Mutational detection of full-length mixed lineage leukemia gene in patients with de novo AML-M4 and M5 / 中国实验血液学杂志

Abnormalities of chromosome 11 involving mixed lineage leukemia (MLL) on 11q23 are often seen in acute myeloid leukemia (AML)-M5 or AML-M4. The fusion gene of MLL-PTD and MLL plays a critical role in the pathogenesis of these AML. However, rare chromosome abnormalities have been identified in this type of leukemia. To explore whether there were other MLL gene mutations at M4 and M5, in this study all of the MLL exons were sequenced at cDNA level. 25 patients with de novo AML-M4 or M5 with normal karyotypes excluding M4eo and MLL fusion gene or MLL-PTD were selected, the amplification and direct sequencing analysis of full length MLL gene exons were carried out, then the mutations found were verified at genomic DNA level. Furthermore, the point mutations were tested in normal samples and a larger group of AML patients using the platform of Mass Array. The results showed that high-frequency deletion/insertion and point mutations in RD, PHD, TAD and SET domains of MLL were found, while these alterations in normal samples and other subtypes of AML samples were also verified, and without significant difference (P > 0.05). It is concluded that a variety of deletions/insertions in MLL mRNA and point mutations are respectively alternative splicing of MLL gene at transcriptional level and single nucleotide polymorphism. These alternations together constituted genetic polymorphisms of MLL. Although these variations may not play a direct role in the molecular pathogenesis of AML-M4 or M5, their correlations to clinical treatment and prognosis need to be further explored.

Recent progress of study on retroviral mediated mouse model of myeloid leukemia --- review / 中国实验血液学杂志

Human leukemia is closely associated with various genetic alterations such as chromosomal translocations and gene mutations. The use of retroviral transduction/bone marrow transplantation mouse model harboring these genetic abnormalities has been critical in understanding the molecular pathogenesis of leukemia and exploring new therapeutic target. Additional genetic events are verified to cooperate with fusion genes resulting from chromosomal translocations in acute myeloid leukemia (AML) to develop a leukemic phenotype in mice, such as C-KIT N822K with AML1-ETO, FLT3-ITD with PML-RARα, Meis1 with NUP98-HOX, and Cdx4 with MLL-AF9. Mouse model shows that BCR/ABL fusion gene induces chronic myeloid leukemia (CML), and suggests that GATA-2 L359V and high expression of Hes1 are key molecules in acute myeloid transformation of CML. Furthermore, combination therapy with Imatinib and arsenic sulfide for CML mice exerts more profound therapeutic effects than either drug as a single agent. This review focuses the recent progress and application of retroviral-mediated mouse models of myeloid leukemia, and discusses some factors influencing the mouse model establishment, including retroviral construction, retrovirus titer and hematopoietic microenvironment.

Exploration of the financing and management model of a children's critical disease security system in China based on the implementation of Shanghai Children Hospital Care Aid / 中华医学杂志(英文版)

This study is designed to serve as a reference for the establishment of health security systems for children’s critical diseases. Through analysis of the operation of Shanghai Children Hospital Care Aid (SCHCA), this study explored the financing model and management of a children’s critical disease healthcare system and analyzed the possibility of expanding this system to other areas. It is found that a premium as low as RMB 7 per capita per year under SCHCA can provide high-level security for children’s critical diseases. With the good experience in Shanghai and based on the current basic medical insurance system for urban residents and the new rural cooperative medical scheme (NRCMS), it is necessary and feasible to build a health security system for children’s critical diseases at the national level.

