Inhibition of protein kinase CK2 facilitates cellular senescence by inhibiting the expression of HO-1 in articular chondrocytes.

Protein kinase casein kinase 2 (CK2) is important in the regulation of cell proliferation and death, even under pathological conditions. Previously, we reported that CK2 regulates the expression of heme oxygenase­1 (HO­1) in stress­induced chondrocytes. In the present study, it was shown that CK2 is involved in the dedifferentiation and cellular senescence of chondrocytes. Treatment of primary articular chondrocytes with CK2 inhibitors, 4,5,6,7­terabromo­2­azabenzimidazole (TBB) or 5,6­dichlorobenzimidazole 1­ß­D­ribofuranoside (DRB), induced an increase in senescence­associated ß­galactosidase (SA­ß­gal) staining. In addition, TBB reduced the expression of type II collagen and stimulated the accumulation of ß­catenin, phenotypic markers of chondrocyte differentiation and dedifferentiation, respectively. It was also observed that the abrogation of CK2 activity by CK2 small interfering RNA induced phenotypes of chondrocyte senescence. The association between HO­1 and cellular senescence was also examined in CK2 inhibitor­treated chondrocytes. Pretreatment with 3­morpholinosydnonimine hydrochloride, an inducer of the HO­1 expression, or overexpression of the HO­1 gene significantly delayed chondrocyte senescence. These results show that CK2 is associated with chondrocyte differentiation and cellular senescence and that this is due to regulation of the expression of HO­1. Furthermore, the findings suggest that CK2 is crucial as an anti­aging factor during chondrocyte senescence.

Induction of heme oxygenase­1 expression protects articular chondrocytes against cilostazol­induced cellular senescence.

Chondrocyte senescence is associated with the aging and degeneration of cartilage, and eventually leads to joint destruction. The aim of this study was to elucidate the mechanisms responsible for the cytoprotective effects of heme oxygenase­1 (HO­1) on chondrocytes in cartilage. Chondrocyte senescence was induced using cilostazol and measured using a specific senescence­associated ß­galactosidase (SA­ß­gal) staining assay. Cilostazol altered the expression of type â…¡ collagen and ß­catenin, which are phenotypic markers of the differentiation and dedifferentiation of chondrocytes. Cilostazol also significantly induced HO­1 expression, and the induction of HO­1 expression was affected by a significant increase in reactive oxygen species (ROS) production caused by cilostazol treatment. Of note, pre­treatment with 3­morpholinosydnonimine hydrochloride (SIN­1), an inducer of HO­1 expression, markedly attenuated cilostazol­induced chondrocyte senescence, and thus, we examined whether HO­1 directly modulates chondrocyte senescence induced by cilostazol. The upregulation of HO­1 was found to suppress cilostazol­induced cellular senescence. In addition, the inhibition of HO­1 activity with the iron chelator, desferrioxamine (DFO), or HO­1 siRNA increased cilostazol­induced chondrocyte senescence. Based on these results, it can be concluded that HO­1 is associated with the suppression of chondrocyte senescence, and that the enforced overexpression of HO­1 protects chondrocytes against stress­induced senescence.

A novel cancer/testis antigen KP-OVA-52 identified by SEREX in human ovarian cancer is regulated by DNA methylation.

SEREX has proven to be a powerful method that takes advantage of the presence of spontaneous humoral immune response in some cancer patients. In this study, immunoscreening of normal testis and two ovarian cancer cell line cDNA expression libraries with sera from ovarian cancer patients led to the isolation of 75 independent antigens, designated KP-OVA-1 through KP-OVA-75. Of these, RT-PCR showed KP-OVA-52 to be expressed strongly in normal testis, in ovarian cancer cell lines (3/9) and in ovarian cancer tissues (1/17). The expression of KP-OVA-52 in cancer cells is also induced by the demethylating agent 5­aza­2'­deoxycytidine (ADC). To test immunogenicity, we used the Serum Antibody Detection Assay (SADA) to analyze anti-IgG antibodies against the 75 antigens that were initially isolated by SEREX. Four of the 75 antigens (KP­OVA­25, KP­OVA­35, KP­OVA­68 and KP­OVA­73) reacted exclusively with sera from cancer patients. However, KP­OVA­52 reacted with 1 of 20 ovarian cancer sera. These data suggest that the KP-OVA-52 can be considered a novel CT antigen that is regulated by DNA methylation.

Protein kinase CK2 mediates peroxynitrite-induced heme oxygenase-1 expression in articular chondrocytes.

