Accumulation of Ku80 proteins at DNA double-strand breaks in living cells

Ku plays a key role in multiple nuclear processes, e.g., DNA double-strand break (DSB) repair. The regulation mechanism of the localizations of Ku70 and Ku80 plays a key role in regulating the multiple functions of Ku. Although numerous biochemical studies in vitro have elucidated the DNA binding mechanism of Ku, no accumulation mechanisms of Ku70 and Ku80 at DSBs have been clarified in detail in vivo. In this study, we examined the accumulation mechanism of Ku80 at DSBs in living cells. EGFP-Ku80 accumulation at DSBs began immediately after irradiation. On the other hand, our data show that Ku70 alone, which has DNA binding activity independent of Ku80, cannot accumulate at the DSBs, whereas Ku70 bound to Ku80 can. The deletion of the C-terminal DNA-PKcs-binding domain and the mutation at the SUMOylation site of Ku80 had no effect on Ku80 accumulation. Unexpectedly, N-terminal deletion mutants of Ku80 fully lost their accumulation activity, although the mutants retained their Ku70 binding activity. Altogether, these data demonstrate that Ku80 is essential for Ku70 accumulation at DSBs. Furthermore, three domains of Ku80, i.e., the N-terminal α/β, the DNA-binding, and Ku70-binding domains, seem to necessary for the accumulation at or recognition of DSBs in the early stage after irradiation.     

Oestradiol inhibits spontaneous and cisplatin-induced apoptosis in epithelial ovarian cancer cells: relationship to DNA repair capacity

A prospective role of sex steroid hormones in the pathogenesis of common epithelial ovarian cancer remains equivocal. We hypothesized that oestradiol can protect ovarian cells from apoptosis by augmenting their DNA repair capacity. Two established oestrogen receptor-positive human cancer cell lines of ovarian surface epithelial origin (OVCAR-3, SKOV-3) were studied during short-term (24 h) subculture in the absence or presence of oestradiol-17β and/or the DNA-damaging chemotherapeutic agent cisplatin. Apoptosis was monitored among individual cells by in situ DNA fragmentation analysis. Basal rates of apoptosis were diminished by exposure to oestradiol (progesterone or testosterone were without effect). Oestradiol also suppressed apoptosis induced by cisplatin and enhanced the repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene transfected into ovarian cells. The ability of oestrogen-responsive ovarian cancer cells to efficiently repair DNA and thereby avoid apoptosis may be related to propensity for clonal expansion and drug resistance. This revised version was published online in November 2006 with corrections to the Cover Date.     

Regulation of homologous integration in yeast by the DNA repair proteins Ku70 and RecQ

The product of the BLM gene, which is mutated in Bloom syndrome in humans, and the Saccharomyces cerevisiae protein Sgs1 are both homologous to the Escherichia coli DNA helicase RecQ, and have been shown to be involved in the regulation of homologous recombination. Mutations in these genes result in genome instability because they increase the incidence of deletions and translocations. We present evidence for a genetic interaction between SGS1 and YKU70, which encodes the S. cerevisiae homologue of the human DNA helicase Ku70. In a yku70 mutant background, sgs1 mutations increased sensitivity to DNA breakage induced either by treatment with camptothecin or by the expression of the restriction enzyme EcoRI. The yku70 mutation caused a fourfold increase in the rate of double-strand break (DSB)-induced target integration as that seen in the sgs1 mutant. The combination of yku70 and sgs1 mutations additively increased the rate of the targeted integration, and this effect was completely suppressed by deletion of RAD51. Interestingly, an extra copy of YKU70 partially suppressed the increase in targeted integration seen in the sgs1 single mutant. These results suggest that Yku70 modulates the repair of DSBs associated with homologous recombination in a different way from Sgs1, and that the inactivation of RecQ and Ku70 homologues may enhance the frequency of gene targeting in higher eukaryotes.     

APPL proteins modulate DNA repair and radiation survival of pancreatic carcinoma cells by regulating ATM

