No abstract

Ku70 is one component of a protein complex, the Ku70/Ku80 heterodimer, which binds to DNA double-strand breaks and activates DNA-dependent protein kinase (DNA-PK), leading to DNA damage repair. Our previous work has confirmed that Ku70 is important for DNA damage repair in that Ku70 deficiency compromises the ability of cells to repair DNA double-strand breaks, increases the radiosensitivity of cells, and enhances radiation-induced apoptosis. Because of the radioresistance of some human cancers, particularly glioblastoma, we examined the use of a radio-gene therapy paradigm to sensitize cells to ionizing radiation. Based on the analysis of the structure-function of Ku70 and the crystal structure of Ku70/Ku80 heterodimer, we designed and identified a candidate dominant negative fragment involving an NH 2 -terminal deletion, and designated it as DNKu70. We generated this mutant construct, stably overexpressed it in Rat-1 cells, and showed that it has a dominant negative effect (i.e., DNKu70 overexpression results in decreased Ku-DNA endbinding activity, and increases radiosensitivity). We then constructed and generated recombinant replication-defective adenovirus, with DNKu70 controlled by the cytomegalovirus promoter, and infected human glioma U-87 MG cells and human colorectal tumor HCT-8 cells. We show that the infected cells significantly express DNKu70 and are greatly radiosensitized under both aerobic and hypoxic conditions. The functional ramification of DNKu70 was further shown in vivo: expression of DNKu70 inhibits radiation-induced DNA-PK catalytic subunit autophosphorylation and prolongs the persistence of ;-H2AX foci. If radiation-resistant tumor cells could be sensitized by down-regulating the cellular level/ activity of Ku/DNA-PK, this approach could be evaluated as an adjuvant to radiation therapy. [Cancer Res 2007;67(2):634-42]

The role of mammalian 70-kDa heat shock protein (hsp7O) in regulating cellular response to heat shock was examined by using three closely related rat cells: control Rat-1 cells, thermotolerant Rat-i (TT Rat-1) cells, and heatresistant M21 cells, a derivative of Rat-1 cells that constitutively overexpress human hsp7O. In all these cells, after a prescribed heat shock, the level of the phosphorylated form of heat shock transcription factor HSF1 and that of HSF1 The heat shock protein hsp70 appears to protect cells from thermal damage (1-3). There is also evidence that hsp7O regulates its own synthesis (4, 5). At the transcriptional level, hsp70 may be involved in modulation of the heat shock transcription factor (HSF) (6-9). HSF is a sequence-specific DNA binding protein that, in its trimeric form, binds tightly to multicopies of a highly conserved sequence motif in the promoter region of heat shock genes, termed the heat shock element (HSE).In Saccharomyces cerevisiae, HSF is a trimer bound to HSE, even in unstressed cells. Upon heat shock, HSF undergoes phosphorylation, concurrent with the transcriptional activation of heat shock genes (10,11 (8,14,(23)(24)(25). In unstressed cells, HSF1 is monomeric and non-DNA binding, probably through its interaction with hsp70. Upon heat shock, the increased level of thermally denatured or malfolded proteins may titrate the available hsp7O, setting free the hsp70-bound HSF1. The released HSF1 then assembles into a trimer, binds to HSE, and becomes phosphorylated. Transcriptional activation of heat shock genes then increases the level of hsp70. When sufficient hsp70 is made, it interacts again with activated HSF1 to form a HSF1-hsp70 complex. Finally, HSF1 dissociates from the DNA and is eventually converted to the inactive, non-DNAbinding HSF1 monomers (25). Consistent with this idea, data suggesting interaction between hsp70 and HSE-bound HSF1 have been reported (8).We have previously shown that rat cell lines that stably and constitutively express a cloned human hsp7O gene are heat resistant and have enhanced recovery of transcriptional and translational activities after heat shock (3,26). This study shows that hsp7O modulates the DNA-binding activity and phosphorylation of HSF1 after heat shock. During post-heat shock recovery, the kinetics of dephosphorylation of HSF1 and loss of the DNA-binding ability of HSF1 appear to be faster in cells overexpressing hsp7O. Treatment of cells with a phosphatase inhibitor, okadaic acid, prolongs both the phosphorylation and the DNA-binding ability of HSF1 in thermalsensitive Rat-1 cells but not in cells overexpressing human hsp70. These results support our hypothesis that hsp70 plays a role in recovery of cells after a thermal insult. MATERIALS AND METHODSCell Cultures and Heat Shock Treatment. Rat fibroblasts (Rat-1) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The transfected Rat-1 cells, constitutively expressing the intact human hsp70 (M21 and HR24) or its deletion derivatives ...

Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSF1. We purified the CHBF to apparent homogeneity from HeLa cells using column chromatographic techniques including an HSE oligonucleotide affinity column. The purified CHBF consists of two polypeptides with apparent molecular masses of 70 and 86 kDa. Immunoblot and gel mobility shift analysis verify that CHBF is identical or closely related to the Ku autoantigen. The DNA binding characteristics of CHBF to double-stranded or single-stranded DNA are similar to that of Ku autoantigen. In gel mobility shift analysis using purified CHBF and recombinant human HSF1, CHBF competes with HSF1 for the binding of DNA sequences containing HSEs in vitro. Furthermore, when Rat-1 cells were co-transfected with human Ku expression vectors and the hsp70-promoter-driven luciferase reporter gene, thermal induction of luciferase is significantly suppressed relative to cells transfected with only the hsp70-luciferase construct. These data suggest a role of CHBF (or Ku protein) in the regulation of heat response in vivo.

The constitutive HSP70 purified from CHO cells, which indicated a single band in SDS-polyacrylamide gel electrophoresis, showed multiple bands in native-polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS-polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1-10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [gamma-32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins.