Mutation P732L in Human DNA Topoisomerase IIβ Abolishes DNA Cleavage in the Presence of Calcium and Confers Drug Resistance

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.

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Gene transfer of multidrug resistance into a factor-dependent human hematopoietic progenitor cell line: in vivo model for genetically transferred chemoprotection

Blood ◽

10.1182/blood.v87.7.2723.bloodjournal8772723 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2723-2731 ◽

Cited By ~ 16

Author(s):

P Schwarzenberger ◽

S Spence ◽

N Lohrey ◽

T Kmiecik ◽

DL Longo ◽

...

Keyword(s):

Drug Resistance ◽

Multidrug Resistance ◽

Gene Transfer ◽

Hematopoietic Progenitor Cells ◽

Mdr1 Gene ◽

In Vivo Model ◽

Hematopoietic Progenitor ◽

Human Dna

To develop a rapid preclinical in vivo model to study gene transfer into human hematopoietic progenitor cells, MO-7e cells (CD-34+, c-kit+) were infected with multidrug resistance (MDR1)-containing retroviruses and then transplanted into nonobese diabetic severe combined immunodeficient mice (NOD SCID). MO-7e cells infected with a retrovirus encoding the human MDR1 cDNA showed integration, transcription, and expression of the transfered MDR1 gene. This resulted in a 20-fold increase in the resistance of MO-7e cells to paclitaxel in vitro. The expression of the MDR1 gene product was stable over a 6-month period in vitro without selection in colchicine. MO-7e and MDR1-infected MO-7e cells were transplanted into NOD SCID mice to determine whether MDR1 could confer drug resistance in vivo. A sensitive polymerase chain reaction method specific for human sequences was developed to quantitate the level of human cell engraftment in NOD SCID bone marrow (BM) cells. The percentage of human DNA in BM cells from MO-7e- transplanted mice was 10.9% and decreased to 0.7% in mice treated with paclitaxel. The percentage of human DNA in infected-MO-7e transplanted mice was 7.6% and that level was unchanged in mice treated with paclitaxel. These results show that expression of the MDR1 gene in human hematopoietic progenitor cells can confer functional drug resistance in an in vivo model.

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