Heme oxygenase-1 (HO-1) is induced as an adaptive mechanism against oxidative stress in chondrocytes, which play an important role in the maintenance and degradation of cartilage. In the present study, we examined the role of protein kinase casein kinase (CK2) on peroxynitrite-induced expression of HO-1 in primary articular chondrocytes. 3-Μorpholinosydnonimine hydrochloride (SIN-1) has been shown to mediate cell death by activating apoptosis-related molecules in cells. In this study, we used a low concentration of SIN-1 that did not induce apoptosis to elucidate the mechanism by which SIN-1 upregulates HO-1 expression. In chondrocytes, SIN-1 induced HO-1 expression with spontaneous downregulation in a different manner than with high concentrations of SIN-1. Importantly, SIN-1 treatment of chondrocytes increased CK2 activation. Additionally, inhibition of CK2 with 4,5,6,7-tetrabromobenzotriazole (TBB) or siRNA did not induce HO-1 expression and reduced NF-E2-related factor 2 (Nrf2) accumulation in chondrocytes. Therefore, we examined whether CK2 directly regulates Nrf2, which is a transcription factor that regulates the expression of HO-1. Indeed, TBB treatment inhibited phosphorylation and nuclear translocation of Nrf2 in SIN-1-treated cells. Moreover, using an immunoprecipitation assay, we confirmed that SIN-1 treatment enhanced the interaction between CK2 and Nrf2. Taken together, our findings suggest that peroxynitrite activates Nrf2 via CK2 signaling, leading to the upregulation of HO-1 in primary chondrocytes.

Cilostazol induces cellular senescence and confers resistance to etoposide-induced apoptosis in articular chondrocytes.

We recently reported that cilostazol protects chondrocytes against stress-induced apoptosis and prevents cartilage destruction in an osteoarthritis (OA) model. In the present study, we elucidate the mechanism underlying the protective effect induced by cilostazol against stress-induced apoptosis in chondrocytes. Cilostazol significantly reduced the expression of type II collagen and stimulated the accumulation of ß-catenin in primary rat articular chondrocytes. Moreover, cilostazol-induced chondrocytes showed induction of senescent phenotypes, such as changes in cell morphology, decrease in cell proliferation and increase in specific senescence-associated ß-galactosidase (SA-ß-gal) staining. Moreover, dedifferentiated chondrocytes obtained by serial subculture showed cellular senescence that increased with passage number. In addition, the percentage of terminal dUTP nick end-labeling (TUNEL)-positive cells was higher when chondrocytes were treated with cilostazol and the apoptosis inducer etoposide than when the cells were treated with etoposide alone. Our findings suggest that cilostazol induces dedifferentiation and senescence in rat articular chondrocytes and renders them resistant to etoposide-induced apoptosis.

Cobalt protoporphyrin induces differentiation of monocytic THP-1 cells through regulation of cytoplasmic Ref-1-related NADPH oxidase activity.

Cobalt protoporphyrin (CoPP) is a potent and effective metalloporphyrin inducer of heme oxygenase-1 (HO-1) activity in many tissues. Here, we report that CoPP induces differentiation of monocytic THP-1 cells into macrophage-like cells. CoPP induced a marked growth inhibition with a slight reduction in viability, and increased adhesion and spreading of THP-1 cells. However, other protoporphyrins did not. CoPP also resulted in expression of CD11b, MMP9, MSR1, CD14 and ICAM-1, which are differentiation markers for macrophages. Interestingly, we observed a decrease of cytoplasmic redox factor-1 (Ref-1) levels in the process of CoPP-induced differentiation of THP-1 cells. In addition, knockdown of Ref-1 by siRNA enhanced cell adhesion induced by CoPP. Furthermore, an inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), completely abolished CoPP-induced adhesion of Ref-1-deficient cells using an siRNA. A cytosolic factor for NADPH oxidase activity, p47phox, was significantly increased in THP-1 cells by CoPP treatment. Κnockdown of Ref-1 increased CoPP-induced p47phox expression in THP-1 cells. Taken together, these results suggest that CoPP induces differentiation of monocytic THP-1 cells, and that the CoPP-induced differentiation is associated with cytoplasmic Ref-1-related NADPH oxidase activity.

Molecular mechanism of diallyl disulfide in cell cycle arrest and apoptosis in HCT-116 colon cancer cells.

