Chi Dug Kang

Bcr-Abl-Independent Imatinib-Resistant K562 Cells Show Aberrant Protein Acetylation and Increased Sensitivity to Histone Deacetylase Inhibitors

Bcr-Abl-independent signaling pathways are known to be involved in imatinib resistance in some patients with chronic myelogenous leukemia (CML). In this study, to find new targets for imatinib-resistant CML displaying loss of Bcr-Abl kinase target dependence, we isolated imatinib-resistant variants, K562/R1, K562/R2, and K562/R3, which showed profound declines of Bcr-Abl levels and its tyrosine kinase activity, from K562 cells. Importantly, the imatinib resistance mechanism in these variants also included aberrant acetylation of nonhistone proteins such as p53, Ku70, and Hsp90 that was due to upregulation of histone deacetylases (HDACs) and down-regulation of histone acetyltransferase (HAT). In comparison with K562 cells, the imatinib-resistant variants showed up-regulation of HDAC1, -2, and -3 (class I HDACs) and class III SIRT1 and down-regulation of CBP/p300 and PCAF with HAT activity, and thereby p53 and cytoplasmic Ku70 were aberrantly acetylated. In addition, these were associated with down-regulation of Bax and up-regulation of Bcl-2. In contrast, the class II HDAC6 level was significantly decreased, and this was accompanied by an increase of Hsp90 acetylation in the imatinib-resistant variants, which was closely associated with loss of Bcr-Abl. These results indicate that alteration of the normal balance of HATs and HDACs leads to deregulated acetylation of Hsp90, p53, and Ku70 and thereby leads to imatinib resistance, suggesting the importance of the acetylation status of apoptosis-related nonhistone proteins in Bcr-Abl-independent imatinib resistance. We also revealed that imatinib-resistant K562 cells were more sensitive to suberoylanilide hydroxamic acid, an HDAC inhibitor, than K562 cells. These findings may have implications for HDAC as a molecular target in imatinib-resistant leukemia cells.

Increased and correlated nuclear factor-kappa B and Ku autoantigen activities are associated with development of multidrug resistance

In this study, we investigated possible engagement of NF-κB and Ku autoantigen (Ku) activation in development of multidrug resistance (MDR) and circumvention of MDR by modulation of NF-κB and Ku. The NF-κB activity and NF-κB p65 subunit level were constitutively higher in MDR cells than in drug-sensitive parental cells. Interestingly, a faster running NF-κB DNA binding complex was identified as Ku, a DNA damage sensor and a key double strand break repair protein, and was positively correlated with the NF-κB activity in MDR cells and Ku- or both subunits of NF-κB-transfected cells. Also both NF-κB and Ku activities were activated or inhibited by treatment with etoposide (VP-16) or MG-132 (a proteasome inhibitor), respectively. Furthermore, PKA inhibitor suppressed markedly the constitutive and drug-induced activities of NF-κB and Ku in MDR cells and subsequently potentiated the cytotoxic activity of anticancer drugs. Our results proposed that the NF-κB and Ku activation could be one of multi-factorial MDR mechanism, and PKA inhibitor, likely via inhibition of NF-κB and Ku activities, could enhance the effectiveness of anticancer drugs against MDR cells with high activities of NF-κB and Ku.

Tissue-specific changes of DNA repair protein Ku and mtHSP70 in aging rats and their retardation by caloric restriction.

To provide an improved understanding of the molecular basis of the aging process, it is necessary to measure biological age on a tissue-specific basis. The role of DNA damage has emerged as a significant mechanism for determination of life span, and DNA repair genes and stress-response genes are also implicated in the aging process. In the present study, we investigated the changes of DNA-PK activity, especially Ku activity, in the various tissues including kidney, lung, testis and liver during aging and its correlation with mtHSP70 expression. We showed that the modulation of Ku activity during the aging process was highly tissue-specific as shown with highly impaired Ku activity in testis and unaffected Ku activity in liver with age, and the level of Ku70 or Ku80 was differentially expressed in each aging tissue. We found also that age-associated alteration of Ku70/80 was prevented or not prevented by caloric restriction (CR) in a tissue-specific manner. Age-related decline in Ku70 during the aging process was associated with the increase of mtHSP70, which could play a role as a predictive marker for aging related to Ku regulation, and CR retarded aging-induced mtHSP70.

Association of DNA-dependent protein kinase with hypoxia inducible factor-1 and its implication in resistance to anticancer drugs in hypoxic tumor cells.

Tumor hypoxia contributes to the progression of a malignant phenotype and resistance to ionizing radiation and anticancer drug therapy. Many of these effects in hypoxic tumor cells are mediated by expression of specific set of genes whose relation to therapy resistance is poorly understood. In this study, we revealed that DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double strand break repair, would be involved in regulation of hypoxia inducible factor-1 (HIF-1). HIF-1β-deficient cells showed constitutively reduced expression and DNA-binding activity of Ku, the regulatory subunit of DNA-PK. Under hypoxic condition, the expression and activity of DNA- PK were markedly induced with a concurrent increase in HIF-1α expression. Our result also demonstrated that DNA-PK could directly interact with HIF- and especially DNA-PKcs, the catalytic subunit of DNA-PK, could be involved in phosphorylation of HIF-1α, suggesting the possibility that the enhanced expression of DNA- PK under hypoxic condition might attribute to modulate HIF-1α stabilization. Thus, the correlated regulation of DNA-PK with HIF-1 could contribute to therapy resistance in hypoxic tumor cells, and it provides new evidence for developing therapeutic strategies enhancing the efficacy of cancer therapy in hypoxic tumor cells.

