Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro.

AIM: Over-expressed CHMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CHMP5 single chain variable fragment antibody (CHMP5-scFv) retrovirus and studied the changes of programmed cell death (PCD) of AML leukemic cells after infection by the retrovirus. METHODS: The anti-CHMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CHMP5. The protein expression of CHMP5 was studied using immunofluorescence analysis. pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot. RESULTS: We obtained a monoclonal antibody of CHMP5, and found the expression of CHMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CHMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor (AIF)-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated. CONCLUSION: CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CHMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.

Blastic plasmacytoid dendritic cell neoplasm: A case report and literature review.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a clinically aggressive tumor, which frequently presents as cutaneous lesions and subsequently progresses to bone marrow (BM) involvement and leukemic dissemination. BPDCN is a rare entity that belongs in the same class as acute myeloid leukemia-associated precursor neoplasms, according to the 2008 World Health Organization classification. The present study reported the case of a 26-year-old female who presented with evident thrombocytopenia, leukocytosis and anemia, but without skin lesions. The results of peripheral blood, BM smear and BM biopsy examinations detected numerous blastic or abnormal cells. In addition, flow cytometric analysis of BM demonstrated the presence of plasmacytoid dendritic cell-neoplastic precursor cells (CD4+, CD56+, CD123+, CD304+ and human leukocyte antigen-DR+ phenotype).

A preliminary study on epigenetic regulation of Acanthopanax senticosus in leukemia cell lines.

Conventional chemotherapy for leukemia inevitably causes systemic toxicity. Acanthopanax senticosus, a naturally occurring herb used in traditional Chinese medicine, has been found to be a multipotent bioflavonoid with great potential in the prevention and treatment of malignant diseases. However, the mechanism underlying the action of A. senticosus in epigenetic regulation is poorly understood. In the study described here, we focused on the efficacy of A. senticosus in inducing apoptosis of leukemia cells and a possible mechanism. By evaluating the inhibition ratio and morphologic changes, we found that A. senticosus can inhibit growth and induce apoptosis of human leukemia HL-60 and HL60/ADM cells in a dose- and time-dependent manner. Furthermore, A. senticosus induced Fas ligand (FasL) expression and blocked the cell cycle in S phase. In addition, A. senticosus exhibited a potential for inhibition of histone deacetylase (HADC), which contributes to histone acetylation. It possibly resulted in the promotion of the expression of FasL. It is suggested that A. senticosus could be recognized as a new HDAC inhibitor which was able to reactivate aberrantly silenced genes. We discuss the clinical aspects of using A. senticosus for treatment of leukemia.

Glycolytic inhibitor 2-deoxy-d-glucose suppresses cell proliferation and enhances methylprednisolone sensitivity in non-Hodgkin lymphoma cells through down-regulation of HIF-1α and c-MYC.

Metabolic reprogramming is linked to tumorigenesis, disease progression, clinical outcome and resistance to chemotherapy. However, the significance of glycolytic metabolism in non-Hodgkin lymphoma (NHL) remains unclear. Here we report that both NHL patient-samples and cell lines exhibited significant up-regulation of glycolytic metabolism. The glycolytic inhibitor 2-deoxy-d-glucose (2-DG) inhibited glucose consumption, lactic acid generation and cell proliferation and induced cell cycle arrest in NHL cell lines under both normoxia and hypoxia, and hypoxia could even enhance the inhibitory effects of 2-DG. Furthermore, 2-DG combined with methylprednisolone synergistically inhibited cell proliferation, induced cell apoptosis and cell cycle arrest, and thus increased the sensitivity of NHL cells to methylprednisolone via down-regulation of HIF-1α and c-MYC. In conclusion, these results present a novel insight into critical roles of glycolytic pathway activation in NHL progression and glucocorticoid resistance. Inhibition of the glycolytic pathway may provide a new therapeutic strategy for the treatment of NHL.