Diallyl disulfide (DADS) is the most prevalent oil-soluble sulfur compound in garlic and inhibits cell proliferation in many cancer cell lines. Here we examined DADS cytotoxicity in a redox-mediated process, involving reactive oxygen species (ROS) production. In the present study, p53-independent cell cycle arrest at G2/M phase was observed with DADS treatment, along with time-dependent increase of cyclin B1. In addition, apoptosis was also observed upon 24-h DADS treatment accompanied by activation of p53. In HCT-116 cells, DADS application induced a dose-dependent increase and time-dependent changes in ROS production. Scavenging of DADS-induced ROS by N-acetyl cysteine or reduced glutathione inhibited cell cycle arrest, apoptosis and p53 activation by DADS. These results suggest that ROS trigger the DADS-induced cell cycle arrest and apoptosis and that ROS are involved in stress-induced signaling upstream of p53 activation. Transfection of p53 small interfering RNA prevents the accumulation of cleaved poly(ADP-ribose) polymerase and sub-G1 cell population by 65% and 35%, respectively. Moreover, DADS-induced apoptosis was also prevented by treatment with oligomycin, which is known to prevent p53-dependent apoptosis by reducing ROS levels in mitochondria. These results suggest that mitochondrial ROS may serve as second messengers in DADS-induced apoptosis, which requires activation of p53.

Differential role of diphenyleneiodonium, a flavoenzyme inhibitor, on p53-dependent and -independent cell cycle progression.

We investigated the differential role of diphenyleneiodonium (DPI), which is widely used as an inhibitor of NADPH oxidase, on the activation of cell cycle regulators in the cell cycle progression. DPI efficiently blocked the transition from G0/G1 to S phase by serum stimulation in quiescent HCT-116 (wild-type p53) and HL-60 (null p53) cells. Concomitant with G0/G1 arrest, HCT-116 cells treated with DPI resulted in strong and sustained upregulation of p53 and p21. p53- or p21-deficient HCT-116 cells using a small interfering RNA (siRNA) significantly increased the progression into S phase by stimulation of DPI, compared with DPI alone. However, the silencing of p53 resulted in more efficient transition into S phase than the silencing of p21 siRNA and significantly inhibited p21 upregulation by DPI stimulation. Interestingly, brief exposure to DPI did not change p53 expression, but showed transient upregulation of p21 and G0/G1 arrest. These results suggest that p53 upregulation sustains G0/G1 cell cycle arrest and p21 upregulation by DPI stimulation in HCT-116 cells. In HL-60 cells, DPI also induced p21 upregulation in a p53-independent manner and the increase of p21 expression seems to be regulated by DPI-mediated ERK activation. Cyclin D1 expression was not significantly affected by DPI treatment in HCT-116 cells. However, in HL-60 cells, DPI irreversibly impaired cyclin D1 upregulation by serum stimulation and a much greater fraction of cells arrested in G0/G1 was observed in HL-60 cells than in HCT-116 cells at 24 h after brief DPI treatment. These results suggest that cyclin D1 is an important regulatory factor in the inhibition of cell cycle progression by DPI in HL-60 cells.

"Dual-scopic" intraoperative radiofrequency ablation for the treatment of a hepatic metastatic tumor located beneath the diaphragm.

Surgical resection leaving negative margins provides the only chance for a cure in hepatic metastasis of colorectal cancer. Tumor ablation techniques are known for their reliability and effectiveness in the treatment of primary and metastatic hepatic tumors. Among these treatment modalities, radiofrequency ablation (RFA) is commonly used for local control of primary and metastatic hepatic tumors with acceptable complication rates. Although the percutaneous approach is the usual route for RFA application, both laparoscopic approach and laparotomy can also be used. Hepatic tumors, located immediately beneath the diaphragm (segment VIII), are not suitable for percutaneous RFA due to the risks of injuring the diaphragm, and poor visualization by ultrasonography. Herein, we present a case of hepatic metastatic tumor located immediately beneath the diaphragm successfully treated by "dual-scopic" approach. A 50-year-old female patient had previously undergone an extended right hemicolectomy for transverse colon cancer, and she visited our department for hepatic metastasis on the dome of liver. She successfully underwent percutaneous transthoracic transdiaphragmatic intraoperative RFA under the guidance of a combination of thoracoscopic and laparoscopic approaches.

Diallyl disulfide induces reversible G2/M phase arrest on a p53-independent mechanism in human colon cancer HCT-116 cells.

