P53 mRNA Transcripts Research Articles

Apoptotic p53 Gene Expression in the Regulation of Persistent Organic Pollutant (POP)-Induced Oxidative Stress in the Intertidal Crab Macrophthalmusjaponicus.

Persistent organic pollutants (POPs), some of the most dangerous chemicals released into the aquatic environment, are distributed worldwide due to their environmental persistence and bioaccumulation. In the study, we investigated p53-related apoptotic responses to POPs such as hexabromocyclododecanes (HBCDs) or 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in the mud crab Macrophthalmus japonicus. To do so, we characterized M. japonicus p53 and evaluated basal levels of p53 expression in different tissues. M. japonicus p53 has conserved amino acid residues involving sites for protein dimerization and DNA and zinc binding. In phylogenetic analysis, the homology of the deduced p53 amino acid sequence was not high (67–70%) among crabs, although M. japonicus p53 formed a cluster with one clade with p53 homologs from other crabs. Tissue distribution patterns revealed that the highest expression of p53 mRNA transcripts was in the hepatopancreas of M. japonicus crabs. Exposure to POPs induced antioxidant defenses to modulate oxidative stress through the upregulation of catalase expression. Furthermore, p53 expression was generally upregulated in the hepatopancreas and gills of M. japonicus after exposure to most concentrations of HBCD or BDE-47 for all exposure periods. In hepatopancreas tissue, significant increases in p53 transcript levels were observed as long-lasting apoptotic responses involving cellular defenses until day 7 of relative long-term exposure. The findings in this study suggest that exposure to POPs such as HBCD or BDE-47 may trigger the induction of cellular defense processes against oxidative stress, including DNA repair, cell cycle arrest, and apoptosis through the transcriptional upregulation of p53 expression in M. japonicus.

Abstract P491: Myosin-7 E848G Mutation Induces P53-associated Cell Death That Leads To Impaired Tissue Contractility In Patient-derived Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy

Introduction: Familial hypertrophic cardiomyopathy (HCM), affecting 1 in 500 adults, is characterized by idiopathic thickening of the heart and occasional impaired systolic function. Mechanisms through which cardiac sarcomeric mutations manifest in HCM are poorly understood. Hypothesis: We previously identified a novel MYH7 E848G mutation associated with HCM. We hypothesize E848G induces cell death that results in impaired tissue contractility in a dose-dependent manner. Methods: We created MYH7 expressing CMs with WT/WT, E848G/WT, or E848G/E848G alleles by CRISPR/Cas9 gene-editing patient-specific induced pluripotent stem cells (hiPSCs). hiPSC-derived cardiomyocytes were metabolically purified and cocultured with stromal cells on PDMS posts to create 3D engineered heart tissues (EHTs) or cultured as a monolayer. Results: Day 65 monolayer E848G/E848G CMs had 48.5% effective cell number relative to WT/WT. p53 (2.80 ± .11-fold), p21 (7.24 ± .18-fold), and BAX (1.64 ± 0.14-fold) mRNA transcripts were upregulated in day 60 monolayer E848G/E848G relative to WT/WT. E848G/E848G EHTs (n = 12) exhibited lower maximum active twitch force (104.6 ± 18.2 μN) and smaller 2D projected area (5.31 ± 0.22 mm 2 ) at day 14 relative to WT/WT (n = 15; 238.0 ± 20.4 μN; 6.87 ± 0.26 mm 2 ). E848G/WT EHTs (n = 7) had intermediate twitch force (168.7 ± 12.2 μN) and 2D area (6.18 ± 0.36 mm 2 ). Conclusion: These results suggest the MYH7 E848G mutation induces p53-associated cell death that leads to reduced tissue contractility. Ongoing studies will elucidate the molecular mechanism through which E848G activates cell death pathways. Figure: Representative EHTs. L-R: WT/WT, E848G/WT, E848G/E848G.

Effects of selenium on the proliferation and apoptosis of sheep spermatogonial stem cells in vitro

The objective of this study was to investigate effects of selenium (Se) on proliferation and apoptosis of sheep spermatogonial stem cells (SSC) in vitro. The SSC were assigned to five treatment groups (0, 2.0, 4.0, 8.0 and 16.0 μmol/L Se). After treatment with Se for 96 h, cell proliferation and apoptosis were evaluated. The relative abundance of P53 mRNA transcript and protein, cell cycle and apoptosis-related genes were detected using real-time PCR and Western blot quantifications, respectively. The results indicate there were the least cell proliferation rates in the Se16.0 group. Treatments with relatively greater Se concentrations (8.0 and 16.0 μmol/L) resulted in a greater percentage of apoptotic cells, which was consistent with the relative abundances of P53, P21, P27 and pro-apoptosis mRNA transcripts. There were relatively greater ROS concentrations in the control, Se8.0 and Se16.0 groups. Compared with the control group, treatment with the Se concentration of 16.0 μmol/L resulted in an increased abundance of P53, P21, P27 and BAX proteins. Treatment with Pifithrin-α suppressed the increase in abundance of P53 and P21 proteins induced by the relatively greater concentration of Se (16.0 μmol/L), however, did not result in a change in abundances of P27 and BAX proteins. These results indicate the regulatory functions of Se on proliferation and apoptosis of sheep SSC is associated with the P21-mediated P53 signalling pathway. The P27 and BAX proteins have limited functions during the apoptotic process of SSC induced by the relatively greater concentrations of Se.

Nonsense mutation-dependent reinitiation of translation in mammalian cells.

In-frame stop codons mark the termination of translation. However, post-termination ribosomes can reinitiate translation at downstream AUG codons. In mammals, reinitiation is most efficient when the termination codon is positioned close to the 5′-proximal initiation site and around 78 bases upstream of the reinitiation site. The phenomenon was studied mainly in the context of open reading frames (ORFs) found within the 5′-untranslated region, or polycicstronic viral mRNA. We hypothesized that reinitiation of translation following nonsense mutations within the main ORF of p53 can promote the expression of N-truncated p53 isoforms such as Δ40, Δ133 and Δ160p53. Here, we report that expression of all known N-truncated p53 isoforms by reinitiation is mechanistically feasible, including expression of the previously unidentified variant Δ66p53. Moreover, we found that significant reinitiation of translation can be promoted by nonsense mutations located even 126 codons downstream of the 5′-proximal initiation site, and observed when the reinitiation site is positioned between 6 and 243 bases downstream of the nonsense mutation. We also demonstrate that reinitiation can stabilise p53 mRNA transcripts with a premature termination codon, by allowing such transcripts to evade the nonsense mediated decay pathway. Our data suggest that the expression of N-truncated proteins from alleles carrying a premature termination codon is more prevalent than previously thought.

Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies

ABSTRACTTranscription initiation sites of Trp53 gene in mice were determined using the 5′RACE method. Based on sequence alignment of the 5′-terminal regions of p53 mRNA in mammals, the site for the most abundant transcript turned out to be essentially identical with that determined for human TP53 gene and slightly differed for the longest transcripts, in mice and humans. Secondary structures of the 5′ -terminal regions of the shorter, most abundant and the longest mouse transcripts were determined in vitro and the shorter transcript was also mapped in transfected mouse cells. For the first time, secondary structure models of the 5′ terminus of two mouse p53 mRNAs were proposed. Comparing these models with the conservativeness of the nucleotide sequence of the 5′-terminal region of mRNA in mouse and other mammals, the possible function of the selected structural domains of this region was discussed. To elucidate the translation mechanisms, the two studied mRNAs were translated in the presence of an increasing concentration of the cap analog. For the longest transcript, the data suggested that IRES element(s) was/were involved in translation initiation. Additionally, changes in p53 synthesis under genotoxic and endoplasmic reticulum stress conditions in mouse cells were analyzed.

