DNA damage, p53 gene expression and p53 protein level in the rat brain aging

ABSTRACT Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53K317R mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53K317R MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53K317R thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53K317R thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

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Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.4.c946 ◽

1999 ◽

Vol 276 (4) ◽

pp. C946-C954 ◽

Cited By ~ 46

Author(s):

Li Li ◽

Ji Li ◽

Jaladanki N. Rao ◽

Minglin Li ◽

Barbara L. Bass ◽

...

Keyword(s):

Gene Expression ◽

Growth Inhibition ◽

P53 Protein ◽

Specific Inhibitor ◽

P53 Gene ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Polyamine Biosynthesis ◽

Fetal Bovine ◽

P53 Mrna

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.

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Anticancer and Gene Expression Analysis of Piper nigrum Extract on Colon Cancer Cell Lines

Bioscience Biotechnology Research Communications ◽

10.21786/bbrc/14.4.87 ◽

2021 ◽

Vol 14 (4) ◽

pp. 1968-1974

Author(s):

Neha Jadhav Giridhar

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Mtt Assay ◽

P53 Gene ◽

Staining Technique ◽

Suppressor Gene ◽

Piper Nigrum ◽

Genetic Syndromes ◽

Anticancer Activities ◽

Dual Staining

One of the most prevalent malignancies among geriatrics is colorectal cancer (CRC), which starts to develop in the forms of genetic syndromes in young adults. The Piper nigrum is one important common spice used in the household having anticancer activities. The current study aims to evaluate P. nigrum seed extracts potency as anticancer against CRC cell line (COLO205). The extract is used to elucidate the MTT assay, DNA damage studies (COMET assay), Acridine Orange/Ethidium Bromide dual staining, cell death, cell cycle arrest using Flow cytometry, and regulation of Bcl-2, Bax & P53 gene regulation. To check the cell cytotoxicity by MTT assay methanolic extract was used. To evaluate anticancer activity the sample was extracted in methanol. RT-PCR was used to elevate gene expression studies of Bcl-2, Bax, and P53. In the dose-dependent mode, the extract inhibited the growth of COLO205 cells and the IC50 value was calculated at 48.2 μg/ml. The DNA fragmentation induced by apoptosis was the primary reason for the cell toxicity as observed by DNA damage studies & AO/EB dual staining technique. The extract concentration ranging from 40 & 80 μg/ml remarkably increased the proportion of cells in the S & G2/M phase. Cells at the late-apoptotic stage were found to be in the range of 22% - 57%. The Bax and P53 were upregulated and Bcl-2 was downregulated when treated with the extract. From this investigation underlying the mechanism of CRC was found to be P. nigrum extract caused to induce apoptosis and upregulation of tumor suppressor gene downregulation of apoptosis-suppressing gene bcl-2.

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High-expression of ROCK1 modulates the apoptosis of lens epithelial cells in age-related cataracts by targeting p53 gene

10.21203/rs.3.rs-48570/v2 ◽

2020 ◽

Author(s):

Shanshan Hu ◽

Dongmei Su ◽

Lei Sun ◽

Zhongying Wang ◽

Lina Guan ◽

...

Keyword(s):

Epithelial Cells ◽

Protein Level ◽

P53 Protein ◽

P53 Gene ◽

Lens Epithelial Cells ◽

Protein Levels ◽

P53 Signaling ◽

Age Related ◽

Gene And Protein Expression

Abstract Background: Age-related cataract (ARC) is a serious visual impairment disease, and its pathogenesis is unclear. This article aims to investigate the role of ROCK1 in the apoptosis of lens epithelial cells (LECs) in age-related cataracts. Methods: We collect anterior capsule samples from normal people, patients with age-related cataracts, young mice and naturally aging cataract mice. The Oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with H2O2. MTT, Hoechst 33342, and TUNEL assay were performed to explore proliferation and apoptosis. HE assay was used to observe cell morphology. The gene and protein expression were assessed by quantitative real-time PCR, western blot, immunofluorescence, and immunohistochemical staining.Result: The results from the clinic and mice experiments showed that the numbers of lens epithelial cells from cataract individuals were less than the control individuals. In vitro, the apoptotic cells were increased in lens epithelial cells under H2O2 treatment. The ROCK1 protein level increased in the lens epithelial cells from age-related cataract patients and the old mice, respectively. Meanwhile, the up-regulation of the ROCK1 gene was associated with H2O2-induced HLE-B3 cells apoptosis. MTT and apoptosis assay showed ROCK1 was necessary in mediating H2O2-induced lens epithelial cells apoptosis through ROCK1 over-expression and knockdown experiment, respectively. Further investigation showed that p53 protein levels had been increased during ROCK1-mediated apoptosis in response to H2O2. Besides, ROCK1 phosphorylated p53 at ser15 to up-regulate its protein level. Conclusions: This study established the novel association of ROCK1/p53 signaling with lens epithelial cells apoptosis and age-related cataract genesis.

