Showing papers on "Promyelocytic leukemia protein published in 1998"
TL;DR: It is shown that, in APL cells, As2O3 triggers rapid degradation of PML–RARα and provokes the restoration of intact nuclear bodies, suggesting that post‐translational modification by SUMO‐1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures.
Abstract: The PML protein, identified first as part of the oncogenic PML-RARalpha chimera in acute promyelocytic leukemia (APL), concentrates within discrete subnuclear structures, corresponding to some types of nuclear bodies. These structures are disrupted in APL cells, and retinoic acid (RA) can trigger their reorganization, correlating with its therapeutic effect in this type of leukemia. Recently, arsenic trioxide (As2O3) was identified as a potent antileukemic agent which, similarly to RA, induces complete remissions in APL patients. Here we show that, in APL cells, As2O3 triggers rapid degradation of PML-RARalpha and provokes the restoration of intact nuclear bodies. In non-APL cells, the ubiquitin-like protein SUMO-1 is covalently attached to a subset of wild-type PML in a reversible and phosphorylation-dependent manner. The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO-1-polymodified forms of PML are compartmentalized exclusively in the PML nuclear bodies. As2O3 administration strikingly increases the pool of SUMO-1-PML conjugates that, subsequently, accumulate in enlarged nuclear bodies. In contrast to PML-RARalpha, the overall amount of PML seems to remain unaltered up to 36 h following As2O3 treatment. These findings indicate that the conjugation of PML with SUMO-1 modulates its intracellular localization and suggest that post-translational modification by SUMO-1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures. They provide additional evidence that the role of 'ubiquitin-like' post-translational modification is not limited to a degradation signal.
682 citations
TL;DR: It is demonstrated that Pml is a mediator of multiple apoptotic signals, and implicate inhibition of apoptosis in the pathogenesis of APL.
Abstract: The PML
gene of acute promyelocytic leukaemia (APL) encodes a cell growth and tumour suppressor, however, the mechanisms by which PML suppresses tumorigenesis are poorly understood. We show here that Pml is required for Fas- and caspase-dependent DNA-damage–induced apoptosis. We also found that Pml is essential for induction of programmed cell death by Fas, tumour necrosis factor α (TNF), ceramide and type I and II interferons (IFNs). As a result, Pml
–/–
mice and cells are protected from the lethal effects of ionizing radiation and anti-Fas antibody. Pml is required for caspase 1 and caspase 3 activation upon exposure to these stimuli. The PML-RARα fusion protein of APL renders haemopoietic progenitor cells resistant to Fas-, TNF- and IFN-induced apoptosis with a lack of caspase 3 activation, thus acting as a Pml dominant-negative product. These results demonstrate that Pml is a mediator of multiple apoptotic signals, and implicate inhibition of apoptosis in the pathogenesis of APL.
587 citations
TL;DR: It is shown that Histone deacetylase inhibitors such as Trichostatin A (TSA), in combination with RA, can overcome the transcriptional repressor activity of PML-RARα and PLZF-Rarα as well as the unresponsiveness of PLZ fusions-expressing leukaemic cells to RA.
Abstract: Acute promyelocytic leukaemia (APL), associated with chromosomal translocations involving the retinoic acid receptor alpha gene (RARA) and the PML gene, is sensitive to retinoic acid (RA) treatment, while APL patients harbouring translocations between RARA and the PLZF gene do not respond to RA. We have generated PML-RARA and PLZF-RARA transgenic mice and show here that these fusion proteins play a critical role in leukaemogenesis and in determining responses to RA in APL, because PLZF-RARA transgenic mice develop RA-resistant leukaemia, while PML-RARA mice are responsive to RA treatment. We demonstrate that both PML-RARalpha and PLZF-RARalpha fusion proteins can act as transcriptional repressors and are able to interact with nuclear receptor transcriptional co-repressors, such as SMRT. PLZF-RARalpha, but not PML-RARalpha, can form, via its PLZF moiety, co-repressor complexes which are insensitive to RA. Histone deacetylase inhibitors such as Trichostatin A (TSA), in combination with RA, can overcome the transcriptional repressor activity of PML-RARalpha and PLZF-RARalpha as well as the unresponsiveness of PLZF-RARalpha-expressing leukaemic cells to RA. Thus, our findings unravel a crucial role for transcriptional silencing in APL pathogenesis and resistance to RA in APL.
