• Journal of Korean Neurosurgical Society
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• v.37 no.1
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• pp.48-53
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• 2005

Objective: The purpose of this study is to elucidate in vitro responses to combined gamma knife irradiation and p53 gene transfection on human malignant glioma cell lines. Methods: Two malignant human glioma cell lines, U87MG (p53-wild type) and U373MG (p53-mutant) were transfected with an adenoviral vector containing p53 (MOI of 50) before and after applying 20Gy of gamma irradiation. Various assessments were performed, including, cell viability by MTT assay; apoptosis by annexin assay; and cell cycle by flow cytometry, for the seven groups: mock, p53 only, gamma knife (GK) only, GK after LacZ, LacZ after GK, GK after p53, p53 after GK. Results: Cell survival decreased especially, in the subgroup transfected with p53 after gamma irradiation. Apoptosis tended to increase in p53 transfected U373 MG after gamma irradiation (apoptotic rate, 38.9%). The G2-M phase cell cycle arrest markedly increased by transfecting with p53, 48 hours after gamma knife irradiation in U373 MG (G2-M phase, 90.8%). Conclusion: These results suggest that the in vitro effects of combined gamma knife irradiation and p53 gene transfection is an augmentation of apoptosis and G2-M phase cell cycle arrest, which are more exaggerated in U373 MG with p53 transfection after gamma knife irradiation.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.15
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• pp.6753-6759
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• 2015

Background: Loss of function of the p53 gene is implicated in defective apoptotic responses of tumors to chemotherapy. Although the pro-apoptotic roles of eugenol and capsaicin have been amply reported, their dependence on p53 for apoptosis induction in gastric cancer cells is not well elucidated. The aim of the study was to elucidate the role of p53 in the induction of apoptosis by eugenol and capsaicin in a human gastric cancer cell line, AGS. Materials and Methods: AGS cells were incubated with or without various concentrations of capsaicin and eugenol for 12 hrs, in the presence and absence of p53 siRNA. Cell cycling, annexin V and expression of apoptosis related proteins Bax, Bcl-2 ratio, p21, cyt c-caspase-9 association, caspase-3 and caspase-8 were studied. Results: In the presence of p53, capsaicin was a more potent pro-apoptotic agent than eugenol. However, silencing of p53 significantly abrogated apoptosis induced by capsaicin but not that by eugenol. Western blot analysis of pro-apoptotic markers revealed that as opposed to capsaicin, eugenol could induce caspase-8 and caspase-3 even in the absence of p53. Conclusions: Unlike capsaicin, eugenol could induce apoptosis both in presence and absence of functional p53. Agents which can induce apoptosis irrespective of the cellular p53 status have immense scope for development as potential anticancer agents.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6039-6045
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• 2012

This study was conducted to assess the predictive value of p-glycoprotein (p-gp) and p53 immunoexpression in human papillomavirus (HPV) infected cases of cervical dysplasia. Expression of both p-gp and p53 proteins was detected in cervical smears from 177 squamous intraepithelial lesions (SIL) cases along with 183 "atypical squamous cells of unknown significance" (ASCUS) and 150 normal cases. HPV 16 and 18 infection was detected by polymerase chain reaction using type-specific primers for HPV sub-types. There were no significant detectable p53 and p-gp expression in the normal cervix smears (p>0.05). In the ASCUS group 10 cases were positive for both p53 and p-gp immunoreactivity. In cervical dysplasia cases, p53 was positive in 86 (48.58%) while p-gp was positive in 93 (52.54%) and the two markers showed a highly significant correlation (r=0.92, p<0.001). Expression of p53 and p-gp was associated with grade of SIL (p<0.001). A positive correlation between the presence of HPV and expression of proteins p53 and p-gp in smears of patients with cervical lesions was also noted (p<0.001). Thus, p53 and p-gp immunostaining in cervical smears may act as an auxiliary biomarker for detection of HPV-associated cervical lesions. Additionally, a significant positive correlation between ascending grades of SIL and labeling indices of markers suggests that p53 and p-gp can be used as an adjunct to cytomorphological interpretation of conventional cervical Pap smears.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3503-3507
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• 2013

