF–p53 axis, indicating that the p53 pathway is the main determinant for tumor suppression in this model system (Talos et al, 2005). To explore the mechanism underlying this MIF–p53 functional interaction, we studied spontaneous tumorigenesis in p53−/− and MIF−/−p53−/− mice. Our study demonstrates that the loss of MIF expression aggravates the tumor-prone phenotype of p53−/− mice and predisposes them to a broader tumor spectrum, including B-cell lymphomas and carcinomas. We show that MIF plays a role in regulating the activity of SCF ubiquitin ligases. Specifically, MIF loss uncouples Chk1/Chk2 DNA damage checkpoints from SCF-dependent degradation of key cell-cycle regulators such as Cdc25A, E2F1 and DP1, creating conditions for the genomic instability of cells. Our results indicate that the involvement of MIF in p53 function is routed through p53-independent mechanisms controlling protein stability, DNA damage checkpoints and genomic integrity. Results DKO mice develop an aggravated tumor phenotype and die prematurely Using the Eμ-Myc lymphoma mouse model, we recently demonstrated that loss of MIF expression induces a p53-dependent block in B-cell proliferation (Talos et al, 2005). Moreover, almost all B-cell lymphomas that arise in MIF-deficient Eμ-Myc mice exhibit mutations within the ARF–p53 axis, identifying p53 pathway as the main determinant for tumor suppression in this model system (Talos et al, 2005). In order to explore the mechanism underlying this MIF–p53 interconnection, we examined spontaneous tumorigenesis in genetically matched p53−/−, MIF+/−p53−/−, and MIF−/−p53−/− (DKO) mice. On the 129Sv genetic background, most p53−/− mice developed spontaneous tumors and died within 2–5 months of birth (Figure 1A, curve I). Loss of one MIF allele had no statistically significant effect on overall survival in a p53-null background (Figure 1A, curve II). By contrast, loss of both MIF alleles shortened the lifespan of p53−/− mice (Figure 1A, curve III), with DKO animals exhibiting a mean survival of 3.3±−0.9 months compared with 4.1±−1.4 months of the corresponding p53−/− mice (P<0.002). Figure 1.Concomitant loss of MIF aggravates the tumor-prone phenotype of p53−/− mice. (A) Kaplan–Meier analysis of survival of p53−/− mice (curve I, n=43 animals), MIF+/−p53−/− mice (curve II, n=41) and MIF−/−p53−/− mice (curve III, DKO, n=45) over time. Statistical significance was determined using the Student's t-test. (B) Tumor spectrum in mice of the indicated genotypes. Download figure Download PowerPoint Previous studies demonstrated that p53-null mice are tumor-prone and sustain high rates of T-cell lymphomas and soft tissue sarcomas (Attardi and Jacks, 1999; Maser and DePinho, 2003). In agreement, the majority of p53-null mice in our cohort also developed T-cell lymphomas (74%) and fibrosarcomas (10%), whereas B-cell lymphomas and carcinomas were rare (Figure 1B). By contrast, T-cell lymphomas accounted for only 45% of DKO tumors (Figure 1B). Moreover, we did not observe fibrosarcomas in any of the MIF+/−p53−/− and DKO mice examined to date (Figure 1B). Thus, connective tissue tumors are profoundly affected by loss of MIF expression, consistent with our results on the defective transforming properties of MIF-deficient MEFs (Petrenko and Moll, 2005). On the other hand, DKO mice developed more frequent B-cell lymphomas and carcinomas (Figure 1B). The latter included four colorectal adenocarcinomas, a gastric carcinoma and a testicular teratocarcinoma. Nearly 20% of DKO mice contained more than one tumor type, a phenomenon rarely seen in p53−/− animals. Therefore, slightly shortened latency of T-cell lymphomas in DKO animals (3.4±−0.7 months; Supplementary Figure 1A) compared with p53−/− mice (3.8±−1.0 months, P=0.2) likely reflects the increased frequency of other tumor types. Analysis of the lymphoid organs of DKO mice revealed a