Elevated Levels Of P53 Protein Research Articles

PROGNOSTIC IMPORTANCE OF MOLECULAR GENETIC MARKERS: Ki67, Bcl2, and p53 IN PATIENTS WITH LOCALLY ADVANCED CERVICAL CANCER

Summary. In biopsy preparations, there are 30 patients with cervical cancer at the IIb-IIIb clinical stages. A study in patients with locally advanced cervical cancer showed that a high expression level of Ki67 proliferation in primary patients, elevated levels of p53 protein before carrying out correlate with an unfavorable prognosis, which makes it possible to use these indicators in monitoring the course of the disease. It was shown that prior to the initiation of antitumor treatment, the expression level of Ki67 was as high as possible in those patients who subsequently showed progression of the disease, and reached 85%. The expression level of Ki67 and p53 protein was shown to correlate with indicators of tumor progression. The aim of the study was: In accordance with the purpose of the study outlined above, the expression level of molecular biological markers Ki67, Bcl2, p53 and their prognostic value in patients with locally advanced cervical cancer who received complex treatment were assessed. Material and methods: 30 patients with locally advanced cervical cancer with IIB – IIIA-B stage who received chemoradiotherapy in ССncer center from 2014 to 2017 were examined. The stages of the disease before the start of treatment were carried out in accordance with the international classification of TNM (seventh revision 2009) and in accordance with the classification of the International Federation of Obstetricians-Gynecologists classification (FIGO, 2011). The diagnosis of the disease in all cases verified histological. Morphologically, all women were diagnosed with squamous cell carcinoma. The possibilities of the proliferative activity marker Ki67, p53 protein, Bcl2 in determining the prognosis of the disease, locally advanced cervical cancer were studied. The material of the study was cervical All patients underwent 3 courses of neoadjuvant polychemotherapy with 21 day intervals, according to the scheme: Cisplatin 100 mg 1 day, Fluorouracil 1000 mg 1-4 days.Then, after computerized topometry of the pelvic organs, planning of combined radiation therapy was carried out, including remote gamma therapy and intracavitary brachytherapy. Remote irradiation was carried out on a gamma therapeutic apparatus (TERABALT type 80 model SCS 2012 Czech Republic) in the standard fractionation mode (daily 5 times a week Single Focal Dose 2 Gy to Total Focal Dose 46 Gy). Intracavitary radiotherapy was performed on a BEBIG brachytherapy device - MULTISOURSE Co60 2013. (Germany) in fractional mode Single Focal Dose 5 Gy, eq Total Focal Dose to point A up to 70-90 Gy, to point B 50-58 Gy. The initial expression of the listed immunohistochemical markers in the tumor and prior to chemo-radiotherapy was evaluated. The immunohistochemical study was carried out according to a standard procedure on dewaxed sections of cervical tissue blocks obtained from resection specimens or cervical biopsies. A pathologist to clarify the histological diagnosis and the correspondence of the blocks to the selected sections reviewed all drugs.

VALUE OF MOLECULAR GENETIC MARKERS IN THE PROGNOSIS OF LOCALLY ADVANCED FORMS OF CERVICAL CANCER

In biopsy preparations, there are 30 patients with cervical cancer at the IIb-IIIb clinical stages. A study in patients with locally advanced cervical cancer showed that a high expression level of VEGF and Ki67 proliferation in primary patients, elevated levels of p53 protein before carrying out correlate with an unfavorable prognosis, which makes it possible to use these indicators in monitoring the course of the disease. It was shown that prior to the initiation of antitumor treatment, the expression level of VEGF and Ki67 were as high as possible in those patients who subsequently showed progression of the disease, and reached 85%. The expression level of VEGF, Ki67 and p53 protein was shown to correlate with indicators of tumor progression.

Prognostic significance of Ki67, Bcl2 and p53 in patients with locally advanced cervical cancer

In biopsy preparations, there are 30 patients with cervical cancer at the IIb-IIIb clinical stages. A study in patients with locally advanced cervical cancer showed that a high expression level of Ki67 proliferation in primary patients, elevated levels of p53 protein before carrying out correlate with an unfavorable prognosis, which makes it possible to use these indicators in monitoring the course of the disease. It was shown that prior to the initiation of antitumor treatment, the expression level of Ki67 was as high as possible in those patients who subsequently showed progression of the disease, and reached 85%. The expression level of Ki67 and p53 protein was shown to correlate with indicators of tumor progression.

Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response.

ABSTRACTThe purpose of study was to explore the role of glutamine-dependent anaplerosis in cell fate determination (proliferation and senescence) and the potential associated mechanism by employing a pharmacological inhibitor of glutamine-dependent anaplerosis, amino-oxyacetate (AOA). Using the WI38 normal human embryonic fibroblast cell line, we found that exposure to AOA induced mTORC1 inactivation−mTORC2 activation (within day 1), cell cycle arrest (day 2–6) and cellular senescence (day 4–6). These AOA effects were blocked by concomitantly providing anaplerotic factors [α-ketoglutarate (αKG), pyruvate or oxaloacetate], and not affected by ROS scavenger N-acetyl-cysteine (NAC). Moreover, AOA-induced cellular senescence in WI38 cells is associated with elevated protein levels of p53, p21CIP1 and p16INK4A and decreased Rb protein level, which was blocked by αKG supplementation. In p16INK4A-deficient U2OS human osteosarcoma cells and p16INK4A-knockdown WI38 cells, AOA exposure also induced similar effects on cell proliferation, and protein level of P-Rb-S807/811 and Rb. Interestingly, no AOA induction of cellular senescence was observed in U2OS cells, yet was still seen in p16INK4A-knockdown WI38 cells accompanied by the presence of p16 antibody-reactive p12. In summary, we disclose that glutamine-dependent anaplerosis is essential to cell growth and closely associated with mTORC1 activation and mTORC2 inactivation, and impedes cellular senescence particularly associated with p16INK4A.

AT‑101 induces G1/G0phase arrest via the β‑catenin/cyclinD1 signaling pathway in human esophageal cancer cells.

AT‑101, an orally available and well‑tolerated natural pan‑Bcl‑2family protein inhibitor, has been reported to be effective against a variety of cancers. However, the mechanisms whereby AT‑101 exhibits anticancer activity have not been fully elucidated. In this study, we demonstrated that AT‑101 reduced the cell viability of human esophageal cancer cells by inducing G1/G0phase arrest and apoptosis. Apoptotic cell death occurred later than cell cycle arrest, as evidenced by an increase in the proportion of AnnexinV‑positive cells and cleaved caspase‑3,‑9 and PARP protein levels. AT‑101 markedly downregulated the protein levels of phospho‑retinoblastoma (Ser780) and cyclinD1, whereas it elevated protein levels of p53 and p21Waf1/Cip1, contributing to the inhibition of cell cycle progression. Moreover, AT‑101 substantially reduced β‑catenin expression. XAV‑939, a small molecule that inhibits the Wnt/β‑catenin signaling pathway by facilitating β‑catenin degradation, lowered β‑catenin and cyclinD1 protein expression to an extent similar to AT‑101. XAV‑939 alone resulted in G1/G0phase arrest and further induced cell cycle arrest in combination with AT‑101, suggesting that the β‑catenin/cyclinD1 signaling pathway mediated, at least in part, the cell cycle arrest induced by AT‑101. The present study may shed new light on the anticancer activity of AT‑101 in relation to cell cycle arrest as well as apoptosis in human esophageal cancer cells.

Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin

Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n=9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n=5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC50<3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents.

MiR-375 is Essential for Human Spinal Motor Neuron Development and May Be Involved in Motor Neuron Degeneration.

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons.

Abstract 3188: Nucleolin phosphorylation mediated regulation of gene expression in determining cellular fate during the DNA damage response (DDR).

