Expression and Clinical Significances of HGFA, Matriptase, HAI-1 and HAI-2 in Acute Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression and clinical significances of HGFA, Matriptase, HAI-1 and HAI-2 in patients with acute myeloid leukemia (AML).</p><p><b>METHODS</b>The bone marrow samples from 91 AML patients, 41 AML patients in complete remission, and 32 normal controls were collected. Real time fluorescence quantitative RT-PCR (qRT-PCR) was used to detect the mRNA expressions levels of HGFA, Matriptase, HAI-1, HAI-2 . The expressions of these genes were compared among AML untreated group, the complete remission group and the healthy control group. The correlation of their expression with clinical characteristics was analyzed.</p><p><b>RESULTS</b>The level of HGFA in the AML untreated group was higher than that in the healthy control group（P<0.05）, while the HAI-2 mRNA level was lower than that in the healthy control group（P<0.05）. The mRNA levels of HAI-1 and Matriptase were not changed significantly in all groups. The HAI-2 mRNA expression level was significantly lower in the high white blood cell group (P<0.05).</p><p><b>CONCLUSION</b>The abnormal activation of HGF/c-Met signaling system in AML may result from the increase of HGFA expression and the decrease of HAI-2 expression of the upstream regulatory factors.</p>

Expression of HIF-1α in primary acute myeloid leukemia cells and its relationship with prognosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to detect the expression of HIF-1α in acute myeloid leukemia (AML) except acute promyelocyte leukemia (APL) and investigate the relationship of its expression levels with clinical parameters and prognosis.</p><p><b>METHODS</b>The primary AML cells were collected from peripheral blood of 53 newly diagnosed AML patients by using CD3 negative sorting. The expression of HIF-1α was measured by real-time fluorescent quantitative PCR (FQ-PCR) , and the relationship between expression level of HIF-1α and clinical parameters (age, sex, WBC count, clinical typing, prognosis) was analysed according to relative expression level. Furthermore, Western blot was used to detect the protein level of HIF-1α in AML patients with or without extramedullary infiltration.</p><p><b>RESULTS</b>The expression level of HIF-1α did not correlate with age, sex, WBC count, Hb level, Plt count and the percentage of blast. There was no significant difference of HIF-1α expression between different AML subtype based on FAB. The higher level of HIF-1α was found in AML patients who did not get complete remission after one or two courses of chemotherapy, however, the difference was not statistically significant. The relapse rate was higher in AML patients with the higher expression of HIF-1α. In addition, the higher level of HIF-1α mRNA and protein were found in bone marrow of AML patients with extramedullary infiltration (P < 0.01). The negative correlation between HIF-1α and PTEN was observed (r = -0.48, P = 0.001).</p><p><b>CONCLUSIONS</b>Overexpression of HIF-1α are closely related with extramedullary infiltration and prognosis of acute myeloid leukemia, and may be used as an early indicator of extramedullary infiltration and prognosis.</p>

Expression of Hydrodynamic Injection-mediated PD-L1 in Myeloablative Conditioning Mouse Model / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish the mouse model for the expression of PD-L1 by hydrodynamic injection and to study the effects of myeloablative conditioning on hydrodynamic injection-mediated PD-L1 expression.</p><p><b>METHODS</b>Plasmid amplification, hydrodynamic injection, collagenase perfusion, real time PCR, ELISA and flow cytometry were applied to test the expression and function of PD-L1. Also, animal models were set up to test the effects of chemical or radiactive myeloablative conditioning on hydrodynamic injection-mediated PD-L1 expression.</p><p><b>RESULTS</b>The expression of PD-L1 mRNA and protein could be detected as early as 8 h after hyrodynamic injection and reached peak expression by 24 h, and returned to baseline level by 7 d after injection. Serum PD-L1 level reached to 100 µg/ml as early as 24 h after injection and plateaued at 7 d after injection. Serum PD-L1 persisted for 3 weeks and declined to baseline after 1 month of hydrodynamic injection. The PD-L1 function induced by hydrodynamic injection was consistent with literature reports. At each time point, the PD-L1 expression was not different significantly between the myeloablative conditioning group and control group; the mice transfected with PD-L1 showed a higher survival rate than that in control group.</p><p><b>CONCLUSION</b>Myeloablative conditioning does not affect hydrodynamic injection-mediated PD-L1 expression, indicating that the PD-L1 can be used in HSCT mouse model.</p>

Expression of CD131 Gene in Acute Myeloid Leukemic Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the expression level and the mutantion of CD131 in acute myeloid leukemic (AML) cells, and to analyze the relationship of CD131 expression level with clinical features.</p><p><b>METHODS</b>The peripheral blood mononuclear cells (PBMNC) from 44 AML patients and 25 healthy donors were collected, and the expression level of CD131 mRNA was detected by RT-PCR. The full length coding sequence of CD131 from 15 patients and 5 healthy donors was amplified by RT-PCR, and linked to pGEM-T vector to clone TA, 10 positive recombinant clones of each sample were analyzed by DNA sequencing. The AML patients were divided into CD131 negative and CD131 positive groups according the expression level of CD131, and white blood cell counts, CD34(+) cells percentage, complete remission rate of patients were compared.</p><p><b>RESULTS</b>CD131 was expressed at a lower level in AML than that in healthy donor. Five kinds of CD131 mutantions could be detected in both AML and healthy donor groups, but the mutation rate in AML (75.33%) was higher than that in healthy donors (18.0%). CD131 negative group showed a higher CD34(+) cells percentage (69.1% ± 20.8%), and lower complete remission rate (33.3%) than that in CD131 positive group (69.1% ± 20.8%, 33.3%). No statistically significant difference of WBC counts was found between 2 groups.</p><p><b>CONCLUSION</b>CD131 is expressed at a low level and shows high frequency of mutation in acute myeloid leukemic cells. CD131 negative AML correlats with high proportion of CD34(+) cells, and showed insensitive to chemotherapy.</p>

Expression of G-CSFR IV isoform in adult acute myeloid leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the expression of granulocyte colony-stimulating factor receptor IV(G-CSFR IV) in adult acute leukemia patients and its clinical significance. The bone marrow hematopoietic stem cells from healthy persons were used as controls. The real-time RT-PCR was used to determine the expression level of G-CSFR I-IV in 99 AML, 34 ALL patients and 19 healthy persons. The results showed that the relative expression level of G-CSFR IV/G-CSFR I in AML patients was obviously elevated, as compared with that in ALL patients and controls, while the relative expression level of G-CSFR IV/G-CSFR I in ALL patients showed no statistical difference from controls. The analysis of clinical features and chemotherapeutic efficacy demonstrated that the clinical remission rate in patients with high expression of G-CSFR IV/G-CSFR I was lower than that in patients with low expression. The relative expression level of G-CSFR IV/G-CSFR I was not related with risk stratification from sex, age, blast ratio, FAB typing, chromosome and fusion gene. It is concluded that the abnormal high expression of G-CSFR IV relates with poor prognosis of AML.

Regulation of all-trans retinoic acid and arsenic trioxide on CD44v6 expression in NB4 cells / 中国实验血液学杂志

The adhesion molecule CD44 variant isoform (CD44v6) closely associates with progress of acute myeloid leukemia (AML). This study was purposed to investigate the effects of all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) on the expression of CD44v6 and the associated signal pathway phosphatidylinositol 3-kinase (PI3K)/Akt in acute promyelocytic leukemia (APL) cell line NB4 cells. The differentiation of NB4 was detected by morphologic observation and flow cytometry; the NB4 cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI double staining; the CD44v6 mRNA expression in NB4 cells was determined by real-time RT-PCR, the CD44v6 protein expression and changes of PI3K/Akt signal pathway in NB4 cells were analysed by Western blot. The results demonstrated that in ATRA-induced differentiation, the transcriptional level of CD44v6 was dominantly down-regulated, the translational level of CD44v6 did not change and the PI3K/Akt signal axis was activated. In As2O3-induced apoptosis, both the transcriptional level and translational level of CD44v6 were remarkably reduced, and the PI3K/Akt pathway was inhibited. It is concluded that the regulation of ATRA on expression of CD44v6 in NB4 cells differs from that of As2O3. The results provide an experimental basis to reveal the different mechanism of ATRA and As2O3 in view of the intercommunication between leukemia cells and hematopoietic microenvironment.

