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[Cancer Research 62, 6621-6624, November 15, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

Selective Inactivation of DNA-dependent Protein Kinase with Antisense Oligodeoxynucleotides

Consequences for the Rejoining of Radiation-induced DNA Double-Strand Breaks and Radiosensitivity of Human Cancer Cell Lines1

Ali Sak2, Martin Stuschke, Reinhard Wurm, Gisela Schroeder, Brigitte Sinn, Gudrun Wolf and Volker Budach

Department of Radiotherapy, University Essen, 45122 Essen [A. S., M. S.], and Department of Radiotherapy, Humboldt-University, Charité, 10117 Berlin [R. W., G. S., B. S., G. W., V. B.], Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inhibition of DNA-dependent protein kinase activity with antisense-oligodeoxynucleotide (As-ODN) and its consequences for the rejoining of DNA-double-strand breaks (Dsbs) and radiation sensitivity was studied in human non-small cell lung cancer (NSCLC) cell lines. Cells were transfected with As-ODNs specific for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). In comparison, cells were treated with Wortmannin, a potent but nonspecific inhibitor of DNA-PK activity. As-ODN efficiently reduced the kinase activity with an IC50 of about 100–200 nM and was attributed to degradation of target mRNA. In comparison, the IC50 of Wortmannin was at ~5–10 µM. Treatment of cells with 300 nM As-ODN increased the fraction of residual Dsb at 4 h after irradiation by a factor of 4.4, 2.6, and 1.7 in A549, H460, and H661 cells, respectively. The respective values after treatment with 20 µM Wortmannin were 5.3, 4.3, and 2.2. Inhibition of DNA-PK activity by As-ODN and Wortmannin also decreased the surviving fraction of the NSCLC cell lines. These data show that kinase activity of DNA-PKcs can be specifically inhibited with As-ODN as effective as Wortmannin and results in marked inhibition of DNA-Dsb rejoining and radiosensitization of NSCLC cell lines.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main deleterious damage induced by ionizing radiation are DNA-Dsbs.3 They can lead to fragmentation, translocation, misrepair, and loss of chromosomes. Such genotoxic events activate a number of signaling pathways that serve to activate DNA repair mechanisms, cell cycle arrest, or trigger cells into apoptosis. The major mechanism underlying the repair of DNA-Dsbs in mammalian cells is nonhomologous end-joining (1 , 2) and requires the DNA-PK. DNA-PK is a serine-threonine protein kinase consisting of three subunits: a Mr ~450,000 catalytic subunit (DNA-PKcs) and a heterodimeric complex composed of the proteins Ku70 (Mr 70,000), and Ku80 (Mr 86,000). Cells lacking DNA-PK activity as a result of mutation in any of the subunits are deficient in the rejoining of radiation-induced DNA-Dsbs and are radiosensitive in the clonogenic assay (3 , 4) . The only clearly identifiable functional motif in DNA-PKcs is the kinase domain and shows a great similarity with catalytic domains of proteins involved in the phosphorylation of PI3ks.

Previous studies showed that the catalytic activities of DNA-PK, as other proteins of the PI3k family (PI3k, ATM, ATR, and mTOR), are inhibited by Wortmannin (5 , 6) . It was shown that Wortmannin irreversibly decreases the kinase activity of PI3k, DNA-PK, ATM, and ATR proteins in intact cells with half-maximal inhibition IC50 of 0.003, 3.6, 5.8, and 100 µM, respectively (6 , 7) . From these studies, it is evident that Wortmannin is primarily a strong and selective inhibitor of PI3k. At concentrations >1 µM, it also decreases the rejoining of DNA-Dsbs and sensitizes cells to ionizing radiation (8 , 9) . Experiments with genetically inactivated DNA-PKcs have shown that DNA-PKcs is most likely the main target of Wortmannin with regard to its effects on Dsb rejoining and cell killing (10 , 11) . However, because Wortmannin is not a specific inhibitor of DNA-PK, the possibility that interaction with other kinases may also contribute to a decrease in inhibition of Dsb repair and radiation sensitization cannot be ruled out.