550 citations
TL;DR: Results indicate that PML is a critical component of the RA pathway and that disruption of its activity by the PML-RARalpha fusion protein may be important in APL pathogenesis.
Abstract: The PML gene is fused to the retinoic acid receptor α (RARα) gene in chromosomal translocations associated with acute promyelocytic leukemia (APL). Ablation of murine PML protein by homologous recombination revealed that PML regulates hemopoietic differentiation and controls cell growth and tumorigenesis. PML function was essential for the tumor-growth–suppressive activity of retinoic acid (RA) and for its ability to induce terminal myeloid differentiation of precursor cells. PML was needed for the RA-dependent transactivation of the p21WAF1/CIP1 gene, which regulates cell cycle progression and cellular differentiation. These results indicate that PML is a critical component of the RA pathway and that disruption of its activity by the PML-RARα fusion protein may be important in APL pathogenesis.
519 citations
TL;DR: The results show that the disruption of ND10 during virus infection correlates with the loss of several PML isoforms and this process is dependent on active proteasomes, suggesting a simple yet powerful mechanism by which Vmw110 might function duringirus infection.
Abstract: The small nuclear structures known as ND10 or PML nuclear bodies have been implicated in a variety of cellular processes including response to stress and interferons, oncogenesis, and viral infection, but little is known about their biochemical properties. Recently, a ubiquitin-specific protease enzyme (named HAUSP) and a ubiquitin-homology family protein (PIC1) have been found associated with ND10. HAUSP binds strongly to Vmw110, a herpesvirus regulatory protein which has the ability to disrupt ND10, while PIC1 was identified as a protein which interacts with PML, the prototype ND10 protein. We have investigated the role of ubiquitin-related pathways in the mechanism of ND10 disruption by Vmw110 and the effect of virus infection on PML stability. The results show that the disruption of ND10 during virus infection correlates with the loss of several PML isoforms and this process is dependent on active proteasomes. The PML isoforms that are most sensitive to virus infection correspond closely to those which have recently been identified as being covalently conjugated to PIC1. In addition, a large number of PIC1-protein conjugates can be detected following transfection of a PIC1 expression plasmid, and many of these are also eliminated in a Vmw110-dependent manner during virus infection. These observations provide a biochemical mechanism to explain the observed effects of Vmw110 on ND10 and suggest a simple yet powerful mechanism by which Vmw110 might function during virus infection.
407 citations
TL;DR: It is shown that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins, which support a model in which the NBs may play a role in certain aspects of chromatin dynamics.
Abstract: The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RARalpha hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.
279 citations
TL;DR: It is shown that overexpression of PML and not Sp100 induces resistance to infections by vesicular stomatitis virus and influenza A virus but not by encephalomyocarditis virus, suggesting that PML can contribute to the antiviral state induced in IFN-treated cells.
Abstract: The interferon (IFN)-induced promyelocytic leukemia (PML) protein is specifically associated with nuclear bodies (NBs) whose functions are yet unknown. Two of the NB-associated proteins, PML and Sp100, are induced by IFN. Here we show that overexpression of PML and not Sp100 induces resistance to infections by vesicular stomatitis virus (VSV) (a rhabdovirus) and influenza A virus (an orthomyxovirus) but not by encephalomyocarditis virus (a picornavirus). Inhibition of viral multiplication was dependent on both the level of PML expression and the multiplicity of infection and reached 100-fold. PML was shown to interfere with VSV mRNA and protein synthesis. Compared to the IFN mediator MxA protein, PML had less powerful antiviral activity. While nuclear body localization of PML did not seem to be required for the antiviral effect, deletion of the PML coiled-coil domain completely abolished it. Taken together, these results suggest that PML can contribute to the antiviral state induced in IFN-treated cells.
251 citations
TL;DR: Results show that As 2 O 3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RAR expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL.
231 citations
TL;DR: Using a COS cell expression system, it is shown that PML is covalently modified by all three members of the sentrin family of ubiquitin-like proteins, which highlights the fine substrate specificity of the sendrinization pathway.
218 citations
TL;DR: ACUTE PROMYELOCYTIC leukemia (APL) is a distinct subtype of acute myelogenous leukemia (AML) identified by the French-American-British classification as AML-M3 and cytogenetically characterized by the balanced reciprocal translocation between chromosomes 15 and 17.