Preliminary structural and functional study on a novel gene HSPCSET / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To characterize the structural and the functional feature of a novel gene HSPCSET isolated from human CD34+ hematopoietic stem/progenitor cells (HS/PCs).</p><p><b>METHODS</b>Bioinformatic technology was used to identify the structural features of the HSPCSET protein and perform the multiple sequence alignment. Yeast-two-hybrid system was used to identify the proteins interacting with the HSPCSET protein. After sequencing, we selected out the positive clones which had clear functions, and carried out beta-gal experiment and GST pull down assay to confirm the results. The cellular location of the HSPCSET was checked by immunofluorescence assay.</p><p><b>RESULTS</b>The HSPCSET protein belongs to a SET domain family, which is evolutionarily conserved across species. It implied that HSPCSET may have biologically important function. Using yeast-two-hybrid system, we showed that the protein sequence with SET domain might bind to 13 proteins, which involved in signaling transduction, transcriptional regulation, apoptosis, tumorigenesis, development, etc. And 4 proteins (GADD34, SIVA, DNAJ and PHF1) were confirmed by one-on-one back of the hybrid experiment, beta-gal test and GST pull down assay. When GADD34 and HSPCSET were co-transfected, they co-localized in the nucleus, suggesting a strong interaction.</p><p><b>CONCLUSION</b>The novel gene HSPCSET is likely to have biologically important function. This study provides the basis for further studies of its function in hematopoiesis and tumorigenesis.</p>

Experimental hematology bridging the gap between laboratory and clinic: hope of hematology / 中国实验血液学杂志

This article summarizes the progress of hematology in the recent tens years to show that experimental hematology used to pick up the 'hints' from clinical problems as the renewal of research directions and targets in experimental studies continuously. As the feedback, the results from lab investigations inserted into clinical practice and eventually made a quick modernization of hematology, which was actually a good model for the "translational research". The past few decades have witnessed tremendous advances in our understanding of normal hematopoiesis where genes dictate, epigenetics regulate, transcription factors mediate, and stem cells self-renew and differentiate. Dissection of disease pathogenesis not only elucidates molecular basis of disorders including hemoglobinopathy, aplastic anemia, hemophilia, hematopoietic malignancies such as leukemia and myeloproliferative disorders, but also provides therapeutic targets for drug development. Introduction of targeted therapies and combinatory targeting therapies greatly benefits hundreds of thousands of patients, and even turns acute promyelocytic leukemia from highly fatal to highly curable. In the 21st century the experimental hematology is entering the era of genomics and system biomedicine, and the pace of progress extrapolates to a prediction of hematologic neoplasms control in this century.

FIP1L1-PDGFRalpha alone or with other genetic abnormalities reveals disease progression in chronic eosinophilic leukemia but good response to imatinib / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The FIP1L1-PDGFRalpha fusion gene plays an important role in the pathogenesis of chronic eosinophilic leukemia (CEL) and is a direct therapeutic target of the tyrosine kinase inhibitor imatinib mesylate.</p><p><b>METHODS</b>In 24 hypereosinophilic syndromes (HES) patients, using reverse transcriptase-polymerase chain reaction (RT-PCR), nested PCR and sequence analysis, we investigated the frequency of FIP1L1-PDGFRalpha and other abnormalities of tyrosine kinase family genes like PDGFRalpha, PDGFRbeta, C-KIT, FGFR1, ABL and FLT3 as well as gene mutation "hotspots", like MPL515 and JAK2V617F, frequently involved in myeloproliferative diseases. Fluorescence in situ hybridization was used to confirm the 4q12 deletion.</p><p><b>RESULTS</b>The FIP1L1-PDGFRalpha fusion transcript was found in 8 (33%) of 24 patients with HES, corresponding to the chromosome 4q12 deletion identified by FISH. The FIP1L1-PDGFRalpha-associated patients diagnosed with CEL, frequently had hepatosplenomegaly, eosinophil-related tissue damage, anemia, thrombocytopenia, myelofibrosis and a short overall survival time. Nevertheless, imatinib mesylate induced rapid and complete hematological responses in treated FIP1L1-PDGFRalpha cases, followed by molecular remission and reversal of myelofibrosis. FIP1L1-PDGFRalpha fusion could co-exist with other mutations of tyrosine kinase family genes, like FLT3 or PDGFRbeta. We also demonstrated that the SNPs of PDGFRbeta were associated with selective splicing of exon 19 in case 20.</p><p><b>CONCLUSIONS</b>Correlating the CEL genotype with phenotype, FIP1L1-PDGFRalpha emerges as a relatively homogeneous clinicobiological entity that co-exists with other abnormalities of tyrosine kinase family genes. It reflects the disease progression and there is a good response to imatinib. Detection of the FIP1L1-PDGFRalpha fusion gene is valid for both CEL diagnosis and therapy surveillance.</p>