To identify more selective inhibitors of DNA-PK, the effect of As-ODNs specific for DNA-PKcs mRNA, in comparison to the effect of Wortmannin on the same end points, i.e., the kinase activity of DNA-PK, steady-state mRNA level, rejoining of DNA-Dsb, and radiosensitivity of NSCLC cell lines were evaluated in this study.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Irradiation, and Clonogenic Survival.
Cell culture conditions and irradiation of the NSCLC cell lines A549, H460, and H661 were as described previously (12) . For the measurement of clonogenic survival, cells were irradiated at 20–24 h after transfection, harvested, and plated in triplicate at 1 x 102-2 x 104 cells/25-cm2 T-flasks (Nunc) depending on the treatment and irradiation dose and analyzed as described elsewhere (12) .

ODNs.
A total of three As-ODNs targeting various regions of the DNA-PKcs mRNA, one ODN with reverse orientation to As-PKcs, and one unrelated ODN were synthesized and purified by BioTez (Berlin, Germany). A BLASTN search of a database containing all sequences in the National Center for Biotechnology information database revealed no homology of the ODNs to other human genes. All oligonucleotides were phosphorothioates purified by high-pressure liquid chromatography. The sequences derived from a cDNA sequence (gene accession no. U47077 from National Center for Biotechnology) were as follows: As-PK-1 (translation initiation region, nucleotides 58–80), 5'-ACACCGGCTCCGGAGCCCGCCAT-3'; As-PK-2 (putative kinase region of DNA-PKcs, nucleotides 11720–11742), 5'-ATAAAGTTGTTCAGATGTCTGTC-3'; As-PK-3 (scid-mutation region, nucleotides 12079–12103), 5'-CTCTTAGCGTAACATATTTTCTG-3'; and unrelated (13) , 5'-AAGAGAGGTCCGAGGAGGGG-3'.

Lipid-mediated Transfection of As-ODNs.
Cells were plated in 9.6-cm2 culture dishes at a density of about 2 x 104 cells/cm2 for H661 and 4 x 104 cells/cm2 for the H460 and A549 cell lines. Transfections were done at 20–24 h after plating when cells reached a confluence of 50–80%. The formation of lipid/ODN complexes was carried out by diluting the appropriate amount of Lipofectamine (6 µg/ml) and ODN (300 nM) in 100 µl of serum-free Opti-MEM for the transfection in 9.6-cm2 culture dishes and 300 µl for 25-cm2 flasks according to the manufacturer’s instructions (Life Technologies, Inc., Paisley, United Kingdom). The overall concentration of ODN was kept constant by filling up with URs. The transfection complexes were added to the cells and incubated for 6 h at 37°C in 5% CO2. One volume of complete growth medium supplemented with 30% (MEM) or 20% (RPMI) FCS without antibiotics was added and incubated 15–18 h before processing cells for additional analysis. After 20 h of incubation at 37°C, FITC-labeled control ODNs were taken up by >90% of the cells.

Cell Extracts.
Exponentially growing cells 20–24 h after transfection were scraped from the dishes in PBS and centrifuged at 4°C for 5 min at 1500 rpm. Cell pellets were resuspended in 1 pellet volume of extraction buffer [50 mM NaF, 450 mM NaCl, 20 mM HEPES (pH 7.6), 25% w/v glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml pepstatin A, 1 mg/ml trypsin inhibitor, 0.5 µg/ml aprotinin, and 40 µg/ml bestatin; all proteinase inhibitors were from Roche, Mannheim, Germany]. The swollen cells were disrupted by incubation alternatively on liquid nitrogen and 30°C (4 times) for 1 min each. The resulting suspension was sedimented by centrifugation (15,000 x g, 10 min, 4°C), and supernatants were stored at -80°C before use.

DNA-PK Kinase Activity Assays.
The SignaTECT DNA-PK assay system (Promega Corp., Madison, WI) was used to measure DNA-PK activity. Kinase reactions were conducted with 5-µl aliquots of the cell extracts containing 20–30 µg of protein in an assay volume of 25 µl of reaction-buffer according to the manufacturer’s instructions. The background kinase activity in the absence of double-stranded DNA was subtracted from that with double-stranded DNA. The difference of both represents the DNA-dependent kinase activity of DNA-PK.