Despite intensive multimodal therapies, the overall survival rate of patients with ductal adenocarcinoma of the pancreas is still poor. The chemo- and radioresistance mechanisms of this tumor entity remain to be determined in order to develop novel treatment strategies. In cancer, endocytosis and membrane trafficking proteins are known to be utilized and they also critically regulate essential cell functions like survival and proliferation. On the basis of these data, we evaluated the role of the endosomal proteins adaptor proteins containing pleckstrin homology domain, phosphotyrosine binding domain and a leucine zipper motif (APPL)1 and 2 for the radioresistance of pancreatic carcinoma cells. Here, we show that APPL2 expression in pancreatic cancer cells is upregulated after irradiation and that depletion of APPL proteins by small interfering RNA (siRNA) significantly reduced radiation survival in parallel to impairing DNA double strand break (DSB) repair. In addition, APPL knockdown diminished radiogenic hyperphosphorylation of ataxia telangiectasia mutated (ATM). Activated ATM and APPL1 were also shown to interact after irradiation, suggesting that APPL has a more direct role in the phosphorylation of ATM. Double targeting of APPL proteins and ATM caused similar radiosensitization and concomitant DSB repair perturbation to that observed after depletion of single proteins, indicating that ATM is the central modulator of APPL-mediated effects on radiosensitivity and DNA repair. These data strongly suggest that endosomal APPL proteins contribute to the DNA damage response. Whether targeting of APPL proteins is beneficial for the survival of patients with pancreatic adenocarcinoma remains to be elucidated.     

The apoptotic effect of intercalating agents on HPV-negative cervical cancer C-33A cells

Summary. Cervical cancer is one of the leading causes of female cancer death worldwide with about 500,000 deaths per year. Both mitomycin C and cisplatin are alkylating agents, which bind and intercalate DNA, and thus used as anti-cancer drugs. In these studies, we focused on investigating the apoptotic effects of intercalating agents on HPV-negative cervical cancer C-33A cells. Accordingly, C-33A cells were treated with carboplatin, mitomycin C or cisplatin. Cell cycle analysis revealed that treatment with mitomycin C and cisplatin but not with carboplatin resulted in apoptosis. Both mitomycin C and cisplatin induced apoptosis in C-33A cells via caspase-8 and -3 processing in a Fas/FasL-dependent manner and also suppressed IL-18 expression, while they down-regulated IκB expression and up-regulated p65 expression. These results suggest that both mitomycin C and cisplatin induce apoptosis, not only via the caspase-8 and -3 dependent Fas/FasL pathway, but also via the regulation of NF-κB activity and IL-18 expression in HPV-negative cervical cancer C-33A cells.     

Profiling signalling pathways of the receptor activator of NF-κB ligand-induced osteoclast formation in mouse monocyte cells,RAW264.7

Summary. Cell-based signal chemical genomics can profile the signalling pathway for certain cellular events by using a target-known chemical library. To ascertain its usefulness, the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse monocyte/macrophage cells RAW264.7 was used as an in vitro experimental model. Of 180 target-known inhibitors/activators formatted in a 384-well plate, 8 chemicals were shown to inhibit the osteoclast formation, but 4 chemicals enhanced this process. A variety of references support, or possibly lead one to expect the effects of these 12 chemicals on the cellular process of osteoclastogenesis in RAW264.7 cells, but several signalling pathways were newly found in this study; for example, CA-074 Me inhibiting cathepsin B and nitrendipine blocking the calcium channel could have the potential to inhibit the osteoclast formation as well as bone resorption. This is a simple but very fast and powerful method of profiling the signalling pathway of certain cellular events. Signal chemical genomics could provide invaluable information for the exploration of new target signalling processes and further target-based drug discovery strategies. Authors’ address: Seong Hwan Kim, PhD, Laboratory of Chemical Genomics, Bio-Organic Science Division, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong-gu, Daejeon 305-600, Korea     

Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells

Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis. The severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. It has been hypothesized that oxidative stress may induce a decrease in the Ku70 and Ku80 levels and apoptosis in pancreatic acinar cells. In this study, it was found that oxidative stress caused by glucose oxidase (GO) acting on beta-d-glucose, glucose/glucose oxidase (G/GO), induced slight changes in cytoplasmic Ku70 and Ku80 but drastically induced a decrease in nuclear Ku70 and Ku80 both time- and concentration-dependently in AR42J cells. G/GO induced apoptosis determined by poly(ADP-ribose) polymerase cleavage, an increase in expression of p53 and Bax, and a decrease in Bcl-2 expression. G/GO-induced apoptosis was in parallel with the loss of nuclear Ku proteins in AR42J cells. Caspase-3 inhibitor prevented G/GO-induced nuclear Ku loss and cell death. G/GO did not induce apoptosis in the cells transfected with either the Ku70 or Ku80 expression gene but increased apoptosis in those transfected with the Ku dominant negative mutant. Pulse and pulse-chase results show that G/GO induced Ku70 and Ku80 syntheses, even though Ku70 and Ku80 were degraded both in cytoplasm and nucleus. G/GO-induced decrease in Ku binding to importin alpha and importin beta reflects possible modification of nuclear import of Ku proteins. The importin beta level was not changed by G/GO. These results demonstrate that nuclear decrease in Ku70 and Ku80 may result from the decrease in Ku binding to nuclear transporter importins and the degradation of Ku proteins. The nuclear loss of Ku proteins may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.     

