P53 Protein In Cells Research Articles

Cytotoxic Activity and Molecular Docking Studies of the Fraction Containing Ergosta-14,22-dien-3-ol (3β, 5α, and 22E) from Dysidea avara Sponges

Background: Cancer is a global health burden, and the discovery of novel therapeutic agents remains a critical pursuit. Marine organisms, including sponges, have emerged as a promising source of structurally diverse natural products with potential anticancer properties. Objectives: In the present study, the cytotoxic activity of the fraction containing Ergosta-14,22-dien-3-ol (3β, 5α, and 22E), a steroid compound derived from Dysidea avara, was evaluated against Jurkat/E6-1 and Hek293 cells. Methods: Multiple analytical techniques, including column chromatography, TLC, and GC-MS, were used to isolate and identify the compound from marine sponges from the Persian Gulf. The XTT assay determined the cytotoxic activity and western blot for P53 expression in the Jurkat/ E6-1 cell line. The compound was also docked within the poly (ADP-ribose) polymerase-1 (PARP1) and E3 ubiquitin-protein ligase (MDM2) to investigate its potential mechanism of action. Furthermore, the pharmacological properties of the compound were predicted using PerADME, SwissADME, and Molinspiration tools. Results: The results showed that Ergosta-14,22-dien-3-ol (3β, 5α, and 22E) exhibited significant cytotoxic activity against Jurkat/E6-1 cells with an IC50 of 26.59 μg/mL. Western blotting analysis demonstrated a noticeable increase in the expression of P53 protein in cells treated with 50 and 100 µg/mL of the compound. In silico analysis revealed adequate binding energy against PARP1 and E3 MDM2 receptors. The compound also demonstrated favorable pharmacokinetic characteristics, specifically in absorption, distribution, metabolism, and excretion (ADME). Conclusions: Ergosta-14,22-dien-3-ol (3β, 5α, and 22E) has the potential to be developed as a promising anti-cancer agent.

Novel compounds that synergize with aminoglycoside G418 or eRF3 degraders for translational readthrough of nonsense mutant TP53 and PTEN

ABSTRACT The TP53 and PTEN tumour suppressor genes are inactivated by nonsense mutations in a significant fraction of human tumours. TP53 nonsense mutant tumours account for approximately one million new cancer cases per year worldwide. We have screened chemical libraries with the aim of identifying compounds that induce translational readthrough and expression of full-length p53 protein in cells with nonsense mutation in this gene. Here we describe two novel compounds with readthrough activity, either alone or in combination with other known readthrough-promoting substances. Both compounds induced levels of full-length p53 in cells carrying R213X nonsense mutant TP53. Compound C47 showed synergy with the aminoglycoside antibiotic and known readthrough inducer G418, whereas compound C61 synergized with eukaryotic release factor 3 (eRF3) degraders CC−885 and CC−90009. C47 alone showed potent induction of full-length PTEN protein in cells with different PTEN nonsense mutations. These results may facilitate further development of novel targeted cancer therapy by pharmacological induction of translational readthrough.

Effect of the BBC3 Gene on the Proliferation and Apoptosis of Hepatocellular Carcinoma Cells Through p53-Regulated Signaling

