1. Volume 150, Issue 3, Pages 563-574 (August 2012)
Tristetraprolin Impairs Myc-Induced Lymphoma and Abolishes the Malignant State 
Robert J. Rounbehler, Mohammad Fallahi, Chunying Yang, Meredith A. Steeves, Weimin Li, Joanne R. Doherty, Franz X. Schaub, Sandhya Sanduja, Dan A. Dixon, Perry J. Blackshear, John L. Cleveland 
Cell 
Volume 150, Issue 3, Pages (August 2012)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Myc Alters the Expression of Hundreds of Genes Containing AREs
(A and B) Gene expression profiling of arrays showing genes from the ARED Organism database whose expression is specifically altered in B220+ B cells from premalignant Eμ-Myc (n = 5) and wild-type (n = 4) mice (A) and in Eμ-Myc lymphomas (n = 13) compared to premalignant Eμ-Myc B cells (B). All probe sets shown have a > 2.0-fold change and are significantly altered by unpaired t test analysis (p < 0.05).
(C) Gene expression profiling comparing ARED genes differentially expressed between 44 human BL and 129 human non-BL samples from GSE4475. All probe sets shown have >2.0-fold change and are significantly altered by unpaired t test analysis (p < 0.05).
See also Figure S1 and Tables S1–S3.
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4. Figure 2 Myc Alters the Expression of AUBPs
(A, B, and E) Gene expression profiling of arrays showing differentially expressed AUBP genes in B220+ B cells from premalignant Eμ-Myc versus wild-type littermates (A), Eμ-Myc lymphomas compared to premalignant Eμ-Myc B cells (B) and human BL and non-BL samples (E) from GSE4475. All probe sets shown have > 2.0-fold change and are significantly altered by unpaired t test analysis (p < 0.05).
(C and F) qRT-PCR analysis of total RNA isolated from B220+ B cells of wild-type and premalignant Eμ-Myc mice and lymphomas that arose in independent Eμ-Myc transgenics (C), and of RNA of CD19+ B cells from a healthy individual compared to human BL samples (F). The relative expression was determined for TTP (ZFP36), TIS11B (ZFP36L1), HUR, AUF1, AUF2, and NCL (Nucleolin). Individual samples are indicated by circles and the mean for each group is represented by a line. Results were normalized to the expression of Ubiquitin (Ub).
(D) Immunoblots comparing the levels of TTP, Actin, and Tubulin in B220+ B cells of wild-type and premalignant Eμ-Myc littermates.
Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S2.
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5. Figure 3 Myc Directly Represses TTP and TIS11B Transcription
(A) qRT-PCR analysis of total RNA isolated from primary wild-type B220+ B cells grown ex vivo in the presence of IL7. IL7 was removed at −18 hr and reintroduced at 0 hr and cells were collected at the indicated intervals. The relative expression was determined for c-Myc, TTP, and Tis11b. Results were normalized to Ubiquitin (Ub) expression.
(B) Expression of c-Myc, TTP, and Tis11b throughout mouse B cell (GSE15907) and T cell (GSE30631) development.
(C) Total RNA was isolated from P493-6 B cells treated with Tet for 72 hr to repress c-Myc expression. Cells were either washed to remove Tet (black lines with circles), allowing Myc activation, or were treated with β-E2 (red lines with squares), allowing proliferation via ER-EBNA2 activation. Cells were collected at the indicated intervals. qRT-PCR analysis was performed to determine the relative expression of c-Myc, TTP, TIS11B, HUR, AUF1, AUF2, and NCL, and results were normalized to Ubiquitin (Ub) expression.
(D and E) ChIP analyses establish that Myc binds to the initiator element (Inr) found in the promoters of TTP and TIS11B and that this is associated with reduced binding of RNA Pol II. Chromatin was immunoprecipitated with an α-c-Myc antibody from P493-6 cells with repressed c-Myc (+Tet) or following 8 hr of c-Myc induction (-Tet) (D), or with an α-RNA Pol II antibody from P493-6 cells when c-Myc was repressed by Tet (0 hr) or following c-Myc induction by removal of Tet (24 hr) (E). Bound chromatin was evaluated by qRT-PCR and compared to the total amount of chromatin. Results show the mean with error bars indicating ± SEM.
