geng Li

Modulation of thermal induction of hsp70 expression by Ku autoantigen or its individual subunits.

Previously, we proposed a dual control mechanism for the regulation of the heat shock response in mammalian cells: a positive control mediated by the heat shock transcription factor HSF1 and a negative control mediated by the constitutive heat shock element-binding factor (CHBF). To study the physiological role of CHBF in the regulation of heat shock response, we purified CHBF to apparent homogeneity and showed it to be identical to the Ku autoantigen, a heterodimer consisting of 70-kDa (Ku-70) and 86-kDa (Ku-80) polypeptides. To study further the functional significance of Ku/CHBF in the cellular response to heat shock, we established rodent cell lines that stably and constitutively overexpressed one or both subunits of the human Ku protein, and examined the thermal induction of hsp70 and other heat shock proteins in these Ku-overexpressing ing cells. We show that expression of the human Ku-70 and Ku-80 subunits jointly or of the Ku-70 subunit alone specifically inhibits heat-induced hsp70 expression. Conversely, expression of human Ku-80 alone does not have this effect. Thermal induction of other heat shock proteins in all of the Ku-overexpressing cell lines appears not to be significantly affected, nor is the state of phosphorylation or the DNA-binding ability of HSF1 affected. These findings support a model in which hsp70 expression is controlled by a second regulatory factor in addition to the positive activation of HSF1. The Ku protein, specifically the Ku-70 subunit, is involved in the regulation of hsp70 gene expression.

Adenovirus-Mediated Expression of a Dominant Negative Ku70 Fragment Radiosensitizes Human Tumor Cells under Aerobic and Hypoxic Conditions

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 end-binding 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 gamma-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.

Stable Expression of Human HSP70 Gene in Rodent Cells Confers Thermal Resistance

When exposed to a non lethal heat shock, a variety of organisms and cell lines acquire a transient resistance to one or more subsequent exposures at elevated temperatures (Gener and Schneider, 1975; Henle and Leeper, 1976). This phenomenon has been termed thermotolerance (Henle and Dethlefsen, 1978). The mechanism for thermotolerance is not well understood. In mammalian systems, several studies suggest that heat shock proteins (HSPs) may be involved in the development of thermotolerance (Lindquist and Craig, 1988; Landry etal., 1982; Li and Werb, 1982; Subjeck etal., 1982). HSP70, a major heat shock protein, is synthesized by cells of many organisms in response to thermal or other environmental stresses (Lindquist and Craig, 1988; Landry etal., 1982; Li and Werb, 1982; Subjeck etal., 1982; Li, 1985;Lasglo and Li, 1985; Hahn and Li, 1990). It has been hypothesized that the transient induction of HSP70 may enable cells to recover from previous thermal stress, and/or to provide cells a transient degree of protection to subsequent heat challenge. A corollary of this hypothesis is that the overexpression of HSP70 in cells confers permanent heat resistance. We have initiated direct tests of the hypothesis that HSP70 protects cells from thermal stress by microinjecting human HSP70 into CHO cells or introducing a cloned human HSP70 gene into rat fibroblasts, and then evaluating the effect of human HSP70 gene expression on transient thermotolerance and permanent thermal resistance.