Quercetin enhances adriamycin cytotoxicity through induction of apoptosis and regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling in multidrug-resistant leukemia K562 cells.

Multidrug resistance (MDR) has become a significant challenge in chemotherapeutic treatment of cancer. Quercetin, a naturally occurring flavonoid, has been found to possess anti-proliferative, anti-inflammatory and immunoregulatory bioactivities. The present study was performed to examine the effect of quercetin on human leukemic MDR K562/adriamycin (ADR) cells. Treatment of K562/ADR cells with a combination of quercetin and ADR resulted in potentiation of cytotoxicity, which was measured using a cell counting kit-8 assay. Flow cytometric analysis revealed that quercetin dose-dependently promoted cell apoptosis and treatment with a combination of quercetin and ADR caused synergistic enhancement of the apoptotic effect. In addition, treatment of K562/ADR cells with quercetin alone or in combination with ADR resulted in loss of mitochondrial membrane potential, activation of caspase-8, -9 and -3, reduced expression of the anti-apoptotic proteins B-cell lymphoma (Bcl)-2 and Bcl-extra large and enhanced expression of the pro-apoptotic proteins Bcl-2-interacting mediator of cell death, Bcl-2-associated death promoter and Bcl-2-associated X protein in the cells. Furthermore, the combined treatment of quercetin and ADR synergistically increased the expression of phosphorylated (p-)c-Jun N-terminal kinase and p-p38 mitogen-activated protein kinase and decreased the expression of p-extracellular signal-regulated kinase in the K562/ADR cells. In addition, the expression of P-glycoprotein was significantly decreased following treatment with quercetin alone or in combination with ADR. These findings demonstrated that quercetin is important in MDR and may be developed into a new reversal agent for cancer chemotherapy.

[The relationship between cytochrome P450, subfamily IIIA, polypeptide 5 gene and drug resistance in leukemia cell lines].

OBJECTIVE: To investigate if the transcription, expression and activities of cytochrome P450, subfamily IIIA, polypeptide 5 gene (CYP3A5) are correlated with drug resistance in leukemia cell lines. METHODS: Using RT-PCR, immunohistochemistry and reverse-phase high-performance liquid chromatography (HPLC) assay, CYP3A5 mRNA protein and activities in leukemia cell lines were detected. Daunorubicin (DNR) sensitivity profiles of leukemia cell lines were obtained with MTT assay. Cell cycle characteristics and apoptosis induced by DNR were observed with flow cytometry (FCM) analysis. RESULTS: K562, U937, HL-60, NB4 and Jurkat cells were chosen as experimental cell line panels. It was found that CYP3A5 mRNA were measured only in K562 and U937 cells. CYP3A4 and CYP3A7 were not detectable in each cell line. The five leukemia cell lines presented sensitivity to DNR (IC(50), mg/L) in an order as follows: NB4 (0.068 +/- 0.036) > Jurkat (0.076 +/- 0.013) > HL-60 (0.092 +/- 0.016) > K562 (0.148 +/- 0.041) > U937 (0.150 +/- 0.035). Interestingly, K562 and U937 cells (cell lines with CYP3A5 gene transcription) were more resistant to DNR as compared with the other three cell lines in a statistically significant way (P < 0.01). Furthermore, expression and activities of CYP3A5 gene and cell cycle characteristics were observed in K562 and NB4 cells, which represented cell lines with or without CYP3A5 gene transcription respectively. In comparison with NB4 cells, K562 cells expressed CYP3A5 protein and showed a statistically significant higher CYP3A5 activity (3.075 +/- 0.036) x 10(-3) versus (1.635 +/- 0.196) x 10(-3), P < 0.05. FCM analysis showed that S phase cell proportions were similar in K562 and NB4 cells. Incubation with DNR (1 mg/L) for 6 hours induced a remarkable apoptosis peak in NB4 cells, while such peak not occur in K562 cells (apoptosis cell percentage 18.4% and 0.8% respectively). CONCLUSION: Only CYP3A5 gene of CYP3A subfamily were detectable in leukemia cell lines, and its transcription, expression and activities were correlated with drug resistance in leukemia cell lines.

