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Ribonucleotide reductase plays a central role in cell proliferation by supplying deoxyribonucleotide precursors for DNA synthesis and repair. The holoenzyme is a protein tetramer that features two large (hRRM1) and two small (hRRM2 or p53R2) subunits. The small subunit contains a di-iron cluster/tyrosyl radical cofactor that is essential for enzyme activity. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a new, potent ribonucleotide reductase inhibitor currently in phase II clinical trials for cancer chemotherapy. Ferric chloride readily reacts with Triapine to form an Fe(III)-(3-AP) complex, which is reduced to Fe(II)-(3-AP) by DTT. Spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide prove that Fe(II)-(3-AP) reduces O 2 to give oxygen reactive species (ROS). In vitro activity assays show that Fe(II)-(3-AP) is a much more potent inhibitor of hRRM2/hRRM1 and p53R2/hRRM1 than Triapine. Electron paramagnetic resonance measurements on frozen solutions of hRRM2 and p53R2 show that their tyrosyl radicals are completely quenched by incubation with Fe(II)-(3-AP). However, the enzyme activity is maintained in protein samples supplemented with catalase alone or in combination with superoxide dismutase. Furthermore, catalase alone or in combination with superoxide dismutase markedly decreases the antiproliferative effect of Triapine in cytotoxicity assays. These results indicate that Triapineinduced inhibition of ribonucleotide reductase is caused by ROS. We suggest that ROS may ultimately be responsible for the pharmacologic effects of Triapine in vivo. [Mol Cancer Ther 2006;5(3):586 -92]

Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes beta, gamma, and delta). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or beta gamma subunits of heterotrimeric GTP binding (G) proteins. Although the PH domains of PLC-delta and PLC-gamma have been studied, the comparable domains of the beta isotypes have not. Here, we have measured the affinities of the isolated PH domains of PLC-beta 1 and -beta 2 (PH-beta 1 and PH-beta 2, respectively) for lipid bilayers and G-beta gamma subunits. Like the intact enzymes, these PH domains bind to membrane surfaces composed of zwitterionic phosphatidylcholine with moderate affinity. Inclusion of the anionic lipid phosphatidylserine or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and inclusion of G-beta gamma subunits had little affect on their membrane affinity. In contrast, binding of PLC-delta 1 or its PH domain was highly dependent on PI(4,5)P2. We also determined whether these domains laterally associate with G-beta gamma subunits bound to membrane surfaces using fluorescence resonance energy transfer. Affinities for G-beta gamma were in the following order: PH-beta 2 >/= PH-beta 1 > PH-delta 1; the affinities of the native enzyme were as follows: PLC-beta 2 >> PLC-delta 1 > PLC-beta 1. Thus, the PH domain of PLC-beta 1 interacts with G-beta gamma in isolation, but not in the context of the native enzyme. By contrast, docking of the PH domain of PLC-beta2 with G-beta gamma is comparable to that of the full-length protein and may play a key role in G-beta gamma recognition.

Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) family, encodes a transmembrane tyrosine kinase receptor in Her-2-positive tumors. Accumulating evidences demonstrate that signaling networks activated by EGFR and transcription factor NFjB are associated with cell response to ionizing radiation (IR). The present study shows that overexpression of ErbB2 enhanced NF-jB activation induced by IR in human breast carcinoma MCF-7 cells transfected with ErbB2 genes (MCF-7/ErbB2). Stable transfection of dominantnegative mutant IjB (MCF-7/ErbB2/mIjB) or treatment with anti-ErbB2 antibody, Herceptin, inhibited NF-jB activation and radiosensitized MCF-7/ErbB2 cells. Consistent with NF-jB regulation, basal and IR-induced Akt, a kinase downstream of ErbB2, was activated in MCF-7/ ErbB2 cells and inhibited by Herceptin. To identify specific genes affected by ErbB2-mediated NF-jB activation, a group of IR-responsive elements Cyclin B1, Cyclin D1, Bcl-2, Bcl/XL, BAD and BAX were evaluated. Basal levels of prosurvival elements Cyclin B1, Cyclin D1, Bcl-2 and Bcl/XL but not apoptotic BAD and BAX were upregulated in MCF-7/ErbB2 cells with striking enhancements in Bcl-2 and Bcl/XL. IR further induced Cyclin B1 and Cyclin D1 expression that was reduced by Herceptin. Bcl-2 kept a high steady level after Herceptin þ IR treatment and, in contrast to control MCF-7/Vector cells, Bcl/XL was inhibited in MCF-7/ErbB2 cells by Herceptin þ IR treatment. However, all four prosurvival proteins were downregulated by inhibition of NF-jB in MCF-7/ErbB2/mIjB cells. These results thus provide evidence suggesting that overexpression of ErbB2 is able to enhance NF-jB response to IR, and that a specific prosurvival network downstream of NF-jB is triggered by treatments using anti-ErbB2 antibody combined with radiation.

Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the ␤␥ subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-␤ 2 (PLC-␤ 2 ), which is regulated by G␤␥, replaces the PH domain of PLC-␦ 1 which binds to, but is not regulated by, G␤␥. We found that attachment of the PH domain of PLC-␤ 2 onto PLC-␦ 1 not only causes the membrane-binding properties of PLC-␦ 1 to become similar to those of PLC-␤ 2 , but also results in a G␤␥-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.Pleckstrin homology (PH) 1 domains are ϳ100 amino acid structural modules which, despite low amino acid sequence homology (i.e. only one conserved residue), appear to have the same overall spatial topology of a barrel-like structure formed from seven ␤-sheets with one side closed off by a C-terminal ␣-helix (for review, see Ref. 1). PH domains have been identified in over 100 diverse proteins involved in cell signaling and/or membrane/cytoskeletal interactions. While it is generally agreed that PH domains help tether their host proteins to membrane surfaces, some PH domains, such as that of the ␤-adrenergic receptor kinase (2, 3), have also been implicated as binding sites for G␤␥ subunits.All mammalian inositol-specific phospholipase C (PLC) enzymes contain a PH domain at their N terminus. PLCs are soluble enzymes that catalyze the hydrolysis of a minor component in membranes, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), releasing the two second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (for review, see Refs. 4 and 5). These enzymes have a multidomain structure (Fig. 1) containing, besides the N-terminal PH domain, four elongation factor (EF) hands, a catalytic X-Y domain with an insertion of varying length, and a C2 domain (6). There are three known families of mammalian PLCs (␤, ␥, and ␦), each with a distinct set of protein regulators. PLC-␤ isoenzymes are activated by ␣ q and ␤␥ subunits of heterotrimeric GTP binding proteins (G proteins). PLC-␥ enzymes are regulated by receptor tyrosine kinases. Protein regulators of PLC-␦ enzymes are unknown although RhoGAP and transglutaminase have been implicated (7,8).PLCs must associate with membranes during catalysis, and their PH domain plays a key role in membrane attachment. The PH domain of PLC-␥ binds specifically to membranes containing PI(3,4,5)P 3 (9) and the PH domain of PLC-␦ binds strongly to membranes containing PI(4,5)P 2 (10, 11). In contrast, the PH domain of PLC-␤ binds strongly and nonspecifically to membranes which helps to promote its lateral association with G protein subunits, and it has been found that the isolated PH domain of PLC-␤ 2 binds to G-␤␥ on membranes with a similar strong affinity as that o...

Although several signaling pathways have been suggested to be involved in the cellular response to ionizing radiation, the molecular basis of tumor resistance to radiation remains elusive. We have developed a unique model system based upon the MCF-7 human breast cancer cell line that became resistant to radiation treatment (MCF+FIR30) after exposure to chronic ionizing radiation. By proteomics analysis, we found that peroxiredoxin II (PrxII), a member of a family of peroxidases, is up-regulated in the radiation-derived MCF+FIR3 cells but not in the MCF+FIS4 cells that are relatively sensitive to radiation. Both MCF+FIR3 and MCF+FIS4 cell lines are from MCF+FIR30 populations. Furthermore, the resistance to ionizing radiation can be partially reversed by silencing the expression of PrxII by PrxII/small interfering RNA treatment of MCF+FIR3 resistant cells, suggesting that PrxII is not the sole factor responsible for the resistant phenotype. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing MCF+FIS4 cells when compared with vector control cells. The upregulation of the PrxII protein in radioresistant cancer cells suggested that human peroxiredoxin plays an important role in eliminating the generation of reactive oxygen species by ionizing radiation. The present finding, together with the observation that PrxII is also up-regulated in response to ionizing radiation in other cell systems, strengthens the hypothesis that the PrxII antioxidant protein is involved in the cellular response to ionizing radiation and functions to reduce the intracellular reactive oxygen species levels, resulting in increased resistance of breast cancer cells to ionizing radiation. (Cancer Res 2005; 65(22): 10338-46)