• KSBB Journal
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• 제31권1호
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• pp.52-57
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• 2016

Mutant p53 (R280K) is highly expressed in MDA-MB-231 triple-negative human breast cancer cells. Currently, we reported the role of mutant p53-R280K in mediating the survival of MDA-MB-231 cells in vitro. The present study was undertaken to determine whether mutant p53-R280K affects breast cancer cell growth in vivo. To this end, we used small interfering RNA to knockdown the level of mutant p53-R280K in MDA-MB-231 cells. Silencing of mutant p53-R280K in MDA-MB-231 cells causes substantial tumor regression of established xenografts in vivo. In xenograft model for breast cancer, silencing of mutant p53-R280K in MDA-MB-231 cells significantly inhibited the tumor growth. Moreover, TUNEL assay showed more occurrence of apoptotic cells in mutant p53-R280K silenced tumors compared to control. Our data indicate that mutant p53-R280K has an important role in mediating tumor growth of MDA-MB-231 cells in vivo. Taken together, this study suggests that endogenous mutant p53-R280K could be used as a therapeutic target for breast cancer cells harboring this TP53 missense mutation.

• The Korean Journal of Physiology and Pharmacology
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• 제24권3호
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• pp.203-212
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• 2020

Promyelocytic leukemia (PML) gene, through alternative splicing of its C-terminal region, generates several PML isoforms that interact with specific partners and perform distinct functions. The PML protein is a tumor suppressor that plays an important role by interacting with various proteins. Herein, we investigated the effect of the PML isoforms on oncostatin M (OSM)-induced signal transducer and activator of transcription-3 (STAT-3) transcriptional activity. PML influenced OSM-induced STAT-3 activity in a cell type-specific manner, which was dependent on the p53 status of the cells but regardless of PML isoform. Interestingly, overexpression of PML exerted opposite effects on OSM-induced STAT-3 activity in p53 wild-type and mutant cells. Specifically, overexpression of PML in the cell lines bearing wild-type p53 (NIH3T3 and U87-MG cells) decreased OSM-induced STAT-3 transcriptional activity, whereas overexpression of PML increased OSM-induced STAT-3 transcriptional activity in mutant p53-bearing cell lines (HEK293T and U251-MG cells). When wild-type p53 cells were co-transfected with PML-IV and R273H-p53 mutant, OSM-mediated STAT-3 transcriptional activity was significantly enhanced, compared to that of cells which were transfected with PML-IV alone; however, when cells bearing mutant p53 were co-transfected with PML-IV and wild-type p53, OSM-induced STAT-3 transcriptional activity was significantly decreased, compared to that of transfected cells with PML-IV alone. In conclusion, PML acts together with wild-type or mutant p53 and influences OSM-mediated STAT-3 activity in a negative or positive manner, resulting in the aberrant activation of STAT-3 in cancer cells bearing mutant p53 probably might occur through the interaction of mutant p53 with PML.

• The Korean Journal of Physiology and Pharmacology
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• 제17권4호
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• pp.291-297
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• 2013

Notch1 has been reported to be highly expressed in triple-negative and other subtypes of breast cancer. Mutant p53 (R280K) is overexpressed in MDA-MB-231 triple-negative human breast cancer cells. The present study aimed to determine whether the mutant p53 can be a potent transcriptional activator of the Notch1 in MDA-MB-231 cells, and explore the role of this mutant p53-Notch1 axis in curcumin-induced apoptosis. We found that curcumin treatment resulted in an induction of apoptosis in MDA-MB-231 cells, together with downregulation of Notch1 and its downstream target, Hes1. This reduction in Notch1 expression was determined to be due to the decreased activity of endogenous mutant p53. We confirmed the suppressive effect of curcumin on Notch1 transcription by performing a Notch1 promoter-driven reporter assay and identified a putative p53-binding site in the Notch1 promoter by EMSA and chromatin immunoprecipitation analysis. Overexpression of mutant p53 increased Notch1 promoter activity, whereas knockdown of mutant p53 by small interfering RNA suppressed Notch1 expression, leading to the induction of cellular apoptosis. Moreover, curcumin-induced apoptosis was further enhanced by the knockdown of Notch1 or mutant p53, but it was decreased by the overexpression of active Notch1. Taken together, our results demonstrate, for the first time, that Notch1 is a transcriptional target of mutant p53 in breast cancer cells and suggest that the targeting of mutant p53 and/or Notch1 may be combined with a chemotherapeutic strategy to improve the response of breast cancer cells to curcumin.

