Potentiation of chemosensitivity in multidrug-resistant human leukemia CEM cells by inhibition of DNA-dependent protein kinase using wortmannin

DNA-dependent protein kinase (DNA-PK) is activated by DNA strand breaks and participates in DNA repair. Its regulatory subunit, Ku autoantigen, binds to DNA and recruits the catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the development of multidrug resistance (MDR). The Ku-DNA binding activity, the levels of Ku70/Ku80 and DNA-PKcs in MDR variants, CEM/VLB(10-2), CEM/VLB(55-8) and CEM/VLB100 were higher than those in their parental drug-sensitive CEM cells in a drug resistance-dependent fashion. Also, CEM/VLB100 cells showed about 3-fold increase of DNA-PK enzyme activity as compared with CEM cells. Similar results were observed in another MDR cell line, FM3A/M mouse mammary carcinoma cells. Moreover, we observed that CEM/VLB100 cells were about 11-fold sensitive to wortmannin, which inhibits DNA-PK, compared with the CEM cells, and sensitized the MDR cells when combined with either bleomycin or vincristine, but have a little effect on CEM cells. Wortmannin was shown to inhibit DNA-PK and Ku-DNA binding activity in CEM/VLB100 cells dose dependently but had a little or no effect on their parental cells. Our results suggested that enhanced expression of DNA-PK participates in the development of MDR, and the use of DNA-PK inhibitors such as wortmannin is likely to improve the effectiveness of anticancer drugs and thus could partially overcome drug resistance in MDR cells, through its ability to inhibit Ku/DNA-PK activity.     

鼻咽癌变过程中DNA依赖蛋白激酶表达水平研究

Objective:To observe the expression of DNA-dependent protein kinase (DNA-PKcs) in the nasopharyngeal tissues during carcinogenesis.Methods:Patients including 30 cases of nasopharyngitis,42 cases of nasopharyngeal atypical hyperplasia and 34 cases of nasopharyngeal carcinoma (NPC) were chosen from Zhongshan area in which there is high incidence rate of NPC.The expression of DNA-PKcs in the nasopharyngeal tissue was examined by immunohistochemistry.Results:The expression rates of DNA-PKcs in the nasopharyngitis tissue,the nasopharyngeal precancerous lesion and the NPC were 6.7%,64.3% and 55.9%,respectively (P＜0.001).Conclusion:The expression of DNA-dependent protein kinase may play an important role in nasopharyngeal tissue carcinogenesis.     

DNA-dependent protein kinase in leukaemia cells and correlation with drug sensitivity

We investigated whether the levels of the DNA-dependent protein kinase (DNA-PK) activity and content correlate with drug sensitivity in different tumour materials and if this can be utilised in predicting treatment outcome. DNA-PK activity and expression were investigated in tumour cells from 8 patients with chronic lymphocytic leukaemia (CLL) and 18 patients with acute myeloid leukaemia (AML), using Western blot and DNA-PK kinase activity assay. Tumour cells from the patients were investigated for their drug sensitivity to topoisomerase II inhibitors (doxorubicin and etoposide), DNA reactive agents (melphalan, 4-hydroxycyclophosphamide and cisplatinum), an antimetabolite (cytosine arabinoside) and an antimicrotubule agent (vincristine) by fluorometric microculture cytotoxicity assay (FMCA). Within each group of leukaemia there was a large variation in both DNA-PK activity and DNA-PKcs expression, while the Ku subunits were expressed more homogeneously. In CLL cells, sensitivity to topoisomerase II inhibitors correlated with DNA-PKcs protein expression (r=0.7174, p=0.0452). In AML samples, sensitivity to DNA cross-linking alkylating agents correlated with Ku86 (r=-0.7512, p=0.0031) and Ku70 (r=-0.6134, p=0.0258) expression. Unexpectedly, DNA-PK activity was found to correlate with sensitivity to vincristine in both CLL (r=0.8557, p=0.0067) and AML (r=0.5480, p=0.0228) cells. The results indicate that DNA-PK is not only involved in the recognition of DNA double-strand breaks (DSB), but also other DNA lesions.     