We examined the effect of the BBC3 gene on hyperplasia and apoptosis in HepG2 cells and its underlying mechanism. Quantitative RT-PCR was used to determine the level of BBC3 expression in HL-7702 normal human liver cells and four different hepatocellular carcinoma cell lines (HepG2, HuH-7, HCCLM3 and MHCC97H). Transfection was performed with Lipofectamine 2000 reagent and the transfectants were divided into three groups: pcDNA-BBC3 group (transfected BBC3 over-expressing plasmid), pcDNA-NC group (transfected empty plasmid), and a Ctrl group (not transfected). Quantitative RT-PCR and western blot analysis were used to measure BBC3 expression. The CCK-8 assay was used to determine the effect of BBC3 on HepG2 cell proliferation. Flow cytometry was used for testing the effect of overexpressing BBC3 on apoptosis in HepG2 cells. The levels of cleaved-Caspase-3 (C-Caspase-3), cleaved-Caspase-9 (C-Caspase-9), and proteins associated with the p53 signaling pathway were assessed by western blot analysis. The level of BBC3 mRNA in HL-7702 normal human liver cells was significantly higher compared with that in human hepatocellular carcinoma cells including HepG2, HuH-7, HCCLM3 and MHCC97H (P < 0.05). The lowest level of BBC3 mRNA was observed in HepG2 cells. The level of BBC3 mRNA and protein in HepG2 cells were significantly higher compared with that of the pcDNA-NC group following transfection with a BBC3 overexpressing plasmid. HepG2 cell proliferation in the pcDNA-NC group was higher compared with that of the pcDNA-BBC3-transfected group (P < 0.05). The apoptotic rate and levels of cleaved-Caspase-3, cleaved-Caspase-9, p53, phospho-p53, and p21 protein in cells were higher compared with that of the pcDNA-NC group. No change was observed in the pcDNA-NC and Ctrl groups. The BBC3 gene was down-regulated in hepatocellular carcinoma cells. HepG2 cell proliferation can be inhibited and HepG2 cell apoptosis can be induced by the overexpression of BBC3 through activation of the p53 signaling pathway.

Effects of obatoclax combined with gemcitabine on breast cancer cells under hypoxia condition

Objective: To explore the effects of obatoclax(OBX) combined with gemcitabine(GEM) on breast cancer cells MCF-7 and BT-20 cell activity, migration, invasion and apoptosis under hypoxia condition.Methods: Breast cancer cells MCF-7 and BT-20 were divided into normal group, hypoxia group, GEM group, OBX+GEM group. Normal group: Cells were cultured at 37℃, 5% CO2 for 24 h and 48 h; Hypoxia group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2 for 24 h and 48 h; GEM group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2, adding 10 μmol/L GEM for 24 h and 48 h; OBX + GEM group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2, adding 10 μmol/L GEM and 50 nmol/L OBX for 24 h and 48 h. Western blot method was used to detect the expressions of HIF-1α in MCF-7 and BT-20 cells under normal oxygen and hypoxia condition. CCK-8 method was used to detect cancer cell activity, each group was provided with 15 compound holes. Scratch experiment was used to detect cells migration ability, each group was provided with 6 compound holes. Western blot method was used to detect the expressions of vimentin, E-Cadherin and p53 protein in cells of each group. Results: Under hypoxia condition, the expression of HIF-1α in MCF-7 and BT-20 cells was much higher than that under normal oxygen(P＜0.05). Compared with hypoxia group, GEM could reduce MCF-7 and BT-20 cells migration ability(P＜0.01)and cell activity(P＜0.05), while decrease the expression of vimentin protein(P＜0.01)and promote the expressions of E-Cadherin (P＜0.01)and p53 protein(P＜0.01) in tumor cells under hypoxia condition. In OBX combined with GEM group, the cell activity and the migration ability of MCF-7 and BT-20 were reduced significantly(P＜0.01). The expression of vimentin in cells was further reduced(P＜0.01). The expressions of E-Cadherin(P＜0.01)and p53(P＜0.01) protein were increased significantly compared with GEM group. Conclusion: Under hypoxia condition, OBX combined with a low-dose of GEM can significantly inhibit the growth, migration and invasion of breast cancer cells, and enhance the pro-apoptotic effect of GEM, but the specific mechanism needs further study.

