Objective:To investigate the molecular interaction between non-structural protein 3 serine protease of hepatitis C virus(HCV)and wild type P53,and to lay the basis for elucidating the mechanism of oncogenesis of hepatocellular carcinoma(HCC)after infection of HCV.Methods:The recombinant plasmids,pGAD424-NS3,pGAD424-NS315aa- and pGAD424-NS330aa-,were constructed and the interaction between NS3 serine protease and its cofactor NS4A,the interaction between wild type P53 and NS3 serine protease and its N-truncated mutants were dectected qualitatively and quantitatively in yeast two-hybrid system.Results:The results indicated that interaction existed not only between full-length NS3 serine protease and P53,but also between N-truncated mutants of NS3 serine protease and P53.Furthermore,the difference between enzyme activity unit(IU)of β-gal induced by these interactions was not significant(P>0.05).Conclusions:NS3 serine protease of hepatitis C virus and its N-truncated mutants can interact with wild type P53,and the region of NS3 serine protease involved in the interaction may be located in its C-terminal,but not in its N-terminal.

OBJECTIVETo study the reversal mechanism of adriamycin (ADM) resistance in human breast cancer cell line MCF-7/ADM by beta-elemene (beta-ELE), a wide spectrum anticancer drug derived from the Chinese herb Curcuma chaeocaulis.

METHODSSensitivity to ADM of MCF-7/ADM cells was studied by MTT assay. Intracellular accumulation of ADM in MCF-7/ADM cells was observed by fluorescent-spectrophotometry. Expression of bcl-2 protein was detected by flow cytometry.

RESULTSA non-cytotoxic dose (6 micro g/ml) and a weakly cytotoxic dose (13 micro g/ml) of beta-ELE could significantly enhance the cytotoxic effects of ADM on MCF-7/ADM cells to 1.4 and 2.2 fold as compared to the beta-ELE untreated control cells. The intracellular concentration of ADM in MCF-7/ADM cells was significantly increased after treatment with beta-ELE (P < 0.01). The expression of bcl-2 protein in MCF-7/ADM cells was reduced from 90.2% to 70.0% (P < 0.05).

CONCLUSIONbeta-ELE could partially reverse the drug resistance to ADM in MCF-7/ADM, which is related to the increased accumulation of intracellular ADM and the decreased expression of bcl-2.

Antineoplastic Agents, Phytogenic ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Curcuma ; chemistry ; Doxorubicin ; metabolism ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; Female ; Humans ; Plants, Medicinal ; chemistry ; Sesquiterpenes ; isolation & purification ; pharmacology

OBJECTIVETo explore the effect of Lignum Sappan (LS) containing serum on the proliferation cycle arrest of human lung cancer cell line PG and its molecular mechanism.

METHODSThe lung cancer PG cells were divided into four groups, i.e., the blank control group, the LS group, the LS plus cisplatin group, and the cisplatin group. They were cultured by RPMI-1640 with 20% blank serum, RPMI-1640 with 20% LS containing serum, RPMI-1640 with 20% LS containing serum plus 1 microg/mL cisplatin, and RPMI-1640 with 20% blank serum plus 1 microg/mL cisplatin, respectively. The morphology of PG cells was observed using light microscope and laser scanning confocal microscope in each group. The cell cycle arrest was observed using flow cytometry. The expression of P16 and Rb1 mRNA was tested by PCR method.

RESULTSUnder the light microscope and laser scanning confocal microscope, the apoptosis degree of PG cells in the LS group was significant, but less than that of the LS plus cisplatin group as well as the cisplatin group. Compared with the blank control group, the proportion of PG cells increased at G0/ G1 and S phases (P < 0.05) and decreased at G2/M phase (P < 0.01) in the LS group; The proportion of PG cells increased at G2/M and S phases (P < 0.05, P < 0.01) and decreased at G0/G1 phase (P < 0.01) in the LS plus cisplatin group as well as the cisplatin group. Compared with the LS group, the proportion of PG cells increased at G2/M and S phases (P < 0.05, P < 0.01) and decreased at G0/G1 phase (P < 0.01) in the LS plus cisplatin group as well as the cisplatin group. There was no statistical difference in PG cells at each phase between the cisplatin group and the LS plus cisplatin group (P > 0.05). The expression of P16 and Rb1 mRNA increased in the LS group, when compared with the blank control group. They also increased in the cisplatin group and the LS plus cisplatin group, higher than that of the LS group (P < 0.05). There was no statistical difference in the expression of P16 and Rb1 mRNA between the cisplatin group and the LS plus cisplatin group (P > 0.05).

CONCLUSIONLS containing serum induced PG cell apoptosis by up-regulating the mRNA transcription levels of P16 and Rb1, thus resulting in PG cell arrest at G0/G1 and S phases, which was different from the manner of cisplatin (achieved by arresting PG cells at G2/M and S phases through regulating cyclinB1 mRNA transcription).

