• Journal of Life Science
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• v.19 no.1
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• pp.1-8
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• 2009

In this paper, to elucidate the further mechanisms of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced growth arrest, we investigated the effect of MNNG on cell cycle and proliferation in U937 cells, a p53-null human myeloid leukemia cell line. It was found that MNNG causes an arrest at the G2/M phase of the cell cycle and induces apoptosis, which is closely correlated to inhibition of cyclin B1 and cyelin-dependent kinase (Cdk) 2-associated kinase activities. MNNG treatment in. creased protein and mRNA levels of the Cdk inhibitor p21(WAF1/CIP1), and activated the reporter construct of a p21 promoter. By using p21 promoter deletion constructs, the MNNG-responsive element was mapped to a region between 113 and 61 relative to the transcription start site. These data indicate that in U937 cells MNNG can circumvent the loss of wild-type p53 function and induce critical downstream regulatory events leading to transcriptional activation of p21. Present results indicate that the p53-independent up-regulation of p21 by MNNG is likely responsible for the inhibition of cyclin/Cdk complex kinase activity rather than the down-regulation of cyclins and Cdks expression. These novel phenomena have not been previously described and provide important new insights into the possible biological effects of MNNG.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.6
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• pp.1530-1537
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• 2006

Platycodi Radix, the root of Platycodon grandiflorum A. DC (Campanulaceae), commonly known as Doraji in Korea (Chinese name, 'Jiegeng', and Japanese name, 'Kikyo') has been used as an expectorant in traditional Oriental medicine. Extracts from the roots of P. grandiflorum have been reported to have wide ranging health benefits. In Korea, Platycodi Radix is also used as a food and employed as a folk remedy for adult diseases, such as bronchitis, asthma and pulmonary tuberculosis, hyperlipidemia, diabetes, and inflammatory diseases, and as a sedative. Several studies on its chemical and immunopharmacological effects including immunostimulation and antitumor activity have been performed. However, the relevant molecular mechanisms are poorly understood. Platycodi Radix, the root of Platycodon grandiflorum A. DC (Campanulaceae), commonly known as Doraji in Korea (Chinese name, 'Jiegeng', and Japanese name, 'Kikyo') has been used as an expectorant in traditional Oriental medicine. Extracts from the roots of P. grandiflorum have been reported to have wide ranging health bensfits. In Korea, Platycodi Radix is also used as a food and employed as a folk remedy for adult diseases, such as bronchitis, asthma and pulmonary tuberculosis, hyperlipidemia, diabetes, and inflammatory diseases, and as a sedative. Several studies on its chemical and immunopharmacological effects including immunostimulation and antitumor activity have been performed. However, the relevant molecular mechanisms are poorly understood. In the present study, we investigated the effects of an aqueous extract from the roots of P. grandiflorum AEPG) on the cell growth of human lung adenocarcinoma NCI-H460 cells in order to understand its anti-proliferative mechanism. AEPG treatment down-regulated the cyclin D1 expression in both transcriptional and translational levels without alteration of cyclin E. In AEPG-treated cells, the levels of cyclin-dependent kinase (C아) 6 mRNA and protein were significantly inhibited, but the levels of Cdk2 and Cdk4 were slightly inhibited by treatment of AEPG. AEPG treatment induced a marked accumulation of Cdk inhibitors, p16 and p27. However, AEPG treatment did not affect not only retinoblastoma protein (pRB) but also tumor suppressor p53 protein expression. The present results indicated that AEPG-induced inhibition of lung cancer cell proliferation is associated with the blockage of G1 phase progression through induction of Cdk inhibitors such as p16 and p27, and inhibition of cyclin D1 and Cdk6. AEPG exposure, as offered by this study, provides cluse for the mechanism of AEPG action. Taken together, these findings suggest that P. grandiflorum has strong potential for development as an agent for prevention and treatiment against human lung cancer.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.4
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• pp.485-492
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• 2011

The influence of high temperature on mammary cell turnover during the dry period is still unclear. The objective of this study was to investigate mammary cell turnover and p53 protein expression in the mammary tissue under high temperature conditions. Mammary gland biopsy samples from 8 dairy cows were obtained at 7, 25, 40, and 53 d during the dry period in summer or spring (n = 4, each season). Cell cycle, cell turnover, and p53 protein expression were analyzed by flow cytometry. During the dry period in summer, the percentage of mammary epithelial cells in the G0/G1 phase was the highest, but those in the S and G2/M phases were lower. However, the proportion of cells in the different stages of the cell cycle was not significantly different among the different biopsy time points, except in the G2/M phase. Under different temperature conditions, the cells were significantly different in their apoptotic rate and proliferation index; moreover, the tendencies of these indicators to change significantly differed. In general, the samples under high temperature conditions showed significantly lower apoptotic rates and proliferation indices. Under high temperature conditions, the apoptotic rate and proliferation index were the lowest (2.17% and 3.26%, respectively) at day 40, and the highest at day 53 (3.67% and 4.61%, respectively). However, under normal temperature conditions, the values of these indicators were the lowest (7.60% and 5.54%, respectively) at day 7, and almost the highest at day 25 (12.85% and 6.47%, respectively). Moreover, p53 protein expression was significantly higher under high temperature conditions than under normal temperature conditions, except at day 25. The level of p53 protein was the lowest (13.10%) under high temperature conditions at day 25, but was the highest (26.07%) under normal temperature conditions. Our findings suggest that high temperature delayed the G2/M phase of the cell cycle and the cell turnover rate, but remarkably increased p53 protein expression. Thus, the results indicate that high temperature extends the recovery period of mammary epithelial cells.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.1
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• pp.347-350
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• 2012

TP53 is the mostly commonly mutated gene in many cancers and the P53 tumor suppressor protein is involved in multiple cellular processes, including transcription, DNA repair, genomic stability, senescence, cell cycle control and apoptosis. A common single nucleotide polymorphism located within the proline rich region of TP53 gene at codon 72 in exon 4 encodes either proline or arginine. TP53 Arg 72 is more active than TP53 Pro 72 in inducing apoptosis. The aim of this study was to understand the association of the 72 codon polymorphism with acute leukemia development and prognosis. A total of 288 acute leukemia cases comprising 147 acute lymphocytic leukemia (ALL) and 141 acute myeloid leukemia (AML), as well as 245 controls were recruited for analysis of the TP53 72 polymorphism using PCR-RFLP method. Significant association of homozygous arginine genotype with AML was observed (${\chi}^2$- 133.53; df-2, p < 0.001. When data were analyzed with respect to clinical variables, elevation in mean WBC, blast %, LDH levels and slight reduction in DFS in ALL cases with the arginine genotype was observed. In contrast, AML patients with Pro/Pro had elevated WBC, Blast%, LDH levels with slightly reduced DFS. Our study indicates that Arg/Arg genotype might confer increased risk to development of acute myeloid leukemia.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.5767-5772
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• 2015

Background: Polymorphisms in the MDM2 309 (T>G) and TP53 72 (G>C) genes are reported to increase the susceptibility to head and neck cancer (HNC) in various populations. The risk for HNC is also strongly associated with etiologic habits such as smoking, alcohol consumption and/or chewing of betel quid (BQ). In a case-control study, we investigated the significance of the above polymorphisms alone, and upon interaction with one another as well as with various etiologic habits in determining HNC risk in a Northeast Indian population. Materials and Methods: Genotyping at 309 MDM2 and 72 TP53 in 122 HNC patients and 86 cancer free healthy controls was performed by PCR using allele specific primers, and the results were confirmed by DNA sequencing. Results: Individuals with the GG mutant allele of MDM2 showed a higher risk for HNC in comparison to those with the TT wild type allele (OR=1.9, 95%CI: 1.1-3.3) (p=0.022). The risk was further increased in females by ~4-fold (OR=4.6, 95% CI: 1.1-19.4) (P=0.04). TP53 polymorphism did not contribute to HNC risk alone; however, interaction between the TP53 GC and MDM2 GG genotypes resulted in significant risk (OR=4.9, 95% CI: 0.2-105.1) (p=0.04). Smokers, BQ- chewers and alcohol consumers showed statistically significant and dose-dependent increase in HNC risk, irrespective of the MDM2 genotype. Conclusions: MDM2 genotype could serve as an important predictive biomarker for HNC risk in the population of Northeast India.

• Tuberculosis and Respiratory Diseases
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• v.65 no.6
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• pp.476-486
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• 2008

Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates $NF-{\kappa}B$ in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate $NF-{\kappa}B$ in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of $NF-{\kappa}B$ activation using proteasome inhibitor MG132 which blocks $I{\kappa}B{\alpha}$ degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study $NF-{\kappa}B$-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgG ${\kappa}-NF-{\kappa}B$ luciferase construct. To investigate DNA binding of $NF-{\kappa}B$ activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 ($3{\mu}M$) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced $NF-{\kappa}B$ transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of $NF-{\kappa}B$ activated by TRAIL and supershift with p65 antibody. $I{\kappa}B{\alpha}$ degradation was proven by western blot. MG132 completely blocked both TRAIL-induced $NF-{\kappa}B$ dependent luciferase activity and DNA binding of $NF-{\kappa}B$. Conclusion: This results suggest that inhibition of $NF-{\kappa}B$ can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Tuberculosis and Respiratory Diseases
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• v.48 no.6
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• pp.909-921
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• 2000

