Topo Link Kit

Analysis of In Vivo Cleavable Complex Formation with Topoisomerases I and II Using A New Kit:

The In Vivo Link-Kit

ABSTRACT

TopoGEN has developed a powerful new technology that allows the investigator to detect topoisomerase/DNA cleavable complexes within cells. The method is based upon the fact that the endogenous enzyme can be trapped in a cleavage complex by rapid denaturation with detergents; however, the cleavable complex cannot be detected without drugs that stabilize the broken intermediate. The covalent topoisomerase/DNA adducts can be resolved from free protein by CsCl gradient centrifugation. The DNA bound enzyme (or cleavable complex) and free enzyme are then quantified by immunoblotting. The advantage of this method is that direct measurement of topo I or II mediated DNA damage is possible in any given cell or tissue setting. The technique is particularly useful for identifying and characterizing new inhibitors in different cell backgrounds; however, more importantly, the method is physiologically relevant since the drugs are tested in a nuclear setting in which transcription, DNA replication and repair processes are ongoing. These processes are known to have a direct influence on drug induced-cleavable complex formation.

INTRODUCTION

Eukaryotic topoisomerases are ubiquitous nuclear enzymes that alter DNA topology by breaking and resealing DNA strands (for reviews see 5-8). There are two classes of topoisomerases; type II enzymes transiently break both strands of DNA in concert, and type I enzymes transiently break one strand at a time. Given that these enzymes are important targets for novel anticancer and antibacterial therapeutics, assays that allow physiological detection and evaluation of novel inhibitors are extremely valuable (4-6). TopoGEN has extensive experience with assays for topo I and topo II inhibition in vivo and we have developed a new kit just for this purpose. The TopoGEN In Vivo Link Kit contains specific antibodies, reagents and instructions for immunodetection of topo I or II cleavable complexes that form in a chromosomal setting in the presence of different test compounds. The kit inlcudes positive and negative controls so that each analysis is rigorously controlled for a given cell line or tissue. The system is user defined, allowing the researcher to test new compounds (that may show promise in a biochemical assay) in different tumors, cell lines or even virus infected cells (1-3).

The utility of this method is evident when one considers that in vitro assays are designed to identify topoisomerase-active agents under conditions that may or may not reflect events within the cell. For example, chemical interconversion or degradation of topo active drugs is common in cells. Furthermore, uptake and/or delivery of a compound to the site of enzyme activity cannot be evaluated using in vitro topo assays. Finally, there is evidence that DNA damage inflicted by topoisomerase poisons is strongly influenced by processes such as nascent DNA chain growth, transcription and DNA repair (5). The next level of sophistication in mechanistic drug design and development is in vivo evaluation of efficacy; the procedure described here in is a novel, tractable and user friendly technique for measuring the formation of the DNA lesion directly responsible for cytotoxicity. The method can be modified for type I or II topoisomerases by simply using the appropriate antibody.

MATERIALS

Antitsera

The eukaryotic type II antibody is an affinity purified peptide antibody directed against the C-terminus of the 170 kDa enzyme (cat #2011-1). The antibody to topo I (Cat# 2012) is a scleroderma antibody (human IgG). Both antibodies are suitable for Western blotting using [125I]-Protein A. Non-radioactive detection can also be performed using the ECL system (Amersham) with antitopo II antibody. Excess antibody is provided with each kit to allow a large number of experiments with the diluted reagents.

General procedures

An outline of the method is shown in Figure 1. Cells to be tested may be a particular cell line, virus infected cell or tumor tissue. Cells are incubated under conditions that favor endogenous topo activity (defined as physiological conditions conducive to cell growth); thus, the endogenous enzyme is engaging the DNA template in a series of breaking and resealing steps. Concurrently, central genetic processes such as DNA replication, transcription and repair are ongoing. The cells are then rapidly lysed with a detergent (sarkosyl). It is important that lysis be carried out while maintaining the cells at 37oC; if the cells are cooled or manipulated prior to lysis, the cleavage complexes tend to re-ligate and yield negative results (2,9-11). The next step requires purification of DNA away from free protein; however, organic extractions or proteinase digestions must be avoided. We use a step CsCl gradient for this purpose. The density steps are designed to resolve DNA from free protein and there is quantitative recovery of both. Covalent complexes containing topo and DNA sediment to the position of DNA. It is known that covalently bound protein can shift the density of DNA in CsCl and the magnitude of the density shift is proportional to the total amount of protein. In vitro, topo I may produce a density shift of DNA under conditions of stoichiometric excess of protein (data not shown); however, in vivo significantly less protein is coupled to DNA. In fact, topoisomerase does not cause any density shift of genomic DNA in this analysis (Fig. 1) for two reasons. First, the number of bound topo molecules per DNA molecule is very low and in these gradients, complexes behave like free DNA (vs. DNA/protein adducts). Second, the gradients are very steep and it is not possible to resolve small density differences anyway. After CsCl centrifugation, the gradients are fractionated and amount of bound and free topo is measured by Western blotting using slot or dot blots and antibodies developed by TopoGEN. The ratio of bound/free is a direct measure of the cleavable complex formation in the particular cell system. We have found that topoisomerases are difficult (if not impossible) to trap as cleavable complexes within cells in the absence of inhibitors; thus, any test compound that results in detection of topo I or II in the DNA peak (Fig. 1) is most likely a topo-active agent. The In Vivo Link-Kit then allows the investigator to evaluate a compound for activity against topo I and II in different tissue settings or with virus infected cells. Finally, it is possible to combine the analysis of topo I and II in the same experiment. In this case, one can evaluate topo I inhibition using the topo I antibody (Cat# 2012) or topo II using that antibody (Cat# 2011-1). Some drugs may conceivably act upon both enzymes (Trask and Muller, 1988).

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