Characterization of the cellular loci of bleomycin-mediated damage

The bleomycins (BLMs) are a family of glycopeptide derived antitumor agents. These agents are thought to elicit their chemotherapeutic activity via their ability to mediate sequence selective DNA strand scission. Currently, BLM treatment results in a large number of side effects unrelated to the desired therapeutic effect. It is these toxic side effects that limit the dose administered. Because the maximally tolerated doses are much lower than that required to effect killing of all tumor cells, a fraction of the tumor cells are able to survive and can evolve mechanisms of resistance.; Resistance to DNA damaging agents can be achieved by the initiation of DNA repair systems. The DNA damage mediated by BLM is believed to be repaired by excision repair. In the specific case of repair of BLM-mediated DNA damage this type of repair is thought to involve short gap filling mediated by DNA polymerase {dollar}beta.{dollar} It was thought that the inhibition of DNA polymerase {dollar}beta{dollar} would render cells dysfunctional to repair BLM-mediated DNA damage. This scenerio was experimentally tested by investigating the ability of an authentic polymerase {dollar}beta{dollar} inhibitor, anacardic acid 1, to potentiate the therapeutic activity of BLM in cultured mammalian cells. In this study it was observed that in the presence of anacardic acid 1 BLM-induced cytotoxicity was increased substantially. Further, from measurements of unscheduled DNA synthesis and inhibition of short gap filling the mechanism of this synergistic effect has been determined to be inhibition of DNA repair.; Although BLM is generally thought to exert its anticancer effects by mediating DNA damage, Fe(II){dollar}bullet{dollar}BLM has also been observed to afford cleavage of RNA substrates in vitro. RNA may constitute a relevant therapeutic target for several reasons. Unlike DNA, RNA is accessible to BLM in the cytoplasm of the cell. Further, there is no known repair mechanism for RNA damage. As a result of the obvious attractive features of RNA as a cellular target for BLM we designed a system to examine the possible effects of BLM on a cellular mRNA. Specifically, a single E. coli messenger RNA, the mRNA encoding for dihydrofolate reductase was examined following BLM treatment of the E. coli cells. Not only was damage to this cellular RNA observed but, interestingly the damage was much more selective than that observed when the same RNA was treated with Fe(II){dollar}bullet{dollar}BLM in a cell free system.; As a result of the similarities between cellular metal catalyzed oxidation systems and activated metallobleomycin, it was of interest to examine the effects of BLM on protein substrates. This effect was studied on two structurally similar dehydrogenase enzymes, glucose-6-phosphate dehydrogenase and yeast alcohol dehydrogenase. Interestingly, both enzymes were deactivated in the presence of activated Fe{dollar}bullet{dollar}BLM, but the apparent cause of BLM-mediated deactivation was different.