Combination of eriocalyxin B and homoharringtonine exerts synergistic anti-tumor effects against t(8;21) AML.

Understanding the molecular pathogenesis of acute myeloid leukemia (AML) with well-defined genomic abnormalities has facilitated the development of targeted therapeutics. Patients with t(8;21) AML frequently harbor a fusion gene RUNX1-RUNX1T1 and KIT mutations as "secondary hit", making the disease one of the ideal models for exploring targeted treatment options in AML. In this study we investigated the combination therapy of agents targeting RUNX1-RUNX1T1 and KIT in the treatment of t(8;21) AML with KIT mutations. We showed that the combination of eriocalyxin B (EriB) and homoharringtonine (HHT) exerted synergistic therapeutic effects by dual inhibition of RUNX1-RUNX1T1 and KIT proteins in Kasumi-1 and SKNO-1 cells in vitro. In Kasumi-1 cells, the combination of EriB and HHT could perturb the RUNX1-RUNX1T1-responsible transcriptional network by destabilizing RUNX1-RUNX1T1 transcription factor complex (AETFC), forcing RUNX1-RUNX1T1 leaving from the chromatin, triggering cell cycle arrest and apoptosis. Meanwhile, EriB combined with HHT activated JNK signaling, resulting in the eventual degradation of RUNX1-RUNX1T1 by caspase-3. In addition, HHT and EriB inhibited NF-κB pathway through blocking p65 nuclear translocation in two different manners, to synergistically interfere with the transcription of KIT. In mice co-expressing RUNX1-RUNX1T1 and KITN822K, co-administration of EriB and HHT significantly prolonged survival of the mice by targeting CD34+CD38- leukemic cells. The synergistic effects of the two drugs were also observed in bone marrow mononuclear cells (BMMCs) of t(8;21) AML patients. Collectively, this study reveals the synergistic mechanism of the combination regimen of EriB and HHT in t(8;21) AML, providing new insight into optimizing targeted treatment of AML.

Transcriptomic profiling of pemphigus lesion infiltrating mononuclear cells reveals a distinct local immune microenvironment and novel lncRNA regulators.

Pemphigus is an autoimmune skin disease. Ectopic lymphoid-like structures (ELSs) were found to be commonly present in the pemphigus lesions, presumably supporting in situ desmoglein (Dsg)-specific antibody production. Yet functional phenotypes and the regulators of Lymphoid aggregates in pemphigus lesions remain largely unknown. Herein, we used microarray technology to profile the gene expression in skin lesion infiltrating mononuclear cells (SIMC) from pemphigus patients. On top of that, we compared SIMC dataset to peripheral blood mononuclear cells (PBMC) dataset to characterize the unique role of SIMC. Functional enrichment results showed that mononuclear cells in skin lesions and peripheral blood both had over-represented IL-17 signaling pathways while neither was characterized by an activation of type I Interferon signaling pathways. Cell-type identification with relative subsets of known RNA transcripts (CIBERSORT) results showed that naïve natural killer cells (NK cells) were significantly more abundant in pemphigus lesions, and their relative abundance positively correlated with B cells abundance. Meanwhile, plasma cells population highly correlated with type 1 macrophages (M1) abundance. In addition, we also identified a lncRNA LINC01588 which might epigenetically regulate T helper 17 cells (Th17)/regulatory T cells (Treg) balance via the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Here, we provide the first transcriptomic characterization of lesion infiltrating immune cells which illustrates a distinct interplay network between adaptive and innate immune cells. It helps discover new regulators of local immune response, which potentially will provide a novel path forward to further uncover pemphigus pathological mechanisms and develop targeted therapy.

Arsenic trioxide replacing or reducing chemotherapy in consolidation therapy for acute promyelocytic leukemia (APL2012 trial).