Expression of tumor suppressor gene PRDM1 in lung cancers / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To determine the expression of tumor suppressor gene PRDM1 in lung cancers.</p><p><b>METHODS</b>Forty-five cases were enrolled in this study, including squamous cell carcinoma (20 cases), adenocarcinoma (15 cases), and small cell cancer (10 cases). PRDM1 protein was detected in paraffin-embedded tissue by immunohistochemistry. Tumor cells in lung cancers were further selected by laser microdissection for RT-PCR analysis. PRDM1 protein in frozen tissue was also detected by Western blot.</p><p><b>RESULTS</b>(1) PRDM1 protein was found in paraffin-embedded tissues in 90.0% (18/20) of squamous cell carcinoma, 13.3% (2/15) of adenocarcinoma, and 0 (0/10) small cell lung cancer. Squamous cell carcinoma predominantly expressed PRDM1 protein ( P < 0.01). (2) Gene product of PRDM1 DNA binding region was not found in microdissected tumor cells, but an abnormal PRDM1 protein about 70 KD was detected simultaneously in whole tumor tissue.</p><p><b>CONCLUSION</b>PRDM1 may be considered as a specific biomarker in pulmonary squamous cell carcinoma. The abnormal PRDM1 expression both at transcriptional and protein levels indicated that this tumor suppressor gene lost its function, which may become a new target in the strategy of treatment for lung cancers.</p>

The molecular cytogenetic aberration analyzed by comparative genomic hybridization and its significance in diffuse large B-cell lymphoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify genetic alterations in diffuse large B-cell lymphoma (DLBCL) and to analyse the relationship between the genetic aberrations and the clinical characteristics.</p><p><b>METHODS</b>Using comparative genomic hybridization (CGH) to investigate the genomic changes in 24 cases of DLBCL and to analyse the relationship between these aberrations and clinical parameters including Ann arbor stage, systemic symptoms, chemotherapy efficacy and survival.</p><p><b>RESULTS</b>Aberrations were detected in 62.5% patients of 24 cases; the most common chromosomal alterations included loss of 6q15-21 as well as gain of 18q11-ter, of which the incidences were 20.8% and 16.7%, respectively; with comparing clinical parameters between patients with normal CGH and abnormal CGH, we found that patients with abnormal CGH suffered more from stage III-IV and had higher incidence of systemic symptoms, poor chemotherapy efficacy and poor survival (P<0.05), but there was no difference observed in the incidence of extranodal involvement between two groups.</p><p><b>CONCLUSION</b>The gains and/or losses of genomic DNA from DLBCL patients are the common molecular cytogenetic aberrations; loss of 6q15-21 and gain of 18q11-ter are nonrandom event to DLBCL patients; abnormal CGH is a clinical parameter reflecting malignant progressive course and poor survival to DLBCL patients.</p>

