Expression of securin promotes colorectal cancer cell death via a p53-independent pathway after radiation

Securin has been shown to regulate genomic stability; nevertheless, the role of securin on the cytotoxicity after radiation is still unclear. Exposure to 1–10 Gy X-ray radiation induced cell death in RKO colorectal cancer cells. The protein levels of securin, p53, and p21 were elevated by radiation. The proteins of phosphorylation of p53 at serine-15, which located on the nuclei of cancer cells, were highly induced by radiation. However, radiation increased securin proteins, which located on both of nuclei and cytoplasma in RKO cells. The p53-wild type colorectal cancer cells were more susceptible on cytotoxicity than the p53-mutant cells following exposure to radiation. Besides, the existence of securin in colorectal cancer cells induced higher apoptosis than the securin-null after radiation. Securin proteins were elevated by radiation in the p53-wild type and -mutant cells; furthermore, radiation raised the p53 protein expression in both the securin-wild type and -null cells. As a whole, these findings suggest that the existence of securin promotes apoptosis via a p53-indpendent pathway after radiation in human colorectal cancer cells.     

VEGF expression and its reguration by p53 gene transfection in endometrial carcinoma cells

Vascular endothelial growth factor (VEGF) that activates endothelial cell growth induces angiogenesis, which is indispensable to tumor igenesis and tumor progression. On the other hand, tumor suppressor gene p53 has been considered to regulate VEGF expression, but the detailed relationship between them remains unclear. In this study, we aimed to study VEGF expression in endometrial carcinoma cells and the effect of p53 gene transfection on VEGF expression using p53-mutated endometrial carcinoma cell line, HEC-50B. Immunoblotting for detecting VEGF protein, p53 protein and beta-actin was performed using 11 endometrial carcinoma cell lines. Levels of VEGF in the cultured media were measured by Enzyme immunoassay(EIA). Transfection of wild p53 gene was carried out by SuperFect method in HEC-50B cells, which had mutant p53 gene and did not express p53 protein. The results of immunoblotting were analyzed by NIH image and expressed as values. The results of EIA were expressed as the relative value. The VEGF value was 0.8 +/- 0.3 (n = 6) in p53-wild group, whereas in p53-mutant group it was 1.6 +/- 0.8 (n = 5). VEGF expression was correlated significantly with p53 status (P < 0.05). VEGF levels in p53 gene-transfected cells and the conditioned medium were decreased in 48 hours after p53 gene transfection. VEGF expression was down-regulated by p53 in endometrial carcinoma cells.     

Celecoxib induces p53-PUMA pathway for apoptosis in human colorectal cancer cells

Celecoxib, a clinical non-steroidal anti-inflammatory drug, displays anticarcinogenic and chemopreventive activities in human colorectal cancers, although the mechanisms of apoptosis by celecoxib are poorly understood. The existence of functional p53 but not securin in colorectal cancer cells was higher on the induction of cytotoxicity than the p53-mutational colorectal cancer cells following celecoxib treatment. The p53-wild type HCT116 cells were more susceptible to increase ∼25% cell death than the p53-null HCT116 cells after treatment with 100 μM celecoxib for 24 h. Transfection with a small interfering RNA of p53 reduced the celecoxib-induced cytotoxicity in the RKO (p53-wild type) colorectal cancer cells. Celecoxib (80-100 μM for 24 h) significantly increased total p53 proteins and the phosphorylated p53 proteins at serine-15, -20, -46, and -392 in RKO cells. However, the phospho-p53 (serine-15, -20, and -392) proteins were presented on the nuclei of cells but the phospho-p53 (serine-46) protein was located on the cytoplasma of apoptotic cells following treatment with celecoxib. Interestingly, the p53 up-regulated modulator of apoptosis (PUMA) protein, which located on the mitochondria, was induced by celecoxib in the p53-functional colorectal cancer cells but not in the p53-mutational cells. Together, this study provides the first time that celecoxib induces the various phosphorylated sites of p53 and activates p53-PUMA pathway, which potentiates the apoptosis induction in human colorectal cancer cells.     

