DNA-dependent Protein Kinase Protects against Heat-induced Apoptosis*

Arsenio NuedaDagger , Farlyn Hudson, Nahid F. Mivechi, and William S. Dynan§

From the Institute of Molecular Medicine and Genetics, Program in Gene Regulation, Medical College of Georgia, Augusta, Georgia 30912

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Purified heat shock transcription factor 1 (HSF1) binds to both the regulatory and catalytic components of the DNA-dependent protein kinase (DNA-PK). This observation suggests that DNA-PK may have a physiological role in the heat shock response. To investigate this possibility, we performed a comparison of cell lines that were deficient in either the Ku protein or the DNA-PK catalytic subunit versus the same cell lines that had been rescued by the introduction of a functional gene. DNA-PK-negative cell lines were up to 10-fold more sensitive to heat-induced apoptosis than matched DNA-PK-positive cell lines. There may be a regulatory interaction between DNA-PK and HSF1 in vivo, because constitutive overexpression of HSF1 sensitized the DNA-PK-positive cells to heat but had no effect in DNA-PK-negative cells. The initial burst of hsp70 mRNA expression was similar in DNA-PK-negative and -positive cell lines, but the DNA-PK-negative cells showed an attenuated rate of mRNA synthesis at later times and, in some cases, lower heat shock protein expression. These findings provide evidence for an antiapoptotic function of DNA-PK that is experimentally separable from its mechanical role in DNA double strand break repair.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The DNA-dependent protein kinase (DNA-PK)1 is composed of a 470-kDa catalytic subunit (DNA-PKcs) and a 70/80-kDa heterodimeric regulatory component known as Ku protein. Ku protein binds avidly to DNA ends (1) and recruits DNA-PKcs to form an active complex (2-4). Mutations in DNA-PKcs or Ku protein have been described in mammals, flies, and yeast, and in each case, the mutant organism is deficient in double strand break repair, sensitive to ionizing radiation, or both (reviewed in Refs. 5 and 6). Binding of DNA-PK components to DNA is believed to be the first step in a repair pathway that involves a number of other gene products (reviewed in Ref. 7).

It is not clear if the ability of DNA-PK to protect against the cytotoxic effect of ionizing radiation is attributable solely to the mechanical role of the enzyme in DNA repair or whether DNA-PK also participates in antiapoptotic signaling. There have been several reports that DNA-PK physically interacts with signaling molecules, including poly(A)DP-ribose polymerase, Ikappa B, Vav, c-Abl, and certain transcription factors (8-12), but the physiological significance of these interactions has not yet been fully explored.

In the present study, we investigate the in vivo role of a previously described interaction between DNA-PK and the heat shock transcription factor, HSF1 (12, 13). These prior studies have shown that purified HSF1 binds to both the Ku protein and DNA-PKcs (13). The binding requires a phylogenetically conserved region of HSF1 that includes amino acids 203-280 and results in a stimulation of DNA-PK phosphorylation activity of up to 20-fold in an in vitro reaction (13). These observations suggest that DNA-PK may be involved in regulating some aspect of the heat shock response in vivo.

Other evidence also suggests an involvement of DNA-PK in the response to heat. Overexpression of human Ku70 in rat cells decreases expression of heat shock protein 70 (hsp70) (14, 15). It was originally proposed that this might reflect competition of HSF1 and Ku protein for binding to heat shock promoter elements (16), although this mechanism has not been supported by later studies (17). Even if the underlying mechanism is unclear, the finding that ectopic expression of a Ku protein subunit perturbs expression of a heat shock protein supports the idea that DNA-PK participates in the heat shock response.

It is also known that heat treatment potentiates the cytotoxic effect of ionizing radiation, a phenomenon called thermal radiosensitization (18-20). Interestingly, some cell lines deficient in Ku protein or DNA-PKcs do not show thermal radiosensitization (i.e. their intrinsically high sensitivity to radiation is not further increased by heat) (21, 22). This implicates DNA-PK in the thermal radiosensitization phenomenon. Ku protein may be intrinsically heat-labile (23, 24), in which case heat treatment may reduce double strand break repair. An alternative possibility is that binding of DNA-PK to HSF1 in heat-treated cells competes with its ability to interact with the DNA repair machinery.

In the present work, we show that DNA-PK protects against heat-induced apoptosis. Protection is abrogated by introduction of a constitutively expressed HSF1 gene. These studies provide evidence for an antiapoptotic function of DNA-PK that is separable from its role in DNA repair. We find that there are some differences in the time course of induction of hsp70 mRNA and protein in DNA-PK-positive and DNA-PK-negative cells. It is possible that a difference in the state of the transcriptional machinery accounts for the differential heat sensitivity. Alternatively, protection from apoptosis may occur by a nontranscriptional mechanism.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture, Clonogenic Survival Assays, and Generation of Stably Transfected Cell Lines-- For cell culture and clonogenic assays, HeLa, scva2, sc(8)-10, xrs-6cvec, xrs-6cKu80, and AA8 cells were grown at 37 °C with 5% CO2 in minimal essential medium (Life Technologies, Inc.), with 10% heat-inactivated fetal bovine serum, nonessential amino acids, and 50 µg/ml gentamycin. Medium for sc(8)-10, xrs-6cvec, and xrs-6cKu80 cells contained 400 µg/ml Geneticin. For clonogenic survival assays, cells were heat-treated as indicated in the figure legends and counted. Equal numbers of cells were plated and allowed to grow for 7 days. Colonies were fixed in 5% glutaraldehyde and stained with crystal violet, and colonies of more than 20 cells were counted.

For transfection, 107 cells were electroporated in 800 µl of minimal essential medium with 20 µg of expression plasmid and 5 µg of pBabe plasmid, which contains a puromycin selection marker (25). scva2 and sc(8)-10 cells were electroporated at 300 V, 1500 microfarads. xrs-6cKu80 and xrs-6cVec cells were electroporated at 300 V, 2500 microfarads. After transfection, cells were plated in minimal essential medium (without Geneticin). After 24 h, Geneticin (400 µg/ml) and puromycin (3 µg/ml for scva2 and sc(8)-10, 5 µg/ml for xrs-6cVec and xrs-6cKu80 cells) were added.

Detection of Apoptosis by Enzyme-linked Immunosorbent Assay and dUTP Labeling-- Apoptosis was quantitated by measuring release of histone-DNA fragments into the cytoplasm using a commercial assay (Roche Molecular Biochemicals). Briefly, cytoplasmic extracts corresponding to 103 cells were incubated for 2 h in microtiter wells that had been precoated with anti-histone mouse monoclonal antibody (clone H11-4). The wells were washed, and mouse anti-DNA peroxidase-conjugated monoclonal antibody (clone MCA-33) was added. Incubation was continued for 2 h, and the wells were developed with ABTS substrate. Signal was read as the difference between A405 and A492.

For immunofluorescence, cells were plated on glass coverslips and allowed to grow for 24 h and then heat-treated as indicated in the figure legends. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature, washed, and incubated for 2 min on ice with a solution containing 0.1% Triton X-100 and 0.1% sodium citrate. Apoptotic death was scored using the TUNEL (TdT-mediated dUTP nick-end labeling) method using fluorescein-dUTP (Roche Molecular Biochemicals) (26). The cells were washed and incubated for 30 min at room temperature with blocking solution containing 5% nonfat dry milk, 0.9% NaCl, 10 mM Tris-HCl, pH 7.2, 0.1% Tween 20, and 0.2% sodium azide. Cells were stained for 1 h each with 1 µg of primary and Texas Red-conjugated secondary antibodies as specified in the figure legends. Cells were washed in PBS containing 1 µg/ml diamidino 2-phenylindole dihydrochloride hydrate (DAPI) (Aldrich) and mounted using 5 µl of mounting medium containing 10% Mowiol (Hoechst), 25% glycerol in 200 mM Tris-HCl, pH 8.5.

Immunoblotting-- To obtain cell extracts, 5-10 × 106 cells were heat-treated as described in the figure legends, washed in PBS, scraped from the plate, suspended in 200 µl of PBS, and lysed by the addition of 200 µl of 2× SDS-polyacrylamide gel electrophoresis sample buffer. Equal quantities of protein from each extract were fractionated by SDS-polyacrylamide gel electrophoresis. The proteins were transferred to a polyvinylidene difluoride or nitrocellulose membrane by electroblotting in a buffer containing 25 mM Tris base, 192 mM glycine, and 20% methanol. The membrane was blocked in PBS containing 0.1% Triton X-100 and 5% nonfat dry milk and sequentially probed with the primary antibody for 2 h, washed with PBS containing 0.1% Triton X-100, probed with alkaline phosphatase-conjugated secondary antibody, and finally incubated for 5 min with enzyme-catalyzed fluorescence substrate (Vistra ECF Substrate, Amersham Pharmacia Biotech) or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Promega). The membrane was dried, and signal was quantitated with a Storm imaging system (Molecular Dynamics, Inc., Sunnyvale, CA).

Electrophoretic Mobility Shift Assay-- Electrophoretic mobility shift assay was performed as described previously (27). Briefly, 5 × 106 cells were heat-treated as indicated in the figure legends, washed with PBS, and lysed in 200 µl of buffer containing 10 mM Pipes, pH 7.9, 0.4 M NaCl, 0.1 mM EGTA, 0.5 mM dithiothreitol, 5% glycerol, and 0.5 mM phenylmethylsulfonyl fluoride. For binding, 20 µg (approximately 10 µl) of cell extract was mixed with 10 µg of tRNA, 1 µg of sheared Escherichia coli DNA, 10 µg of poly(dI-dC), and 1 ng of [alpha -32P]dCTP-labeled double-stranded oligonucleotide in a total volume of 25 µl. The sample was incubated for 15 min at 25 °C and analyzed by nondenaturing gel electrophoresis in 4.5% acrylamide. After electrophoresis, the gel was fixed in 7% acetic acid for 5 min and dried under vacuum, and complexes containing radiolabeled DNA were detected by PhosphorImager analysis.

Nuclear Run-on and Northern Blot Analysis of RNA Synthesis-- Total RNA was isolated from untreated or heat-treated HeLa, scva2, or sc(8)-10 cells by the guanidine isothiocyanate method, fractionated by formaldehyde-agarose electrophoresis, and transferred to a nitrocellulose membrane as described (28). The filter was probed with a cDNA corresponding to the complete sequence of human hsp70D (kindly provided by Dr. Richard I. Morimoto).

