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Growth arrest of BCR-ABL positive cells with a sequence-specific polyamide-chlorambucil conjugate.

Chou CJ, O'Hare T, Lefebvre S, Alvarez D, Tyner JW, Eide CA, Druker BJ, Gottesfeld JM - PLoS ONE (2008)

Bottom Line: Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML.However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML.Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively active Abl kinase, which is the product of a chimeric BCR-ABL gene, caused by the genetic translocation known as the Philadelphia chromosome. Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML. However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML. Although alternative Bcr-Abl tyrosine kinase inhibitors have been developed to overcome drug resistance, a cocktail therapy of different kinase inhibitors and additional chemotherapeutics may be needed for complete remission of CML in some cases. Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease. Here we report that a DNA sequence-specific pyrrole-imidazole polyamide-chlorambucil conjugate, 1R-Chl, causes growth arrest of cells harboring both unmutated BCR-ABL and three imatinib resistant strains. 1R-Chl also displays selective toxicities against activated lymphocytes and a high dose tolerance in a murine model.

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Chemical structure of 1R-Chl, imatinib, dasatinib, and their effects on BCR-ABL unmutated and mutated genes transduced into murine BM cells.(A) 1R-Chl (left) targets the DNA sequences 5′-WGGWGW-3′. Bold, imidazole rings. imatinib (middle) targets Bcr-Abl kinase, and dasatinib (right) targets 14 out of 15 Bcr-Abl mutants. (B) Murine BM cells transduced with unmutated BCR-ABL and single point mutation Y253H, E255K, and T315I genes were tested for the effectiveness of 1R-Chl (125 nM to 1000 nM), 1S-Chl (500 nM and 1000 nM), imatinib (500 nM and 5000 nM; IC50 = 260 nM for the native BCR-ABL transduced cells) [6], and dasatinib (10 nM and 100 nM; IC50 = 0.8 nM for the native BCR-ABL transduced cells) [6]. Triplicate experiments were done, and the numbers of colonies were quantified 7 days after initial plating.
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pone-0003593-g001: Chemical structure of 1R-Chl, imatinib, dasatinib, and their effects on BCR-ABL unmutated and mutated genes transduced into murine BM cells.(A) 1R-Chl (left) targets the DNA sequences 5′-WGGWGW-3′. Bold, imidazole rings. imatinib (middle) targets Bcr-Abl kinase, and dasatinib (right) targets 14 out of 15 Bcr-Abl mutants. (B) Murine BM cells transduced with unmutated BCR-ABL and single point mutation Y253H, E255K, and T315I genes were tested for the effectiveness of 1R-Chl (125 nM to 1000 nM), 1S-Chl (500 nM and 1000 nM), imatinib (500 nM and 5000 nM; IC50 = 260 nM for the native BCR-ABL transduced cells) [6], and dasatinib (10 nM and 100 nM; IC50 = 0.8 nM for the native BCR-ABL transduced cells) [6]. Triplicate experiments were done, and the numbers of colonies were quantified 7 days after initial plating.

Mentions: Chlorambucil (Chl) is an effective first-line single-agent treatment for chronic lymphocytic leukemia and as a combined chemotherapeutic in low-grade non-Hodgkin's and Hodgkins' disease [12], [13]. However, Chl has poor affinity toward its known target DNA, with efficacy only at ∼100 µM concentration [14], [15]. When conjugated to pyrrole-imidazole (Py-Im) polyamides, Chl has shown increased affinity and specificity toward DNA [16], [17], [18]. By screening a small library of polyamide-Chl conjugates, we identified one specific conjugate, termed 1R-Chl (Figure 1A), which blocks proliferation of various cancer cell lines in culture. 1R-Chl has anti-tumor activity in mice bearing human SW620 colon carcinoma, K562 CML, Calu-1 lung cancer and 22Rv1 prostate cancer xenografts [19], [20]. Microarray analysis indicated that limited numbers of genes are significantly down regulated by 1R-Chl. One such gene encodes histone H4 (H4c), and western blotting experiments confirmed that total H4 protein levels are reduced in 1R-Chl treated cells. 1R-Chl is expected to bind the general DNA sequence 5′-WGGWGW-3′, where W = A or T., This sequence is found in the histone H4 gene, which is bound by the parent compound lacking Chl and alkylated by 1R-Chl. Alkylation within the coding region of the H4c gene was confirmed in cell culture by ligation-mediated PCR. Such alkylation leads to down-regulation of H4 gene expression, with a concomitant loss of H4 protein. This leads to nucleosome disruption, widespread alkylation within genomic DNA and cell cycle arrest at G2/M. Changing the chirality of the “turn” amino acid to S-2,4-diaminobutyric acid (1S-Chl) eliminates the biological activity of the conjugate due to a loss of binding and alkylation of this target DNA sequence. 1R-Chl has also been shown to inhibit proliferation of the CML cell line K562 at nanomolar concentrations [20]. Most importantly, polyamide-Chl conjugates that do not target the H4c gene fail to down-regulate this gene or block cell proliferation [19], [20].


