Disrupt P53 Function Research Articles

Insulin-like Growth Factor-1 Impacts p53 Target Gene Induction in UVB-irradiated Keratinocytes and Human Skin.

The tumor suppressor protein p53 limits mutagenesis in response to ultraviolet-B (UVB) light exposure by activating the transcription of genes that mitigate the damaging effects of UVB radiation on DNA. Because most nonmelanoma skin cancers (NMSCs) occur in older individuals, it is important to understand the process of mutagenesis in the geriatric skin microenvironment. Based on previous studies demonstrating that geriatric skin expresses lower levels of the growth factor insulin-like growth factor-1 (IGF-1) than young adult skin, a role for IGF-1 in the regulation of p53 target genes was investigated in both human keratinocytes in vitro and human skin explants ex vivo. The products of the p53 target genes p21 and DNA polymerase eta (pol η) were found to be increased by UVB exposure in both experimental systems, and this induction was observed to be partially abrogated by depriving keratinocytes of IGF-1 in vitro or by the treatment of keratinocytes in vitro and human skin explants with an IGF-1 receptor antagonist. Because p21 and pol η function to limit mutagenic DNA replication following UVB exposure, these results suggest that NMSC risk in geriatric populations may be due to age-dependent decreases in IGF-1 signaling that disrupt p53 function in the skin.

Nutlin-3 downregulates p53 phosphorylation on serine392and induces apoptosis in hepatocellular carcinoma cells

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser392-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser392-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser392-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser392 presents an alternative for HCC chemotherapy. [BMB Reports 2014; 47(4): 221-226]

Comparative proteome analysis to explore p53 pathway disruption in head and neck carcinogenesis

The 5-year-survival rate of head and neck squamous cell carcinoma (HNSCC) has been only moderately improved over the last few decades. HNSCC develops in precursor fields of genetically altered mucosal cells, typically characterized by p53 pathway disruption, that mostly do not give any clinical symptoms. Patients present therefore often with invasive carcinomas in an advanced stage. After tumor resection, part of these fields frequently stays behind unnoticed, causing secondary tumors. Identification of these precursor fields would allow screening and early detection of both primary and secondary tumors. Our aim was to identify differential proteins related to p53 dysfunction. These proteins may serve as valuable biomarkers that can predict the presence of a precursor field. We used a squamous cell model for p53 inactivation, which was analyzed by 2D-DIGE and LC-MS/MS. This approach enabled us to identify a set of 74 proteins that were differentially expressed in cells with normal versus disrupted p53 function. For six proteins the major changes in expression were verified with immunohistochemical staining. The most promising result was the identification of peroxiredoxin-1 which allowed immunohistochemical discrimination between normal epithelium and precursor field tissue with a TP53 mutation.

Modification of serine 392 is a critical event in the regulation of p53 nuclear export and stability

Although it has been shown that phosphorylations of p53 serine its residues are critical events for the regulation of their function, the specific biological effects of each of these phosphorylations, especially at serine 392, remain to be elucidated. Serine 392 has been proposed to play a role in the tetramerization of p53 and in the enhancement of its DNA-binding affinity. However, this is not consistent with other reports showing that substitution of serine 392 does not disrupt p53 function. These discrepancies suggest that modification of serine 392 may contribute to p53 activity through other transactivating pathways. In this study, we demonstrate that this C-terminal serine residue (p53-392S) in fact plays a critical role in the regulation of p53 stability such that substitution with alanine (p53-392A) strongly enhances p53 stability without disrupting mouse double minute 2 binding. Additionally, the p53-392A mutant is localized mainly in the nucleus, whereas both wild-type p53 and a glutamic acid mutant, p53-392E, are evenly distributed throughout the cytoplasm and nucleus. However, each of these p53 species had similar effects on both cell cycle inhibition and apoptosis, in response to either UV or adriamycin treatment. Moreover, p53-392A protein was resistant to E6-mediated degradation. Our results suggest that although serine 392 is not essential for the transactivation and nuclear import of p53, it exerts important effects upon p53 stability via the inhibition of its nuclear export mechanism.

Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is one of the most commonly diagnosed leukemias managed by practicing hematologists. For many years patients with CLL have been viewed as similar, with a long natural history and only marginally effective therapies that rarely yielded complete responses. Recently, several important observations related to the biologic significance of V(H) mutational status and associated ZAP-70 overexpression, disrupted p53 function, and chromosomal aberrations have led to the ability to identify patients at high risk for early disease progression and inferior survival. Concurrent with these investigations, several treatments including the nucleoside analogues, monoclonal antibodies rituximab and alemtuzumab have been introduced. Combination of these therapies in clinical trials has led to high complete and overall response rates when applied as initial therapy for symptomatic CLL. Thus, the complexity of initial risk stratification of CLL and treatment has increased significantly. Furthermore, when these initial therapies do not work, approach of the CLL patient with fludarabine-refractory disease can be quite challenging. This session will describe the natural history of a CLL patient with emphasis on important decision junctures at different time points in the disease. In Section I, Dr. Stephan Stilgenbauer focuses on the discussion that occurs with CLL patients at their initial evaluation. This includes a review of the diagnostic criteria for CLL and prognostic factors utilized to predict the natural history of the disease. The later discussion of risk stratification focuses on molecular and genomic aberrations that predict rapid progression, poor response to therapy, and inferior survival. Ongoing and future efforts examining early intervention strategies in high risk CLL are reviewed. In Section II, Drs. Ian Flinn and Jesus G. Berdeja focus on the discussion of CLL patients when symptomatic disease has developed. This includes an updated review of monotherapy trials with nucleoside analogs and recent trials that have combined these with monoclonal antibodies and/or alternative chemotherapy agents. Appropriate application of more aggressive therapies such as autologous and allogeneic immunotherapy and less aggressive treatments for appropriate CLL patient candidates are discussed. In Section III, Dr. John Byrd focuses on the discussion that occurs with CLL patients whose disease is refractory to fludarabine. The application of genetic risk stratification in choosing therapy for this subset of patients is reviewed. Available data with conventional combination based therapies and monoclonal antibodies are discussed. Finally, alternative promising investigational therapies including new antibodies, kinase inhibitors (CDK, PDK1/AKT, PKC) and alternative targeted therapies (DNA methyltransferase inhibitors, histone deacetylase inhibitors, etc.) are reviewed with an emphasis on the most promising agents for this patient population.

Arsenite cocarcinogenesis: an animal model derived from genetic toxicology studies.

Although epidemiologic evidence shows an association between inorganic arsenic in drinking water and increased risk of skin, lung, and bladder cancers, no animal model for arsenic carcinogenesis has been successful. This lack has hindered mechanistic studies of arsenic carcinogenesis. Previously, we and others found that low concentrations (< or =5 microm) of arsenite (the likely environmental carcinogen), which are not mutagenic, can enhance the mutagenicity of other agents, including ultraviolet radiation (UVR) and alkylating agents. This enhancing effect appears to result from inhibition of DNA repair by arsenite, but not via inhibition of DNA repair enzymes. Rather, low concentrations of arsenite disrupt p53 function and upregulate cyclin D1. Failure to find an animal model for arsenic carcinogenesis might be because arsenite is not a carcinogen per se but acts as an enhancing agent (cocarcinogen) with a genotoxic partner. We tested this hypothesis with solar UVR in hairless but immunocompetent Skh1 mice. Mice were given 10 mg/L sodium arsenite in drinking water (or not) and irradiated with 1.7 KJ/m(2) solar UVR 3 times weekly. As expected, no tumors appeared in any organs in control mice or in mice given arsenite alone. After 26 weeks irradiated mice given arsenite had a 2.4-fold increase in skin tumor yield compared with mice given UVR alone. The tumors were mostly squamous cell carcinomas, and those occurring in mice given UVR plus arsenite were much larger and more invasive. These results are consistent with the hypothesis that arsenic acts as a cocarcinogen with a second (genotoxic) agent by inhibiting DNA repair and/or enhancing positive growth signaling. Skin cancers in populations drinking water containing arsenic may be caused by the enhancement by arsenic compounds of carcinogenesis induced by UVR (or other environmental agents). It is possible that lung and bladder cancers associated with arsenic in drinking water may also require a carcinogenic partner.