Behavioral, molecular and physiological responses of embryo-larval zebrafish exposed to types I and II pyrethroids

Pyrethroids are potent neurotoxicants that may elicit multiple pathways of toxicity in non-target organisms. Comparative studies on the mechanistic and developmental effects of types I and II pyrethroids against non-target aquatic species are limited. This study assessed the effects of the two pyrethroid types against embryo-larval zebrafish (Danio rerio) at environmentally relevant and laboratory concentrations. Zebrafish embryos were exposed to type-I (permethrin, bifenthrin) and type-II (deltamethrin, λ-cyhalothrin, fenvalerate, esfenvalerate) pyrethroids at 1000, 10, 0.1, 0.01, 0.0 μg/L, starting at 5-h post-fertilization (hpf) through 5-d post-fertilization (dpf) under static exposure conditions. Swimming behavior (distance traveled and velocity) was assessed at 5-dpf. The relative expression of Nrf2a, GST, Casp-9 and p53 mRNA transcripts, carboxyl esterase (CES) activity and total reactive oxygen species (ROS) were measured. The stability of the pyrethroids across 5 days was analyzed. Bifenthrin-(10 μg/L) and esfenvalerate-(1000 μg/L) significantly (p < 0.05) reduced total distance traveled by larvae while 1000 μg/L deltamethrin and λ-cyhalothrin were lethal causing body axis curvature and pericardial edema. At environmentally relevant concentrations-(μg/L) compared to control, permethrin-(0.122) upregulated Nrf2a and Casp-9 expressions while λ-cyhalothrin-(0.053) downregulated Nrf2a and fenvalerate-0.037 downregulated GST. At laboratory concentrations-(μg/L), permethrin-(1000) upregulated Nrf2a, Casp-9 and p53 expressions, bifenthrin-(10) upregulated Casp-9 while fenvalerate-(0.1) and esfenvalerate-(1000) downregulated GST. There was concentration dependent increase in CES activity which correlated positively with total ROS. Pyrethroid concentrations decreased significantly by day 5. This study showed disparity in the mechanistic effects across the pyrethroids types and their instability in aqueous media may underestimate toxicity against non-target aquatic species when exposed in their natural environment.

Variants of the 5\u2032-terminal region of p53 mRNA influence the ribosomal scanning and translation efficiency

The p53 protein is one of the major cell cycle regulators. The protein is expressed as at least twelve protein isoforms resulting from the use of alternative promoters, alternative splicing or downstream initiation codons. Importantly, there is growing evidence that translation initiation of p53 mRNA may be regulated by the structure and length of the naturally occurring variants of the 5′-terminal region of p53 mRNA transcripts. Here, several mRNA constructs were synthesized with variable length of the p53 5′-terminal regions and encoding luciferase reporter protein, and their translation was monitored continuously in situ in a rabbit reticulocyte lysate system. Moreover, four additional mRNA constructs were prepared. In two constructs, the structural context of AUG1 initiation codon was altered while in the other two constructs, characteristic hairpin motifs present in the p53 5′-terminal region were changed. Translation of the last two constructs was also performed in the presence of the cap analogue to test the function of the 5′-terminal region in cap-independent translation initiation. Superposition of several structural factors connected with the length of the 5′-terminal region, stable elements of the secondary structure, structural environment of the initiation codon and IRES elements greatly influenced the ribosomal scanning and translation efficiency.

The role of the 5' terminal region of p53 mRNA in the p53 gene expression.

The p53 tumour suppressor protein is one of the major factors responsible for cell cycle regulation and protection against cancer development. This is why it is often referred to as "the guardian of the genome". On the other hand, mutations in the p53 gene are connected with more than 50% of tumours of various types. The thirty-six years of extensive research on the p53 gene and its protein products have shown how sophisticated the p53-based cell system control is. An additional level of complexity of the p53 research is connected with at least twelve p53 isoforms which have been identified in the cell. Importantly, disturbance of the p53 isoforms' expression seems to play a key role in tumorigenesis, cell differentiation and cell response to pathogenic bacteria, and RNA and DNA viruses. Expression of various p53 isoforms results from the usage of different transcription promoters, alternative splicing events and translation initiation from alternative AUG codons. The importance of the 5'-terminal regions of different p53 mRNA transcripts in the multi-level regulation of the p53 gene has recently been documented. In this review we focus on the structural features of these regions and their specific role in the p53 translation initiation process.