BCL-XL expression and mutation in non-Hodgkin's lymphoma / 中国实验血液学杂志

The study was aimed to investigate the BCL-XL expression and mutation, and its clinical significance in non-Hodgkin's lymphoma. Lymphoma cells were selectively isolated by laser microdissection. BCL-XL expression from lymphoma tissue and microdissected lymphoma cells was measured by using real-time quantitative reverse transcription-polymerase chain reaction. BCL-XL mutation was analyzed by using direct sequencing of PCR products. The results showed that compared to 15 patients with reactive hyperplasia, BCL-XL was overexpressed in follicular lymphoma (n = 30), both in lymphoma tissue (P = 0.0064) and in microdissected lymphoma cells (P < 0.0001). No significant rise of BCL-XL expression was observed in patients with T-cell lymphoma (n = 24) and diffuse large B cell lymphoma (n = 24). In follicular lymphoma, high BCL-XL level was associated with multiple extranodal involvement (P = 0.0004), elevated lactate dehydrogenase level (P = 0.0019), high-risk international prognostic index (P = 0.0013) and a short overall survival time (P = 0.0451). Mutation analysis revealed one synonymous mutation (Codon 109 ACA-->ACC) in one case of follicular lymphoma patient. It is concluded that BCL-XL expression is closely correlated with progress of follicular lymphoma and prognosis of patients with follicular lymphoma. The value of BCL-XL expression as a prognostic marker in follicular lymphoma should be considered.

Basic and clinical studies of the gene product-targeting therapy based on leukemogenesis--editorial / 中国实验血液学杂志

In the last twenty years, using all-trans retinoic acid (ATRA) as a differentiation inducer, Shanghai Institute of Hematology has achieved an important breakthrough in the treatment of acute promyelocytic leukemia (APL), which realized the theory of reversing phenotype of cells and provided a successful model of differentiation therapy in cancers. Our group first discovered in the world the variant chromosome translocation t(11;17)(q23;q21) of APL, and cloned the PML-RAR alpha, PLZF-RAR alpha and NPM-RAR alpha fusion genes corresponding to the characterized chromosome translocations t(15;17); t(11;17) and t(5;17) in APL. Moreover, establishment of transgenic mice model of APL proved their effects on leukemogenesis. The ability of ATRA to modify the recruitment of nuclear receptor co-repressor with PML-RAR alpha but not PLZF-RAR alpha caused by the variant chromosome translocation elucidated the therapeutic mechanism of ATRA from the molecular level and provides new insight into transcription-modulating therapy. Since 1994, our group has successfully applied arsenic trioxide (As(2)O(3)) in treating relapsed APL patients, with the complete remission rate of 70% - 80%. The molecular mechanism study revealed that As(2)O(3) exerts a dose-dependent dual effect on APL. Low-dose As(2)O(3) induced partial differentiation of APL cells, while the higher dose induced apoptosis. As(2)O(3) binds ubiquitin like SUMO-1 through the lysine 160 of PML, resulting in the degradation of PML-RAR alpha. Taken together, ATRA and As(2)O(3) target the transcription factor PML-RAR alpha, the former by retinoic acid receptor and the latter by PML sumolization, both induce PML-RAR alpha degradation and APL cells differentiation and apoptosis. Because of the different acting pathways, ATRA and As(2)O(3) have no cross-resistance and can be used as combination therapy. Clinical trial in newly diagnosed APL patients showed that ATRA/As(2)O(3) in combination yields a longer disease-free survival time. With the median survival of 18 months, none of the 20 cases in combination treatment relapsed, whereas 7 relapsed in 37 cases in mono-treatment. This is the best clinical effect achieved in treating adult acute leukemia to this day, possibly making APL the first adult curable leukemia. Based on the great success of the pathogenetic gene target therapy in APL, this strategy may extend to other leukemias. Combination of Gleevec and arsenic agents in treating chronic myeloid leukemia has already make a figure both in clinical and laboratory research, aiming at counteracting the abnormal tyrosine kinase activity of ABL and the degradating BCR-ABL fusion protein. In acute myeloid leukemia M(2b), using new target therapy degradating AML1-ETO fusion protein and reducing the abnormal tyrosine kinase activity of c-kit will also lead to new therapeutic management in acute leukemias.

hCG-PLZF-RARalpha/hCG-RARalpha-PLZF transgenic mice developing into leukemia / 中国实验血液学杂志

To investigate the potential role and the mechanism of PLZF-RARalpha/RARalpha-PLZF double fusion gene in the pathogenesis of acute promyelocytic leukemia (APL) in vivo at systematic biological level, PLZF-RARalpha/RARalpha-PLZF double transgenic mouse model was established by intercross; the integration and expression of fusion genes were analyzed by PCR and RT-PCR; the disease phenotype was detected by morphological and pathological examination of peripheral blood and bone marrow cells, as well as flow cytometry assays; the effects of ATRA with or without tricostatin A on bone marrow blast cells from PLZF-RARalpha/RARalpha-PLZF double TM were observed. The results showed that leukemia occurred in 5 PLZF-RARalpha/RARalpha-PLZF double TM 7, 7, 9, 11 and 11 months respectively, out of them two (40%) with classic APL features, the others (60%) with chronic myeloid leukemia through an observation period of 18 months. The leukemia occurrence of PLZF-RARalpha/RARalpha-PLZF TM was about 10%, which was similar to PLZF-RARalpha TM as that reported before. The latency was over 6 months, not earlier than PLZF-RARalpha TM only. No morphologic changes of PLZF-RARalpha/RARalpha-PLZF double TM blast cells to ATRA were observed, but increased cytoplasmic-nuclear ratio and nuclear condensation in bone marrow blast cells were found in combination of ATRA with tricostatin A. It is concluded that PLZF-RARalpha/RARalpha-PLZF double fusion gene transgenic mice have heterogeneity of pathogenesis. HDAC inhibitors such as trichostatin A, in combination with ATRA, induce differentiation of the blast/promyelocytic cells from PLZF-RARa/RARa-PLZF double TM, but not ATRA alone.

Detection of MRD and its clinical application--review / 中国实验血液学杂志

With recent development and progress achieved in the diagnosis and treatment of leukemia, relapse of disease still remains as the major problem in clinical management and the minimal residual disease (MRD) has been confirmed to be associated with leukemia relapse in the past decades. Due to the low sensitivity, morphology-based assays have limitation in the MRD monitoring. With the development of molecular assays, especially the real-time RT-PCR method have been a sensitive, precise and reliable tool to MRD detection. Numerous clinical studies demonstrated that the existence of MRD reflects the clinical and molecular response, and remain as a major prognostic factor for leukemia. Another important application of MRD detection is to evaluate the efficacy of different therapy at molecular level. In this paper, the different methods and their clinical application for MRD detection were systemically reviewed and it is confident that the establishment of standardized MRD detection system will be important in the clinical prevention for relapse of leukemia.

Establishment of CPP-SOM integrated cDNA microarray technology / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To get an insight into the molecular mechanisms of diseases development and targeted therapy at the transcriptome level and search for potential therapeutic targets.</p><p><b>METHODS</b>The present researchers established a cDNA microarray platform and applied component plane presentation integrated self-organizing map (CPP-SOM) to the microarray data obtained from a differentiation model, all trans retinoic acid-induced differentiation in NB4 cells.</p><p><b>RESULTS</b>The platform included 12630 unique clones, including 9436 known genes. By CPP-SOM, the researchers were able to not only well classify the regulated genes into functionally distinct categories but also depict transcriptional changes throughout the process of the development of diseases or drug treatment.</p><p><b>CONCLUSION</b>The platform has proven to be steady and reliable, and the CPP-SOM could serve as an important and good tool for analysis of microarray data.</p>

Influence of different conditions on two-dimensional gel electrophoresis of proteins from human acute promyelocytic leukemia cell line NB4 / 中国实验血液学杂志

The aim of this study was to explore the effect of different conditions on two-dimensional gel electrophoresis of proteins from human acute promyelocytic leukemia cell line NB4. The 24 cm pH 3-10 linear immobilized pH gradient (IPG) strips were chosen, the isoelectric focusing was carried out by using IPGphor. Then, the second-dimensional SDS-PAGE was performed. After silver staining, the gel was analyzed by ImageMaster 2D Elite. The results showed that low ion intensity sample washing buffer improved the performance of isoelectric focusing. The lysis buffer containing 7 mol/L urea and 40 mmol/L DTT could solubilize the most proteins from NB4 cell line. The rehydration solution containing thiourea and urea increased the low molecular weight protein points to be resolved in the area of basic end. The reasonable sample load and Volt/hour of NB4 were about 100 micro g and 63 200 V/h for the 24 cm pH 3-10 IPG strips. It is concluded that the proteins from NB4 and similar cell line are complicated and affected by many factors, so that, it is very important to select the right methods for sample preparation and the conditions of two-dimensional gel electrophoresis.