196 citations
TL;DR: Results indicate that PML/RARalpha is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PMl/Raralpha.
TL;DR: It is reported that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RARα expression.
Abstract: Acute promyelocytic leukemia (APL) is characterized by a clonal expansion of myeloid precursors blocked at the promyelocytic stage. A chromosome translocation involving the PML gene on chromosome 15 and the retinoic acid receptor α (RARα) gene on chromosome 17 is found in over 95% of APL cases (1, 7, 16). The resulting PML-RARα fusion gene encodes a PML-RARα fusion protein that is implicated in the pathogenesis of the disease (17, 41, 42, 58). In fact, PML-RARα transgenic mice develop abnormal myelopoiesis with phenotypic features of APL (8, 31), and in vitro, PML-RARα expression has biological activities which are consistent with the promyelocytic leukemia phenotype, e.g., block of terminal differentiation and increased survival (29).
PML is a ubiquitously expressed, matrix-associated nuclear phosphoprotein whose overexpression induces growth suppression (11, 23, 55). However, its physiological function and biochemical activities remain unknown. PML is a member of a growing family of proteins characterized by the presence of a RING domain, two additional Cys/His-rich regions (B1 and B2 boxes), and an α-helical coiled-coil domain (5, 6, 50, 61). Within the latter, four clusters of heptads of hydrophobic amino acids define a dimerization interface through which PML forms homodimers and, in APL cells, heterodimers with PML-RARα (42, 59).
PML is localized within discrete nuclear structures referred to as nuclear bodies (NBs), ND10, Kr bodies, or promyelocytic leukemia oncogenic domain (19, 45, 71). Other components of the PML NBs are Sp100 (68), NDP55 (2), Int-6 (15), and PIC-1 (4). The integrity of the PML NBs is lost in APL cells: PML-RARα localizes to novel nuclear structures (so-called microspeckles) and causes the delocalization of PML and other components of the NBs (19, 45, 71). The disorganization of the NB structure is thought to be relevant to the pathogenesis of the APLs since retinoic acid treatment, which reverts the differentiation block of the APL blasts in vitro and causes disease remission in vivo, induces degradation of the PML-RARα protein and the consequent assembly of the PML NBs (for a review, see reference 28).
Altered localization of PML and structural changes of the NBs have also been shown to occur during DNA virus infection (10, 18, 21, 43). Some viral proteins, such as herpes simplex virus type 1 Vmw110 and adenovirus E4-ORF3, have been described to be directly involved in the redistribution of NB components (10, 18, 21), whereas Epstein-Barr virus (EBV) EBNA-5 protein has been described to colocalize with PML within morphologically intact NBs (66). Interestingly, PML expression as well as the size and number of the PML NBs increase after treatment of cells with the antiviral agent interferon (12, 46). In summary, a number pieces of indirect evidence suggest that PML and/or the PML NBs are involved in growth control and are the targets of DNA viral infection. Identification of proteins that interact with PML within NBs might help in defining the role of PML and PML NBs in normal and leukemic cells. We investigated the physical and functional interactions of PML with the retinoblastoma gene product (pRB).
pRB regulates cell proliferation by controlling a set of transcription factors (the E2F family of proteins) that activate genes involved in the G1/S transition (70). In the early G1 phase of the cell cycle, pRB is unphosphorylated and stably complexed with E2F; as cells pass the G1/S boundary, pRB becomes phosphorylated, resulting in the functional release of E2F (70). pRB has also been described to regulate the activity of promoters that depend on other transcription factors, such as SP1 or glucocorticoid receptor (44, 65, 69). However, the physiological consequences of these activities of pRB remain unknown.
The analysis of pRB subcellular localization suggests that pRB is distributed in at least two distinct subnuclear compartments, one diffuse and one corresponding to circumscribed granules (54, 67) which morphologically resemble NBs. Interestingly, the EBNA-5 protein has been demonstrated to colocalize with pRB within distinct nuclear foci in EBV-infected lymphoblastoid cells (38).
The similarity in subnuclear localization, together with the shared property of inducing growth suppression, prompted us to investigate the physical and functional interactions between PML and pRB. We report here that PML and pRB colocalize within the PML NBs and that PML forms complexes with the unphosphorylated form of pRB. Functionally, PML and pRB do not appear to be mutually necessary to exert their respective growth suppressor activities, while PML displays an inhibitory effect on pRB-regulated transcriptional activation of glucocorticoid receptor (GR)-responsive promoters. PML-RARα expression causes the delocalization of pRB from the PML NB and shows stimulatory activity on the GR-responsive promoters.