Arecoline, a major alkaloid of areca nut, inhibits p53, represses DNA repair, and triggers DNA damage response in human epithelial cells

The International Agency for Research on Cancer declared that areca nut was carcinogenic to human. Areca nut is the main component of betel quid (BQ), which is commonly consumed in Asia. Epidemiological studies have shown that BQ chewing is a predominant risk factor for oral and pharyngeal cancers. It has been known that areca nut is genotoxic to human epithelial cells. However, the molecular and cellular mechanisms underlying areca nut-associated genotoxicity are not fully understood. Here we showed that arecoline, a major alkaloid of areca nut, might contribute to oral carcinogenesis through inhibiting p53 and DNA repair. We found, on the biological aspect, that arecoline could induce γ-H2AX phosphorylation, a sensitive DNA damage marker, in KB, HEp-2, and 293 cells, suggesting that DNA damages were elicited by arecoline. This phenomenon was supported by the observations of arecoline-induced hyperphosphorylation of ATM, Nbs1, Chk1/2, p53, and Cdc25C, as well as G2/M cell cycle arrest, indicating that a cellular DNA damage response was activated. To explore the possible mechanism accounting for arecoline-elicited DNA damages, we found that arecoline could inhibit p53 by its expression and transactivation function. As a result, the expression of p53-regulated p21 WAF1 and the p53-activated DNA repair were repressed by arecoline. Finally, we showed that p53 mRNA transcripts were frequently down-regulated in BQ-associated oral cancer, suggesting that arecoline-mediated p53 inhibition might play a role in BQ-associated tumorigenesis.

Effects of Xenoestrogens on T Lymphocytes: Modulation of bcl-2, p53, and Apoptosis

Endogenous estrogens have significant immunomodulatory effects characterized as suppression of cell mediated immunity and stimulation of humoral immunity. Xenoestrogens are environmental estrogens that have endocrine impact, acting as estrogen agonists and antagonists but whose immune effects are not well characterized. Using CD4+ Jurkat T cells as a model, the effects of representative xenoestrogens on T proliferation, cell cycle, and apoptosis were examined. Coumestrol (CM), a phytoestrogen, and tetrachlorodioxin (TCDD) in concentrations of 10-4 to 10-6M significantly inhibited Jurkat T cell lymphoproliferation, whereas bisphenol A (BPA) and DDT had minimal effect, but did antagonize 17-β-estrtadiol induced effects. Xenoestrogens, especially CM, produced accumulation of Jurkat T cells in G2/M phase, and subsequently induced apoptosis, particularly CM (% apoptotic cells = 30 ± 12 vs. control = 5 ± 2). These changes were associated with DNA fragmentation. BPA and DDT also induced DNA fragmentation but not significant DNA hypoploidy. Xenoestrogen – CM, BPA, DDT, and TCDD - exposure suppressed bcl-2 protein and mRNA transcript levels but augmented p53 protein and mRNA transcripts. Human purified peripheral blood lymphocytes responded with similar significant cell cycle changes (G0/G1 exodus and G2/M accumulation) for CM, BPA, and DDT exposure. These preliminary data, taken together, suggest that xenoestrogens have direct, compound-specific T lymphocyte effects that enhance our understanding of environmental modulation of immune and autoimmune responses.

The role of p53, bcl-2 and bax network in dexamethasone induced apoptosis in multiple myeloma cell lines

