Impaired DNA damage checkpoint response in MIF-deficient mice

Abstract

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.

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.

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.

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.

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.

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.

Figure 6

The genomic instability of DKO B-lymphoma cells is partially counteracted by E2F-dependent apoptosis. (A) Immunoprecipitation of E2F–DP1 complexes from p53−/− cells (lines B5 and B8) and DKO cells (lines 399 and 928) treated for 6 h with adriamycin or hydroxyurea. (B) ChIP analysis of DNA binding by E2F1 in p53−/− cells (line B5) and DKO cells (line 399). Immunoprecipitations with E2F1-specific Abs, followed by PCR amplification with primers specific for endogenous replication origin (Ori). Neo-specific primers were used to control for equal DNA input. (C) Apoptosis induction in DB–E2F1-expressing p53−/− and DKO cells exposed for 12 h to the indicated drugs. Error bars represent the average of two experiments. Inset, immunoblot analysis of wild-type E2F1 and DB-E2F1 expression in p53−/− (line B5) and DKO cells (lines 399 and 928) transduced with the corresponding retroviruses. Cdk4 is a loading control. (D) Apoptosis induction in normal splenocytes from mice of the indicated genotypes before and after adriamycin treatment for 12 h. Error bars represent the average of two experiments.

Figure 7

A proposed model of MIF involvement in Jab1/CSN5 regulation and G2M checkpoint control. MIF interacts with Jab1 and thereby promotes SCF-mediated proteolysis in vivo.