TL;DR: Acute promyelocytic leukaemia (APL), characterized by a specific PML‐RARα fusion gene resulting from translocation t(15;17) and by a high response rate to differentiation therapy with all‐trans retinoic acid, presents clinical, morphological and molecular heterogeneity.
Abstract: Acute promyelocytic leukaemia (APL), characterized by a specific PML-RARalpha fusion gene resulting from translocation t(15;17) and by a high response rate to differentiation therapy with all-trans retinoic acid, presents clinical (varying WBC counts, age and treatment outcome), morphological (hypergranular M3 and hypogranular M3V) and molecular (three isoforms of PML breakpoint) heterogeneity. We correlated leukaemic immunophenotype with these aspects in 196 molecularly confirmed APLs (63 children and 133 adults) in Italy. The bcr3 isoform (P = 0.05) and FAB M3V (P = 0.05) were more frequent in children. We confirmed in APL an immunophenotype characterized by frequent expression of CD13, CD33 and CD9 and rare expression of HLA-DR, CD10, CD7 and CD11b. However, we recognized CD2 in 28%, CD34 in 23% and CD19 in 11% of cases and demonstrated by double labelling that CD34 and CD2 may be co-expressed. CD2, CD34 and CD19 were significantly intercorrelated, and variably associated to other features: CD2 and CD34 with PML bcr3 (P < 0.001 and P < 0.001, respectively) and with M3V (P < 0.001 and P = 0.002), whereas only CD19 was directly correlated with WBC counts and only CD2 positively influenced CR rate (logistic model) and event-free survival (Cox model). We conclude that immunophenotype plays a role in the determination of the biological and clinical heterogeneity of childhood and adult APL.
TL;DR: The interphase nucleus compartmentalizes its components to give rise to a highly organized and tightly controlled environment as mentioned in this paper, where individual chromosomes occupy discrete areas, termed "chromosome territories", that are separated from each other by a channel called the "interchromosomal domain" (reviewed in Lamond and Earnshaw 1998).
Abstract: The interphase nucleus compartmentalizes its components to give rise to a highly organized and tightly controlled environment. Individual chromosomes occupy discrete areas, termed “chromosome territories,” that are separated from each other by a channel called the “interchromosomal domain” (reviewed in Lamond and Earnshaw 1998). Actively transcribed genes tend to be at the periphery of chromosomal territories, whereas newly made RNA transcripts localize into the interchromosomal domain, where they can undergo further processing and transport. Movement within the nucleus (Ferreira et al. 1997) may permit chromosomes to enter “factories” that contain all the necessary enzymatic machinery for replication (reviewed in Jackson 1995). Of the many discrete domains identified throughout the nucleus, the largest are nucleoli, sites of ribosomal RNA synthesis and processing, and sites of preribosomal particle assembly (reviewed in Scheer and Weisenberger 1994). Other subnuclear bodies that appear as punctate structures under immunofluorescence (IF) microscopy include various dynamic structures involved in the maintenance and replication of DNA and RNA synthesis, processing, and transport (reviewed in Nickerson et al. 1995): replication foci, transcript foci, speckled domains, coiled bodies, gems, and promyelocytic leukemia protein (PML) nuclear bodies. Spliceosomal small nuclear (sn) ribonucleoprotein (RNP) components and a subset of non-snRNP splicing factors can be found concentrated in discrete subnuclear domains called “coiled bodies” (Matera and Frey 1998 [in this issue]). It is becoming increasingly apparent that the nucleus has an organization and contains a number of discrete macromolecular domains that coordinate a variety of nuclear processes.
TL;DR: The Z protein acts dominantly to overcome the diffuse phenotype observed in several PML mutants and the interaction between PML and Z may influence certain unique characteristics of arenavirus infection.