Abstract The phosphoprotein nucleolin integrates several critical cellular processes, such as cell growth, proliferation, cell cycle arrest, apoptosis as well as DDR. Elevated levels of nucleolin are found in highly proliferative cells including a variety of tumors. Nucleolin is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase (Cdk). Earlier we have demonstrated that the N-terminus of nucleolin associates with Hdm2 to stabilize p53 protein and causes p53-mediated apoptosis. Additionally, nucleolin via its RNA-binding properties has been demonstrated to regulate the stability of several mRNAs and enhance translation. Besides, nucleolin post-translational modifications are linked to its role as an RNA-binding stress-responsive protein. Based on these studies, we hypothesize a role for nucleolin phosphorylation in regulating mRNA stability of different target genes during DDR. To test this we have engineered a novel system with tet-off promoter in human osteosarcoma cells to express 3xFlag-tagged nucleolin-wt or phospho-mutant [6/S*A, defective in undergoing phosphorylation at six consensus CK2 sites] that allow us to control expression of nucleolin using doxycycline. Cell proliferation assays with these inducible cells indicated that although nucleolin phosphorylation by CK2 was required for cell proliferation in unstressed cells, a hypo-phosphorylated nucleolin mutant caused higher cell viability upon genotoxic stress. Further, nucleolin phospho-variants differentially elevated p53 protein levels and hypo-phosphorylated nucleolin associated with p53 even under non-stressed conditions. Our co-immunoprecipitation (co-IP) assays also showed that nucleolin associated with poly(A)-specific ribonuclease (PARN), an mRNA decay enzyme that controls mRNA stability of different genes during DDR. Importantly, hypo-phosphorylated nucleolin mutant decreased PARN deadenylase activity in vitro. Interestingly, nucleolin also interacted with cellular p53-mRNA as detected by RNA immunoprecipitation assays with nucleolin antibodies followed by qRT-PCR. Our data for the first time provides evidential support that nucleolin phosphorylation can play a regulatory role in driving the cell cycle and controlling gene expression during DDR. Direct associations of nucleolin with p53 and PARN probably affect p53-signaling as well as PARN-deadenylase activity. Understanding the control of gene expression that is regulated by nucleolin phosphorylation during DDR, will provide valuable insights into the mechanism(s) behind the cellular decisions of cell survival or death upon DNA damage. Citation Format: Xiaokan Zhang, Shu Xiao, Emral Devany, Zaineb Nadeem, Elif Caglar ýþýn, Frida Kleiman, Anjana D. Saxena. Nucleolin phosphorylation mediated regulation of gene expression in determining cellular fate during the DNA damage response (DDR). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3188. doi:10.1158/1538-7445.AM2013-3188

Identification of HEXIM1 as a Positive Regulator of p53

Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which regulates the transcription elongation of RNA polymerase II and controls 60-70% of mRNA synthesis. Our previous studies show that HEXIM1 interacts with two key p53 regulators, nucleophosmin and human double minute-2 protein (HDM2), implying a possible connection between HEXIM1 and the p53 signaling pathway. Here we report the interaction between p53 and HEXIM1 in breast cancer, acute myeloid leukemia, and colorectal carcinoma cells. The C-terminal regions of p53 and HEXIM1 are required for the protein-protein interaction. Overexpression of HEXIM1 prevents the ubiquitination of p53 by HDM2 and enhances the protein stability of p53, resulting in up-regulation of p53 target genes, such as Puma and p21. Induction of p53 can be achieved by several means, such as UV radiation and treatment with anti-cancer agents (including doxorubicin, etoposide, roscovitine, flavopiridol, and nutlin-3). Under all the conditions examined, elevated protein levels of p53 are found to associate with the increased p53-HEXIM1 interaction. In addition, knockdown of HEXIM1 significantly inhibits the induction of p53 and releases the cell cycle arrest caused by p53. Finally, the transcription of the p53 target genes is regulated by HEXIM1 in a p53-dependent fashion. Our results not only identify HEXIM1 as a positive regulator of p53, but also propose a novel molecular mechanism of p53 activation caused by the anti-cancer drugs and compounds.