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Lung Neoplasms ; pathology

Objective：To investigate the correct expression of dengue 2 virus 43 strain NS1 gene in transfected BHK-21 cell. Methods：The D2-43 DNA fragment coding for signal peptide plus NS1 protein was cloned between KpnⅠ site and EcoR Ⅰ site of expression plamid pcDNA3.1. The obtained recombinant vector pcDNA-NS1 was transfected into BHK-21 cells with electroporation technique. After selection by G418, resistant clones were screened by RT-PCR and Western blotting test. Results：The RT-PCR results of four in five randomly selected cell clones were positive. Western blotting test showed that NS1 gene could be expressed in BHK-21 cells. Conclusions：NS1 protein was capable of being expressed and appropriately processed in pcDNA-NS1 transfected BHK-21 cells. The present results suggest the feasibility of NS1-based DNA immunization.

OBJECTIVETo study the effect of tanshinone microemulsion (Tan-M) on the cytotoxicity to human leukemia-cell-line (K562/ADM) and the reversion of MDR in vitro.

METHODMicroemulsion being supposed as the control group, MT method is adopted to test cytotoxicity and the reverse of MDR.

RESULTObvious cytotoxicity to K562/ADM was observed for tan-M. Cell non-toxic dosage (growth quotiety > 95%) of Tan-M is 0.2 microg x mL(-1). Low toxic dosage (growth quotiety 85-90%) was 0.7 microg x mL(-1). Cell non-toxic dosage of was 0.7 microg x mL(-1) and low toxic dosage was 1.2 microg x mL(-1). Cell non-toxic dosage of Tan-M (0.2 microg x mL(-1)) significantly lowered the IC50 of K562/ADM by ADM (P < 0.01), and reversed MDR was 3.88 times. Low toxic dosage of Tan-M reversed MDR was 3.97 times. E-M (0.2 microg x mL(-1)) reversed MDR was 2.62 times.

CONCLUSIONThe result indicates that tanshinone microemulsion possesses cell-toxic effects on human leukemia cell-line and may reverse MDR of tumor cells.

Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Cell Proliferation ; drug effects ; Diterpenes, Abietane ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Emulsions ; Humans ; K562 Cells ; drug effects ; Phenanthrenes ; administration & dosage ; pharmacology

AIMTo investigate the genetic polymorphism of several species of Fritillaria and to develop a DNA chip for the genotyping and identification of the origin of various species of Fritillaria at molecular level.

METHODSGenomic DNA from bulbs of several Fritillaria species was extracted and the polymorphisms of the D2 and D3 regions inside the 26S rDNA gene were identified by direct sequencing. Oligonucleotide probes specific for these polymorphisms were designed and printed on the poly-lysine coated slides to prepare the DNA chip. PCR products from the Fritillaria species were labeled with fluorescence by incorporation of dye-labeled dideoxyribonucleotides and hybridized to the immobilized probes on the chip.

RESULTSThe polymorphisms were used as markers for discrimination among various species. Specific oligonucleotide probes were designed and immobilized on a DNA chip. Differentiation of the various Fritillaria species was accomplished based on hybridization of fluorescent labeled PCR products with the DNA chip.

CONCLUSIONThe results demonstrated the reliability of using DNA chips to identify different species of Fritillaria, and the DNA chip technology can provide a rapid, high throughput tool for genotyping and quality assurance of the plant species verification.

Base Sequence ; DNA, Plant ; analysis ; Fritillaria ; classification ; genetics ; Genotype ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; methods ; Plants, Medicinal ; genetics ; Polymorphism, Single Nucleotide ; RNA, Ribosomal ; genetics ; Species Specificity

OBJECTIVETo assess the prevalence of several tyrosine kinases (TKs) gene mutations including c-Kit, FLT3 and JAK2 V617F in core binding factor related acute myeloid leukemia (CBF-AML), and analyze their impact on clinical characteristics and prognosis.

METHODSMutations of c-Kit, FLT3-ITD and FLT3-TKD were detected by genomic DNA PCR and sequencing, and JAK2 V617F mutation screening by allele-specific PCR in 58 newly diagnosed CBF-AML patients [28 AML with inv(16) and 30 with t(8;21)], and analyze the patients clinical characteristics and prognoses.

RESULTSc-Kit aberrations were detected in 32.8% cases, including 6 cases mutated in exon 8 (mutKIT8) and 13 mutated in exon 17 (mutKIT17). MutKIT8 was more prominent in inv(16) than in t(8;21) patients (21.4% vs 0, P = 0.009). Only 2 cases had FLT3-ITD and 7 (12.1%) FLT3-TKD mutations. The result of JAK2 V617F mutation screenings in these CBF-AML patients was negative. The frequency of receptor tyrosine kinases(RTK) mutations was 46.6% and only one case had two kinds of missense mutations (mutKIT8 & TKD(+)). Median age of onset was higher for mutKIT17 than for wide-type c-Kit (wtKIT) patients (55 vs 31, P = 0.003). c-Kit mutations were significantly associated with decreased overall survival (OS) and continuous complete remission (CCR) rates (P = 0.053, and 0.048 respectively), and so did more for exon17 mutated patients reduced (P = 0.005, and 0.013 respectively). FLT3-TKD mutation showed no effects on prognosis of CBF-AML patients.