As all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are widely accepted in treating acute promyelocytic leukemia (APL), deescalating toxicity becomes a research hotspot. Here, we evaluated whether chemotherapy could be replaced or reduced by ATO in APL patients at different risks. After achieving complete remission with ATRA-ATO-based induction therapy, patients were randomized (1:1) into ATO and non-ATO groups for consolidation: ATRA-ATO versus ATRA-anthracycline for low-/intermediate-risk patients, or ATRA-ATO-anthracycline versus ATRA-anthracycline-cytarabine for high-risk patients. The primary end point was to assess disease-free survival (DFS) at 3 y by a noninferiority margin of -5%; 855 patients were enrolled with a median follow-up of 54.9 mo, and 658 of 755 patients could be evaluated at 3 y. In the ATO group, 96.1% (319/332) achieved 3-y DFS, compared to 92.6% (302/326) in the non-ATO group. The difference was 3.45% (95% CI -0.07 to 6.97), confirming noninferiority (P < 0.001). Using the Kaplan-Meier method, the estimated 7-y DFS was 95.7% (95% CI 93.6 to 97.9) in ATO and 92.6% (95% CI 89.8 to 95.4) in non-ATO groups (P = 0.066). Concerning secondary end points, the 7-y cumulative incidence of relapse (CIR) was significantly lower in ATO (2.2% [95% CI 1.1 to 4.2]) than in non-ATO group (6.1% [95% CI 3.9 to 9.5], P = 0.011). In addition, grade 3 to 4 hematological toxicities were significantly reduced in the ATO group during consolidation. Hence, ATRA-ATO in both chemotherapy-replacing and -reducing settings in consolidation is not inferior to ATRA-chemotherapy (https://www.clinicaltrials.gov/, NCT01987297).

[Post-translational Modifications of PML in Regulating the Functions of Nuclear Bodies --Review].

Abstract　　The promyelocytic leukemia (PML) gene encoded PML protein as a tumor suppressor protein, plays important roles in the occurrence and development of various cancers including acute promyelocytic leukemia. Recent studies have indicated that there are a variety of post-translational modifications of the PML protein, such as SUMOylation, ubiquitination, phosphorylation, and acetylation in cells. These modifications of the PML protein can directly affect the formation of PML nuclear bodies (PML-NBs), repair DNA damage, and modulate cell apoptosis. Furthermore, the abnormal modifications of PML not only result in the occurrence of hematopoietic tumors, but also are closely related to the drug-resistance of cancer. Therefore, investigating the post-translational modifications of PML is significant to uncover the mechanism of formation and functions of PML-NBs, thus contributing to the prevention and treatment of related hematopoietic tumors. In this review, the characteristics of the post-translational modifications of PML protein and the relationship between these modifications and functions of PML-NBs are summarized so as to provide the potential targets for the treatment of related cancers.

Homoharringtonine deregulates MYC transcriptional expression by directly binding NF-κB repressing factor.

Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+; LICs) in a t(8;21) murine leukemia model and exerts a down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). We discovered that NF-κB repressing factor (NKRF) is bound directly by HHT via the second double-strand RNA-binding motif (DSRM2) domain, which is the nuclear localization signal of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding sites to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from the nucleus (including nucleoli) to the cytoplasm by occupying the DSRM2 domain, strengthens the p65-NKRF interaction, and interferes with p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on the MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in concert with MYC down-regulation. Our work thus identifies a mechanism of action of HHT that is different from, but acts in concert with, the known mode of action of this compound. These results justify further clinical testing of HHT in AML.

Arsenic trioxide at conventional dosage does not aggravate hemorrhage in the first-line treatment of adult acute promyelocytic leukemia.

OBJECTIVES: The arsenic trioxide (ATO) plus all-trans retinoic acid (ATRA) therapy has demonstrated a tremendous success in the first-line treatment of acute promyelocytic leukemia (APL). Actually, early death (ED) is currently thought as a major challenge in APL. ATO has been reported to inhibit platelet function in vitro, and whether it increases the ED rate by exacerbating the hemorrhagic symptoms remains to be investigated. METHODS: Effects of ATO on platelet aggregation and adhesion were evaluated in vitro and in thirty-two complete remission (CR) and four newly diagnosed APL patients. Furthermore, concentrations of plasma total arsenic were monitored in APL patients via ICP-MS. RESULTS: The inhibition of platelet function, either aggregation or adhesion, did occur in vitro when the concentration of ATO reached 2 µmol/L. However, in CR APL patients receiving ATO with normal platelet count, the platelets responded normally when being activated and so did those in the newly diagnosed patients with thrombocytopenia. Our data further showed that the conventional dosage of ATO reached a plasma concentration substantially below the required concentration to inhibit platelets. CONCLUSIONS: In the first-line treatment of APL, the use of ATO is safe and effective and does not compromise the hemostatic potential that may eventually increase ED rate.

