OBJECTIVETo construct a vector containing protein transduction domain (PTD) and bcr/abl fusion gene of chronic myelogenous leukemia and express PTD-bcr/abl fusion protein in E. Coli.

METHODSDNA fragment encoding PTD was synthesized and fused to PCR-amplified bcr/abl gene fragment, then inserted into plasmid pET-16b to get the expression vector pEPb containing PTD-bcr/abl fusion gene, which was transfected and expressed in E. Coli LB21. PTD-bcr/abl fusion protein was purified by affinity chromatography.

RESULTS523 bp bcr/abl fusion gene was effectively amplified. The PTD-bcr/abl gene sequencing showed the same sequence as scheduled. The fusion peptide was successfully expressed in E. Coli and purified.

CONCLUSIONThe results may provide a new PTD-bcr/abl fusion peptide for the immunotherapy of CML.

Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Gene Expression ; Gene Products, tat ; genetics ; metabolism ; Genetic Vectors ; genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo detect bcr/abl fusion gene at DNA level in K562 cell line and mononuclear cells from patients with chronic myelogenous leukemia (CML).

METHODSBased on previous research, a set of DNA-PCR primers was redesigned. DNA from K562 cells and mononuclear cells of CML patients was extracted respectively. After DNA-PCR for bcr/abl fusion gene the amplified fragments were then sequenced.

RESULTAt DNA level the bcr/abl fusion gene in K562 cells and mononuclear cells of 2 CML patients was amplified. Furthermore, the DNA breakpoint of fusion gene in the above samples through sequencing of amplified fragments was localized.

CONCLUSIONDNA-PCR offers a new detection technology for bcr/abl fusion without the expression of fusion gene.

Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Leukocytes, Mononuclear ; metabolism ; Polymerase Chain Reaction ; methods

OBJECTIVETo explore the effects of As(2)O(3) on BCR-ABL protein level and signal transduction in chronic myeloid leukemia (CML) cells.

METHODSImmunoprecipitation, protein tyrosine kinase (PTK) activity assay, real-time Taqman quantitative PCR and Western blot were used.

RESULTSAs(2)O(3) downregulated BCR-ABL protein and STAT1 protein of CML mononuclear cells in the concentrations of 1.0 approximately 2.0 micro mol/L and 0.5 approximately 2.0 micro mol/L after 48 h exposure, respectively. However, p27 protein level was not affected. The PTK activity of BCR-ABL protein was also mildly decreased in CML monouclear cells at 60 h. The bcr-abl mRNA level remained unchanged.

CONCLUSIONAs(2)O(3) downregulats BCR-ABL protein, STAT1 protein, BCR-ABL signal transduction and PTK activity in CML cells.

Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Phosphorylation ; drug effects ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction

OBJECTIVETo investigate the possibility of multi-unit ribozymes to purge bone marrow of chronic myelocytic leukemia (CML), its in vitro cleavage ability and the reversal effect on CML cell's malignant phenotype.

METHODSAs bcr-abl fusion gene plays an important role in CML pathology, three single-unit ribozymes were designed and synthesized in 44 base pairs near the fusion point, two enzyme cleavage sites on bcr gene and one on abl gene. Multi-unit ribozymes' in vitro transcription and retroviral vector through gene recombination were constructed. Then, its in vitro cleavage ability was tested and the retroviral vector was transfected into K562 cell. Through MTT assay, the incorporation rate of (3)H-TdR, RT-PCR, Southern and Northern blot hybridization, flow cytometry, transmission and scanning electron microscopy were used to study the effect of multi-unit ribozymes on CML cell proliferation, cell structure, cell cycle and the induction of apoptosis.

RESULTSMulti-unit ribozymes had in vitro cleavage efficiency of 70.8%. After the transfection of multi-unit ribozymes retroviral vector into K562 cell, cell proliferation and DNA synthesis were greatly reduced with an inhibition rate of about 50% after 96 hours of transfection. Multi-unit ribozymes could cleave K562 cell's RNA with a reduction rate about one 1 000 th of the original. By flow cytometry (FCM), 18.4% cells underwent apoptosis after 72 hours transfection with most of the cells blocked in the G phase. Here, the ratio in S phase was lowered by 41.9%. Under transmission and scanning electron microscope, compaction of nuclear chromation and apoptosis bodies were observed in the transfected cells.

CONCLUSIONMulti-unit ribozymes possess high cleavage ability in vitro. The ribozymes, whose retroviral vector being transfected into CML cell, are able to express a lasting ability to cleave the fusion gene, induce apoptosis, reduce cell proliferation, revert the malignant phenotype. It is possible to make use of multi-unit ribozymes to purge CML bone marrow. Therefore, multi-unit ribozymes may very well be valuable in the gene therapy of CML.

