Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0023473 (chronic myeloid leukemia)
 18,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2', 5'-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210(bcr/abl) kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of beta-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.
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PMID:2',5'-Oligoadenylate-antisense chimeras cause RNase L to selectively degrade bcr/abl mRNA in chronic myelogenous leukemia cells. 983 40

We describe a one-tube multiplex reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of bcr-abl fusion mRNA in analysis of patients with chronic myeloid leukaemia and acute lymphoblastic leukaemia. The assay provides a quick and reliable method for the detection and analysis of chromosome translocations resulting in formation of the fusion proteins p210 (b3a2/b2a2) and p190 (e1a2). The method is based on the use of magnetic beads and sequence-specific reverse transcription primers. By combining direct mRNA isolation, reverse transcription and first-stage PCR we have reduced the number of manipulations, maintained sensitivity, and minimized the risk of contamination. A nested primer strategy is used to secure sensitivity. We also introduce a competitive one-tube RT-PCR to be able to monitor the relative quantity of transcripts using in vitro transcribed RNA as competitor.
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PMID:One-tube multiplex RT-PCR of BCR-ABL transcripts in analysis of patients with chronic myeloid leukaemia and acute lymphoblastic leukaemia. 1008 1

Because of the probable causal relationship between constitutive p210(bcr/abl) protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210(bcr/abl)-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain-containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210(bcr/abl)-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4, 5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210(bcr/abl)-mediated myeloid expansion.
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PMID:A novel SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells. 1019 51

The chimeric oncogene bcr-abl is detected in virtually every case of chronic myelogenous leukemia. It has been shown that cells (such as K562) expressing Bcr-Abl/p210, a protein tyrosine kinase, not only undergo cellular transformation but also demonstrate multiple drug resistance. Recent studies also demonstrate that the proteasome is involved in the survival signaling pathway(s). In the current study, we tested the hypothesis that the proteasome might play a role in regulating Bcr-Abl function. We have demonstrated by using a variety of inhibitors that inhibition of the proteasome, but not of the cysteine protease, activity is able to activate the apoptotic cell death program in K562 cells. Proteasome inhibition-induced apoptosis is demonstrated by condensation and fragmentation of nuclei, appearance of an apoptotic population with sub-G1 DNA content, the internucleosomal fragmentation of DNA, and cleavage of poly(ADP-ribose) polymerase, and can be blocked by a specific caspase-3-like tetrapeptide inhibitor. Western blot analysis with specific antibodies to c-Abl and Bcr proteins show that treatment of K562 cells with a proteasome inhibitor results in significant reduction of Bcr-Abl protein expression, which occurs several hours before the onset of apoptotic execution. Levels of c-Abl/p145 and Bcr/p160 proteins, however, remain essentially unaltered at that time. Furthermore, reduced Bcr-Abl expression is reflected in significantly attenuated Bcr-Abl-mediated protein tyrosine phosphorylation. Taken together, these results indicate that proteasome inhibition is sufficient to inactivate Bcr-Abl function and subsequently activate the apoptotic death program in cells that are resistant to apoptosis induced by chemotherapy.
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PMID:Proteasome inhibition leads to significant reduction of Bcr-Abl expression and subsequent induction of apoptosis in K562 human chronic myelogenous leukemia cells. 1021 53

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210(BCR/ABL) tyrosine phosphorylation. We report here that preincubation of CML or normal CD34(+) cells with graded concentration of AG957 (1 to 100 micromol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 micromol/L; P =.008), burst-forming unit-erythroid (BFU-E; 29 v 89 micromol/L; P =.004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 micromol/L; P =.004), and LTC-IC (43 v 181 micromol/L; P =.004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 micromol/L significantly reduced the mean (+/-SD) percentage of BCR/ABL-positive progenitors (92% +/- 10% v 33 +/- 5%; P =.001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v 9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34(+) cells with AG957 (1 micromol/L) and CH11 (1 microgram/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34(+) cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti-Fas-R antibody CH11. These data may have significant therapeutic applications.
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PMID:Effects of the tyrosine kinase inhibitor AG957 and an Anti-Fas receptor antibody on CD34(+) chronic myelogenous leukemia progenitor cells. 1033 7

