In this paper, the situation on antisense oligonucleotide as a means of gene therapy was outlined, and the main factors impeding its progress at present was summarized. The one of main factors is the efficiency of antisense oligonucleotide as a drug and the other is the side-effect in clinical use. At the level of cell and gene, these influential factors were analyzed in detail. The main factor that makes side-effect in using antisense oligonucleotide is the difficulty to distinguish effectively homologous-gene from target gene. The another factor is the secondary structure and three-dimensional structure of target gene that seriously affect antisense oligonucleotide to arrive at target position. The third problem is what can affect antisense oligonucleotide transmission and quick annealing. How use computer technique to analyze fully the target gene of antisense oligonucleotide including the secondary structure and homology of target gene, and to design effective antisense oligonucleotide, in order to reduce its side-effect in clinical use of antisense oligonucleotide as a drug of gene therapy, and the computer-aid design method were described.
Computer-Aided Design ; Fusion Proteins, bcr-abl ; genetics ; Genetic Therapy ; Humans ; Nucleic Acid Conformation ; Oligonucleotides, Antisense ; therapeutic use ; RNA ; chemistry

No abstract available.
Aged, 80 and over ; Base Sequence ; Bone Marrow/pathology ; DNA/chemistry/metabolism ; Female ; Fusion Proteins, bcr-abl/*genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/*genetics ; Multiplex Polymerase Chain Reaction ; Protein Isoforms/genetics ; Sequence Analysis, DNA

The objective of this study was to investigate the specificity of detecting the phosphotyrosine level with anti-phosphotyrosine monoclonal antibody PY20 for diagnosis and prognosis of patients with chronic myeloid leukemia (CML) and the possibility of its clinical application. The positive rate of PY20 in 28 newly diagnosed CML patients was detected by flow cytometry using anti-PY20 antibody, the bcr-abl fusion gene was detected by nested RT-PCR, the Ph chromosome was measured by R-banding cytogenetic analysis, and the coincidence of PY20 positive rate with results of bcr-abl fusion gene and Ph chromosome detection was compared. In addition, the positive rate of PY20, the changes of bcr-abl fusion gene and Ph chromosome were determined in follow up 7 CML patients after allo-hematopoietic stem cell transplantation. The results indicated that the positive rates of PY20 in 28 newly diagnosed CML patients in groups of chronic phase (CP), accelerated phase (AP), and blast phase (BP) were (40.31% +/- 1.22)%, (77.28 +/- 1.14)% and (78.12 +/- 1.32)% respectively. The positive rate of PY20 in CP was lower than that in AP and BP (p < 0.05). There was no difference in positive rate of PY20 between AP and BP (p > 0.05). PY20 expression level of leukocytes from peripheral blood and bone marrow showed no difference (p < 0.05). The positive rates of PY20 in patients with CR, PR and NR were (15.56% +/- 1.51)%, (38.73% +/- 2.31)% and (60.43% +/- 2.04)% respectively. The positive and negative coincidence between PY20 and RT-PCR was 92.31% and 95.45% respectively. The positive and negative coincidence between PY20 and Ph Chromosome in newly diagnosed patients was 88.46% and 95.46% respectively. Ph chromosome and PY20 were all negative in 7 CML patients after allo-HSCT. Bcr-abl fusion gene was negative persistently in 5 patients, but in the other 2 patients, the fusion gene was persistently positive. In conclusion, the detection of the level of phosphotyrosine in CML cells has high sensitivity and specificity. The results of PY20 cell positive rate combined with detection of bcr/abl fusion gene and Ph chromosome might be useful in diagnosis as a good index of monitoring.
Adolescent ; Adult ; Antibodies, Monoclonal ; chemistry ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; Male ; Middle Aged ; Philadelphia Chromosome ; Phosphotyrosine ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Young Adult

The study was purposed to analyze the relationship between the content of T-cell receptor excision DNA circles (TREC) and bcr-abl mRNA levels in CML patients and to evaluate the prognostic significance of recent thymic output function detection in patients with chronic myelogenous leukemia (CML). Quantitative detection of TREC and bcr-abl fusion gene transcripts in peripheral blood from 15 CML patients were preformed by real-time PCR. The change of bcr-abl levels in 6 patients was followed-up for two years. The results showed that there was no significant correlation between TREC and bcr-abl mRNA levels in peripheral blood from CML patients for the first attack. Patients who had higher TREC at diagnosis had a larger reduction of bcr-abl after 2 years of follow-up. While out of 2 patients who underwent haemopoietic stem cell transplantation (HSCT), one patient with higher level of TREC before transplantation was confirmed to express undetectable level of TREC by three consecutive detections after transplantation, other one patient was identified to express low level of bcr-abl. It is concluded that high thymic output function in CML patients can be beneficial for killing the residual CML cells.
Adolescent ; Adult ; Aged ; Female ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; Gene Rearrangement ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; Male ; Middle Aged ; Proto-Oncogene Proteins c-abl ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcr ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Antigen, T-Cell ; analysis ; immunology ; T-Lymphocytes ; chemistry ; immunology ; Thymus Gland ; immunology

OBJECTIVETo discover BCR-ABL tyrosine kinase inhibitors through structure based virtual screening.