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High-expression of ROCK1 modulates the apoptosis of lens epithelial cells in age-related cataracts by targeting p53 gene

Molecular Medicine ◽

10.1186/s10020-020-00251-6 ◽

2020 ◽

Vol 26 (1) ◽

Author(s):

Shanshan Hu ◽

Dongmei Su ◽

Lei Sun ◽

Zhongying Wang ◽

Lina Guan ◽

...

Keyword(s):

Epithelial Cells ◽

Protein Level ◽

P53 Protein ◽

P53 Gene ◽

Lens Epithelial Cells ◽

Protein Levels ◽

P53 Signaling ◽

Age Related ◽

Gene And Protein Expression

Abstract Background Age-related cataract (ARC) is a serious visual impairment disease, and its pathogenesis is unclear. This article aims to investigate the role of ROCK1 in the apoptosis of lens epithelial cells (LECs) in age-related cataracts. Methods We collect anterior capsule samples from normal people, patients with age-related cataracts, young mice and naturally aging cataract mice. The oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with H2O2. MTT, Hoechst 33342, and TUNEL assay were performed to explore proliferation and apoptosis. HE assay was used to observe cell morphology. The gene and protein expression were assessed by quantitative real-time PCR, western blot, immunofluorescence, and immunohistochemical staining. Result The results from the clinic and mice experiments showed that the numbers of lens epithelial cells from cataract individuals were less than the control individuals. In vitro, the apoptotic cells were increased in lens epithelial cells under H2O2 treatment. The ROCK1 protein level increased in the lens epithelial cells from age-related cataract patients and the old mice, respectively. Meanwhile, the up-regulation of the ROCK1 gene was associated with H2O2-induced HLE-B3 cells apoptosis. MTT and apoptosis assay showed ROCK1 was necessary in mediating H2O2-induced lens epithelial cells apoptosis through ROCK1 over-expression and knockdown experiment, respectively. Further investigation showed that p53 protein levels had been increased during ROCK1-mediated apoptosis in response to H2O2. Besides, ROCK1 phosphorylated p53 at ser15 to up-regulate its protein level. Conclusions This study established the novel association of ROCK1/p53 signaling with lens epithelial cells apoptosis and age-related cataract genesis.

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In utero radiation-induced apoptosis and p53 gene expression in the developing rat brain

International Journal of Radiation Biology ◽

10.1080/095530001453131 ◽

2001 ◽

Vol 77 (1) ◽

pp. 71-81 ◽

Cited By ~ 21

Author(s):

S. Bolaris, E. Bozas, A. Benekou, H. P

Keyword(s):

Gene Expression ◽

Rat Brain ◽

P53 Gene ◽

In Utero ◽

Radiation Induced ◽

Induced Apoptosis ◽

Developing Rat

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Mutation of Mouse p53 Ser23 and the Response to DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.22.8.2441-2449.2002 ◽

2002 ◽

Vol 22 (8) ◽

pp. 2441-2449 ◽

Cited By ~ 76

Author(s):

Zhiqun Wu ◽

John Earle ◽

Shin'ichi Saito ◽

Carl W. Anderson ◽

Ettore Appella ◽

...

Keyword(s):

Dna Damage ◽

Ionizing Radiation ◽

Radiation Treatment ◽

Es Cells ◽

P53 Protein ◽

Embryonic Stem ◽

P53 Gene ◽

Wild Type ◽

Embryonic Fibroblasts ◽

Phosphorylation Event

ABSTRACT Recent studies have suggested that phosphorylation of human p53 at Ser20 is important for stabilizing p53 in response to DNA damage through disruption of the interaction between MDM2 and p53. To examine the requirement for this DNA damage-induced phosphorylation event in a more physiological setting, we introduced a missense mutation into the endogenous p53 gene of mouse embryonic stem (ES) cells that changes serine 23 (S23), the murine equivalent of human serine 20, to alanine (A). Murine embryonic fibroblasts harboring the p53S23A mutation accumulate p53 as well as p21 and Mdm2 proteins to normal levels after DNA damage. Furthermore, ES cells and thymocytes harboring the p53S23A mutation also accumulate p53 protein to wild-type levels and undergo p53-dependent apoptosis similarly to wild-type cells after DNA damage. Therefore, phosphorylation of murine p53 at Ser23 is not required for p53 responses to DNA damage induced by UV and ionizing radiation treatment.