See also Figure S3.
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6. Figure 4 Generation of Eμ-TTP and Eμ-Tis11b Transgenic Mice
(A) Schematic of Eμ-TTP and Eμ-Tis11b transgenes. The complete coding region for mouse TTP or Tis11b, was cloned into the pEμSR plasmid downstream of the SRα promoter. The transgenes also include the mouse Igh 5′ enhancer (Eμ), the rabbit globin poly A and an SV40 tag region.
(B) qRT-PCR analysis of TTP or Tis11b expression in total RNA isolated from B220+ B cells from wild-type and Eμ-TTP littermates from two different founders (Line 1 and Line 2) or from Eμ-Tis11b littermates. Results were normalized to Ubiquitin (Ub) expression.
(C) Immunoblots analyses of TTP and Actin levels in bone marrow and splenic B220+ B cells of wild-type and Eμ-TTP littermates.
Results show the mean with error bars indicating ± SEM. See also Figure S4.
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7. Figure 5 TTP Suppresses Myc-driven Lymphomagenesis
(A) Survival curves of Eμ-Myc;Eμ-TTP-1 (n = 19), Eμ-Myc;Eμ-TTP-2 (n = 25), and Eμ-Myc;Eμ-Tis11b (n = 23) transgenic mice were compared to Eμ-Myc littermates (n = 16, 30 or 22, respectively). Tick marks above survival curve indicate mice that were still alive when the analysis was completed. The p values were determined by using Mantel-Cox log rank test.
(B) Immunoblots analyses of Myc and Actin levels in Eμ-Myc versus Eμ-Myc;Eμ-TTP-1 lymphomas.
(C) Splenic mass and numbers of B220+ B cells in the spleens of 5-week-old wild-type, Eμ-TTP-1, Eμ-Myc, and Eμ-Myc;Eμ-TTP-1 littermates.
(D and E) Analysis of TUNEL-positive (D) and BrdU+ (E) B220+ cells, separated based upon their IgM expression, from BM and spleen of 5-week-old wild-type, Eμ-TTP-1, Eμ-Myc, and Eμ-Myc;Eμ-TTP-1 mice.
Results show the mean with error bars indicating ± SEM. Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S5.
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8. Figure 6 TTP Impairs Maintenance of Eμ-Myc Lymphoma
(A and B) Nude mice were injected with unsorted Eμ-Myc lymphoma cells infected with MSCV-I-GFP versus MSCV-TTP-I-GFP retrovirus (left panel) or with Eμ-Myc lymphoma cells infected with MSCV-I-GFP versus MSCV-Tis11b-I-GFP retrovirus (right panel). Shown is analysis of the change in the percentage of GFP+ B cells found in tumors compared to the percentage of GFP+ lymphoma cells that were injected (A) and the percentage of GFP+ B cells found in peripheral blood (B) at the time of sacrifice. Individual samples are indicated by circles and the mean for each group is represented by a line.
(C) Percentage of GFP+ unsorted ex vivo Eμ-Myc lymphoma cells infected with MSCV-I-GFP versus MSCV-TTP-I-GFP retrovirus (left panel) or with MSCV-I-GFP versus MSCV-Tis11b-I-GFP retrovirus (right panel) was determined at the indicated intervals.
(D) Changes in cell cycle distribution of unsorted ex vivo Eμ-Myc lymphoma cells infected with MSCV-I-GFP versus MSCV-TTP-I-GFP retrovirus based upon the presence or absence of GFP within the lymphoma cells (i.e., GFP+ cells minus GFP-negative cells). Cells were labeled with BrdU, harvested and analyzed by FACS. The graph represents the average of three individually infected plates of Eμ-Myc lymphoma cells for each retrovirus.
