Novel MYC-driven medulloblastoma models from multiple embryonic cerebellar cells.

Group3 medulloblastoma (MBG3) that predominantly occur in young children are usually associated with MYC amplification and/or overexpression, frequent metastasis and a dismal prognosis. Physiologically relevant MBG3 models are currently lacking, making inferences related to their cellular origin thus far limited. Using in utero electroporation, we here report that MBG3 mouse models can be developed in situ from different multipotent embryonic cerebellar progenitor cells via conditional expression of Myc and loss of Trp53 function in several Cre driver mouse lines. The Blbp-Cre driver that targets embryonic neural progenitors induced tumors exhibiting a large-cell/anaplastic histopathology adjacent to the fourth ventricle, recapitulating human MBG3. Enforced co-expression of luciferase together with Myc and a dominant-negative form of Trp53 revealed that GABAergic neuronal progenitors as well as cerebellar granule cells give rise to MBG3 with their distinct growth kinetics. Cross-species gene expression analysis revealed that these novel MBG3 models shared molecular characteristics with human MBG3, irrespective of their cellular origin. We here developed MBG3 mouse models in their physiological environment and we show that oncogenic insults drive this MB subgroup in different cerebellar lineages rather than in a specific cell of origin.

Cross-species epigenetics identifies a critical role for VAV1 in SHH subgroup medulloblastoma maintenance.

The identification of key tumorigenic events in Sonic Hedgehog (SHH) subgroup medulloblastomas (MBSHH) will be essential for the development of individualized therapies and improved outcomes. However, beyond confirmation of characteristic SHH pathway mutations, recent genome-wide sequencing studies have not revealed commonly mutated genes with widespread relevance as potential therapeutic targets. We therefore examined any role for epigenetic DNA methylation events in MBSHH using a cross-species approach to candidate identification, prioritization and validation. MBSHH-associated DNA methylation events were first identified in 216 subgrouped human medulloblastomas (50 MBSHH, 28 Wnt/Wingless, 44 Group 3 and 94 Group 4) and their conservation then assessed in tumors arising from four independent murine models of Shh medulloblastoma, alongside any role in tumorigenesis using functional assessments in mouse and human models. This strategy identified widespread regional CpG hypo-methylation of VAV1, leading to its elevated expression, as a conserved aberrant epigenetic event, which characterizes the majority of MBSHH tumors in both species, and is associated with a poor outcome in MBSHH patients. Moreover, direct modulation of VAV1 in mouse and human models revealed a critical role in tumor maintenance, and its abrogation markedly reduced medulloblastoma growth. Further, Vav1 activity regulated granule neuron precursor germinal zone exit and migration initiation in an ex vivo model of early postnatal cerebellar development. These findings establish VAV1 as a critical epigenetically regulated oncogene with a key role in MBSHH maintenance, and highlight its potential as a validated therapeutic target and prognostic biomarker for the improved therapy of medulloblastoma.

Binding of carbon nanotube to BMP receptor 2 enhances cell differentiation and inhibits apoptosis via regulating bHLH transcription factors.

Biomaterials that can drive stem cells to an appropriate differentiation level and decrease apoptosis of transplanted cells are needed in regenerative medicine. Nanomaterials are promising novel materials for such applications. Here we reported that carboxylated multiwalled carbon nanotube (MWCNT 1) promotes myogenic differentiation of mouse myoblast cells and inhibits cell apoptosis under the differentiation conditions by regulating basic helix-loop-helix transcription factors. MWCNT 1 attenuates bone morphogenetic protein receptor (BMPR) signaling activity by binding to BMPR2 and attenuating the phosphorylation of BMPR1. This molecular understanding allowed us to tune stem cell differentiation to various levels by chemical modifications, demonstrating human control of biological activities of nanoparticles and opening an avenue for potential applications of nanomaterials in regenerative medicine.

Ink4c is dispensable for tumor suppression in Myc-induced B-cell lymphomagenesis.

p18(Ink4c) functions as a dedicated inhibitor of cyclin-D-dependent kinases. Loss of Ink4c predisposes mice to tumor development and, in a dose-dependent manner, complements the tumor-promoting effects of various oncogenes. We have now addressed whether Ink4c loss impacts B-cell tumor development in the Emu-Myc transgenic mouse, a model of human Burkitt lymphoma. Loss of one or both alleles did not influence the onset of lymphoma in Emu-Myc transgenics, and did not appreciably affect Myc's proliferative or apoptotic responses in precancerous B cells. Nevertheless, Ink4c loss modulated the effects of Myc-induced transformation by decreasing the frequency of Arf loss, an ordinarily common event in Emu-Myc-induced lymphomas.

p53-Dependent and -independent functions of the Arf tumor suppressor.

