Small ubiquitin-like modifier (SUMO) modification has emerged as an important posttranslational control of protein functions. Daxx, a transcriptional corepressor, was reported to repress the transcriptional potential of several transcription factors and target to PML oncogenic domains (PODs) via SUMO-dependent interactions. The mechanism by which Daxx binds to sumoylated factors mediating transcriptional and subnuclear compartmental regulation remains unclear. Here, we define a SUMO-interacting motif (SIM) within Daxx and show it to be crucial for targeting Daxx to PODs and for transrepression of several sumoylated transcription factors, including glucocorticoid receptor (GR). In addition, the capability of Daxx SIM to bind SUMO also controls Daxx sumoylation. We further demonstrate that arsenic trioxide-induced sumoylation of PML correlates with a change of endogenous Daxx partitioning from GR-regulated gene promoter to PODs and a relief of Daxx repression on GR target gene expression. Our results provide mechanistic insights into Daxx in SUMO-dependent transcriptional control and subnuclear compartmentalization.

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor a (RARa) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RARa, which is able to form a heterodimer with PML. It was hypothesized that PML-RARa is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RARa, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RARoa resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RARot resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RARao, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.Acute promyelocytic leukemia (APL) represents a clonal proliferation and expansion of the hematologic precursors at the promyelocyte stage of myeloid differentiation. A nonrandom chromosomal translocation, t(15;17), can be found in over 95% of patients with APL (3, 42), suggesting that this translocation plays a critical role in leukemogenesis. In recent attempts to explore this role further, genes involved in this translocation have been cloned and characterized (4,7,17,37,48). The t(15;17) breakpoint occurs within the second intron of the retinoic acid (RA) receptor ox (RARox) gene and within two major sites of the PML (or MYL) gene (1,8,10,28,47 (VDR) to its target sequence and prevent vitamin D3-dependent activation of VDR-responsive genes. Furthermore, it was shown that transfection of PML-RARot into U937 human myeloid leukemia cells inhibits its ability to respond to vitamin D3 and transforming growth factor 13 and so induce differentiation. These results strongly suggest that the fusion protein encoded from the breakpoint of the t(15;17) translocation plays a critical role in APL pathogenesis. Recently, Doucas et al. (19) reported that PML-RARot, instead of being an inhibitor, is an RA-dependent activator of the transcription factor AP-1. However, since, as Kastner et al. (38)

Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.

This study aimed to investigate the role of AR-V7 in development of castration-resistant prostate cancer (CRPC) and to determine whether the AR-V7 expression in CRPC tissues can predict cancer-specific survival. We enrolled 100 localized prostate cancer (PCa) (cohort 1), 104 newly diagnosed metastatic PCa (cohort 2), and 46 CRPC (cohort 3) patients treated at our institution. The expression of AR-V7 in PCa was assessed by immunohistochemistry. Cox regression models were used to evaluate the predictive role of all covariates for the development of CRPC in cohort 2 and for cancer-specific survival in cohort 3. Time to CRPC and cancer-specific survival curves were estimated using the Kaplan-Meier method. AR-V7 expression rate in cohort 3 was significantly elevated compared with other two cohorts (p < 0.001). Multivariate analysis revealed that AR-V7 was an independent predictive factor for CRPC development (HR = 2.627, p = 0.001) and for cancer specific survival (HR = 2.247, p = 0.033). Furthermore, the AR-V7 expression was associated with shorter survival in CRPC patients. Our results demonstrated protein AR-V7 levels in primary tumors can be used as a predictive marker for the development of CRPC and as a prognostic factor in CRPC patients. Therapy targeting AR-V7 may help prevent PCa progression and improve the prognosis of CRPC patients.

The promyelocytic leukemia gene (PML) involved in the t(15;17) (q22;q12) translocation in acute promyelocytic leukemia is a growth suppressor. To elucidate the functional domains of PML, several mutants lacking the nuclear localization signal (PMLnls-), the dimerization domain (PMLdim-), the proline-rich domain at the N-terminal (PMLpro-), the proline-rich RING finger motif (PMLpr-), the proline-rich RING finger B-box-1 (PML-prb-), the serine-proline-rich domain at the C-terminal (PMLsp-), and the double mutant (PMLprb-nls-) have been constructed. Immunofluorescence staining of transiently transfected NIH3T3 cells demonstrated that the RING finger motif, dimerization domain, and nuclear localization signal are all required for the formation of PML oncogenic domains (PODs). Immunofluorescence staining of transiently transfected GM637D human fibroblasts indicated that expression of PMLprb-, PM-Lnls-, and PMLprb-nls- led to a significant reduction or, in some cases, complete elimination of PODs. PMLdim-, PMLnls-, PMLpr-, PMLprb-, and PMLprb-nls- mutants were found to lose their ability to suppress transformation of NIH3T3 cells by activated neu, while PMLpro- and PMLsp- mutants did not. These results suggest that the ability of PML to form a POD is essential for suppression of growth and transformation. Furthermore, since PMLprb-, PMLnls-, and PMLprb-nls- mutants could block the suppression effect of wild-type PML on transformation of NIH3T3 cells by the neu oncogene, these PML mutants are potential dominant negative inhibitors of PML. Our study also suggests that the RING finger motif may interact with other nuclear proteins.

By means of the polymerase chain reaction (PCR) technique, DNA sequences were amplified that flank the crossover sites of a characteristic chromosomal translocation for follicular lymphomas, t(14;18)(q32;q21). This technique permitted the detection of cells carrying the t(14;18) hybrid DNA sequences at a dilution of 1:100,000. The remission marrow and blood samples of a patient with follicular lymphoma and the t(14;18) failed to show any abnormality by morphological examination and conventional Southern blot analysis. However, the t(14;18) hybrid DNA sequences were detected by the PCR technique. Thus, this technique is a highly sensitive tool to detect minimal residual cells carrying the t(14;18) and has the potential to identify a subpopulation of patients with subclinical disease.

Our previous studies demonstrated that the promyelocytic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing e ect in breast cancer cells were examined. We showed that Ad-PML e ectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h postinfection and a detectable level remained at day 16. Ad-PML signi®cantly suppressed the growth rate, clonogenicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the e ect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a signi®cant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphorylated 48 h post-infection. These ®ndings indicate that PML exerts its growth suppressing e ects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppressing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy.

Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxideinduced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation.