[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.

[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.

[Effect of daunorubicin and cytarabine on cell line NB4].

OBJECTIVE: To explore whether daunorubicin (DNR) combined with cytosine arabinoside (Ara-C) and DNR alone have similar effect on acute promyelocytic leukemia (APL) cell line NB4 and acute myeloblastic leukemia cell line HL-60 in vitro. METHODS: Cell morphology, cells viability, and cell apoptosis (Annexin-V by flow cytometry assay) were analysed. RESULTS: After incubation with DNR plus Ara-C for 24 hours,NB4 cell viability [(36.75 +/- 3.82)%] (n = 6) and cell apoptosis rate [(21.24 +/- 5.82)%] (n = 3) did not change significantly compared to that treated with DNR alone for 24 hours [(35.73 + 6.28 )%, (22.55 +/- 3.26)%, respectively] (P > 0.05). However, HL-60 cell viability [(67.17 +/- 2.07)%] and cell apoptosis rate [(48.05 +/- 0.92)%] changed significantly in DNR plus Ara-C group compared with DNR alone [(63.31 +/- 1.80)% ,(41.51 +/- 0.89)%, respectively] (P < 0.01 and < 0.05, respectively). CONCLUSION: DNR plus Ara-C and DNR alone have similar effect on NB4 cells, but have different effect on HL-60 cells.

[Effects of quercetin on morphology and VEGF secretion of leukemia cells NB4 in vitro].

OBJECTIVE: To investigate the effects of quercetin on cell morphology and VEGF expression of acute myeloblastic leukemia cells NB4 in vitro. METHODS: The cytomorphology of NB4 cells was assessed by Wright-stain, apoptosis rate by apoptotic marker Annexin V, and VEGF secretion level by ELISA. RESULTS: Typical apoptosis was found in NB4 cells after treatment with quercetin. Apoptotic marker Annexin V analysis showed that the apoptotic rate of NB4 cells was increased after treatment with quercetin. The secretion of VEGF of NB4 cells was significantly decreased after treatment with quercetin. CONCLUSION: Quercetin can induce apoptosis and inhibit secretion of VEGF in NB4 leukemia cells.

PNAS-2: a novel gene probably participating in leukemogenesis.

OBJECTIVE: As(4)S(4) is an effective drug for the treatment of acute promyelocytic leukemia but its mechanism of action remains largely unknown. In a previous study, we identified PNAS-2, a human apoptosis-related protein gene, using gene expression profiling. In this study, we tried to clarify the role of PNAS-2 in apoptosis and leukemogenesis. METHODS: NB4 and U937 leukemia cell lines and serial clinical samples were studied. RNA interference (RNAi) and RNA overexpression were used to address the potential role of PNAS-2 in apoptosis. PNAS-2 expression was examined using Northern blot in multiple tissues, and real-time PCR was applied to analyze PNAS-2 expression in various patient samples. RESULTS: Functional analyses of PNAS-2 by RNAi and RNA overexpression indicate PNAS-2 is an anti-apoptosis gene. PNAS-2 expression is significantly increased in de novo or relapsed acute leukemia, but in patients in complete remission PNAS-2 levels decrease to levels comparable to those found in normal controls. In carcinomas, PNAS-2 expression was not upregulated, indicating that PNAS-2 overexpression was specific for leukemia. CONCLUSION: Based on the preliminary data, we suggest that the PNAS-2 gene functions as an anti-apoptotic gene and probably participates in leukemogenesis.

[Transfection of HL-60 cells with CYP3A5 gene induces drug-resistant phenotype].