METHODSDocking screening against the distinctive inactive conformation of the catalytic domain of BCR-ABL tyrosine kinase was performed on 3D database. The MTT assay was performed to assess the viability of the tumor cells treated with selected compounds. The amount and kinase activity of BCR-ABL protein were detected in the presence of compounds by Western blot analysis and immunoprecipitation.

RESULTSFrom the top 1,000 compounds with the best DOCK energy score, 15 compounds were selected for biological assay. Eight out of 15 compounds showed notable inhibitory activity against Ph+ human K562 cells with IC50 values ranging from 10 to 200 micromol/L. In cell-based assays of ABL tyrosine phosphorylation, the ability of two kinds of novel, structurally diverse, lead compounds to inhibit ABL kinase activity was observed. However, no significant differences in the amount of BCR-ABL protein were noted on the ABL immunoblot in the presence of these lead compounds.

CONCLUSIONSTwo promising lead compounds were discovered to inhibit BCR-ABL tyrosine kinase activity. Virtual screening technique has been proven to narrow down the size of screening compound libraries to the most prospective drug candidates with high success rates.

Computer Simulation ; Drug Evaluation, Preclinical ; Enzyme Inhibitors ; chemical synthesis ; Fusion Proteins, bcr-abl ; Humans ; K562 Cells ; Models, Molecular ; Molecular Conformation ; Phosphorylation ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; chemistry ; genetics

No abstract available.
Aged ; Base Sequence ; Bone Marrow/metabolism/pathology ; Chromosome Aberrations ; DNA/chemistry/genetics/metabolism ; Fusion Proteins, bcr-abl/*genetics ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Male ; Myelodysplastic Syndromes/diagnosis/*genetics ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA

No abstract available.
Antineoplastic Agents/therapeutic use ; Base Sequence ; DNA/chemistry/genetics/metabolism ; DNA Mutational Analysis ; Fusion Proteins, bcr-abl/*genetics ; Genotype ; Humans ; Imatinib Mesylate/therapeutic use ; Karyotyping ; Male ; Multiplex Polymerase Chain Reaction ; Mutation ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*diagnosis/drug therapy/*genetics

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the overproduction of granulocytes, which leads to high white blood cell counts and splenomegaly in patients. Based on clinical symptoms and laboratory findings, CML is classified into three clinical phases, often starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis. Blast crisis phase of CML is clinically similar to an acute leukemia; in particular, B-cell acute lymphoblastic leukemia (B-ALL) is a severe form of acute leukemia in blast crisis, and there is no effective therapy for it yet. CML is induced by the BCR-ABL oncogene, whose gene product is a BCR-ABL tyrosine kinase. Currently, inhibition of BCR-ABL kinase activity by its kinase inhibitor such as imatinib mesylate (Gleevec) is a major therapeutic strategy for CML. However, the inability of BCR-ABL kinase inhibitors to completely kill leukemia stem cells (LSCs) indicates that these kinase inhibitors are unlikely to cure CML. In addition, drug resistance due to the development of BCRABL mutations occurs before and during treatment of CML with kinase inhibitors. A critical issue to resolve this problem is to fully understand the biology of LSCs, and to identify key genes that play significant roles in survival and self-renewal of LSCs. In this review, we will focus on LSCs in CML by summarizing and discussing available experimental results, including the original studies from our own laboratory.
5-Lipoxygenase-Activating Proteins ; metabolism ; Animals ; Benzamides ; Disease Models, Animal ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; chemistry ; metabolism ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; enzymology ; genetics ; pathology ; Male ; Mice ; Neoplastic Stem Cells ; enzymology ; pathology ; PTEN Phosphohydrolase ; metabolism ; Philadelphia Chromosome ; Piperazines ; therapeutic use ; Point Mutation ; Protein Structure, Tertiary ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; chemistry ; metabolism ; Pyrimidines ; therapeutic use

Imatinib mesylate is a selective Bcr/Abl kinase inhibitor and an effective anticancer agent for Bcr/Abl-positive chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Mutations within the BCR/ABL kinase domain are the most commonly identified mechanism associated with relapse. To overcome the imatinib resistance in CML, many investigators have tried to clarify molecular mechanism for imatinib resistance in cells of patients who failed to respond to imatinib. Our aim was to invesitigate underlying mechanism for imatinib resistance in SR-1 cells, which were derived from a CML patient in blast crisis. We detected the new mutation of BCR/ABL, resulting in premature termination and loss of BCR/ABL fusion protein expression, which might be possible mechanism for the resistance to imatinib in SR-1 cells.
Amino Acid Sequence ; Base Sequence ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*genetics ; Fusion Proteins, bcr-abl/chemistry/*genetics ; Humans ; Leukemia, Myeloid/*genetics ; Molecular Sequence Data ; Mutagenesis, Insertional/*genetics ; Nucleotides/genetics ; Piperazines/*pharmacology ; Point Mutation/*genetics ; Pyrimidines/*pharmacology ; Research Support, Non-U.S. Gov't