Checkpoint kinase 2 (Chk 2) is involved in maintaining cellular genomic integrity by regulating cell entry into mitosis at the G2/M phase of the cell cycle. Specifically, it is involved in the ATM directed signaling pathway where it phosphorylates many substrates including p53, Brca 1, Cdc25A, and Cdc25C in response to DNA damage. Protein tyrosine phosphatases (PTPs) are key components of certain survival pathways and are responsible for their inactivation. Inappropriate cell survival responses, by overriding cell cycle checkpoints, may potentially produce death resistant cells and propagate DNA damage. Certain forms of hexavalent chromium [Cr(VI)] are human respiratory carcinogens and genotoxins. We have previously shown that a broad range PTP inhibitor, sodium orthovanadate (SOV) abrogates Cr (VI)-induced growth arrest and bypasses the G2/M checkpoint, coincident with AKT activation. We have also shown that Cr (VI) activates ATM and induces Chk 2 phosphorylation. Therefore, our aim was to examine the effect of PTP inhibition by SOV on Chk 2 cellular localization. Diploid human lung fibroblasts (HLF) were seeded in 8-well chamber slides at a concentration of 104 cells/well and were treated with 0-6 μM Cr (VI) in the presence and absence of 10 μM SOV for 1, 4, and 24 h. The cells were then incubated with a primary antibody to Chk 2 followed by an anti-rabbit secondary antibody conjugated to the Alexa 488 fluorochrome. The cells were then visualized by flourescence microscopy. In the absence of Cr (VI) and SOV, Chk 2 exhibited a distinct cytosolic localization. Cr (VI) induced a dose dependent increase in Chk 2 nuclear localization, which was partially abrogated in the presence of SOV. The relationship of Chk 2 localization to its activation is presently unclear. Our results indicate the ability of PTP inhibition, and potentially AKT activation, to interfere with checkpoint signaling upstream of cell cycle checkpoints. The potential override of DNA damage response signaling by survival pathways may lead to genomic instability and neoplastic transformation. Supported by NIH grants CA107972 to SC, and ES05304 and ES09961 to SRP.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]