Induction and Repair of DNA-Dsbs.
Uniformly labeled cells (1.85 kBq [2-14C]thymidine/ml (1.92 MBq/mmol) were subcultured at a density of about 2 x 104 cells/cm2 for H661 and 4 x 104 cells/cm2 for the H460 and A549 cell lines, and transfections were carried out 24 h later as described. Cells were irradiated with 30 Gy at 4°C 20–24 h after transfection and were serially sampled at 0, 1, and 4 h after incubation at 37°C and cast into plug molds. Cell lysis and electrophoresis were performed as described previously (12) .

Detection of DNA-PKcs mRNA by Northern Blot Analysis.
At 20–24 h after transfection, cells were harvested mechanically and washed once with PBS. Total cellular RNA was extracted according to the manufacturer’s instruction with TRIzol solution (Life Technologies, Inc.). Equal amounts of total cellular RNA were denatured and analyzed by electrophoresis. After electrophoresis, RNA was transferred to nylon membranes. The following oligonucleotide probes were chosen for northern hybridization: 5'-AACTCCTTATTCTTATGACCAGTAGAAGTATCCTTGAAGG-3' (14) was the specific probe for DNA-PKcs mRNA and corresponds to the complement of nucleotides 10676–10715 of GenBank entry U47077. To control for unspecific effects of the As-ODN to the expression level of other kinases with homology to DNA-PKcs, a probe for PI3k was used (5'-CCATCGCTAGTTCTTTGCGGCTGATGCCCACAGACTCCAG-3') and corresponds to the nucleotides 555–594 of GenBank entry Y11312. As a control for loading error and overall mRNA transcription activity, a probe for gylceraldehyde-3-phosphate dehydrogenase was used (5'-TGTTGAAGTCAGAGGAGACCACCTGGTGCTCAGTGTAGCC-3') and corresponds to the complement of nucleotides 883–922 of GenBank locus HSGA PDR.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of As-ODN and Wortmannin on Kinase Activity and mRNA Expression of DNA-PKcs.
To verify the specificity of the As-ODNs used and to exclude unspecific effects, levels of DNA-PKcs activity and expression of DNA-PKcs mRNA were investigated after treatment with As-ODNs. The specificity of the DNA-PK assay was tested by determination of 32P transfer onto p53-derived peptide in the presence and absence of double-stranded DNA for background evaluation. The ratio between kinase activities in the presence and absence of double-stranded DNA represents the DNA-dependent kinase activity of DNA-PK and was 2.6 ± 0.5, 2.0 ± 0.5, and 2.6 ± 0.4 (pmol ATP/min/µg protein) for untreated A549, H460, and H661 cells, respectively.

Treatment of cells with Wortmannin as shown in Fig. 1aCitation significantly reduced the DNA-PK activity in all cell lines studied. The IC50 for Wortmannin-mediated inhibition of kinase activity was at ~5 µM for A549 and H460 and at ~10 µM for H661 cells. For comparison, transfection with As-ODNs specific for the translational start of DNA-PKcs mRNA efficiently inhibited the kinase activity of DNA-PK in A549, H460, and H661 cells (Fig. 1b)Citation . The IC50 for As-PK-1 was at about 100, 200, and 150 nM in A549, H460, and H661, respectively. The specificity of the As-ODN used was tested using unrelated ODN, which showed no effect on DNA-PK activity. To test the hypothesis that As-ODNs can initiate an RNase H-mediated degradation of target mRNAs, we quantified the steady-state level of DNA-PKcs mRNA in A549 cells after transfection with As-PK-1. As shown in Fig. 2Citation , As-ODNs decreased the level of DNA-PKcs mRNA in a concentration-dependent manner. The specificity of As-PK-1 was additionally explored by probing the blots with PI3k-specific probes. As-PK-1 has no significant effect on the steady-state level of PI3k mRNA. These data show that As-ODNs directed against the translational start of DNA-PKcs mRNA specifically inhibits the kinase activity of DNA-PK and is mainly the result of mRNA degradation with the same IC50 of ~100 nM. In comparison, Wortmannin had no effect on mRNA stability of DNA-PKcs but inactivates the DNA-PKcs protein.



