The promyelocytic leukemia protein isoform PML1 is an oncoprotein and a direct target of the antioxidant sulforaphane (SFN)

The gene encoding promyelocytic leukemia protein (PML) generates several spliced isoforms. Ectopic expression of PML1 promotes the proliferation of ERα-positive MCF-7 breast cancer (BC) cells, while a loss of PML by knockdown or overexpression of PML4 does the opposite. PML is an essential constituent of highly dynamic particles called PML nuclear bodies (NBs). PML NBs are heterogenous multiprotein subnuclear structures that are part of cellular stress sensing machinery. The antioxidant sulforaphane (SFN) inhibits the proliferation of BC cells and causes a redistribution of the subcellular localization of PML, a disruption of disulfide-bond linkages in nuclear PML-containing complexes, and a reduction in the number and size of PML NBs. Mechanistically, SFN modifies several cysteine residues, including C204, located in the RBCC domain of PML. PML is sumoylated and contains a Sumo-interacting motif, and a significant fraction of Sumo1 and Sumo2/3 co-localizes with PML NBs. Ectopic expression of the mutant C204A selectively inhibits the biogenesis of endogenous PML NBs but not PML-less Sumo1-, Sumo2/3, or Daxx-containing nuclear speckles. Importantly, PML1 (C204A) functions as a dominant-negative mutant over endogenous PML protein and promotes anti-proliferation activity. Together, we conclude that SFN elicits its cytotoxic activity in part by inactivating PML1's pro-tumorigenic activity.     

PML2‐mediated thread‐like nuclear bodies mark late senescence in Hutchinson–Gilford progeria syndrome

Progerin accumulation disrupts nuclear lamina integrity and causes nuclear structure abnormalities, leading to premature aging, that is, Hutchinson–Gilford progeria syndrome (HGPS). The roles of nuclear subcompartments, such as PML nuclear bodies (PML NBs), in HGPS pathogenesis, are unclear. Here, we show that classical dot‐like PML NBs are reorganized into thread‐like structures in HGPS patient fibroblasts and their presence is associated with late stage of senescence. By co‐immunoprecipitation analysis, we show that farnesylated Progerin interacts with human PML2, which accounts for the formation of thread‐like PML NBs. Specifically, human PML2 but not PML1 overexpression in HGPS cells promotes PML thread development and accelerates senescence. Further immunofluorescence microscopy, immuno‐TRAP, and deep sequencing data suggest that these irregular PML NBs might promote senescence by perturbing NB‐associated DNA repair and gene expression in HGPS cells. These data identify irregular structures of PML NBs in senescent HGPS cells and support that the thread‐like PML NBs might be a novel, morphological, and functional biomarker of late senescence.     

PML与基因组稳定性

基因组稳定性同肿瘤的发生、发展密切相关,维护基因组稳定性对于细胞行使正常的生理功能是至关重要的.早幼粒细胞白血病蛋白PML(promyelocytic leukemia)主要借助分子中RBCC结构,同近50种有重要功能的蛋白相互作用而形成PML-NBs(PML nuclear bodies).PML-NBs是与核基质结合的、动态的、亚核多蛋白复合物,它作为区室化核结构(compartmentalized nuclear architecture)——染色质间区室(interchromatin compartment)的功能单位,满足了真核基因高层次表达调控模式的时空要求.最新的研究证明：PML是基因组稳定性“守门人”——p53分子的搭档分子,同样在基因组稳定性调控中发挥着重要的功能作用.它协同p53参与了DNA损伤反应所诱发的细胞凋亡,还可组织多种DNA修复分子参与DNA损伤修复,在DNA损伤反应中具有重要作用;此外,PML还通过调控aurora A的活性参与中心体复制检查点调控,借助调控survivin的表达参与有丝分裂纺锤体组装检查点调控,在染色体复制和细胞分裂中均显示了重要的调控作用.而当PML表达缺失或不足时则与多种肿瘤的发生、发展相关联,因此PML分子在维护基因组稳定性中具有重要功能作用,本文仅就相关的最新研究进展予以概述     

