Consecuencias para la progenie celular de la pérdida de actividad de la diana antitumoral topoisomerasa 2, bien mediante mutaciones adquiridas durante la selección de resistencia tumoral, bien mediante el uso de agentes anticancerígenos

Resumen

Cancer is characterized by abnormal cell growth produced by genetic instability. The activation of programmed cell death or the induction of mitotic catastrophe that would put an end to the multiplication of cancer cells is the goal of many anticancer treatments. Type II topoisomerases (Top2) disentangle the DNA molecules at the time of mitosis, and thus, their inactivation produces a catastrophic event whereby anaphase bridges, which are resolved by creating massive DNA damage, lead to cell death. Furthermore, Top2 is one of the most important targets in anticancer therapy, yet not through its role in DNA disentanglement but because many antitumor drugs block the Top2 catalytic cycle in a step that breaks the DNA into pieces. Resistance to these antitumor drugs, known as Top2 poisons, can develop through mutations that reduce Top2 activity. This might render these resistant cancer cells hypersensitive to another class of anti-Top2 compounds known as catalytic inhibitors. Herein, we have studied the consequences for the cell progeny of the transitory inactivation of Top2 in the model organism Saccharomyces cerevisiae. We have compared the phenotypes of two thermosensitive alleles, top2-4 and top2-5, which differ in their intrinsic resistance to anti-Top2 poisons. We have found that they also differ in the response to confront an anaphase bridge, as well as they have different means to overcome the subsequent mitotic catastrophe. Thus, top2-4 shows a high percentage of cells arrested with anaphase bridges, whereas top2-5 is able to resolve them almost instantly, but at the expense of generating much more DNA damage and cell death than top2-4. Interestingly, in both cases we found patterns of programmed cell death, such as the production of reactive oxygen species, chromatin condensation and fragmentation, or cell swelling. These phenotypes however, seem to be independent of the yeast metacaspase 1 (Yca1), although the deletion of YCA1 improves viability. It is noteworthy that the genetic study of the survivors after a transitory inactivation of Top2 in hybrid diploids showed that a high proportion of them have uniparental disomy, a genetic alteration frequently observed in tumour cells. In addition, we also characterized the effects of the antitumour drug β-lapachone (β-lap), currently undergoing clinical trials for its antitumour use. It is known that β-lap generates reactive oxygen species, but it has been proposed that β-lap, as many other quinones, could contribute to the creation of DNA damage by acting against Top2. In this work, we have studied the determinants of β-lap cytotoxicity in Saccharomyces cerevisiae by downregulating pathways of anti-oxidant response, DNA and microtubule integrity responses and Top2 itself. We found that β-lap creates toxicity in yeast solely by a massive reactive oxygen species production and that neither β-lap nor some of its chemical derivatives have any effect on Top2. This is supported by the fact that, under β-lap treatment, reactive oxygen species scavengers protect cells, aerobic conditions increase toxicity, and the deletion of Yap1, the main transcription factor in oxidative response, worsens the response. We also show that cells suffer from an apoptotic/necrotic phenotype. Altogether, we conclude that the toxicity of β-lap in yeast is due to the massive production of oxidative stress that ultimately kills the cells or induces apoptotic pathways.