The Ink4a-Arf locus encodes two closely wedded tumor suppressor proteins (p16(Ink4a) and p19(Arf)) that inhibit cell proliferation by activating Rb and p53, respectively. With few exceptions, the Arf gene is repressed during mouse embryonic development, thereby helping to limit p53 expression during organogenesis. However, in adult mice, sustained hyperproliferative signals conveyed by somatically activated oncogenes can induce Arf gene expression and trigger a p53 response that eliminates incipient cancer cells. Disruption of this tumor surveillance pathway predisposes to cancer, and inactivation of INK4a- ARF by deletion, silencing, or mutation has been frequently observed in many forms of human cancer. Although it is accepted that much of Arf's tumor-suppressive activity is mediated by p53, more recent genetic evidence has pointed to additional p53- independent functions of Arf, including its ability to inhibit gene expression by a number of other transcription factors. Surprisingly, the enforced expression of Arf in mammalian cells promotes the sumoylation of several Arf-interacting proteins, implying that Arf has an associated catalytic activity. We speculate that transcriptional down-regulation in response to Arf-induced sumoylation may account for Arf's p53-independent functions.

Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis.

The ARF and p53 tumor suppressors mediate Myc-induced apoptosis and suppress lymphoma development in E mu-myc transgenic mice. Here we report that the proapoptotic Bcl-2 family member Bax also mediates apoptosis triggered by Myc and inhibits Myc-induced lymphomagenesis. Bax-deficient primary pre-B cells are resistant to the apoptotic effects of Myc, and Bax loss accelerates lymphoma development in E mu-myc transgenics in a dose-dependent fashion. Eighty percent of lymphomas arising in wild-type E mu-myc transgenics have alterations in the ARF-Mdm2-p53 tumor suppressor pathway characterized by deletions in ARF, mutations or deletions of p53, and overexpression of Mdm2. The absence of Bax did not alter the frequency of biallelic deletion of ARF in lymphomas arising in E mu-myc transgenic mice or the rate of tumorigenesis in ARF-null mice. Furthermore, Mdm2 was overexpressed at the same frequency in lymphomas irrespective of Bax status, suggesting that Bax resides in a pathway separate from ARF and Mdm2. Strikingly, lymphomas from Bax-null E mu-myc transgenics lacked p53 alterations, whereas 27% of the tumors in Bax(+/-) E mu-myc transgenic mice contained p53 mutations or deletions. Thus, the loss of Bax eliminates the selection of p53 mutations and deletions, but not ARF deletions or Mdm2 overexpression, during Myc-induced tumorigenesis, formally demonstrating that Myc-induced apoptotic signals through ARF/Mdm2 and p53 must bifurcate: p53 signals through Bax, whereas this is not necessarily the case for ARF and Mdm2.

Differential effects of p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages.

Establishment of cell lines from primary mouse embryo fibroblasts depends on loss of either the Arf tumor suppressor or its downstream target, the p53 transcription factor. Mouse p19(Arf) is encoded by the Ink4a-Arf locus, which also specifies a second tumor suppressor protein, the cyclin D-dependent kinase inhibitor p16(Ink4a). We surveyed bone marrow-derived cells from wild-type, Ink4a-Arf-null, or Arf-null mice for their ability to bypass senescence during continuous passage in culture. Unlike preB cells from wild-type mice, those from mice lacking Arf alone could be propagated indefinitely when placed onto stromal feeder layers engineered to produce IL-7. The preB cell lines remained diploid and IL-7-dependent and continued to express elevated levels of p16(Ink4a). By contrast, Arf-null bone marrow-derived macrophages that depend on colony-stimulating factor-1 for proliferation and survival in culture initially grew at a slow rate but gave rise to rapidly and continuously growing, but still growth factor-dependent, variants that ceased to express p16(Ink4a). Wild-type bone marrow-derived macrophages initially expressed both p16(Ink4a) and p19(Arf) but exhibited an extended life span when p16(Ink4a) expression was extinguished. In all cases, gene silencing was accompanied by methylation of the Ink4a promoter. Therefore, whereas Arf loss alone appears to be the major determinant of establishment of murine fibroblast and preB cell lines in culture, p16(Ink4a) provides an effective barrier to immortalization of bone marrow-derived macrophages.

Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukemic cells.

Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT+) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT+ versus the MOCK cell lines (P < 0.001). TPMT+ cells were less sensitive to thioguanine than MOCK cells (IC(50) = 1.10+/- 0.12 microM versus 0.55 +/- 0.19 microM; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC(50) = 0.52 +/- 0.20 microM versus 1.50 +/- 0.23 microM; P < 0.01). The lower sensitivity of TPMT+ versus MOCK cells to thioguanine was associated with lower thioguanine nucleotide concentrations (917 +/- 282 versus 1515 +/- 183 pmol/5 x 10(6) cells; P = 0.01), higher methylthioguanine nucleotide concentrations (252 +/- 34 versus 27 +/- 10 pmol/5 x 10(6) cells; P = 0.01), less inhibition of de novo purine synthesis (13 versus 95%; P < 0.01), and lower deoxythioguanosine incorporation into DNA (2.0 +/- 0.6% versus 7.2 +/- 2.0%; P < 0.001). The higher sensitivity of TPMT+ cells to mercaptopurine was associated with higher concentrations of methylmercaptopurine nucleotide (2601 +/- 1055 versus 174 +/- 77 pmol/5 x 10(6) cells; P = 0.01) and greater inhibition of de novo purine synthesis (>99% versus 74%; P < 0.01) compared with MOCK cells. We conclude that methylation of mercaptopurine contributes to the antiproliferative properties of the drug, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotides, whereas thioguanine is inactivated primarily by TPMT.

Apoptosis triggered by Myc-induced suppression of Bcl-X(L) or Bcl-2 is bypassed during lymphomagenesis.

Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively suppresses the expression of the Bcl-X(L) or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-X(L) RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-X(L) and Bcl-2 expression is inhibited by Myc in precancerous B cells from Emu-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-X(L) by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-X(L) levels are markedly elevated in over one-half of all lymphomas arising in Emicro-myc transgenic mice. Bcl-2 and/or Bcl-X(L) overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-X(L) or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.

c-Myc-mediated regulation of telomerase activity is disabled in immortalized cells.

Myc overexpression is a hallmark of human cancer and promotes transformation by facilitating immortalization. This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), as ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells. Paradoxically, Myc overexpression also triggers the ARF-p53 apoptotic program, which is activated when MEFs undergo replicative crises following culture ex vivo. The rare immortal variants that arise from these cultures generally suffer mutations in p53 or delete Ink4a/ARF, and Myc greatly increases the frequency of these events. Alternative reading frame (ARF)- and p53-null MEFs have increased telomerase activity, as do variant immortal clones that bypass replicative crisis. Similarly, immortal murine NIH-3T3 fibroblasts and myeloid 32D.3 and FDC-P1.2 cells do not express ARF and have robust telomerase activity. However, Myc overexpression in these immortal cells results in remarkably discordant regulation of TERT and telomerase activity. Furthermore, in MEFs and 32D.3 cells TERT expression and telomerase activity are regulated independently of endogenous c-Myc. Thus, the regulation of TERT and telomerase activity is complex and is also regulated by factors other than Myc, ARF, or p53.

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

Male mice lacking both the Ink4c and Ink4d genes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18(Ink4c) and p19(Ink4d) are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking both Ink4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lacking Ink4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast, Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed in Ink4c/Ink4d double-null males. Our data indicate that p18(Ink4c) and p19(Ink4d) are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

The MN1-TEL fusion protein, encoded by the translocation (12;22)(p13;q11) in myeloid leukemia, is a transcription factor with transforming activity.

The Tel gene (or ETV6) is the target of the translocation (12;22)(p13;q11) in myeloid leukemia. TEL is a member of the ETS family of transcription factors and contains the pointed protein interaction (PNT) domain and an ETS DNA binding domain (DBD). By contrast to other chimeric proteins that contain TEL's PNT domain, such as TEL-platelet-derived growth factor beta receptor in t(5;12)(q33;p13), MN1-TEL contains the DBD of TEL. The N-terminal MN1 moiety is rich in proline residues and contains two polyglutamine stretches, suggesting that MN1-TEL may act as a deregulated transcription factor. We now show that MN1-TEL type I, unlike TEL and MN1, transforms NIH 3T3 cells. The transforming potential depends on both N-terminal MN1 sequences and a functional TEL DBD. Furthermore, we demonstrate that MN1 has transcription activity and that MN1-TEL acts as a chimeric transcription factor on the Moloney sarcoma virus long terminal repeat and a synthetic promoter containing TEL binding sites. The transactivating capacity of MN1-TEL depended on both the DBD of TEL and sequences in MN1. MN1-TEL contributes to leukemogenesis by a mechanism distinct from that of other chimeric proteins containing TEL.

p53-independent functions of the p19(ARF) tumor suppressor.