Arsenic trioxide induces apoptosis in the THP1 cell line by downregulating EVI-1.

Acute leukemia is a malignant clonal hematopoietic stem cell disease. In the current study, the effects of arsenic trioxide (ATO) on the ecotropic viral integration site-1 (EVI-1) gene were investigated in the THP1 cell line. THP-1 cells were treated with different concentrations of ATO (0, 1, 3 and 5 µM) for 24, 48 or 72 h, then tested for cell viability by CCK-8 kit, cell morphology by cytospin smear, cell apoptosis by flow cytometry, EVI-1 mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) and protein quantity by western blot. ATO treatment was shown to inhibit proliferation and induce apoptosis in THP1 cells in a dose- and time-dependent manner. ATO downregulated the mRNA and protein expression of EVI-1 in the THP1 cell line. In addition, ATO significantly decreased the expression of antiapoptotic proteins, B-cell lymphoma 2 (Bcl-2) and B cell lymphoma-extra large (Bcl-xL), but markedly increased the expression of proapoptotic proteins, including c-Jun N-terminal kinase (JNK), phosphorylated-JNK, Bax, full length caspase-3 and cleaved caspase-3. These results indicated that ATO inhibited the proliferation and induced apoptosis in THP1 cells partially via blocking the inhibitory effects of EVI-1 on the JNK signaling pathway with the involvement of apoptosis-associated proteins, including Bax, Bcl-2, Bcl-xL and caspase-3. These novel observations may be used to elucidate the mechanism by which ATO induces apoptosis in acute leukemia cells, and provide rationales to develop a personalized medicine strategy for ATO via targeting EVI-1 positive neoplasm.

Using modified whole-mount in situ hybridization to study mpo expression in zebrafish.

In this study, we cloned the myeloperoxidase (mpo) gene of zebrafish and prepared a digoxigenin-labeled mpo RNA probe to investigate mpo gene expression in zebrafish during embryonic development by whole-mount in situ hybridization (WISH). The earliest mpo expression was detected in cells of the intermediate cell mass (ICM) at 18 h post-fertilization (hpf). It was detected 1 to 2 h later in cells in the rostral blood island (RBI) and strong signals were observed in the anterior ICM. Then, it spread over the yolk sac. By 72 hpf these mpo-expressing cells were in the circulation and distributed throughout the embryo. We identified that the level of mpo expression detected by WISH at an early stage was consistent with the data of cytological analyses of adult fish. The use of this method enabled us to track the gene changes that took place before morphological phenotypes were detected, as well to as investigate the hematopoietic cell fate in mutational or transgenic models in vivo. In this study, we modified several steps of WISH. The improved hybridization results demonstrated high specificity, distinct coloration and low background figures.

[Mitochondria-mediated apoptosis induced by 5-aminolevulinic acid-based photodynamic therapy in HL-60 cells].

This study was purposed to investigate the changes of mitochondrial membrane potential (MMP) and apoptosis-related gene Bcl-2 expression of HL-60 cells treated with 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). HL-60 cell line was used as a model and divided into 4 groups: ALA group, PDT group, ALA+PDT group and control group. The change of MMP was detected by flow cytometry with JC-1 (lipophilic cation 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-benzimidazol-carbocyanine iodide); the mRNA expression of Bcl-2 was determined by semi-quantitative RT-PCR and real-time PCR. The results demonstrated that MMP significantly decreased after treatment with ALA-PDT and the ratio of cells with disrupted MMP obviously increased in ALA+PDT group in time-dependence manner, as compared with control, ALA and PDT groups (P < 0.05), while no difference between ALA and PDT groups was found. The semi-quantitative RT-PCR and real-time PCR showed that the expression level of Bcl-2 was obviously down regulated at 2 h after ALA-PCT, further down-regulated at 4 h, and lasted in low level at 24 h. It is concluded that ALA-PDT-induced apoptosis of HL-60 cells is associated with its effect on MMP, that is ALA-PDT promotes cell apoptosis through effect on mitochondrial function.

