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.
Humans ; Intranuclear Inclusion Bodies ; Leukemia, Promyelocytic, Acute ; Nuclear Proteins ; Promyelocytic Leukemia Protein ; Protein Processing, Post-Translational

The promyelocytic leukemia (PML) was originally identified and named as acute promyelocytic leukaemia (APL) . The PML, encoded by PML gene, locates in the nuclear body (NB) and shuttles in the cell nucleus-cytoplasm, so that PML completes many regulation functions. There are many research on the function of nuclear PML, but in recent years the foreign data indicate that cytoplasmic PML gene plays an important role in hematologic malignancies and solid tumors. In this article, the biological functions of PML gene in cytoplasm are reviewed.
Cytoplasm ; genetics ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; Nuclear Proteins ; genetics ; Promyelocytic Leukemia Protein ; Transcription Factors ; genetics ; Tumor Suppressor Proteins ; genetics

Acute promyelocytic leukemia (APL) is characterized by a specific t (15;17) translocation which produce a PML/RAR-alpha fusion messenger RNA and by effectiveness of all-trans retinoic acid (ATRA) differentiation therapy. Breakpoints within PML intron 3 (bcr 3) produce a short PML/RAR-alpha isoform (S-isoform), whereas breakpoints within PML intron 6 (bcr 1) result in a longer form (L-isoform). Additionally, breakpoints within PML exon 6 (bcr 2) make a variable length transcript (V-isoform) in a small number of patients. The influence of breakpoint site on patient outcome remains controversial. Previous reports showed that patients with S-isoform have an increased incidence of clinical relapse and shorter survival compared to those with L-isoform. Others reported no difference in DFS between these patients groups. In this issue, Lee et al. reported that there were 58 L-isoform (62.1%), 32 S-isoform (34.0%), 4 V-isoform (4.3%) and, no significant prognostic factor for EFS from induction therapy using anthracycline plus ATRA among 94 patients with APL. They concluded pretreatment clinical characteristics and treatment outcomes were not significantly different according to PML/RAR-alpha isoform types in this induction group. Recently, it was reported that FLT3/ITD mutation was frequently associated with S-isoform and with the M3v form of leukemia and CNS relapse in APL was mostly related to S-isoform. With previous studies including this article, outcomes of different types of PML/RAR-alpha isoforms are not conclusive. Future researches need to be focused not only on clinical outcomes of different types of PML/RAR-alpha isoforms, but also clinical relevance of PML/RARA-alpha mRNA isoforms with other prognostic factors and particular clinical characteristics.
Exons ; Humans ; Incidence ; Introns ; Leukemia ; Leukemia, Promyelocytic, Acute ; Protein Isoforms ; Recurrence ; RNA Isoforms ; RNA, Messenger ; Tretinoin

OBJECTIVE: To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR). METHODS: By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique. RESULTS: The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected. CONCLUSION A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.
Exosomes ; Gene Expression ; Humans ; Leukemia, Promyelocytic, Acute ; Oncogene Proteins, Fusion ; analysis ; Polymerase Chain Reaction ; Protein Isoforms

BACKGROUND/AIMS: There are three types of PML-RAR alpha mRNA fusion transcripts associated with acute promyelocytic leukemia (APL): the short (S)-form, the long (L)-form and the variable (V)-form. No study on the Korean population has addressed the clinical significance of the specific types of PML-RAR alpha mRNA fusion transcripts for APL patients who receive the combination therapy of all-trans-retinoic-acid and idarubicin (AIDA regimen). METHODS: We performed a retrospective analysis on 94 patients with APL to evaluate differences in the therapeutic outcomes, such as the response rate, an event-free survival (EFS), and overall survival (OS), after remission following the induction of chemotherapy. We also analyzed whether differences in the pretreatment clinical characteristics depend on the PML-RAR alpha isoform. RESULTS: The median age of the patients was 41 years (range 15-85). Among the 94 patients, there were 58 L-form cases (62.1%), 32 S-form cases (34.0%), and 4 V-form cases (4.3%). The CR rate following remission induction treatment was 84.9%. The CR rate was higher in patients with an initial WBC <10.0x109/L, as compared to patients with an initial WBC higher than 10.0X109/L (93.5% vs. 65.4%, p=0.001). The AIDA induction regimen was associated with a better EFS than non-AIDA induction regimens (81.9% vs. 49.6%, p=0.006). The induction group was also a significant prognostic factor for EFS in the multivariate analysis (p=0.020). There were no differences in OS and EFS in patients with either isoform L or isoform S in the AIDA induction group. CONCLUSIONS: This retrospective study demonstrated that pretreatment clinical characteristics and treatment outcomes were not significantly different among patients with varying PML-RAR alpha isoform types in the AIDA induction group.
Disease-Free Survival ; Humans ; Idarubicin ; Leukemia, Promyelocytic, Acute ; Multivariate Analysis ; Protein Isoforms ; Remission Induction ; Retrospective Studies ; RNA, Messenger