The p19(ARF) tumor suppressor antagonizes Mdm2 to induce p53-dependent cell cycle arrest. Individual TKO (triple knock out) mice nullizygous for ARF, p53, and Mdm2 develop multiple tumors at a frequency greater than those observed in animals lacking both p53 and Mdm2 or p53 alone, demonstrating that p19(ARF) can act independently of the Mdm2-p53 axis in tumor surveillance. Reintroduction of ARF into TKO mouse embryo fibroblasts (MEFs), but not into those lacking both p53 and ARF, arrested the cell division cycle in the G1 phase. Inhibition of the retinoblastoma protein had no effect on the ability of ARF to arrest TKO MEFs. Thus, in the absence of Mdm2, p19(ARF) interacts with other targets to inhibit cell proliferation.

Stat5a/b contribute to interleukin 7-induced B-cell precursor expansion, but abl- and bcr/abl-induced transformation are independent of stat5.

The cytokines interleukin 7 (IL-7) and interleukin 4 (IL-4) regulate lymphoid differentiation and function and activate the transcription factor Stat5. Using mice deficient for the 2 highly related transcription factors, Stat5a and Stat5b (Stat5a/b(-/-)), we investigated the role of Stat5 for B-cell differentiation, expansion, and function. Peripheral blood B cells of Stat5-deficient mice are significantly reduced, but no proliferation defects in response to various mitogenic stimuli are found. Also, IgM and IgG1 antibody production and immunoglobulin class switching are not affected. Pre- and pro-B cells of Stat5-deficient animals were found to have reduced responses to IL-7. Pro- and pre-B cells are the target cells of the abl oncogene and numerous studies have suggested that Stat5a/b is essential for transformation by derivatives of the Abelson (abl) gene. To assess the role of Stat5a/b in transformation, we have evaluated the ability of various abl derivatives to transform cells from Stat5a/b-deficient mice in vitro or in vivo. We demonstrate that the absence of Stat5a/b is not essential for the induction of lymphoid or myeloid tumors in vivo or on the ability to transform bone marrow cells in vitro.

Expression of p19INK4d, CDK4, CDK6 in glioblastoma multiforme.

Deregulation of the G1/S checkpoint is a frequent event in the development of glioblastoma multiforme (GBM). Previous studies have shown more than 50% of primary GBM tumours contain either complete loss of the p16INK4a locus or amplification of the CDK4 gene. Moreover, many heterozygosity studies have shown deletion on human chromosome 19p13.2, where the p19INK4d gene has been localized. We examined the expression of p19INK4d and its two CDK substrates in a series of glioma-derived cell lines and tumours. No gene rearrangement or deletion was observed in the p19INK4d gene in these cell lines; however, expression of CDK4 and CDK6 was elevated relative to matched normal brain tissue in eight of 18 GBM tumours (44%). Furthermore, CDK6 expression level was increased in 12/14 glioblastomas, but undetectable in tumour samples of a previous lower grade tumour from the same patient. These data attest to the functional importance of both CDK4 and CDK6 in astrocytic tumourigenesis, particularly during the later stages of tumour progression.

Disruption of the ARF transcriptional activator DMP1 facilitates cell immortalization, Ras transformation, and tumorigenesis.