Ribosomal proteins S13 and L23 promote multidrug resistance in gastric cancer cells by suppressing drug-induced apoptosis

Ribosomal proteins (RP) S13 and RPL23 were previously identified as two upregulated genes in a multidrug-resistant gastric cancer cell line SGC7901/VCR compared to its parental cell SGC7901 by differential display PCR. The aim of this study was to explore the roles of RPS13 and RPL23 in multidrug resistance (MDR) in gastric cancer cells. RPS13 and RPL23 were genetically overexpressed in SGC7901 cells, respectively. Either RPS13 or RPL23 enhanced resistance of SGC7901 cells to vincristine, adriamycin, and 5-fludrouracil. RPL23 also enhanced resistance of SGC7901 cells to cisplatin. Overexpression of either RPS13 or RPL23 did not alter the population doubling time, [³H]leucine incorporation, and intracellular adriamycin accumulation of SGC7901 cells. However, either RPS13 or RPL23 could protect SGC7901 cells from undergoing vincristine-induced apoptosis. Western blot analysis revealed that both RPS13 and RPL23 significantly increased the expression level of Bcl-2 and Bcl-2/Bax ratio in SGC7901 cells. In addition, overexpression of RPL23 enhanced glutathione S-transferase (GST) activity and intracellular glutathione content in SGC7901 cells. Together, this work demonstrates that either RPS13 or RPL23 can promote MDR in gastric cancer cells by suppressing drug-induced apoptosis, and that RPL23 may also promote MDR in gastric cancer cells through regulation of glutathione S-transferase-mediated drug-detoxifying system.     

Inducible expression of maize polyamine oxidase in the nucleus of MCF-7 human breast cancer cells confers sensitivity to etoposide

Summary. In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H₂O₂, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine. Authors’ address: Dr. Paraskevi Tavladoraki, Department of Biology, University ‘Roma Tre’, Viale G. Marconi 446, 00146 Rome, Italy     

The role of growth hormone and amino acids on brain protein synthesis in aged rats given proteins of different quantity and quality

Summary. The purpose of the present study was to determine whether the regulation of brain protein synthesis was mediated through changes in the plasma concentrations of insulin and growth hormone (GH), and whether the concentrations of amino acids in the brain and plasma regulate the brain protein synthesis when the quantity and quality of dietary protein is manipulated. Two experiments were done on three groups of aged rats given diets containing 20% casein, 5% casein or 0% casein (Experiment 1), and 20% casein, 20% gluten, or 20% gelatin (Experiment 2) for 1 d (only one 5-h period) after all rats were fed the 20% casein diet for 10 d (only 5-h feeding per day). The aggregation of brain ribosomes, the concentration in plasma GH, and the branched chain amino acids in the plasma and cerebral cortex declined with a decrease of quantity and quality of dietary protein. The concentration of plasma insulin did not differ among groups. The results suggest that the ingestion of a higher quantity and quality of dietary protein increases the concentrations of GH and several amino acids in aged rats, and that the concentrations of GH and amino acids are at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.     

The rates of deceleration of nuclear and organellar DNA syntheses differ in the progenitor cells of the apical meristems during carrot somatic embryogenesis

 The synthesis of DNA in nuclei and organellar nucleoids at the various stages of somatic embryogenesis in carrot (Daucus carota L. cv. Kurodagosun) was analyzed using anti-5-bromo-2′-deoxyuridine (BrdU) immunofluorescence microscopy. The active syntheses of both nuclear and organellar DNA started in the cells forming the embryo proper 3 d after the initiation of embryogenesis, but not in cells forming suspensor-like cell aggregates. In the early globular embryo, active DNA syntheses were continuously observed in the whole embryo proper, except for the progenitor cells of the root apical meristem (RAM) and shoot apical meristem (SAM). These were recognized as slowly cycling cells with a non-BrdU-labelled nucleus and strongly BrdU-labelled organellar nucleoids. At the heart- and torpedo-shaped embryo stages, both nuclear and organellar DNA syntheses were inactive in the presumptive RAM and SAM. Thus, slowing down of organellar DNA synthesis is not coupled with, but is later than, that of nuclear DNA synthesis in the progenitor cells of the embryonic RAM and SAM. These findings clearly indicate that the timing of DNA synthesis is similar in the progenitor cells of both the RAM and SAM in the early stages of somatic embryogenesis. Received: 18 January 2000 / Accepted: 2 March 2000     