Genomic Profiling of Adult and Pediatric B-cell Acute Lymphoblastic Leukemia.

Genomic landscapes of 92 adult and 111 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) were investigated using next-generation sequencing and copy number alteration analysis. Recurrent gene mutations and fusions were tested in an additional 87 adult and 93 pediatric patients. Among the 29 newly identified in-frame gene fusions, those involving MEF2D and ZNF384 were clinically relevant and were demonstrated to perturb B-cell differentiation, with EP300-ZNF384 inducing leukemia in mice. Eight gene expression subgroups associated with characteristic genetic abnormalities were identified, including leukemia with MEF2D and ZNF384 fusions in two distinct clusters. In subgroup G4 which was characterized by ERG deletion, DUX4-IGH fusion was detected in most cases. This comprehensive dataset allowed us to compare the features of molecular pathogenesis between adult and pediatric B-ALL and to identify signatures possibly related to the inferior outcome of adults to that of children. We found that, besides the known discrepancies in frequencies of prognostic markers, adult patients had more cooperative mutations and greater enrichment for alterations of epigenetic modifiers and genes linked to B-cell development, suggesting difference in the target cells of transformation between adult and pediatric patients and may explain in part the disparity in their responses to treatment.

Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways.

RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.

DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells.

The gene encoding DNA methyltransferase 3A (DNMT3A) is mutated in ∼20% of acute myeloid leukemia cases, with Arg882 (R882) as the hotspot. Here, we addressed the transformation ability of the DNMT3A-Arg882His (R882H) mutant by using a retroviral transduction and bone marrow transplantation (BMT) approach and found that the mutant gene can induce aberrant proliferation of hematopoietic stem/progenitor cells. At 12 mo post-BMT, all mice developed chronic myelomonocytic leukemia with thrombocytosis. RNA microarray analysis revealed abnormal expressions of some hematopoiesis-related genes, and the DNA methylation assay identified corresponding changes in methylation patterns in gene body regions. Moreover, DNMT3A-R882H increased the CDK1 protein level and enhanced cell-cycle activity, thereby contributing to leukemogenesis.

[Unraveling the chromosome higher-order structures in the transcriptional regulation of hematopoiesis by chromosome conformation capture].

Emerging evidence has demonstrated that genomes are organized into higher-order structures in vivo and long range interactions between genomic regions largely contribute to the regulation of gene expression. Hematopoiesis, orchestrated by the precise spatial regulation and organization of hematopoietic transcription factors, serves as a good model for exploring these issues. The chromosome conformation capture (3C) methodology and its high throughput based technology provide an innovative solution for analyzing the regulation of functional elements through inter-chromosomal and intra-chromosomal interactions, and contacts of functional components in nuclei, thus leading to a more comprehensive understanding of human genome and gene expression. This review focuses on the recent progress of 3C and its derivatives, and their applications in unraveling the mechanisms of transcriptional regulation in hematopoiesis.

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide.

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34(+) cells, especially the LSC-enriched CD34(+)CD38(-) subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34(+) cells but not those from normal CD34(+) cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide-down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.

[Transcriptional regulation of BHLHB2 induced by PML-RARα].

Objective of this study was to investigate the transcriptional regulation of BHLHB2 gene by the PML-RARα fusion protein in APL cells and reveal the pathogenesis of APL. RT-PCR was performed to detect the expression change of BHLHB2 before and after the induction of PML-RARα in PR9 cells, and its expression level after the treatment of ATRA in PR9 and APL patient derived NB4 cells. Chromatin immunoprecipitation (ChIP)-based PCR was used to analyze whether the BHLHB2 promoter could be bound by PML-RARα in vivo. A large-scale gene expression profile dataset was used to observe the expression pattern of BHLHB2 in AML. The results showed that the expression level of BHLHB2 was significantly reduced with the induction of PML-RARα and ATRA could reverse this inhibition in both PR9 and NB4 cells and increase the expression of BHLHB2. However, the expression of BHLHB2 could not be induced by ATRA in U937 cells which do not express PML-RARα. Mechanism study revealed that PML-RARα could bound to the promoter of BHLHB2 in vivo to regulate the the expression of BHLHB2. It was found that the expression of BHLHB2 was relatively lower in APL as compared with other subtypes of AML and normal bone marrow cells. It is concluded that BHLHB2 is the target of PML-RARα, and the expression of BHLHB2 is inhibited by PML-RARα through binding to its promoter in APL.