The role of bcl-2 in protection against apoptosis is well established in a great variety of cells and has become a hallmark of drug-resistance in many tumor cell lines, notably in lymphomas and leukemias. Conflicting results have been reported as for the role of bcl-2 in spontaneous and drug induced apoptosis in multiple myeloma (MM), although high expression of bcl-2 was observed, in spite of relatively low frequency of t(14:18). We, therefore, decided to conduct a detailed study of the role of the bcl-2/bax/p53 network in dexamethasone (DEX) induced apoptosis in MM cells. Eight myeloma cell lines were screened for their expression of mRNA transcripts for p53, bcl-2 and bax, and the levels were correlated for sensitivity to DEX induced apoptosis. Two cell lines (HS-Sultan and ARH-77) expressed relatively high levels of bcl-2 transcripts, and were highly resistant to DEX induced apoptosis (up to 10 mu M). Two cell lines (8226 and ARP-1) expressed relatively low-levels of bcl-2 mRNA transcripts, and were highly sensitive to DEX (ID50= 0.1 mu M, at 24-48 h). Fax mRNA transcripts were abundant, were expressed ubiquitously in all cell lines, and, thus, did not correlate with sensitivity to DEX. The level of expression of mRNA transcripts for p53 varied among the various cell lines, and did not always correlate with resistance to DEX. Induction of apoptosis in 8226 and ARP-1 cells (DEX sensitive) resulted, within 24 h, in a transient but marked down-regulation of mRNA transcripts for bcl-2 and p53, whereas the level of expression of bax mRNA transcripts were unchanged, except for ARP-1 cells (lacking p53), where slight down-regulation of mRNA transcripts for bax, was observed. In contrast to the DEX sensitive cell lines, the level of expression of bcl-2, bax and p53 mRNA transcripts in the DEX resistant cell lines, were unchanged during 72 h of treatment with DEX (up to 10 mu M). We, therefore, conclude that bcl-2 and perhaps p53 are involved in resistance to DEX in myeloma cell lines.

Overexpression and point mutations of p53 tumor suppressor gene in hepatocellular carcinomas in Hong Kong Chinese people.

Gene deletion, point mutations, and abnormalities in expression of the tumor suppressor gene p53 in hepatocellular carcinoma have been reported to occur with varying frequency in different geographic regions. To assess the expression and point mutation of the p53 gene, 31 patients with hepatocellular carcinomas were examined using Northern blotting, immunohistochemical methods, and DNA sequencing. All patients were Chinese, and 90.3% were positive for hepatitis B surface antigen (HBsAg). p53 transcript or protein was found in 14 (48.4%) of the 31 patients. Detectable p53 mRNA transcripts were found in 10 patients, and p53 protein was detected in 8 patients. In most cases of patients who had detectable p53 mRNA transcripts, the transcripts in the tumors were exhibited at a higher level than they were in the corresponding nontumorous livers. No p53 protein was detected in the nontumorous livers in all 31 patients. Six (23.1%) of the 26 tumors sequenced showed point mutation scattered in exons 5-9. Of these, only two were at codon-249, and the nature of these two mutations was G-to-T transversions. All but one of the six patients with point mutations had overexpression of the gene. Our results show that scattered point mutations are not uncommon in hepatocellular carcinomas in patients from Hong Kong. The distribution pattern of the mutations seems to have no particular correlation with HBsAg status despite a high prevalence rate of HBsAg positivity in our patients. Consistent with a low aflatoxin exposure, aflatoxin-related specific mutation at codon-249 is much less related to the pathogenesis of hepatocellular carcinoma in Hong Kong Chinese people than in other regions with a high-aflatoxin exposure.

Changes in p53 mRNA expression during terminal differentiation of murine erythroleukemia cells

The protein p53 is functionally implicated in the normal regulation of cell proliferation. We have previously reported that the rate of p53 protein synthesis is reduced during the cessation of cellular proliferation which accompanies the in vitro induced differentiation of Friend-erythroleukemia cells. In this work we followed the p53 mRNA expression during the differentiation of these cells. We report on a new type of p53 mRNA with a slower electrophoretic mobility on gels, which appeared in the cytoplasmic fraction of the erythroleukemia cells between 1 to 3 days following induction of differentiation and persisted in the cells until Day 7. The larger type of p53 mRNA was found associated with polysomes, suggesting that it is translatable in cells. The difference in size between the noninduced and the differentiation-specific type of p53 mRNAs (about 200 nucleotides) was not abrogated following the deadenylation of the mRNAs, thus excluding the possibility that the altered size might result from a longer poly(A) tract. S1 nuclease mapping of the 3′ termini of the p53 mRNAs revealed that the 3' ends of both p53 mRNA types were identical, suggesting that either alternative splicing or a longr 5' noncoding region could cause this heterogeneity in p53 mRNA transcripts.