Inhibition of constitutively activated nuclear factor-kappaB induces reactive oxygen species- and iron-dependent cell death in cutaneous T-cell lymphoma.

Aberrant signaling of the nuclear facotr (NF-kappaB) pathway has been identified as a mediator of survival and apoptosis resistance in leukemias and lymphomas. Here, we report that cell death of cutaneous T-cell lymphoma cell lines induced by inhibition of the NF-kappaB pathway is independent of caspases or classic death receptors. We found that free intracellular iron and reactive oxygen species (ROS) are the main mediators of this cell death. Antioxidants such as N-Acetyl-l-cysteine and glutathione or the iron chelator desferrioxamine effectively block cell death in cutaneous T-cell lymphoma cell lines or primary T cells from Sézary patients. We show that inhibition of constitutively active NF-kappaB causes down-regulation of ferritin heavy chain (FHC) that leads to an increase of free intracellular iron, which, in turn, induces massive generation of ROS. Furthermore, direct down-regulation of FHC by siRNA caused a ROS-dependent cell death. Finally, high concentrations of ROS induce cell death of malignant T cells. In contrast, T cells isolated from healthy donors do not display down-regulation of FHC and, therefore, do not show an increase in iron and cell death upon NF-kappaB inhibition. In addition, in a murine T-cell lymphoma model, we show that inhibition of NF-kappaB and subsequent down-regulation of FHC significantly delays tumor growth in vivo. Thus, our results promote FHC as a potential target for effective therapy in lymphomas with aberrant NF-kappaB signaling.

Immunoscreening of a cutaneous T-cell lymphoma library for plasma membrane proteins.

Cutaneous T-cell lymphomas (CTCL) belong to non-Hodgkin lymphomas, which are primarily manifested in the skin and mostly exhibit a T-helper memory phenotype. Mycosis fungoides (MF) and the leukemic variant Sézary syndrome (SzS) are the most common forms of CTCL. The aim of this study was to identify CTCL surface proteins with a tumor specific expression profile. A plasma membrane enriched fraction of the CTCL cell line HuT78 was used for immunization of two rabbits. Subsequently, a CTCL cDNA phage library was screened by a new variant of the SEREX method (serological identification of antigens by recombinant expression cloning) using the polyspecific rabbit antisera instead of patients' sera. Isolated reactive transfectants were sequenced and 42 different genes identified including four known plasma membrane proteins: Ligatin, HLA-A, integrin alpha4 and MT5-MMP. The level of transcripts of the matrix metalloproteinase MT5-MMP was diminished in MF tumor specimens. MT5-MMP normally occurs in several different protein variants. Western blot analysis revealed that activated MT5-MMPs were reduced in tumor specimens, whereas the amounts of most of the inactivated variants were unchanged. The amount of mRNA coding for the adhesion protein integrin alpha4 was not altered in tumor specimens in comparison to controls when analyzed by quantitative real-time PCR analysis. Ku86, known to be predominantly located in the nucleus and cytosol, was frequently detected during the SEREX screening. Western blot analysis revealed higher protein amounts of Ku86 in HuT78 than in control cells. In addition, we could show, that Ku86 can also be detected in lipid rafts of CTCL cells as it has been described for other tumor types. Thus, Ku86 might be involved in homo- and heterotypic adhesion steps of CTCL tumor cells and might protect these cells against apoptosis triggered by irradiation as it was suggested for multiple myeloma cells. The design of this study enabled screening for all proteins on the plasma membrane, irrespectively of whether these are directly anchored within the membrane or associated with other membrane proteins. Further analysis will unravel whether the list of identified proteins harbors candidates, which might be accessible for antibodies from outside the cell.