[Expression of human Jagged-1 protein on eukaryotic cells and establishment of stable transfectant cell line].

Jagged-1 protein is one of the ligands belonging to Notch signaling pathway. Notch signaling pathway is one of the major signaling pathways mediated by contact between cells and plays an important role to regulate the process of proliferation and differentiation of hematopoietic cells in the hematopoietic microenvironment. To study the biological effect after the combination of receptor and ligand in Notch signaling pathway and the mechanism of Notch signaling pathway in bone marrow stromal cells mediated-drug resistance, a NIH-3T3 cell line over-expressing Jagged-1 protein was constructed for further research purposes. A full coding region of Jagged-1 gene was cloned and inserted into eukaryotic expression plasmid to construct pEGFP-IRES2-Jagged-1 eukaryotic expression vector, then transfected into NIH-3T3 cell line, a mammalian cells. As a result Western blot analysis confirmed that the transfectant NIH-3T3 cells highly expressed Jagged-1 protein and flow cytometry analysis confirmed that the NIH-3T3-pEGFP-IRES2-Jagged-1 cell line over-expressed Jagged-1 protein was monoclonal after screened by selective medium and limiting dilution analysis. It is concluded that the pEGFP-IRES2-Jagged-1 eukaryotic expression vector and a stable transfectant monoclonal NIH-3T3 cell line are successfully established. The construction of the stable transfectant monoclonal NIH-3T3 cell line which overexpressed Jagged-1 protein, provides the conditions to further study the mechanism of the bone marrow stromal cell-mediated drug resistance and to discover the new drug targets.

[Influence of AML1 and AML1-ETO on transcriptional regulation of nucb2 gene].

This study was purposed to investigate the effect of AML1-ETO fusion protein resulted from hematopoietic transcription factor (AML1) and acute myeloid leukemia M(2b)(AML-M(2b)) on transcription activity of nucleobindin 2 (nucb2) promoter, and to explore the role of AML1-ETO in molecular pathogenesis of AML-M(2b). The real-time RT-PCR was used to study the regulation of AML1-ETO on nucb2 at transcription level in AML1-ETO inducible leukemia cell line, the chromatin immunoprecipitation (ChIP)-based qPCR was used to investigate the direct in vivo interaction between the AML1, AML1-ETO and nucb2 promoter in AML1-ETO positive leukemia cell line, the luciferase report gene assay was used to detect the regulation of AML1, AML1-ETO on the transcription activity of nucb2 promoter. The results showed that the expression level of nucb2 was reduced with the increase of AML1-ETO. The promoter of nucb2 could be bound by both AML1 and AML1-ETO. The promoter of nucb2 was trans-repressed by AML1 and AML1-ETO respectively. It is concluded that the nucb2 is the direct target gene of AML1 and AML1-ETO, the transcription regulation of AML1, AML1-ETO on nucb2 is carried out via repressing its promoter activity.

Gene expression profiles of thymic neuroendocrine tumors (carcinoids) with ectopic ACTH syndrome reveal novel molecular mechanism.

Although there has been increased knowledge about the molecular biology of neuroendocrine tumors (NETs), little is known about thymic carcinoids and even less about those with excessive hormone disorders, such as ectopic ACTH syndrome. This study was designed to gain insights into the molecular networks underlying the tumorigenesis of thymic carcinoids with ACTH secretion. By an approach integrating cDNA microarray and methods of computational biology, we compare gene expression profile between ACTH-producing thymic carcinoids and the normal thymus. In total, there are 63 biological categories increased and 108 decreased in thymic carcinoids. Cell proliferation was stimulated, which may explain the relatively uncontrolled cell growth of the tumor. Dysregulation of the Notch-signaling pathway was likely to be underlying the neuroendocrine features of this type of tumors. Moreover, inhibition of immunity and increased neuropeptide signaling molecules (POMC and its sorting molecule CPE) made the clinical manifestation reasonable and thus validated the array data. In conclusion, thymic carcinoids have a distinct gene expression pattern from the normal thymus, and they are characterized by deregulations of a series of biofunctions, which may be involved in the development of NETs. Hence, this study has provided not only a detailed comprehension of the molecular pathogenesis of thymic carcinoids with ectopic ACTH syndrome, but also a road map to approach thymic NETs at the system level.