frequent presence of splenomegaly that was already detectable at a young age and became more evident over time (Supplementary Figure 1B and C). Splenomegaly with effacement of splenic architecture in these mice was caused by abnormally proliferating B-lymphoid cells (Supplementary Figure 1D). Most B-cell lymphomas developed by DKO animals were also manifest in the spleen, resulting in splenomegaly that ranged from 2-fold to 25-fold (Supplementary Figure 1C). Eventually, some of these tumors spread to peripheral sites, including lymph nodes and liver (Supplementary Figure 1D). Phenotypically, cell lines established from such DKO lymphomas were immature CD19+B220+CD25+IgM−/− B-cells (Supplementary Figure 1E). Based on this phenotype, the cellular target for transformation in these mice could not have passed the pro-B stage. Most thymic lymphomas developed by DKO mice expressed T-cell-specific markers CD3ε, CD4 and/or CD8, and lacked expression of B-cell markers (e.g. tumors 630 and 645, Supplementary Figure 1F). However, some thymic tumors stained positive for B-cell markers CD19 and B220, but lacked the expression of T-cell markers (tumor 928; Supplementary Figure 1F). Therefore, these tumors were also B-cell lymphomas. Several other DKO thymic tumors coexpressed markers of B-lymphoid and myeloid lineage cells (tumor 947; Supplementary Figure 1F). On the other hand, abnormally expanded T-cell populations were frequently observed in DKO B-cell lymphomas (tumor 768; Supplementary Figure 1F). As a consequence, the two diseases were sometimes difficult to distinguish, as they bore similarities to human T-cell-rich B-cell lymphoma, an aggressive subgroup of diffuse large B-cell lymphoma (Harris, 1999; Rossi and Gaidano, 2002) (Figure 1B). To determine if DKO tumors were transplantable, B-cell lines established from representative lymphomas were injected into the tail vein of athymic nude mice. After 3 weeks, reconstituted animals were killed and examined. Splenomegaly was manifest in all reconstituted mice (Supplementary Figure 2A and B). Histopathology analysis revealed that inoculated cells had also infiltrated the livers, lymph nodes and thymus anlagen of reconstituted animals, and grew as aggressive lymphomas (Supplementary Figure 2C and D). Collectively, these results indicate that concomitant loss of MIF aggravates the tumor-prone phenotype of p53−/− mice by broadening tumor spectrum to B-lymphomas and carcinomas, and significantly shortens lifespan. Notably, we did not observe spontaneous tumors in any of the MIF−/− and MIF−/−p53+/− mice examined to date (more than 50 animals of each genotype at 1 year of age). Thus, presence of one or both p53 alleles blocks tumorigenesis associated with MIF deficiency. Chromosomal aberrations in DKO B-cell lymphomas Although p53-deficient mice sustain high rates of T-cell lymphomas, these tumors lack characteristic chromosomal anomalies and exhibit modest levels of aneuploidy (Maser and DePinho, 2003). By contrast, advanced B-cell lymphomas invariably possess characteristic chromosomal rearrangements that point to a defective antigen receptor assembly or DNA repair process. Thus, reciprocal translocations between the IgH locus and c-Myc, Bcl2 or Bcl6 genes are present in human B-cell malignancies such as Burkitt's lymphoma, follicular lymphoma and diffuse large B-cell lymphoma (Kuppers, 2005). Likewise, chromosomal translocations involving the IgH region and the c-Myc proto-oncogene represent initiating events in the development of pro-B-cell lymphomas in mice (Bassing and Alt, 2004; Casali and Zan, 2004). Consistent with this, more than 50% of primary lymphoma isolates from DKO mice overexpressed c-Myc or N-Myc (Figure 2A). Cell lines established from DKO B-cell lymphomas also showed increased levels of c-Myc or N-Myc expression (Figure 2B). Of note, there was no detectable N-Myc