OBJECTIVE: The purpose of the study was to examine a possible physiological function of p32-mediated apoptosis signaling in ovarian cancer cells. METHODS: SK-OV-3 cells were transfected with respective plasmid DNAs, and cell viability was measured. By immunoprecipitation and immunofluorescence staining analysis, we confirmed that p32 interacts with Harakiri in ovarian cancer cells. RESULTS: In SK-OV-3 cells, p32 interacted with Harakiri and both p32 and Harakiri were colocalized in the mitochondria. In addition, overexpression of p32 induced apoptosis of ovarian cancer cells and augmented Harakiri-mediated apoptosis. CONCLUSION: Our results demonstrated p32 as an apoptosis inducer and helped to provide the better understanding of the function of p32 in ovarian cancer cells and a possibility of p32 in the application of cancer therapeutics.
Apoptosis ; Cell Death ; Cell Survival ; DNA ; Fluorescent Antibody Technique ; Humans ; Immunoprecipitation ; Mitochondria ; Ovarian Neoplasms ; Plasmids

Osteoblasts are affected by TNF-alpha overproduction by immune cells during inflammation. It has been suggested that functional NF-kappaB sites are involved in TNF-alpha-induced bone resorption. Thus, we explored the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor (NF-kappaB), on the induction of TNF-alpha-induced activation of JNK/SAPK, AP-1, cytochrome c, caspase and apoptosis in MC3T3E1 osteoblasts. Pretreatment of the cells with PDTC blocked TNF-alpha-induced NF-kappaB activation. TNF-alpha-induced activation of AP-1, another nuclear transcription factor, was suppressed by PDTC. The activation of c-Jun N-terminal kinase, implicated in the regulation of AP-1, was also down regulated by PDTC. TNF-alpha-induced apoptosis, release of cytochrome c and subsequent activation of caspase-3 were abolished by PDTC. TNF-alpha-induced apoptosis was partially blocked by Ac-DEVD-CHO, a caspase-3 inhibitor, suggesting that caspase-3 is involved in TNF-alpha- mediated signaling through NF-kappaB in MC3T3E1 osteoblasts. Thus, these results demonstrate that PDTC, has an inhibitory effect on TNF-alpha-mediated activation of JNK/SAPK, AP-1, cytochrome c release and subsequent caspase-3, leading to the inhibition of apoptosis. Our study may contribute to the treatment of TNF-alpha-associated immune and inflammatory diseases such as rheumatoid arthritis and periodontal diseases.
Apoptosis* ; Arthritis, Rheumatoid ; Bone Resorption ; Caspase 3 ; Cytochromes c ; Inflammation ; JNK Mitogen-Activated Protein Kinases ; NF-kappa B ; Osteoblasts ; Periodontal Diseases ; Transcription Factor AP-1 ; Transcription Factors ; Tumor Necrosis Factor-alpha

OBJECTIVE: Promoter methylation of Bcl-2 family genes in cancer cells were studied to verify possible correlation between DNA methylation pattern of Bcl-2 family members and cancer. METHODS: The genomic DNAs were extracted from different cancer cell lines, HeLa, CaSki and K562, and ovarian cancer tissue from patients. The cytosine residues were converted to uracil by sodium bisulfite treatment. MSP (methylation specific PCR) was performed to determine the methylation status of Bcl-2, Mcl-1, Noxa, and Harakiri promoters. Using primers that distinguish methylated DNA from unmethylated DNA after bisulfite modification of DNA, MSP was conducted to observe the methylation pattern of Bcl-2 family genes in different cancer cells. RESULTS: The promoter regions of Bcl-2 family genes including Mcl-1, Bcl-2, and Noxa were not methylated in cancer cells, whereas the proapoptotic Bcl-2 family gene Harakiri was detected as methylated in the cancer cell lines and hypomethylated in the ovarian cancer tissue. CONCLUSION: The present study demonstrated the differential methylation profiles of Bcl-2 family genes in cancerous cells, which suggests a possible connection between the methylation pattern of some of Bcl-2 family genes and ovarian cancer.
Cell Line ; Cytosine ; DNA Methylation* ; DNA* ; Humans ; Methylation ; Ovarian Neoplasms ; Promoter Regions, Genetic ; Sodium ; Uracil