Since the successful introduction of all-trans-retinoic acid (ATRA) and its combination with anthracycline-containing chemotherapy, the prognosis for acute promyelocytic leukemia (APL) has markedly improved. With ATRA and anthracycline-based-chemotherapy, the complete remission rate is greater than 90%, and the long-term survival rate is 70-89%. Moreover, arsenic trioxide (ATO), which was introduced for APL treatment in 1994, resulted in excellent remission rates in relapsed patients with APL, and more recently, several clinical studies have been designed to explore its role in initial therapy either alone or in combination with ATRA. APL is a rare disease in children and is frequently associated with hyperleukocytosis, which is a marker for higher risk of relapse and an increased incidence of microgranular morphology. The frequency of occurrence of the promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) isoforms bcr 2 and bcr 3 is higher in children than in adults. Although recent clinical studies have reported comparable long-term survival rates in patients with APL, therapy for APL in children is challenging because of the risk of early death and the potential long-term cardiac toxicity resulting from the need to use high doses of anthracyclines. Additional prospective, randomized, large clinical trials are needed to address several issues in pediatric APL and to possibly minimize or eliminate the need for chemotherapy by combining ATRA and ATO. In this review article, we discuss the molecular pathogenesis, diagnostic progress, and most recent therapeutic advances in the treatment of children with APL.
Adult ; Anthracyclines ; Arsenic ; Arsenicals ; Child ; Humans ; Incidence ; Leukemia, Promyelocytic, Acute ; Oxides ; Prognosis ; Protein Isoforms ; Rare Diseases ; Recurrence ; Survival Rate ; Tretinoin

The promyelocytic leukemia protein (PML), encoded by PML gene, plays a tumor suppressor in acute promyelocytic leukemia and other hematologic malignancies. Recent evidence indicates that PML involves in regulating multiple cell biological function, regulates self-renewal and maintains stable function in stem cell/cancer stem cell of multiple tissues, leading to drug resistance of cancer. This review summarizes the latest research advances about the relationship and therapeutic options between PML and cancer stem cell of hematologic neoplasms, aiming to propose a new avenue for blood cancer treatment.
Hematologic Neoplasms ; blood ; Humans ; Neoplastic Stem Cells ; Nuclear Proteins ; Promyelocytic Leukemia Protein ; Transcription Factors ; Tumor Suppressor Proteins

OBJECTIVE: To investigate the expression of peptidylarginine deiminase 4 (PADI4) during the process of differentiation into granulocyte of NB4 cells induced by all-trans-retinoic acid (ATRA) and whether PADI4 is involved in the inflammatory cytokines expression. METHODS: Granulocyte differentiation model of NB4 cells induced by ATRA was established. The cell morphology changes were observed by Wright-Giemsa staining. The expression of cell differentiation marker CD11b was analyzed by flow cytometry. The mRNA and protein expression of PADI4 was detected by RT-PCR and Western blot, respectively. The expression of tumor necrosis factor (TNF) α and interleukin (IL) 1β was analyzed by ELISA, and also examined with the knockdown of PADI4 expression by siRNA. RESULTS: After NB4 cells induced by ATRA, the cytoplasm increased and the ratio of nuclear to cytoplasmic was reduced. Nuclear dented, and rod-shaped nucleus, lobulated phenomenon increased (P<0.05). Flow cytometry analysis results showed that the cell surface molecule CD11b expression increased (P<0.01). RT-PCR and Western blot showed the expression of PADI4 increased at both transcriptional and translational levels during the process of the differentiation. ELISA showed TNF-α and IL-1β secretion increased in differentiated macrophages, while they could be inhibited by PADI4-specific siRNA. CONCLUSION During the differentiation into granulocyte of NB4 cells induced by ATRA, PADI4 expression increased. Furthermore, PADI4 appeared to play a critical role in inflammatory cytokines secretion.
Cell Differentiation ; Cell Line, Tumor ; Cytokines/metabolism* ; Granulocytes ; Humans ; Leukemia, Promyelocytic, Acute ; Protein-Arginine Deiminase Type 4/metabolism* ; Tretinoin/pharmacology*

