Wild-type P53 Protein Research Articles

Transcriptional Repression of Epithelial Cell Adhesion Molecule Contributes to p53 Control of Breast Cancer Invasion

p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis, and maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We show by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function and loss-of-function experimental systems. Induction of wild-type p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (a) wild-type p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (b) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion.

Alterations in gene expression and sensitivity to genotoxic stress following HdmX or Hdm2 knockdown in human tumor cells harboring wild-type p53

While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. Our laboratory has been interested in tumor cells possessing wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. In this study we utilized RNAi to knockdown HdmX or Hdm2 in MCF7 human breast cancer cells, which harbor wild-type p53 and elevated levels of HdmX and Hdm2 then examined gene expression changes and effects on cell growth.Cell cycle and growth assays confirmed that the loss of either HdmX or Hdm2 led to a significant growth inhibition and G1cell cycle arrest. Although the removal of overexpressed HdmX/2 appears limited to an anti-proliferative effect in MCF7cells, the loss of HdmX and/or Hdm2 enhanced cytotoxicity in these same cells exposed to DNA damage. Through the use of Affymetrix GeneChips and subsequent RT-qPCR validations, we uncovered a subset of anti-proliferative p53 target genes activated upon HdmX/2 knockdown. Interestingly, a second set of genes, normally transactivated by E2F1 as cells transverse the G1-S phase boundary, were found repressed in a p21-dependent manner following HdmX/2 knockdown.Taken together, these results provide novel insights into the reactivation of p53 in cells overexpressing HdmX and Hdm2.

High-efficiency transfer and expression of AdCMV-p53 in human cervix adenocarcinoma cells induced by subclinical-dose carbon beam radiation

The aim of this study is to evaluate the effect of carbon-beam irradiation on adenovirus-mediated p53 transfer in human cervix adenocarcinoma. The HeLa cells pre-exposed to carbon-beam or gamma-ray, were infected with replication-deficient adenovirus recombinant vectors, containing human wild-type p53 (AdCMV-p53) and green fluorescent protein (GFP) (AdCMV-GFP), respectively. The GFP transfer and p53 expression were detected by flow cytometric analysis. The GFP transfer frequency in C-beam with AdCMV-GFP groups was 38-50% more than that in gamma-ray with AdCMV-GFP groups. The percentage of p53 positive cells in the C-beam with AdCMV-p53 groups was 34-55.6% more than that in gamma-ray with AdCMV-p53 groups (p < 0.05), suggesting that subclinical-dose C-beam irradiation could significantly promote exogenous p53 transfer and p53 expression, and extend the duration of p53 expression in the HeLa cells. The expression of p21 increased with p53 expression in HeLa cells. The survival fractions for the 0.5-1.0 Gy C-beam with AdCMV-p53 groups were 38-43% less than those for the isodose gamma-ray with AdCMV-p53 groups, and 31-40% less than those for the C-beam only groups (p < 0.05). The subclinical-dose C-beam irradiation could significantly promote the transfer and expression of exogenous p53, extend the duration of p53 expression, and enhance the suppression of p53 on cervix adenocarcinoma cells.

Structural and Functional Basis for Therapeutic Modulation of p53 Signaling

Effective modulation of structural features and/or functional properties of the major tumor suppressor p53 as a wild-type or cancer-associated mutant protein represents a major challenge in drug development for cancer. p53 is an attractive target for therapeutic design because of its involvement as a mediator of growth arrest and apoptosis after exposure to chemoradiotherapy and/or radiotherapy. Although most clinically used cytotoxic agents target stabilization of wild-type p53, there are a number of approaches that hold promise for reactivation of mutant p53. On the other hand, brief blockade of p53 may reduce toxicity from systemic cytotoxic therapy. Screens for restoration of p53 transcriptional responses in p53-deficient cells may provide a functional means to develop anticancer therapeutics. Structure-based modulation continues to hold promise for development of peptides or small molecules capable of modulation of either wild-type or mutant p53 proteins.