Direct contact with bone stromal HS-5 cells reduces the sensitivity of HEL to drugs / 中国实验血液学杂志

This study was proposed to investigate the sensitivity and resistence of HEL cells co-cultured with bone marrow stromal HS-5 cells to chemotherapeutic drugs. HEL cells were cultured in direct contact with HS-5 cells for 6, 12, and 24 h. Cell Counting Kit-8 (CCK-8) was used to determine the sensitivity of HEL cell to cytarabine, methotrexate, VP16, and daunomycin. Cell cycle distribution was determined by using flow cytometry. Real-time RT-PCR was performed to detect the transcription levels of p19, p21, p27, MDR1, ABCG2 and bcl-2. Western blot was performed to determine the protein levels of p-Akt(Ser473), p-glycogen synthase kinase 3β (p-GSK3β(Ser9)), p-signal transducer and activator of transcription (p-STAT3(Tyr705)), Bcl-2, cleaved-Notch1(V1754), and Hes1. The results showed that chemo-sensitivity of HEL cells was remarkably reduced when co-cultured with HS-5 cells. HEL cells were arrested in the G(0)/G(1) phase after co-culture for 24 h. Transcription of p21 was significantly up-regulated at 6 h. Transcription of p19 decreased at 12 h and returned to baseline at 24 h. No significant changes in the mRNA expression of other genes were found. The expressions of p-Akt(Ser473), p-GSK3β(Ser9), cleaved-Notch1(V1754) and Bcl-2 proteins were significantly up-regulated in HEL cells, and Hes1 protein was significantly down-regulated. There was no change in p-STAT3(Tyr705) expression. It is concluded that the direct contact with HS-5 cells can reduce the chemo-sensitivity of HEL cells.

microRNAs expression profile in acute promyelocytic leukemia cell differentiation induced by all-trans retinoic acid and arsenic trioxide / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression profile of microRNAs in acute promyelocytic leukemia (APL) cells during differentiation.</p><p><b>METHODS</b>Differentiation of APL cell line NB4 cells was induced by all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3). Morphological and immunological assay was performed by Wright-Giemsa staining and flow-cytometric analysis of CD11b surface expression. During in vitro NB4 differentiation induced by ATRA and As2O3, microRNA expression profiles (miR-15b, miR-16, miR-34a, miR-107, miR-124a, miR-146, miR-155, miR-181a, miR-223, miR-342, let7c) were detected by real time RT-PCR, and the relative expression level of microRNAs were quantitatively analyzed by using 2(-ΔΔCt), and compared with that of control group. Meanwhile, the microRNA expression profiles were also detected in 15 newly diagnosed APL patients and 15 complete remission (CR) APL cases by real time RT-PCR, and the relative expression level of microRNA was quantitated by using 2(-ΔCt), and compared with that of control group (newly diagnosed APL as control group). These data were expressed as x(-) ± s, and differences between groups were examined using t test. P < 0.05 was considered statistically significant.</p><p><b>RESULTS</b>The expression levels of miR-15b, miR-16, miR-107, miR-223 and miR-342 in NB4 differentiation group were obviously up-regulated (3.40, 4.22, 5.41, 20.03 and 5.29 folds higher in ATRA treated NB4 cells than that of control group respectively, and 3.62, 2.49, 2.58, 4.27 and 1.94 folds higher in AS2O3 treated NB4 cells than that of control group respectively), except for miR-15b, the expression levels of miR-16, miR-107, miR-223 and miR-342 in ATRA treated group was significantly higher than that in As2O3 treated group. The relative expression levels of miR-15b, miR-16, miR-107, miR-181a, miR-223 and miR-342 were 0.4137, 0.6367, 0.1260, 0.0522, 0.6611, 0.0280 in APL CR group, and 0.0751, 0.2022, 0.0425, 0.3064, 0.1733, 0.0090 in newly diagnosed APL group, respectively. The expression level of miR-15b, miR-16, miR-107, miR-223 and miR-342 in APL CR group were significantly upregulated compared with that of newly diagnosed APL groups (P < 0.05), while the expression level of miR-181a was significantly downregulated (P < 0.05).</p><p><b>CONCLUSION</b>Specific expression of microRNA profiles is a key contributing factor in the differentiation of APL.</p>