Effect of new olivacine derivatives on p53 protein level

BackgroundThe p53 protein is a transcription factor for many genes, including genes involved in inhibiting cell proliferation and inducing apoptosis in genotoxically damaged and tumor-transformed cells. In more than 55% of cases of human cancers, loss of the essential function of p53 protein is found. In numerous reports, it has been shown that small molecules (chemical compounds) can restore the suppressor function of the mutant p53 protein in tumor cells. The aim of this study was to evaluate the potential anticancer activity of three newly synthesized olivacine derivatives.MethodsThe study was performed using two cell lines—CCRF/CEM (containing the mutant p53 protein) and A549 (containing a non-mutant, wild-type p53 protein). The cells were incubated with olivacine derivatives for 18 h and then assays were carried out: measurement of the amount of p53 and p21 proteins, detection of apoptosis, cell cycle analysis, and rhodamine 123 accumulation assay (evaluation of P-glycoprotein inhibition). Multiple-criteria decision analysis was used to compare the anticancer activity of the tested compounds.ResultsEach tested compound caused the reconstitution of suppressor activity of the p53 protein in cells with the mutant protein. In addition, one of the compounds showed significant antitumor activity in both wild-type and mutant cells. For all compounds, a stronger effect on the level of the p53 protein was observed than for the reference compound—ellipticine.ConclusionsThe observed effects of the tested new olivacine derivatives (pyridocarbazoles) suggest that they are good candidates for new anticancer drugs.

The nucleoside analog clitocine is a potent and efficacious readthrough agent.

Nonsense mutations resulting in a premature stop codon in an open reading frame occur in critical tumor suppressor genes in a large number of the most common forms of cancers and are known to cause or contribute to the progression of disease. Low molecular weight compounds that induce readthrough of nonsense mutations offer a new means of treating patients with genetic disorders or cancers resulting from nonsense mutations. We have identified the nucleoside analog clitocine as a potent and efficacious suppressor of nonsense mutations. We determined that incorporation of clitocine into RNA during transcription is a prerequisite for its readthrough activity; the presence of clitocine in the third position of a premature stop codon directly induces readthrough. We demonstrate that clitocine can induce the production of p53 protein in cells harboring p53 nonsense-mutated alleles. In these cells, clitocine restored production of full-length and functional p53 as evidenced by induced transcriptional activation of downstream p53 target genes, progression of cells into apoptosis, and impeded growth of nonsense-containing human ovarian cancer tumors in xenograft tumor models. Thus, clitocine induces readthrough of nonsense mutations by a previously undescribed mechanism and represents a novel therapeutic modality to treat cancers and genetic diseases caused by nonsense mutations.

Hepatocellular carcinoma: immunohistochemical characteristics of apoptosis and cell proliferation activity.

Background. Hepatocellular carcinoma which takes about 85% of primary liver cancer, is the most aggressive and prognostically unfavorable tumor. Current information about the level of apoptosis and cell proliferative activity of HCC are contradictory. Objective. To determine the apoptotic and proliferative activity of hepatocellular carcinoma of the liver by the immunohistochemically level of p53, caspase-3 and Ki-67 expression. Methods. Trephine biopsies of 53 patients with hepatocellular carcinoma were studied. Results. It was found that in 62.26% of cases the hyperexpression of nuclear p53 protein in cells was detected, in 20.75% of cases - a high level of expression of this protein, and in 16.98% of cases - a low level of p53 expression. The average area of p53-immunopositive cells was 52,07±33,97%. Simultaneously, in 47.16% of hepatocellular carcinoma it was detected a weak level of cytoplasmic expression of caspase-3, in 26.42% of the patients moderate level of expression of caspase-3 by hepatocellular carcinoma cells was determined, pronounced expression of caspase-3 was detected in hepatocellular carcinoma cells 26, 42%. Caspase-3-immunopositive cells occupied 48,21±27,66% of the total area of hepatocellular carcinoma. It is established that in the hepatocellular carcinoma prevailed moderate level of expression of Ki-67, which was observed in 66.04% of patients, in 22.64% it was high and in 11.32% of cases – it was low. Immupositive Ki-67 cells occupied 53,39±23,25% of the total area of the tumor. Conclusion. Hyperexpression of nuclear p53 protein in hepatocellular carcinoma was associated with weak and low level of apoptosis of tumor cells and moderate level of their proliferation.