Tissue transglutaminase induces the release of apoptosis inducing factor and results in apoptotic death of pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignant disease with poor long-term survival rates. Major reason for poor disease outcome is the profound intrinsic resistance of PDAC cells to currently available treatment regimens. We recently found that a great majority of PDAC tumors and tumor cell lines express high basal level of tissue transglutaminase (TG2), a multifunctional protein implicated in apoptosis, cell attachment, cell survival, and cell motility functions. Based on these observations, we hypothesized that activation of endogenous TG2 can induce spontaneous apoptosis in PDAC cells. The results obtained suggested that activation of endogenous TG2 by calcium ionophore A23187 induced rapid and spontaneous apoptosis in PDAC cells. TG2-induced apoptosis was associated with release of apoptosis-inducing factor (AIF). The release of AIF from mitochondria led to its translocation to the nucleus and subsequent apoptosis of PDAC cells in caspase-independent manner. In conclusion, our results provide first evidence that TG2 can induce apoptosis in PDAC cells in an AIF-dependent and caspase-independent manner.     

Characterization of human and rat immortalized clones of parotid acinar cells with respect to specific proteins and their mRNAs,and receptor-linked adenylate cyclase

Summary This study reports the isolation and characterization of a rat nontumorigenic parotid acinar cell clone (2RSG), a human nontumorigenic parotid acinar cell clone (2HPC8), and a human tumorigenic acinar clone (2HP₁G). The levels ofα-amylase mRNAs detected when usingα-amylase cDNA of 1176 and 702 bp for hybridization were higher in 2RSG and 2HPC8 cells than their respective whole parotid glands. The level of these mRNAs decreased in 2HP₁G cells. In contrast toα-amylase mRNAs levels, theα-amylase activity in cultured acinar cells was extremely low in comparison to whole glands, irrespective of species or cell status. The levels of proline-rich protein (PRP) mRNA and parotid secretory protein (PSP) mRNA detected when using PRP cDNA of 600 bp and PSP cDNA of 805 bp for hybridization were higher in 2RSG cells than those in rat parotid glands; the reverse was observed in 2HPC8 cells and human parotid glands. The levels of PRP mRNA and PSP mRNA in 2HPC8 and 2HP₁G acinar cells were similar. The level of mRNA was not detectable in murine neuroblastoma cells (NBP2) using the sameα-amylase cDNA, PRP cDNA and PSP cDNA for hybridization. The PSP level in rat parotid gland was lower than that found in 2RSG cells; the reverse was observed in 2HPC8 cells and human parotid glands. The level of PSP in 2HP₁G cells was higher than that found in 2HPC8 cells. Isoproterenol increased the cAMP level in 2RSG, 2HPC8, and 2HP₁G clones, being most effective in 2RSG cells, and least effective in 2HPG cells. Prostaglandin E₁ (PGE₁) also increased cAMP level, being most effective in 2HPC8 cells and ineffective in 2HP₁G cells, suggesting that the PGE₁ receptor-linked adenylate cyclase becomes inactive upon transformation. These results suggest that the three clonal acinar cells from rat and human parotid glands reported here can be useful in comparative studies on regulation of growth, differentiation, and transformation.     

Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins

Summary. Thermophilic proteins show substantially higher intrinsic thermal stability than their mesophilic counterparts. Amino acid composition is believed to alter the intrinsic stability of proteins. Several investigations and mutagenesis experiment have been carried out to understand the amino acid composition for the thermostability of proteins. This review presents some generalized features of amino acid composition found in thermophilic proteins, including an increase in residue hydrophobicity, a decrease in uncharged polar residues, an increase in charged residues, an increase in aromatic residues, certain amino acid coupling patterns and amino acid preferences for thermophilic proteins. The differences of amino acids composition between thermophilic and mesophilic proteins are related to some properties of amino acids. These features provide guidelines for engineering mesophilic protein to thermophilic protein. Authors’ addresses: Yuan-Jiang Pan, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Zhejiang University Road 38, Hangzhou 310027, China; Wei-Fen Li, Microbiology Division, College of Animal Science, Zhejiang University, Hangzhou 310029, China     

Physiological significance of taurine and the taurine transporter in intestinal epithelial cells

Summary. Taurine transport in human intestinal epithelial Caco-2 cells was down-regulated by culturing the cells in taurine-containing media and was up-regulated in a taurine-free medium. This adaptive regulation was associated with changes in both the Vmax and Km values of taurine transport. A change in the mRNA level of the taurine transporter (TAUT) in this regulation was also observed. The presence of such a regulatory mechanism for maintaining the intracellular taurine content at a certain level suggests that taurine plays an important role in the intestinal cell functions. The intracellular taurine content was increased when Caco-2 cells were exposed to a hypertonic stress. TAUT was up-regulated via the increased expression of TAUT mRNA in the hypertonic cells, suggesting that taurine serves as an osmolyte and protects the cells from osmotic stress. Similar up-regulation of TAUT was observed in the small intestine of water-deprived rats. Received January 25, 2000/Accepted January 31, 2000