Nuclear run-on assays were performed as described (29) with modifications. Nuclei were prepared from 5 × 106 cells that were heat-treated as described in the figure legends. Nuclei that had been stored in 200 µl of glycerol storage buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40) were mixed with 200 µl of 2× reaction buffer (10 mM Tris- HCl, pH 8.0, 5 mM MgCl2, 0.3 M KCl, 10 mM dithiothreitol, 1 mM concentration each of ATP, GTP, and UTP), and 10 ml of [alpha -32P]CTP (10 µCi/ml) was added to begin the labeling of nascent RNA. The mixture was incubated for 30 min at 30 °C; the reaction was stopped with 540 µl of 0.5 M NaCl, 50 mM MgCl2, 2 mM CaCl2,10 mM Tris-Cl, pH 7.4; and 60 µl of DNase I (1 unit/ml, Promega) were added. A 180-µl aliquot of 5% SDS, 0.5 M Tris-Cl, pH 7.4, 0.125 M EDTA and a 20-µl aliquot of 20 mg/ml Proteinase K were added to the reactions, which were then incubated for 30 min at 42 °C. Samples were extracted with phenol/chloroform and precipitated with sodium acetate/ethanol for 20-30 min at -20 °C before centrifugation. The resulting pellet was resuspended in 225 µl of 20 mM Hepes, pH 7.5, 5 mM MgCl2, 1 mM CaCl2, and 25 µl of DNase I (1 unit/ml, Promega), and the mixture was incubated for 20 min at 37 °C. A 10-µl aliquot of 0.5 M EDTA, 30 µl of 10% SDS, and a 10-µl aliquot of 20 mg/ml Proteinase K were added, and the mixture was further incubated for 30 min at 30 °C. Samples were extracted with phenol/chloroform and precipitated with ammonium acetate/ethanol. After 2 h at -20 °C, samples were centrifuged, resuspended in 150 µl of TES buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.2% SDS), and treated with 40 µl of 1 M NaOH for 10 min at 4 °C. The reactions were stopped by the addition of 75 µl of 1 M Hepes, pH 7.8, and precipitated again with NH4OAc and ethanol. Samples were centrifuged, resuspended in 150 µl of TES, heated for 5 min at 90 °C, and added directly to the hybridization solution described below.

To prepare membranes for hybridization with the radiolabeled run-on RNA, 20 µg of plasmid DNA were digested with restriction endonucleases. Plasmid containing human beta -actin cDNA was digested with EcoRV and ScaI to release the actin structural gene; pT7Zpm70.1, containing the mouse hsp70.1 gene, was digested with HindIII, StuI, and BglII; and pT7hsp27, containing the hamster hsp27 cDNA, was digested with ClaI and BamHI. DNA was fractionated by agarose gel electrophoresis and transferred to a nylon membrane using 0.4 NaOH (30). Membrane strips were prehybridized for 2 h at 42 °C in a solution containing 5× SSC, 5× Denhardt's solution, 50% formamide, 1% SDS, and 100 µg/ml denatured salmon sperm DNA. Membranes were hybridized for 24 h at 42 °C in 10 ml of the same buffer containing radiolabeled RNA. After hybridization, membranes were washed for 1 h at 42 °C and 1 h at 65 °C in a solution containing 2× SSC and 0.1% SDS. Hybridization of radiolabeled RNA was detected by PhosphorImager analysis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DNA-PK-deficient Cells Have an Increased Sensitivity to Heat-- To test whether the heat shock response is abnormal in DNA-PK-deficient cells, we made use of mutant cell lines that lack either DNA-PKcs or Ku80, as listed in Table I. The DNA-PKcs-negative cell line, scva2, derived from a severe combined immunodeficiency (scid) mouse (31), is homozygous for an allele of DNA-PKcs that has a nonsense mutation, causing a small truncation at the C terminus of the protein (32-34). This results in loss of kinase activity and decreased levels of antigenically reactive DNA-PKcs (35). A matched DNA-PKcs-positive cell line, sc(8)-10, was derived from scva2 by introduction of a centromeric fragment of human chromosome 8 containing the DNA-PKcs structural gene, which rescues radiation resistance and DNA-PK activity (35). The Ku80-negative cell line is derived from xrs-6 (36), a Chinese hamster ovary cell line that has a mutation in the Ku80 structural gene that affects RNA splicing and abolishes Ku80 protein expression (37). The xrs-6cKu80 line was derived from xrs-6 by transfection with a human Ku80 cDNA, which rescues Ku protein expression and radiation resistance, and the isogenic xrs-6cvec line was derived by transfection with the same expression vector lacking the Ku80 cDNA (38). The unmutated, Ku80-positive, AA8 CHO cell line was also used in some experiments.

                              
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Table I
Cell lines used in this study

Cells were subjected to heat treatment for various times at 43 °C and tested for reproductive viability, i.e. the ability to form colonies in a monolayer cloning assay. The surviving fraction was calculated by counting the number of colonies formed by the heated cells and dividing by the number of colonies formed by unheated control cells plated at the same time. The data reveal a substantial difference in heat sensitivity (Fig. 1). The DNA-PKcs- and Ku80-negative cells (sva2, xrs-6cvec) are up to 30-fold more sensitive to heat-induced cell death than their genetically matched counterparts (sc(8)-10, xrs-6cKu80). The Ku80-negative cells (xrs-6cvec) were also more heat-sensitive than the unmutated AA8 Chinese hamster ovary cell line, although the difference was not as great, perhaps reflecting a level of endogenous Ku protein in AA8 cells that is somewhat lower than in the rescued xrs-6cKu80 cell line. Differences in cell survival were seen at all heat doses. The observed differences in heat sensitivity are of the same magnitude as the differences in ionizing radiation sensitivity previously reported with these cell lines (31, 38). These data indicate that DNA-PK provides substantial protection against heat-induced cell killing under the conditions of these experiments and that both the Ku and DNA-PKcs components are required.