P53 protein in proliferation, repair and apoptosis of cells

The p53 protein is an important factor of many intra- and extracellular processes. This protein regulates the repair of cellular DNA and induces apoptosis. It is also responsible for the regulation of the senescence and the cell entering the subsequent stages of the cellular cycle. The protein p53 is also involved in inhibiting angiogenesis and the induction of oxidative shock. In our study, we examined the activity of p53 protein in the uterine epithelial cells in rats treated with cladribine. Its action is mainly based on apoptosis induction. We compared the activity of p53 protein in cells with a high apoptosis index and in cells with active repair mechanisms and high proliferation index. We observed stronger p53 protein expression in the epithelial cells of the materials taken 24 h after the last dose of 2-CdA associated with the active process of apoptosis and inhibition of proliferation. After 4 weeks from the last dose of cladribine, the stronger expression of p53 protein was associated with both the existing changes in the cell's genome, the effects of the ongoing repair mechanisms, as well as the high proliferation activity.

Cytotoxicity of curcumin, resveratrol and plant extracts from basil, juniper, laurel and parsley in SH-SY5Y and CV1-P cells

The health-promoting effects of natural phenolic compounds are attracting growing interest. In this study, we tested four plant extracts, namely basil, juniper, laurel and parsley, and two well-known pure phenolic compounds of natural origin, curcumin and resveratrol, for their possible cytotoxic effects in vitro. AraC was used as an apoptotic reference compound. The pure compounds were studied in two cell lines, SH-SY5Y neuroblastoma and CV1-P fibroblast cells, and the plant extracts in SH-SY5Y cells. Cytotoxicity was examined by MTT and LDH assays, and the molecular mechanisms by Western blot analysis of p53 protein in cells. Juniper extract decreased the cell viability and increased the amount of p53 in SH-SY5Y cells at lower concentrations than did other plant extracts, and its effects on the amount of p53 in cells were comparable to the treatment with 50 μM AraC. The actions of curcumin and resveratrol were dependent on the concentration and cell line. Curcumin decreased the cell viability and increased the amount of p53 in SH-SY5Y cells more effectively than in CV1-P cells. The comparison between the results of AraC and pure curcumin showed a similar effect on the amount of p53. Our results indicate a potential role of plant extracts and plant-derived compounds as health-promoting food constituents, as well as candidates for drug development.

P-Phenylenediamine induces p53-mediated apoptosis in Mardin–Darby canine kidney cells

The mechanism of toxicity p-phenylenediamine ( p-PD), a component of human permanent hair dye and a suspected carcinogen, on the growth of Mardin–Darby canine kidney cells (MDCK) was investigated. With the analysis of flow cytometry, a dose-dependent accumulation of the sub-G1 peak and the G0/G1-phase arrested in cell cycle, and time-dependent induction of apoptosis after staining with Annexin V-Fluorescein and propidium iodide were observed. After the treatment of cells with p-PD, dose dependent DNA fragmentation shown by gel electrophoresis, the reduction of membrane potential (Δ Ψ m) by mitochondria membrane depolarization and the increase of the expression of p53 protein in cells, suggested that the effect of p-PD on overall viability and cell numbers is mediated by an increase in apoptosis.

A Chromatin-associated and Transcriptionally Inactive p53-Mdm2 Complex Occurs in mdm2 SNP309 Homozygous Cells

In cancer cells, the function of the tumor suppressor protein p53 is usually blocked. Impairment of the p53 pathway results in tumor cells with endogenous overexpression of Mdm2 via a naturally occurring single nucleotide polymorphism (SNP) in the mdm2 gene at position 309. Here we report that in mdm2 SNP309 cells, inactivation of p53 results in a chromatin-associated Mdm2-p53 complex without clearance of p53 by protein degradation. Nuclear accumulation of p53 protein in mdm2 SNP309 cells results after 6 h of camptothecin, etoposide, or mitomycin C treatment, with the p53 protein phosphorylated at Ser15. Chromatin immunoprecipitation demonstrated p53 and Mdm2 bound to p53 responsive elements. Interestingly, although the p53 protein was able to bind to DNA, quantitative PCR showed compromised transcription of endogenous target genes. Additionally, exogenously introduced p53 was incapable of activating transcription from p53 responsive elements in SNP309 cells, confirming the trans-acting nature of the inhibitor. Inhibition of Mdm2 by siRNA resulted in transcriptional activation of these p53 targets. Our data suggest that overproduction of Mdm2, resulting from a naturally occurring SNP, inhibits chromatin-bound p53 from activating the transcription of its target genes.