Abstract: The promyelocytic leukemia protein (PML) forms nuclear bodies which are altered in some disease conditions. We report that the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV) influences the distribution of PML bodies. In cells infected with LCMV, the Z protein and PML form large bodies primarily in the cytoplasm. Transient transfection studies indicate that Z alone is sufficient to redistribute PML to the cytoplasm and that PML and Z colocalize. Coimmunoprecipitation studies show specific interaction between PML and Z proteins. A similar result was observed with a Z protein from another arenavirus, Lassa virus, suggesting that this is a general feature of the Arenaviridae. Genetically engineered mutations in PML were used to show that the Z protein binds the N-terminal region of PML and does not need the PML RING or the nuclear localization signal to colocalize. The Z protein acts dominantly to overcome the diffuse phenotype observed in several PML mutants. The interaction between PML and Z may influence certain unique characteristics of arenavirus infection.
TL;DR: The interaction of IE1(491aa) with chromatin was therefore not required for the disruption of PML-bodies, and both PML and IE1 were observed to associate with mitotic chromosomes in cells infected with HCMV or transfected with the IE1 gene.
Abstract: The PML gene product is associated with a defined nuclear structure (10-20 per cell) known variously as PML-bodies, ND10, PODs or Kr bodies. Certain conditions are known to compromise the integrity of PML-bodies; these include environmental stress (e.g. heat shock), a chromosomal translocation-associated acute promyelocytic leukaemia, and infection with certain viruses [including human cytomegalovirus (HCMV), herpes simplex virus type 1 and adenovirus]. Expression of the HCMV major immediate early (IE) protein (IE1(491aa)) is by itself sufficient to cause disruption of PML-bodies, resulting in the dispersal of the PML antigen uniformly throughout the nucleus. In uninfected cells undergoing mitosis PML is excluded from chromatin. However, both IE1(491aa) and PML were observed to associate with mitotic chromosomes in cells infected with HCMV or transfected with the IE1 gene. A series of in-frame IE1 deletion mutants was used in DNA transfection experiments to identify two large sequence elements (aa 132-274 and the C-terminal aa 347-491) not required for dispersal of the PML antigen. However, a putative leucine-zipper domain (aa 105-139), a putative zinc-finger domain (aa 267-286) and exon 2 and 3 coding sequences (aa 6-85) were required. The association of the IE1 gene product with chromatin required an acidic domain near the C terminus (aa 421-486). The interaction of IE1(491aa) with chromatin was therefore not required for the disruption of PML-bodies. Exon 2 (aa 1-24) was shown to encode a nuclear localization signal.
TL;DR: Analysis of the effects of PML on Sp1 DNA binding by electrophoretic mobility shift assay showed that PML could specifically disrupt the binding of Sp1 to DNA, and suggest that the association ofPML and Sp1 represents a novel mechanism for negative regulation of EGFR and other Sp1 target promoters.
Abstract: The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein with growth- and transformation-suppressing 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 vitro-translated 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.
TL;DR: 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 effect in Breast cancer cells were examined.
Abstract: 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 effect in breast cancer cells were examined. We showed that Ad-PML effectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post-infection and a detectable level remained at day 16. Ad-PML significantly 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 effect 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 significant 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 findings indicate that PML exerts its growth suppressing effects 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.
TL;DR: PML and ICP8 staining are both observed within replication compartments, indicating a potential role for PML in HSV-1 replication.
Abstract: Herpes simplex virus type 1 (HSV-1) infection results in the disruption of ND10 (also called nuclear bodies, PODs, or PML-associated bodies), which are nuclear matrix domains of unknown function present in mammalian cells. After ND10 disruption, viral transcription and DNA replication occur in globular nuclear domains called replication compartments. In this report we define four stages of infection by using antibodies to ICP8 (also called SSB and UL29) and the ND10 antigen PML. Immediately after infection, cells contain intact ND10 as detected by staining for PMLs (stage I); within 1 hour, however, ND10 are disrupted and cells begin to exhibit diffuse staining for the major viral DNA binding protein, ICP8 (stage II). After all ND10 have been disrupted, foci which resemble but are not equivalent to ND10 appear, containing both PML and ICP8 (stage III). Cells infected with mutants defective in the helicase-primase or origin binding protein are unable to form stage III foci. Cells infected with a mutant that is null for the polymerase catalytic subunit, however, form stage III-like ICP8 foci which do not contain PML. Thus, stage III foci recruit the cellular PML protein in the presence but not the absence of HSV polymerase. PML was recruited to stage III foci in some but not all cells infected with a mutant defective in the polymerase accessory protein, UL42. Thus, UL42 is not required for the recruitment of PML to viral foci. In wild-type infection, stage III cells are quickly replaced by cells containing replication compartments (stage IV). PML and ICP8 staining are both observed within replication compartments, indicating a potential role for PML in HSV-1 replication. Models for the role of ND10 proteins in the formation of replication compartments are discussed.