Disruption of PML nuclear bodies is mediated by ORF61 SUMO-interacting motifs and required for varicella-zoster virus pathogenesis in skin

Promyelocytic leukemia protein (PML) has antiviral functions and many viruses encode gene products that disrupt PML nuclear bodies (PML NBs). However, evidence of the relevance of PML NB modification for viral pathogenesis is limited and little is known about viral gene functions required for PML NB disruption in infected cells in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes cutaneous lesions during primary and recurrent infection. Here we show that VZV disrupts PML NBs in infected cells in human skin xenografts in SCID mice and that the disruption is achieved by open reading frame 61 (ORF61) protein via its SUMO-interacting motifs (SIMs). Three conserved SIMs mediated ORF61 binding to SUMO1 and were required for ORF61 association with and disruption of PML NBs. Mutation of the ORF61 SIMs in the VZV genome showed that these motifs were necessary for PML NB dispersal in VZV-infected cells in vitro. In vivo, PML NBs were highly abundant, especially in basal layer cells of uninfected skin, whereas their frequency was significantly decreased in VZV-infected cells. In contrast, mutation of the ORF61 SIMs reduced ORF61 association with PML NBs, most PML NBs remained intact and importantly, viral replication in skin was severely impaired. The ORF61 SIM mutant virus failed to cause the typical VZV lesions that penetrate across the basement membrane into the dermis and viral spread in the epidermis was limited. These experiments indicate that VZV pathogenesis in skin depends upon the ORF61-mediated disruption of PML NBs and that the ORF61 SUMO-binding function is necessary for this effect. More broadly, our study elucidates the importance of PML NBs for the innate control of a viral pathogen during infection of differentiated cells within their tissue microenvironment in vivo and the requirement for a viral protein with SUMO-binding capacity to counteract this intrinsic barrier.     

Cross talk between PML and p53 during poliovirus infection: implications for antiviral defense

PML nuclear bodies (NBs) are dynamic intranuclear structures harboring numerous transiently or permanently localized proteins. PML, the NBs' organizer, is directly induced by interferon, and its expression is critical for antiviral host defense. We describe herein the molecular events following poliovirus infection that lead to PML-dependent p53 activation and protection against virus infection. Poliovirus infection induces PML phosphorylation through the extracellular signal-regulated kinase pathway, increases PML SUMOylation, and induces its transfer from the nucleoplasm to the nuclear matrix. These events result in the recruitment of p53 to PML NBs, p53 phosphorylation on Ser15, and activation of p53 target genes leading to the induction of apoptosis. Moreover, the knock-down of p53 by small interfering RNA results in higher poliovirus replication, suggesting that p53 participates in antiviral defense. This effect, which requires the presence of PML, is transient since poliovirus targets p53 by inducing its degradation in a proteasome- and MDM2-dependent manner. Our results provide evidence of how poliovirus counteracts p53 antiviral activity by regulating PML and NBs, thus leading to p53 degradation.     

p53 and p21(Waf1) Are Recruited to Distinct PML‐Containing Nuclear Foci in Irradiated and Nutlin‐3a‐Treated U2OS Cells

Promyelocytic leukemia nuclear bodies (PML‐NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML‐NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53‐dependent growth inhibition. ALT‐associated PML bodies (APBs) are specialized PML‐NBs that include telomere‐repeat binding‐factor TRF1 and are exclusively in telomerase‐negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell‐cycle and p53 responses in ALT‐positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin‐3a. Both IR and Nutlin‐3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin‐3a. p53 was recruited to PML‐NBs 3–4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML‐NBs marked sites of apparently unrepaired DNA double‐strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin‐3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin‐3a‐treated cells. These data indicate (1) p53 is recruited to PML‐NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53–p21 pathway activation increases the percentage of APB‐positive cells, (3) p21 and p53 are recruited to ALT‐associated PML‐NBs after Nutlin‐3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance. J. Cell. Biochem. 111: 1280–1290, 2010. © 2010 Wiley‐Liss, Inc.