Promyelocytic leukemia (PML) protein, a tumor suppressor, plays an important role in patients with acute promyelocytic leukemia (APL) receiving arsenic trioxide (AsO) therapy. APL is a M3 subtype of acute myeloid leukemia (AML), which is characterized by expression of PML-RARα (P/R) fusion protein, leading to the oncogenesis. AsO is currently used as the first-line drug for patients with APL, and the mechanism may be:AsO directly binds to PML part of P/R protein and induces multimerization of related proteins, which further recruits different functional proteins to reform PML nuclear bodies (PML-NBs), and finally it degraded by SUMOylation and ubiquitination proteasomal pathway. Gene mutations may lead to relapse and drug resistance after AsO treatment. In this review, we discuss the structure and function of PML proteins; the pathogenesis of APL induced by P/R fusion protein; the involvement of PML protein in treatment of APL patient with AsO; and explain how PML protein mutations could cause resistance to AsO therapy.
Antineoplastic Agents ; therapeutic use ; Arsenic Trioxide ; therapeutic use ; Drug Resistance, Neoplasm ; genetics ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; Mutation ; Oncogene Proteins, Fusion ; metabolism ; Promyelocytic Leukemia Protein ; chemistry ; genetics ; metabolism

OBJECTIVETo study the changes in expression and activity of protein phosphatases type 2A (PP2A ) during differentiation of NB4 and NB4-MR2 cells induced by all-trans retinoic acid (ATRA), and evaluate the role of PP2A in MR2 resistance to ATRA.

METHODSATRA, okadaic acid (OKA) and ATRA + OKA at the same dosage were incubated with NB4 and MR2 cells respectively. Wright's staining and NBT reduction test were employed to evaluate the change in the cells. The CD11b expression was measured by flow cytometry. The activity of PP2A was evaluated by serine/threonine phosphatase assay system, and the level of PP2A subunits was detected by Western blot.

RESULTS1) Wright's staining, NBT reduction test and flow cytometry results showed OKA could augment the differentiation of NB4 induced by ATRA, and OKA + ATRA induced slight differentiation of MR2 cells. 2) Phosphatase assay showed a decrease in PP2A phosphatase activity [(534 +/- 43) pmol x min(-1) x microg protein(-1)] in NB4 after ATRA treatment, accompanied with that activity [(959 +/- 83) pmol x min(-1) x microg protein(-1)] in untreated NB4 cells. OKA enhanced the inhibitory effect of ATRA on the activity in NB4. When OKA + ATRA was incubated with MR2, PP2A in the cells was significantly decreased [(229 +/- 23) pmol x min(-1) x microg protein(-1)]. 3) Western blot analysis showed that the level of PP2A catalytic subunit (PP2A/C) was decreased during the course of ATRA-induced NB4 cell differentiation, whereas expressions of every subunits of PP2A in MR2 cells were somewhat unaltered.

CONCLUSIONExpression of PP2A/C and activity of PP2A is decreased during differentiation of NB4 induced by ATRA, and no repression of the PP2 activity maybe related to MR2 resistance to ATRA.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Okadaic Acid ; pharmacology ; Phosphoprotein Phosphatases ; metabolism ; Protein Phosphatase 2 ; antagonists & inhibitors ; metabolism ; Tretinoin ; pharmacology