Tyrosine kinase inhibitor, methyl 2,5-dihydromethylcinnimate, induces PML nuclear body formation and apoptosis in tumor cells

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs) are the nuclear structure consisting of various proteins such as PML, SUMO-1, and p53. PML-NBs are implicated in the regulation of tumor suppression, antiviral responses, and apoptosis. In this study, we searched for bioactive metabolites that would promote the formation of PML-NBs in tumor cells. As a result, methyl 2,5-dihydromethylcinnimate (2,5-MeC), a tyrosine kinase inhibitor, enhanced expression and/or stability of PML proteins and induced PML-NB formation in p53 null H1299 cells established from non-small cell lung cancer (NSCLC) and wild-type p53-expressing U2OS cells derived from osteosarcoma. Furthermore, it enhanced apoptosis by exogenously expressed wild type p53 and the expression of p53-responsive genes, such as PUMA and p21, in H1299 cells. 2,5-MeC also activated endogenous p53 and induced apoptosis in U2OS cells. The results suggest that 2,5-MeC is likely to be a promising candidate drug for the clinical treatment of terminal cancer-expressing wild-type p53.     

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     

The role of PML in tumor suppression

The PML gene, involved in the t(15;17) chromosomal translocation of acute promyelocytic leukemia (APL), encodes a protein which localizes to the PML-nuclear body, a subnuclear macromolecular structure. PML controls apoptosis, cell proliferation, and senescence. Here, we review the current understanding of its role in tumor suppression.     

Promyelocytic leukemia is a direct inhibitor of SAPK2/p38 mitogen-activated protein kinase

The promyelocytic leukemia gene (PML) encodes a growth/tumor suppressor protein that is essential for the induction of apoptosis in response to various apoptotic signals. The mechanism by which PML plays a role in the regulation of cell death is still unknown. In the current study, we demonstrate that PML negatively regulated the SAPK2/p38 signaling pathway by sequestering p38 from its upstream kinases, MKK3, MKK4, and MKK6, whereas PML did not affect the SAPK1/c-Jun NH(2)-terminal kinase pathway. PML associated with p38 both in vitro and in vivo and the carboxyl terminus of PML mediated the interaction. In contrast to other studies of PML and PML-nuclear bodies (NB), our study shows that the formation of PML-NBs was not required for PML to suppress p38 activity because PML was still able to bind and inhibit p38 activity under the conditions in which PML-NBs were disrupted. In addition, we show that the promotion of Fas-induced cell death by PML correlated with the extent of p38 inhibition by PML, suggesting that PML might regulate apoptosis through manipulating SAPK2/p38 pathways. Our findings define a novel function of PML as a negative regulator of p38 kinase and provide further understanding on the mechanism of how PML induces multiple pathways of apoptosis.     

SAHA treatment overcomes the anti-apoptotic effects of Bcl-2 and is associated with the formation of mature PML nuclear bodies in human leukemic U937 cells

Bcl-2 protects tumor cells from the apoptotic effects of various antineoplastic agents. Increased expression of Bcl-2 has been associated with poor response to chemotherapy in various malignancies, including leukemia. Therefore, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. We undertook this study to examine whether SAHA (suberoylanilide hydroxamic acid) overcomes the resistance by Bcl-2 in human leukemic cells, with a specific focus on the involvement of PML-NBs. Experiments were conducted with Bcl-2-overexpressing human leukemic U937 cells. Since we previously demonstrated that overexpression of Bcl-2 attenuates resveratrol-induced apoptosis in human leukemic U937 cells, resveratrol-treated U937 cells were used as a negative control. The present study indicates that SAHA at 1-7 μM, the dose range known to induce apoptosis in various cancer cells, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2-overexpressing human leukemic U937 cells. Notably, we observed that SAHA-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with overcoming the anti-apoptotic effects of Bcl-2 in human leukemic U937 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that could help bypass the resistance to apoptosis conferred by Bcl-2. Elucidating exactly how PML regulates Bcl-2 will require further work.     

The herpesvirus associated ubiquitin specific protease, USP7, is a negative regulator of PML proteins and PML nuclear bodies

The PML tumor suppressor is the founding component of the multiprotein nuclear structures known as PML nuclear bodies (PML-NBs), which control several cellular functions including apoptosis and antiviral effects. The ubiquitin specific protease USP7 (also called HAUSP) is known to associate with PML-NBs and to be a tight binding partner of two herpesvirus proteins that disrupt PML NBs. Here we investigated whether USP7 itself regulates PML-NBs. Silencing of USP7 was found to increase the number of PML-NBs, to increase the levels of PML protein and to inhibit PML polyubiquitylation in nasopharyngeal carcinoma cells. This effect of USP7 was independent of p53 as PML loss was observed in p53-null cells. PML-NBs disruption was induced by USP7 overexpression independently of its catalytic activity and was induced by either of the protein interaction domains of USP7, each of which localized to PML-NBs. USP7 also disrupted NBs formed from some single PML isoforms, most notably isoforms I and IV. CK2α and RNF4, which are known regulators of PML, were dispensable for USP7-associated PML-NB disruption. The results are consistent with a novel model of PML regulation where a deubiquitylase disrupts PML-NBs through recruitment of another cellular protein(s) to PML NBs, independently of its catalytic activity.     

Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.     

Promyelocytic leukemia protein 4 induces apoptosis by inhibition of survivin expression

The promyelocytic leukemia protein (PML) plays an essential role in multiple pathways of apoptosis. Our previous study showed that PML enhances tumor necrosis factor-induced apoptosis by inhibiting the NFkappaB survival pathway. To continue exploring the mechanism of PML-induced apoptosis, we performed a DNA microarray screening of PML target genes using a PML-inducible stable cell line. We found that Survivin was one of the downstream target genes of PML. Cotransfection experiments demonstrated that PML4 repressed transactivation of the Survivin promoter in an isoform-specific manner. Western blot analysis demonstrated that induced PML expression down-regulated Survivin. Inversely, PML knockdown by siRNA up-regulated Survivin expression. A substantial increase in Survivin expression was found in PML-deficient cells. Re-expression of PML in PML-/- mouse embryo fibroblasts down-regulated the expression of Survivin. Furthermore, cells arrested at the G2/M cell cycle phase expressed a high level of Survivin and a significantly lower level of PML. Overexpression of PML in A549 cells reduced Survivin expression leading to massive apoptotic cell death associated with activation of procaspase 9, caspase 3, and caspase 7. Together, our results demonstrate a novel mechanism of PML-induced apoptosis by down-regulation of Survivin.     

The IL-6 family of cytokines modulates STAT3 activation by desumoylation of PML through SENP1 induction

Post-translational modification by small ubiquitin-like modifier (SUMO) plays an important role in the regulation of different signaling pathways and is involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies following sumoylation of PML. In the present study, we found that IL-6 induces desumoylation of PML and dissociation between PML and SUMO1 in hepatoma cells. We also found that IL-6 induces mRNA expression of SENP1, a member of the SUMO-specific protease family. Furthermore, wild-type SENP1 but not an inactive SENP1 mutant restored the PML-mediated suppression of STAT3 activation. These results indicate that the IL-6 family of cytokines modulates STAT3 activation by desumoylation and inactivation PML through SENP1 induction.     

Leukemia: beneficial actions of retinoids and rexinoids

Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia, is the prototype of a cancer that can be cured by differentiation therapy using combined retinoic acid (RA) and chemotherapy. Acute promyelocytic leukemia is caused by chromosomal translocations, which in the large majority of cases generate the prototypic promyelocytic leukemia-retinoic-acid receptor alpha (PML-RARalpha) an oncogenic fusion protein formed from the retinoic-acid receptor alpha and the so-called PML protein. The fusion protein leads to the deregulation of wild type PML and RARalpha function, thus inducing the differentiation block and an altered survival capacity of promyelocytes of affected patients. A plethora of studies have revealed molecular details that account for the oncogenic properties of acute promyelocytic leukemia fusion proteins and the events that contribute to the therapy-induced differentiation and apoptosis of patients' blasts. Illustrating the beneficial mechanisms of action of retinoids for acute promyelocytic leukemia patients this review goes on to discuss a plethora of recently recognized molecular paradigms by which retinoids and rexinoids, alone or in combination with other compounds, regulate growth, differentiation and apoptosis also in non-acute promyelocytic leukemia cells, highlighting their potential as drugs for cancer therapy and prevention.     

The alphaherpesvirus serine/threonine kinase us3 disrupts promyelocytic leukemia protein nuclear bodies

Us3, a serine/threonine kinase encoded by all alphaherpesviruses, plays diverse roles during virus infection, including preventing virus-induced apoptosis, facilitating nuclear egress of capsids, stimulating mRNA translation and promoting cell-to-cell spread of virus infection. Given this diversity, the full spectrum of Us3 function may not yet be recognized. We noted, in transiently transfected cells, that herpes simplex virus type 2 (HSV-2) Us3 disrupted promyelocytic leukemia protein nuclear bodies (PML-NBs). However, PML-NB disruption was not observed in cells expressing catalytically inactive HSV-2 Us3. Analysis of PML-NBs in Vero cells transfected with pseudorabies virus (PRV) Us3 and those in Vero cells infected with Us3-null or -repaired PRV strains indicated that PRV Us3 expression also leads to the disruption of PML-NBs. While loss of PML-NBs in response to Us3 expression was prevented by the proteasome inhibitor MG132, Us3-mediated degradation of PML was not observed in infected cells or in transfected cells expressing enhanced green fluorescent protein (EGFP)-tagged PML isoform IV. These findings demonstrate that Us3 orthologues derived from distantly related alphaherpesviruses cause a disruption of PML-NBs in a kinase- and proteasome-dependent manner but, unlike the alphaherpesvirus ICP0 orthologues, do not target PML for degradation.