PICOT is a Positive Regulator of DNA Damage Response Pathways in T lymphocytes

Introduction

PICOT, also termed glutaredoxin-3 (GLRX3), was discovered as a protein kinase C (PKC) theta-binding partner in human T lymphocytes, but in contrast to PKC-theta, which is selectively expressed in certain hematopoietic cells and smooth muscle, PICOT is a ubiquitously expressed protein. It resides predominantly in the cytosol, but upon exposure of T cells to hydrogen peroxide, can also translocate to the nucleus. Immunohistochemical studies demonstrated high expression levels of PICOT in proliferating cells in inflamed human lymph nodes, as well as in Hodgkin’s lymphoma Reed Sternberg cells and anaplastic large cell lymphoma. PICOT plays a critical role in embryogenesis, and PICOT-deficient mice die in utero approximately at embryonic day 12.5. In cardiac muscles, PICOT was found to attenuate pressure overload-induced hypertrophy by disrupting the calcineurin–NFAT signaling pathway. Here, we tested the potential role of PICOT in cell responses to genotoxic stress.

Materials and methods

Silencing of PICOT in Jurkat T cells was obtained by constitutive expression of PICOT shRNA and selection of clones expressing reduced levels of PICOT. Cell survival analysis was carried out using the MTT assay, intracellular reactive oxygen species (ROS) levels were measured using the oxidant-sensing fluorescent probe, DCFH-DA and DNA damage-induced phosphorylation of H2AX and upstream kinases following genotoxic stress was tested in whole cell lysates, gel fractionation and immunoblot, using phosphoprotein-specific antibodies.

Results and discussion

Silencing of PICOT in Jurkat T cells resulted in a retarded in vitro growth rate. Exposure of these cells to genotoxic drugs resulted in increased production of intracellular reactive oxygen species, augmented fragmentation of DNA and reduced cell viability. In addition, phosphorylation of the DNA damage sensor histone protein, H2AX, following exposure to genotoxic agents, was downregulated in Jurkat.1A, which expresses reduced levels of PICOT. A similar reduction in the phosphorylation and activation of the H2AX upstream kinases, ATR and ATM was observed in Jurkat.1A cells. This was accompanied by reduced phosphorylation and activation of Chk1 and Chk2 Ser/Thr-kinases which regulate DNA repair and cell cycle arrest and prevent damaged cells from progressing through the cell cycle.

Conclusions

Silencing of PICOT increases the susceptibility of Jurkat T cells to genotoxic stress suggesting a positive role for PICOT in the activation of DNA damage response mechanisms.