ABSTRACT
The aim of this study was to improve the dyeing method of hydroperoxidase (HPO), to analyze the morphologic features of Phi bodies and to evaluate the clinical application of this method. 128 bone marrow or peripheral blood smears from patients with myeloid and lymphoid malignancies were stained by improved HPO staining. The Phi bodies were observed with detection rate of Phi bodies in different leukemias. 69 acute myeloid leukemia (AML) specimens were chosen randomly, the positive rate and the number of Phi bodies between the improved HPO and POX stain based on the same substrate of 3, 3'diaminobenzidine were compared. The results showed that the shape of bundle-like Phi bodies was variable, long or short. while the nubbly Phi bodies often presented oval and smooth. Club-like Phi bodies were found in M(3). The detection rates of bundle-like Phi bodies in AML M(1)-M(5) were 42.9% (6/14), 83.3% (15/18), 92.0% (23/25), 52.3% (11/21), 33.3% (5/15) respectively, and those of nubbly Phi bodies were 28.6% (4/14), 66.7% (12/18), 11.1% (3/25), 33.3% (7/21), 20.0% (3/15) respectively. The detection rate of bundle-like Phi bodies in M(3) was significantly higher than that in (M(1) + M(2)) or (M(4) + M(5)) groups. The detection rate of nubbly Phi bodies in (M(1) + M(2)) group was higher than that in M(3) group. In conclusion, after improvement of staining method, the HPO stain becomes simple, the detection rate of Phi bodies is higher than that by the previous method, the positive granules are more obvious, and the results become stable. This improved method plays an important role in differentiating AML from ALL, subtyping AML, and evaluating the therapeutic results.
Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Bone Marrow , Pathology , Coloring Agents , Leukemia, Myeloid, Acute , Diagnosis , Pathology , Staining and LabelingABSTRACT
<p><b>OBJECTIVE</b>To construct a short hairpin RNA (shRNA) eukaryotic expression vector specific to MDR1 gene in multidrug resistance (MDR) human leukemia cell line K562/A02 to observe its silencing effect on MDR1 and P-glycprotein (P-gp) expression.</p><p><b>METHODS</b>The shRNA expression vector was constructed by gene recombination, then transfected into the cultured K562/A02 cells. The transcription of MDR1 gene was detected by semi-quantitative RT-PCR and the expression level of P-gp was determined by Western blot. 50% inhibition concentration (IC50) of ADM in K562/A02 cells was determined by MTT method. The intracellular doxorubicin (ADM) concentration was determined by HPLC.</p><p><b>RESULTS</b>The introduction of pEGFP-C1/U6/MDR1-A or pEGFP-C1/U6/MDR1-B expression vector was shown to efficiently and specifically inhibit the expression of P-gp according to results of Western blot, with an inhibitory rate of 50.67%. Semi-quantitative RT-PCR showed that mRNA transcription of MDR1 gene was reduced by (48.2 +/- 2.5)%. On the contrast, the control plasmid did not exhibit inhibitory effect on the protein expression and mRNA transcription of MDR1. The relative efficiency of K562/A02 to ADM was 40.8% or 62.4%, respectively, and the intracellular accumulation of ADM increased after shRNA treatment.</p><p><b>CONCLUSION</b>The shRNA expression vector targeting MDR1 gene showed dramatic inhibition on RNA transcription and protein expression. It could effectively restore the sensitivity of K562/A02 cells to conventional chemotherapeutic agents.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Antibiotics, Antineoplastic , Metabolism , Pharmacology , Blotting, Western , Cell Survival , Dose-Response Relationship, Drug , Doxorubicin , Metabolism , Pharmacology , Drug Resistance, Multiple , Genetics , Drug Resistance, Neoplasm , Genetics , Gene Silencing , Genetic Vectors , Genetics , K562 Cells , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
To construct a plasmid expressing MDR1 short hairpin RNA (shRNA) mediated by pEGFP-C1/U6 vector, two coding sequences of 19 nucleotides were selected from MDR. Two pairs of oligonucleotides were designed for these two fragments. After annealing the formed double-stranded DNAs were ligated with plasmid pEGFP-C1/U6 (pEGFP-C1 vector with U6 promoter). The plasmids producing MDR1 shRNA were constructed from the inverted motif containing 9 spacers and four Ts. The results showed that the constructed plasmids were named pEGFP-C1/U6/A and pEGFP-C1/U6/B, and the constructs were identified by restriction and sequence analysis, no any base mutation was observed. It is concluded that plasmids of pEGFP-C1/U6/A and pEGFP-C1/U6/B expressing MDR1 shRNA were successfully constructed, providing a highly effective method for reversing the multidrug resistance in clinic.
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , DNA-Binding Proteins , Drug Resistance, Multiple , Genetics , Drug Resistance, Neoplasm , Genetics , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Plasmids , Genetics , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , RNA, Small Nuclear , GeneticsABSTRACT
To investigate the chemosensitizing effect of pyrroledithiocarbomate (PDTC) on daunorubicin in drug-resistant leukemic cells in vitro, MTT method was used to observe the changes of the proliferation of intractable leukemia MNC treated with daunorubicin (30 microg/ml) combined with PDTC (25, 50 or 100 micromol/L). The results showed that inhibiting rate of daunorubicin combined with PDTC(25, 50 or 100 micromol/L) on drug-resistant leukemic cells was significantly higher than that of daunorubicin alone (P < 0.05). Among the three different doses of PDTC, the concentration of 50 micromol/L of PDTC inhibited the proliferation of drug-resistant leukemic cells significantly. In conclusion, PDTC can sensitize anti-tumor effect of daunorubicin in vitro. The concentration of 50 micromol/L of PDTC has stronger chemosensitizing effect on daunorubicin than that of the other concentrations of PDTC (25 micromol/L or 100 micromol/L) in vitro.
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Bone Marrow Cells , Pathology , Cell Proliferation , Daunorubicin , Pharmacology , Dose-Response Relationship, Drug , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Synergism , Leukemia , Blood , Pathology , Leukocytes, Mononuclear , Pathology , Proline , Pharmacology , Thiocarbamates , Pharmacology , Tumor Cells, CulturedABSTRACT
OBJECTIVE@#To explore the effect of neferine on the chemotherapic sensitivity of STI 571 to K562/A02 cells and to reveal its mechanism.@*METHODS@#MTT method was used to observe the alteration of the proliferation of K562/A02 cell line treated with STI571 alone or combined with neferine. The transcription of mdrl gene was detected by semi-quantitative RT-PCR and the P-gp expression was determined by Western blot after STI 571 alone or combined with neferine treatment.@*RESULTS@#The cytotoxic effect of STI 571 (1 micromol/L) combined with neferine (IC50 = 3.02 micromol/L) on K562/A02 cell line was significantly higher than that of STI 571 alone (IC50 = 0.689 micromol/L). After treating with STI571 combined with neferine, the synergistic interaction on K562/A02 cells increased 4.38 folds (P < 0.05); the mdrl mRNA expression by semi-quantitative RT-PCR was significantly reduced by (45.4 +/- 2.5) % (P < 0.01); and the P-gp expression by Western blot was deregulated by 40.58% (P < 0.05).@*CONCLUSION@#Neferine significantly enhances the antineoplastic effect of STI 571 on K562/A02 cells by reducing mdrl mRNA transcription and blocking P-gp expression.