Koster DA, Palle K, Bot ES, Bjornsti MA, Dekker NH.

Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.

Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils. Topotecan is thought to stabilize a covalent topoisomerase-DNA complex, rendering it an obstacle to DNA replication forks. Here we use single-molecule nanomanipulation to monitor the dynamics of human topoisomerase I in the presence of topotecan. This allowed us to detect the binding and unbinding of an individual topotecan molecule in real time and to quantify the drug-induced trapping of topoisomerase on DNA. Unexpectedly, our findings also show that topotecan significantly hinders topoisomerase-mediated DNA uncoiling, with a more pronounced effect on the removal of positive (overwound) versus negative supercoils. In vivo experiments in the budding yeast verified the resulting prediction that positive supercoils would accumulate during transcription and replication as a consequence of camptothecin poisoning of topoisomerase I. Positive supercoils, however, were not induced by drug treatment of cells expressing a catalytically active, camptothecin-resistant topoisomerase I mutant. This combination of single-molecule and in vivo data suggests a cytotoxic mechanism for camptothecins, in which the accumulation of positive supercoils ahead of the replication machinery induces potentially lethal DNA lesions.

PMID: 17589503 [PubMed - indexed for MEDLINE]
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NCI’s Alliance for Nanotechnology in Cancer is reporting about an interesting discovery, recently reported in the journal Nature:

The annoying bulges of an overwound telephone cord that shorten its reach and limit a caller’s motion help explain why drugs called camptothecins are so effective in killing cancer cells, according to investigators led by Mary-Ann Bjornsti, Ph.D., at St. Jude Children’s Research Hospital, and Nynke Dekker, Ph.D., at Delft Technology University. Using nanoscale magnetic tweezers (nanotweezers), the researchers showed that a camptothecin drug called topotecan kills cancer cells by preventing an enzyme called DNA topoisomerase I from uncoiling double-stranded DNA in those cells. Instead, the DNA becomes locked in tight twists called supercoils, which bulge out from the side of the overwound DNA molecule much like the bulges in an overwound telephone cord. If these supercoils accumulate and persist while the cell is trying to separate the two strands of DNA to make exact copies of the chromosomes during cell division, the cells will die.
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The enzyme superoxide dismutase (SOD), catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide.

We can’t live without oxygen. Our cells rely on oxygen as the final acceptor of electrons in respiration, allowing us to extract far more energy from food than would be possible without oxygen. But oxygen is also a dangerous compound. Reactive forms of oxygen, such as superoxide (oxygen with an extra electron), leak from the respiratory enzymes and wreak havoc on the cell. This superoxide can then cause mutations in DNA or attack enzymes that make amino acids and other essential molecules. This is a significant problem: one study showed that for every 10,000 electrons transferred down the respiratory pathway in Escherichia coli cells, about 3 electrons end up on superoxide instead of the proper place. To combat this potential danger, most cells make superoxide dismutase (SOD, shown here from PDB entry 2sod), an enzyme that detoxifies superoxide.
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“for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells”
1. Gene modification in mice
Introduction

The 2007 Nobel Prize in physiology or medicine is awarded to Drs Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells. Their work has made it possible to modify specific genes in the germline of mammals and to raise offspring that carry and express the modified gene. The toolbox of experimental genetic methods developed by Capecchi, Evans and Smithies, commonly called the knockout technology, has permitted scientists to determine the role of specific genes in development, physiology, and pathology. It has revolutionized life science and plays a key role in the development of medical therapy.

The discoveries
Martin Evans identified and isolated the embryonic stem cell of the early embryo, the cell from which all cells of the adult organism are derived. He established it in cell culture, modified it genetically, and reintroduced it into foster mothers in order to generate a genetically modified offspring. Mario Capecchi and Oliver Smithies, independently of each other, discovered how homologous recombination between segments of DNA molecules can be used to target genes in the mammalian genome and developed methods to generate genetically modified mice. Such animals have become indispensable in medical research. Furthermore, the knowledge concerning stem cell biology and gene technology obtained during the research that led to the “knockout mouse” has changed our understanding of normal development and disease processes and identified new avenues for medical therapy. Fig. 1 shows the general strategy for gene targeting in mice.
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