Overexpression of miR-22 reverses paclitaxel-induced chemoresistance through activation of PTEN signaling in p53-mutated colon cancer cells

Chemoresistance is a key cause of treatment failure in colon cancer. MiR-22 is a tumor-suppressing microRNA. To explore whether miR-22 is an important player in the development of chemoresistance in colon cancer, we overexpressed miR-22 and subsequently tested its role in cell proliferation, apoptosis, survival, and associated signaling in p53-mutated HT-29 and HCT-15 cells, and p53 wild-type HCT-116 cells. We further investigated the role of miR-22 on cytotoxicity of paclitaxel in both the p53-mutated and p53 wild-type colon cancer cells. Results showed that HT-29 and HCT-15 cells were resistant to paclitaxel-induced cytotoxicity, which normally inhibits cell proliferation and survival, and induces apoptosis. Conversely, HCT-116 was relatively sensitive to the cytotoxicity of paclitaxel. Overexpression of miR-22 significantly decreased cell proliferation and survival, and induced cell apoptosis in the p53-mutated colon cancer cells, but played no role in the p53 wild-type cells. Importantly, miR-22 overexpression enhanced the cytotoxic role of paclitaxel in p53-mutated HT-29 and HCT-15 cells, but not in p53 wild-type HCT-116 cell. We further demonstrated that the tumor-suppressive role of miR-22 in p53-mutated colon cancer cells was mediated by upregulating PTEN expression, which negatively regulated Akt phosphorylation at Ser(473) and MTDH expression, and subsequently increased Bax and active caspase-3 levels. Our study is the first to identify the tumor-suppressive role of miR-22 and its associated signaling in the p53-mutated colon cancer cells and highlighted the chemosensitive role of miR-22.     

Purvalanol A enhances cell killing by inhibiting up-regulation of CDC2 kinase activity in tumor cells irradiated with high doses of X rays

To clarify the relationship between CDC2 kinase activity and radiation-induced apoptosis, we examined whether the cyclin-dependent kinase (CDK) inhibitor purvalanol A enhanced radiation-induced apoptosis in gastric tumor cells. MKN45 cells exposed to 20 Gy of X rays increased the CDC2 kinase activity and the expression of regulatory proteins (phospho-CDC2 and cyclin B1) of the G2/M phase, followed by activation of the G2/M checkpoint, whereas the treatment of X-irradiated MKN45 cells with 20 microM purvalanol A suppressed the increase in the CDC2 kinase activity and expression of the G2/M-phase regulatory proteins and reduced the fraction of the cells in the G2/M phase in the cell cycle. Furthermore, this treatment resulted in not only a significant increase in radiation-induced apoptosis but also the loss of clonogenicity in both MKN45 (p53-wild) and MKN28 (p53-mutated) cells. The expression of anti-apoptosis proteins, inhibitor of apoptosis protein (IAP) family members (survivin and XIAP) and BCL2 family members (Bcl-X(L) and Bcl-2), in purvalanol A-treated cells with and without X rays was significantly lower than for cells exposed to X rays alone. These results suggest that the inhibition of radiation-induced CDC2 kinase activity by purvalanol A induces apoptosis through the enhancement of active fragments of caspase 3.     

The p53-mediated G1 checkpoint is retained in tumorigenic rat embryo fibroblast clones transformed by the human papillomavirus type 16 E7 gene and EJ-ras.

Rat embryo fibroblast clones transformed with the human papillomavirus type 16 E7 gene and the H-ras oncogene (ER clones) fall into two groups on the basis of endogenous p53 genotype, wild type or mutant. We have compared these clones with the aim of indentifying physiological differences that could be attributed to p53 protein function. We show that all ER clones, regardless of p53 gene status, are tumorigenic and metastatic in severe combined immunodeficiency mice. We demonstrate that only the wild-type p53 protein expressed in ER clones is functional on the basis of its site-specific double-stranded DNA-binding activity and its ability to confer a G1 delay on cells following treatment with ionizing radiation. These data indicate that disruption of the p53 growth-regulatory pathway is not a prerequisite for the malignant conversion of rat embryo fibroblasts expressing the E7 gene and mutant ras. Differences in phenotype that were correlated with loss of p53 protein function included the following: serum-independent growth of ER clones in culture, decreased tumor doubling time in vivo, and increased radioresistance. In addition, we demonstrate the p53-dependent G1 checkpoint alone does not determine radiosensitivity.     