Clinical and prognostic significance of miR-155 and miR-146a expression levels in formalin-fixed/paraffin-embedded tissue of patients with diffuse large B-cell lymphoma.

It has been found that aberrant expression of microRNAs (miRNAs) is strongly associated with carcinogenesis. In the present study, we investigated the expression of miR-155 and miR-146a in diffuse large B-cell lymphoma (DLBCL) patients (n=90). The expression levels of miR-155 and miR-146a were significantly higher in de novo DLBCL patients. miR-146a expression levels were associated with miR-155, lactate dehydrogenase, ß 2 microglobulin, c-myc, International Prognostic Index status and Eastern Cooperative Oncology Group performance status. We found that patients with low miR-155 and miR-146a expression levels achieved a higher complete remission rate, higher overall response rate and longer progression-free survival time. Moreover, a high expression level of miR-155, but not miR-146a, was an independent indicator for chemotherapy protocol selection in our study. Patients with high expression of miR-155 received more survival benefits from rituximab treatment. These data suggest that miR-155 and miR-146a have potential as diagnostic and prognostic markers in DLBCL.

[Expression of alternatively spliced human tissue factor in acute leukemia cells].

The high expression of tissue factor (TF) is related to the coagulation disorder in acute leukemia. TF in blood circulation is mainly expressed in cells, microparticles (MP) and alternatively spliced human tissue factor (asHTF). To elucidate the role of TF in the coagulation disorder of acute myeloid leukemia (AML), RT-PCR was performed on 6 common AML cell lines NB4, HL-60, Kasumi-1, U937, K562 and THP-1. The results showed that only NB4 and U937 cells expressed baseline full-length TF and asHTF which were proved by sequencing. The flow cytometric detection, TF activity and TF antigen tests in NB4 and U937 cells revealed that the asHTF was expressed in trace amount and almost had no activity, while the TF antigen and activity in microparticles were significantly higher than that in asHTF. It is concluded that asHTF may play an unimportant role in the coagulation disorder of AML. Microparticle associated tissue factor (MP-TF) is the predominant source of TF activity released from AML cells.

Targeting Smad4 links microRNA-146a to the TGF-beta pathway during retinoid acid induction in acute promyelocytic leukemia cell line.

The expression pattern of microRNAs (miRNAs) and their potential target genes were investigated in acute promyelocytic leukemia (APL) cell line NB4 cells during all-trans-retinoid acid (ATRA) treatment by using a miRNA microarrays platform and real-time quantitative PCR (RTQ-PCR). MiR-146a as one of the miRNAs down-regulated by ATRA during APL differentiation was identified. Direct interaction between miR146a and its predictive target gene Smad4 were confirmed by Luciferase assay. Down-regulation of miR-146a and upregulation of Smad4 at protein levels were demonstrated. These data suggested that miR-146a might influence proliferation of APL cells through TGF-beta1/Smad signal transduction pathway during ATRA induction.

Modification of TGF-beta1 signaling pathway during NB4 cells differentiation by all-trans retinoid acid induction.