expression in tumors with high levels of c-Myc, whereas upregulation of c-Myc and Bcl6 concurred (Figure 2B and data not shown). Figure 2.Chromosomal aberrations in DKO B-cell lymphomas. (A) Immunoblot analysis of c-Myc and N-Myc expression in primary lymphoma isolates from DKO mice. Cdk4 is a loading control. (B) Immunoblot analysis of cell lines established from DKO tumors. B5 and M3 are E-μMyc B-cell lymphomas. (C) Representative SKY and FISH analyses of DKO B-cell lymphomas. Arrows indicate N-Myc (green) and IgH (red) hybridization signals. Inset (tumor 928) shows derivative chromosome 12 carrying the IgH and duplicated N-Myc genes. Download figure Download PowerPoint Tumors with evidence of significant c-Myc or N-Myc upregulation were further analyzed by spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). The analysis revealed that DKO lymphomas harbor clonal gene amplifications and chromosomal translocations, including a characteristic reciprocal translocation t(12;15) that juxtaposes the c-Myc gene to antigen receptor loci (tumor 399; Figure 2C) and non-reciprocal translocation t(12;16) carrying the amplified N-Myc gene at the fusion of chromosomes 12 and 16 (tumor 928; Figure 2C). In addition, tumor 928 contained dicentric chromosome 12 carrying the IgH and duplicated N-Myc genes (Figure 2C, inset). Other translocations revealed by SKY harbored portions of chromosomes 6, 7, 13, 14, and 17 (Figure 2C). Similar cytogenetic features are found in pro-B-cell lymphomas that arise in mice with dual impairment of p53 and DNA double-strand break repair or non-homologous end joining (Maser and DePinho, 2003; Bassing and Alt, 2004), suggesting a potential role for MIF in maintenance of genomic stability. To test this hypothesis, we performed the following experiments. MIF loss impairs the DNA damage checkpoint response Mutations compromising the DNA damage pathway allow cell proliferation under conditions of replication stress (Nyberg et al, 2002; Bartek and Lukas, 2003). On the other hand, constitutive expression of aberrant c-Myc under conditions that inhibit cell growth, including serum deprivation, exposure to cytotoxic agents or microtubule inhibitors, can trigger apoptosis in many human and rodent cell types (Hoffman and Liebermann, 1998). To assess the effects of MIF deficiency on cellular responses to DNA damage and checkpoint activation, cell lines established from DKO tumors were incubated for 12 h in the presence of genotoxic drugs adriamycin, etoposide, or campthothecin. In addition, checkpoint activation resulting from stalled DNA replication was achieved by hydroxyurea or aphidicolin treatment (Brown and Baltimore, 2003). Three assays were then used to quantitate checkpoint response: inhibition of DNA synthesis (cell-cycle delay), Cdc25A inactivation, and apoptosis induction (a default pathway resulting from a combination of DNA damage, deficient cell-cycle checkpoints, and mitotic catastrophe). Because B-cell lymphomas are rare in p53−/− mice, we used p53-null B-lymphoma cells established from Eμ-Myc transgenic animals for controls (Talos et al, 2005). Although both cell types exhibited comparable levels of c-Myc expression (Figure 3A), flow cytometric analysis of DNA content and BrdU incorporation assays showed that p53−/− B-lymphoma cells exposed to DNA damage responded with a cell-cycle block, whereas DKO cells failed to do so (Figures 3A–C). Instead, a large proportion of DKO cells continued S-phase progression (Figures 3A–C), resulting in extensive apoptosis (Figure 3D). Likewise, inhibition of the cell cycle by serum deprivation or by pharmacological inhibitors hydroxyurea and aphidicolin caused more extensive apoptosis in DKO than in p53−/− cells (Figure 3D and data not shown). Figure 3.MIF loss impairs DNA damage checkpoint response. (A) S-phase proportion of p53−/− and DKO lymphoma cells exposed