(E) Survival curves of Nude mice injected with Eμ-Myc lymphoma cells sorted for GFP+ following infection with MSCV-I-GFP or MSCV-TTP-I-GFP retrovirus. P values were determined using the Mantel-Cox log rank test.
Results show the mean with error bars indicating ± SEM. Student's t test (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗∗p < ). See also Figure S6.
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9. Figure 7 TTP-Dependent Myc Targets Associated with Tumorigenesis
(A and E) Gene expression profiling of arrays showing ARED genes whose expression is specifically altered in B220+ BM B cells from premalignant Eμ-Myc versus Eμ-Myc;Eμ-TTP-1 mice (A) or all genes whose expression is specifically altered in lymphomas from Eμ-Myc versus Eμ-Myc;Eμ-TTP-1 mice (E). Genes labeled in (A) have known roles in cancer. All probe sets shown have >2.0-fold change and are significantly altered by unpaired t test analysis (p < 0.05).
(B and C) qRT-PCR analysis of total RNA isolated from B220+ BM B cells of wild-type and Eμ-TTP-1 transgenics and from premalignant Eμ-Myc and Eμ-Myc;Eμ-TTP-1 mice (B) or from wild-type B220+ splenic B cells and from Eμ-Myc and Eμ-Myc;Eμ-TTP-1 lymphomas (C). The relative expression of Ccnd1, Fstl1, Gabarapl1, Tes, Tns3, and Uaca is shown. Results were normalized to Ubiquitin (Ub) expression.
(D) qRT-PCR (top left panel) and immunoblot (top right panel) analyses show the mRNA and protein levels of TTP-Flag, Cyclin D1 and Actin in HeLa Tet-Off/TTP-Flag cells that were grown +Dox (repressed TTP-Flag) or −Dox (induced TTP-Flag) for 48 hr. For RNP-IP analyses (bottom panel), RNPs were immunoprecipitated with α-Flag antibody from control or TTP-Flag-expressing HeLa cells and used for qPCR detection of cyclin D1 mRNA.
Results show the mean with error bars indicating ± SEM. Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S7 and Tables S4–S7.
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10. Figure S1
ARED Gene Expression in Myc-driven Eμ-Myc Lymphoma and MYCN-amplified Neuroblastoma, Related to Figure 1
(A) Gene expression profiling of arrays showing those ARED genes that are induced or repressed in B220+ premalignant Eμ-Myc versus wild-type B cells (from Figure 1A), compared to their expression in malignant B cells of Eμ-Myc lymphomas. Note that most ARED genes repressed or induced in premalignant Eμ-Myc B cells have a similar profile in Eμ-Myc lymphoma, although the magnitude of the response is often amplified in tumors.
(B) The expression of genes from ARED Organism database (Halees et al., 2008) whose expression is specifically altered in primary human MYCN-amplified (n = 20) and MYCN nonamplified (n = 81) neuroblastoma samples from Gene Expression Omnibus Series GSE3960 (Wang et al., 2006). Probe sets shown have a > 2.0-fold change and are significantly altered by unpaired t test analysis (p < 0.05).
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11. Figure S2
The Expression of Select AUBPs is Altered in Malignancies with MYC Involvement, Related to Figure 2
(A and C) AUBP genes whose expression is specifically altered in primary human MYCN-amplified (n = 20) and MYCN nonamplified (n = 81) neuroblastoma samples (A) from Gene Expression Omnibus Series GSE3960 (Wang et al., 2006), and in normal colon (n = 17) and colorectal cancer (n = 77) (C) from Gene Expression Omnibus Series GSE18105 (Matsuyama et al., 2010) are shown. Probe sets shown have a > 1.5-fold change and are significantly altered by unpaired t test analysis (p < 0.05).