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Fig. 1.   Analysis of reproductive viability DNA-PK-positive and -negative cells subjected to heat treatment. Monolayer cloning assays were performed as described under "Materials and Methods." A, flasks of DNA-PKcs-positive (sc(8)-10) and DNA-PKcs-negative (scva2) cells were treated for the indicated times at 43 °C, allowed to recover for 2 h at 37 °C, trypsinized, and plated. Colonies with 20 or more cells were counted after 1 week. The surviving fraction was calculated as described under "Results" section of the text. B, flasks of Ku-positive (xrs-6cKu80) and Ku-negative (xrs-6cvec) cells were treated for the indicated times at 43 °C, trypsinized immediately, and plated. Colonies were counted, and the surviving fraction was calculated as in A.

Heat Sensitivity Reflects Increased Apoptosis in DNA-PK-deficient Cells-- Heat-induced cell death can occur by either apoptosis or necrosis, depending on the heat dose and the cell type. At temperatures ranging between 42 and 45 °C, many types of cells die by apoptosis. At higher temperatures, necrosis is the prevalent form of cell death (39-46).

To determine whether the DNA-PK-deficient cells in our experiments were undergoing cell death by an apoptotic mechanism, we used a quantitative immunoassay to detect release of histone-DNA complexes into the cytoplasm, which is diagnostic of apoptotic death (47). Heat treatment resulted in an increase in apoptotic complexes in all cell lines. The increase was greater, however, in the DNA-PKcs-negative and Ku80-negative cells, as compared with the matched controls (Fig. 2). For all cell lines, the level of apoptotic histone-DNA complexes reached a maximum within 4 h after heat treatment. This rapid onset of cell death is consistent with results of previous studies using heat-treated cells (45, 46).


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Fig. 2.   Quantitative immunoassay of apoptotic histone-DNA complexes. A, equal numbers of DNA-PKcs-positive (sc(8)-10) and DNA-PKcs-negative (scva2) cells were plated in individual wells of a multiwell plate. Cells were left unheated (control (ctrl)) or were treated for 60 min at 43 °C and allowed to recover at 37 °C for the indicated times (heat shock (HS)). Individual wells were harvested, and cell extracts were assayed by enzyme-linked immunosorbent assay as described under "Materials and Methods." Points are the average of duplicates with S.E. indicated. B, same as A but with Ku80-positive (xrs-6cKu80) and Ku80-negative (xrs-6cvec) cells. Experiments were performed at least three times for each cell line with similar results.

The difference in the amount of apoptotic histone-DNA complexes measured in DNA-PK-positive and -negative cell populations was on the order of 2-fold. This is a smaller difference than what was seen in the monolayer cloning assay. It is likely that the immunoassay underestimates the differences between cell lines because it measures only complexes recovered from intact cells that remain attached to the culture flask. Many of the DNA-PK-deficient cells disintegrated or detached from the flask prior to harvesting, reducing the amount of apoptotic complexes that were detected.

We also used a different assay that allowed us to visualize apoptotic cells individually and thus measure the fraction of apoptotic cells that were present in the population at a given time. In this assay, fluorescein-labeled dUTP was incorporated into the ends of apoptotic DNA fragments by terminal deoxynucleotidyl transferase and visualized by fluorescence microscopy. The same samples were also stained with the DNA binding dye DAPI to allow visualization of all cells present in the preparation.

Heat treatment of DNA-PKcs-negative cells resulted in a marked increase in the fraction of fluorescein-labeled cells, as compared with the unheated control population (Fig. 3). Quantitation showed that the fraction of apoptotic cells in the DNA-PKcs-negative population increased more than 10-fold, whereas there was little change in the fraction of apoptotic cells in the DNA-PKcs-positive population (Fig. 3B). Similar results were obtained in experiments using Ku80-negative and -positive cells and will be presented below.


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Fig. 3.   Immunofluorescence analysis and quantitation of apoptotic DNA fragmentation in DNA-PKcs-positive and DNA-PKcs-negative cells. A, cells were left untreated (control) or heated at 43 °C for 60 min and allowed to recover at 37 °C for 4 h (heat shock). Staining was performed sequentially using fluorescein dUTP/terminal deoxynucleotidyl transferase labeling and DAPI DNA staining dye. Images in each row show the same microscope field using different filters to allow DNA and dUTP to be visualized separately. The experiment was performed three times, and representative results are shown. B, quantitation of apoptotic cells was carried out using microscope fields from two different experiments, including the field shown in A. Bright dUTP-labeled cells were counted, and the number was divided by the total number of cells counted in the DAPI-stained field. Between 365 and 560 total cells were counted for each condition.

The difference in cell death measured by the immunofluorescence assay (about 10% in scva2 cells) compared with the colony forming assays (99% under similar conditions) may be in part attributed to inherent underestimation by the immunofluorescence method, because only cells that are undergoing apoptosis at a single time point are scored. Some apoptotic cells may float away before they can be fixed and detected, and others may be destined to undergo apoptosis at later times and also escape detection. By contrast, the colony forming assay provides a measure of cumulative death in the entire population. In addition, the colony formation assay scores cells that do not undergo apoptosis but nevertheless lose reproductive viability.

Overexpression of HSF1 Constructs in xrs-6cKu80 Cells Results in Increased Apoptosis-- The preceding experiments establish that DNA-PK protects cells against heat-induced apoptosis. We wished to determine whether this protection was modulated by HSF1. Previous studies have shown that HSF1 binds to and regulates DNA-PK in vitro (12, 13). If overexpression of wild-type or mutant HSF1 could be shown to affect DNA-PK-mediated protection against heat-induced apoptosis, this would be evidence for a regulatory interaction between these proteins in vivo. Accordingly, we established populations of transfected cells expressing HSF1 derivatives in a Ku80-positive and -negative background.