for 12 h to cell-cycle inhibition or DNA damage. Error bars represent the standard deviation. Inset, immunoblot analysis of c-Myc expression in p53−/− and DKO lymphoma cells. Cdk4 is a loading control. (B) BrdU incorporation (6-h pulse) by p53−/− and DKO cells before or after exposure for 2 h to the indicated cell-cycle inhibitors. Error bars represent the average of two experiments. (C) Flow cytometric analysis of cell-cycle distribution of p53−/− and DKO lymphoma cells exposed to adriamycin for 12 h. (D) Induction of apoptosis in p53−/− and DKO cells exposed for 12 h to DNA damage or cell-cycle inhibitors. Error bars represent the standard deviation. (E) Induction of apoptosis in p53−/− and DKO cells after 12 h of adriamycin treatment in the presence of caffeine, UCN-01, Chk2 inhibitor II, BN2002, NSC663284 or NU6102. Error bars represent the average of two experiments. Download figure Download PowerPoint One of the major biological responses to DNA damage or stalled replication is induction of the Chk1- and Chk2-dependent checkpoints that inhibit proliferation of cells with damaged templates (Bartek and Lukas, 2003). Among the key functional targets of Chk1/Chk2 regulation is the Cdc25 protein phosphatase family, which plays a critical role in activating Cdk2 and Cdk1 kinase complexes (Nyberg et al, 2002). To further assess the functional status of DNA damage checkpoints in MIF-deficient cells we used a series of pharmacological inhibitors directed against Cdk1/2 and their upstream regulators. Specifically, the ATM/ATR inhibitor caffeine and Chk2 inhibitor II increased susceptibility to DNA damage-induced apoptosis of p53−/− cells, while having little effect in DKO cells (Figure 3E). On the other hand, Chk1 inhibitor UCN-01 eliminated the difference between p53−/− and DKO cells, causing extensive apoptosis upon DNA damage (Figure 3E). Furthermore, UCN-01 and Chk2 inhibitor II displayed a similar effect on p53−/− and DKO B-lymphoma cells that were challenged with cisplatin or hydroxyurea (Supplementary Figures 3A and B). Conversely, Cdk inhibitor NU6102 reduced susceptibility to DNA damage-induced apoptosis of DKO cells, while having little effect in p53−/− cells (Figure 3E). Likewise, specific Cdc25 inhibitors BN82002 and NSC663284 reduced susceptibility to apoptosis of DKO cells to levels seen in similarly treated p53−/− cells (Figure 3E). Of note, reconstitution of MIF expression in DKO B-lymphoma cells also reduced DNA damage-induced apoptosis (Supplementary Figure 3C). Together, these results suggest that MIF-deficient tumor cells harbor a defect in DNA damage checkpoints that regulate S-phase progression and mitotic entry. Moreover, this defect maps downstream of Chk1/Chk2 regulation. MIF is required for DNA-damage-induced Cdc25A degradation In support of the latter conclusion, total cellular levels of Chk1 and Chk2 were similar in unstressed B-lymphoma cells of both genotypes, as was the dynamics of their regulatory phosphorylation upon DNA damage (Figure 4A and Supplementary Figure 4A). The phosphorylation of histone H2AX, a downstream target of ATM/ATR regulation (Brown and Baltimore, 2003), was also similar between the two cell types, particularly in response to stalled replication (Supplementary Figure 4B). However, p53−/− and DKO lymphoma cells showed differences in the levels of proteins that regulate the activity of Cdk1/2 complexes, most noticeably Cdc25A. Thus, treatment with adriamycin, etoposide, or cisplatin caused a more rapid decline of Cdc25A levels in p53−/− than in DKO cells (Figures 4A and B). Likewise, DKO cells failed to rapidly downregulate Cdc25A in response to serum deprivation or cell-cycle inhibitors like aphidicolin, hydroxyurea or nocodazole (Figure 4A–C and Supplementary Figures 4A and C). In consequence, the levels of Cdk2 were markedly increased in DKO lymphoma cells (Figure 4A and