TL;DR: It is proposed that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.
TL;DR: It is suggested that nucleophosmin/B23 is one of the key elements in the down-regulation of nucleolar function for cellular differentiation and significantly potentiated RA-induced cellular differentiation.
Abstract: Human promyelocytic leukemia HL-60 cells were induced to undergo granulocytic differentiation by treatment with retinoic acid (RA, 10 μM, 1–5 days). The steady-state level of nucleophosmin/B23 mRNA decreased during the RA-induced differentiation. There was also decrease in the level of total cellular nucleophosmin/B23 protein during the RA-induced differentiation. Stabilization and nuclear run-on assays indicate that the decrease in nucleophosmin/B23 mRNA in RA-treated HL-60 cells was transcriptionally regulated. Unlike c-myc mRNA, there was virtually no decline of nucleophosmin/B23 mRNA during the growth arrest by serum-starvation. The decrease in nucleophosmin/B23 mRNA expression in HL-60 cells subsequent to retinoic acid treatment can thus be attributed to cellular differentiation rather than the growth arrest induced by RA. Nucleophosmin/B23 antisense oligomer treatment significantly potentiated RA-induced cellular differentiation. Results of this study suggest that nucleophosmin/B23 is one of the key elements in the down-regulation of nucleolar function for cellular differentiation.
TL;DR: It is demonstrated that PML is involved in the AP-1 complex and can modulate Fos-mediated transcriptional activity, which may contribute to its growth suppressor function.
Abstract: The growth and transformation suppressor function of promyelocytic leukemia (PML) protein are disrupted in acute promyelocytic leukemia (APL) as a result of its fusion to the RARalpha gene by t(15;17) translocation. There is significant sequence homology between the dimerization domain of PML and the Fos family of proteins, which imply that PML may be involved in AP-1 activity. Here we show that PML can cooperate with Fos to stimulate its AP-1-mediated transcriptional activity. Cotransfection of PML with GAL4/Fos strongly induced Fos-mediated activation of GAL4-responsive reporters, indicating a functional interaction between Fos and PML in vivo. Deletion analysis of Fos and PML demonstrated that the intact C-terminal domain of Fos (containing the dimerization domain), and the RING-finger, B1 box and nuclear localization domains of PML are involved in the cooperative activity of Fos and PML. Immunoprecipitation and electrophoretic mobility shift assay showed that PML is associated with the AP-1 complex. PMLRARalpha was also found to enhance the transcriptional activity of GAL4/Fos. The addition of retinoic acid abrogated the PMLRARalpha, but not PML-induced stimulation of GAL4/Fos activity in a dose-dependent manner. This study demonstrated that PML is involved in the AP-1 complex and can modulate Fos-mediated transcriptional activity, which may contribute to its growth suppressor function.
TL;DR: The observation that retinoic acid induces the terminal differentiation of human acute promyelocytic leukemia (APL) cells both in vitro and in vivo is confirmed.
TL;DR: Overall the results suggest that the PML- and PLZF-NBs are distinct functional nuclear domains, but that they may share common regulatory pathways and/or targeting sequences, as revealed by the common localization of their corresponding fusion proteins.