Accumulation of an inactive form of p53 protein in cells treated with TNF alpha.

In MCF-7 cells, TNF alpha induces a G1 arrest with an increased expression of p21/Waf1, an activation of NF-kappa B and an accumulation of p53. NF-kappa B and p53 are two transcriptional factors known to activate p21/Waf1 gene expression. Here we show that p53 inhibition has no effect on p21/Waf1 mRNA accumulation following TNF alpha treatment. In contrast, inactivation of NF-kappa B inhibits p21/Waf1 expression without affecting G1 arrest. The fact that p21/Waf1 gene expression is still stimulated when p53 is inactivated strongly suggests that TNF alpha induces accumulation of an inactive form of p53 protein. This assumption was further supported by the following observations: (i) the p53 DNA-binding activity to its consensus sequence was not stimulated following TNF alpha treatment, (ii) phosphorylation at Ser-15, -20 or -392 was not detected in response to TNF alpha, (iii) the transcription rate of Ddb2, another p53 target gene, was not stimulated by TNF alpha. Finally, the accumulation of p53 in the nuclei of TNF alpha-treated MCF-7 cells was concomitant with an increase in p53 mRNA level, suggesting a regulation at the transcription level.

Stoichiometric Phosphorylation of Human p53 at Ser315Stimulates p53-dependent Transcription

p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser(315) phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser(315) can be detected in vivo, whether steady-state Ser(315) phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser(315) where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser(315) phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser(315) site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser(315) after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

A translation repressor element resides in the 3' untranslated region of human p53 mRNA.

The 3' untranslated region of human p53 mRNA represses translation both in vitro and in vivo. Here, we identify a cis-acting 66-nucleotide U-rich sequence in the human p53 mRNA 3' untranslated region that mediates translational repression. Using UV cross-linking, we detect a 40 kDa protein that interacts specifically with the p53 3'UTR containing the repressor element. Enhanced translation of p53 mRNA contributes to the accumulation of p53 protein in cells exposed to gamma-radiation and could be a consequence of relieving the inhibition mediated by the repressor element.

Nuclear exclusion of wild-type p53 in immortalized human retinoblastoma cells.

Retinoblastoma is the most common childhood tumor of the eye, arising from cells that are defective in both copies of the retinoblastoma susceptibility gene (RB1). Most retinoblastoma tumor cells eventually undergo programmed cell death (i.e., apoptosis); however, some cells can acquire the ability to metastasize and become immortal. Transfection of immortal retinoblastoma cells with DNA sequences encoding wild-type p53 protein induces cell death, suggesting that the loss of both RB1 and p53 functions may be required for cell immortalization. We have examined this possibility by characterizing the p53 protein and messenger RNA in six independently isolated, immortalized retinoblastoma cell lines. Western blotting methods were used to assess p53 protein level in each cell line, and Cleavase Fragment-Length Polymorphism analysis of complementary DNAs was used to screen for mutations in p53 messenger RNA. Localization of p53 protein in cells of the immortalized lines and in specimens of retinoblastoma tumors was achieved by means of indirect immunofluorescence and immunocytochemistry, respectively. All six immortalized cell lines expressed wild-type p53 messenger RNA and high levels of p53 protein. Although p53 is normally a nuclear protein, the p53 in four of the six cell lines was located predominately in the cytoplasm; in the remaining two cell lines, p53 was localized in both the nucleus and the cytoplasm. Cytoplasmic localization of p53 in retinoblastoma tumor specimens was rare and usually restricted to cells that had invaded adjacent ocular tissues, indicative of the early stages of metastasis. Some immortalized retinoblastoma cells may exhibit p53 dysfunction through nuclear exclusion of wild-type p53 protein.

