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5003-1 In Vivo Screen for Topo I Agents

5003-2 In Vivo Screen for Topo II Agents

  Topo I or II targeting agents that induce cleavage complexes may be extremely effective anticancer drugs; however, they need to be tested in cells. The Level III (in vivo) screens will fulfill this need. The method is based on the “In Vivo Link Kit” developed exclusively by TopoGEN. Level III screens are very rigorous and quantitative methods to define efficacy of action of an unknown drug relative to the industry standards. The method will also expose whether a test agent can enter cells and act on the target (topo I or topo II). TopoGEN has extensive experience with assays for topoisomerase poisons in vivo. These experiments allow the investigator to ascertain whether a novel agent is active against endogenous topo in a chromosomal setting in nuclei. An important benefit to this analysis is that one can use any tumor cell or tissue in order to establish clinical efficacy of a test drug against a specific tumor cell line. We use essentially the same basic approach for Topo I as for Topo II. The methods are based upon physically separating the topo/DNA adducts from free DNA and using antibodies to measure bound topo I or II. In this analysis, tissue culture cells are treated with a test compound along with negative controls (no drug) and positive controls (with known inhibitors). The cells are drug treated and rapidly lysed with Sarkosyl which traps some fraction of the endogenous topo on DNA in a covalent cleavage complex. Following detergent lysis, the lysate is diluted to fully dissociate non-covalent DNA/protein complexes. Covalent topo/DNA complexes are resolved on a step CsCl gradient (ionic DNA/protein interactions are also prevented by 5 M CsCl). The gradient resolves DNA, chromatin aggregates, and protein, respectively. Gradients are centrifuged overnight and fractionated. The amount of topo coincident with the DNA peak is a measure of covalent DNA/topo complexes. Topo concentration in the DNA peak is determined by immunoblotting using antibody to topo I or II as probe. In the absence of agents that stabilize the cleavable complex (etoposide, camptothecin) only low levels of topo are found in the DNA peak; this is particularly obvious with topo II since the type II enzyme is not trapped by this method unless a specific poison is used. The ratio of topo at the DNA density and the protein density reflects the relative efficiency of stabilization of the cleavable complexes. It is important to stress that this method is ANTIBODY based and therefore specific for topo I or II. 



In Vitro Screening Services