Phosphorothioate oligonucleotides, suramin and heparin inhibit DNA-dependent protein kinase activity

Phosphorothioate oligonucleotides and suramin bind to heparin binding proteins including DNA polymerases, and inhibit their functions. In the present study, we report inhibition of DNA-dependent protein kinase activity by phosphorothioate oligonucleotides, suramin and heparin. Inhibitory effect of phosphorothioate oligonucleotides on DNA-dependent protein kinase activity was increased with length and reached a plateau at 36-mer. The base composition of phosphorothioate oligonucleotides did not affect the inhibitory effect. The inhibitory effect by phosphorothioate oligodeoxycytidine 36-mer can be about 200-fold greater than that by the phosphodiester oligodeoxycytidine 36-mer. The inhibitory effect was also observed with purified DNA-dependent protein kinase, which suggests direct interaction between DNA-dependent protein kinase and phosphorothioate oligonucleotides. DNA-dependent protein kinase will have different binding positions for double-stranded DNA and phosphorothioate oligodeoxycytidine 36-mer because they were not competitive in DNA-dependent protein kinase activation. Suramin and heparin inhibited DNA-dependent protein kinase activity with IC(50) of 1.7 microM and 0.27 microg ml(-1) respectively. DNA-dependent protein kinase activities and DNA double-stranded breaks repair in cultured cells were significantly suppressed by the treatment with suramin in vivo. Our present observations suggest that suramin may possibly result in sensitisation of cells to ionising radiation by inactivation of DNA-dependent protein kinase and the impairment of double-stranded breaks repair.     

低剂量放射超敏感性和放射拒抗与DNA依赖蛋白激酶的表达

低剂量放射超敏感性是最近十几年来研究的热点课题之一。是继放射敏感性之后的又一重大发现,是对传统放射生物学的一个有益补充和发展。有助于解决肿瘤细胞的放射耐受和正常组织的放射损伤问题。对放射治疗分割方法的设计、物理计划设计和生物学优化都具有重要指导意义。低剂量放射超敏感性主要包括放射超敏感期和放射拒抗期。放射超敏感期与细胞凋亡密切相关,放射拒抗期主要以DNA损伤修复后非同源末端连接相关,DNA-PK(DNA-PKcs、Ku70、Ku80)等蛋白参与DNA非同源末端连接,在超敏中具有重要作用。本文对DNA-PK在低剂量超敏感性中的作用做一综述。     

Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.

Replication protein A (RPA, also known as human single-stranded DNA-binding protein) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair, and recombination. Phosphorylation of the RPA2 subunit is observed after exposure of cells to ionizing radiation (IR) and other DNA-damaging agents, which implicates the modified protein in the regulation of DNA replication after DNA damage or in DNA repair. Although ataxia telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported. Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these kinases and completely abolished in wild-type, ATM, or DNA-PK-deficient cells after treatment with wortmannin at a concentration-inhibiting ATM and DNA-PK. Caffeine, an inhibitor of ATM and ATM-Rad3 related (ATR) but not DNA-PK, generates an ataxia-telangiectasia-like response in wild-type cells, prevents completely RPA2 phosphorylation in DNA-PKcs deficient cells, but has no effect on ataxia-telangiectasia cells. These observations rule out ATR and implicate both ATM and DNA-PK in RPA2 phosphorylation after exposure to IR. UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation. Because UCN-01 abrogates checkpoint responses, this observation dissociates RPA2 phosphorylation from checkpoint activation. Phosphorylated RPA has a higher affinity for nuclear structures than unphosphorylated RPA suggesting functional alterations in the protein. In an in vitro assay for DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM. Because RPA2 phosphorylation kinetics are distinct from those of the S phase checkpoint, we propose that DNA-PK and ATM cooperate to phosphorylate RPA after DNA damage to redirect the functions of the protein from DNA replication to DNA repair.     