Packaging of lentivirus carrying gene hβc and overexpression of gene hβc in NB4 cells / 中国实验血液学杂志

This study was aimed to overexpress gene hβc in NB4 cells via the method of lentivirus-mediated gene transfer, to observe the differentiation behaviour change of hβc over-expressing NB4 cells treated with IL-3 or GM-CSF, to explore the relationship between hβc gene and the differentiation behaviour of NB4 cells. The targeted hβc gene was amplified by PCR from the cloned vector carrying ORF of hβc. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying hβc, named pLV-hβc. And the pLV-hβc plasmid was confirmed by restriction and sequencing. The recombinant lentivirus was produced by co-transfecting three plasmids into 293T packing cells. After transfection, the lentiviral supernatant was collected to transfect NB4 cells. GFP expression was examined by fluorescent microscope and the expression of hβc gene was detected by Western blot. Then, the NB4 cells over-expressing hβc were treated with IL-3 (10 ng/ml), GM-CSF (10 ng/ml), ATRA (1 µmol/L) respectively, and the CD11b expression, morphology and differentiation behaviour changes of every groups were observed by flow cytometry and microscopy, while NB4 cells transfected with blank lentivirus (NB4-blank cells) were used as controls. The results showed that the recombinant lentivirus vector carrying hβc gene could efficiently transfect NB4 cells and made NB4 cells to stably over-express hβc gene. The expression of CD11b was up-regulated in NB4-hβc cells treated with of IL-3 or GM-CSF, but it was not as obvious as the effect of ATRA, and no morphological change was observed in NB4 hβc cells treated with the IL-3 or GM-CSF. It is concluded that IL-3 or GM-CSF can induce NB4 cells over-expressing hβc to differentiate to neutrophils, but can not make them fully matured.

Expression pattern of myeloid differentiation-related transcription factor mRNA in differentiation of NB4 and HL-60 cells induced by all-trans retinoic acid / 中国实验血液学杂志

Hematopoiesis is coordinated by a complex regulatory network of transcription factors that involves proliferation, differentiation and maturation of a very small population of pluripotent hematopoietic stem cells with self-renewing and differentiating into various specialized and distinct blood cell types. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML). The purpose of this study was to investigate the expression pattern of transcription factor mRNA in acute myeloid leukemia (AML) cells during in vitro differentiation. The 2 human leukemic cell lines HL-60 and NB4 had been used as model cell lines. Differentiation of HL-60 and NB4 cells was induced by all-trans retinoic acid (ATRA) for 4 days. Morphological changes were observed by May-Grunwald Giemsa stainings, the CD11b expression level was detected by flow cytometry. Transcription factor mRNA profiles (PU.1, C/EBPα, ε, γ, GATA-1, GATA-2) were determined by real time RT-PCR during in vitro HL-60 and NB4 differentiation; The expression level of transcription factor mRNA was relatively quantitatively analyzed by using 2(-ΔΔCT) and compared with control group. The results showed that the expression levels of PU.1 and C/EBP ε mRNA in NB4 differentiation group were 5.75 and 6.16, respectively, which were significantly higher than those in untreated group; while the expression level of C/EBPα, γ, GATA-1, GATA-2 mRNA in NB4 differentiation group were 62%, 31%, 63% and 8.7% respectively, which were significantly lower than those in untreated group; In HL-60 differentiation group, the expression levels of PU.1, C/EBPα, ε were 1.97, 1.95 and 2.35 respectively, which were significantly higher than those in untreated group; while the expression levels of C/EBPγ, GATA-1, GATA-2 in HL-60 differentiation group were 20%, 21% and 18% respectively, which were significantly lower than those in untreated group. It is concluded that dysregulation of transcription factors is a key contributing factor in the pathogenesis of acute myeloid leukemia.