Background : Defects in apoptotic signaling pathways play important role in tumor initiation, progression, metastasis and resistance to treatment. Several proteins which may promote tumorigenesis by inhibiting apoptosis were identified. The survivin protein is the member of inhibitor of apoptosis protein(IAPs) family which inhibits apoptosis. Unlike other IAPs, it is expressed in during the fetal period but not in adult differentiated tissues. Many reports have stated that survivin is selectively expressed in many cancer cell lines and cancer tissues. We performed immunohistochemical analysis for survivin expression in non-mall cell lung cancer to get evaluate its clinical implication. Methods : Twenty nine surgically resected lung cancers were examined. Immunohistochemical staining were performed by immuno-peroxidase technique using avidin-biotinylated horseradish pemxidase complex in the formalin-fixed, paraffin-embedded tissue $4{\mu}m$ section. Anti-survivin polyclonal antibody was used for primary antibody and anti-p53 monoclonal antibody was also used to analyze the correlation between survivin and p53 expression. The survivin expression scores were determined by as the sum of the stained area and intensity. Results : Immunohistochemical analysis showed cancer specific expression of survivin in 20 of 29 cases (69.0%). Western blot analysis also showed the selective survivin expression in tumor tissue. There was no correlation between survivin expression and clinicopathological parameters and prognosis. We analyzed the ∞π'elation between survivin expression and p53 expression, but found none. Conclusion: We confirmed the tumor specific expression of survival in non-small cell lung canær. But this expression was not correlated with clinical parameters as well as histology, tumor stage, recurrence, and survival rate. Also it was not statistically correlated with the expression of p53.

• Molecules and Cells
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• v.42 no.5
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• pp.397-405
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• 2019

The regulatory role of long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in both cancerous and noncancerous cells have been widely reported. This study aimed to evaluate the role of lncRNA GAS5 in heart failure caused by myocardial infarction. We reported that silence of lncRNA GAS5 attenuated hypoxia-triggered cell death, as cell viability was increased and apoptosis rate was decreased. This phenomenon was coupled with the down-regulated expression of p53, Bax and cleaved caspase-3, as well as the up-regulated expression of CyclinD1, CDK4 and Bcl-2. At the meantime, the expression of four heart failure-related miR-NAs was altered when lncRNA GAS5 was silenced (miR-21 and miR-142-5p were up-regulated; miR-30b and miR-93 were down-regulated). RNA immunoprecipitation assay results showed that lncRNA GAS5 worked as a molecular sponge for miR-142-5p. More interestingly, the protective actions of lncRNA GAS5 silence on hypoxia-stimulated cells were attenuated by miR-142-5p suppression. Besides, TP53INP1 was a target gene for miR-142-5p. Silence of lncRNA GAS5 promoted the activation of PI3K/AKT and MEK/ERK signaling pathways in a miR-142-5p-dependent manner. Collectively, this study demonstrated that silence of lncRNA GAS5 protected H9c2 cells against hypoxia-induced injury possibly via sponging miR-142-5p, functionally releasing TP53INP1 mRNA transcripts that are normally targeted by miR-142-5p.

• Natural Product Sciences
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• v.13 no.1
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• pp.1-5
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• 2007

Dietary flavonoids have antioxidant and antitumor promoting effects. Rhus verniciflua Stokes (RVS) is a flavonoid-rich herbal medicine and has long been used as a food additive and an antitumor agent in Korea. Previous study demonstrated that a purified flavonoid fraction prepared from RVS, herein named RCMF (the RVS chloroform-methanol fraction), exhibited growth inhibition and induced apoptosis in human osteosarcoma(HOS) cells. This study evaluated if p53-mediated pathway is associated with the RCMF-induced apoptosis in HOS cells. RCMF was shown to be capable of inducing apoptosis of the cells, as expected, and transparently increased p53 expression in the cells. However, the RCMF-induced cytotoxicity was suppressed by transfecting the cells with antisense p53 oligonucleotide, which also inhibited the decrease of Bcl-2 and the increase of Bax in mitochondria, and the release of cytochrome c into cytosol. This finding suggests that p53-mediated mitochondrial stress is required for RCMF-induced apoptosis in HOS cells.