The aim of the study was to present the possible mechanisms of transforming growth factor beta 1(TGF-beta1) signal pathway during cell differentiation by studying the expression levels of six components of TGF-beta1 pathway (TGF-beta1, two TGF-beta1 receptors and three Smad proteins). The morphology change, the CD11 expression levels, and the mRNA and protein expression levels of TGF-beta1, TGF-beta ReceptorI (TbetaRI), TGF-beta ReceptorII (TbetaRII), Smad2, Smad4 and Smad7 were assessed by exposing NB4 cells to all-trans retinoid acid (ATRA) using Wright's stain, flow cytometry, real-time PCR assay and Western blot analysis. The mRNA and protein expression levels of all six components increased during NB4 cells differentiation induced by ATRA. They were most significantly increased after 24-72 h individually when cells were induced by ATRA (the mRNA and protein expression levels of TGF-beta1, TbetaRI, TbetaRII and Smad2 reached their peaks at 48 and 48 h individually after the treatment, Smad4 at 48 and 72 h, and Smad7 at 72 and 72 h). The change in mRNA expression levels was earlier than the change in the same gene controlling protein. These results indicate that the upregulation of TGF-beta1 pathway plays an important role in NB4 cells differentiation induced by ATRA.

[Preparation and identification of monoclonal antibody against PNAS-2 protein].

This study was purposed to prepare and primarily identify the specific monoclonal antibodies (McAbs) against the apoptosis related protein PNAS-2 so as to provide the essential tool for study of PNAS-2 function. The McAbs against PNAS-2 were prepared via the immunization of mice, cell fusion and cloning using synthetic peptide of PNAS-2 as immunogen; the specificity, titer and subtype of McAb were detected by Western blot, ELISA and immunofluorescence. The results showed that the stable hybridoma cell line S-31-7 producing McAbs against PNAS-2 protein was successfully obtained. The immunoglobulin of the McAb was identified to be IGg1lambda. The titer of ascetic fluid fled McAb were 1:8,000. A single specific band with 28 kD was shown in Western blot test, and the antigen recognized was present in cell cytoplasm by immunofluorescence. In conclusion, the obtained McAb against PNAS-2 displays strong specificity and high titer, which may be applied to the advanced research on PNAS-2 protein.

[Polymorphisms of CYP3A5 gene in acute leukemia patients and their role in chemotherapy and prognosis].

The objective of study was to investigate the role of the polymorphisms and protein expression of CYP3A5 gene in the therapy and prognosis of acute leukemia (AL) patients, the polymorphisms of CYP3A5 gene and the expression of protein CYP3A5 were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and immunohistochemistry method respectively. The results showed that there were three CYP3A5 genotypes in the 88 cases, namely CYP3A5*1/*1, CYP3A5*1/*3 and CYP3A5*3/*3 with frequencies of 26%, 50% and 24%, respectively. There were no significant differences in clinic data between the three groups, but the expressions of CYP3A5 of three groups were (36.6+/-19.2)%, (7.8+/-9.2)%, (0.5+/-0.9)%, the OS were (11.6+/-2.1) months, (30.5+/-12.2) months, (52.3+/-8.5) months, and the DFS were (7.5+/-1.8), (27+/-15.8), (52.3+/-8.1) months, respectively (p<0.05). It is concluded that the polymorphism of CYP3A5 gene is not related with the morbidity of AL, but closely associated with the expression of CYP3A5 in AL patients, and the latter are closely associated with the chemotherapeutic effect and prognosis. CYP3A5 genotype may be used as a new predictor to the chemotherapeutic effect and prognosis in AL cases.

[Expression spectra of apoptosis-related gene pnas-2].

To explore the expression spectra of apoptosis-related gene pnas-2 in normal tissues and acute leukemia (AL) patient tissues, the expressions of pnas-2 gene in tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, lymph node, thymus, leukocyte, bone marrow and fetal liver were detected by Northern blot. The expressions of pnas-2 in samples including 44 de novo, 9 non-CR, 27 CR and 12 relapsed AL patients were measured by real-time RT-PCR and Northern blot, and the expression levels of pnas-2 in normal and tumor tissues from 31 patients with malignancies were also detected. The results showed that pnas-2 was not expressed in the most tissues except in placenta. The results of real-time PCR indicated that pnas-2 expressions in samples of de novo, non-CR and relapsed patients ware significantly higher than that in CR, tumor tissues and normal tissues. In serial monitoring of 7 AL patients, the expression level of pnas-2 was high at first visit examination, but remarkably decreased after remission, and the pnas-2 expression level increased again when relapsed. It is concluded that the pnas-2 is specifically up-regulated in acute leukemia patients, which might be an oncogene and participate in leukemogenesis.

