Abstract 1027: Thioredoxin-interacting protein-reactive oxygen species (TXNIP/ROS) relationship defines a stress response gene signature in patients with CLL

Abstract

Thioredoxin-interacting protein (TXNIP) binds and inactivates thioredoxin (TRX) leading to increased reactive oxygen species (ROS) production and apoptosis in many cell types. TXNIP expression has never been evaluated in CLL. Since it has been previously shown that patients with CLL have high levels of ROS compared to normal individuals and that TRX has a protective function in CLL, our goal was to measure the levels of TXNIP and determine whether there is any correlation with ROS. Furthermore, we also studied whether this correlation was associated with any particular stress response gene signature. Venous blood samples were obtained from consecutive patients with definite diagnosis of CLL during routine clinic visits over a 12-week period. Patients were selected independently from demographics, stage of disease or prior treatment. Seventeen patient samples were analyzed for this study, 8 under observation, 6 receiving fludarabine-based regimen and 3 receiving other regimens. Gene expression levels were measured by semiquantitative PCR and expressed as relative intensity. ROS was measured by DCFDA fluorescence and expressed as mean fluorescence. TRX in plasma was assessed by ELISA. Statistical analysis included student t-test (p values) and Pearson's correlation, (ρ values), with ρ ≥ 0.7 considered high positive correlation and ρ ≤ −0.7 strong inverse correlation. TXNIP levels (avg. 0.82 ± 0.18, range 0.45-1.15) and ROS (avg. 7967 ± 2515, range 2340-12340) were found to be highly positively correlated (ρ = 0.92). Neither TRX message nor protein in serum correlated with TXNIP or ROS. TXNIP expression was generally higher in our observation group than treated (0.89 ± 0.18 vs 0.76 ± 0.15) although not statistically significant. Four samples with highest levels of TXNIP (3 obs, 1 treat; avg TXNIP 1.06 ± 0.06) and 4 with lowest TXNIP (3 treat, 1 obs; avg TXNIP 0.59 ± 0.1) were further evaluated by using a stress response PCR assay. We found 50 genes with differing expression pattern between the groups. We chose BAX, GSK3-B and Bcxl to further analyze in all samples. Whereas BAX, Gsk3-B and Bclx all showed an inverse correlation with TXNIP expression (BAX: ρ = −0.67, GSK3-B ρ = −0.66, Bclx: ρ = −0.64), Bcl-2 showed no correlation. We then grouped these genes into treated vs observation for further analysis. BAX (0.45 ± 0.18 vs 0.26 ± 0.06, p < 0.05), GSK-3B (0.55 ± 0.17 vs 0.38 ± 0.17, p < 0.05) and Bclx gene expression levels (0.46 ± 0.21 vs 0.27 ± 0.05, p < 0.03) were all significantly higher in the treated vs observation group only. In conclusion, we have measured the expression of TXNIP in 17 CLL patients and found that TXNIP expression significantly correlates with ROS in the circulating CLL cells. By analyzing genes that may be associated with TXNIP levels in these cells we have identified BAX, GSK-3B and Bclx as genes of interest in a platform for further analysis in patients with CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1027.