OBJECTIVE: To investigate if CYP3A5 gene is involved in the molecular mechanisms for multiple drug resistance in leukemia cells. METHODS: A full length cDNA of CYP3A5 gene was cloned, and a recombinant eukaryotic expression plasmid was constructed, then stably transfected cell lines were established. Furthermore, the sensitivity of those cell lines to several anticancer drugs were assessed by MTT and FCM assay. RESULTS: The recombinant plasmid was designated as pcDNA3-CYP3A5. Transfecting HL-60 cells (which didn't show transcript of CYP3A5 gene) with recombinant plasmid pcDNA3-CYP3A5 generated HL-60/CYP3A5 cell line, and transfecting of HL-60 cells with the parental pcDNA3 vector served as control HL-60/pc cell line. Daunorubicin induced remarkable apoptosis peaks in HL-60 and HL-60/pc cells, while such effect did not occur in HL-60/CYP3A5 cells (apoptosis cell percentage were 7.3%, 6.3% and 1.2%, respectively). Compared with HL-60 and HL-60/pc cells, HL-60/CYP3A5 cells were statistically significantly resistant to daunorubicin, aclacinomycin A, vincristine and harringtonine (resistance multiples were 2.89, 2.01, 4.05 and 2.79 times, respectively, P < 0.05), however the sensitivity to teniposide didn't change (resistance multiple was 1.04 times). CONCLUSION: Transcription of CYP3A5 gene in leukemia cells directly induces resistance to anthracyclines and alkaloids, however the cells are still sensitive to epipodophyllotoxins. Therefore, our findings confirmed a new mechanism of multidrug resistance.

[Reversal of multidrug resistance of HL-60 adriamycin resistant leukemia cell line by quercetin and its mechanisms].

OBJECTIVE: Quercetin, a widely distributed natural flavonoid with a variety of biological functions, can reverse multidrug resistance (MDR) in leukemia according to recent researches. The aim of this study was to investigate the mechanisms of reversal of multi-drug resistance by quercetin mainly in respect of membrane transporters. METHODS: MTT cell viability assay was used to verify the chemo-sensitization to daunorubicin (DNR) by quercetin in HL-60/ADM cell line and determine the effective reversal concentration, the expression of MRP(1) gene and its protein product, multidrug resistant associated protein 1 by RT-PCR and flow cytometry By confocal laser scanning microscopy, the subcellular distribution of DNR in HL-60/S and HL-60/ADM cells was examined before and after quercetin exposure. RESULTS: Compared with HL-60/S, 20-40 micromol/L quercetin in vitro remarkably enhanced the sensitivity of HL-60/ADM cells to daunorubicin, down-regulated the expression of MRP(1) gene and its protein product MRP(1), restored the abnormal subcellular distribution of daunorubicin, so as to reverse MDR. Moreover, such an effective concentration of quercetin was non-toxic to the cells. CONCLUSION: Quercetin could be a candidate of effective multidrug resistance-reversing agent with low toxicity in leukemia chemotherapy.

[Restorative effect of quercetin on subcellular distribution of daunorubicin in multidrug resistant leukemia cell lines K562/ADM and HL-60/ADM].

BACKGROUND & OBJECTIVE: Quercetin, a widely distributed natural flavonoid with a variety of biological functions, can reverse multidrug resistance (MDR) in leukemia according to recent researches. This study was to investigate the mechanism of quercetin restoring subcellular distribution of daunorubicin (DNR) in multidrug resistant leukemia cell lines, K562/ADM and HL-60/ADM, and reversing their MDR. METHODS: MTT cell viability assay was used to verify the sensitization of DNR by quercetin in K562/ADM and HL-60/ADM cells,and determine the reverse concentration extent,confocal laser scanning microscope was used to observe the subcellular distribution of DNR in K562/ADM and HL-60/ADM cells,and relevant sensitive cell lines, K562/S and HL-60/S,before and after quercetin exposion. RESULTS: Compared with K562/S and HL-60/S cells,20-40 micromol/L of quercetin in vitro remarkably enhanced the sensitivity of K562/ADM and HL-60/ADM cells to DNR, restore the subcellular distribution of DNR, so as to reverse MDR. CONCLUSION: quercetin could be a candidate of effective multidrug resistance-reversing agent in leukemia 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.

[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.