CONCLUSIONSRTK mutations are common in patients with CBF-AML. c-Kit mutations frequently and JAK2V617F mutation rarely appear in CBF-AML. c-Kit mutations, especially mutKIT17 confers higher relapse risk and poorer prognosis.

Adolescent ; Adult ; Aged ; Core Binding Factors ; DNA Mutational Analysis ; Female ; Humans ; Janus Kinase 2 ; genetics ; Leukemia, Myeloid, Acute ; diagnosis ; etiology ; genetics ; Male ; Middle Aged ; Mutation ; Prognosis ; Protein-Tyrosine Kinases ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Young Adult ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVETo study the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and detect JAK2 mutation in BMSCs from myeloproliferative neoplasms (MPN) patients.

METHODSJAK2 V617F mutation and exon 12 mutation in 70 MPN patients' blood or bone marrow samples were detected. Isolated BMSCs were then characterized their phenotype, mesenchymal differentiation capacity and existence of JAK2 mutation.

RESULTSBMSCs derived from the patients were similar with healthy donors in terms of morphology, surface antigen and differentiation ability. Of them, 38 patients' blood or bone marrow samples harbored JAK2 V617F, and identified that 3 V617F-negative-patients' samples existed JAK2 exon 12. No patients' BMSC harbored JAK2 mutation though their blood or bone marrow samples carried JAK2 mutation.

CONCLUSIONBMSCs from MPN patients had similar biological characteristics with healthy donors. BMSCs from MPN patients known to bear JAK2 mutation in blood or bone marrow cells didn't carry the mutation.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow Cells ; cytology ; Bone Marrow Neoplasms ; genetics ; Case-Control Studies ; Child ; DNA Mutational Analysis ; Female ; Humans ; Janus Kinase 2 ; genetics ; Male ; Mesenchymal Stromal Cells ; cytology ; Middle Aged ; Mutation ; Myeloproliferative Disorders ; genetics ; Young Adult

Despite the chemotherapy is successful in inducing remission of hematologic malignancy, this disease also has a high probability of relapse; besides, the toxicity of chemotherapy for these patients can not be avoided. Researchers have been attempting to eliminate tumor cells by immunotherapy. Recently, various leukemia-associated antigens (LAA) that are recognized by cytotoxic T cell (CTL) in the context of HLA class I molecules have been identified. These LAA include WT1, PR-3, RHAMM, BCR-ABL and Aur-A. On the basis of these findings, various clinical trials of immunotherapy for hematologic malignancy including tumor peptide vaccination, adoptive T cell therapy, NK cell therapy and dendritic cells-cytokine induced killer (DC-CIK) cell therapy are on going. In this review, the current status and future feasibility of cellular immunotherapy for leukemia are discussed.
Humans ; Immunotherapy, Adoptive ; Leukemia ; therapy ; T-Lymphocytes, Cytotoxic ; immunology

This study was purposed to culture murine compact bone-derived mesenchymal stem cell (MSC) and analyze the immunological and trilineage differentiation potential. Tibia and femur were extracted. Bone marrow cells were flushed out and compact bone fragments were digested with collagenase. The digested cells were cultured in 6-well plates. The immunophenotype, immunosuppressive function and trilineage differentiation potential were analysed by flow cytometry, mixed lympocyte reaction and Oil red O, von Kossa and alcian blue straining, respectively. The results indicated that the pure compact bone MSC could be isolated with in 3 weeks. The resulting MSC had trilineage differentiation potential and immunosuppressive effect on mixed lymphocyte reaction. The count per minute (CPM) value in control group of BALB/c T cells cocultured with irradiated C57BL/6 T cells was (2.56 ± 0.31) × 10(4), while CPM values of mixed lymphocyte cocultured with C57BL/6 compact bone MSC at ratios of 100:1 and 10:1 were (0.47 ± 0.12) × 10(4) and (0.28 ± 0.09) × 10(4). The CPM value of control group was higher than those of MSC cocultured group (P < 0.001). Compact bone-MSC had an immunosuppressive effect on mixed lymphocyte reaction in a dose dependent manner. It is concluded that murine compact bone has rich MSC and the primary MSC is contaminated with less hematopoietic cells. Murine compact bone-MSC have immunosuppressive effect on mixed lymphocyte reaction and trilineage differentiation potential. Compact bone-MSC have promising experimental study value.
Animals ; Bone Marrow Cells ; cytology ; immunology ; Bone and Bones ; cytology ; Cells, Cultured ; Female ; Immunophenotyping ; Lymphocyte Culture Test, Mixed ; Mesenchymal Stromal Cells ; cytology ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL