Effect of cations on purine.purine.pyrimidine triple helix formation in mixed-valence salt solutions.
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The effect of various monovalent, divalent and oligovalent cations on the reaction of triplex formation by GT and AG motif triplex-forming oligonucleotides, designed to bind to biologically relevant polypurine-polypyrimidine sequences occurring in the promoters of the murine Ki-ras and human bcr genes, has been investigated by means of electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments. We found that in the presence of 10 mm MgCl2 the triple helices were progressively destabilized by adding increasing amounts of NaCl, from 20 to 140 mm, to the solution. We also observed that, while the total monovalent-ion concentration was constant at 100 mm, the exchange of sodium with potassium, but not lithium, results in a further destabilization of the triple helices, due to self-association equilibria involving the G-rich triplex-forming oligonucleotides. Potassium was found to destabilize triplex DNA even when the triple helices are preformed in the absence of K+. However, footprinting experiments also showed that the inhibitory effect of K+ on triplex DNA is partially compensated for by millimolar amounts of divalent transition metal ions such as Mn2+ and Ni2+, which upon coordinating to N7 of guanine are expected to enhance hydrogen-bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G-rich triplex-forming oligonucleotides. Triplex enhancement in the presence of potassium was also observed, but to a lesser extent, when spermine was added to the reaction mixture. Here, the ion effect on triplex DNA is rationalized in terms of competition among the different valence cations to bind to triplex DNA, and differential cation stabilization of unusual quadruplex structures formed by the triplex-forming oligonucleotides. (+info)
Thiotepa, busulfan and cyclophosphamide as a preparative regimen for allogeneic transplantation for advanced chronic myelogenous leukemia.
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Thirty-six adults with chronic myelogenous leukemia (CML) in second or greater chronic phase, accelerated phase, or blast crisis underwent marrow or blood stem cell transplantation from an HLA-matched sibling using high-dose thiotepa, busulfan and cyclophosphamide (TBC) as the preparative regimen. All evaluable patients engrafted and had complete donor chimerism. One patient failed to clear meningeal leukemia, and one patient had one of 30 metaphases positive for the Philadelphia chromosome at 2 months post transplant. The remainder of the patients studied had eradication of CML documented by cytogenetics and/or Southern blot for BCR gene rearrangement, and 13 of 15 patients studied became negative for the BCR gene rearrangement by polymerase chain reaction. Three-year relapse rate is 42% (95% CI, 19-64%). The relapse rate was significantly lower for patients transplanted without blast crisis (9% vs 100%, P < 0.001). Eight (22%, 95% CI, 10-39%) patients had severe or fatal veno-occlusive disease (VOD). Elevated liver enzymes within 1 month prior to transplantation and transplantation using marrow were significantly associated with the occurrence of VOD. Three-year survival is 28% (95% CI, 13-43%). Survival was significantly higher for patients transplanted without blast crisis (45% vs 0%, P = 0.01). TBC is an effective preparative regimen for CML in accelerated phase but not refractory blast crisis, and it should be used with caution in patients with prior hepatopathy who have an increased risk of severe VOD. (+info)
Bcr: a negative regulator of the Bcr-Abl oncoprotein.
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Chronic myelogenous leukemia is typically characterized by the presence of the Philadelphia chromosome (Ph) in which 5' portions of the BCR gene are fused to a large portion of the ABL gene. Our studies and those of others indicate that Bcr sequences within the Bcr-Abl oncoprotein are critically involved in activating the Abl tyrosine kinase and actively participate in the oncogenic response, which is generated by the Bcr-Abl oncoprotein. We investigated the role of the Bcr protein in the oncogenic effects of Bcr-Abl. Reduction of the level of the Bcr protein by incubating cells with a 3' BCR anti-sense oligodeoxynucleotide increased the growth rate and survival of hematopoietic cell lines expressing Bcr-Abl. Also, enforced expression of Bcr in Bcr-Abl cell lines strongly reduced transformation efficiency. Induction of Bcr expression drastically reduced the phosphotyrosine content of Bcr-Abl in Rat-1 fibroblasts transformed by P185 BCR-ABL and in hematopoietic cells expressing P210 Bcr-Abl within days following induction of Bcr. Rat-1/P185 cells maintained for three weeks after Bcr induction had dramatically reduced amounts of phosphotyrosine proteins compared to cells in which Bcr expression was repressed by the addition of Tet. In contrast Bcr expression did not decrease the phosphotyrosine content of either v-Src or activated Neu tyrosine kinase. Importantly, the phosphotyrosine content of total P160 BCR (induced plus endogenous) was strongly reduced by inducing expression of Bcr, indicating that the induced Bcr protein was not a target of the tyrosine kinase activity of Bcr-Abl but instead functioned as an inhibitor of Bcr-Abl. These results show that the Bcr protein can function as a negative regulator of Bcr-Abl, but that the inhibitory effects of Bcr are dependent on achieving an elevated level of Bcr expression relative to Bcr-Abl. (+info)
Cyclin D2 is essential for BCR-mediated proliferation and CD5 B cell development.
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Progression into G(1) in B lymphocytes is regulated by cyclins D2 and D3, components of the cell cycle machinery currently believed to have overlapping and potentially redundant roles in cell cycle control. To study the specific role of cyclin D2 in B lymphocyte proliferation, we examined B cells from cyclin D2(-/-) mice and demonstrate a specific requirement for cyclin D2 in BCR- but not CD40- or lipopolysaccharide-induced proliferation. Furthermore, conventional B cell development proceeds normally in the mutant mice; however, the CD5 B cell compartment is dramatically reduced, suggesting that cyclin D2 is important in CD5 B cell development as well as antigen-dependent B cell clonal expansion. (+info)
Exon-skipping in BCR/ABL is induced by ABL exon 2.