Apoptosis ; Cell Division ; drug effects ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; K562 Cells ; RNA, Catalytic ; metabolism ; pharmacology

Bcr-abl antisense oligodeoxynucleotides (AS-ODNs) have provided evidence of an antileukemia effect when tested in vitro against Philadelphia-positive cells. In order to investigate the efficacy of AS-ODNs as purging agents in chronic myeloid leukemia (CML) patients, K562 cells, a human CML cell line, were treated in vitro with various types of AS-ODNs and interferon-alpha. Cells were treated in vitro for 0 and 36 hr with 40 microgram/mL of AS-ODNs, respectively, and incubated at 37 degrees C for 36 hr. Cytotoxic effects were measured by counting the number of viable cells as well as by MTT test. Clonogenic activities were evaluated by methylcellulose culture for 2 weeks. The effects of purging agents on the rearrangement of bcrabl gene were evaluated by RT-PCR. AS-ODNs inhibited the proliferation of K562 cells with time in cell count assay and MTT test. AS-ODNs were superior to INF-alpha in inhibiting clonogenic activity (recovery rate; 26.3% vs 64.0%). After incubation with bcr-abl AS-ODNs primers and mRNA isolated from K562 cells, positive bands were abolished, especially of b3a2 type and phosphorothioate type. Our results suggest that AS-ODNs mediated purging may be one of the efficient methods and that autograft may be an alternative treatment for allograft in high-risk group patients of CML if they do not have a stem cell donor.
Bone Marrow Purging* ; Colony-Forming Units Assay ; Fusion Proteins, bcr-abl/genetics ; Fusion Proteins, bcr-abl/metabolism ; Hematopoietic Stem Cells/physiology* ; Human ; Leukemia, Myeloid, Chronic/therapy ; Neuroblastoma/therapy* ; Oligonucleotides, Antisense/metabolism* ; Oligonucleotides, Antisense/therapeutic use ; Transplantation, Autologous* ; Tumor Cells, Cultured

This study was aimed to investigate the chimerism and fusion gene expression in patients with CML after allo-HSCT, to analyse engraftment and minimal residual disease by using STR-PCR combined with RT-PCR qualitative and quantitative assays, and to evaluate their clinical value for predicting disease relapse. 4 relapsed patients with CML after allo-HSCT were dynamically investigated. Qualitative analysis of donor chimerism was performed by multiplex PCR amplification of STR markers and capillary electrophoresis with fluorescence detection, qualitative detection of bcr/abl transcripts was performed by RT-PCR. The results showed that the 100% donor chimerism appeared in 4 patients on day 28 after transplantation and bcr/abl expression was negative, but the 4 patients were in status of unstable mixed chimerism (DC: 0% - 80.4%) at the different time points during the following up with bcr/abl gene positive. 2 patients of them were continuously mixed chimerism after relapse of CML, the other 2 changed from MC to CC by intervention of clinical treatment. Decreasing values of donor chimerism were detected prior to the occurrence of graft rejection and CML relapse, and bcr/abl gene expression was positive. It is concluded that the results of STR-PCR in the range of its sensitivity fully correspond with bcr/abl tests in patients. The combination of STR-PCR with RT-PCR will provide a highly sensitive and valuable tool for evaluating engraftment, graft rejection, and relapse and predicting GVHD. Furthermore, it can provide a basis for early intervention of clinical treatment, and can identify these high risk patients with molecular or cytogenetic relapse after allo-HSCT.
Fusion Proteins, bcr-abl ; genetics ; metabolism ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; therapy ; Neoplasm Recurrence, Local ; genetics ; Neoplasm, Residual ; diagnosis ; genetics ; RNA, Messenger ; genetics ; metabolism ; Transplantation Chimera ; Transplantation, Homologous

P210(bcr/abl) fusion gene is indispensable for generation and progression of chronic myeloid leukemia (CML). Small molecule inhibitors, such as imatinib, are effective for P210(bcr/abl) gene mediated CML, but drug resistance may occur. The unique fusion junction of P210(bcr/abl) gene is an attractive target for therapeutic intervention using RNA interference (RNAi). This study was purposed to constructed the BaF3 cell line by viral vector which can stably express P210(bcr/abl) shRNA and P210(bcr/abl) mRNA at the same time, and investigate the effect of lentiviral-victor-delivered shRNA on P210(bcr/abl) gene expression. The infective rate of lentiviral vector on BaF3 cells with P210(bcr/abl) gene was assayed by fluorescent microscopy; the cell proliferation ability was determined by trypan blue exclusion; the P210(bcr/abl) mRNA and protein expressions were detected by RT-PCR and Western blot respectively. The results found that stable expression of the P210(bcr/abl) shRNA resulted in obvious inhibition of P210(bcr/abl) mRNA and protein expression and increased sensitivity of these P210(bcr/abl) gene transformed Ba/F3 cells to imatinib. The IC(50) to imatinib in these cells decreased < 50% as compared with Ba/F3-P210(bcr/abl) cells which did not express P210(bcr/abl) mRNA. The survival time of the lethal dose irradiated mice induced by intravenous injection of these Ba/F3 cells was longer than the other group induced by Ba/F3-P210(bcr/abl). It is concluded that stable expression of shRNA targeting the P210(bcr/abl) gene fusion junction may potentiate the effects of conventional therapy for CML.
Animals ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Lentivirus ; genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; Mice ; NIH 3T3 Cells ; RNA, Small Interfering ; genetics