Development of human myeloid leukemia-like phenotype in NUP98-PMX1 transgenic mice / 中华血液学杂志

<p><b>OBJECTIVE</b>In order to investigate the leukemogenic potential of NUP98-PMX1 fusion gene in vivo.</p><p><b>METHODS</b>NUP98-PMX1 transgenic mice were generated, in which the fusion gene was driven by hCG promoter and expressed in myeloid cells at early stage of differentiation. Molecular cloning technology was used to construct NUP98-PMX1 transgenic plasmid. The genotype and phenotype of the NUP98-PMX1 transgenic mice were analyzed by PCR, RT-PCR, peripheral blood count (PBC), bone marrow (BM) cells morphology and pathological examination.</p><p><b>RESULTS</b>NIH3T3 cells transfected with NUP98-PMX1 fusion gene grew faster, formed colonies in soft agar, and developed tumors in 10 inoculated nude mice. Among 8 disordered NUP98-PMX1 transgenic mice, 4 developed myeloid leukemia-like phenotype, including 3 resembling human chronic myeloid leukemia.</p><p><b>CONCLUSION</b>NUP98-PMX1 has oncogenic activity and plays a crucial role in leukemogenesis.</p>

Gene expression profile of yolk sac and fetal liver in mouse / 中华血液学杂志

<p><b>OBJECTIVE</b>To better understand the mechanisms of the fetal hematopoiesis turn over from primitive to definitive hematopoiesis through the expression level of c-kit(+) and sca-1(+), and major characters of gene expression profile of these cells.</p><p><b>METHODS</b>c-kit and sca-1 expression level were monitored with fluorescence activated cell sorting (FACS) of the mononuclear cells from mouse yolk sac and fetal liver, while gene expression profile was carried out with EST sequencing strategy.</p><p><b>RESULTS</b>The Sca-1(+) cells were increased while the c-kit(+) cells decreased with the embryonic development. Through profiling the functionally identified known genes, most of the highly expressed were globin genes, especially of embryonic types.</p><p><b>CONCLUSION</b>The erythropoiesis played a key role in early fetal hematopoiesis in mammalian.</p>

Establishment of transgenic mice for HRX-EEN fusion gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the biological function of fusion gene HRX-EEN and its role in leukemogenesis, and to provide an ideal animal model for anti-leukemia drug screening.</p><p><b>METHODS</b>HRX-EEN fusion gene was constructed by use of three different DNA fragments, and it was inserted into hCG transgenic vector. G(0) transgenic mice were obtained by microinjection of the recombined DNA into the pronucleus of zygotes, followed by implantation of the injected zygotes into pseudopregnant mice. The integration of the transgene was tested by PCR and its expression by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The sequence of recombined HRX-EEN gene was confirmed by sequencing. PCR testing revealed a total of 7 G(0) transgenic mice, these mice were then mated with C57 wild type mice. Except mouse No. 35 that died, the others all had their F1 offsprings. From these 6 lines of transgenic mice, HRX-EEN gene was found to be stably expressed in 3 lines by RT-PCR. Up to now, all transgenic mice expressing the fusion gene have no obvious abnormal phenotypes.</p><p><b>CONCLUSION</b>A transgenic mice model in which the HRX-EEN fusion gene can be stably expressed has been established.</p>