Background: Although the majority of investigations concerned with TP53 and its protein have focused on coding regions, recently a set of studies highlighted significant roles of regulatory elements located in p53 mRNA, especially 5'UTR. The wrap53${\alpha}$ transcript is one of those that acts as a natural antisense agent, forming RNA-RNA hybrids with p53 mRNA and protecting it from degradation. Materials and Methods: In this study, we focused on the mutation status of exon $1{\alpha}$ of the WRAP53 gene (according to exon 1 of p53) in 160 breast tumor tissue samples and conducted a bioinformatics search for probable miRNA binding site in the p53/wrap53 overlapping region. Mutations were detected, using single stranded conformation polymorphism (SSCP) and sequencing. We applied the miRBase database for prediction of miRNAs which target overlapping region of p53/wrap53 transcripts. Results: Our results showed all samples to have wild type alleles in exon 1 of TP53 gene. We could detect a novel and unreported intronic mutation (IVS1+56, G>C) outside overlapping regions of p53/wrap53 genes in breast cancer tissues and also predict the presence of a binding site for miR-4732-5p in the 5'UTR of Wrap53 mRNA. Conclusions: From our findings we propose designing further studies focused on overexpression of miRNA-4732-5p and introducing different mutations in the overlapping region of wrap53 and p53 genes in order to study their effects on p53 and its ${\Delta}N$ isoform (${\Delta}$40p53) expression. The results may provide new pieces in the p53 targeting puzzle for cancer therapy.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.279-285
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• 1993

Alterations of the p53 gene arc among the most frequent genetic changes in human cancer and often result in increased levels of p53 protein within the malignant cells. Detection of accumulated p53 protein can be a useful prognostic tool in human cancer. In order to make polyclonal antibodies for immunohistochemical screening. human p53 gene was expressed in E. coli in the form of GST (glutathione S-transfi.:rase) fusion proteins. Two p53 gene fragments. which were N('()I small fragment encoding amino acid residues of 1-151-: and Ncol large fragment of 159-393. were subeloned into the unique BamHI site present within the pGEX-2T vector using BamHI linker and recombinant plasmids pGTNS and pGTNL were constructed. respectively. The p53 cDNA fragment (from pC53-$SN_3$,) encoding amino acid 38-145 (proline at residue 72) was amplified by polymerase chain reaction(PCR). The amplified DNA was digested with BamHI and Prull and inserted into the BamHI-Smal sites of pG EX-2T and recombinant plasmid pGTBP was constructed. After IPTG induction of these plasmids for 4 hours. fusion proteins were purified from E. coli extracts with glutathione Sepharose beads. The bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and the molecular weights were 54 kDa. 53 kDa and 40 kDa. respectively. Approximately one milligram of fusion proteins were purified from 1 -liter cultures.

• Journal of Life Science
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• v.25 no.2
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• pp.158-163
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• 2015

Rice embryo is more abundant than endosperms in nutrients such as proteins, lipids, and vitamin B1. In this study, we constructed p53 plasmid that could be expressed in a plant system, and investigated optimal germination conditions in a variety of media. For construction of p53 plasmid, we performed p53 amplification from pCDNA-p53, subcloned to TA cloning vector, and then reconstructed into pGEM-CaMV plant expression vector. On the other hand, we prepared a variety of imbibition buffers and complete media for efficient germination of the rice embryo. Imbibition buffers prepared with different concentrations of salt or detergent showed no significant effect on germination efficiency. We prepared further culture media, such as solid agar, liquid media, and paper towel to establish the optimal conditions. Rice embryo showed germination rates of more than 70% in the solid medium, more than 60% in the paper towel medium, but less than 25% in liquid media, although germination rate did not differ with varying concentrations of salt and sucrose in culture media. Under the optimal germination conditions, we introduced the p53 plasmid using imbibition method, and finally detected human p53 gene expression in the germinated rice embryo. This method might present a novel, practical approach for evaluating efficient gene expression utilizing imbibition method in rice embryo.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.6
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• pp.365-369
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• 2010