Immunohistochemical and Biogenetic Features of Diffuse-Type Tenosynovial Giant Cell Tumors: The Potential Roles of Cyclin A, P53, and Deletion of 15q in Sarcomatous Transformation

Diffuse-type tenosynovial giant cell tumor (D-TSGCT) is an aggressive proliferation of synovial-like mononuclear cells with inflammatory infiltrates. Despite the COL6A3-CSF1 gene fusion discovered in benign lesions, molecular aberrations of malignant D-TSGCTs remain unidentified. We used fluorescent in situ hybridization and in situ hybridization to evaluate CSF1 translocation and mRNA expression in six malignant D-TSGCTs, which were further immunohistochemically compared with 24 benign cases for cell cycle regulators involving G(1) phase and G(1)-S transition. Comparative genomic hybridization, real-time reverse transcription-PCR, and a combination of laser microdissection and sequencing were adopted to assess chromosomal imbalances, cyclin A expression, and TP53 gene, respectively. Five of six malignant D-TSGCTs displayed CSF1 mRNA expression by in situ hybridization, despite only one having CSF1 translocation. Cyclin A (P = 0.008) and P53 (P < 0.001) could distinguish malignant from benign lesions without overlaps in labeling indices. Cyclin A transcripts were more abundant in malignant D-TSGCTs (P < 0.001). All malignant cases revealed a wild-type TP53 gene, which was validated by an antibody specifically against wild-type P53 protein. Chromosomal imbalances were only detected in malignant D-TSGCTs, with DNA losses predominating over gains. Notably, -15q was recurrently identified in five malignant D-TSGCTs, four of which showed a minimal overlapping deletion at 15q22-24. Deregulated CFS1 overexpression is frequent in malignant D-TSGCTs. The sarcomatous transformation involves aberrations of cyclin A, P53, and chromosome arm 15q. Cyclin A mRNA is up-regulated in malignant D-TSGCTs. Non-random losses at 15q22-24 suggest candidate tumor suppressor gene(s) in this region. However, P53 overexpression is likely caused by alternative mechanisms rather than mutations in hotspot exons.

High-efficiency transfer and expression of AdCMV-p53 in human hepatocellular carcinoma cells induced by low-dose carbon-ion radiation

To investigate whether the irradiation with C-beam could enhance adenovirus-mediated transfer and expression of p53 in human hepatocellular carcinoma. HepG2 cells were exposed to C-beam or gamma-ray and then infected with replication-deficient adenovirus recombinant vectors containing human wild-type p53 or green fluorescent protein, respectively. The transfer efficiency and expression level of the exogenous gene were detected by flow cytometric analysis. Cell survival fraction was detected by clonogenic assay. The transfer frequency in C-beam or gamma-irradiated groups increased by 50-83% and 5.7-38.0% compared with the control, respectively (P<0.05). Compared with C-beam alone, p53 alone, and gamma-ray with p53, the percentages of p53 positive cells for 1 Gy C-beam with p53 increased by 56.0-72.0%, 63.5-82.0%, and 31.3-72.5% on first and third day after the treatments, respectively (P<0.05). The survival fractions for the 2 Gy C-beam and AdCMV-p53 infection groups decreased to approximately 2%. C-beam irradiation could significantly promote AdCMV-green fluorescent protein transfer and expression of p53.

Enhancement of p53 expression in keratinocytes by the bioflavonoid apigenin is associated with RNA‐binding protein HuR

We have reported previously that apigenin, a naturally occurring nonmutagenic flavonoid, increased wild-type p53 protein expression in the mouse keratinocyte 308 cell line by a mechanism involving p53 protein stabilization. Here we further demonstrated that the increase in p53 protein level induced by apigenin treatment of 308 keratinoyctes was not the result of enhanced transcription, mRNA stabilization or cytoplasmic export of p53 mRNA. Instead, biosynthetic labeling showed that apigenin increased nascent p53 protein synthesis by enhancing p53 translation. The AU-rich element (ARE) within the 3'-untranslated region (UTR) of p53 mRNA was found to be responsible for apigenin's ability to increase p53 translation, as demonstrated in studies wherein the 3'-UTR of p53 mRNA containing the ARE was fused downstream of a luciferase reporter gene. Furthermore, apigenin treatment increased the level of association of the RNA binding protein HuR with endogenous p53 mRNA. Apigenin treatment also augmented HuR translocation into the cytoplasm. Overexpression of HuR enhanced apigenin-induced p53 protein expression in 308 keratinocytes, whereas siRNA-mediated HuR reduction suppressed apigenin-induced p53 protein expression and de novo translation of p53. Moreover, apigenin treatment of cells induced p16 protein expression, which in turn was correlated with cytoplasmic localization of HuR induced by apigenin. Overall, these findings indicate that, in addition to modulating p53 protein stability, one of the mechanisms by which apigenin induces p53 protein expression is enhancement of translation through the RNA binding protein HuR.