Diallyl disulfide (DADS), a major organosulfur compound of garlic oil, is known to have an anticancer effect on human cancer cells. However, the exact mechanisms of this anticancer activity remain unclear. Here, we investigate the effects of DADS on cell cycle progression in human colon cancer HCT-116 cells by exploring the role played by regulatory molecules such as p53 and cyclin B1. Treatment of HCT-116 cells by DADS induced a marked growth inhibition with a slight reduction in viability and induced transient cell cycle arrest in the G2/M phase. Cyclin B1 is thought to play an important role in this process, as the DADS-induced G2/M phase arrest occurs with the increase of cyclin B1 expression. DADS also significantly induced the expression of p53, which contributes to cell cycle arrest in cancer cells, at a late time-point of 24 h. In addition, knockdown of p53 by siRNA did not affect cell cycle arrest, its reversibility, or the expression of cyclin B1 in the G2/M phase induced by DADS. Based on these results we conclude that, with the dynamic expression of cyclin B1, DADS induces reversible cell cycle arrest in the G2/M phase of HCT-116 cells through a p53-independent mechanism.

Redox factor-1 mediates NF-kappaB nuclear translocation for LPS-induced iNOS expression in murine macrophage cell line RAW 264.7.

Redox-sensitive transcriptional regulator redox factor-1 (Ref-1) is induced by oxidative stress and protects cells against it. However, the function of Ref-1 in regulating nitric oxide (NO) synthesis in macrophages has not been defined. We investigated the role of Ref-1 related to the regulation of NO synthesis in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. LPS stimulates the up-regulation and nuclear translocation of Ref-1 in macrophages. Importantly, Ref-1-deficient macrophages using a small interfering RNA did not stimulate inducible NO synthase (iNOS) expression as well as nuclear factor-kappaB nuclear translocation by stimulation with LPS. When the cells were pretreated with diphenyleneiodonium or p47(phox) small interfering RNA for inhibition of NADPH oxidase activity, LPS did not stimulate the nuclear translocation of Ref-1. We next asked whether reactive oxygen species are sufficient for the nuclear translocation of Ref-1 in macrophages. The direct use of H2O2 stimulated the translocation to the nucleus of nuclear factor-kappaB, but not Ref-1 and antioxidant N-acetyl cysteine did not inhibit the LPS-stimulated nuclear translocation of Ref-1. These data suggest that Ref-1 nuclear translocation in LPS-stimulated macrophages requires the activation of other signalling molecules aside from reactive oxygen species followed by the activation of NADPH oxidase.

Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line.

Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.

[Educational issues and strategies to improve APN education].

PURPOSE: This study was aimed at exploring the current status of graduate programs for an advanced practice nurse(APN) to recommend future directions of APN education. METHODS: A total of 142 students enrolled in seven APN specialty programs, 67 professors who were involved in APN education, and nine nurse administrators participated in the study. Data was collected by questionnaires and focus group interviews. RESULTS: The current definition of APN was found not to be specific enough to represent expected roles of APN in regards to knowledge, attitudes, roles, and skills. Standard curricula employed regardless of the area of APN specialty, lack of qualified clinical practice settings, as well as prepared instructors were found to be problematic. CONCLUSION: The following needs to be addressed: 1. redefining of APN roles, 2. tailoring specialty areas of APN, 3. consolidating educational programs, and 4. ensuring APN role models and faculty. Suggesting a CNS role in Korean APN, areas of APN should be rearranged to clarify their roles and educational programs need to be further developed to meet the expectations and quality of APNs. It is necessary to ensure APN's employment in the health care system by laws and policies to perform advanced nursing roles.

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells.

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.

Genetic polymorphism of CYP2C9 in a Vietnamese Kinh population.

Cytochrome P450 2C9 (CYP2C9) shows genetic polymorphism with high interethnic variation, but no report has addressed the genetic polymorphism in the Vietnamese population. In the present study, the distribution of 2 common allelic variations of CYP2C9 was investigated in Vietnamese Kinh population, a major ethnic group in Vietnam. Genomic DNA from 157 Vietnamese subjects was amplified by polymerase chain reaction, and the presence of CYP2C9*2 and CYP2C9*3 allelic variants was determined by pyrosequencing. Among 157 Vietnamese subjects, no subject with the CYP2C9*2 allele was detected, but 7 subjects were heterozygous for the CYP2C9*3 allele. The allele frequency of CYP2C9*3 was 2.2% in the Vietnamese Kinh population. This genotype distribution was well correlated with previous reports suggesting no occurrence of CYP2C9*2 in Asians. These results suggest that CYP2C9*2 may be absent in Vietnamese Kinh population and that CYP2C9*3 is major allelic variant that causes interindividual variation of drug responses to CYP2C9 substrate drugs in the Vietnamese Kinh population.