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.     

Cyclic AMP-dependent and -independent protein kinases and protein phosphorylation in human promyelocytic leukemia (HL60) cells induced to differentiate by retinoic acid

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8-bromo-cyclic adenosine 3:5 monophosphate (8-Br-cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate-dependent protein kinase (cyclic AMP-dPK), we investigated the role of cyclic AMP-dPK and adenosine 3:5 cyclic monophosphate-independent protein kinase (cyclic AMP-iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP-dPK and cyclic AMP-iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP-dPK and cyclic AMP-iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type I cytosolic cyclic AMP-dPK and cyclic AMP-iPK (protamine kinase) while no such increases are noted in cells treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8-azido-cyclic AMP demonstrate marked increases in type I cyclic AMP-dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA-differentiated cells. (4) The appearance and disappearance of specific cyclic AMP-dependent and -independent protein phosphorylations are associated with the induced myeloid differentiated state.     

Microarray analysis revealing common and distinct functions of promyelocytic leukemia protein (PML) and tumor necrosis factor (TNF alpha) signaling in endothelial cells

ABSTRACT: BACKGROUND: Promyelocytic leukemia protein (PML) is a tumor suppressor that is highly expressed in endothelial cells nonetheless its role in endothelial cell biology remains elusive. Tumor necrosis factor alpha TNF-a is an important cytokine associated with many inflammation-related diseases. We have previously demonstrated that TNF-a induces PML protein accumulation. We hypothesized that PML may play a role in TNF-a signaling pathway. To identify potential PML target genes and investigate the putative crosstalk between PML's function and TNF-a signaling in endothelial cells, we carried out a microarray analysis in human primary umbilical endothelial cells (HUVECs). RESULTS: We found that PML and TNF-a regulate common and distinct genes involved in a similar spectrum of biological processes, pathways and human diseases. More importantly, we found that PML is required for fine-tuning of TNF-a-mediated immune and inflammatory responses. Furthermore, our data suggest that PML and TNF-a synergistically regulate cell adhesion by engaging multiple molecular mechanisms. Our biological functional assays exemplified that adhesion of U937 human leukocytes to HUVECs is co-regulated by PML and TNF-a signaling. CONCLUSIONS: Together, our study identified PML as an essential regulator of TNF-a signaling by revealing the crosstalk between PML knockdown-mediated effects and TNF-a-elicited signaling, thereby providing novel insights into TNF-a signaling in endothelial cells.     

Anticancer effect of retinoic acid via AP-1 activity repression is mediated by retinoic acid receptor alpha and beta in gastric cancer cells

To uncover the mechanisms relating to the anticancer effect of retinoic acids in gastric cancer cells, the mediation of activator protein-1 (AP-1) activity repression by retinoic acid receptors (RARs) was investigated. All-trans retinoic acid (ATRA) inhibited AP-1 activity in BGC-823 cells (RARalpha(+), RARbeta(+)), but not in MKN-45 cells (RARalpha(lo), RARbeta(-)). Transient transfection of RARbeta expression vector into MKN-45 cells significantly resulted in direct repression of AP-1 activity in a receptor concentration-dependent manner, and this could be strengthened by ATRA. Stable transfection of RARbeta into MKN-45 cells directly inhibited cell growth and colony formation, and ATRA also enhanced these effects. Transient transfection of RARalpha into MKN-45 cells however, displayed receptor concentration-dependent AP-1 activity inhibition only in the presence of ATRA. Stable transfection of RARalpha into MKN-45 cells resulted in ATRA-dependent inhibition of cell growth and colony formation. For AP-1 binding activity induced by TPA, the repressive effect of ATRA was only observed in BGC-823 and RARalpha and RARbeta stably transfected MKN-45 cells, but not in intact MKN-45 cells. This indicates the necessity for sufficient cellular RARalpha and/or RARbeta in order for AP-1 activity repression to occur. Deletion of DNA binding domain (DBD) of RARbeta, but not ligand binding domain (LBD), eliminated the anti-AP-1 function of RARbeta. It is therefore concluded that both RARalpha and RARbeta are mediators in the anticancer function of ATRA via AP-1 activity inhibition, and that RARbeta, not RARalpha, can inhibit AP-1 activity to a certain extent directly by itself. Thus DBD, not LBD, is critical for anti-AP-1 activity.     

CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.     

The phosphorylation site located in the A region of retinoic X receptor alpha is required for the antiproliferative effect of retinoic acid (RA) and the activation of RA target genes in F9 cells

Mouse F9 embryocarcinoma cells constitute a well established cell autonomous model system for investigating retinoic acid (RA) signaling in vitro. RA induces the differentiation of F9 cells grown as monolayers into endodermal-like cells and decreases their rate of proliferation. Knock-out of the retinoic X receptor alpha (RXRalpha) gene abolishes endodermal differentiation and the induction of several endogenous RA-responsive genes. RXRalpha null cells are also drastically impaired in their antiproliferative response to RA. The role of the RXRalpha phosphorylation site located in the N-terminal A region (Ser(22)) has been investigated here by establishing cell lines re-expressing RXRalpha either wild type or mutated at the phosphorylation site (RXRalphaS22A) in a RXRalpha-null background. We show that Ser(22) is dispensable for RA-induced endodermal differentiation but is crucial for the expression of several RA-responsive genes. Ser(22) is also indispensable for the antiproliferative effect of RA and necessary for the RA-induced down-regulation of p21(CIP) and p27(KIP) CKIs proteins that are known to be involved in the control of cell cycle progression.     

DNA recognition by the aberrant retinoic acid receptors implicated in human acute promyelocytic leukemia.

Human acute promyelocytic leukemias (APLs) are associated with chromosomal translocations that replace the NH2 terminus of wild-type retinoic acid receptor (RAR) alpha with portions of the promyelocytic leukemia protein (PML) or promyelocytic leukemia zinc-finger protein (PLZF). The wild-type RARalpha readily forms heterodimers with the retinoid X receptors (RXRs), and these RAR/RXR heterodimers appear to be the principal mediators of retinoid signaling in normal cells. In contrast, PML-RARalpha and PLZF-RARa display an enhanced ability to form homodimers, and this enhanced homodimer formation is believed to contribute to the neoplastic properties of these chimeric oncoproteins. We report here that the DNA recognition specificity of the RXRalpha/RARa heterodimer, which is presumed to be the dominant receptor species in normal cells, differs from that of the PML-RARalpha and PLZF-RARalpha homodimers, which are thought to prevail in the oncogenic cell. We suggest that differences in target gene recognition by the normal and oncogenic RARalpha proteins may contribute to the leukemogenic phenotype.     

The prevention of adipose differentiation of 3T3-L1 cells caused by retinoic acid is elicited through retinoic acid receptor alpha

Retinoids, especially all-trans retinoic acid (RA), have been shown to inhibit the differentiation of preadipose cells. It is important to human health, especially to obesity, that the regulatory system for the differentiation of adipocytes is well defined. Previously, we have shown that retinoic acid receptor (RAR) γ2 gene expression is up-regulated by RA in 3T3-L1 preadipose cells. In this study, the RAR system was dissected and the RA-regulated function in 3T3-L1 cells was assigned to one given receptor. We used three synthetic retinoids; (1) Ro 41–5253, a selective RAR α antagonist, (2) Ch 55, an RAR α, β and γ agonist, and (3) Am 80, an RAR α and β agonist, which has less affinity to RAR γ. Ro 41–5253 reverted RA-induced inhibition of the differentiation of 3T3-L1 cells. However, there was no significant reversion in RA-induced RAR γ mRNA level by treatment with Ro 41–5253. In the case of RAR agonists, both Am 80 and Ch 55 strongly inhibited the differentiation of 3T3-L1 cells. However, Am 80 weakly increased RAR γ mRNA content less than did Ch 55. These findings suggest, that RAR α is involved in the prevention of adipose differentiation by RA in 3T3-L1 cells. Moreover, there seems no causal relationship between the prevention of adipose differentiation by RA and the up-regulation of RAR γ2 gene expression by RA in 3T3-L1 cells. We have shown the functional heterogeneity of RA action through different RARs in 3T3-L1 cells.     