Abstract: Acute promyelocytic leukaemia is characterized by translocations that involve the retinoic acid receptor α (RARα) locus on chromosome 17 and the PML locus on 15 or the PLZF locus on 11. The resulting abnormal translocation products encode for PML/RARα or PLZF/RARα fusion proteins. There is increasing experimental evidence that the APL-specific fusion proteins have similar biologic activities on differentiation and survival and that both components of the fusion proteins (PML or PLZF and RARα) are indispensable for these biological activities. The physiologic function of PML or PLZF or whether PML and PLZF contribute common structural or functional features to the corresponding fusion proteins is not known. We report here immunofluorescence studies on the cellular localization of PLZF and PLZF/RARα and compare it with the localization of PML and PML/RARα. PLZF localizes to nuclear domains of 0.3-0.5 microns, approximately 14 per cell in the KG1 myeloid cell line. These PLZF-bodies are morphologically similar to the domains reported for PML (PML-NBs). There is tight spatial relationship between about 30% of PLZ-NBs and PML-NBs: they partially overlap. However, PML and PLZF do not form soluble complexes in vivo. PLZF- and PML-NBs are functionally distinct. Adenovirus E4-ORF3 protein expression alters the structure of the PML-NBs and interferon increases the number of PML-NBs and neither has any effect on PLZF NBs. The localization of PLZF/RARα is different to that of PLZF and RARα. The nuclear distribution pattern of PLZF/RARα is one of hundreds of small dots (microspeckles) less than 0.1 micron. Expression of PLZF/RARα did not provoke disruption of the PML-NBs. Co-expression of PML/RARα and PLZF/RARα in U937 cells revealed apparent colocalization. Overall the results suggest that the PML- and PLZF-NBs are distinct functional nuclear domains, but that they may share common regulatory pathways and/or targeting sequences, as revealed by the common localization of their corresponding fusion proteins.
TL;DR: The promyelocytic leukaemia (protein) (PML) localizes to multiprotein complexes known as PML nuclear bodies and it is found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-immunoprecipitates with PML and co-localizes withPML in nuclear bodies, indicating that the association betweenPML and GAPDH depends on the presence of RNA.
Abstract: The promyelocytic leukaemia (protein) (PML) localizes to multiprotein complexes known as PML nuclear bodies. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-immunoprecipitates with PML and co-localizes with PML in nuclear bodies. RNase treatment disrupts the ability of PML and GAPDH to both co-localize and co-immunoprecipitate, indicating that the association between PML and GAPDH depends on the presence of RNA. Disruption of PML bodies contributes towards reduced apoptosis in acute promyelocytic leukaemia and GAPDH induces apoptotic neuronal death. The GAPDH-PML interaction may be involved in the regulation of apoptosis.
TL;DR: Results show that these retinoid-resistant NB4 cells require persistent PML/RAR expression for leukemic cell growth, which can account for why these cells do not respond to ATRA and how reduction of PML-RAR-related protein abrogates the antiapoptotic effect it confers to theseLeukemic cells.
TL;DR: The molecular mechanisms by which IFNs and RA can cooperate in controlling cell growth and differentiation of normal hemopoietic cells and leukemic cells are reviewed, focusing on APL as a model system.
Abstract: Acute promyelocytic leukemia (APL) has been regarded as the paradigm for therapeutic approaches utilizing differentiating agents, due to the fact that almost 95% of patients undergo complete remission when treated with all-trans retinoic acid (ATRA). However, complete clinical remission with ATRA alone is always transient, and relapse in APL is almost invariably associated with the acquisition of resistance to ATRA. Acquired resistance to ATRA in APL cell lines and in some APL clinical cases can be partially overcome by interferons (IFNs), cytokines which have well established tumor-growth suppressive activities. APL is associated in 99% of cases with a 15;17 translocation that fuses the PML and Retinoic Acid Receptor alpha (RARa) genes. RARa is one of the Retinoic Acid (RA) nuclear receptors which mediates, at the transcriptional level, ATRA differentiating and growth suppressive activity. PML is a tumor-growth suppressor whose expression is directly regulated by IFNs. Here we review the molecular mechan...
TL;DR: The altered expression of PML indicates that this nuclear protein may play an important role in cellular response to stress and inflammation, as well as in compensatory cell growth.
TL;DR: A patient with acute promyelocytic leukemia (APL), in hematological and molecular remission who developed T-ALL three years after the diagnosis of APL, shows signs of having originated from a different clone from that of the APL cells.
Abstract: Secondary acute lymphoblastic leukemia (sALL) following acute myeloid leukemia (AML) is a rare event; only eight cases have been reported. We report a patient with acute promyelocytic leukemia (APL), in hematological and molecular remission who developed T-ALL three years after the diagnosis of APL. The morphological, cytochemical, phenotypical and molecular features of this T-ALL were different from those of the previous APL. The absence of t(15;17), negative PML/RAR alpha at reverse transcription polymerase chain reaction (RT-PCR) analysis and presence of TcR delta support the hypothesis that the T-ALL in this sALL case originated from a different clone from that of the APL cells.
TL;DR: The results confirmed that the PML/RAR alpha gene on the der(15)t(15;17), not the RAR alpha/PML gene, must be essential to leukemogenesis in APL.