Establishment and characterization of highly tumorigenic leukemia cell line HL-60 transplanted through repeated passages into nude mice / 中国实验血液学杂志

This study was purposed to investigate the change of biological characteristics of HL-60 cells with high tumorigenicity transplanted through repeated passages into nude mice and to explore the tumorigenic mechanisms of the cell line. The human highly tumorigenic leukemia cell line HL-60 model in nude mice were established by serial passages in vivo and in vitro, and their biological features were compared. The trypan blue staining assay was used to detect the cell growth, the flow cytometry was used to analyze the cell cycle, the transmission electron microscopy and laser scanning confocal microscopy were used to observe the cell ultrastructures and cell fluorescence level respectively. The results indicated that the cell growth velocity was quickened, cell doubling time was shortened in high tumorigenic leukemia cell line HL-60; the cell count in S phase increased; the amount of mitochondria in HL-60 cells obviously decreased, furthermore the dilation of interspace, decrease of the number of ridges, vacuolation of mitochondria, significant reduction of fluorescence level in microfilament and enhancement of cell cloning efficacy were observed. It is concluded that the high tumorigenicity of HL-60 cells multiple-passaged in nude mice is associated with enhancement of proliferative ability, changes of number and structure of mitochondria in HL-60 cells, and alteration of microfilament in cytoskeleton.

Preparation of recombinant polypeptide of N-terminal heparin-binding domain of fibronectin and its effect on disseminated intravascular coagulation in rats / 中国实验血液学杂志

This study was aimed to prepare the polypeptide of N-terminal heparin-binding domain of fibronectin(rhFNHN-29 polypeptide) with pichia expression system, to detect biological activity of recombinant polypeptide and investigate its effect on disseminated intravascular coagulation (DIC) in rats. The sequence of N-terminal heparin-binding domain of fibronectin was amplified from FNcDNA by PCR. The aim gene was cloned into T vector for selection. Then it was cloned into pAo815SM and pPIC9K vectors.Lined pPIC9K vectors were transformed into GS115 Pichia cells so as to express the aim polypeptide in Pichia expression system. The fermentation liquid were precipitated by 80% ammonium sulfate, and the further dissolved sediment were purified using S-100 column and SP column. Its activity of binding with heparin were detected by Western-blot. The established DIC rats (40 rats) were randomly divided into two groups. One group was treated with rhFNHN-29 polypeptide, and the other was treated with normal saline. The rats in the former group were injected with rhFNHN-29 polypeptide (10 mg/kg) through tail vein at 0.5 hour before, 2 hours and 4 hours after injection of LPS respectively. The rats in latter group were injected with equal volume saline. In addition, 20 normal rats injected with normal saline were as normal controls. 500 microl blood was taken from the rat vein, at 6 hours after the injection of LPS. White blood cell (WBC), hemoglobin (Hb) and platelets were tested from 50 microl blood. The rest 450 microl blood was used to isolate plasma for detecting TNFa level and coagulogram. The rats were killed at 24 hours after injection with LPS. Their livers, lungs, hearts, kidneys, and brain tissues were taken for histopathologic examination. The results showed that the aim polypeptide was successfully expressed in Pichia expression system. The expression level reached approximately 30 mg/L. The polypeptide had activity of binding with heparin antibody. In the experiment study of polypeptide effect on DIC in rats, the plasma TNFa level in polypeptide-treated group was lower than that in saline control group, the hemogram, coagulogram and histopathology were more obviously improved in polypeptide-treated group as compared with saline control group. It is concluded that the rhFNHN-29 polypeptide is successfully prepared, this polypeptide can antagonize DIC induced by endotoxin in rats.