[Correlation between expression of apoptosis-related gene pnas-2 and leukemia].

The study was purposed to explore the correlation between apoptosis-related gene pnas-2 and leukemia. The RT-PCR was performed to detect the expression levels of pnas-2 gene in NB4, K562, U937 cells before and after treatment with AS(4)S(4), and to analysis the expression change of pnas-2 gene in bone marrow cells from patients with acute leukemia before and after chemotherapy. The results showed that the expression of pnas-2 gene in arsenic sulfide treated NB4 cells was down regulated in time-dependent manner, but the same outcome in K562 and U937 cells after being treated with AS(4)S(4) was not found. The positive expression rate of pnas-2 in cells from untreated patients with acute leukemia was 100%, and was significantly higher than that in normal control group. After chemotherapy, the expression was negative in complete remission patients, whereas in no-remission patients there were no significant differences of expression of pnas-2 before and after treatment. It is concluded that the pnas-2 gene may be closely related with apotosis of arsenic sulfide treated APL cells, and may consider as a molecular biological remission marker in acute leukemia.

[Effect of recombinant human granulocyte colony-stimulating factor on neutrophil morphology, function and phenotype in patients with acute leukemia undergoing chemotherapy].

This study was to explore the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on neutrophil morphology, function and phenotype in patients with acute leukemia undergoing chemotherapy. Neutrophil morphology was observed under microscope with oil immersion; phagocytotic function was examined by measuring the amount of hydrogen peroxide produced by neutrophil; chemotaxis was analyzed by agarose method; oxidative burst was analyzed by flow cytometry using immunofluorescence technique; neutrophil phenotype was analyzed by flow cytometry and immunofluorescence techniques. The results showed that after rhG-CSF administration, the increased "toxic" granulation, vacuoles and Döhle bodies were observed in neutrophils of patients with acute leukemia. Compared with normal control, the functions of phagocytosis, chemotaxis, oxidative burst of neutrophil were impaired after chemotherapy, while these functions were enhanced and returned to normal level or even to be exceeded after administration of rhG-CSF. In patients with acute leukemia the neutrophil presented significantly higher expression of CD64 and CD62L than that in normal control, and a mild increase of CD64 expression and significant increase of CD62L expression were found in patients after rhG-CSF treatment. No modifications of CD16, CD32, CD14 and CD11b expression were detected in these patients before or after G-CSF administration. It is concluded that rhG-CSF administration can modify the morphology, function and phenotype of neutrophils in the patients with acute leukemia undergoing chemotherapy, and these modifications of neutrophil behavior may be supposed to be a reason for the enhancement of organism anti-infection ability.

[Effect of ATRA and DNR on the expression and secretion of VEGF in leukemic cells].

OBJECTIVE: To investigate the expression of VEGF mRNA and secretion of VEGF protein in NB4 and HL-60 cells affected by all-trans retinoic acid (ATRA) and daunorubincin (DNR) respectively. METHODS: Semi-quantitative RT-PCR and ELISA were used to study the expression of VEGF mRNA and secretion of VEGF protein in NB4 and HL-60 cell lines treated by ATRA and DNR respectively. RESULTS: VEGF was expressed in both NB4 and HL-60 cells. The expression of VEGF mRNA and secretion of VEGF protein could be down-regulated by ATRA and DNR respectively in a time and dose dependent manner. CONCLUSION: Besides inducing apoptosis and restraining proliferation of leukemic cells, ATRA and DNR exerted their anti-leukemia effects by reducing angiogenesis via reduction of angiogenic reaction stimulating signals.