Positive regulation of retinoic acid receptor alpha by protein kinase C and mitogen-activated protein kinase in sertoli cells

Mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) regulate oocyte meiosis, yet little is known regarding their mechanisms of action. This study addressed the functional importance of active MAPK and PP2A in regulating oocyte meiosis. Experiments were conducted to identify MAPK activation, PP2A activity, intracellular enzyme trafficking, and ultrastructural associations during meiosis. Questions of requisite kinase and/or phosphatase activity and chromatin condensation, microtubule polymerization, and spindle formation were addressed. At the protein level, MAPK and PP2A were present in constant amounts throughout the first meiotic division. Both MAPK and PP2A were activated following germinal vesicle breakdown (GVBD) in conjunction with metaphase I development. Immunocytochemical studies confirmed the absence of active MAPK in germinal vesicle-intact (GVI) and GVBD oocytes. At metaphase I and during the metaphase I/metaphase II transition, activated MAPK colocalized with microtubules, poles, and plates of meiotic spindles. Protein phosphatase 2A was dispersed evenly throughout the GVI oocyte cytoplasm. Throughout the metaphase I/metaphase II transition, PP2A colocalized with microtubules of meiotic spindles. Both active MAPK and PP2A associated with in vitro-polymerized microtubules, suggesting that active MAPK and PP2A locally regulate spindle formation. Inhibition of MAPK activation resulted in compromised microtubule polymerization, no spindle formation, and loosely condensed chromosomes. Treatment with okadaic acid (OA) or calyculin-A (CL-A), which inhibits oocyte cytoplasmic PP2A, caused an absence of microtubule polymerization and spindles, even though MAPK activity was increased under these treatment conditions. Thus, active MAPK is required, but is not sufficient, for normal meiotic spindle formation and chromosome condensation. In addition, the oocyte OA/CL-A-sensitive PP, presumably PP2A, is essential for microtubule polymerization and meiotic spindle formation.     

Modulation of ionizing radiation-induced apoptosis and cell cycle arrest by all-trans retinoic acid in promyelocytic leukemia cells (HL-60)

Acute promyelocytic leukemia is characterized by a block of myeloid differentiation. The incubation of cells with 1 micromol/l all-trans retinoic acid (ATRA) for 72 h induced differentiation of HL-60 cells and increased the number of CD11b positive cells. Morphological and functional changes were accompanied by a loss of proliferative capacity. Differentiation caused by preincubation of leukemic cells HL-60 with ATRA is accompanied by loss of clonogenicity (control cells: 870 colonies/10(3) cells, cells preincubated with ATRA: 150 colonies/10(3) cells). D0 for undifferentiated cells was 2.35 Gy, for ATRA differentiated cells 2.46 Gy. Statistical comparison of clonogenity curves indicated that in the whole range 0.5-10 Gy the curves are not significantly different, however, in the range 0.5-3 Gy ATRA differentiated cells were significantly more radioresistant than non-differentiated cells. When HL-60 cells preincubated with 1 micromol/l ATRA were irradiated by a sublethal dose of 6 Gy, more marked increase of apoptotic cells number was observed 24 h after irradiation and the surviving cells were mainly in the G1 phase of the cell cycle, while only irradiated cells were accumulated in G(2) phase. Our results imply that preincubation of cells with ATRA accelerates apoptosis occurrence (24 h) after irradiation by high sublethal dose of 6 Gy. Forty-eight hours after 6 Gy irradiation, late apoptotic cells were found in the group of ATRA pretreated cells, as determined by APO2.7 positivity. This test showed an increased effect (considering cell death induction) in comparison to ATRA or irradiation itself.