In this study, we examined whether melatonin promotes apoptotic cell death via p53 in prostate LNCaP cells. Melatonin treatment significantly curtailed the growth of LNCaP cells in a dose- and time-dependent manner. Melatonin treatment (0 to 3 mM) induced the fragmentation of poly(ADP-ribose) polymerase (PARP) and activation of caspase-3, caspase-8, and caspase-9. Moreover, melatonin markedly activated Bax expression and decreased Bcl-2 expression in dose increments. To investigate p53 and p21 expression, LNCaP cells were treated with 0 to 3 mM melatonin. Melatonin increased the expressions of p53, p21, and p27. Treatment with mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor), confirmed that the melatonin-induced apoptosis was p21-dependent, but ERK-independent. With the co-treatment of PD98059 and melatonin, the expression of p-p53, p21, and MDM2 did not decrease. These effects were opposite to the expression of p-p53, p21, and MDM2 observed with SP600125 and SB202190 treatments. Together, these results suggest that p53-dependent induction of JNK/p38 MAPK directly participates in apoptosis induced by melatonin.

• BMB Reports
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• v.47 no.3
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• pp.167-172
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• 2014

Reactivating the p53 pathway in tumors is an important strategy for anticancer therapy. In response to diverse cellular stresses, the tumor suppressor p53 mediates apoptosis in a transcription-independent and transcription-dependent manner. Although extensive studies have focused on the transcription-dependent apoptotic pathway of p53, the transcription-independent apoptotic pathway of p53 has only recently been discovered. Molecular interactions between p53 and Bcl-2 family proteins in the mitochondria play an essential role in the transcription-independent apoptosis of p53. This review describes the structural basis for the transcription-independent apoptotic pathway of p53 and discusses its potential application to anticancer therapy.

• BMB Reports
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• v.50 no.7
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• pp.373-378
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• 2017

The Jun activation-domain binding protein 1 (Jab1) induces p53 nuclear export and cytoplasmic degradation, but the underlying mechanism is poorly understood. Here, we show that phosphorylation at the threonine 155 residue is essential for Jab1-mediated p53 nuclear export. Jab1 stimulated phosphorylation of p53 at T155 was inhibited by curcumin, an inhibitor of COP9 signalosome (CSN)-associated kinases. The T155E mutant, which mimics phosphorylated p53, exhibited spontaneous cytoplasmic localization in the absence of Jab1. This process was prevented by leptinomycin B (LMB), but not by curcumin. The substitution of threonine 155 for valine (T155V) abrogated Jab1-mediated p53 nuclear export, indicating that phosphorylation at this site is essential for Jab1-mediated regulation of p53. Although T155E can be localized in the cytoplasm in the absence of Mdm2, the translocation of T155E was significantly enhanced by ectopic Hdm2 expression. Our data suggests that Jab1-mediated phosphorylation of p53 at Thr155 residue mediates nuclear export of p53.

• Archives of Pharmacal Research
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• v.25 no.2
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• pp.208-213
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• 2002

The human polyomavirus JC virus is the etiologic agent of progressive multifocal leukoencephalopathy (PML). As the JC virus early promoter directs cell-specific expression of the viral replication factor large T antigen, transcriptional regulation constitutes a major mechanism of glial tropism in PML. It has been demonstrated that SV4O or JC virus large T antigen interacts with p53 protein and regulates many viral and cellular genes. In this study we founts that p53 represses the JC virus early promoter in both glial and nonglial cells To identify the cis-regulatory elements responsible for p53-mediated repression, deletional and site-directed mutational analyses were performed . Deletion of the enhancer region diminished p53-mediated transcriptional repression. However, point mutations of several transcription factor binding sites in the basal promoter region did not produce any significant changes. In support of this observation, when the enhancer was fused to a heterologous promoter, p53 red reduced the promoter activity about three fold. These results indicate that the enhancer region is important for tole repression of JC virus transcription by p53. Furthermore, coexpression of JC virus T antigen with a p53 protein abolished p53-mediated repression of the JC virus early promoter in non-glial cells, but not in glial cells. This finding suggests that T antigen interacts with p53 and regulates JC virus transcription in a cell-specific manner.