Restoration of Peroxiredoxins and Catalase Is Closely Correlated with the Level of Philadelphia Chromosome during Imatinib Therapy in CML.

Background: Several investigators have recently shown that activated growth factor receptors increase the relative levels of intracellular ROS and that bcr/abl kinase induces the production of ROS in hematopoietic cells. In addition, bcr-abl kinase induces self-mutagenesis via ROS to encode IM resistance. Meanwhile, two members of the peroxiredoxin family, Prx 1 or Prx 2, efficiently lowered the intracellular level of H₂O₂ and blocked the induction of apoptosis by ceramide, suggesting that the Prx enzymes contribute to intracellular signaling by removing H₂O₂. In this study, we investigated the changse in the levels of H₂O₂-removing enzymes like Prxs, glutathione peroxidase 1 (Gpx1), and catalase during Imatinib (IM) therapy in CML.

Methods: Mononuclear cells(MNC) were isolated by standard Ficoll-Hypaque from the bone marrow aspiration at the time of diagnosis and during treatment with IM (Gleevec, Novartis, East Hanover, NJ). Standard cytogenetics analysis and RT-PCR for Philadelphia chromosomes were performed. For immunoblot analysis of antioxidant enzymes, cell lysates were fractionated by SDS-PAGE, and the separated proteins were transferred electrophoretically to a nitrocellulose membrane (Protran, Germany) and were probed with antibodies specific for Prx I, II, VI, GPx1, or catalase (AbFrontier, South Korea).

Results: Samples from the diagnosis CML patients showed significantly decreased levels of Prx I, Prx II, Prx IV, and Gpx I, but also revealed an increased level of catalase. Especially prominent was the diminished ratio of Prx II to catalase in CML patients when compared with those of normal individuals. As the level of Philadelphia chromosomes decreased to that of normal individuals as the result of IM treatment, the expression levels of Prx(s) (P=0.018) and catalase (P=0.009) were restored to the levels of normal individuals.

Conclusions: Decreased Prx 2 and elevated catalase levels at the time of diagnosis are closely correlated with the elevated bcr/abl kinase level in CML. The aberrant expression of those antioxidant enzymes returned to normal with IM treatment. Now, we will attempt to develop these method(s) to assess the changes of Prx(s) and catalase on the single cell level using immunohistochemistry with monoclonal Ab(s). Understanding the molecular mechanisms of changes on antioxidant might be a potent tool in developing more effective new drugs in CML.