Expression change of IL-3 receptor system in all-trans retinoic acid induced differentiation of NB4 cells / 中国实验血液学杂志

Interleukin-3 receptor (IL-3R) is a heterodimeric membrane receptor. The α subunit is essential for ligand binding and confers ligand specificity to the receptor. The common beta chain (βc) subunit, which is shared by the granulocyte macrophage-colony stimulating factor (GM-CSF), IL-3 and IL-5 receptors, is required for high-affinity ligand binding and signal transduction, mediating growth and survival of hematopoietic progenitor cells and the production and activation of mature hematopoietic cells. In order to investigate the role of IL-3 receptor system (IL-3Rα, GM-CSFRα and hβc) in myeloid differentiation, the expression level of IL-3 receptor system gene in all-trans retinoic acid (ATRA)-induced NB4 cell differentiation was detected by quantitative real time RT-PCR. At the same time, DNA sequence change was analyzed by cDNA sequencing. The results showed that the expression level of IL-3Rα mRNA was obviously down-regulated in NB4 cells treated with ATRA for 24 hours, but during differentiation of ATRA induced NB4 cells, the expression level of IL-3Rα mRNA was gradually restored, while the expression levels of GM-CSFRα mRNA and hβc mRNA were gradually up-regulated. The sequence of IL-3Rα and GM-CSFRα gene did not change before and after NB4 cells differentiation, but the sequence of hβc gene changed when NB4 cells were treated with ATRA, the expression of hβc mRNA sequence before NB4 cell differentiation taken truncated mutation as dominant, as regards expression of hβc mRNA sequence after NB4 cell differentiation, the truncated mutation of hβc mRNA had restored to wild type. It is concluded that the IL-3 receptor abnormality exists in NB4 cells, over expression of IL-3Rα and truncated mutation of hβc may be involved in proliferation and differentiation block in NB4 cells.

Selective effect of nispex in inhibiting human cancer cell proliferation and inducing cell apoptosis / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the in vitro anticancer effect of Nispex, the total flavonoids extract from Scurrula parasitic L.</p><p><b>METHODS</b>The cell proliferation inhibitory effects of Nispex on various kinds of tumor cells or non-tumor cells in human and rats were detected with MTT assay and colony forming assay respectively, the cell apoptosis induced by Nispex was detected by AO/EB fluorescence staining, TUNEL assay and AnnexinV-FITC/PI flow cytometry.</p><p><b>RESULTS</b>Nispex could significantly inhibit human cancer cell proliferation and induce human cancer cell apoptosis, especially to the proliferative cell group, but its inhibition on human non-tumor cell was insignificant, and showed no effect on murine cancer cells in the tested scope.</p><p><b>CONCLUSION</b>Nispex is a nature plant extract which shows good selectivity for killing human cancer cell.</p>

Gene expression profile of human TGF-beta signal transduction pathway of B cell type acute lymphocytic leukemia / 中国实验血液学杂志

This study was aimed to investigate the gene expression profile of TGF-beta signal transduction pathway in B-cell type acute lymphocytic leukemia (B-ALL). The gene expression profiles in B-ALL primary cells and cell lines (NALM6 cells, Raji cells), B-lymphocyte of control were detected by cDNA microarray including 113 different genes in human TGF-beta/BMP signal transduction pathway, and TGF-beta(1) mRNA expression was detected by real time RT-PCR. The B lymphocytes in peripheral blood of heacthy persons sorted by flow cytometry were used as control. The difference between them was compared. The results showed that as compared with B lymphocytes in peripheral blood of heacthy persons, the TGF-beta(1) expression in B-ALL cells, NALM6 cells and Raji cells were down-regulationed, myc and smad1 gene expressions were up-regulated, IL-6, smad 7 gene expressions were down-regulated. It is concluded that TGF-beta signal transduction is abnormal in B-cell type acute lymphocytic leukemia.