The DMP1 transcription factor induces the ARF tumor suppressor gene in mouse fibroblasts, leading to cell cycle arrest in a p53-dependent manner. We disrupted sequences encoding the DNA-binding domain of DMP1 in mouse embryonic stem cells and derived animals lacking the functional protein. DMP1-null animals are small at birth, and males develop more slowly than their wild-type littermates. Some adult animals exhibit seizures and/or obstuctive uropathy, each of unknown cause. The growth of explanted DMP1-null mouse embryo fibroblasts (MEFs) is progressively retarded as cells are passaged in culture on defined transfer protocols; but, unlike the behavior of normal cells, p19(ARF), Mdm2, and p53 levels remain relatively low and DMP1-null MEFs do not senesce. Whereas the establishment of cell lines from MEFs is usually always accompanied by either p53 or ARF loss of function, continuously passaged DMP1-null cells readily give rise to established 3T3 and 3T9 cell lines that retain wild-type ARF and functional p53 genes. Early-passage DMP1-null cells, like MEFs from either ARF-null or p53-null mice, can be morphologically transformed by oncogenic Ha-Ras (Val-12) alone. Splenic lymphocytes harvested from both DMP1-null and ARF-null mice exhibit enhanced proliferative responses in long-term cultures when stimulated to divide with antibody to CD3 and interleukin-2. Although only 1 of 40 DMP1-null animals spontaneously developed a tumor in the first year of life, neonatal treatment with dimethylbenzanthracene or ionizing radiation induced tumors of various histologic types that were not observed in similarly treated DMP1(+/+) animals. Karyotypic analyses of MEFs and lymphomas from DMP1-null animals revealed pseudodiploid chromosome numbers, consistent with the retention of wild-type p53. Together, these data suggest that ARF function is compromised, but not eliminated, in animals lacking functional DMP1.

Oncogenic Ras induces p19ARF and growth arrest in mouse embryo fibroblasts lacking p21Cip1 and p27Kip1 without activating cyclin D-dependent kinases.

Oncogenic Ras induces two products of the INK4a/ARF tumor suppressor locus (p16(INK4a) and p19(ARF)) in primary human and rodent fibroblasts, ultimately leading to a permanent state of cell cycle arrest resembling replicative senescence. Whereas p16(INK4a) antagonizes the activities of cyclin D-dependent kinases, p19(ARF) activates the p53 transcription factor. Immortalized rodent fibroblast cell lines that lack INK4a/ARF function, ARF alone, or p53 are resistant to the growth inhibitory effects of oncogenic Ras and instead continue to proliferate and undergo morphological transformation. Primary mouse embryo fibroblasts lacking Cip1 and Kip1 genes encoding inhibitors of cyclin-dependent kinase-2 were used to further explore the effects of oncogenic Ras on arrest of the cell division cycle. Although early passage primary fibroblast strains that lack both p21(Cip1) and p27(Kip1) fail to assemble cyclin D-dependent kinases, oncogenic Ras retained its ability to induce p19(ARF), but not p16(INK4a), protecting Cip/Kip-null cells from proliferating and undergoing transformation. Under these conditions, Ras did not induce G(1) phase arrest but instead triggered DNA synthesis, abnormal nuclear divisions, failure of cytokinesis, and emergence of polyploid cells. Therefore, in the absence of p16(INK4a), p21(Cip1), and p27(Kip1), oncogenic Ras affects the functions of genes required for completion of the cell cycle.

Stat3-dependent induction of p19INK4D by IL-10 contributes to inhibition of macrophage proliferation.

We have previously reported that IL-10 inhibits proliferation of normal bone marrow-derived macrophages and of the monocyte/macrophage cell line J774. Activation of Stat3 was shown to be necessary and sufficient to mediate inhibition of proliferation. To investigate further the mechanism of growth arrest, we examined the effect of IL-10 on expression of cell cycle inhibitors. We found that IL-10 treatment increases expression of the cyclin-dependent kinase inhibitors p19INK4D and p21CIP1 in macrophages. IL-10 cannot induce p19INK4D expression or block proliferation when Stat3 signaling is blocked by a dominant negative Stat3 or a mutant IL-10Ralpha which does not recruit Stat3 in J774 cells, whereas p21CIP1 induction is not affected. An inducibly active Stat3 (coumermycin-dimerizable Stat3-Gyrase B), which suppresses J774 cell proliferation, also induced p19INK4D expression. Sequencing of the murine p19INK4D promoter revealed two candidate Stat3 binding sites, and IL-10 treatment activated a reporter gene controlled by this promoter. These data suggest that Stat3-dependent induction of p19INK4D mediates inhibition of proliferation. Enforced expression of murine p19INK4D cDNA J774 cells significantly reduced their proliferation. Use of antisense p19INK4D and analysis of p19INK4D-deficient macrophages confirmed that p19INK4D is required for optimal inhibition of proliferation by IL-10, and indicated that additional IL-10 signaling events contribute to this response. These data indicate that Stat3-dependent induction of p19INK4D and Stat3-independent induction of p21CIP1 are important components of the mechanism by which IL-10 blocks proliferation in macrophages.

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex.

The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.