A map of the HSF1 derivatives used in this experiment is shown in Fig. 4A. All of these derivatives form trimers and bind to DNA in vitro (13). They differ in their ability to interact with DNA-PK. HSF1-(1-450), which contains the first 450 of the 529 amino acids of human HSF1, binds strongly to DNA-PK components in vitro and stimulates DNA-PK activity (13). HSF1 Delta 24, which has a deletion from amino acid 280 to 370, binds to DNA-PK but does not stimulate activity (13). HSF1 Delta 01, which has a deletion from amino acid 203 to 224, does not bind to DNA-PK in vitro (13). These HSF1 derivatives were subcloned into the pcDNA3 vector, which allows expression under the control of a cytomegalovirus promoter.


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Fig. 4.   Generation of cell lines stably transfected with HSF1 derivatives. A, schematic representation of the transfected constructs with the normal human HSF1 sequence shown as open boxes and the deleted regions in each construct shown as solid lines. The known functional and structural domains of human HSF1 are indicated. B, expression of the transfected HSF1 constructs in xrs-6cKu80 cells. Immunofluorescence assays were performed with cells that were unheated (control) or treated for 1 h at 43 °C and allowed to recover for 2 h at 37 °C (heat shock). The pattern of expression in corresponding xrs-6cvec derivatives was essentially identical (not shown).

Plasmids expressing each of these constructs, a plasmid expressing full-length HSF1, as well as the pcDNA3 vector alone, were cotransfected with a puromycin selection marker into Ku80-positive and -negative cells. Populations were established and tested for HSF1 expression by immunofluorescence. As shown in Fig. 4B, xrs-6cKu80 cells transfected with pcDNA3 alone expressed a low level of endogenous HSF1. Cells transfected with expression constructs expressed higher levels of HSF1. As expected, the endogenous HSF1 was predominantly cytoplasmic in unheated control cells and partially relocalized to the nucleus after heat treatment. The HSF1 Delta 01 derivative behaved similarly. Unexpectedly, the HSF1 Delta 24 and HSF1-(1-450) derivatives were localized in the nucleus in both control and heat-shocked cells. Full-length HSF1 had a pattern of localization similar to HSF1-(1-450) (not shown). The reason for these differences in localization is unknown but presumably relates to the combination of overexpression and the presence of the DNA-PK binding site. Similar experiments were performed to measure expression of HSF1 derivatives in xrs-6cvec transfectants. Results were comparable with those with xrs-6cKu80 cells (not shown).

Heat shock-induced apoptosis was measured in each transfected population using the fluorescein-dUTP incorporation assay. Representative data are shown in Fig. 5A, with quantitation of these and other experiments in Fig. 5, B and C. The Ku80-positive cells transfected with pcDNA3 vector alone (xrs-6cKu80 pcDNA3) were relatively heat-resistant, as expected (Fig. 5A, top row). The same cells transfected with the HSF1-(1-450) showed an increase in heat sensitivity of about 8-fold (Fig. 5A, second row). The Ku80-negative cells transfected with pcDNA3 vector alone (xrs-6cvec pcDNA3) were about 20-fold more sensitive to heat than the Ku80-positive cells (Fig. 5A, third row). Interestingly, transfection of these cells with HSF1-(1-450) produced no change in heat sensitivity (Fig. 5A, fourth row). That is, constitutive overexpression of HSF1 under the conditions of this experiment specifically suppresses the protective effect of DNA-PK. The simplest interpretation is that HSF1 is a negative regulator of DNA-PK in vivo. The implications of this finding will be considered further under "Discussion."


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Fig. 5.   Immunofluorescence analysis and quantitation of apoptosis in Ku80-positive and -negative cells stably transfected with HSF1 deletion constructs. A, representative results using xrs-6cKu80 cells transfected with pcDNA3 or HSF1-(1-450) as indicated, and xrs-6cvec cells transfected with pcDNA3 or HSF1-(1-450) as indicated. Cells were unheated (control) or heat-treated for 1 h at 43 °C and allowed to recover for 4 h at 37 °C (heat shock). Cells were labeled with dUTP-fluorescein/terminal deoxynucleotidyl transferase and DAPI DNA staining dye. The same field was analyzed using two different filters. B, quantitation of apoptosis in xrs-6cKu80 transfectants. Results are included from cells shown in A, as well as cells transfected with HSF1 Delta 01, HSF1 Delta 24, and full-length, wild type HSF1 (wt). Bright dUTP-labeled cells were counted, and the number was divided by the total number of cells counted in the corresponding DAPI-stained fields. In most cases, results from two different experiments were averaged (except for HSF1 wild type transfectants, where multiple fields from a single experiment were averaged). C, the same analysis as in B using xrs-6cvec cells. In B and C, between 232 and 872 total cells were counted for each condition.

As a further test of the specificity of the interaction between HSF1 and DNA-PK, we evaluated cells that had been transfected with mutant HSF1 derivatives. Expression of HSF1 Delta 01, which does not interact with DNA-PK in vivo, did not sensitize xrs-6cKu80 cells to heat (Fig. 5B). By contrast, expression of two other HSF1 derivatives that bind DNA-PK in vitro, HSF Delta 24 and full-length HSF1, resulted in an increase in heat sensitivity similar to that seen with HSF1-(1-450). None of these HSF1 derivatives had any effect in xrs-6cvec cells (Fig. 5C). In addition, none of the HSF1 derivatives increased apoptosis in unheated control cells, indicating that their effects are not due to general toxicity.

It is of interest that, while some of the HSF1 derivatives sensitized cells to heat, neither the full-length HSF1 nor any of the HSF1 derivatives exhibited a protective effect. This finding must be considered in the context of the absence of a functional inducible hsp70 gene in these cells (see "Discussion").

