The Level of Peroxiredoxin and Catalase On Single Cell Level Is Correlated with the Positivity of Ph Chromosome in the Bone Marrow of CML Patients During Imatinib Therapy.

: It has been proposed that treating cancer cells with vectors that cause the over expression of MnSOD and catalase could inhibit tumor cell growth by disrupting the redox signaling involved with cell division as well as, therapeutically, by providing protection for normal cells. These results support the hypotheses that increasing the dismutation of mitochondrial superoxide alters redox signaling in cancer cells that can result in growth suppression. Peroxiredoxins (Prxs) are a ubiquitous family of multifunctional antioxidant thioredoxin-dependent peroxidases that have roles in the reversible inactivation of PTPs and PTEN in cells stimulated by growth factors and protect cells against oxidative stress and modulate intracellular signaling cascades (Rhee et al, Cell Biology 17:183-189, 2005). In our study, we investigated the changes of the levels of H₂O₂-removing enzymes (ie Prxs, glutathione peroxidase 1 (Gpx1), and catalase) on a single cell during IM therapy in newly diagnosed CML.

Six paired CML samples (two blast crisis, one accelerated phase, three chronic phases) were analyzed in a study was approved by the institutional review board. Patients gave written informed consent according to institutional guidelines. 400 mg or 600 mg of imatinib was given to the chronic phase and accelerated/blast crisis samples respectively. Following encircling cells from marrow BM aspiration from patients or normal individuals with the Dako Pen (Dako, Denmark) on a dry slides, cells were incubated with blocking solution (10% FBS in PBS) for 30minutes to block non-specific staining. Then, cells were incubated with specific primary antibodies to antioxidant enzymes in 0.2% TritonX-100/10% FBS/PBS solution overnight (1:100). After washing with PBS, cells were incubated with Alexa-488 or 594-conjugated goat antibodies to mouse or rabbit IgG, respectively for an hour. Confocal fluorescence images of cells were obtained with an LSM510 microscope (Carl Zeiss, Tornwood, NJ).

Samples of newly diagnosed CML patients showed significantly decreased levels of Prx 2, but revealed increased level of catalase on single cell level. The immunohistochemical expression was well correlated with western blot results. As the level of Philadelphia chromosomes decreased with IM treatment, the expression levels of Prx II and catalase were restored to the levels of normal individuals. Using our technique, we are able to quantify the changes of Prx and catalase on each myeloid, erythroid and megakaryocytic lineages at the single cell level.

Decreased Prx 2 and elevated catalase levels at the time of diagnosis are closely correlated with the elevated bcr/abl kinase level in CML at the each single cell level. The aberrant expression of those antioxidant enzymes were back to the level of normal individuals after IM treatment. This method might be accurate and convenient to assess the changes of the level of Prx and Catalase especially in the situation of cytopenia with IM treatment. Understanding the molecular mechanisms of changes on antioxidant might be potent tools to develop more effective new drugs in CML patients especially for Imatinib resistant patients.

No relevant conflicts of interest to declare.