Programmed Cell Death-4 Tumor Suppressor Protein Contributes to Retinoic Acid–Induced Terminal Granulocytic Differentiation of Human Myeloid Leukemia Cells

Abstract Programmed cell death 4 (PDCD4) is a recently identied novel tumor suppressor protein that inhibits cap-dependent mRNA translation. PDCD4 inhibits tumor promoter incuced carcinogenesis and transformation by suppressing the helicase activity of eIF4A, leading to translational inhibition. Recently we found that PDCD4 is required for all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of acute promyelocytic leukemia (APL) cells (Ozpolat&Akar et al, Mol Cancer Res, in press), type of acute myeloid leukemia characterized by a t(15;17) and a differentiation block. Here we investigated the downstream mediators or targets of PDCD4 in leukemia cell differentiation. ATRA is currently used as a first line standard treatment in APL. Recently, we reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms that involve suppression of translation initiation, a rate limiting step of protein synthesis (Harris&Ozpolat et al, Blood, 104 (5) 2004). We found that ATRA treatment induced PDCD4 expression in NB4 APL, HL60 AML, primary APL patient leukemia cells and normal human CD34+ bone marrow progenitors cells during granulocytic differentiation. However, ATRA/maturation resistant NB4.R1 and HL60R cells failed to express and translocate PDCD4 into nucleus after ATRA treatment. To identify downstream targets of PDCD4 we knock downed PDCD4 expression by siRNA and examined changes in the expression of target proteins that are known to be regulated by ATRA by Western blot analysis in NB4 cells. We found that PDCD4 represses c-myc, and WT1 expression however, it is required for the expression of DAP5 (death associated protein 5), and cyclin dependent kinase inhibitor p27KIP1, and but not c-jun, p21Cip1, and tissue transglutaminase (TG2). Inhibition of PDCD4 by siRNA resulted in upregulation of phospho-P70S6K, suggesting that PDCD4 inhibits activity of PI3K/Akt pathway. RT-PCR analysis revealed that mRNA of these proteins did not change suggesting that PDCD4 tumor suppressor protein regulates expression of these important cellular proteins at translational level and suppresses PI3K/Akt pathway. Furthermore we rapamycin, a specific mTOR inhibitor currently in clinical trials in AML, induced a marked expression of PDCD4, which regulates c-myc and p27 Kip1, revealing a novel mechanism of action of rapamycin, providing new rationale for targeting translational pathways as a therapeutic intervention in the treatment of AML. Overall, data suggest that PDCD4 regulates expression of critical cellular proteins involved in differentiation of leukemia cells and PDCD4 mediated translational control may be an important regulatory mechanism for regulation of gene expression.

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Redox Regulation of Retinoic Acid Receptor Function in Myeloid Leukemia Cell Differentiation and Apoptosis.

Blood ◽

10.1182/blood.v106.11.2483.2483 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2483-2483

Author(s):

Kent A. Robertson ◽

Meihua Luo ◽

Ashley Chastain ◽

Scott Colvin ◽

April Reed ◽

...

Keyword(s):

Cell Death ◽

Retinoic Acid ◽

Programmed Cell Death ◽

Growth Inhibition ◽

Myeloid Leukemia ◽

Redox Regulation ◽

Retinoic Acid Receptor ◽

Leukemia Cell ◽

Granulocytic Differentiation ◽

Acid Receptor

Abstract Ape1/ref-1 is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFkB, PAX, HIF-1a, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that Ape1/ref-1 expression is related to apoptosis. To further define this relationship, we used retroviral gene transduction to over-express HA-tagged Ape1/ref-1 in HL-60 myeloid leukemia cells. We observed that the RA-induced growth inhibition of HL-60 cells over-expressing Ape1/ref-1 was significantly enhanced compared to wild type HL-60 cells. To determine if the growth inhibition was related to enhanced programmed cell death and differentiation, we treated Ape1/ref-1 transduced and vector-only (LXSN) transduced HL-60 cells with RA and evaluated the expression of Ape1/ref-1 and the development of apoptosis and markers of differentiation. Results: 1) RA induced expression of the retroviral Ape1/ref-1 construct as determined by Western blot resulting in a higher (ie retroviral + endogenous Ape1/ref-1) overall expression of Ape1/ref-1 compared to control cells; 2) analysis of RA-treated cells for apoptosis by propidium iodide, TUNEL, and Annexin V staining as well as morphology, unexpectedly demonstrated enhanced programmed cell death in cells expressing the transduced Ape1/ref-1; 3) Ape-1 over-expression enhanced the retinoid differentiation response by morphology and expression of CD11b. Additional mobility shift experiments demonstrated the redox dependence of retinoic acid receptor binding to retinoid response elements mediated by Ape-1/ref-1. In conclusion, our data supports the contention that Ape1/ref-1 expression may be important for mediating RA-induced myeloid differentiation and programmed cell death.