• IMMUNE NETWORK
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• 제1권3호
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• pp.221-229
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• 2001

Background: Inactivation of tumor suppressor genes is believed to be important in the development of many human malignancies. Recently, several lines of evidence have indicated that the wild type p53 gene located at 17p13.3, may function as a tumor suppressor gene and that a mutant p53 gene could promote transformation by inactivating normal p53 function in a dominant negative fashion. These broad spectrum of p53 mutation in human cancers provide that mutant p53 and their protein may be potential targets of tumor diagnostic and therapeutic interventions. Method: Colony formation was performed to investigate growth suppressional ability. p53 expression pattern was examined by western blot and p53-mediated transactivation ability was assessed by CAT activity. SNU C2A cells were observed in apoptotic aspects induced by etoposide and $H_2O_2$ treatment, detecting sensitivity on agent, DNA fragmentation through agarose gel, chromatin condensation by fluorescence microscope, and cell cycle distribution by FACS. Result: 1) p53 mutant his179arg ($histidine{\rightarrow}arginine$) detected in SNU C2A cells lost transcriptional activity and growth suppression ability, showing dominant negative effect on its wild type p53. 2) Etoposide-treated SNU C2A cells induced apoptosis, exhibiting dramatic reduction of cell growth, DNA fragmentation, nuclear condensation formation of apoptotic body and increment of sub-G1 cell fraction. 3) Etoposide and $H_2O_2$-treated SNU C2A cells have no high increase of p53 expression and overexpressed p53 protein changed localization, from cytoplasm to nucleus. Also, p53-mediated transcriptional activity was increased by agents-treatment. Conclusion: SNU C2A cells coexpress wild-type and mutant p53 protein induced apoptosis in the condition on DNA damage, through localizational shift from cytoplasm to nucleus of p53 protein rather than the induction of p53 protein. SNU C2A cells derived mutant p53 his179arg abrogated both the growth supression ability and transactivational activity, showing inhibition effect on transcriptional activity of wild type p53, but did not repress the activity of wild type p53 in SNU C2A cells owing to dominant activity of wild type. These cell condition may provide new gene therapeutic implications leading effective antiproliferation of cell when mutant and wild-type p53 protein were co-expressed in cell.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.844-848
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• 2002

In more than half of human tumors, the p53 tumor suppressor gene is mutated. Thus, restoration of wild-type p53 activity by repair of mutant RNA could be a potentially promissing approach to cancer treatment. To explore the potential use of RNA repair for cancer therapy, trans-splicing group I ribozymes were developed that could replace mutant p53 RNA with RNA sequence attached to the 3'end of ribozymes. By employing a mapping library of ribozymes, we first determined which regions of the p53 RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, trans-splicing ribozymes were generated that specifically recognized the sequences around these accessible regions. Subsequently, the ribozymes reacted with and altered the p53 transcripts by transferring a 3'exon tag sequence onto the targeted p53 RNA with high fidelity. Thus, these ribozymes could be utilized to repair mutant p53 in tumors, which would revert the neoplastic phenotype.

• 한국식품위생안전성학회지
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• 제25권3호
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• pp.258-262
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• 2010

Radiotherapy is one of the major therapies for cancer treatment. p53 acts as a central mediator of the cellular response to stressful stimuli, such as radiation. Recently it has been known that activation of the phosphatidylinositol-3-kinase (PI3K) pathway is associated with radioresistance. In this study, we investigated whether X-irradiation up-regulates PI3K in a p53-dependent manner in human colon cancer cells. In order to study this phenomenon, we have treated p53-wild type and p53-mutant type HCT116 cells with X-ray. Treatment of wild type HCT116 cells with 8 Gy resulted in a marked increase in PI3K (p85), which paralleled an increase in PTEN, a counterpart of PI3K. However, these effects of X-rays in the p53-mutant cells were not observed. These results suggest that the X-irradiation-induced up-regulation of PI3K/PTEN pathway is p53-dependent.

• Journal of Chest Surgery
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• 제31권12호
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• pp.1134-1146
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• 1998

배경:종양억제 유전자인 p53은 폐암을 비롯하여 인체에 발생될 수 있는 다양한 종양들에 있어서 가장 빈번히 변이를 일으키는 유전자로 알려져 왔으며, 정상적인 p53은 세포분화 과정 중 G1 arrest 또는 세포자멸을 통하여 세포 성장을 억제한다는 증거들이 발견되고있다. 더불어 유전자 치료에 p53을 사용할 수 있는지에 대해서도 많은 연구가 진행 중이다 대상 및 방법: 이런 p53 단백질의 기능을 이해하기 위한 한 가지 방법으로 정상세포나 종양세포 안에 과량의 p53을 발현시킬 수 있는 p53 표현 중개물(expression vectors)을 이용할 수 있다. 우리는 각기 다른 p53 성질을 지니고 있는 네가지 비소세포폐암 세포를 대상으로 정상적인 p53을 지닌 adenovirus를 이용하여 비소세포폐암에 Adenovirus-p53을 이입시킬 경우 p53 단백질이 증가하는지의 여부와 Adenovirus-p53이 종양세포의 성장에 미치는 영향을 관찰하였으며, p53에 의한 세포 독성에 있어서 세포자멸과의 관련성 여부 및 실험관 안에서 Adenovirus-p53이 비소세포폐암의 종양형성 능력에 미치는 영향을 관찰하였다. 결과: 이번 실험 결과는 다음과 같이 요약될 수 있다. 변이성 p53을 갖고 있거나 p53 유전자가 없는 비소세포폐암 세포는 정상적인 p53을 가지고 있는 종양세포에 비하여 Adenovirus-p53에 의한 세포성장 억제정도가 높았으며, p53을 과발현시킬 경우 세포자멸을 관찰할 수 있었다. 그리고 Adenovirus-p53은 비소세포폐암의 집락 형성 능력을 억제할 수 있고, 또 이미 형성된 집락도 그 성장을 억제할 수 있다는 사실도 확인하였다. 결과: 이번 실험의 결과로 adenovirus는 비소세포폐암 세포에 종양억제유전자를 이입시키는데 매우 효과적인 매개물이며, 특히 p53이 소실되거나, 변이를 일으킨 종양세포들은 Adenovirus-p53에 의하여 쉽게 세포자멸이 유발된다는 사실을 알게 되었다.