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  		Fig. 1. DNA-PK activities of NSCLC cell lines. DNA-PK activities from at least three independent experiments (mean ± SE) are shown for cells treated with 0, 5, 10, and 20 µM (W0, W5, W10, and W20) Wortmannin (a) or transfected with 0, 150, and 300 nM As-PK-1 (b). L (Lipofectamin), PK0 (300 nM UR), PK150 (150 nM As-PK-1 + 150 nM UR), and PK300 (300 nM As-PK-1).

 


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  		Fig. 2. Expression of DNA-PKcs and PI3k mRNA in A549. Representative hybridization blot of A549 cells treated with 0 (W0) and 20 (W20) µM Wortmannin and As-PK-1 (0, 150, and 300 nM) are shown. L (Lipofectamin), PK0 (300 nM UR), PK150 (150 nM UR +150 nM As-PK-1), and PK300 (300 nM As-PK-1).

 
Effect of As-ODN and Wortmannin on Induction and Rejoining of DNA-Dsb.
The kinetics of Dsb repair were assessed over a 4-h period after exposure of cells to 30 Gy at 0 h (initial induction), 1 h (fast rejoining fraction), and 4 h (slow rejoining fraction) after treatment with Wortmannin or transfection with As-ODNs specific for DNA-PKcs. The respective results for Dsb rejoining for all cell lines are shown in Fig. 3Citation . In all cell lines studied, no change in the initial (0 h) induction levels of Dsb after treatment with Wortmannin or transfection with ODN was found. In comparison, the fraction of Dsb repaired after 1 and 4 h was significantly reduced if cells were pretreated for 1 h with Wortmannin (Fig. 3a)Citation or transfected with As-ODN (Fig. 3b)Citation specific for the translational start of DNA-PKcs mRNA (As-PK-1). A dose-dependent increase in the fraction of nonrepaired Dsb at 1 and 4 h, both for Wortmannin and As-PK-1, was found. The proportion of damage remaining at 1 and 4 h to the initial Dsb at 0 h was determined as a measure for the fast and slow rejoining fraction. Treatment of cells with 20 µM Wortmannin increased the fraction of nonrejoined Dsb at 1 h by a factor of 2.1, 2.0, and 1.5 and at 4 h by a factor of 5.3, 4.3, and 2.2 in A549, H460, and H661 cells, respectively. In comparison, As-ODN at 300 nM increased the fraction of nonrejoined Dsb at 1 h by a factor of 2.3, 1.7, and 1.4 and at 4 h by a factor of 4.4, 2.6, and 1.7 in A549, H460, and H661 cells, respectively. These data show that As-PK-1 at 300 nM was as effective as 20 µM Wortmannin in decreasing the fraction of Dsb rejoined at 1 h after irradiation. However, the effect of Wortmannin on the fraction of Dsb remaining after 4 h was higher. Initially, other As-ODNs directed against sequences within exon regions of DNA-PKcs mRNA, As-PK-2 (kinase region of DNA-PKcs), As-PK-3, (mutation region of DNA-PKcs in scid cells) and As-PK1-rev (reverse orientation of As-PK-1) were tested and had no significant effect on Dsb rejoining (data not shown). An URs, which was used to fill up the overall ODN concentrations up to 300 nM, also had no significant effect on Dsb repair.



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  		Fig. 3. Rejoining of ionizing radiation induced DNA-DSB in NSCLC cell lines. The fraction of activity released (FAR) into the gel as a measure for DNA-Dsb at 0, 1, and 4 h after irradiation with 30 Gy is shown for A549, H460, and H661 treated with 0 (W0), 5 (W5), 10 (W10), and 20 (W20) µM Wortmannin or transfected with AS-ODN. L (Lipofectamin), PK0 (300 nM UR), PK150 (150 nM As-PK-1 + 150 nM UR), and PK300 (300 nM As-PK-1).