Expression of Heat Shock Genes in DNA-PKcs- and Ku-deficient Cells-- Heat shock initiates a well characterized program of gene expression in mammalian cells. HSF1 plays a central role in this process. Heat treatment induces the conversion of HSF1 from a latent to an active form, which is followed by binding of HSF1 to promoters of heat shock genes, transcription of heat shock mRNAs, and synthesis of heat shock proteins. Because of the previous experiments showing that there is a regulatory interaction between DNA-PK and HSF1, it was of interest to examine various steps in this pathway in DNA-PK-positive and -negative cell lines.

Electrophoretic mobility shift assays were performed with extracts of various cell lines in order to detect HSF1 DNA binding activity (Fig. 6A). All of the cell lines showed induction of binding activity upon heat treatment for 20 min at 43 °C, as evidenced by formation of a characteristic HSF1-DNA complex. In each case, the complex was specific for the heat shock element sequence, as demonstrated by its sensitivity to competition with a nonradiolabeled oligonucleotide. These data show that Ku and DNA-PKcs are not required for conversion of endogenous HSF1 to the DNA-binding form.


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Fig. 6.   Induction of endogenous HSF DNA binding activity and heat shock mRNA synthesis. A, measurement of HSF DNA binding activity in different cell lines. Equal numbers of cells were plated in replicate flasks. Cells were left unheated (control) or were heated for 20 min at 43 °C (H. shock). Cell extracts were prepared, and equal amounts of protein were added to binding reactions containing a radiolabeled double-stranded DNA oligonucleotide with one HSF1 binding site. Where indicated, reactions also contained a 200-fold excess of nonradiolabeled oligonucleotide containing a single heat shock element (200× HSE). Protein-DNA complexes were analyzed by native gel electrophoresis. The positions of the HSF-DNA complexes are marked by an arrowhead. B, analysis of nuclear run-on transcription in DNA-PKcs-positive (sc(8)-10) and DNA-PKcs-negative (scva2) cells. Equal numbers of cells were plated in replicate flasks. Cells were left unheated (control) or were subjected to heat treatment, with times and temperatures as indicated. Nuclei were prepared, and nascent RNA was radiolabeled by extension in the presence of [alpha -32P]CTP. This RNA was purified and hybridized to membranes to which various plasmid DNA fragments had been blotted as indicated. The positions of relevant DNA fragments are marked by arrows. C, analysis of steady state hsp70 mRNA levels. DNA-PKcs-positive (sc(8)-10) and DNA-PKcs-negative (scva2) cells were left untreated (control) or heated for 30 min at 43 °C and allowed to recover for 2 h at 37 °C (H. shock). Total RNA was purified and quantitated. Equal amounts of RNA were analyzed by formamide-agarose gel electrophoresis and blotted to a nitrocellulose membrane as described under "Materials and Methods." The membrane was hybridized with a probe containing the coding sequence of human hsp70. The same membrane was stripped and rehybridized to a probe containing the cDNA coding sequence for human beta -actin.

The induction of mRNA for the major inducible heat shock protein, hsp70, was examined in detail in DNA-PKcs-positive and -negative cell lines. To assess the rate of hsp70 gene transcription, we performed nuclear run-on assays. Nuclei were isolated, run-on RNA was synthesized in the presence of [alpha -32P]CTP, and RNA was hybridized to a restriction digest of a plasmid containing the mouse hsp70.1 gene (48). The restriction digest produces three bands, corresponding to the 5'-untranslated region of the mRNA, the coding region, and the 3'-untranslated region, as marked in Fig. 6. We saw a progressive increase in the hsp70 transcription rate upon heat treatment for 10 or 30 min at 43 °C (Fig. 6B). This initial induction was similar in both cell lines. The DNA-PKcs-negative cells, however, showed a somewhat lower rate of hsp70 transcription at the final time point, after 30 min of heat shock and 2 h of recovery.

We also measured hybridization of run-on RNA to a cDNA encoding another heat shock protein, hsp27 (49), and to a cDNA encoding a housekeeping protein, beta -actin. The hsp27 signal was near the limit of detection in the run-on assay but did not appear to differ in DNA-PKcs-positive and -negative cells (Fig. 6B). Transcription of beta -actin was shut down in response to heat treatment in both DNA-PKcs-negative and DNA-PKcs-positive cells (Fig. 6B), consistent with prior work showing that heat treatment reduces synthesis of non-heat shock mRNAs (reviewed in Refs. 50 and 51).

Steady state hsp70 mRNA levels were measured by Northern blot analysis (Fig. 6C). Heat shock reduced the yield of total RNA from DNA-PK-negative cells, and in order to compensate, we loaded an equal mass of RNA, rather than RNA from an equal number of cells, in each lane. Under these conditions, the amount of hsp70 transcript was essentially identical in DNA-PKcs-positive and -negative cell lines. Hybridization to beta -actin mRNA was performed as a control (Fig. 6C).

To determine whether there was a defect in production of heat shock proteins in DNA-PK-positive and -negative cells, we performed immunoblotting. The major inducible form of hsp70 (hsp72) was detected in both DNA-PKcs-positive and -negative cell lines (Fig. 7A). Induction in the DNA-PKcs-negative cells was somewhat delayed, however, and reached a 3-4-fold lower steady state level. To examine the expression of hsp70 at the individual cell level, we performed immunofluorescence assays. The level of hsp70 was uniformly reduced throughout the DNA-PKcs-negative population (Fig. 7B). Overlay of the anti-hsp staining with fluorescein/dUTP staining showed no apparent correlation between the level of hsp70 proteins and the probability of apoptotic DNA fragmentation in individual cells (data not shown).


