The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RAR␣ encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.The nonrandom chromosomal translocation t(15;17), a cytogenetic hallmark of acute promyelocytic leukemia (APL), fuses the retinoic acid receptor ␣ gene (RAR␣) and the promyelocytic leukemia gene (PML) (8,17,34). The fusion gene PML-RAR␣ encodes a fusion protein that has been shown to interfere with leukemia cell differentiation (25,26) and to cause leukemia in animal models (11,27,32,33). Disruption of PML's growth suppressor function in APL is also believed to play a role in leukemogenesis (51). PML is a nuclear-matrixassociated protein localized in the nucleus in a distinct nuclear speckled pattern designated the PML nuclear body (NB), which is disrupted in the leukemic blasts of APL (14,15,20,75). A significant number (Ͼ90%) of APL patients can be induced to complete clinical remission by high-dose all-transretinoic acid (ATRA) or arsenic trioxide (As 2 O 3 ) therapy (16,59,60,72,74). Retinoic acid (RA) treatment induces differentiation of the leukemic blasts, rapid degradation of the fusion protein PML-RAR␣, and restoration of a normal PML NB (20,75). Recent studies demonstrated that PML-RAR␣ recruits histone deacetylase (HDAC) by directly interacting with the N-CoR-Sin3 complex through the RAR␣ portion of the fusion protein, turning the fusion protein into a strong transcription repressor for RA-responsive genes. Treating APL cells with high-dose ATRA reverses the binding of PML-RAR␣ to the N-CoR-Sin3 corepressor complex and reactivates RA-responsive genes (24,32,45). PML belongs to a family of nuclear proteins consisting of the RING finger motif and two other Cys-His domains designated the B-box motif. The region following is the ␣-helical domain, which is responsible for dimerization (57). PML is the major component of this novel NB, and many proteins associated with PML have been identified. For example, the ubiquitin-like protein modifier SUMO-1 (PIC-1 or sentrin) (7,35,36,53,62), interferon-induced protein ISG20 (23), the im...

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.

The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein with growth-and transformationsuppressing ability. Having previously shown it to be a transcriptional repressor of the epidermal growth factor receptor (EGFR) gene promoter, we have now shown that PML's repression of EGFR transcription is caused by inhibition of EGFR's Sp1-dependent activity. On functional analysis, the repressive effect of PML was mapped to a 150-bp element (the sequences between ؊150 and ؊16, relative to the ATG initiation site) of the promoter. Transient transfection assays with Sp1-negative Drosophila melanogaster SL2 cells showed that the transcription of this region was regulated by Sp1 and that the Sp1-dependent activity of the promoter was suppressed by PML in a dose-dependent manner. Coimmunoprecipitation and mammalian two-hybrid assays demonstrated that PML and Sp1 were associated in vivo. In vitro binding by means of the glutathione S-transferase (GST) pull-down assay, using the full-length and truncated GST-Sp1 proteins and in vitrotranslated PML, showed that PML and Sp1 directly interacted and that the C-terminal (DNA-binding) region of Sp1 and the coiled-coil (dimerization) domain of PML were essential for this interaction. Analysis of the effects of PML on Sp1 DNA binding by electrophoretic mobility shift assay (EMSA) showed that PML could specifically disrupt the binding of Sp1 to DNA. Furthermore, cotransfection of PML specifically repressed Sp1, but not the E2F1-mediated activity of the dihydrofolate reductase promoter. Together, these data suggest that the association of PML and Sp1 represents a novel mechanism for negative regulation of EGFR and other Sp1 target promoters.The promyelocytic leukemia gene, PML, was first identified at the breakpoint of the t(15;17) translocation in acute promyelocytic leukemia (APL) (10,14,19,35,37). PML encodes a nuclear phosphoprotein that functions as a transcriptional regulator (9, 50, 58) and belongs to the RING family of proteins, which share a cysteine-rich motif at the N terminus. This motif is divided into a RING finger (C 3 C 4 zinc binding) motif and two B-box (B1 and B2) motifs (18). This region is followed by a predicted ␣-helical coiled-coil (dimerization) domain, which allows PML to homodimerize and form heterodimer complexes with the APL fusion protein PMLRAR␣ and the promyelocytic leukemia zinc finger (PLZF) protein (37,40). PML localizes to distinct domains in the nucleus called PML nuclear bodies, or PML oncogenic domains (PODs) (16,60). In addition to PML, there are several other POD-associated factors, including SP100, the ubiquitin-like protein PIC1, and the interferon-stimulated 20-kDa gene product called ISG20 (3,6,20). PODs are frequently targeted and/or reorganized by viral proteins, such as the herpes simplex virus type 1 (HSV-1) gene product Vmw110 (17), the adenoviral proteins E1A and E4-ORF3 (8), the Epstein-Barr virus-encoded nuclear antigen EBNA-5 (53), and the human cytomegalovirus major immediate-early proteins IE1 and IE2 (1).PMLRAR␣, which r...

Our previous studies demonstrated that PML is a growth suppressor that suppresses oncogenic transformation of NIH/3T3 cells and rat embryo fibroblasts. PML is a nuclear matrix-associated phosphoprotein whose expression is regulated during the cell cycle. Disruption of PML function by t(15;17) in acute promyelocytic leukemia (APL) plays a critical role in leukemogenesis. To further study the role of PML in the control of cell growth, we have stably overexpressed PML protein in the HeLa cell line. This overexpression of PML significantly reduced the growth rate of HeLa cells and suppressed anchorage-independent growth in soft agar. We consequently investigated several parameters correlated with cell growth and cell cycle progression. We found that, in comparison with the parental HeLa cells, HeLa/PML stable clones showed proportionally more cells in G1 phase, fewer cells in S phase and about the same number in G2/M phase. The HeLa/PML clones showed a significantly longer doubling time as a result of a lengthening of the G1 phase. No effect on apoptosis was found in HeLa cells overexpressing PML. This observation indicates that PML suppresses cell growth by increasing cell cycle duration as a result of G1 elongation. To further understand the mechanism of the effect of PML on HeLa cells, expression of cell cycle-related proteins in HeLa/PML and parental HeLa cells was analyzed. We found that Rb phosphorylation was significantly reduced in PML stable clones. Expression of cyclin E, Cdk2 and p27 proteins was also significantly reduced. These studies indicate that PML affects cell cycle progression by mediating expression of several key proteins that normally control cell cycle progression. These results further extend our current understanding of PML function in human cells and its important role in cell cycle regulation.

The distribution for collagen type VI is different from normal in the skin of trisomy 21 fetuses, and there is overexpression of COL6A1 compared with COL6A3.