[Analysis of gene expression profiles in gastric cancer cell cycle].

OBJECTIVE: To detect the gene expression profile in gastric cancer cell cycle and explain the mechanism of gastric cancer cell proliferation by a genomic study. METHODS: Gastric cancer cells MKN45 were synchronized at G2/M and G1/S point by nocodazole-thymidine and double thymidine methods. The synchronizing degree of cells was monitored by flow cytometry. The gene expression profiles at G2/M point, M/G1 transition, G1 early phase, G1 late phase, G1/S point, S early phase, S late phase, G2 early phase and G2 late phase in MKN45 cell cycling were examined using cDNA microarray chips. Hierarchy analysis was conducted with a professional software package and the up-regulated genes at G1 late and G2 phase were analyzed according to gene database. Furthermore, the mRNA level of cyclin E, cyclin B, plk1 and STK15 in above mentioned nine points were measured by quatitative PCR. RESULTS: 2001 genes were detected to be available at all 9 points via software processing, out of which 959 appeared up-regulated or down-regulated. 379 genes showed to be up-regulated at late G1 (147) or G2 phases (232), 40 at S and M phases (also up-regulated at G1 late and G2 phases). The 147 up-regulated genes at G1 late phase are involved in DNA metabolism, transcription and translation, protein transportation, ubiquitination and signal transduction, etc. The 232 up-regulated genes in G2 phase are involved in RNA synthesis and processing, intracellular protein transportation, cytoskeleton synthesis, signal transduction, apoptosis and anti-apoptosis, transcription regulation, ubiquitination, mitosis regulation and oncogene expression, etc. The mRNA level of 4 genes detected by quantitative PCR during cell cycle was in agreement with that detected by microarray. CONCLUSION: During MKN45 cell cycling, the preparation for DNA synthesis and chromosome separation are conducted in G1 and G2, which are implicated in multiple genes, may be the main impetus of driving MKN45 cell cycle. Some of these genes may be related to tumor over-proliferation. The cDNA microarray technique has characteristic features such as reliability and can provide a great deal for future research on cell cycle related genes in gastric cancer.

[Analysis of gene expression profile of G1/S transition in gastric cancer cell cycle].

OBJECTIVE: To detect the variation of gene expression profile of G1/S transition and elucidate the role of related genes regulating cell cycle from G1 to S phase in gastric cancer. METHODS: Nocodazole-thymidine and double thymidine methods were used to synchronize gastric cells at G2/M and G1/S point, cDNA microarray chips was applied to examine the gene expression profile at G1 early and late phase, S early and late phase during the cell cycle, hierarchy analysis was conducted by a professional software package. RESULTS: A total of 2001 genes were detected available, 959 genes appeared to be upregulated or downregulated, including 147 genes upregulated at G1 late phase. These 147 genes are involved in DNA metabolism, transcription and translation,posttranslational modification, ubiquitination, signal transduction etc, which all affected cell cycle from different aspects. CONCLUSION: Complex multiple gene processes, such as DNA metabolism, transcription and translation, posttranslational modification, ubiquitination, signal transduction etc,are implicated in and also essential for G1/S transition during gastric cancer cell cycle, part of these genes are significantly associate with overproliferation in gastric cancer.

Benzodithiophenes potentiate differentiation of acute promyelocytic leukemia cells by lowering the threshold for ligand-mediated corepressor/coactivator exchange with retinoic acid receptor alpha and enhancing changes in all-trans-retinoic acid-regulated gene expression.

