• Journal of Life Science
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• v.15 no.3 s.70
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• pp.406-414
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• 2005

The genetic instability of cancer cells may be related to inappropriately activated DNA repair pathways. In present study, the modulated expression of DNA-dependent protein kinase (DNA-PK), a major DNA repair protein, in human cancer metastatic cells was tested. The expressions of Ku70/80, regulatory subunit of DNA-PK, and the Ku DNA-binding activity in various highly metastatic cell lines were higher than those in each parental cell line. Also, the expression of DNA-PKcs, catalytic subunit of DNA-PK, and the kinase activity of the whole DNA-PK complex in highly metastatic cells were significantly increased as compared to those of parental cells, suggesting that the enhanced DNA repair capacity of metastatic cells could be associated with aberrant use of DNA repair, which may mediate tumor progression and metastatic potential. Increased EGFR (epidermal growth factor receptor) signaling has been associated with tumor invasion and metastasis, and the linkage between EGFR-mediated signaling and DNA-PK has been suggested. This study showed that PKI166, the new EGFR tyrosine kinase inhibitor, modulated the expressions of Ku70/80 and DNA-PKcs and also revealed the chemosensitization effect of PKI166 against metastatic cells may be in part due to inhibition of Ku70/80. These results suggest that interference in EGFR signaling by EGFR inhibitor resulted in the impairment of DNA repair activity, and thus DNA-PK could be possible molecular targets for therapy against metastatic cancer cells.

• Journal of Life Science
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• v.17 no.6 s.86
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• pp.748-755
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• 2007

The in vitro activity of ST1571, an inhibitor of the Abl group of protein-tyrosine kinases, alone or in combination with camptothecin (CPT), a specific topoisomerase I inhibitor, was evaluated against human cancer cells with different metastatic capacity and drug resistance potency. These cell lines showed different sensitivity to ST157 on growth inhibition, and the expression of DNA-dependent protein kinase (DNA-PK), which interacts constitutively with c-Abl, was significantly decreased in drug sensitive CEM and MCF-7 cells and poorly metastatic PC3 and KMl2 cells as compared with that of multidrug resistant CEM/MDR and MCF-7/MDR cells and highly metastatic PC3-MM2 and KM/L4a cells, respectively. These results suggest differential modulation of DNA-PK by ST1571 treatment in drug resistance and metastatic degree dependent manner. We showed that CPT as well as ST1571 significantly inhibits the expression of DNA-PK. The combined treatment with ST15fl and CPT revealed synergistic effect, and the effect was accompanied by inhibition of cell proliferation due to significant reduced expression of DNA-PK components, which resulted in CPT sensitizes human cancer cells resistant to ST1571. Therefore, the results of our study suggested that the suppression of DNA-PK using combination of ST1571 and CPT could be a novel molecular target for against drugresistant and metastatic cancer cells.

• BMB Reports
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• v.40 no.3
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• pp.432-438
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• 2007

V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the $365^{th}$ serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.

• Journal of Veterinary Clinics
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• v.21 no.3
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• pp.253-258
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• 2004

DNA double-strand break (DSB) is a serious treat for the cells including mutations, chromosome rearrangements, and even cell death if not repaired or misrepaired. Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) bound to double strand DNA breaks are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and the interaction is essential for DNA-dependent protein kinase (DNA-PK) activity. The Ku80 mutants were designed to bind Ku70 but not DNA end binding activity and the peptides were treated in breast cancer cells for co-therapy strategy to see whether the targeted inhibition of DNA-dependent protein kinase (DNA-PK) activity sensitized breast cancer cells to ionizing irradiation or chemotherapy drug to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. We designed domains of Ku80 mutants, 26 residues of amino acids (HN-26) as a control peptide or 38 (HNI-38) residues of amino acids which contain domains of the membrane-translocation hydrophobic signal sequence and the nuclear localization sequence, but HNI-38 has additional twelve residues of peptide inhibitor region. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, resulting in inactivation of DNA-PK complex activity in breast cancer cells (MDA-465 and MDA-468). Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to irradiation or chemotherapy drugs. The growth of breast cancer cells was also inhibited. These results demonstrate the possibility of synthetic peptide to apply breast cancer therapy to induce apoptosis of cancer cells.

• Journal of Life Science
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• v.19 no.8
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• pp.1023-1032
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• 2009

Despite the fact that many cancer cells are sensitive to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, some cancer cells show either partial or complete resistance to TRAIL. Human leukemic K562 and CEM cells also show resistance to TRAIL-induced apoptosis. Novel molecular target and treatment strategies are required to overcome TRAIL resistance of human leukemia cells. Therefore, the purpose of this study was to target key anti-apoptotic molecules deciding TRAIL resistance for sensitization of TRAIL-resistant K562 and CEM cells, and to evaluate the effect of quercetin as a TRAIL sensitizer on these TRAIL-resistant cells. We found that quercetin acted in synergy with TRAIL to enhance TRAIL-induced apoptosis in K562 cells by inhibition of the DNA-PK/Akt signaling pathway, which leads to enhancement of TRAIL-mediated activation of caspases and concurrent cleavage of PARP and up-regulation of Bax. The findings suggest that the DNA-PK/Akt signaling pathway plays an essential role in regulating cells to escape from TRAIL-induced apoptosis, and quercetin could act in synergy with TRAIL to increase apoptosis by inhibition of the DNA-PK/Akt signaling pathway, which overcomes TRAIL-resistance of K562 and CEM cells. This study suggests that DNA-PK might interfere with TRAIL-induced apoptosis in human leukemic cells through activation of the Akt signaling pathway.