 
To test whether Wortmannin can increase the effect of As-ODN, H460 cells were transfected with 150 nM As-PK-1 and treated thereafter with 5 µM Wortmannin. The fraction of nonrejoined Dsb at 1 h was increased by a factor of 1.4, 1.6, and 2.3 and at 4 h by a factor of 2.2, 2.9, and 2.9 for cells treated with 150 nM As-PK-1, 5 µM Wortmannin, and 150 nM As-PK-1 + 5 µM Wortmannin, respectively. These data show that combination of both treatment schedules in comparison to the effect of As-PK-1 or Wortmannin alone increased the fraction of nonrejoined Dsb at 1 h but has no effect on the fraction of nonrejoined Dsb at 4 h.

In conclusion, these data show that treatment of NSCLC cell lines with As-ODN specific for the translational start of DNA-PKcs mRNA was as effective as Wortmannin with respect to rejoining of radiation-induced Dsb rejoining.

Influence of As-ODN and Wortmannin on Radiation Sensitivity.
To examine the relation between DNA-PK activity and radiation sensitivity, survival after a radiation dose of 2 Gy (SF2) was determined by the colony forming assay. The SF2 values were 64.6 ± 4.3, 43.7 ± 1.1, and 58.6 ± 5.3% for untreated A549, H460, and H661 cells, respectively. The cell lines with higher surviving (A549, H661) fraction also show a high DNA-PK activity. Transfection with 300 nM As-PK-1 significantly reduced the percentage of cells surviving a radiation dose of 2 Gy, as compared with control cultures treated with Lipofectamin only. Treatment of cells with unrelated ODN did not change the surviving fraction after irradiation with 2 Gy. In comparison, treatment of cells with Wortmannin also effectively reduced the surviving fraction after irradiation. The survival of the cell lines in the absence of radiation was not significantly affected by Wortmannin. As a measure for the effectiveness of treatment schedules, we defined the SER as the ratio of SF2 without treatment divided by the SF2 when cells are treated with Wortmannin or As-ODN. The SER was 4.7, 1.5, and 4.3 for treatment with 300 nM As-PK-1 and 10.8, 7.3, 6.5 for treatment with 20 µM Wortmannin for A549, H460, and H661 cells, respectively. These data show an effective decrease in clonogenic survival after transfection with As-ODN targeting the mRNA of DNA-PK. In comparison, Wortmannin was much more effective on the clonogenic survival of NSCLC cell lines.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In previous studies, radiation sensitive mutants defective in Dsb repair were used to establish the involvement of DNA-PK in the rejoining of DNA-Dsb (15) . The Dsb rejoining deficiency of cells from Ku80 and Ku70 knockout mice confirms the importance of DNA-PK activity in the repair process. The existence of many other proteins related to Dsb repair such as the gene products of ATM, ATR and NBS1 complicated the interpretation of the results. To evaluate the effect of modulation of DNA-PKcs expression in Dsb repair and radiation sensitivity, As-ODNs were used. The data obtained in this study show a specific As-ODN-mediated degradation of the target mRNA. In all cell lines studied, As-PK-1 effectively inhibited the kinase activity of DNA-PK with an IC50 of ~100–200 nM. The initial repair fraction within the first 60 min was inhibited by a factor 1.5–2 with 300 nM As-ODN. The effect increases if the residual damage at 4 h was measured with a factor of ~2–4. The most effective sequence is one that includes the initiation codon in its sequence. In comparison, two other As-ODNs directed to different exons of the DNA-PKcs mRNA were not effective (data not shown). Nonspecific effects of As-ODNs have been observed if two or more contiguous guanosines are present in ODN sequences (16) . To control for unspecific effects of the two GG strings present in the sequence of As-PK-1, an unrelated oligo (UR) with two GG and one GGGG strings was chosen. The data show no effect of the unrelated ODN on DNA-repair, DNA-PK kinase activity, and cell survival after irradiation.

In comparison, Wortmannin known to bind covalently to DNA-PKcs protein (17) has been used to inhibit the kinase activity of DNA-PK protein. Our results show that Wortmannin efficiently inhibits the kinase activity of DNA-PK with an IC50 of ~5 µM in the cell lines A549 and H460 and 10 µM in H661. In addition, Wortmannin effectively reduced the rejoining of DNA-Dsbs with an increase in the residual damage at 1 h after irradiation by a factor of ~2 in all cell lines studied. The effect of Wortmannin on Dsb rejoining was more obviously at 4 h after irradiation with an increase in residual damage by a factor of 3–6. Consequently, Wortmannin led markedly to a reduced survival rate after irradiation.