Growth arrest of BCR-ABL positive cells with a sequence-specific polyamide-chlorambucil conjugate.

Chou CJ, O'Hare T, Lefebvre S, Alvarez D, Tyner JW, Eide CA, Druker BJ, Gottesfeld JM - PLoS ONE (2008)

Chemical structure of 1R-Chl, imatinib, dasatinib, and their effects on BCR-ABL unmutated and mutated genes transduced into murine BM cells.(A) 1R-Chl (left) targets the DNA sequences 5′-WGGWGW-3′. Bold, imidazole rings. imatinib (middle) targets Bcr-Abl kinase, and dasatinib (right) targets 14 out of 15 Bcr-Abl mutants. (B) Murine BM cells transduced with unmutated BCR-ABL and single point mutation Y253H, E255K, and T315I genes were tested for the effectiveness of 1R-Chl (125 nM to 1000 nM), 1S-Chl (500 nM and 1000 nM), imatinib (500 nM and 5000 nM; IC50 = 260 nM for the native BCR-ABL transduced cells) [6], and dasatinib (10 nM and 100 nM; IC50 = 0.8 nM for the native BCR-ABL transduced cells) [6]. Triplicate experiments were done, and the numbers of colonies were quantified 7 days after initial plating.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2571993&req=5

pone-0003593-g001: Chemical structure of 1R-Chl, imatinib, dasatinib, and their effects on BCR-ABL unmutated and mutated genes transduced into murine BM cells.(A) 1R-Chl (left) targets the DNA sequences 5′-WGGWGW-3′. Bold, imidazole rings. imatinib (middle) targets Bcr-Abl kinase, and dasatinib (right) targets 14 out of 15 Bcr-Abl mutants. (B) Murine BM cells transduced with unmutated BCR-ABL and single point mutation Y253H, E255K, and T315I genes were tested for the effectiveness of 1R-Chl (125 nM to 1000 nM), 1S-Chl (500 nM and 1000 nM), imatinib (500 nM and 5000 nM; IC50 = 260 nM for the native BCR-ABL transduced cells) [6], and dasatinib (10 nM and 100 nM; IC50 = 0.8 nM for the native BCR-ABL transduced cells) [6]. Triplicate experiments were done, and the numbers of colonies were quantified 7 days after initial plating.
Mentions: Chlorambucil (Chl) is an effective first-line single-agent treatment for chronic lymphocytic leukemia and as a combined chemotherapeutic in low-grade non-Hodgkin's and Hodgkins' disease [12], [13]. However, Chl has poor affinity toward its known target DNA, with efficacy only at ∼100 µM concentration [14], [15]. When conjugated to pyrrole-imidazole (Py-Im) polyamides, Chl has shown increased affinity and specificity toward DNA [16], [17], [18]. By screening a small library of polyamide-Chl conjugates, we identified one specific conjugate, termed 1R-Chl (Figure 1A), which blocks proliferation of various cancer cell lines in culture. 1R-Chl has anti-tumor activity in mice bearing human SW620 colon carcinoma, K562 CML, Calu-1 lung cancer and 22Rv1 prostate cancer xenografts [19], [20]. Microarray analysis indicated that limited numbers of genes are significantly down regulated by 1R-Chl. One such gene encodes histone H4 (H4c), and western blotting experiments confirmed that total H4 protein levels are reduced in 1R-Chl treated cells. 1R-Chl is expected to bind the general DNA sequence 5′-WGGWGW-3′, where W = A or T., This sequence is found in the histone H4 gene, which is bound by the parent compound lacking Chl and alkylated by 1R-Chl. Alkylation within the coding region of the H4c gene was confirmed in cell culture by ligation-mediated PCR. Such alkylation leads to down-regulation of H4 gene expression, with a concomitant loss of H4 protein. This leads to nucleosome disruption, widespread alkylation within genomic DNA and cell cycle arrest at G2/M. Changing the chirality of the “turn” amino acid to S-2,4-diaminobutyric acid (1S-Chl) eliminates the biological activity of the conjugate due to a loss of binding and alkylation of this target DNA sequence. 1R-Chl has also been shown to inhibit proliferation of the CML cell line K562 at nanomolar concentrations [20]. Most importantly, polyamide-Chl conjugates that do not target the H4c gene fail to down-regulate this gene or block cell proliferation [19], [20].

Bottom Line: Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML.However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML.Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively active Abl kinase, which is the product of a chimeric BCR-ABL gene, caused by the genetic translocation known as the Philadelphia chromosome. Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML. However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML. Although alternative Bcr-Abl tyrosine kinase inhibitors have been developed to overcome drug resistance, a cocktail therapy of different kinase inhibitors and additional chemotherapeutics may be needed for complete remission of CML in some cases. Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease. Here we report that a DNA sequence-specific pyrrole-imidazole polyamide-chlorambucil conjugate, 1R-Chl, causes growth arrest of cells harboring both unmutated BCR-ABL and three imatinib resistant strains. 1R-Chl also displays selective toxicities against activated lymphocytes and a high dose tolerance in a murine model.

Show MeSH
Related in: MedlinePlus