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Fig. 7.   Expression of heat shock proteins in DNA-PK-positive and -negative cells. A, analysis by immunofluorescence. Cells were left unheated (control) or heat-treated for 1 h at 43 °C and allowed to recover for 4 h at 37 °C (heat shock). They were then fixed and stained sequentially with anti-hsp72/73 antibody, Texas Red-conjugated secondary antibody, and DAPI DNA staining dye. The same field was analyzed with two different filters. B, analysis by immunoblotting. Equal numbers of cells were plated in replicate flasks and were left untreated (control) or heated for 1 h at 43 °C and allowed to recover at 37 °C. At the indicated times, individual flasks were harvested, and cells were lysed in SDS-polyacrylamide gel electrophoresis sample buffer. Equal amounts of protein were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting using an anti-hsp72/73 antibody, as described under "Materials and Methods."

Taken together, the results of these experiments suggest that the initial steps in the induction of heat shock transcriptional response, including the activation of HSF1 DNA binding and the onset of hsp70 mRNA synthesis, occur normally in DNA-PKcs-negative cells. There is an attenuation of the later steps of the heat shock response, including a reduction in the rate of hsp70 transcription after 2 h of recovery and in levels of translated hsp70 protein. These later effects could be secondary, however, and attributable to the imminent onset of cell death in the DNA-PKcs-negative population.

Experiments were also performed to measure the induction of hsp70 mRNA and protein in xrs-6c-derived cell lines. Surprisingly, there was no detectable synthesis of the major inducible hsp70 mRNA or protein in either the xrs-6cvec or the xrs-6cKu80 cell line (data not shown). This apparent transcriptional silencing may be attributable to a secondary mutation in the xrs-6 cells, which were originally derived from a chemically mutagenized population (36), or it may be some other clonotypic effect. Silencing of the major inducible form of hsp70 has been previously described in several murine cell lines, and it is attributable in some cases to promoter methylation (52, 53). Immunoblotting with a broad specificity polyclonal antibody showed that the hsp73 heat shock cognate protein was induced by heat in the xrs-6cvec and the xrs-6cKu80 cell lines (not shown), indicating that the overall heat shock transcription response is operative, although the major hsp70 gene is silenced.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we present evidence that DNA-PK protects cells against heat-induced cell death. Under the conditions of our experiments, this protective effect ranged up to 30-fold, depending on the type of assay. Protection was detected using three different assays of cell death, which measured reproductive viability, the release of apoptotic histone-DNA complexes, and apoptotic DNA fragmentation, respectively. The effect of DNA-PK on survival after heat shock is similar in magnitude to the effect on survival after exposure to ionizing radiation. This surprising result suggests that the effect of DNA-PK on cell survival may be separable, under some conditions, from its mechanical role in DNA double strand break repair.

It is unlikely that the heat doses used in our experiments induce significant DNA damage. Previous work has suggested that mild heat treatment does not induce DNA damage in mammalian cells (54, 55). Moreover, immunoblotting of xrs-6c derivatives (not shown) indicates that heat shock is not accompanied by induction of p53. This argues that significant DNA damage is not present. Mutants of Saccharomyces cerevisiae that lack Ku protein arrest at cell cycle checkpoints when grown at a nonpermissive temperature (56). This growth arrest is accompanied by accumulation of DNA damage-inducible genes, however, and thus may not be comparable with the heat sensitivity seen in mammalian cells that lack Ku or DNA-PKcs.

The finding that Ku-deficient and DNA-PKcs-deficient cells have similar phenotypes, together with the knowledge that these proteins cooperate biochemically, makes it plausible to assume that both components of DNA-PK work together to establish resistance to heat-induced apoptosis. A formal genetic test to distinguish whether Ku protein and DNA-PKcs work together, rather than independently, will require analysis of double mutants, which are not yet available in the mammalian system. We have also not yet explored whether overexpression of DNA-PK components in wild-type cells would further increase resistance to heat-induced apoptosis. It is of interest to consider, however, that HeLa cells, which are a rich source of DNA-PK for biochemical purification, are highly resistant to heat-induced apoptosis, even at temperatures as high as 45 °C.2

Experiments that measure cell death as their end point do not distinguish whether the effect of DNA-PK is specific to heat or reflects a broader antiapoptotic function. Other data suggest, however, that the protective effect of DNA-PK relates in some way to its physical interaction with HSF1. This interaction has been demonstrated previously in vitro, using purified proteins (13). The interaction requires an HSF1 sequence defined, in part, by the HSF1 Delta 01 mutant, which deletes amino acids 204-223 (13). The present results extend previous findings by suggesting that there may be a regulatory interaction between HSF1 and DNA-PK in vivo. Constitutive overexpression of HSF1-(1-450) and certain other derivatives that are capable of binding DNA-PK suppressed the ability of DNA-PK to protect against heat-induced apoptosis in vivo. There was no effect when any of the derivatives were expressed in cells lacking functional DNA-PK holoenzyme. By contrast, overexpression of HSF1 Delta 01 had no effect. Although we cannot rule out pleiotropic effects of the HSF1 Delta 01 mutation on other HSF1 functions, the lack of an effect in our experiments is in concordance with the results of in vitro binding studies.