P53 gene mutations are associated with poor survival in low and low-intermediate risk diffuse large B-cell lymphomas

Backgroundp53 alterations have been associated with a poor prognosis in aggressive B-cell lymphoma. We investigated the clinical relevance of p53 status in diffuse large B-cell lymphoma (DLBCL), focusing on patients who belong to lower risk groups of the international prognostic index and were uniformly treated. We aimed to determine whether this biological marker could identify among such patients those with a pejorative outcome who could benefit from a distinct therapeutic approach. Patients and methodsWe studied 69 patients presenting with no, one (low-risk, n = 40) or two (low-intermediate risk, n = 29) risk factors treated with an anthracyclin-containing induction regimen. p53 exons 5–8 mutations were screened for using denaturing gradient gel electrophoresis and confirmed by direct sequencing. Immunohistochemical detection of p53 protein and of its downstreamtarget p21 were also evaluated in 60 of 69 cases. Resultsp53 mutations were detected in 16 of 69 (23%) lymphoma samples. The presence of a p53 gene mutation affected survival (P = 0.01), with a 6-year survival rate estimated to be 44% in mutated patients, compared with 79% in non-mutated ones. Using a stepwise Cox model, p53 mutation constituted the only parameter affecting survival (relative risk = 2.7, P = 0.03). A p53+/p21– immunohistochemical pattern (n = 15), suggestive of a disrupted p53 function, strongly correlated with p53 gene status and was associated with a lower 6-year survival rate when compared with a p53– or p53+/p21+ phenotype (47% versus 74%, P = 0.05). Conclusionsp53 alterations constitute a pejorative biological indicator able to discriminate among clinically defined lower risk patients with DLBCL.

Expression of Different Mutant p53 Transgenes in Neuroblastoma Cells Leads to Different Cellular Responses to Genotoxic Agents

The involvement of p53 as a determinant of chemosensitivity or radiosensitivity is not well understood and is complicated by numerous contradictory reports. Here we have addressed this issue using a series of isogenic clones derived from two neuroblastoma cell lines that express wild-type p53 genes, Nub7 and IMR32. Two different mutant p53 transgenes were used in an attempt to disrupt p53 function in the clones. Our findings indicate that the cellular response is dependent on the genotoxic agent used as well as on the specific p53 transgene used. Cellular radiosensitivity showed no association with apoptosis or with the ability of the cells to arrest in G1 after irradiation. An association was observed, however, between γ-radiation sensitivity and DNA double-strand break rejoining activity.

Arsenite Is a Cocarcinogen with Solar Ultraviolet Radiation for Mouse Skin: An Animal Model for Arsenic Carcinogenesis

Although epidemiological evidence shows an association between arsenic in drinking water and increased risk of skin, lung, and bladder cancers, arsenic compounds are not animal carcinogens. The lack of animal models has hindered mechanistic studies of arsenic carcinogenesis. Previously, this laboratory found that low concentrations of arsenite (the likely environmental carcinogen) which are not mutagenic can enhance the mutagenicity of other agents, including ultraviolet radiation (UVR). This enhancing effect appears to result from inhibition of DNA repair by arsenite. Recently we found that low concentrations of arsenite disrupted p53 function and upregulated cyclin D1. These results suggest that the failure to find an animal model for arsenic carcinogenesis is because arsenite is not a carcinogen per se, but rather acts as an enhancing agent (cocarcinogen) with a genotoxic partner. We tested this hypothesis with solar UVR as carcinogenic stimulus in hairless Skh1 mice. Mice given 10 mg/l sodium arsenite in drinking water for 26 weeks had a 2.4-fold increase in yield of tumors after 1.7 KJ/m2 UVR three times weekly compared with mice given UVR alone. No tumors appeared in mice given arsenite alone. The tumors were mostly squamous cell carcinomas, and those occurring in mice given UVR plus arsenite appeared earlier and were much larger and more invasive than in mice given UVR alone. These results are consistent with the hypothesis that arsenic acts as a cocarcinogen with a second (genotoxic) agent by inhibiting DNA repair and/or enhancing positive growth signaling.