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome resulting from the translocation t(9-22) producing the chimeric 190 and 210 kDa BCR-ABL fusion proteins. Evolution of the CML to the more agressive acute myelogenous leukemia (AML) is accompanied by increased cellular proliferation and genomic instability at the cytogenetic level. We hypothezised that genomic instability at the nucleotide level and spontaneous error in DNA replication may also contribute to the evolution of CML to AML. Murine Ba/F3 cell line was transfected with the p190 and p210-encoding BCR-ABL oncogenes, and spontaneous mutation frequency at the Na-K-ATPase and the hypoxanthine guanine phosphoribosyl transferase (HPRT) loci were measured. A significant 3-5-fold increase in mutation frequency for the transfected cells relative to the untransfected control cells was found. Furthermore, we observed that BCR-ABL transfection induced an overexpression of DNA polymerase beta, the most inaccurate of the mammalian DNA polymerases, as well as an increase in its activity, suggesting that inaccuracy of DNA replication may account for the observed mutator phenotype. These data suggest that the Philadelphia abnormality confers a mutator phenotype and may have implications for the potential role of DNA polymerase beta in this process.
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PMID:Mutator phenotype of BCR--ABL transfected Ba/F3 cell lines and its association with enhanced expression of DNA polymerase beta. 1034 41

Improvement in diagnostic cytogenetic techniques has led to the recognition of an increasing number of leukemia-associated chromosomal translocations and inversions. These genetic lesions frequently are associated with the disruption of putative transcription factors and the production of hybrid transcripts that are implicated in leukemogenesis. Epidemiologic evidence suggests that some, but not all, individuals with a history of gamma-irradiation exposure are at increased risk of developing chronic myeloid leukemia (CML). CML is characterized by the Philadelphia chromosome and transcription of the resulting hybrid BCR-ABL gene. Utilizing the leukemia-associated BCR-ABL p210 transcript as a marker, we sought differences in the induction of illegitimate genetic recombination following high-dose gamma-irradiation of karyotypically normal lymphoblastoid cell lines (LCL) derived from individuals with and without a history of myeloid leukemias. Six LCL [4 leukemia patient derived [2 acute myeloid leukemia and 2 CML] and 2 from normal individuals were analyzed with reverse transcriptase polymerase chain reaction for BCR-ABL under stringent conditions following exposure to 0, 50, or 100 Gy of LET gamma-irradiation delivered via a Varian linear accelerator at 4 MV. Transcripts identical to disease-associated b2a2 and b3a2 transcripts were detected both spontaneously (background illegitimate genetic recombination) and following gamma-irradiation. Background BCR-ABL positivity was demonstrable in 4 of the 6 LCL, with no significant difference in detection between leukemic- and nonleukemic-derived LCL. Overall, increasing gamma-irradiation dose resulted in an increased frequency of BCR-ABL transcript detection (0 Gy vs 50 Gy vs 100 Gy,p = 0.0023, Chi-square test). Within the leukemic- but not the nonleukemic-derived LCL there was significantly greater BCR-ABL positivity after gamma-irradiation compared to unirradiated equivalents. Furthermore, the BCR-ABL positivity of both the AML- and CML-derived LCL after gamma-irradiation was significantly greater than that of the nonleukemic-derived LCL after gamma-irradiation. We speculate that this difference in the detection of illegitimate after gamma-irradiation recombination may be due to aberrant DNA double strand break repair mechanisms in individuals predisposed to the development of myeloid leukemias.
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PMID:Leukemia patient-derived lymphoblastoid cell lines exhibit increased induction of leukemia-associated transcripts following high-dose irradiation. 1048 Apr 30

The Philadelphia chromosome is present in a heterogeneous group of leukemias. It is most commonly associated with chronic myelogenous leukemia (CML) and B-lineage acute lymphoblastic leukemia (ALL) being found in more than 95% and 15-25% of cases respectively. We undertook a study to determine the morphologic, phenotypic and molecular diversity of Philadelphia positive de novo acute leukemia patients seen at our institution over the past 3 1/2 years. Twenty-one patients with de novo acute leukemia were found to have the Philadelphia chromosome by cytogenetic studies. They consisted of 3 patients with acute myelogenous leukemia (AML), 1 biphenotypic leukemia and 17 ALL patients. Of the patients with ALL, 16 were of B-lineage while 1 had a T-cell phenotype. Ten patients expressed the p210 BCR-ABL transcript alone and 10 expressed only the p190 BCR-ABL transcript. One patient had co-expression of p190 and p210 b3a2 BCR-ABL transcripts. Thus the Philadelphia chromosome can be found in a diverse cohort of morphologic and immunologic subtypes of de novo acute leukemia reflecting the heterogeneity of lineage involvement in this disease.
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PMID:Molecular and phenotypic spectrum of de novo Philadelphia positive acute leukemia. 1056 81