EDD induces cell cycle arrest by increasing p53 levels

Tight regulation of p53 is essential for its central role in maintaining genome stability and tumor prevention. Here, EDD/ UBR5/hHyd, hereafter called EDD, is identified as a novel regulator of p53. Downregulation of EDD results in elevated p53 protein levels both in transformed and untransformed cells. Concomitant with a rise in p53, the levels of p21, a critical p53 target, are also elevated in these conditions. Surprisingly, EDD knockdown does not affect p53 protein stability, and p53 mRNA levels do not increase significantly upon EDD depletion. Consistent with the function of p53, EDD downregulation triggers a senescent phenotype in fibroblasts at later time points. In addition, the increased p53 levels upon EDD depletion cause a G1 arrest, as co-depletion of EDD and p53 completely rescues this effect on cell cycle progression.

P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,

Abstract 3432Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation. Disclosures:No relevant conflicts of interest to declare.

Inhibition of p53 by Adenovirus Type 12 E1B-55K Deregulates Cell Cycle Control and Sensitizes Tumor Cells to Genotoxic Agents

Adenovirus E1B-55K represses p53-mediated transcription. However, the phenotypic consequence of p53 inhibition by E1B-55K for cell cycle regulation and drug sensitivity in tumor cells has not been examined. In HCT116 cells with constitutive E1B-55K expression, the activation of p53 target genes such as the p21, Mdm2, and Puma genes was attenuated, despite markedly elevated p53 protein levels. HCT116 cells with E1B-55K expression displayed a cell cycle profile similar to that of the isogenic HCT116p53(-/-) cells, including unhindered S-phase entry despite DNA damage. Surprisingly, E1B-55K-expressing cells were more sensitive to drug treatment than parental cells. Compared to HCT116 cells, HCT116p53(-/-) cells were more susceptible to both doxorubicin and etoposide, and E1B-55K expression had no effects on drug treatment. E1B-55K expression increased the rate of cell proliferation in HCT116 but not in HCT116p53(-/-) cells. Thus, deregulation of p53-mediated cell cycle control by E1B-55K probably underlies sensitization of HCT116 cells to anticancer drugs. Consistently, E1B-55K expression in A549, A172, and HepG2 cells, all containing wild-type (wt) p53, also enhanced etoposide-induced cytotoxicity, whereas in p53-null H1299 cells, E1B-55K had no effects. We generated several E1B-55K mutants with mutations at positions occupied by the conserved Phe/Trp/His residues. Most of these mutants showed no or reduced binding to p53, although some of them could still stabilize p53, suggesting that binding might not be essential for E1B-55K-induced p53 stabilization. Despite heightened p53 protein levels in cells expressing certain E1B-55K mutants, p53 activity was largely suppressed. Furthermore, most of these E1B-55K mutants could sensitize HCT116 cells to etoposide and doxorubicin. These results indicate that E1B-55K might have utility for enhancing chemotherapy.

P53 Down-Regulates Phosphatase and Tensin Homologue Deleted on Chromosome 10 Protein Stability Partially through Caspase-Mediated Degradation in Cells with Proteasome Dysfunction

There has been intense investigation regarding the interaction between the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and p53 tumor suppressors. p53 has been shown to up-regulate PTEN expression as a transcriptional activator. However, clinical observations by immunohistochemistry studies indicate that significant increases in p53 protein levels coexist with reduced or absent expression of PTEN protein in a variety of neoplasias. In this study, we propose a mechanism that begins to explain how p53 can both up-regulate and down-regulate PTEN. We have found that PTEN protein is down-regulated under proteasome dysfunction induced by proteasome inhibitor MG132 in both human lymphoblast cells and MCF7 cells. The reduction of PTEN is coincident with elevated p53 protein levels and the association between PTEN and p53 but independent of its phosphatase activities. Quantitative reverse transcription-PCR indicates that proteasome inhibition does not reduce PTEN message levels but affects PTEN protein stability. The p53 inhibitor, pifithrin-alpha, is able to attenuate the effect of proteasome inhibition. Using ectopic expression studies in p53-null mouse embryonic fibroblasts and p53/PTEN-null PC3 cells, we show that PTEN is more stable in p53-null cells compared with p53-expressing cells. Inhibition of caspases, the downstream targets of p53, particularly caspase-3, can partially restore the stability of PTEN. This study provides the first evidence that p53 is able to down-regulate PTEN protein stability in stressed cells. Our study sheds some light on the mechanisms that regulate PTEN protein stability, which is important to fully elucidate to comprehend the broad neoplastic manifestations of Cowden syndrome/Bannayan-Riley-Ruvalcaba and sporadic cancers.