• Toxicological Research
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• v.24 no.3
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• pp.175-182
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• 2008

DNA-dependent protein kinase(DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination and is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKcs. It has been suggested that DNA-PK might be $2^{nd}$ upstream kinase for protein kinase B(PKB). In this report, we showed that Ser473 phosphorylation in the hydrophobic-motif of PKB is blocked in DNA-PK knockout mouse embryonic fibroblast cells(MEFs) following insulin stimulation, while there is no effect on Ser473 phosphorylation in DNA-PK wild type MEF cells. The observation is further confirmed in human glioblastoma cells expressing a mutant form of DNA-PK(M059J) and a wild-type of DNA-PK(M059K), indicating that DNA-PK is indeed important for PKB activation. Furthermore, the treatment of cells with doxorubicin, DNA-damage inducing agent, leads to PKB phosphorylation on Ser473 in control MEF cells while there is no response in DNA-PK knockout MEF cells. Together, these results proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.1
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• pp.9-14
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• 2003

Recent studies indicated that cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs by enhanced DNA repair of the lesions. Therefore, it is expected to increase the killing of cancer cells and reduce drug resistance by inhibiting DNA repair pathways that tumor cells rely on to escape chemotherapy. There are a number of key human DNA repair pathways which depend on multimeric polypeptide activities. For example, Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) on binding to double strand DNA breaks (DSBs) are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and are essential for DNA-dependent protein kinase (DNA-PK) activity. It has been known that DNA-PK is an important factor for DNA repair and also is a sensor-transmitting damage signal to downstream targets, leading to cell cycles arrest. Our ultimate goal is to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. This would greatly facilitate tumor cell cytotoxic activity and programmed cell death through DNA damaging drug treatment. Therefore, we designed a domain of Ku80 mutants that binds to Ku70 but not DNA end binding activity and used the peptide in co-therapy strategy to see whether the targeted inhibition of DNA-PK activity sensitized breast cancer cells to irradiation or chemotherapy drug. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, thus resulting in inactivation of DNA-PK activity. Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to IR or chemotherapy drugs, and the growth of breast cancer cells was inhibited. Additionally, the results obtained in the present study also support the physiological role of resistance of cancer cells to IR or chemotherapy.

• Journal of Life Science
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• v.13 no.3
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• pp.241-247
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• 2003

While the DNA-protein kinase (DNA-PK) complex, comprised of DNA-PKcs and Ku80, is primary involved in the repair of DNA double-strand breaks, it is also believed to participate in additional cellular processes. Here, treatment of embryo fibroblasts (MEFs) derived from either wild-type (Wt) or DNA-PKcs-null (DNA-$PKcs^{-/-}$) mice with various stress inducing agents revealed that adriamycin was markedly more cytotoxic for $Ku80^{-/-}MEFs$ and led to their long-term accumulation in the $G_2$/M phase. This differential response was not due to differences in DNA repair, since adrimycin-triggered DNA damage was repaired with comparable efficiency in both Wt and $Ku80^{-/-}MEFs$, but was associated with differences in the expression of important cell cycle regulatory genes. Our results support the notion that Ku80-mediated cytoprotection and $G_2$/M-progression are not only dependent on the cell's DNA repair but also may reflect Ku80's influence on additional cellular processes such as gene expression.

• The Plant Pathology Journal
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• v.20 no.2
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• pp.115-126
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• 2004

The actinomycete strain PK-A41 was isolated from a soil sample from pepper fields in Ko-yang, Korea. The strain PK-A41 inhibited the mycelial growth of some plant pathogenic fungi and oomycete, Alternaria mali, Colletotrichum orbiculare, Fusarium oxysporum f.sp. lycopersici, Magnaporthe grisea, Rhizoctonia solani, and Phytophthora capsici. The presence of LL-diaminopi-melic acid in the cell wall extract and the nucleotide sequence of the 16S rDNA region of the strain PK-A41 was assigned to Streptomyces scabiei. Further morpho-logical, biochemical, and pathological analyses also confirmed the strain PK-A41 to be S. scabiei, which is pathogenic to potato tubers. The maximum antibiotic production of the strain PK-A41 was achieved when grown on the glycerol peptone broth (GPB) medium for 9 days.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1199-1206
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• 2006

Plasminogen kringle 5 is a potent inhibitor of endothelial tell proliferation like an endogenous angiogenesis inhibitor, angiostatin consisting of plasminogen kringles 1-4. In this study, we produced the recombinant protein of plasminogen kringle 5 (PK5) employing an Pichia expression system and examined its. effect on~endothelial cell migration and its possible inhibitory mechanism. PK5 was expressed in Pichia pastoris GS115 by fusion of the cDNA spanning from Thr456 to Phe546 to the secretion signal sequence of a-factor prepro-peptide. After methanol induction, the secreted PK5 was purified by using S-spin column. SDS-PACE analysis of the purified protein showed one major band of approximately 10kDa. In in vitro migration assays, the purified protein inhibited dose-dependently the migration of human umbilical endothelial cells (HUVECs) induced by basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) with an $IC_{50}$ of approximately 500nM. Accordingly, it inhibited bfGF-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in HUVECs at 500nM. In addition, it also potently inhibited bFGF-induced cytoskeletal rearrangement of HUVECs. Thus, these results suggest that Pichia-produced PK5 effectively inhibits endothelial cell migration, in part by suppression of ERK1/2 activation and blocking cytoskeleton rearrangement.