Induction of micronuclei by a new non-peptidic mimetic farnesyltransferase inhibitor RPR-115135: role of gene mutations.

To investigate the relationship between oncogene activation and induction of micronuclei by a new non-peptidic mimetic farnesyltransferase inhibitor, RPR-115135, two isogenic cell lines, human colon cancer line HCT-116, which harbors a K-ras mutation, and spontaneously immortalized human breast epithelial cell line MCF-10A, were utilized. HCT-116 cells were transfected with an empty control pCMV vector (clone CMV-2) or with a dominant negative mutated p53 transgene (clone Mu-p53-2) to disrupt p53 function. In both clones RPR-115135 induced a significant increase in the frequency of micronucleation at concentrations that did not affect cell membrane integrity. RPR-115135 produced a significant increase in the ratio of CREST+ to CREST- micronuclei. MCF-10A cells were stably transfected with either c-Ha-ras or c-erbB-2 or both H-ras + c-erbB-2. No induction of micronuclei was observed. No induction of micronuclei was reported in human lymphocytes and in primary spinal cells obtained from 7-day chick embryos. In conclusion, RPR-115135 acts as an aneugenic agent in a complex manner, dependent upon the complement of mutations in cell regulatory genes in tumour cells and this activity may be independent of ras genotype.

RPR-115135, a new non peptidomimetic farnesyltransferase inhibitor, induces G0/G1 arrest only in serum starved cells.

A new non peptidomimetic farnesyltransferase inhibitor, RPR-115135, was studied in an isogenic cell model system consisting of human colon cancer HCT-116 line. HCT-116 cells were transfected with an empty control pCMV vector or with a dominant-negative mutated p53 transgene to disrupt p53 function. Growth inhibitory effects of RPR-115135 were evaluated on cells growing under different conditions (serum starvation, serum starvation and recovery, nocodazole treatment). The cytotoxic activity of RPR-115135 was independent of the cell cycle status of the target cells. Addition of RPR-115135 only to cells exposed to reduced serum conditions (0.1% FCS) resulted in an enhanced ability of HCT-116 cells to arrest in the G0/G1 phase. This arrest response appeared independent of p53/p21cip1/waf-1 function. A reduction of Cyclin A protein amount by RPR-115135 was observed in both clones. These latter results suggest that RPR-115135 might down-regulate the cell cycle factor that would normally impede G0/G1 arrest.

Characterization of p53 oligomerization domain mutations isolated from Li-Fraumeni and Li-Fraumeni like family members.

p53 is a tumour suppressor gene which functions as a transcription factor to upregulate genes for growth arrest and apoptosis following DNA damage. p53 mutations are associated with Li-Fraumeni and Li-Fraumeni like syndromes. Recently mutations of the oligomerization domain have been isolated from an LFS and an LFL family affecting respectively codon 344 (Leu to Pro) and 337 (Arg to Cys). The present study was designed to determine the affect of these mutations on the function of p53 protein. p53 344 Leu to Pro existed only in a monomeric form and could not bind to DNA. It was inactive at inducing apoptosis, transactivating luciferase from a bax promoter and inhibiting cell growth. In contrast, p53 337 Arg to Cys could form tetramers and could bind to DNA. However, p53 337 Arg to Cys was not fully active and could only induce apoptosis, transactivate luciferase from a bax promoter and inhibit cell growth with approximately 60% of the ability of wild-type p53. Both mutant proteins had reduced ability to bind to MDM2, p53 337 Arg to Cys being more reduced than p53 344 Leu to Pro. These results indicate that point mutations in the oligomerization domain can disrupt p53 function. In addition, the value of LFS and LFL families for the further understanding of the biological and biochemical properties of p53 is demonstrated.

Mdm 2 mRNA expression is associated with survival in ovarian cancer.

Expression of mdm-2 mRNA was measured in 90 ovarian-cancer tissue specimens using the S1 nuclease assay, to investigate a possible association between MDM2 expression and prognosis. mdm-2 mRNA expression was an independent prognostic factor for patients with primary ovarian cancer, FIGO (International Federation of Gynecology and Obstetrics) stages III and IV (n = 57), who all received chemotherapy with carboplatin or cisplatin and cyclophosphamide. Median survival time for patients (FIGO stages III and IV) with no detectable expression of mdm-2 mRNA (n = 14) was 171 days, as compared with 839 days for patients (n = 43) with detectable mdm-2 mRNA (p = 0.0194; log-rank test). However, no association between mdm-2 mRNA expression and survival was observed for patients with FIGO stages I and II who did not receive chemotherapy. mdm-2 expression was not associated with FIGO stage, residual disease, histologic grade and type. Our results suggest that mdm-2, which is known to disrupt p53 function, sensitizes ovarian-cancer cells to cisplatin/cyclophosphamide, possibly by inhibition of p53-mediated G1 cell-cycle arrest and p53-stimulated nucleotide-excision repair.

UCN-01: a Potent Abrogator of G2 Checkpoint Function in Cancer Cells With Disrupted p53

Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G2 arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G2 checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent. We studied the effect of UCN-01 on G2 checkpoint control in human lymphoma CA46 cells, whose sensitivity to various DNA-damaging agents and G2 response to DNA damage have been characterized. We also assessed the ability of UCN-01 to enhance the cytotoxicity of gamma irradiation in CA46 cells and human colon carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01-mediated abrogation of the G2 checkpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either expressed or did not express the human papillomavirus type-16 E6 protein. MCF-7 cells have normal p53 function, and the E6 protein binds p53 protein and promotes its destruction. The effect of UCN-01 on cell cycle arrest induced by gamma irradiation was studied in CA46 cells and in transfected MCF-7 cells by use of flow cytometry. A histone H1 phosphorylation assay was employed to measure cyclin B1/Cdc2 kinase activity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphorylation status of Cdc2 kinase protein in the same extracts was determined by use of western blotting. The effect of UCN-01 on the cytotoxicity of gamma irradiation in CA46 and HT-29 cells was determined by use of MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respectively; a clonogenic assay was also used to measure the effect of UCN-01 on the cytotoxicity of cisplatin in transfected and nontransfected MCF-7 cells. G2 arrest induced in CA46 cells by gamma irradiation was minibited by treatment with UCN-01 in a dose-dependent manner; arrest in G2 was completely abrogated by exposure to 300 nM UCN-01. Biochemical markers indicative of the G2/M transition, including the activation of cyclin B1/Cdc2 kinase and the suppression of Cdc2 threonine-14 and tyrosine-15 phosphorylation, were detected in irradiated cells treated with UCN-01. UCN-01 enhanced the cytotoxicity of gamma irradiation in CA46 and HT-29 cells. MCF-7 cells with functional p53 protein were more resistant to G2 checkpoint abrogation by UCN-01 than MCF-7 cells with disrupted p53 function. UCN-01 markedly enhanced the cell-killing activity of cisplatin in MCF-7 cells defective for p53 function. UCN-01 is a potent abrogator of G2 checkpoint control in cancer cells with disrupted p53 function. UCN-01 might be capable of enhancing the effectiveness of DNA-damaging agents in the treatment of tumors with cells lacking normal p53 function.