Several studies have shown that the tumor endothelial cells are different from the normal tissue endothelial cells. These tumor endothelial cells may contribute to tumor neo-vasculogenesis. This study was purposed to analyze the biologic features and determine the expression level of CD133 and BCR/ABL fusion gene in circulating endothelial cells (CEC) isolated from peripheral blood of CML patients, as well as to investigate the role of CEC in disease progression. Mononuclear cells were isolated from peripheral blood by density gradient centrifugation; CEC were sorted by MACS and harvested in the endothelial growth medium. The morphologic features of CEC were observed by microscopy, the cell growth rate was calculated by cell counting, and the cells were identified by immunofluorescence staining for the expression of CD31,CD34,VWF and CD133. The expression of BCR/ABL fusion gene was examined by FISH in 12 CML patients. The results indicated that the isolated CEC displayed the typical cobble-stone morphology. These cells could be identified by the positive immunofluorescence staining for CD31, CD34 and VWF, and showed more increased proliferative potential as compared to that of healthy donors. It was found that the positive rate of CD133 was 31.29% in CML patients, which was significantly different from that of healthy donors (P < 0.05). In 12 CML patients, CEC carried the same chromosome aberration as the leukemia cells (10.77%). Higher expression level of CD133 and BCR/ABL fusion gene positively correlated with progression of disease. It is concluded that the CEC may participate in invasion and angiogenesis in patients with CML and possibly correlate to the spreading and progression of the disease.
AC133 Antigen ; Adult ; Antigens, CD ; metabolism ; Cell Proliferation ; Endothelial Cells ; metabolism ; Female ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Glycoproteins ; metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; Male ; Middle Aged ; Neovascularization, Pathologic ; Peptides ; metabolism ; Young Adult

OBJECTIVETo establish a real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) for quantitative detection of the common molecular markers that have affirmative clinical significance in the acute and chronic leukemia patients, and evaluate its significance in diagnosing leukemias and monitoring minimal residual disease (MRD).

METHODSPrimers and TaqMan probes were designed for detecting various fusion transcripts and normal abl gene was used as the internal control. The expression level of fusion transcripts in 202 newly diagnosed leukemias were determined.

RESULTSIn absolute quantity, expression level of the fusion transcripts in various leukemias was b3a2 (b2a2) 47614.63, e1a2 98847.53, AML1-ETO 300029.51, PML-RAR alpha 25506.28, respectively, while in relative quantity to abl, the levels were 1.05, 0.91, 5.33 and 0.55, respectively.

CONCLUSIONThe relative quantification of gene expression level by using RQ-RT-PCR to abl control gene is more accurate and direct viewing. Different levels of transcription of corresponding fusion genes are found in various subtypes of leukemias at diagnosis, among which the level of AML1-ETO was higher and PML-RAR alpha lower.

Biomarkers, Tumor ; genetics ; metabolism ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Leukemia ; diagnosis ; genetics ; metabolism ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic

This paper is aimed to investigate the transcription and expression of BCR-ABL-pIRES-SEA fusion gene vaccines in vivo in mice. The reconstructed plasmids (BCR-ABL-pIRES-SEA) which were developed previously in our laboratory were injected into the skeletal muscles of BALB/c mice at 14d intervals for three cycles. The transcription and expression of BCR-ABL and staphylococcal enterotoxin A (SEA) in injection site were detected using RT-PCR and immunohistological methods. The BCR-ABL/SEA mRNA and protein could be identified in the injection site of BCR-ABL-pIRES-SEA vaccinated mice. The reconstructed BCR-ABL-pIRES-SEA plasmids can effectively express gene production in the skeletal muscles of mice and have the common features of DNA vaccine.
Animals ; Enterotoxins ; genetics ; immunology ; metabolism ; Fusion Proteins, bcr-abl ; genetics ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal ; metabolism ; Plasmids ; immunology ; RNA, Messenger ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, DNA ; administration & dosage ; immunology