Study on cytotoxic activities on human leukemia cell line HL-60 by flavonoids extracts of Scurrula parasitica from four different host trees / 中国中药杂志

<p><b>OBJECTIVE</b>To compare the anticancer effects of flavonoids extracts of Scurrula parasitica from different host trees in vitro.</p><p><b>METHOD</b>80% ethanol extracts of S. parasitica parasitizing on Nernium indicum, Morus alba, Opsmanthus fragrans, and Sapindus mulorossi were purified by polyamides column chromatography, and the eluates of 30%, 50%, 70% and 90% ethanol were mixed as flavonoids extracts. Human acute myeloid leukemia cell line HL-60 was used to evaluate the cytotoxicity induced by flavonoids extracts of S. parasitica L with MTT assay. Apoptosis was detected by AO/EB fluorescence staining and DNA fragmentation analysis, apoptosis rates and cell cycle distribution were detected by flow cytometry analysis.</p><p><b>RESULT</b>Extract of S. parasitica parasitizing on N. indicum (NISPEX) was the most sensitive to HL-60 cells of the 4 different host trees, the IC50 value being 0.60 mg x L(-1); and extract of S. parasitica parasitizing on M. alba took the second place, the IC50 value, being 2.49 mg x L(-1); extract of S. parasitica parasitizing on O. fragrans had no effectiveness as high as 50 mg x L(-1) concentration. NISPEX induced HL-60 cell apoptosis and inhibited the cell proliferation in dose and time-dependent manner. Cell cycles were arrested at G0-G1 phase after treated with NISPEX.</p><p><b>CONCLUSION</b>Anticancer effects of S. parasitica correlated with the host trees. Flavonoids extracts of S. parasitica parasitizing on N. indicum exhibited comparatively better anticancer activity in vitro among the host trees studied. NISPEX is found to be a good candidate for anticancer.</p>

Effects of recombinant human tumor necrosis factor-alpha on HL-60 cells in vitro and in vivo / 中国实验血液学杂志

To study the effects of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on HL-60 cells in vitro and in vivo, MTT and colony forming assay were used to examine the effects of rhTNF-alpha on proliferation of HL-60 cells; AO/EB (acridine orange-ethidium bromide) staining, Annexin-V flow cytometry analysis and TUNEL assay were used to detect apoptotic cells. The effect of rhTNF-alpha on xenograft growth of HL-60 cells was evaluated by tumor inhibition rate, histology, ultrastructure and TUNEL assay. The results showed that rhTNF-alpha inhibited the proliferation of HL-60 cells in a dose-dependent manner. Staining of cells with AO/EB revealed that rhTNF-alpha induced nuclear chromatin condensation and fragmentation. Positive Annexin V-FITC on cell membrane showed that rhTNF-alpha induced apoptosis of HL-60 cells in a dose-dependent manner. TUNEL assay showed that the apoptotic percentage of HL-60 cells reached 37.5% when incubated with 3200 U/ml rhTNF-alpha for 48 hours. In vivo rhTNF-alpha inhibited xenograft growth of HL-60 cells with the highest inhibition rate of 60.33%. Pathologically it was found that there were necrotic areas in the tumors of groups treated with rhTNF-alpha. There were more apoptotic cells in treatment groups than in that control group by transmission electron microscopy (TEM) and TUNEL assay. It is concluded that rhTNF-alpha is able to inhibit the proliferation of HL-60 cells and to induce apoptosis of HL-60 cells in vitro and in vivo.

Identification of the isoform in type II receptor of transforming growth factor-beta in patients with acute leukemia and its clinical significance / 中国实验血液学杂志

Recent research indicates that TGF-beta and type II receptor (TbetaR-II) play an important role in the pathogenesis of tumor. A high frequency of abnormalities in TbetaR-II has been demonstrated in various cancers. To identify the mutation of TbetaR-II in patients with acute leukemia, the bone marrow samples from 6 patients with acute leukemia and 11 normal individuals as control were detected by long-range RT-PCR. To detect a deletion in sequence of the TbetaR-IIgene, the PCR products were cloned to T vector and then sequenced. The results showed that there was existance of the isoform of TbetaR-II in 2 cases out of 6 patients with acute leukemia. These two patients had more poor prognosis than others. In conclusion, there was the isoform of TbetaR-II in partial patients with acute leukemia, and the isoform may be related with prognosis.