Decreased expression of DNA-dependent protein kinase, a DNA repair protein, during human colon carcinogenesis.

DNA-dependent protein kinase (DNA-PK), consisting of a catalytic subunit (DNA-PKcs) and the Ku70 and Ku86 proteins, participates in the repair of DNA double-strand breaks (DSBs). We assessed its expression immunohistochemically in normal human colon tissue, colon adenomas, colon carcinomas, and normal tissue distant from carcinomas. Normal colonocytes expressed all DNA-PK proteins. Compared with the expression in normal tissue [176.62 +/- 18.56 (the intensity of expression x the percentage of cells expressing this protein), mean + SE], the expression of Ku70 was significantly reduced in adenomas (36.62 +/- 11.09; P < 0.001) and carcinomas (85.68 +/- 15.76; P < 0.01), as was the expression of Ku86 [(113.10 +/- 10.22 versus 41.66 +/- 14.71 in adenomas (P < 0.01) or versus 85.68 +/- 15.76 in carcinomas (P < 0.05)]. The expression of DNA-PKcs was not significantly changed. The marked underexpression of Ku70 and Ku86 starting at the adenoma stage may be crucial to the development of colon cancer.     

Role of DNA-dependent protein kinase catalytic subunit in cancer development and treatment

DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of the non-homologous end-joining (NHEJ) pathway, is involved in DNA double-strand break repair, immunocompetence, genomic integrity, and epidermal growth factor receptor signaling. Clinical studies indicate that expression and activity of DNA-PKcs is correlated with cancer progression and response to treatment. Various anti-DNA-PKcs strategies have been developed and tested in preclinical studies to exploit the benefit of DNA-PKcs inhibition in sensitization of radiotherapy and in combined modality therapy with other antitumor agents. In this article, we review the association between DNA-PKcs and cancer development and discuss current approaches and mechanisms for inhibition of DNA-PKcs. The future challenges are to understand how DNA-PKcs activity is correlated with cancer susceptibility and to identify those patients who would most benefit from DNA-PKcs inhibition.     

DNA依赖蛋白激酶与大肠癌淋巴转移相关性研究

Objective:To investigate DNA-dependent protein kinase(DNA-PK)expression,and its relationship with lymphatic metastasis in colorectal cancer.Methods:Tumor tissues from 60 patients,divided into two groups according to lymphatic metastasis,were immunohistochemically stained to detect the DNA-PK expression including Ku70,Ku80 and PKcs proteins.Results:Positivity of both Ku70 and Ku80 in colorectal cancer was negatively correlated with lymphatic metastasis with an rvalue of-0.57 and -0.38,respectively.Similar correlation was found between Ku expression,especially Ku70.and long-term survival.PKcs,however,displayed no significant correlation.Statistical analysis failed to detect any correlation between DNA-PK expression,and clinical characteristics,such as age,sex,tumor Jocation,tumor thickness and distant metastasis(P＞0.05).Conclusion:DNA-PK expression,especially Ku70 expression,is negatively correlated with lymphatic metastasis,and the survival of patients with colorectal cancer.Ku70 expression may be a potential indicator for the preoperative evaluation,and prognosis in colorectal cancer.     

Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro.

It has been suggested that DNA-dependent protein kinase (DNA-PK) is a central component of DNA double-strand-break repair. The mechanism of DNA-PK action, however, has not been fully understood. Poly(ADP-ribose) polymerase (PARP) is another nuclear enzyme which has high affinity to DNA ends. In this study, we analysed the interaction between these two enzymes. First, DNA-PK was found to suppress the PARP activity and alters the pattern of poly(ADP-ribosyl)ation. Although DNA-PK phosphorylates PARP in a DNA-dependent manner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP. Conversely, PARP was found to ADP-ribosylate DNA-PK in vitro. However, the auto-phosphorylation activity of DNA-PK was not influenced by this modification. In a competitive electrophoretic mobility shift assay, Ku 70/80 complex, the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP. Furthermore, co-immunoprecipitation analysis suggested direct or close association between Ku and PARP. Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or sequestration of DNA-ends which serve as an important stimulator for both enzymes.     

Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1

The DNA repair enzymes, DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1), are key determinants of radio- and chemo-resistance. We have developed and evaluated novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) for use in anticancer therapy. PARP-1- and DNA-PK-deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR) in G(0) cells, compared with their proficient counterparts. NU7026 (10 micro M) potentiated IR cytotoxicity [potentiation factor at 90% cell kill (PF(90)) = 1.51 +/- 0.04] in exponentially growing DNA-PK proficient but not deficient cells. Similarly, AG14361 (0.4 micro M) potentiated IR in PARP-1(+/+) (PF(90) = 1.37 +/- 0.03) but not PARP-1(-/-) cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive (e.g., PF(90) = 2.81 +/- 0.19 in PARP-1(+/+) cells). Both inhibitors alone reduced PLDR approximately 3-fold in the proficient cell lines. Furthermore, the inhibitor combination completely abolished PLDR. IR-induced DNA double strand break (DNA DSB) repair was inhibited by both NU7026 and AG14361, and use of the inhibitor combination prevented 90% of DNA DSB rejoining, even 24-h postirradiation. Thus, there was a correlation between the ability of the inhibitors to prevent IR-induced DNA DSB repair and their ability to potentiate cytotoxicity. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radiosensitizers and show potential as tools for anticancer therapeutic intervention.     

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.     

Investigation of the relationship between DNA-dependent protein kinase and lymphatic metastasis in colorectal cancer

Objective  To investigate DNA-dependent protein kinase (DNA-PK) expression, and its relationship with lymphatic metastasis in colorectal cancer. Methods  Tumor tissues from 60 patients, divided into two groups according to lymphatic metastasis, were immunohistochemically stained to detect the DNA-PK expression including Ku70, Ku80 and PKcs proteins. Results  Positivity of both Ku70 and Ku80 in colorectal cancer was negatively correlated with lymphatic metastasis with an r value of −0.57 and −0.38, respectively. Similar correlation was found between Ku expression, especially Ku70, and long-term survival. PKcs, however, displayed no significant correlation. Statistical analysis failed to detect any correlation between DNAPK expression, and clinical characteristics, such as age, sex, tumor location, tumor thickness and distant metastasis (P > 0.05). Conclusion  DNA-PK expression, especially Ku70 expression, is negatively correlated with lymphatic metastasis, and the survival of patients with colorectal cancer. Ku70 expression may be a potential indicator for the preoperative evaluation, and prognosis in colorectal cancer. Supported by a grant from the Scientific Research Project of the Bureau of Health of Jiading in Shanghai (No. KYXM-2004-11-07).     

Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.     

DNA-dependent protein kinase: a major protein involved in the cellular response to ionizing radiation

DNA-dependent protein kinase (DNA-PK) is a DNA-activated nuclear serine/threonine protein kinase. DNA-PK consists of a regulatory sub-unit, the heterodimeric Ku protein (composed of a 70- and a 86-kDa subunit) which binds DNA ends and targets the catalytic sub-unit, DNA-PKcs to DNA strand breaks. DNA-PK plays a major role in the repair of double-strand breaks induced in DNA after exposure to ionizing radiation as shown by the extreme radiosensitivity of cells with mutations in Ku86, Ku70 or DNA-PKcs genes. Cells deficient in DNA-PK activity also exhibit hypersensitivity to genotoxic drugs such as cisplatin and nitrogen mustards. In the first part of this review, the current knowledge on the biochemical characteristics of DNA-PK, its mechanism of action in DNA repair and the phenotype of DNA-PK deficient cells is summarized. These results suggest that DNA-PK might play a role in the acquisition of a resistant phenotype of human tumors to radiotherapy, chemotherapy using genotoxic drugs or to both treatments. In the second part of this review, the studies devoted to inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.