Tissue microarrays for comparing molecular features with proliferation activity in breast cancer

Tissue microarrays (TMAs) are potentially suited to find associations between molecular features and clinical outcome. Enhanced cell proliferation, as measured by Ki67 immunohistochemistry, is related to poor patient prognosis in many different tumor types. Ki67 expression shows considerable intratumoral heterogeneity. It is unclear if the TMA format is suitable for the analysis of potentially heterogeneous markers because of the small size of TMA spots. We have analyzed a breast cancer TMA containing 2,517 breast tissues, including 2,222 neoplastic and 295 normal or premalignant samples, for Ki67 labeling index (Ki67 LI) and additional markers with a known relationship to Ki67 LI by immunohistochemistry (ER, PR, Bcl-2, Egfr, p16, p53) and Fluorescence in situ hybridization (HER2, MDM2, CCND1, MYC). A high Ki67 LI was linked to tumor phenotype including grade (p < 0.0001), stage (p < 0.0001), nodal stage (p = 0.0018), and patient prognosis (p < 0.0001), elevated protein levels of p53, p16 and Egfr, reduced levels of Bcl2, ER, and PR (p < 0.0001 each), as well as amplifications of HER2, MYC, CCND1 and MDM2 (p < 0.0001 each). In summary, all expected associations between Ki67 and the analyzed molecular markers could be reproduced with high statistical significance using a TMA containing only one tissue sample per tumor, measuring 0.6 mm in diameter. We conclude that associations with cell proliferation can be reliably analyzed in a TMA format.

Accumulation of p53 protein in pterygia is not accompanied by TP53 gene mutation

Previously, we reported that pterygial epithelial cells show positive p53 staining by immunohistochemistry, and that they do not demonstrate apoptosis. We wished to determine whether the accumulation of p53 protein was caused by missense mutations in exons 5–8 of the TP53 gene, as is frequently the case in malignant tumours that contain high levels of abnormal p53. From 11 pterygia, epithelial cells were isolated by laser capture microdissection, or manually, in order to reduce the contribution of TP53 from normal cells. DNA from pterygial epithelial cells was amplified across exons 5–8 in 10 pterygia and across exons 5,7 and 8 in another pterygium. In 2 pterygia, all translated exons (2–11) were sequenced. No mutations were found, although normal polymorphisms in codon 72 were readily detected in 2 pterygia. RT-PCR was used to compare amounts of TP53 mRNA isolated from normal conjunctiva and pterygia from eight additional patients. We detected an approximate two-fold increase of TP53 RNA in pterygia compared to that in normal conjunctiva. Western blotting was used to compare amounts of p53 protein in pterygia and normal conjunctiva. Consistent with our previous immunohistochemical studies, amounts of p53 protein in pterygia, detected by the western blotting, were elevated compared to those detected in normal conjunctiva and corneal limbal epithelium. However, the TP53 gene in pterygia is not mutated, and therefore, the elevated levels of p53 protein must result from a different mechanism than that seen in malignant tumours containing TP53 missense mutations. The increased amount of p53 protein in pterygial cells does not cause apoptosis or block cell proliferation, suggesting that these normal p53 functions are inactivated in pterygia.

HDM2 phosphorylation by MAPKAP kinase 2

p53 stability is regulated by HDM2, a RING domain protein that acts as an E3 ligase to ubiquitinate p53 and target its degradation. Phosphorylation of HDM2 on serine 166 by AKT has been shown to enhance HDM2 activity and promote the degradation of p53. Here, we show that MAPKAP kinase 2 (MK2) can phosphorylate HDM2 on serine 157 and 166 in vitro. Treatment of cells with anisomycin, which activates MK2, also results in phosphorylation of HDM2 on serine 157 and 166 in vivo. Mutation of the MK2 phosphorylation sites in HDM2 to aspartic acid renders HDM2 slightly more active in the degradation of p53, and mouse cells deficient for MK2 show reduced Mdm2 phosphorylation and elevated levels of p53 protein. Together, our results suggest that MK2 may act to dampen the extent and duration of the p53 response.