Differentiation induction is an effective therapy for acute promyelocytic leukemia (APL), which dramatically responds to all-trans-retinoic acid (ATRA). Recent studies have indicated that combinatorial use of retinoid and nonretinoid compounds, such as histone deacetylase inhibitors, arsenics, and PKA agonists, has higher therapeutic value in this disease and potentially in other malignancies. In a screen of 370 compounds, we identified benzodithiophene analogues as potent enhancers of ATRA-induced APL cell differentiation. These effects were not associated with changes in global histone acetylation and, for the most potent compounds, were exerted at very low nanomolar concentrations, and were paralleled by enhancement of some, but not all, ATRA-modulated gene expressions. Investigating the mechanism underlying the effects of these drugs on ATRA-induced APL cell differentiation, we have shown that benzodithiophenes enhance ATRA-mediated dissociation and association of corepressor N-CoR and coactivator p300 acetyltransferase, respectively, with retinoic acid receptor (RAR) alpha proteins. These data suggest that benzodithiophenes act at the level of receptor activation, possibly by affecting posttranslational modification of the receptor (and/or coregulators), thus leading to an enhancement in ATRA-mediated effects on gene expression and APL cell differentiation. Given the specificities of these low benzodithiophene concentrations for PML-RARalpha and RARalpha, these drugs may be useful for combinatorial differentiation therapy of APL and possibly other acute myelogenous leukemia subtypes in which the overall ATRA signaling is suppressed.

Systems analysis of transcriptome and proteome in retinoic acid/arsenic trioxide-induced cell differentiation/apoptosis of promyelocytic leukemia.

Understanding the complexity and dynamics of cancer cells in response to effective therapy requires hypothesis-driven, quantitative, and high-throughput measurement of genes and proteins at both spatial and temporal levels. This study was designed to gain insights into molecular networks underlying the clinical synergy between retinoic acid (RA) and arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), which results in a high-quality disease-free survival in most patients after consolidation with conventional chemotherapy. We have applied an approach integrating cDNA microarray, 2D gel electrophoresis with MS, and methods of computational biology to study the effects on APL cell line NB4 treated with RA, ATO, and the combination of the two agents and collected in a time series. Numerous features were revealed that indicated the coordinated regulation of molecular networks from various aspects of granulocytic differentiation and apoptosis at the transcriptome and proteome levels. These features include an array of transcription factors and cofactors, activation of calcium signaling, stimulation of the IFN pathway, activation of the proteasome system, degradation of the PML-RARalpha oncoprotein, restoration of the nuclear body, cell-cycle arrest, and gain of apoptotic potential. Hence, this investigation has provided not only a detailed understanding of the combined therapeutic effects of RA/ATO in APL but also a road map to approach hematopoietic malignancies at the systems level.

[Mechanisms of fenretinide-triggered apoptosis in leukemic cells].

The retinoid N-4-hydroxyphenyl retinamide (4-HPR also known as fenretinide), a synthetic derivative of all trans retinoic acid (ATRA), has shown as an efficient chemopreventive, chemotherapeutic agent and a potent inducer of apoptosis in various cancer cell types in vitro, including leukemic cells. However the mechanisms by which 4-HPR has the apoptotic effects is not completely elucidated. This study was aimed to investigate the effect of 4-HPR on several leukemic cells and explore its mechanisms of effect on U937 cells. The cell growth and proliferation experiments were performed [corrected] cell apoptosis was detected by annexin V; reactive oxygen species (ROS) and mitochondrial transmembrane potential (DeltaPsim) were determined; protein [corrected] expression was detected by Western blot. The results showed that 4-HPR inhibited the proliferation of U937 cells in a dose- and time-dependent manner. 4-HPR markedly [corrected] induced apoptosis in U937 cells, triggered the generation of ROS, induced the loss of mitochondrial transmembrane potential, decreased the expression of procaspase-8 and procaspase-3. Pretreatment of L-ascorbic acid suppressed the generation of ROS, disruption of mitochondrial potential, activation of caspases and apoptosis. It is concluded that the generation of ROS followed by the disruption of mitochondrial transmembrane potential plays an important role on 4-HPR-induced apoptosis in leukemic cells, suggesting that 4-HPR may be one of mitochondrial-targeted agents with clinical potential in treating cancer.