[Dynamic observation of vascular endothelial growth factor (VEGF)/VEGF-receptors expression in acute leukemia].

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF)/VEGF-receptors and its relation to bone marrow angiogenesis in acute leukemia patients before and after chemotherapy. METHODS: Bone marrow biopsies from 122 cases with different stages of human acute leukemia were immunostained with anti-vascular endothelial growth factor, anti-fms-like tyrosine kinase (Flt-1) and anti- kinase-domain insert containing receptor (KDR) antibodies with envision two-step immunohistochemical staining method. RESULTS: The expression of VEGF and KDR protein in remission patients was 5.3 (3.3 - 9.0) and 2.0 (1.0 - 4.0) being significantly lower than newly diagnosed untreated patients 6.0 (3.3 - 12.0) and 5.3 (3.3 - 8.0) (P < 0.05, 0.01). However nonsignificant decrease was shown among non-remission patients. In relapsed patients the expression of VEGF and KDR was significantly increased as compared with that in newly diagnosed patients. Flt-1 staining levels were in the same range between the newly diagnosed untreated patients and control group (P > 0.05), but significantly increased in remission or relapse patients, being 3.3 (1.7 - 5.3) in the remission group and 3.3 (2.0 - 5.3) in the relapse group (P < 0.01). Expression of VEGF and KDR protein was significantly higher in patients with a high degree of bone marrow microvessel counts as compared with those with a low degree and the expression correlated well with microvessel counts (P < 0.01). There was a positive correlation of the percentage of bone marrow blasts with VEGF and KDR expression in AML patients (r = 0.429, 0.359; P = 0.005, 0.02), and with VEGF expression in ALL patients (r = 0.522; P = 0.03). CONCLUSION: VEGF and its two specific cellular receptors are expressed in both haematopoietic cells and endothelial cells. VEGF may be a autocrine factor and modulates the angiogenic reaction in bone marrow as a paracrine factor. VEGF and its cellular receptor KDR may constitute promising targets for antiangiogenic and antileukemic treatment strategies.

[Effects of Red Orpiment on Cell Morphology and Expression of PML mRNA and Protein in NB4, and HL-60 Cells]

In this study the effects of red orpiment on NB4 and HL-60 cells were tried to determine. Semi-quantitative RT-PCR to determine the PML mRNA expression, immuno-fluorcscence study, together with the fluorescence stain and morphological observations were used. The results showed that: (1) red orpiment induces apoptosis morphologically in NB4 and HL-60 cells, the morphology of typical apoptosis can be seen in NB4 and HL-60 cells after 12 hours of treatment with red orpiment. Through the Wright's stain, we can see the extensive membrane blebbing, condensation of heterochromatin, and cell fragmentation, apoptotic body appearing. Many dead cells can be found on the second day. (2) in NB4 cells, red orpiment is shown to induce the PML-RARalpha chimera disappearance and to reorganize then to degradation of PML nuclear bodies which also seen in HL-60 cells, indirect immunofluorescence staining of PML with a specific monoclonal antibody was performed in control and treated cells. In NB4 cells, the control was diffusely microspeckled pattern of immunoreactivity. Upon red orpiment treatment, the microspeckled pattern disappeared, PML protein reversed into normal location. and the the size and the brightness of the particles were increased obviously. The normal nuclear distribution of PML protein was seen in untreated HL-60 cells. After treatment with red orpiment, in the nuclei of HL-60 cells, the size and the brightness of the particles were also increased. After two days of treatment with red orpiment, the immunofluorescent particles in cells almost disappeared. (3) the expression of PML mRNA is not changed in red orpiment-treated cells, RT-PCR to determine the PML mRNA expression in NB4 and HL-60 cells treated with red orpiment, the expression results are similar to the controls, that to say, the PML mRNA lever is unaffected. It was concluded that, red orpiment induced PML to play the effects of induce apoptosis in leukemia cells at the translational level and inhibited the proliferation of leukemia cells.