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Programmed cell death 4 and BCR-ABL fusion gene expression are negatively correlated in chronic myeloid leukemia

Oncology Letters ◽

10.3892/ol.2016.4942 ◽

2016 ◽

Vol 12 (4) ◽

pp. 2976-2981 ◽

Cited By ~ 2

Author(s):

Xia Zhang ◽

Riming Liu ◽

Baohua Huang ◽

Xiaolu Zhang ◽

Weijuan Yu ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

Chronic Myeloid Leukemia ◽

Programmed Cell Death ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Programmed Cell Death 4

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MicroRNA (miRNA)-to-miRNA Regulation of Programmed Cell Death 4 (PDCD4)

Molecular and Cellular Biology ◽

10.1128/mcb.00086-19 ◽

2019 ◽

Vol 39 (18) ◽

Cited By ~ 4

Author(s):

Pamela Ajuyah ◽

Meredith Hill ◽

Alireza Ahadi ◽

Jing Lu ◽

Gyorgy Hutvagner ◽

...

Keyword(s):

Cell Death ◽

Tumor Suppressor ◽

Programmed Cell Death ◽

Tumor Suppressor Genes ◽

Untranslated Region ◽

Inhibitory Concentration ◽

Mutational Analysis ◽

Mirna Regulation ◽

Programmed Cell Death 4 ◽

The Relationship

ABSTRACT The regulation of tumor suppressor genes by microRNAs (miRNAs) is often demonstrated as a one-miRNA-to-one-target relationship. However, given the large number of miRNA sites within a 3′ untranslated region (UTR), most targets likely undergo miRNA cooperation or combinatorial action. Programmed cell death 4 (PDCD4), an important tumor suppressor, prevents neoplastic events and is commonly downregulated in cancer. This study investigates the relationship between miRNA 21 (miR-21) and miR-499 in regulating PDCD4. This was explored using miRNA overexpression, mutational analysis of the PDCD4 3′ UTR to assess regulation at each miRNA site, and 50% inhibitory concentration (IC50) calculations for combinatorial behavior. We demonstrate that the first miR-499 binding site within PDCD4 is inactive, but the two remaining sites are both required for PDCD4 suppression. Additionally, the binding of miR-21 to PDCD4 influenced miR-499 activity through an increase in its silencing potency and stabilization of its mature form. Furthermore, adjoining miRNA sites more than 35 nucleotides (nt) apart could potentially regulate thousands of 3′ UTRs, similar to that observed between miR-21 and miR-499. The regulation of PDCD4 serves as a unique example of regulatory action by multiple miRNAs. This relationship was predicted to occur on thousands of targets and may represent a wider mode of miRNA regulation.

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Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

PLoS ONE ◽

10.1371/journal.pone.0052143 ◽

2013 ◽

Vol 8 (1) ◽

pp. e52143 ◽

Cited By ~ 9

Author(s):

Mahban Irandoust ◽

Julian Alvarez Zarate ◽

Isabelle Hubeek ◽

Ellen M. van Beek ◽

Karin Schornagel ◽

...

Keyword(s):

Cell Death ◽

Acute Myeloid Leukemia ◽

Programmed Cell Death ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

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Inhibition of the Redox Function of Ape-1/ref-1 in Myeloid Leukemia Cells Results in Enhanced Sensitivity to Retinoic Acid Induced Differentiation and Apoptosis.

Blood ◽

10.1182/blood.v110.11.4217.4217 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4217-4217

Author(s):

Kent A. Robertson ◽

Edwin S. Colvin ◽

Mark R. Kelley

Keyword(s):

Flow Cytometry ◽

Retinoic Acid ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Minimal Amount ◽

Morphologic Change ◽

Similar Response ◽

Moderate Increase ◽

Granulocytic Differentiation ◽

Cell Counts

Abstract Ape-1/ref-1 is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFκB, PAX, HIF-1α, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that elevated Ape-1/ref-1 expression is related to differentiation and apoptosis. To further define the role of the redox function of Ape-1/ref-1 in this relationship, redox function was blocked using two techniques. First, we used retroviral gene transduction to over-express a redox-inactive C-65 mutant of Ape1/ref-1 in HL-60 myeloid leukemia cells and examined the response to retinoic acid. In a second set of experiments we used an Ape-1/ref-1 specific small molecule inhibitor to pharmacologically block the redox function and again examined the response to retinoic acid. Differentiation was evaluated by morphologic change in differential cell counts and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis was examined with propidium iodide flow cytometry. Results: HL-60 cells expressing high levels of C-65 Ape-1/ref-1 responded to retinoic acid with a significantly higher level of differentiation and a moderate increase in apoptosis. Pharmacologic blockade of Ape-1/ref-1 redox function resulted in a profound increase in differentiation and a moderate increase in apoptosis compared to controls. dose dependent studies with retinoic acid demonstrated a similar degree of differentiation (CD11b expression) in cells treated with 10 μmolar retinoic acid and those treated with the redox inhibitor + 0.1 μmolar retinoic acid; alllowing HL-60 cells in the presence of the redox inhibitor to give a similar response to a 100 fold lower dose of retinoic acid. The redox inhibitor alone did not induce differentiation and induced only a minimal amount of apoptosis but did increase the number of cells in S phase significantly. In conclusion, our data supports the contention that redox function of Ape-1/ref-1 may be important for controlling RA-induced myeloid differentiation and programmed cell death. The implication of these findings is that myeloid leukemia cells may be sensitized to retinoids by manipulation of the redox status of Ape-1/ref-1.

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PKCδ Regulates Eukaryotic Initiation Factor eIF2α through PKR during Retinoic Acid-Induced Myeloid Cell Differentiation.

Blood ◽

10.1182/blood.v108.11.1928.1928 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1928-1928

Author(s):

Bulent Ozpolat ◽

Ugur Akar ◽

Magaly Barria ◽

Gabriel Lopez-Berestein

Keyword(s):

Retinoic Acid ◽

Cell Differentiation ◽

Protein Kinase ◽

Translation Initiation ◽

Myeloid Leukemia ◽

Mrna Translation ◽

Leukemia Cells ◽

Granulocytic Differentiation ◽

Nb4 Cells

Abstract Dysregulation of mRNA translation can contribute to malignant transformation. Translation initiation is a rate limiting step of mRNA translation and protein synthesis and plays a critical role in regulation of cell growth, proliferation and differentiation. We previously reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms during terminal cell differentiation detected by proteomic analysis (Harris et al, Blood, 104 (5) 2004). Here we investigated the regulation of translation initiation and the role of eIF2α during terminal differentiation of myeloid leukemia cells. We found that ATRA and other granulocytic differentiation inducing agents, such as dimethyl sulfoxide (DMSO), arsenic trioxide (ATO) induce phosphorylation of eIF2α on serine 51 in promyelocytic leukemia (NB4) cells, indicating the suppression of translation initiation. However, monocytic/macrophagic differentiation of NB4 cells by phorbol 12-myristate 13-acetate (phorbol ester, PMA), or by ATRA in U937 and THP-1 myelomonoblastic myeloid leukemia (AML) cells, was not accompanied with induction of eIF2α phosphorylation. ATRA, ATO or DMSO-induced granulocytic differentiation closely correlated with induction of expression and phosphorylation/activation of protein kinase C-delta (PKCδ) on threonin 505 and serine 643 in NB4 cells. The specific PKCδ inhibitor, rottlerin, markedly inhibited ATRA-induced expression and phosphorylation (serin 51) of eIF2a in NB4 cells. Rottlerin reduced phosphorylation of eIF2α expression not only in the leukemia cells but also in solid tumor cells such as breast (MCF7) and pancreatic (Panc28) cancer cells. Because protein kinase R (PKR) has been shown to inhibit mRNA translation by inducing phosphorylation of eIF2α, we also examined whether this pathway is involved in ATRA-induced phosphorylation of eIF2α and whether it is downstream of PKCδ. We observed that ATRA induces expression and phosphorylation/activation of PKR in NB4 cells. Rottlerin inhibited ATRA-induced expression and activity of PKR , suggesting that activity of PKR is regulated by PKCδ in response to ATRA in NB4 cells. Overall, our data suggest that retinoic acid suppresses translation initiation through PKCδ/PKR/eIF2α pathway during granulocytic but not monocytic differentiation of acute myeloid leukemia cells. These results revealed a novel role of ATRA in granulocytic cell differentiation of myeloid cells. Because malignant cells usually have hyperactivated mRNA translation, targeting translational factors/regulators of initiation may offer new strategies for the treatment of myeloid leukemia cells.

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Induction of Both CCAAT/Enhancer Binding Protein β and ε Are Required for All Trans Retinoic Acid-Induced Granulocytic Differentiation of Myeloid Leukemia Cells.