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The BCR/ABL fusion gene is pathognomonic for chronic myelogenous leukaemia (CML). We have previously reported alternative splicing of BCR/ABL, as indicated by the detection of both p190- and p210-encoding transcripts, in about 60% of CML patient samples. These exon-skipping events involved the joining of ABL exon 2 to variable upstream BCR exons. Similarly, ABL exon 2 is alternatively spliced to either of two upstream ABL exons (1a or 1b) in c-ABL. We have constructed BCR and BCR/ABL minigenes to study this phenomenon in more detail. These constructs were transfected into various cell types and splicing was assessed by reverse transcriptase PCR. Whereas the basic BCR minigene expressed exon-inclusive transcripts only, insertion of genomic DNA spanning ABL exon 2 induced exon-skipping but only when expressed in the CML cell lines K562 and EM3. In this study we localized the required sequence element to ABL exon 2 itself. These results mimic the splicing phenotype displayed by most CML patients. We propose a model where a trans-factor present in some CML cells interacts with ABL exon 2 pre-mRNA to promote skipping of upstream BCR exons. (+info)
Efficacy of STI571, an abl tyrosine kinase inhibitor, in conjunction with other antileukemic agents against bcr-abl-positive cells.
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Chronic myelogenous leukemia (CML), a malignancy of a hematopoietic stem cell, is caused by the Bcr-Abl tyrosine kinase. STI571(formerly CGP 57148B), an Abl tyrosine kinase inhibitor, has specific in vitro antileukemic activity against Bcr-Abl-positive cells and is currently in Phase II clinical trials. As it is likely that resistance to a single agent would be observed, combinations of STI571 with other antileukemic agents have been evaluated for activity against Bcr-Abl-positive cell lines and in colony-forming assays in vitro. The specific antileukemic agents tested included several agents currently used for the treatment of CML: interferon-alpha (IFN), hydroxyurea (HU), daunorubicin (DNR), and cytosine arabinoside (Ara-C). In proliferation assays that use Bcr-Abl-expressing cells lines, the combination of STI571 with IFN, DNR, and Ara-C showed additive or synergistic effects, whereas the combination of STI571 and HU demonstrated antagonistic effects. However, in colony-forming assays that use CML patient samples, all combinations showed increased antiproliferative effects as compared with STI571 alone. These data indicate that combinations of STI571 with IFN, DNR, or Ara-C may be more useful than STI571 alone in the treatment of CML and suggest consideration of clinical trials of these combinations. (+info)
A novel four zinc-finger protein targeted against p190(BcrAbl) fusion oncogene cDNA: utilisation of zinc-finger recognition codes.
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A three zinc-finger protein that binds specifically to the cDNA representing the unique fusion gene BCR:Abl, associated with acute lymphoblastic leukaemia, has previously been characterised. At this breakpoint, a sequence homology of 8/9 bp exists between the BCR:Abl (fusion) and c-ABL: (parental) target sequences. We show that the three zinc-finger protein discriminates poorly between the fusion (BCR:Abl) and parental (ABL:) sequence (K:(d)s of 42.8 and 65.1 nM, respectively). In order to improve the discriminatory properties of this protein, and to demonstrate the utility of current zinc-finger databases, we have added a fourth zinc-finger to the original three zinc-finger protein. This fourth finger recognises a 3 bp subsite derived from the BCR: portion of the breakpoint and is not present in c-ABL: This novel four finger protein, which now recognises a 12 bp sequence, demonstrates improved specific binding to BcrAbl (K:(d )= 17 nM). More significantly we have shown that there is now enhanced discrimination between BcrAbl and ABL: sequences by the four finger protein than the original three finger protein. (+info)
Targeting neighbouring poly(purine.pyrimidine) sequences located in the human bcr promoter by triplex-forming oligonucleotides.
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Most poly(purine.pyrimidine) [poly(R.Y)] sequences in eukaryotic genomes are interrupted by one or more base pair inversions. When the inversions are centrally located, the poly(R.Y) sequences can be regarded as the sum of two abutting sites, each potentially capable of forming a triple helix. Employing band-shift, footprinting and modeling methods we examined the formation of triple helices at a critical 27 bp poly(R.Y) sequence interrupted by two adjacent CG inversions, and located in the promoter of the human bcr gene at transcription initiation. We designed several 13-mer and 14-mer triplex-forming oligonucleotides (TFOs) capable of binding the bcr abutting sites, thereby generating different base juxtapositions at the triple helical junction, to examine whether triplex formation occurs in a cooperative manner. It is found that in 50 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 2 mM spermine, 37 degrees C, the 13-and the 14-mer TFOs bind to one half of the bcr site with Delta G between -30 and -35 kJ x mol-1. However, when different 13-mer/14-mer combinations of TFOs were directed against the abutting poly(R x Y) sites, triplex formation has been found to be enhanced only for the triple helical junction formed by the 5'-A-T-3' base juxtaposition, in keeping with a partial stacking suggested from modeling analysis. On the other hand, a longer 24-mer TFO, binding noncooperatively to the same abutting sites, forms a much more stable triplex (Delta G = -51 kJ x mol-1), notwithstanding the two T x CG triads in the middle. Modeling investigations reveal that there is no continuity or propagation of base stacking involving adjacent bases of the third strand at the site of base inversion as well as on the 5' side. The data indicate that the entropy penalty of forming a triplex with two oligonucleotides is much higher than the energy gained from base stacking interactions at the triplex junction formed between the two TFOs. (+info)