Ginsenoside-Rs 4, a new type of ginseng saponin concurrently induces apoptosis and selectively elevates protein levels of p53 and p21 WAF1 in human hepatoma SK-HEP-1 cells

In this paper, we present evidence that ginsenoside-Rs 4 (G-Rs4; an acetylated analogue of ginsenoside-Rg 5), a new ginseng saponin isolated from Panax ginseng C. A. Meyer, elevates protein levels of p53 and p21 WAF1, which are associated with the induction of apoptosis in SK-HEP-1 cells. Flow cytometric analyses showed that G-Rs4 initially arrested the cell cycle at the G1/S boundary, but consequently induced apoptosis as evidenced by generating an apoptotic peak. The induction of apoptosis was confirmed by the results of DNA fragmentation assays and alterations in cell morphology after treatment of the cells with G-Rs4. Immunoblot assays showed that G-Rs4 significantly elevated protein levels of p53 and p21 WAF1, concurrently with the downregulation of both cyclins E- and A-dependent kinase activities and induction of apoptosis. We suggest that G-Rs4 induces apoptosis, the effect of which is closely related to the downregulation of both cyclins E- and A-dependent kinase activity as a consequence of selectively elevating protein levels of p53 and p21 WAF1 in SK-HEP-1 cells.

Participation of the human p53 3'UTR in translational repression and activation following gamma-irradiation.

p53 protein levels have been shown to increase in a number of cells after treatment with genotoxic agents through a post-transcriptional mechanism. In gamma-irradiated human cells, the accumulation of p53 protein is accompanied by an increase in the association of p53 mRNA with large polysomes without any change in the level of p53 mRNA. This redistribution of p53 mRNA on polysomes in response to irradiation is consistent with enhanced translational activity of p53 mRNA. We demonstrate that a region of the p53 3'-untranslated region (3'UTR) inhibits translation of a chimeric reporter mRNA in vivo. Induced elevation of reporter activity after gamma-irradiation was seen in cells expressing chimeric reporter-p53 3'UTR transcripts. These data taken together demonstrate translational control of p53 gene expression after gamma-irradiation and denote a previously unsuspected and novel role for the p53 3'UTR in controlling translation.

Two functional assays employed to detect an unusual mutation in the oligomerisation domain of p53 in a Li-Fraumeni like family.

Previous investigations of a Li - Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was confirmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.

Modulation of (+/-)-anti-BPDE mediated p53 accumulation by inhibitors of protein kinase C and poly(ADP-ribose) polymerase.

The rapid accumulation of the p53 gene product is considered to be an important component of the cellular response to a variety of genotoxins. In order to gain insights on the biochemical pathways leading to p53 stabilization, the effect of (+/-) 7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene [(+/-)-anti-BPDE] induced DNA damage on p53 protein levels was investigated in various repair-proficient and repair-deficient human cells. Brief exposure of normal human fibroblasts to 0.05-1 microM (+/-)-anti-BPDE resulted in elevated p53 protein levels as compared to the constitutive levels of control cells. The rapid induction response, detectable within a few hours, was sustained up to a period of at least 24 h. Repair-proficient and repair-deficient (XPA) human lymphoblastoid cells showed a similar response. The poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3-AB), diminished the p53 induction response by concomitantly decreasing the extent of (+/-)-anti-BPDE induced DNA damage in cells pretreated with the inhibitor. However, the direct involvement of poly ADP-ribosylation was also apparent as 3-AB was able to attenuate (approximately 50%) the p53 response by post-damage inhibitor treatment of the cells. Inhibition of cellular DNA replication by hydroxyurea and AraC, in the presence or absence of DNA damage, also resulted in rapid p53 accumulation in repair-deficient cells. On the contrary, inhibition of protein kinase C (PKC) by calphostin-C led to an abrogation of (+/-)-anti-BPDE mediated p53 induction. Analysis of the downstream effects of carcinogen treatment showed that the lymphoblastoid cells undergo DNA fragmentation indicative of apoptosis while fibroblasts exhibit cell cycle arrest at the G1-S boundary.