The role of p53 deacetylation in p21Waf1 regulation by laminar flow

Laminar flow arrests vascular endothelial cells at the G0/G1 phase with concurrent increase in p53 and p21Waf1. We investigated the molecular mechanism by which laminar flow activates p53 and p21Waf1 in endothelial cells. The application of a laminar flow (12 dyn/cm2) increased the deacetylation at Lys-320 and Lys-373 of p53 and the acetylation at Lys-382 in human umbilical vein endothelial cells. Laminar flow increased the activity of histone deacetylase (HDAC) and the association of p53 with HDAC1. Treating human umbilical vein endothelial cells with trichostatin A (TSA), an HDAC inhibitor, abolished the flow-induced p53 deacetylation at Lys-320 and Lys-373. To investigate the role of the HDAC-deacetylated p53 in the flow activation of p21Waf1, we found that TSA inhibited the activation at both the mRNA and protein levels. Deletion and mutation analyses of the p21Waf1 promoter revealed that flow activated p21Waf1 through p53 and TSA abrogated this p53-dependent activation. The expression plasmid encoding the p53 mutant, with Lys-320 and Lys-373 replaced by Arg, increased the activity of the co-transfected p21Waf1 promoter, which demonstrates that HDAC-deacetylated p53 can transactivate the p21Waf1 gene. The regulation of the p53-p21Waf1 pathway by laminar flow was further supported by observations that flow caused an increase of p21Waf1 level in the wild-type HCT116 (p53+/+) cells but not in the p53-null HCT116 cells.     

Nucleotide Biosynthetic Enzyme GMP Synthase Is a TRIM21-Controlled Relay of p53 Stabilization

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Expression of a Homeostatic Regulator, Wip1 (Wild-type p53-induced Phosphatase), Is Temporally Induced by c-Jun and p53 in Response to UV Irradiation

Wild-type p53-induced phosphatase (Wip1) is induced by p53 in response to stress, which results in the dephosphorylation of proteins (i.e. p38 MAPK, p53, and uracil DNA glycosylase) involved in DNA repair and cell cycle checkpoint pathways. p38 MAPK-p53 signaling is a unique way to induce Wip1 in response to stress. Here, we show that c-Jun directly binds to and activates the Wip1 promoter in response to UV irradiation. The binding of p53 to the promoter occurs earlier than that of c-Jun. In experiments, mutation of the p53 response element (p53RE) or c-Jun consensus sites reduced promoter activity in both non-stressed and stressed A549 cells. Overexpression of p53 significantly decreased Wip1 expression in HCT116 p53^+/+ cells but increased it in HCT116 p53^−/− cells. Adenovirus-mediated p53 overexpression greatly decreased JNK activity. Up-regulation of Wip1 via the p38 MAPK-p53 and JNK-c-Jun pathways is specific, as demonstrated by our findings that p38 MAPK and JNK inhibitors affected the expression of the Wip1 protein, whereas an ERK inhibitor did not. c-Jun activation occurred much more quickly, and to a greater extent, in A549-E6 cells than in A549 cells, with delayed but fully induced Wip1 expression. These data indicate that Wip1 is activated via both the JNK-c-Jun and p38 MAPK-p53 signaling pathways and that temporal induction of Wip1 depends largely on the balance between c-Jun and p53, which compete for JNK binding. Moreover, our results suggest that JNK-c-Jun-mediated Wip1 induction could serve as a major signaling pathway in human tumors in response to frequent p53 mutation.     

Expression of the p53 Gene and Activation of p53-Dependent Transcription in Melanoma Cell Lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type _p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope is not derived from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, suggesting a differential effect of camptothecin on tumor-derived and primary cells.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 445–456.Original Russian Text Copyright © 2005 by Razorenova, Agapova, Chumakov.     

MicroRNA 155 Control of p53 Activity Is Context Dependent and Mediated by Aicda and Socs1

In biological processes, the balance between positive and negative inputs is critical for an effective physiological response and to prevent disease. A case in point is the germinal center (GC) reaction, wherein high mutational and proliferation rates are accompanied by an obligatory suppression of the DNA repair machinery. Understandably, when the GC reaction goes awry, loss of immune cells or lymphoid cancer ensues. Here, we detail the functional interactions that make microRNA 155 (miR-155) a key part of this process. Upon antigen exposure, miR-155^−/− mature B cells displayed significantly higher double-strand DNA break (DSB) accumulation and p53 activation than their miR-155^+/+ counterparts. Using B cell-specific knockdown strategies, we confirmed the role of the miR-155 target Aicda (activation-induced cytidine deaminase) in this process and, in combination with a gain-of-function model, unveiled a previously unappreciated role for Socs1 in directly modulating p53 activity and the DNA damage response in B lymphocytes. Thus, miR-155 controls the outcome of the GC reaction by modulating its initiation (Aicda) and termination (Socs1/p53 response), suggesting a mechanism to explain the quantitative defect in germinal center B cells found in mice lacking or overexpressing this miRNA.     

Phosphorylation of p53 by TAF1 Inactivates p53-Dependent Transcription in the DNA Damage Response

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Prolonged progestin treatment induces the promoter of CDK inhibitor p21 Cip1,Waf1 through activation of p53 in human breast and endometrial tumor cells

Progestins are frequently used in the treatment of advanced breast and endometrial cancer. The human breast carcinoma cell line T47D shows a biphasic response to progestins. Short-term progestin treatment leads to enhanced DNA synthesis, while this line is growth inhibited upon prolonged exposure. An important protein involved in growth regulation by progestins in this cell is the CDK inhibitor p21(Cip1,Waf1). We show that after 1 day of progestin treatment in T47D cells, the p21 promoter-proximal region containing Sp1 binding sites is crucial in the induction by progestins. However, after 3 days the activity of the promoter-distal region becomes predominant in T47D cells or the endometrial carcinoma cell line ECC1. This is dependent upon two domains within this region that contain p53 response elements. In ECC1 and T47D cells 3-day progestin treatment induces a reporter containing a p53 response element, but not a mutated version. This induction is due to activation of p53 by progestin, which may be caused by nuclear translocation of p53. These data indicate that upon prolonged exposure, progestins activate p53, in human breast and endometrial tumor cells, which up-regulates the p21(Cip1,Waf1) promoter. This may be an important mechanism involved in progestin-inhibited cellular proliferation in these cells.     

p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.