DNA‐dependent protein kinase is involved in heat shock protein‐mediated accumulation of hypoxia‐inducible factor‐1α in hypoxic preconditioned HepG2 cells

Hypoxic preconditioning may afford protection against subsequent lethal hypoxia. As hypoxic tolerance induces changes in the expression of genes involved in DNA damage and repair response pathways, we investigated whether DNA‐dependent protein kinase (DNA‐PK), one of the DNA double‐strand break repair proteins, could be involved in hypoxic preconditioning‐induced protective signaling cascades. We showed that induction of hypoxia‐inducible factor‐1α expression during hypoxic preconditioning by repeated hypoxic exposure was associated with increased mRNA and protein levels of DNA‐PK catalytic subunit (DNA‐PKcs) and Ku70/Ku80, the DNA‐PK components, in human hepatoma HepG2 cells, followed by upregulation of Hsp70/Hsp90 and Bcl‐2 and concurrent downregulation of Bax. Additionally, loss of DNA‐PKcs led to attenuated expression of Hsp70/Hsp90, accelerated hypoxia‐inducible factor‐1α degradation, and increased susceptibility to hypoxia‐induced cell death. We also found that the mRNA and protein levels of heat shock factor‐1 (HSF1) were progressively increased with DNA‐PK activation during hypoxic preconditioning, and inhibition of HSF1 function by KNK437 resulted in a significant decrease in the level of protein kinase Akt as well as of DNA‐PKcs, with downregulation of Hsp70/Hsp90 and HIF‐1α. Our results suggest the possibility that DNA‐PK‐mediated signaling pathway is required for the increase in HIF‐1α expression through activation of HSF1 and subsequent upregulation of heat shock proteins after hypoxic reconditioning.

Radiosensitization by targeting radioresistance-related genes with protein kinase A inhibitor in radioresistant cancer cells

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-κB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.

Involvement of DNA-dependent protein kinase in regulation of the mitochondrial heat shock proteins.

Since DNA-dependent protein kinase (DNA-PK) has been known to play a protective role against drug-induced apoptosis, the role of DNA-PK in the regulation of mitochondrial heat shock proteins by anticancer drugs was examined. The levels of basal and drug-induced mitochondrial heat shock proteins of drug-sensitive parental cells were higher than those of multidrug-resistant (MDR) cells. We also demonstrated that the development of MDR might be correlated with the increased expression of Ku-subunit of DNA-PK and concurrent down-regulation of mitochondrial heat shock proteins. The basal mtHsp70 and Hsp60 levels of Ku70(-/-) cells, which were known to be sensitive to anticancer drugs, were higher than those of parental MEF cells, but conversely these mitochondrial heat shock proteins of R7080-6 cells over-expressing both Ku70 and Ku80 were lower than those of parental Rat-1 cells. Also, the mtHsp70 and Hsp60 levels of DNA-PKcs-deficient SCID cells were higher than those of parental CB-17 cells. Our results suggest the possibility that mitochondrial heat shock protein may be one of determinants of drug sensitivity and could be regulated by DNA-PK activity.

Fractionated Irradiation Leads to Restoration of Drug Sensitivity in MDR Cells that Correlates with Down-regulation of P-gp and DNA-Dependent Protein Kinase Activity

Abstract Ryu, J. S., Um, J. H., Kang, C. D., Bae, J. H., Kim, D. U., Lee, Y. J., Kim, D. W., Chung, B. S. and Kim, S. H. Fractionated Irradiation Leads to Restoration of Drug Sensitivity in MDR Cells that Correlates with Down-regulation of P-gp and DNA-Dependent Protein Kinase Activity. Radiat. Res. 162, 527–535 (2004). We showed that the drug sensitivity of multidrug-resistant (MDR) cells could be enhanced by fractionated irradiation. The molecular changes associated with fractionated radiation-induced chemosensitization were characterized. Irradiated cells of the multidrug-resistant CEM/MDR sublines (CEM/ MDR/IR1, 2 and 3) showed a loss of P-glycoprotein (P-gp) and concurrent reduction of Ku DNA binding and DNA-PK activities with decreased level of Ku70/80 and increased level of DNA-PKcs, and these changes were followed by an increased susceptibility to anticancer drugs. These irradiated MDR cells also exhibited the reduction of other chemoresistance-related proteins, including BCL2, NF-κB, EGFR, MDM2 and Ku70/80, and the suppression of HIF-1α expression induced by hypoxia. In contrast, fractionated irradiation increased the levels of these proteins and induced drug resistance in the parental drug-sensitive CEM cells. These results suggest that the chemoresistance-related proteins are differentially modulated in drug-sensitive and MDR cells by fractionated irradiation, and the optimized treatment with fractionated radiation could lead to new chemoradiotherapeutic strategies to treat multidrug-resistant tumors.

Anti-inflammatory effects of Artemisia princeps in antigen-stimulated T cells and regulatory T cells.

Objectives The aim was to investigate the anti‐inflammatory effects of Artemisia princeps extract on the activity of anti‐CD3/CD28‐stimulated CD4+CD25‐ T cells and antigen‐expanded regulatory T cells.

Synergistic antitumor activity of STI571 and camptothecin in human cancer cells

The in vitro activity of STI571, 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 STI571 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 KM12 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 STI571 treatment in drug resistance and metastatic degree dependent manner. We showed that CPT as well as STI571 significantly inhibits the expression of DNA-PK. The combined treatment with STI571 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 STI571. Therefore, the results of our study suggested that the suppression of DNA-PK using combination of STI571 and CPT could be a novel molecular target for against drug resistant and metastatic cancer cells.