The Molecular Epidemiology of Oncoproteins: Serum p53 Protein in Patients With Asbestosis

The Molecular Epidemiology of Oncoproteins: Serum p53 Protein in Patients With Asbestosis

P53 protein accumulation and gene mutation in the progression of human prostate carcinoma.

Nuclear accumulation of p53 protein has been shown to be strongly associated with missense p53 mutations. Studies of nuclear accumulation of p53 protein in prostate carcinoma cells have to date been confined to material from primary tumors. We studied the accumulation of p53 protein in specimens obtained from primary and metastatic sites of prostate carcinoma. By examining the accumulation of this protein as a function of stage, histologic grade, and androgen responsiveness of the tumor, we hoped to determine the role of p53 mutation in the progression of prostate carcinoma. The accumulation of the p53 protein in the cell nuclei was determined by immunohistochemical methods using polyclonal antibody to human p53 CM-1. The material studied consisted of formalin-fixed, paraffin-embedded tissue obtained from primary tumors and metastases of 92 patients with prostate carcinoma. Twelve samples from 11 patients were analyzed for the presence of mutations within exons 5-8 of the p53 gene (also known as TP53) by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) analysis. Sequence analysis was subsequently performed on DNA obtained by polymerase chain reaction amplification of PCR-SSCP reactions produced from six different specimens. The chi-square test, Fisher's exact test, and the Freeman Halton test were used for statistical analyses of the results. All tumors with p53 accumulation were metastatic (stage D), poorly differentiated, and androgen independent. Nuclear accumulation of p53 protein was strongly associated with stage (D2 versus D1 versus A-C, P < .0001), grade (Gleason score 8-10 versus 5-7, P < .003), and androgen sensitivity (androgen independent versus dependent, P < .0001). Logistic regression analysis demonstrated that androgen sensitivity predicted p53 outcome better than did stage (P < .0001) or grade alone (P < .006). There was a perfect concordance between the results obtained by PCR-SSCP analysis and the p53 protein accumulation determined by immunohistochemistry in the 12 samples studied. Mutation of the p53 gene was confirmed by sequencing DNA obtained from six specimens positive in the PCR-SSCP assay. p53 gene mutation is a late event in the progression of prostate cancer and is associated with advanced (metastatic) stage, loss of differentiation, and the transition from androgen-dependent to androgen-independent growth. Testing of prostate cancer biopsy specimens from metastatic sites for p53 protein accumulation and gene mutation may provide useful prognostic information and could influence the recommended course of treatment.

Different levels of v‐Ha‐ras p21 expression in primary keratinocytes transformed with harvey sarcoma virus correlate with benign versus malignant behavior

Primary mouse keratinocytes transformed with an activated ras oncogene transduced by helper-free Harvey sarcoma virus (HaSV) form predominantly benign tumors. In contrast, keratinocytes transformed with helper-associated HaSV form malignant tumors. We report here that this different tumorigenic behavior correlated with a much higher level of v-Ha-ras p21 protein in cells transformed with the helper-associated virus. The high level of v-ras expression in these cells was due to viral spread beyond the initial infection. The low level of v-ras p21 expression that resulted from single-hit infection with helper-free virus, together with the intrinsic heterogeneity of primary keratinocytes, explains, at least in part, the different tumorigenic behavior of keratinocytes transformed by the two types of viruses.

Expression of the cellular p53 protein in cells transformed by adenovirus 12 and viral DNA fragments

Expression of the p53 cellular phosphoprotein was examined in rat cells transformed by adenovirus 12 (Adl 2) virions and by fragments of Adl2 DNA. p53 was detected in all the cell lines examined. Steady-state levels of p53 were highest in cells transformed by the entire El region rather than by El A alone. Physical association between p53 and the Adl2 El B 55K protein was not detected. The Ad12 El V-encoded 55K protein, but not the Ad12 El B 17K and 19K proteins, appears to participate in regulating p53 protein levels.