Functional Modulation of the P53 Gene and its Protein in Human Brain Tumors

The primary human brain tumors account for less than 2% of all human cancers but yet cause a disproportionate burden of cancer related morbidity and mortality. These are the second most common form of tumors in the pediatric population, next only to leukemia and hence the second leading cause of death due to cancer in children. Among the adults they rank 6 th and 8 th in frequency of all neoplasms and form 2 nd and 5 th leading cause of death in men and women respectively who belong to the age group of 20 to 39 years. The survival rates for brain tumor patients have not changed over the past several decades. The p53 gene is one of the most important and intensively studied human tumor suppressor genes. It has been shown to play a major role in cell proliferation as well as cell death. Because of its varied functions the p53 gene and its pathways have become important therapeutic target. Mutations in the p53 gene and the oncogenic function of its protein product have been well documented. However, numerous evidences indicate that based on its conformation and post translational modification the function of the wild type p53 protein can be modulated to be similar to that of the mutant form. In this paper we have reviewed the functional modulation of p53 gene in human brain tumor development. doi: 10.5214/ans.0972.7531.2008.150304 Competing interests: None. Source of Funding: DBT, UGC Received Date: 04 April 2008 Revised Date: 22 May 2008 Accepted Date: 19 June 2008

Identification and Functional Characterization of the Human Glutathione S-Transferase P1 Gene as a Novel Transcriptional Target of the p53 Tumor Suppressor Gene

The glutathione S-transferase P1 (GSTP1) is involved in multiple cellular functions, including phase II metabolism, stress response, signaling, and apoptosis. The mechanisms underlying the significantly high GSTP1 expression in many human tumors are, however, currently not well understood. We report here that the GSTP1 gene is a heretofore unrecognized downstream transcriptional target of the tumor suppressor p53. We identified a p53-binding motif comprising two consecutive half-sites located in intron 4 of the GSTP1 gene and is highly homologous to consensus p53-binding motifs in other p53-responsive genes. Using a combination of electrophoretic mobility shift assay and DNase I footprinting analyses, we showed that wild-type p53 protein binds to the GSTP1 p53 motif and luciferase reporter assays showed the motif to be transcriptionally functional in human tumor cells. In a temperature-sensitive p53-mutant cells, levels of both p21/WAF1 and GSTP1 gene transcripts increased time dependently when cells were switched from the inactive mutant state to the wild-type p53 state. Small interfering RNA-mediated reduction of p53 expression resulted in a specific decrease in GSTP1 expression and in tumor cells with mutated p53; adenovirally mediated expression of wild-type p53 increased GSTP1 expression significantly. In a panel of early-passage brain tumor cultures from patients, high levels of GSTP1 transcripts and protein were associated with wild-type p53 and, conversely, low GSTP1 levels with mutant p53. p53 expression knockdown by small interfering RNA increased cisplatin sensitivity. The ability of wild-type p53 to transcriptionally activate the human GSTP1 gene defines a novel mechanism of protecting the genome and, potentially, of tumor drug resistance.

Similar Extent of Apoptosis Induction at Doses of X-Rays and Neutrons Isoeffective for Cell Inactivation

The relative biological effectiveness (RBE) of neutrons differs for various biological endpoints, and for various cell and tissue types. With respect to the apoptosis induction, a whole range of values can be found in the literature, but the decisive factors are not clear. Most previous studies have used apoptosis-prone hematopoietic cells, whereas tumor cells have received little attention. The authors therefore decided to investigate apoptosis induction caused by X-rays and neutrons in a line of human melanoma cells, at doses which are isoeffective for the loss of colony-forming ability. Human melanoma cells Be11, expressing p53 wild-type protein, were used throughout. Exponentially growing cells were exposed to two pairs of isoeffective doses (at surviving levels 10% and 1%) of 240-kV X-rays and 5.8-MeV neutrons. 1-8 days after irradiation, the frequency of apoptosis in adherent cells was assessed by two-parameter flow cytometric analysis with a DNA-dye-exclusion annexin-V-binding assay as well as by morphological examination with DAPI staining. Apoptosis was induced most significantly 6-7 days after irradiation. The time courses, as well as the magnitudes of apoptosis induction, after isoeffective doses of X-rays and neutrons with respect to loss of colony-forming ability appeared to be comparable. RBE values in the range of 4-5 were estimated for apoptosis 4-8 days after irradiation by both the annexin V assay and morphological examination. Radiation-induced apoptosis depends on ionization density in the same way as cell inactivation in general does, i.e., the RBE is similar, and the ratio of cells dying by apoptosis to cells dying otherwise does not depend on radiation quality.

DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites

The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (-sigma). At -sigma < or = 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher -sigma, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For -sigma = 0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at -sigma > or = 0.05 correlates with the extra enhancement of p53-DNA binding.