• IMMUNE NETWORK
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• 제1권2호
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• pp.151-161
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• 2001

Background: Inactivation in p53 tumor suppressor gene through a point mutation and deletion is one of the most frequent genetic changes found in human cancer, with 50% of an incidence. This high rate of mutation mostly suggests that the gene plays a central role in the development of cancer and the mutations detected so far were found in exons 5 to 8. Mutation of p53 locus produced accumulation of abnormal p53 protein, and negative regulation of cell proliferation and transcriptional activation as a suppressor of transformation were lost. In addition, inhibition of its normal cellular function of wild-type by mutant is an important step in tumorigenesis. Method: 4 colon cancer cell lines (SNU C1, C2A, C4, C5) were examined for mutation in exons 5 to 8 of the p53 tumor suppressor gene by PCR-SSCP analysis and expression pattern by western blotting and immunoprecipitation. p53-mediated transactivation ability were examined by CAT assay and base substitution of p53 in SNU C2A cell were detected by DNA sequencing. Results: 1) SNU C2A cell and SNU C5 cell were detected mobility shifts each in exon 5 and exon 7 of p53 gene by the PCR-SSCP method, implicating being of p53 mutation. 2) 3 colon cancer cell lines (SNU C1, SNU C2A, SNU C5) expressed wild type and mutant type p53 protein. 3) In northern blot experiment, SNU C2A and SNU C5 cell expressed high level of p53 mRNA. 4) Results of p53-mediated transactivation in colon cancer cell lines by CAT assay represented only SNU C2A cell has transcriptional activity. 5) DNA sequencing in SNU C2A cell showed missense mutation in codon 179 of one allele, histidine to arginine and wild type p53 in the other allele. Conclusion: Colon cancer cell lines showed correlation with mutation in p53 gene and accumulation of abnormal p53 protein. Colon cancer cell SNU C2A retained p53-mediated transactivation as heterozygous p53 with one mutant allele in 179 codon and the other wild-type allele.

• Journal of Chest Surgery
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• 제29권11호
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• pp.1223-1231
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• 1996

72예의 원발성 폐암조직에서 두 종양억제유전자 RB와 p53의 단백질 발현 빈도를 면역조직화학적 방법으로 검출한 결과 RB 단백질 발현은 38예 (52.8%)에서 감소하거나 소실되었고, 변이형 p53 단백질은 35예(48.6%)에서 발현을 보였다. 이들의 발현 빈도는 폐암의 조직형에 따라 유의한 차이를 보여 RB는 선암에서 그리고 p53은 편평세포암에서 발현율이 높았다(p＜0.05). 그러나 임상 병기와는 두 단백질의 발현율과는 상관이 없었다. 폐암 환자의 2년 생존율은 두 단백질 발현의 변화가 동반된 군(RB-/p53+)에서 22.4%, 두 단백질 발현의 변화가 전혀 없는 군(RB+/p53-)에서 63.1%로 두 군 사이에 통계적으로 유의한 차이를 보였다(p＜0.05).본 연구에서 RB와 p53단백질의 발현의 유무가 원발성 폐암 환자의 생존율과 관련이 있음을 나타내었고, 특히 RB가 원발성 폐암의 예후에 가장 큰 영향을 미치는 인자임을 나타내었다.

• Molecules and Cells
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• 제45권11호
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• pp.806-819
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• 2022

Synaptic accumulation of α-synuclein (α-Syn) oligomers and their interactions with VAMP2 have been reported to be the basis of synaptic dysfunction in Parkinson's disease (PD). α-Syn mutants associated with familial PD have also been known to be capable of interacting with VAMP2, but the exact mechanisms resulting from those interactions to eventual synaptic dysfunction are still unclear. Here, we investigate the effect of α-Syn mutant oligomers comprising A30P, E46K, and A53T on VAMP2-embedded vesicles. Specifically, A30P and A53T oligomers cluster vesicles in the presence of VAMP2, which is a shared mechanism with wild type α-Syn oligomers induced by dopamine. On the other hand, E46K oligomers reduce the membrane mobility of the planar bilayers, as revealed by single-particle tracking, and permeabilize the membranes in the presence of VAMP2. In the absence of VAMP2 interactions, E46K oligomers enlarge vesicles by fusing with one another. Our results clearly demonstrate that α-Syn mutant oligomers have aberrant effects on VAMP2-embedded vesicles and the disruption types are distinct depending on the mutant types. This work may provide one of the possible clues to explain the α-Syn mutant-type dependent pathological heterogeneity of familial PD.