Experimental studies of the effects of ZnPcS2P2-based-photodynamic therapy on bone marrow purging / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>An effective purging technique plays an important role in autologous hematopoietic stem cells transplantation. Photodynamic therapy (PDT) provides a novel approach for this purpose. This study dealt with the purging effects of di-sulfo-di-phthalimidomethyl phthalolcyanine zinc (ZnPcS2P2)-based photodynamic therapy (ZnPc-PDT).</p><p><b>METHODS</b>Fluorescence intensity of cell extracts was measured using a fluorescence spectrophotometry. The proliferative potency of K562 cells and HL60 cells was detected using MTT colorimetric assay, Typan blue dye exclusion method, colony formation test. The proliferative potency of normal hematopoietic cells was evaluated using mixture colony-forming unit (CFU-Mix), granulocyte-macrophage colony-forming unit (CFU-GM), and erythrocyte colony-forming unit (CFU-E) assays. K562 cells were mixed with normal mononuclear cells (MNCs) at ratios of 1:100 and 1:1000 for creating the model of simulated remission bone marrow. Colony formation test and nested-RT-PCR were carried out to detect the residual K562 cells in cell mixture.</p><p><b>RESULTS</b>After a 5-hour incubation with ZnPcS2P2, the content of ZnPcS2P2 in normal MNCs was the lowest value. At the same time, the content in K562 cells and HL60 cells was very high. Therefore, the time point was selected as the optimal one for irradiating the cell suspensions. ZnPc-PDT could significantly kill proliferative K562 cells and HL60 cells in a dose-dependent manner. At the concentration of 1.0 microg/ml, the inhibitory rate of ZnPc-PDT on the colony formation was 100% for K562 cells, 89.7% for HL60 cells. 0.25 microg/ml ZnPc-PDT could completely photoinactivate residual K562 cells in the simulated remission bone marrow. Under an identical condition, the inhibitory rates of CFU-Mix, CFU-GM, CFU-E were 18.0%, 18.6%, and 17.8% respectively.</p><p><b>CONCLUSION</b>ZnPc-PDT appears to be a promising approach for bone marrow purging.</p>

Effect of endogenous TGF-beta1 and TNF-alpha on the As(2)O(3) inducing apoptosis of HL-60 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of endogenous TGF-beta(1) and TNF-alpha on As(2)O(3) inducing apoptosis of HL-60 cells.</p><p><b>METHODS</b>The expressions of endogenous TGF-beta(1) and TNF-alpha in apoptotic HL-60 cells induced by As(2)O(3) were assayed by RT-PCR, quantitative RT-PCR, ELISA, DNA fragmentation and TUNEL. The effect of TGF-beta(1) and TNF-alpha antisense phosphorothioate oligodeoxynucleotides (PSODNs) on As(2)O(3) inducing apoptotic HL-60 cells was further studied.</p><p><b>RESULTS</b>(1) Expressions of endogenous TGF-beta(1) and TNF-alpha were significantly up-regulated in As(2)O(3) inducing apoptotic HL-60 cells (from 13,546 +/- 124 and 497,216 +/- 187 before treatment to 23,273 +/- 229 and 674,217 +/- 189 after treatment, respectively), accompanied with down-regulated bcl-2 mRNA expression (from 10,424 +/- 274 before treatment to 3,361 +/- 89 after treatment). (2) TGF-beta(1) and TNF-alpha antisense PSODNs could rescue As(2)O(3) induced apoptosis of HL-60 cells, with a restoration of bcl-2 gene expression.</p><p><b>CONCLUSIONS</b>Endogenous TGF-beta(1) and TNF-alpha played an important role in As(2)O(3) inducing HL-60 cells apoptosis through down-regulation of bcl-2 expression.</p>