B). On the other hand, phosphorylation of Cdk2 on its inhibitory Tyr15 site was not detectably altered between the two genotypes (data not shown), suggesting a net increase in Cdk2 activity. Comparison of independently derived B-lymphoma lines of each genotype confirmed that overall, MIF-deficient tumor cells, while exhibiting some clonal variation, had elevated Cdc25A and Cdk2 levels when exposed to genotoxic drugs (Supplementary Figures 4D and E). To rule out the possibility that DKO cells have adapted the loss of MIF by complementing mutations, we reintroduced MIF into DKO B-lymphoma cells by retroviral infection. Reconstitution of MIF expression in DKO cells accelerated DNA damage-induced degradation of Cdc25A (Figure 4D). Figure 4.MIF is required for DNA-damage-induced Cdc25A degradation. (A) Immunoblot analysis of whole-cell lysates of p53−/− (line B5) and DKO B-lymphoma cells (line 399) treated with adriamycin or hydroxyurea for the indicated hours. Jab1 is a loading control. (B) Immunoblot analysis of Cdc25A and Cdk2 expression in p53−/− and DKO cells exposed for 6 h to serum deprivation or indicated cell-cycle inhibitors. (C) Induction of Cdc25A expression in total lysates from p53−/− and DKO cells that were maintained in serum-free media for 12 h before serum stimulation for the indicated hours. Erk1/ 2 is the loading control. (D) Immunoblot analysis of DKO cells (line 399) transduced with MIF-expressing retroviruses, and then challenged for 2 h with the indicated drugs. (E) Immunoblot analysis of p53−/− and DKO cells preincubated for 2 h with MG132, ALLN, caffeine, UCN-01 or Chk2 inhibitor II before treatment with hydroxyurea for 6 h. (F) The proteasomal activity of whole-cell lysates of p53−/− and DKO B-lymphoma cells treated with adriamycin or hydroxyurea was determined by the cleavage of Z-LRGG-AMC, a substrate for isopeptidase T and ubiquitin C-terminal hydrolaze activity. For controls, cell lysates were incubated in the presence of 10 μM MG132 to determine the nonspecific activity, which was subtracted from each measurement. The results represent an average of three experiments. (G) In vitro kinase assays performed on p53−/− and DKO B-lymphoma cells exposed to adriamycin for the indicated hours. Error bars represent the standard deviation. Download figure Download PowerPoint Importantly, RT–PCR analysis of Cdc25A expression failed to detect any MIF-dependent transcriptional differences between p53−/− and DKO cells (data not shown), suggesting a post-transcriptional cause. Cdc25A protein levels are tightly controlled by the ubiquitin-dependent proteasomal degradation. The anaphase-promoting/cyclosome complex (APC/C) mediates degradation of Cdc25A starting at the end of mitosis throughout the G1 phase, whereas the Skp–Cullin–F-box (SCF) ubiquitin E3 ligase complex plays a critical role in Cdc25A degradation in response to DNA damage (Donzelli and Draetta, 2003; Busino et al, 2004). Consistent with MIF-dependent mode of degradation, proteasomal inhibitors MG132 and ALLN prevented degradation of Cdc25A equally well in both p53−/− and DKO cells exposed to S-phase block or adriamycin treatment (Figure 4E and Supplementary Figure 4F). Moreover, exposure of p53−/− B-lymphoma cells to caffeine or UCN-01 also stabilized Cdc25A levels to those observed in untreated DKO cells (Figure 4E and Supplementary Figure 4F). These results imply that MIF loss uncouples Chk1/Chk2 checkpoints from proteasomal degradation of cell-cycle regulators such as Cdc25A. In support, in vitro assays demonstrated that chymotryptic and ubiquitin C-terminal hydrolaze proteasome activities were notably lower in DKO than p53−/− B-lymphoma cells exposed to stress conditions (Figure 4F and Supplementary Figure 4G). Concordant with deregulated Cdc25A and Cdk2 levels, cyclin A levels were also consistently higher in DKO than p53−/− lymphoma cells (Supplementary Figure 4B). Accordingly, the cyclin A-associated kinase activity was markedly increased in DKO lymphoma cells, particularly upon induction of DNA damage or S-phase block (Figure 4G and Supplementary Figure 4H). A similar pattern, albeit on a lower magnitude, was observed in primary splenocytes from premalignant p53−/− and DKO mice (data not shown). Collectively, these data indicate that DKO B-lymphoma cells exhibit constitutive deregulation of cyclin A-Cdk activity, enabling DNA replication under conditions of DNA damage. MIF loss impairs Cdk2-dependent degradation of DP1 We recently showed that MIF-deficient cells are hypersensitive to E2F1 upregulation (Petrenko and Moll, 2005; Talos et al, 2005). Given that after completion of S phase E2F1 is targeted for SCF-dependent degradation (Marti et al, 1999), we reasoned that its unscheduled presence could promote the growth of DKO lymphoma cells with damaged templates, negating the effect of checkpoint activation and rendering cells more susceptible to apoptosis. Indeed, although phosphorylation by ATM/ATR or Chk2 can stabilize E2F1 (Lin et al, 2001; Stevens et al, 2003), the exposure of DKO cells to genotoxic drugs or cell-cycle inhibitors caused higher E2F1 accumulation than in the p53−/− controls (Figure 5A and Supplementary Figure 5A). The expression of Rb family proteins was also altered in DKO lymphoma cells. Whereas p53−/− cells exposed to DNA damage accumulated the growth-inhibitory p130, the DKO cells showed continuous expression of S-phase marker p107 (Supplementary Figure 5B). This coincided with a more robust expression of pro-apoptotic TAp73 (Supplementary Figure 5A), suggesting that MIF loss is associated with an increased E2F1 transcriptional activity. Figure 5.DNA damage and stalled replication induce coordinated activity of Chk1 and the SCF complex. (A) Immunoblot analysis of E2F1 and DP1 expression in p53−/− and DKO B-lymphoma cells exposed for 6 h to the indicated inhibitors. (B) Immunoblot analysis of DP1 expression in p53−/− and DKO cells treated for 6 h with adriamycin in the presence of Cdk inhibitor NU6102 or Cdc25 inhibitors BN2002 and NSC663284. Cdk4 is a loading control. (C) Immunoblot analysis of DP1 expression in p53−/− and DKO cells preincubated for 2 h with MG132, caffeine, UCN-01 or Chk2 inhibitor II before 6-h treatment with hydroxyurea. (D) p53−/− and DKO cells were treated with adriamycin or hydroxyurea for 2 h. Lysates were immunoprecipitated with Cul1-specific Abs, followed by probing with the indicated Abs. (E) p53−/− and DKO cells were treated with hydroxyurea for the indicated hours, followed by immunoprecipitation performed as in (D). (F) p53−/− and DKO cells were labeled for 2 h with 32P orthophosphate in the absence or presence of hydroxyurea, 50 nM UCN-01, or 2 nM NU6102. Cul1 protein was immunoprecipitated and its phosphorylation was detected by autoradiography. Four-hour (top) and overnight exposures (bottom) are shown. Download figure Download PowerPoint E2F factors bind to DNA primarily as heterodimers with DP1 (DeGregori, 2002; Trimarchi and Lees, 2002). Immunoblot analysis revealed a remarkable correlation between imposed cell-cycle arrest and the induction of DP1 degradation in p53−/− B-lymphoma cells (Figure 5A). By contrast, no such correlation was apparent in DKO B-lymphoma cells (Figure 5A). Given that cyclin A–Cdk2 activity is constitutively higher in DKO than p53−/− tumor cells (Figure 4F), this result was counterintuitive. Thus, previous work identified DP1 as a physiological substrate of Cdk2 regulation. As cells progress from G1 into S phase, E2F1 forms stable complexes with cyclin A–Cdk2, which in turn phosphorylates E2F1-bound DP1. This suppresses the DNA-binding activity of E2F1 and triggers SCF-mediated degradation of both E2F1 and DP1 (Krek et al,
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