Blood ◽

10.1182/blood.v110.11.3459.3459 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3459-3459

Author(s):

Min Lu ◽

Lijuan Xia ◽

Alan D. Friedman ◽

Samuel Waxman ◽

Yongkui Jing

Keyword(s):

Retinoic Acid ◽

Myeloid Leukemia ◽

K562 Cells ◽

Leukemia Cells ◽

Atra Treatment ◽

Granulocytic Differentiation ◽

Differentiation Induction ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Enhancer Binding Protein

Abstract All trans retinoic acid (ATRA) induces remission in patients with acute promyelocytic leukemia (APL) by induction of granulocytic differentiation. Since CCAAT/enhancer binding protein (C/EBP) α, β and ε play important roles in normal granulocytic differentiation we compared their expression and regulation in ATRA differentiation inducible NB4 and HL-60 cells to their ATRA differentiation resistant subclones R4 and HL-60/Res cells. All four cell lines robustly express C/EBPα but have low or absent C/EBPβ and ε expression. ATRA treatment increases the levels of C/EBPβ and ε protein in NB4 and HL-60 cells but not in the R4 and HL-60/Res cells which is correlated with the degree of differentiation induction. Knockdown of C/EBPβ or ε using shRNA decreases ATRA differentiation induction of HL-60 cells. HL-60 cells with BCR-ABL stable transfection lose expression of C/EBPα, and ATRA-induced differentiation and expression of C/EBPβ and ε are no longer seen. K562 cells which express BCR-ABL do not have detectable or inducible C/EBPα, β and ε after ATRA treatment and are resistant to ATRA differentiation induction. Ectopic expression of C/EBPα-estrogen receptor (ER) or C/EBPβ-ER, but not C/EBPε-ER, induces granulocytic differentiation in K562 cells after addition of estradiol or tamoxifen which activates these fusion factors. C/EBPα-ER or C/EBPβ-ER infected K562 cells is followed by induction of C/EBPε expression and knockdown of C/EBPε in C/EBPα-ER or C/EBPβ-ER infected K562 cells decreases induction of differentiation. The induction of C/EBPα, β, and ε expression by ATRA is investigated in ten additional myeloid leukemia cell lines and it is found that the expression of C/EBPβ and ε, but not C/EBPα, are induced in THP-1 and ML-1 cells which are responsive to ATRA differentiation induction. These results indicate that induction of C/EBPβ is required and C/EBPε plays a collaborative role with C/EBPβ in ATRA-induced differentiation of myeloid leukemia cells.

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PKCδ Regulates Retinoic Acid-Induced Myeloid Cell Differentiation through Eukaryotic Initiation Factor Alpha (eIF2α).

Blood ◽

10.1182/blood.v110.11.1814.1814 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1814-1814

Author(s):

Bulent Ozpolat ◽

Ugur Akar ◽

Magaly Barria ◽

Gabriel Lopez-Berestein

Keyword(s):

Retinoic Acid ◽

Cell Differentiation ◽

Myeloid Leukemia ◽

Initiation Factor ◽

Leukemia Cells ◽

Eukaryotic Initiation Factor ◽

Granulocytic Differentiation ◽

Nb4 Cells ◽

Factor Alpha ◽

Expression And Activity

Abstract Overactivity of eukaryotic initiation factor-alpha (eIF2α) has been shown to be oncogenic and induces malignant transformation. Here we investigated the regulation and the role of eIF2α in the terminal differentiation of myeloid leukemia cells. We found that all-trans-retinoic acid (ATRA) and other granulocytic differentiation inducers, such as dimethylsulfoxide and arsenic trioxide inhibited activity of eIF2α by inducing serine 51 phosphorylation in promyelocytic leukemia cells (NB4). In contrast, activity of eIF2α was unaffected during ATRA-induced monocytic differentiation of U937 and THP-1 myelomonocytic cells and phorbol 12-myristate 13-acetate-induced monocytic/macrophagic differentiation of NB4 cells. Knockdown of eIF2α by RNA interference (siRNA) significantly inhibited (p<0.05) ATRA-induced differentiation, indicating that eIF2α is critical for the induction of granulocytic differentiation. ATRA-induced eIF2α phosphorylation was correlated with the expression and activity/phosphorylation (Thr505 and Ser643) of protein kinase Cδ (PKCδ) and eIF2a kinase PKR (Thr446). The specific PKCδ inhibitor Rottlerin significantly reduced phosphorylation of eIF2α, activity of PKR and blocked ATRA-induced granulocytic differentiation of NB4 cells (p<0.05). Knockdown of PKCδ by siRNA decreased PKR activity and increased eIF2α activity while knockdown of PKR increased eIF2α activity. We also observed that PKCδ regulates eIF2a activity in normal CD34+ bone marrow progenitor cells and breast and pancreatic cancer cell lines. Furthermore, we found that eIF2α regulated the expression and activity of important targets of ATRA, including c-myc, p21Waf1/Cip1, DAP5, GADD153, ATF-2, TG2, and p-P70S6K. In conclusion, our findings indicate that ATRA-induced granulocytic differentiation of myeloid leukemia cells is regulated by PKCδ through the activity of eIF2α, revealing a novel mechanism of granulocytic cell differentiation. Figure Figure

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