Deoxyribozymes, or DNA enzymes (DNAzymes), are novel nucleic acids that have the ability to bind to specific sequences of RNA, and to cleave the target site catalytically. DNAzymes are smaller and more efficient enzymatically than ribozymes (RZs), which are catalytic nucleic acids synthesized from ribonucleotides. We have designed three DNAzymes that specifically target the two variants of the p210 bcr-abl gene (splice 1, b3a2; splice 2, b2a2) and the p190 variant (ela2). The cleavage sites for these DNAzymes are located 5 nucleotides (nt) 5' from the fusion site for b3a2, and only 1 nt 5' from the fusion sites for b2a2 and e1a2. We have shown in cell-free in vitro cleavage assays that these DNAzymes efficiently cleave their respective substrates. Mutated DNAzymes, in which only one critical base has been altered, do not cleave these targets. We have used a serum-resistant cytofectin (GS 2888; Gilead) to transfect the DNAzymes into target K562 cells, which express p210bcr-abl. In short-term transfection assays, the DNAzymes specifically inhibited p210bcr-abl protein expression by K562 cells by about 40%, and inhibited cell growth by more than 50% in a 6-day liquid culture assay. We have also transfected freshly isolated CD34+ bone marrow cells from patients with CML with the DNAzymes, which specifically inhibited the growth of bcr-abl-positive CFU-Mix colonies by 53-80%. The potential advantages of anti-bcr-abl DNAzymes over RZs include the following: DNAzymes are much less expensive to synthesize; they are more resistant to serum; and the anti-b2a2 DNAzyme cleaves at a site only 1 nt away from the fusion site, whereas its hammerhead RZ counterpart cleaves this target at a site 8 nt 3' to the fusion site, well within abl exon 2. DNAzymes are novel RNA-cleaving molecules that may significantly improve our ability to inhibit bcr-abl oncogene expression in Ph-positive target cells.
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PMID:Inhibition of bcr-abl oncogene expression by novel deoxyribozymes (DNAzymes). 1058 30

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is a chemoattractant for CD34(+) progenitor cells, in vitro and in vivo. The receptor for SDF-1alpha, CXCR-4, is a 7 transmembrane domain receptor, which is also a coreceptor for human immunodeficiency virus (HIV). Here we show that transformation of hematopoietic cell lines by BCR/ABL significantly impairs their response to SDF-1alpha. Three different hematopoietic cell lines, Ba/F3, 32Dcl3, and Mo7e, were found to express CXCR-4 and to respond to SDF-1alpha with increased migration in a transwell assay. In contrast, after transformation by the BCR/ABL oncogene, the chemotactic response to SDF-1alpha was reduced in all 3 lines. This effect was directly due to BCR/ABL, because Ba/F3 cells, in which the expression of BCR/ABL could be regulated by a tetracycline-inducible promoter, also had reduced chemotaxis to SDF-1alpha when BCR/ABL was induced. The reduced response to SDF-1alpha was not due to an inability of BCR/ABL-transformed cell lines to migrate in general, as spontaneous motility of BCR/ABL-transformed cells was increased. In mice, injection of SDF-1alpha into the spleen resulted in a transient accumulation of untransformed Ba/F3 cells, but not Ba/F3. p210(BCR/ABL) cells administered simultaneously. The mechanism may involve inhibition of CXCR-4 receptor function, because in BCR/ABL-transformed cells, CXCR-4 receptors were expressed on the cell surface, but SDF-1alpha calcium flux was inhibited. Because SDF-1alpha and CXCR-4 are felt to be involved in progenitor cell homing to marrow, the abnormality decribed here could contribute to the homing and retention defects typical of immature myeloid cells in chronic myelogenous leukemia.
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PMID:The BCR/ABL oncogene alters the chemotactic response to stromal-derived factor-1alpha. 1059 68


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