Genotoxic Stress–Induced Expression of p53 and Apoptosis in Leukemic Clam Hemocytes with Cytoplasmically Sequestered p53

In nature, the soft shell clam, Mya arenaria, develops a fatal blood cancer in which a highly conserved homologue for wild-type human p53 protein is rendered nonfunctional by cytoplasmic sequestration. In untreated leukemic clam hemocytes, p53 is complexed throughout the cytoplasm with overexpressed variants for both clam homologues (full-length variant, 1,200-fold and truncated variant, 620-fold above normal clam hemocytes) of human mortalin, an Hsp70 family protein. In vitro treatment with etoposide only and in vivo treatment with either etoposide or mitoxantrone induces DNA damage, elevates expression (600-fold) and promotes nuclear translocation of p53, and results in apoptosis of leukemic clam hemocytes. Pretreatment with wheat germ agglutinin followed by etoposide treatment induces DNA damage and elevates p53 expression (893-fold) but does not overcome cytoplasmic sequestration of p53 or induce apoptosis. We show that leukemic clam hemocytes have an intact p53 pathway, and that maintenance of this tumor phenotype requires nuclear absence of p53, resulting from its localization in the cytoplasm of leukemic clam hemocytes. The effects of these topoisomerase II poisons may result as mortalin-based cytoplasmic tethering is overwhelmed by de novo expression of p53 protein after DNA damage induced by genotoxic stress. Soft shell clam leukemia provides excellent in vivo and in vitro models for developing genotoxic and nongenotoxic cancer therapies for reactivating p53 transcription in human and other animal cancers displaying mortalin-based cytoplasmic sequestration of the p53 tumor suppressor, such as colorectal cancers and primary and secondary glioblastomas, though not apparently leukemias or lymphomas.

Effect of siRNA targeting centromere protein-A gene on biological behavior of HepG2 cells

To study the influence of siRNA inhibition of CENP-A expression on the biological behavior of HepG2 cells. Three pairs of 21 bp reverse repeated motifs of CENP-A target sequence with 9 spacer were synthesized and inserted into vector pSilencer 2.1-U6 neo to generate siRNA eukaryotic expression plasmids. After stable transfection into HepG2 cells, cell growth, apoptosis, cell cycles and plate clone forming efficiency were investigated. Expressions of CENP-A mRNA was monitored by the reverse transcriptase polymerase chain reaction (RT-PCR). The protein expression of CENP-A, bcl-2, Bax, p53, p21waf1 and mdm2 were detected by Western-blotting. Two eukaryotic expression plasmids with significant siRNA specific inhibition to the CENP-A gene were created. Compared with control cells, HepG2 cells transfected with the constructs showed G1 phase delay (P < 0.01) and cell number decrease in the S phase (P < 0.001), along with an increased apoptotic rate (P = 0.003), significant increase of Bax expression and decreased bcl-2 expression (P< or =0.001). The protein expressions of p21waf1 was higher and mdm2 was lower than those of the control groups. However, the wild type p53 protein expression was not effected by CENP-A siRNA. An altered expression of CENP-A may be related to the proliferation of hepatocellular carcinoma through cell cycle regulation involving an altered bcl-2/Bax expression, that may be p53 independent.

Hsp70- and p53-reponses after heat treatment and/or X-irradiation mediate the susceptibility of hematopoietic cells to undergo apoptosis

Purpose: The effect of heat treatment in combination with X-irradiation was examined with regard to expression of p53, a tumor suppressor gene product, and Hsp70, a heat-shock protein, in association with the occurrence of programmed cell death (apoptosis).Materials and methods: Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1), which differ in p53 status, were exposed to 42.5°C during one hour and/or X-radiation (total dose 8 Gy). After exposure, both mRNA and protein expression levels of Hsp70 and p53 were investigated by real-time PCR (polymerase chain reaction) and Western blotting. Apoptosis was simultaneously analyzed by observation of cell morphology as well as flowcytometric determination of Annexin V binding to phosphatidylserine and propidium iodide exclusion.Results: Both HL60 and HSB2 cell lines with a low p53 status and a quick response to heat treatment with Hsp70 over-expression are less susceptible to heat-induced apoptosis compared to Kasumi-1 cells with wild-type p53 protein and no Hsp70 response. The combination of first applying X-irradiation followed by heat treatment resulted in the most effective induction of apoptosis due to impairment of the Hsp70 response in all three cell lines.Conclusion: These results indicate that the Hsp70 response and p53 status mediate the susceptibility of hematopoietic cells to undergo heat-induced apoptosis. Therefore, these parameters can be used as markers to predict the effectiveness of hyperthermia in cancer treatment.

