Expression of Ku70 and Ku80 mediated by NF-kappa B and cyclooxygenase-2 is related to proliferation of human gastric cancer cells.

Abstract

Cyclooxygenase-2 (COX-2) expression is mediated by constitutive NF-κB and regulates human gastric cancer cell growth and proliferation. Inactivating Ku70 or Ku80 suppresses cell growth and induces apoptosis. It has been hypothesized that Ku70 and Ku80 expression may be associated with NF-κB activation and COX-2 expression and is involved in cell proliferation. In this study, we found that inhibition of constitutive NF-κB (by transfecting a mutated IκBα gene) and of COX-2 (by treatment with indomethacin and NS-398) suppressed Ku70 and Ku80 expression in cells. Treatment with prostaglandin E2 adenocarcinoma gastric (AGS) increased expression of these Ku proteins in cells with low constitutive NF-κB levels. Inhibition of the Ku DNA end-binding activity by transfection with the C-terminal Ku80 expression gene suppressed cell proliferation. Ku70 or Ku80 overexpression by transfection with the Ku70 or Ku80 expression gene, respectively, enhanced proliferation of cells with low NF-κB levels. These results demonstrate that Ku70 and Ku80 expression is mediated by constitutively activated NF-κB and constitutively expressed COX-2 in gastric cancer cells and that the high Ku DNA end-binding activity contributes to cell proliferation. Ku70 and Ku80 expression may be related to gastric cell proliferation and carcinogenesis.

NF-κB is an inducible transcription factor that regulates the activation of a wide variety of genes that respond to immune or inflammatory signals (1). In resting cells, NF-κB is localized to the cytoplasm as a hetero- or homodimer, which is noncovalently associated with the cytoplasmic inhibitory protein IκBα. Upon stimulation with a variety of pathogenic inducers such as viruses, mitogens, bacteria, agents providing oxygen radicals, and inflammatory cytokines, IκBα is phosphorylated, ubiquitinated, and degraded in the cytoplasm, and the NF-κB complex migrates into the nucleus and binds the DNA recognition sites in the regulatory regions of the target genes (2). However, constitutive NF-κB is aberrantly activated in lymphomas and human breast and gastric cancer cells in the resting state (3-6). There are several reports on the role of NF-κB gene products in cell proliferation, transformation, and tumor development (7, 8). Cyclooxygenase-2 (COX-2),1 an inducible isoform of cyclooxygenase, is also constitutively expressed in certain groups of cancers (9-11) and is related to cell proliferation (12, 13). COX-2 inhibition by specific COX-2 inhibitors suppresses cell proliferation, induces apoptosis, and down-regulates the expression of the anti-apoptotic protein Bcl-2 in pancreatic and colorectal cancer cells (14-19). Prostaglandins that are produced via COX-2 include prostaglandin E2 (PGE2) (20, 21) and prostaglandins A1, A2, and D2(22). They are believed to be the major contributors to cell proliferation and the inflammatory process (23, 24). A previous study demonstrated that COX-2 and prostaglandin syntheses are regulated by constitutive NF-κB, which is related to gastric cancer AGS cell proliferation (11).

The DNA repair protein Ku acts as a heterodimer of the two 70-kDa (Ku70) and 80-kDa (Ku80) subunits and binds to DNA ends, nicks, or single- to double-strand transition (25, 26). It serves as a DNA-binding component of a DNA-dependent protein kinase (DNA-PK) that phosphorylates certain chromatin-bound proteins in vitro (27, 28). Both Ku and the catalytic subunit of DNA-PK have been shown to be crucial for DNA double-strand break repair and V(D)J recombination (29-31). The Ku heterodimer binds to the double-strand DNA break and appears to stabilize the binding of the DNA-PK catalytic subunit to the DNA (32-35). Once bound, this complex stimulates DNA repair and signals the damage/stress responses, which might affect apoptosis and cell proliferation (36, 37). In addition, Umet al. (38) showed that Ku activity positively correlates with NF-κB activity in multidrug-resistant leukemia cells. Therefore, Ku activity can be regulated by NF-κB activity and affect cell growth and proliferation. Recent studies revealed growth retardation in both Ku70 and Ku80 knockout mice. Nussenzweig et al. (39, 40) demonstrated that the Ku80−/− embryonic stem cell line and Ku80−/− mutant primary embryonic fibroblasts display a reduction in cell growth and induction of cell apoptosis compared with Ku80+/− and Ku80+/+ control cells. Sadjiet al. (41) and Li et al. (42) showed that human Ku80 knockout colon cells exhibit slower growth than the corresponding control cells. The growth rate of murine embryonic fibroblasts derived from Ku70−/− embryos is lower than that of control murine embryonic fibroblasts (43). These studies show that inactivation of Ku70 or Ku80 drastically reduces the expression of other Ku subunits, resulting in inactivation of Ku DNA end-binding and DNA-PK activities. Moreover, the loss of one subunit destabilizes the other. Therefore, growth inhibition of Ku70- or Ku80-deficient cells would result from inactivation of Ku70 or Ku80 and inhibition of Ku DNA end-binding and DNA-PK activities. However, the phenotype of Ku70 knockout mice is somewhat different from that of Ku80 knockout mice (41, 43, 44). These studies suggest that either Ku70 or Ku80 might have a unique function that is independent of the other Ku subunit.

As described above, the expression of the COX-2 and Ku proteins (Ku70 and Ku80) is related to cell proliferation. Therefore, COX-2 expression mediated by constitutive NF-κB might be associated with the expression of both Ku70 and Ku80. This aim of this study was to investigate the role of Ku70 and Ku80 in cell proliferation, which may be mediated by constitutively activated NF-κB and constitutively expressed COX-2 in gastric cancer cells. This study examined whether or not constitutive NF-κB would be inhibited by transfection of the mutated IκBα gene and whether constitutively expressed COX-2 inhibited by treatment with the COX-2 inhibitors indomethacin and NS-398 would suppress Ku70 and Ku80 expression in gastric cancer AGS cells. To clarify the roles of the Ku DNA end-binding activity and Ku70 and Ku80 in cell proliferation, either AGS cells were transfected with the Ku dominant-negative gene to inactivate the Ku DNA end-binding activity, or the cells were transfected with either the Ku70 or Ku80 expression gene to overexpress Ku70 and Ku80, respectively. Cell proliferation was determined in the transfected cells. In addition, cells with low constitutive NF-κB levels were treated with PGE2 (a COX-2 product), and the expression of Ku70 and Ku80 in the cells was determined. A low constitutive NF-κB level was confirmed by Western blotting for NF-κB p65 in cytoplasmic extracts and nuclear extracts of the cells.