The loss of heat resistance in xrs-6 Chinese hamster ovary cell derivatives that overexpress HSF1 contrasts with a previously reported increase in heat resistance in mouse cells that overexpress HSF1 (57). This may reflect differences in the cell lines that were used. Transcriptional silencing at the endogenous hsp70 locus in xrs-6 derivatives precludes confounding effects mediated by HSF1-induced changes in hsp70 expression. Moreover, technical differences may have influenced the outcome of the experiments. In particular, the present work measured the fraction of the population in which rapid onset of apoptotic death occurred after mild heat doses. The other work measured loss of reproductive viability, which may occur by both apoptotic and nonapoptotic mechanisms, and found differences between transfected and control cells primarily at higher heat doses.

Although our results suggest that there is an interaction between HSF1 and DNA-PK, they do not inherently distinguish whether HSF1 modulates DNA-PK activity, whether DNA-PK modulates HSF1 activity, or both. We shall consider first the model that HSF1 regulates DNA-PK. There is evidence that such regulation occurs in vitro: purified HSF1 stimulates DNA-PK phosphorylation activity (12, 13). The in vivo data argue for an effect in the opposite direction, however. Overexpression of HSF1 suppresses the ability of DNA-PK to protect against apoptosis. It may be that binding of HSF1 in vivo hinders the ability of DNA-PK to interact with critical target proteins or sequesters DNA-PK in a location where it is not able to perform its antiapoptotic function. Also, prolonged stimulation of DNA-PK might deplete intracellular pools of active enzyme by an autophosphorylation mechanism. Previous work has established that DNA-PK can be negatively regulated by autophosphorylation (58). At present, we do not know if suppression of the DNA-PK protective effect reflects a natural mode of regulation or is a consequence of HSF1 overexpression.

An alternative model is that DNA-PK modulates HSF1 activity. A primary function of HSF1 is to activate synthesis of heat shock mRNAs. It appears that we can rule out a direct role of DNA-PK as a mediator of HSF1 transcriptional activation. The interaction between HSF1 and DNA-PK does not occur through the transcriptional activation domain (13). Moreover, the initial burst of heat shock mRNA synthesis occurs normally in DNA-PKcs-deficient cells. DNA-PK could have more subtle effects on HSF1 activity, however. HSF1 exists in an equilibrium between active and inactive forms. Phosphorylation of HSF1 at serines 303 and 307 shifts the equilibrium toward the inactive form (59-61). This phosphorylation appears to be mediated, in part, by glycogen synthase kinase and members of the MAP kinase family (61-63). DNA-PK phosphorylates HSF1 at multiple sites throughout the C-terminal portion of the protein, at least some of which are distinct from the sites that are phosphorylated by glycogen synthase kinase and MAP kinases (compare results in Refs. 62 and 13). It is possible that one or more of the DNA-PK-mediated phosphorylation events help to maintain HSF1 in its active form and prolong its transcriptional activity. This would be consistent with the higher sustained levels of hsp70 mRNA transcription that we observed in DNA-PK-positive cells.

A more intriguing possibility is suggested by an analysis of the sequence defined by the HSF1 Delta 01 mutant. This sequence, GVKRKIPLMLNDSGSAHSM, contains a match (underlined) to a targeting domain, the D domain, which acts as a binding site for extracellular signal-regulated kinase and c-Jun N-terminal mitogen-activated protein kinases (64, 65). The D domain, which is distinct from the actual sites of phosphorylation, is found in Elk-1 and a number of other transcription factors (64). It is possible that the binding of DNA-PK to a site overlapping the D domain blocks the interaction of mitogen-activated protein kinases with HSF1, preventing inactivation by these kinases. This model could be further evaluated by competitive binding studies.

If there is competition between DNA-PK and c-Jun N-terminal kinases for interaction with a common targeting sequence, this could have broad significance. Hyperthermia causes an increase in ceramide levels, which in turn activates the stress-activated protein kinase/c-Jun N-terminal kinase signaling pathway (66-68). This pathway may be the primary mechanism by which heat induces apoptosis. Interestingly, ionizing radiation also triggers ceramide release, and sphingomyelinase-deficient human lymphoblasts and mice are incapable of undergoing apoptosis even after lethal doses of radiation (69). The involvement of ceramide-dependent signaling pathways is thus a common feature of heat- and radiation-induced apoptosis. Investigation of a possible link between DNA-PK and ceramide-mediated cell signaling will be a potentially fruitful area for further investigation.

    ACKNOWLEDGEMENTS

We thank Dr. R. Morimoto for the human hsp70D cDNA. We thank Drs. S. Peterson and D. Chen for providing the cell lines used in this study. We thank Stephen A. Jesch, Wilhelm Woessman, Mark Anderson, Sunghan Yoo, Juren Huang, and Xinbin Chen for helpful suggestions and reagents and Nancy Miller for expert technical assistance. We thank Rhea-Beth Markowitz for comments on the manuscript.

    FOOTNOTES

* This work was supported in part by U.S. Public Health Service Grants GM 35866 and CA 62130.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a postdoctoral fellowship from Spain's Ministerio de Educación y Ciencia. Present address: CENG DBMS/BRCE, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.

§ Georgia Research Alliance Eminent Scholar and recipient of American Cancer Society Faculty Research Award FRA-418. To whom correspondence should be addressed: Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th St., Augusta, GA 30912. Tel.: 706-721-8756; Fax: 706-721-8752; E-mail: dynan{at}immag.mcg.edu.

2 A. Nueda, F. Hudson, N. F. Mivechi, and W. S. Dynan, unpublished results.

    ABBREVIATIONS

The abbreviations used are: DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; HSF1, heat shock transcription factor 1; hsp, heat shock protein; PBS, phosphate-buffered saline; DAPI, diamidino 2-phenylindole dihydrochloride hydrate; Pipes, 1,4-piperazinediethanesulfonic acid.

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