(B and D) Expression profiles of MYC and TTP in normal colon (n = 24) and colon adenocarcinoma (n = 45) (B) from GSE20916 (Skrzypczak et al., 2010), and in normal prostate (n = 3), prostate cancer (n = 23) and metastatic prostate cancer (n = 9) (D) from (LaTulippe et al., 2002) are shown. TTP probe sets shown have a > 1.5-fold change and are significantly altered by unpaired t test analysis (p < 0.05).
(E) Expression profiles of MYC and TTP in lymph-node-negative breast cancer (n = 286) from GSE2034 (Wang et al., 2005) are shown.
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12. Figure S3
Myc Directly Binds to TTP and TIS11B Inr Elements, Related to Figure 3
(A) ChIP analyses establish that Myc binds to the Initiator element (Inr) found in the promoters of TTP and TIS11B, but not to downstream control regions. 48 hr following c-Myc activation, chromatin from P493-6 cells was immunoprecipitated with either α-Myc or α-IgG antibody. Bound chromatin was evaluated by qRT-PCR and compared to the total amount of chromatin.
(B) ENCODE Analysis (Rosenbloom et al., 2010) was utilized to identify MYC-binding sites in the TTP and TIS11B genes based on ChIP and direct sequencing (ChIP-seq) in five human cancer cell lines, HeLa, K562 BO, K562 Sig, HepG2, and GM12878, and in HUVEC cells. ENCODE Transcription Factor Binding Sites by ChIP-seq from Yale tracks (Yale TFBS) were visualized.
(C) Primary transcript qRT-PCR analysis of nascent, unspliced TTP pre-mRNA from total RNA isolated from P493-6 B cells that were treated with Tet for 72 hr to repress c-Myc expression. Cells were washed to remove Tet and collected at the indicated intervals. Results were normalized to the expression of Ubiquitin (Ub).
Results show the mean with error bars indicating ± SEM.
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13. Figure S4
Enforced TTP Expression in B Cells Controls a Very Select Cast of ARED Genes, Related to Figure 4
Expression profiling of all genes whose expression is specifically altered in B220+ bone marrow B cells from Eμ-TTP-1 mice compared to wild-type littermates. Genes marked with (∗) are ARED genes. All probe sets shown have > 2.0-fold change and are significantly altered by unpaired t test analysis (p < 0.05). Note that the expression of only 16 genes is affected by enforced TTP expression in Eμ-TTP-1 B cells.
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14. Figure S5
Enforced TTP Expression Does Not Affect the Frequencies of p53 or Arf Alterations During Myc-induced Lymphomagenesis, Nor Myc Expression, Related to Figure 5
(A and D) Immunoblots comparing the levels of TTP and Actin (A) and of p53, p19Arf, and Actin (C) in Eμ-Myc versus Eμ-Myc;Eμ-TTP-1 lymphomas.
(B and C) qRT-PCR analysis of total RNA isolated from wild-type B220+ splenic B cells and from Eμ-Myc and Eμ-Myc;Eμ-TTP-1 lymphomas (B) or from B220+ bone marrow B cells of wild-type and Eμ-TTP-1 transgenics and from premalignant Eμ-Myc and Eμ-Myc;Eμ-TTP-1 mice (C) to determine the relative expression of c-Myc. Results were normalized to the expression of Ubiquitin (Ub).
(D) Southern blot analysis of Arf loss (as noted by asterisk∗) in Eμ-Myc and Eμ-Myc;Eμ-TTP-1 lymphomas.
(E) Immunoblots comparing the levels of Bim and Actin in B220+ bone marrow B cells of wild-type and Eμ-TTP-1 littermates.
Results show the mean with error bars indicating ± SEM.
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15. Figure S6
Gene Copy Analysis of the MSCV-TTP-I-GFP Retrovirus in Unsorted Eμ-Myc Lymphoma Transplants, Related to Figure 6
(A) Top: The percentage of GFP+ cells present in different tumors from Nude mice injected with unsorted Eμ-Myc lymphoma cells infected with either MSCV-I-GFP (top left) or MSCV-TTP-I-GFP (top right) retrovirus was determined by FACS. Bottom: The relative copy number of Myc (blue line with squares) from the Eμ-Myc transgene and of GFP (green lines with circles) from either MSCV-I-GFP (bottom left) or MSCV-TTP-I-GFP (bottom right) retrovirus from the same tumor samples as above, were compared to an arbitrarily chosen tumor sample (Tumor #1) and measured by qPCR.
(B) Immunoblots comparing the levels of Myc, TTP and Actin in different lymphomas from Nude mice injected with unsorted Eμ-Myc lymphoma cells infected with MSCV-I-GFP or with MSCV-TTP-I-GFP retrovirus. Lymphomas from the MSCV-TTP-I-GFP group are split into those that had a modest amount (∼25%) of GFP+ cells in the tumor and those that had undetectable levels of GFP by FACS.
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16. Figure S7
TTP-Dependent Targets in Wild-Type B cells and Effects of Acute TTP Expression in Eμ-Myc Lymphoma, Related to Figure 7
(A) qRT-PCR analysis of total RNA isolated from wild-type B220+ B cells grown ex vivo in the presence of IL7. IL7 was removed at −18 hr and reintroduced at 0 hr and cells were collected at the indicated intervals. The relative expression was determined for Ccnd1, Fstl1, Gabarapl1, Tes, Tns3 and Uaca. Results were normalized to the expression of Ubiquitin (Ub).
(B-C) Ex vivo Eμ-Myc lymphoma cells were infected with MSCV-I-dsRed2 or MSCV-D1a-I-dsRed2 retrovirus and dsRed2+ cells were sorted by FACS. These dsRed2+ lymphoma cells were then infected with either MSCV-I-GFP or MSCV-TTP-I-GFP retrovirus, and the percentage of GFP+ versus GFP-negative cells in the culture were determined by FACS analyses of GFP cells and cell counting. The fold change in the percentage of GFP+ at the indicated intervals following the second round of infection (B) or the changes in cell cycle distribution based upon the presence or absence of GFP within the lymphoma cells (i.e., GFP+ cells minus GFP-negative cells) (C) are shown. For cell cycle analysis, cells were harvested, labeled with DAPI and analyzed by FACS. Both graphs represent the average of three individual experiments where sorted Eμ-Myc-dsRed2+ or Eμ-Myc-D1a-dsRed2+ lymphoma cells were infected with either MSCV-I-GFP or MSCV-TTP-I-GFP retrovirus.
(D) Eμ-Myc lymphoma cells infected with MSCV-I-dsRed2 or MSCV-D1a-I-dsRed2 retrovirus shown in (B) were sorted by FACS for the presence of dsRed2, and then infected with MSCV-TTP-I-GFP retrovirus. After 24 hr cells were sorted for GFP presence (GFP+) or absence (GFP−). Total RNA was isolated from these cells and used for qRT-PCR analysis to determine the relative expression of endogenous Ccnd1 (Ccnd1 3′ UTR) or exogenous D1a (D1a-IRES). Results were normalized to the expression of Ubiquitin (Ub).
(E) Gene expression profiling showing genes that are induced or repressed in ex vivo Eμ-Myc lymphoma cells that were infected with MSCV-I-dsRed2 retrovirus, sorted by FACS for the presence of dsRed2, then infected with MSCV-TTP-I-GFP retrovirus and after 24 hr cells were sorted for GFP+ or GFP− cells. All probe sets shown have > 2.0-fold change and are significantly altered by unpaired t test analysis (p < 0.05).
(F) Model showing the biological processes, along with some of the ARED genes that are possibly involved, which are TTP dependent in Myc-induced lymphoma.
Results show the mean with error bars indicating ± SEM. Student's t test (∗p < 0.05, ∗∗p < 0.01).
Cell  , DOI: ( /j.cell )
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