Influence of irradiation on the dynamic three-dimension distribution of abl and bcr genes in the interphase nuclei of IM-9 cell / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the material foundation of the fusion of bcr and abl genes, and to explore the pathogenesis of chronic myeloid leukemia.</p><p><b>METHODS</b>By FISH combined with laser confocal scanning microscopy, the three-dimension (3D) distribution of bcr and abl genes in the interphase nuclei of normal and irradiated IM-9 cells was studied in each cell cycle phases.</p><p><b>RESULTS</b>abl and bcr genes distributed non-randomly in the interphase nuclei of IM-9 cells. abl gene preferably located at the outer layer and bcr near the core of the nucleus. The two genes were drawn near each other most in G(0) phase. The relative distance between the homologous genes was greater at proliferation phase than at quiescence phase. After irradiation, the relative distances from the two genes to the core and between the two genes were shortened, with the shortest distance between the two genes in S phase.</p><p><b>CONCLUSION</b>Irradiation could change the 3D-distribution of abl and bcr genes in the interphase nuclei of IM-9 cell and accelerate them to draw near each other.</p>

In vitro study of the effects of arsenic trioxide combined with 8-CPT-cAMP on differentiation induction in retinoic acid resistant acute promyelocytic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the potential effects of arsenic trioxide (As(2)O(3)) combined with 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells.</p><p><b>METHODS</b>The RA resistant APL cell lines NB4-R1 and NB4-R2 were used as in vitro models. The effect of As(2)O(3) and/or 8-CPT-cAMP was evaluated according to cellular morphology, cell surface antigen and nitroblue-tetrazolium (NBT) assay. Meanwhile, immunofluorescence analysis and Western blot assay were used to detect the degradation of PML-RAR alpha fusion protein and the change of several key cell cycle regulatory proteins in these cells before and after the treatment.</p><p><b>RESULTS</b>Low dose of As(2)O(3) (0.25 micromol/L) synergized with 8-CPT-cAMP (200 micromol/L) in inducing differentiation of NB4-R1 and NB4-R2 cells, while neither of these two drugs alone could induce differentiation of these cells. In addition, 8-CPT-cAMP was able to inhibit the cell growth by modulating the expression of some important cell cycle regulators and to facilitate the As(2)O(3)-mediated degradation of PML-RAR alpha fusion protein.</p><p><b>CONCLUSIONS</b>As(2)O(3) combined with 8-CPT-cAMP could induce differentiation of RA-resistant APL cells.</p>

Clinical observation of the short-term efficacy of the treatment with combination of all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) in newly diagnosed acute promyelocytic leukemia (APL) / 中华血液学杂志

<p><b>OBJECTIVE</b>To study whether all-trans retinoic acid (ATRA) combined with arsenic trioxide (As(2)O(3)) in acute promyelocytic leukemia (APL) treatment could further improve the clinical and molecular remission rate.</p><p><b>METHOD</b>Thirty one newly-diagnosed APL patients of whom 15 were males, 16 females and median age 35.4 years entered into the study. They were treated with ATRA 25 mg x m(-2) x d(-1) combined with As(2)O(3) 0.16 mg x kg(-1) x d(-1) until complete remission (CR). The doses were adjusted according to white blood cell (WBC) counts, occurrence of RA syndrome and the status of liver function. CR rate, time of reaching clinical and molecular remission and side effects were observed.</p><p><b>RESULT</b>Two patients died 2 approximately 3 days after the treatment due to intracranial hemorrhage, and 29 (93.5%) achieved CR. The average time for achieving CR was 25.1 +/- 3.9 days. Hyperleukocytosis emerged in 66.5% and hepatic damages in 65.5% of the patients, they were ameliorated within one week after reduction of the As(2)O(3) dose or its suspension. The PML/RAR alpha fusion gene that was positive in all 29 patients before treatment turned negative only in 3 cases (10.3%) after obtaining CR (CR1) and in 10/13 cases (77%) after consolidation treatment. Up to now (1-8 months follow-up), all 29 patients remain in CR1.</p><p><b>CONCLUSION</b>ATRA combined with As(2)O(3) in de novo APL treatment can yield a high CR rate without intolerable side effects. Long term effect needs further observation.</p>