In all cell lines studied, As-PK-1 inhibited the kinase activity of DNA-PK and the initial repair fraction within the first 60 min as effectively as Wortmannin. However, in comparison to Wortmannin, reduced efficacy of As-ODN on inhibition of Dsb repair at 4 h after irradiation was evident. This reduced effectiveness of As-ODN at later times after irradiation also led to a smaller effect on the radiation sensitivity of the cells, with a SER of ~4 for A549 and H661 and 1.5 for H460 cells treated with 300 nM As-ODN. The respective SER factor for A549 and H460 cells treated with 20 µM Wortmannin was 6–7 for H460, H661, and 10 for A549. The effect of As-PK-1 and Wortmannin on Dsb rejoining and surviving fraction after irradiation was comparable with that of DNA-PKcs-deficient cell lines with an increase in residual damage at 4 h by a factor of about 3–6 and a radiation sensitization factor of about 4–10 in comparison to DNA-PK proficient cell lines (11 , 18) . The enhanced effect of Wortmannin on the radiation sensitivity of NSCLC cell lines, in comparison to As-PK-1, may result from the pleotropic effect of Wortmannin on the members of the PI3k family (6) . The existence of a slow, DNA-PKcs-independent rejoining pathway that can be partially inhibited with Wortmannin (11) , but not with As-PK-1 targeting DNA-PKcs, can also explain the reduced effect of As-ODN at 4 h after irradiation. An additional repair pathway, homologous recombination repair, is recruited to restore the DNA sequence after the initial fast (DNA-PKcs dependent) or slow (DNA-PKcs independent) rejoining of radiation-induced Dsb (19 , 20) . The contribution of regulatory kinases in homologous recombination repair such as ATM and ATR, which can be inhibited with Wortmannin, can also explain the increased radiosensitization effect of Wortmannin in comparison to As-PK-1. Alternatively, the higher effect of Wortmannin on DNA repair and surviving may be the result of noncompetitive and covalent binding of Wortmannin to the kinase region of DNA-PKcs protein (17) . This formation of covalent adducts can arrest DNA-PK repair complexes at an intermediate stage on damaged DNA, which may be inaccessible for repair by other repair pathways. By contrast, antisense ODN, which physically removes DNA-PKcs, leaves DNA ends accessible to DNA-PK-independent repair pathways (slow repair). In this case, one would expect interference for the slow repair, rather than synergy if both treatment schedules were combined. Our data show that combination of antisense and Wortmannin treatment have an additive effect on the fast rejoining fraction, but we failed to demonstrate an additive effect on the slow repair fraction at 4 h.

These data demonstrate that destabilization of DNA-PKcs mRNA with As-ODNs specifically inhibits the kinase activity of DNA-PK, the rejoining of DNA-Dsb, and consequently increases the radiosensitivity of human NSCLC cell lines. The efficacy of As-ODN on repair inhibition at early times after irradiation opens a possible approach to specifically manipulating the activity of repair proteins for the evaluation of repair pathways. These data also provide an encouraging basis for additional investigations into the therapeutic potential of As-ODN, particularly for the combined radiochemotherapy treatment of solid tumors.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Deutsche Krebshilfe. Back

2 To whom requests for reprints should be addressed, at University of Essen, Department of Radiotherapy, Hufelandstr. 55, 45122 Essen, Germany. Phone: 49-201-723-2055/3946; Fax: 49-201-723-5960/5908; E-mail: ali.sak{at}uni-essen.de Back

3 The abbreviations used are: Dsb, double-strand breaks; DNA-PK, DNA-dependent protein kinase; PI3k, phosphoinositide 3'-kinase; As-ODN, antisense-oligodeoxynucleotide; NSCLC, non-small cell lung cancer; UR, unrelated oligodeoxynucleotide; SER, sensitization enhancement factor; ATR, ATM-Rad3 related; mTOR, mammalian target of rapamycin; ATM, ataxia telangiectasia mutated; NBSA, nijmegen breakage syndrome. Back

Received 2/18/02. Accepted 9/20/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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