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Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.3.c941 ◽

2001 ◽

Vol 281 (3) ◽

pp. C941-C953 ◽

Cited By ~ 61

Author(s):

Li Li ◽

Jaladanki N. Rao ◽

Xin Guo ◽

Lan Liu ◽

Rachel Santora ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Growth Inhibition ◽

Intestinal Mucosa ◽

P53 Protein ◽

P53 Gene ◽

Small Intestinal Mucosa ◽

Small Intestinal ◽

The Stability ◽

P53 Mrna

The p53 nuclear phosphoprotein plays a critical role in transcriptional regulation of target genes involved in growth arrest and apoptosis. The natural polyamines, including spermidine, spermine, and their precursor putrescine, are required for cell proliferation, and decreasing cellular polyamines inhibits growth of the small intestinal mucosa. In the current study, we investigated the mechanisms of regulation of p53 gene expression by cellular polyamines and further determined the role of the gene product in the process of growth inhibition after polyamine depletion. Studies were conducted both in vivo and in vitro using rats and the IEC-6 cell line, derived from rat small intestinal crypt cells. Levels for p53 mRNA and protein, transcription and posttranscription of the p53 gene, and cell growth were examined. Depletion of cellular polyamines by treatment with α-difluoromethylornithine (DFMO) increased p53 gene expression and caused growth inhibition in the intact small intestinal mucosa and the cultured cells. Polyamine depletion dramatically increased the stability of p53 mRNA as measured by the mRNA half-life but had no effect on p53 gene transcription in IEC-6 cells. Induction of p53 mRNA levels in DFMO-treated cells was paralleled by an increase in the rate of newly synthesized p53 protein. The stability of p53 protein was also increased after polyamine depletion, which was associated with a decrease in Mdm2 expression. When polyamine-deficient cells were exposed to exogenous spermidine, a decrease in p53 gene expression preceded an increase in cellular DNA synthesis. Inhibition of the p53 gene expression by using p53 antisense oligodeoxyribonucleotides significantly promoted cell growth in the presence of DFMO. These findings indicate that polyamines downregulate p53 gene expression posttranscriptionally and that growth inhibition of small intestinal mucosa after polyamine depletion is mediated, at least partially, through the activation of p53 gene.

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A Glance of p53 Functions in Brain Development, Neural Stem Cells, and Brain Cancer

Biology ◽

10.3390/biology9090285 ◽

2020 ◽

Vol 9 (9) ◽

pp. 285

Author(s):

Yuqing Xiong ◽

Yun Zhang ◽

Shunbin Xiong ◽

Abie E. Williams-Villalobo

Keyword(s):

Gene Expression ◽

Brain Development ◽

P53 Protein ◽

P53 Gene ◽

Stem Cell Regulation ◽

Cellular Events ◽

Damage Repair ◽

Brain Tumor Cells ◽

Metabolic Remodeling

p53 is one of the most intensively studied tumor suppressors. It transcriptionally regulates a broad range of genes to modulate a series of cellular events, including DNA damage repair, cell cycle arrest, senescence, apoptosis, ferroptosis, autophagy, and metabolic remodeling, which are fundamental for both development and cancer. This review discusses the role of p53 in brain development, neural stem cell regulation and the mechanisms of inactivating p53 in gliomas. p53 null or p53 mutant mice show female biased exencephaly, potentially due to X chromosome inactivation failure and/or hormone-related gene expression. Oxidative cellular status, increased PI3K/Akt signaling, elevated ID1, and metabolism are all implicated in p53-loss induced neurogenesis. However, p53 has also been shown to promote neuronal differentiation. In addition, p53 mutations are frequently identified in brain tumors, especially glioblastomas. Mechanisms underlying p53 inactivation in brain tumor cells include disruption of p53 protein stability, gene expression and transactivation potential as well as p53 gene loss or mutation. Loss of p53 function and gain-of-function of mutant p53 are both implicated in brain development and tumor genesis. Further understanding of the role of p53 in the brain may provide therapeutic insights for brain developmental syndromes and cancer.

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