No abstract

We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor B (NF-B) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90 rsk signaling cascade in a p53-independent fashion. In turn, p90 rsk interacts with the IB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-B activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-B pathway that opposes the apoptotic response following DNA damage.

To survive winters, woody perennials of temperate zones must enter into endodormancy. Resumption of spring growth requires sufficient exposure to low temperature (chill units, CUs) in winter (chilling requirement), which also plays a role in the development of cold hardiness (cold acclimation). Physiological studies on dormancy breaking have focused on identifying markers, such as appearance or disappearance of proteins in response to varying degrees of chill unit accumulation. However, whether these changes are associated with dormancy transitions or cold acclimation is not clear. In the present study, greenhouse‐grown blueberry (Vaccinium section Cyanococcus) plants were used to address this question. Three blueberry cultivars, Bluecrop, Tifblue, and Gulfcoast having chilling requirement of approximately 1 200, 900 and 600 CUs, respectively, were first exposed to 4°C for long enough to provide chill units equivalent to one‐half of their respective chilling requirement. This treatment was expected to result in cold acclimation. A fraction of plants was then subjected to a 15/12°C (light/dark) regime for 2 weeks, a treatment expected to be “dormancy‐neutral” but cause deacclimation. Before and after each treatment, cold hardiness and dormancy status of floral buds were determined; proteins were extracted from the buds collected on the same sampling date, and separated by one‐dimensional SDS‐PAGE. Dehydrin‐like proteins were identified by immunoblotting, using anti‐dehydrin antiserum. Results indicate that the chilling treatment resulted in cold acclimation as indicated by increased bud hardiness in all three cultivars. Data also indicate a distinct accumulation of three dehydrin‐like proteins of 65, 60, and 14 kDa during cold acclimation. The cold hardiness and levels of dehydrin proteins decreased during the exposure to 15/12°C for 2 weeks. Results also confirmed that this treatment had no negative effect on chill unit accumulation. Densitometric scans of protein gels indicated a close association between the abundance of dehydrins and degree of cold hardiness in these cultivars. In addition, levels of the dehydrin proteins and cold hardiness remained about the same between 100% and >100% satisfaction of chilling requirement. These results suggest that changes in dehydrin expression are more closely associated with cold hardiness than with dormancy transitions.

NF-B regulates liver cell death during development, regeneration, and neoplastic transformation. For example, we showed that oncogenic Ras-or Raf-mediated transformation of rat liver epithelial cells (RLEs) led to altered NF-B regulation through IKK complex activation, which rendered these cells more resistant to TGF-␤1-induced apoptosis. Thus, based on these findings, we sought to determine whether NF-B could also be in- NF-B was first identified as a nuclear factor specific to B cells that bound to the B site of the light chain gene enhancer. 1 NF-B is now known to be a family of dimeric transcription factors with subunits that contain an amino terminal stretch of approximately 300 amino acids termed the Rel homology domain (RHD) because it shares homology with the v-Rel oncoprotein. 2 The RHD is involved in DNA binding and dimerization of the various subunits. 3 Classical NF-B is composed of a p50 (NF-B1) and a p65 (RelA) subunit, 4,5 and is ubiquitously expressed. In non-B cells, NF-B is sequestered in the cytoplasm by specific inhibitory proteins termed IBs, 6,7 of which IB-␣ is the most well characterized.NF-B can be activated by many inducers including proinflammatory cytokines, phorbol esters, bacterial endotoxins, and oxidative stress. Recently the Akt/protein kinase B (PKB) kinase has been shown to play a role in this signaling in response to TNF-␣ or PDGF signalling. 8,9 The most well-characterized pathway leading to NF-B activation involves the IKK complex, which includes 2 catalytic kinase subunits IKK-1 (IKK-␣) and IKK-2 (IKK-␤), 10-13 and a third regulatory subunit termed IKK-␥ or NEMO. 14,15 Knock-out studies have underscored the nonoverlapping functions of IKK-1 and IKK-2 during vertebrate limb development and immune response, respectively. 16 Following signal-induced IKK activation, sequestered NF-B/IB-␣ is recruited to the IKK complex where the IB-␣ is phosphorylated on serine residues 32 and 36. As a consequence of phosphorylation, IB-␣ protein is rapidly ubiquitinated and degraded through the proteasome pathway, 17-19 allowing for migration of NF-B to the nucleus.Recently, NF-B/Rel factors have been strongly implicated in the regulation of malignant cell growth, survival, and transformed phenotype. 20,21 As we first showed, TGF-␤1-mediated cell killing of murine B cell lymphomas is mediated via inhibition of NF-B, and direct repression of this activity can lead to death of these cells. 22,23 Furthermore, we found that human and rodent breast cancer cells are typified by aberrant NF-B/Rel expression, and inhibition of this activity can similarly lead to cell death. 24 To date, constitutive activation of NF-B has been observed in a variety of tumors including Hodgkin's and T-cell lymphomas, 25,26 melanomas, 27 breast and pancreatic adenocarcinomas, 24,[28][29][30] and primary adult Tcell leukemias. 31 Inhibition of NF-B activity potentiated cell killing of human breast cancer and fibrosarcoma cell lines by TNF-␣, ionizing radiation, and daunorubicin [32][33][34][35] and led to sensitization of...

N uclear factor B (NF-B) was first identified as a nuclear factor specific to B cells bound to the B site of the light chain gene enhancer. 1 Classic NF-B, which is composed of an NF-B1 (p50) and RelA (p65) subunit, belongs to a family of dimeric transcription factors typified by an amino terminal stretch of approximately 300 amino acids, named the Rel homology domain, involved in DNA binding and dimerization of the various subunits. 2 In resting non-B cells, NF-B is sequestered in the cytoplasm through association with a family of inhibitory molecules, of which inhibitor B (IB)-␣ is the best characterized. In response to proinflammatory cytokines, growth factors, or genotoxic stress, inducible phosphorylation of IB-␣ involves the IB kinase (IKK) complex, which is formed by the IKK-␣ and IKK-␤ catalytic subunits and by a scaffold subunit termed IKK-␥/NEMO. 3 Following phosphorylation by IKKs, IB-␣ is degraded through the proteasome pathway, thereby promoting nuclear translocation of NF-B, which then regulates genes involved in immune response, cell adhesion, cell cycle, transformation, and cell survival. 4 In addition, IB-␣ can be phosphorylated at the C-terminal PEST sequence of IB-␣ by the protein kinase CK2 (formerly casein kinase II). 5-8 CK2 is a ubiquitous tetrameric serine-threonine kinase composed of 2 ␣ and ␣Ј catalytic subunits of 43 and 38 kd, respectively, and 2 regulatory ␤ subunits. 9 CK2 has been implicated in the regulation of cell growth and survival of normal and neoAbbreviations: NF-B, nuclear factor B; IB, inhibitor B; IKK, inhibitor B kinase;