Ubiquitination and Degradation of Mutant p53

While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. In this study, we show that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild-type p53, our data suggest that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. Our initial attempts to identify ubiquitin ligases that are responsible for the ubiquitination of mutant p53 have suggested a role for the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein), although other unidentified ubiquitin ligases also appear to contribute. The contribution of Mdm2 to the degradation of mutant p53 may reflect the ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome.

MAGED2: A novel p53-dissociator

The tumor suppressor protein p53 is a transcription factor that is frequently mutated in human cancers. In response to DNA damage, unmutated or wild-type (wt) p53 protein is stabilized and activated by post-transcriptional modifications that enable it to induce either apoptosis or cell cycle arrest. Using a yeast p53-dissociator assay, we identified MAGED2 as a potential negative regulator of wt p53 activity. Subsequently, using co-immunoprecipitation and reporter gene assays in human cultured cells that are often adopted for functional analysis of p53 we demonstrated that MAGED2 interacted physically with p53 and modified its activity. Finally, we were able to illustrate expression of both p53 and MAGED2 within the same subcellular compartment, i.e. either nucleus or cytoplasm, in 2,682 human cancer tissue specimens using a common cancer tissue microarray and antibodies against MAGED2 and p53. The present results implicate MAGED2, a novel protein, as a p53-dissociator.

Re-activation of the p53 pathway inhibits in vivo and in vitro growth of hormone-dependent human breast cancer cells

Mutations in wild-type p53 (wtp53) protein lead to loss of its tumor suppressor function in breast cancer cells, facilitating uncontrolled tumor growth. Consequently, procedures to repair defective p53 functions in tumor cells are being actively pursued. We sought to determine whether expression of wtp53 protein, or conversion of endogenous mutant p53 (mtp53) into a functional p53 protein with small molecule PRIMA-1, can override the tumor-promoting effects of naturally occurring mtp53 protein in hormone-responsive T47-D human breast cancer cells. We show that transfection of wtp53 gene into T47-D cells suppresses their proliferation in regular media, and inhibits estrogen-dependent cell proliferation in media containing dextran-coated charcoal treated serum. Growth inhibition was not due to the absence of estrogen receptor-alpha or estrogen receptor-beta though receptor levels for estrogen receptor-alpha were drastically reduced in wtp53 expressing cells. Focused microarray analysis of wtp53 expressing cells revealed suppression of PCNA cell-cycle regulatory mRNA and protein. Wild-type p53 transfected T47-D cells also failed to grow in vivo in estrogen supplemented nude mice. Furthermore, xenografts obtained with parental T47-D cells expressing mtp53 grew poorly in nude mice treated with PRIMA-1. PRIMA-1 treated tumors exhibited a low proliferation index, even though mice were estrogen-supplemented. PRIMA-1 treatment of tumor cells suppressed VEGF and induced expression of estrogen receptor-beta though expression of estrogen receptor-alpha and progesterone receptors was unaffected. These data indicate that alteration of the p53 signal transduction pathway by re-expression of wtp53 protein in T47-D cells, or treatment of parental cells with PRIMA-1, can prevent in vivo and in vitro proliferation of T47-D breast cancer cells.

Frequency of TP53 Mutations in Relation to Arg72Pro Genotypes in Non–Small Cell Lung Cancer

Mutations in the TP53 gene are important events during human lung carcinogenesis. The TP53 gene harbors several polymorphisms, and functional studies have shown that the Arg72Pro polymorphism alters both wild-type and mutant p53 protein activity. Thus, we hypothesized that certain Arg72Pro genotypes may influence the frequency and pattern of somatic mutations in TP53. We therefore examined the status of the Arg72Pro polymorphism and TP53 mutations in 260 non-small-cell lung cancer cases. Here we report a significant trend toward lower frequency of TP53 mutations with increasing number of Pro72 alleles (P = 0.02). Overall, Pro72 allele carriers had significantly lower frequency of TP53 mutations compared with Arg72 homozygotes (P = 0.02). In addition, carriage of the Pro72 variant was related to a lower frequency of mutations affecting the hotspot codon 273. Mutations at codon 273 accounted for 10.6% of the mutations in Arg72 homozygotes and 1.7% of the mutations in Pro72 allele carriers. Our results suggest that the genotype of the Arg72Pro polymorphism may modulate the frequency of TP53 mutations in non-small-cell lung cancer.

Signaling of DNA damage is not sufficient to induce p53 response: (Re)Activation of wt p53 protein strongly depends on cellular context

It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS(3) cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS(3) cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS(3) cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS(3) cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.

Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.