Protein interaction domains of the ubiquitin-specific protease, USP7/HAUSP

USP7 or HAUSP is a ubiquitin-specific protease in human cells that regulates the turnover of p53 and is bound by at least two viral proteins, the ICP0 protein of herpes simplex type 1 and the EBNA1 protein of Epstein-Barr virus. We have overexpressed and purified USP7 and shown that the purified protein is monomeric and is active for cleaving both a linear ubiquitin substrate and conjugated ubiquitin on EBNA1. Using partial proteolysis of USP7 coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we showed that USP7 comprises four structural domains; an N-terminal domain known to bind p53, a catalytic domain, and two C-terminal domains. By passing a mixture of USP7 domains over EBNA1 and ICP0 affinity columns, we showed that the N-terminal p53 binding domain was also responsible for the EBNA1 interaction, while the ICP0 binding domain mapped to a C-terminal domain between amino acids 599-801. Tryptophan fluorescence assays showed that an EBNA1 peptide mapping to residues 395-450 was sufficient to bind the USP7 N-terminal domain and did so with a dissociation constant of 0.9-2 microM, whereas p53 peptides spanning the USP7-binding region gave dissociation constants of 9-17 microM in the same assay. In keeping with these relative affinities, gel filtration analyses of the complexes showed that the EBNA1 peptide efficiently competed with the p53 peptide for USP7 binding, suggesting that EBNA1 could affect p53 function in vivo by competing for USP7.     

MiR-34a in age and tissue related radio-sensitivity and serum miR-34a as a novel indicator of radiation injury

MiR-34a, a direct target of p53, has shown to exert potent anti-proliferative effects. It has also been found that miR-34a can be induced by irradiation in vitro and in vivo. However, the relationship between miR-34a and radio-sensitivity, and its potential diagnostic significance in radiation biology, remain unclear. This study found that differing responses to ionizing radiation (IR) of young and adult mice were related to miR-34a. First, we found that miR-34a could be induced in many organs by radiation of both young and adult mice. However, the level of miR-34a induced by young mice was much higher when compared to adult mice. Next, we found that miR-34a played a critical role in radio-sensitivity variations of different tissues by enhancing cell apoptosis and decreasing cell viability. We also found that the induction of miR-34a by radiation was in a p53 dependent manner and that one possible downstream target of miR-34a that lead to different radio-sensitivity was the anti-apoptosis molecular Bcl-2. However, over-expression of miR-34a and knockdown of Bcl-2 could significantly enhance the radio-sensitivity of different cells while inhibition of miR-34a could protect cells from radiation injury. Finally, we concluded that miR-34a could be stable in serum after IR and serve as a novel indicator of radiation injury. Taken together, this data strongly suggests that miR-34a may be a novel indicator, mediator and target of radiation injury, radio-sensitivity and radioprotection.     

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.     

Enhanced gene transfer and cell death following p53 gene transfer using photochemical internalisation of glucosylated PEI-DNA complexes

BACKGROUND: p53 is frequently mutated in many cancers including human head and neck squamous cell carcinoma and pancreatic cancer. In tumor models, wild-type (wt) p53 gene transfer induces apoptosis and tumor regression in vivo, justifying the extensive clinical investigation of p53 gene therapy. METHODS: p53 nonviral-mediated gene transfer was achieved using glucosylated polyethylenimine (PEI) in conjunction with photochemical internalisation (PCI). Experimental conditions were optimised using the green fluorescent protein (GFP) as a reporter. p53 gene transfer was then evaluated using semi-quantitative RT-PCR in p53-deleted PANC3 and p53-mutated FaDu cell lines. Following gene transfer, induction of apoptosis was investigated using phosphatidylserine externalisation and nuclear fragmentation assays. Induction of long-term cell death was analysed using colony-forming assays. RESULTS: PCI was found to enhance GFP gene transfer after 48 h in both cell lines. Whether using glucosylated-PEI alone or associated with PCI, p53 gene transfer was achieved with subsequent recovery of p53 mRNA expression in PANC3 cells and a significant 4-fold increase in p53 mRNA expression in FaDu cells. PCI was found to further enhance p53 mRNA expression by 2.3-fold in PANC3 cells. Spontaneous induction of apoptosis following wt-p53 gene transfer was achieved in both cell lines. PCI was found to enhance apoptosis up to levels similar to those achieved with chemotherapy. As a consequence, long-term cell death was significantly enhanced after wt-p53 gene transfer when PCI was used in both cell lines, yielding up to 60% cell death. CONCLUSIONS: PCI increases glucosylated-PEI-mediated p53 gene transfer, apoptosis as well as cell death in mutant p53 human cancer cells.     

Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein-Barr nuclear antigen 1 implications for EBV-mediated immortalization

USP7/HAUSP is a key regulator of p53 and Mdm2 and is targeted by the Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV). We have determined the crystal structure of the p53 binding domain of USP7 alone and bound to an EBNA1 peptide. This domain is an eight-stranded beta sandwich similar to the TRAF-C domains of TNF-receptor associated factors, although the mode of peptide binding differs significantly from previously observed TRAF-peptide interactions in the sequence (DPGEGPS) and the conformation of the bound peptide. NMR chemical shift analyses of USP7 bound by EBNA1 and p53 indicated that p53 binds the same pocket as EBNA1 but makes less extensive contacts with USP7. Functional studies indicated that EBNA1 binding to USP7 can protect cells from apoptotic challenge by lowering p53 levels. The data provide a structural and conceptual framework for understanding how EBNA1 might contribute to the survival of Epstein-Barr virus-infected cells.     

TSPYL5 suppresses p53 levels and function by physical interaction with USP7

We have previously reported a gene expression signature that is a powerful predictor of poor clinical outcome in breast cancer. Among the seventy genes in this expression profile is a gene of unknown function: TSPYL5 (TSPY-like 5, also known as KIAA1750). TSPYL5 is located within a small region at chromosome 8q22 that is frequently amplified in breast cancer, which suggests that TSPYL5 has a causal role in breast oncogenesis. Here, we report that high TSPYL5 expression is an independent marker of poor outcome in breast cancer. Mass spectrometric analysis revealed that TSPYL5 interacts with ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease; HAUSP). USP7 is the deubiquitylase for the p53 tumour suppressor and TSPYL5 reduces the activity of USP7 towards p53, resulting in increased p53 ubiquitylation. We demonstrate that TSPYL5 reduces p53 protein levels and inhibits activation of p53-target genes. Furthermore, expression of TSPYL5 overrides p53-dependent proliferation arrest and oncogene-induced senescence, and contributes to oncogenic transformation in multiple cell-based assays. Our data identify TSPYL5 as a suppressor of p53 function through its interaction with USP7.     

Distinct patterns of gene expression induced by viral oncogenes in human embryonic brain cells

1. The limited lifespan of human embryonic brain (HEB) cells hampers their therapeutic use for the treatment of neurodegenerative diseases.2. Stable expression of SV40 large T antigen (LTA) or E6E7 genes of human papillomavirus type 16 significantly increased the lifespan of HEB cells, but did not induce transformation.3. The extended lifespan was triggered by changes in the expression of antiproliferative genes. We found that changes in the expression of p16 (INK4a), p21 (WAF1), p14ARF, and p53 tumor suppressor gene, but not p27 (Kip1), differed between the LTA- and E6E7-HEB cells.4. Despite the induction of p53 RNA, p53 protein was undetectable in HEB-E6E7 cells. In contrast, p53 protein was increased in HEB-LTA cells as compared with the parental cells. Expression of p21 was, however, reduced in both cell lines.5. While p16 was decreased in HEB-E6E7 cells, its expression was increased in HEB-LTA cells.6. Despite these changes, HEB cell lines showed neuron-like morphological differentiation when the intracellular level of cAMP was elevated.7. This suggests that the mechanisms for inducing neuronal differentiation are still intact in HEB-E6E7 and HEB-LTA cells. More importantly, differentiation signals can override the effects of viral oncogenes in HEB cells.     

Molecular recognition of p53 and MDM2 by USP7/HAUSP

The ubiquitin-specific protease, USP7, has key roles in the p53 pathway whereby it stabilizes both p53 and MDM2. We show that the N-terminal domain of USP7 binds two closely spaced 4-residue sites in both p53 and MDM2, falling between p53 residues 359-367 and MDM2 residues 147-159. Cocrystal structures with USP7 were determined for both p53 peptides and for one MDM2 peptide. These peptides bind the same surface of USP7 as Epstein-Barr nuclear antigen-1, explaining the competitive nature of the interactions. The structures and mutagenesis data indicate a preference for a P/AXXS motif in peptides that bind USP7. Contacts made by serine are identical and crucial for all peptides, and Trp165 in the peptide-binding pocket of USP7 is also crucial. These results help to elucidate the mechanism of substrate recognition by USP7 and the regulation of the p53 pathway.     

The herpesvirus associated ubiquitin specific protease, USP7, is a negative regulator of PML proteins and PML nuclear bodies

The PML tumor suppressor is the founding component of the multiprotein nuclear structures known as PML nuclear bodies (PML-NBs), which control several cellular functions including apoptosis and antiviral effects. The ubiquitin specific protease USP7 (also called HAUSP) is known to associate with PML-NBs and to be a tight binding partner of two herpesvirus proteins that disrupt PML NBs. Here we investigated whether USP7 itself regulates PML-NBs. Silencing of USP7 was found to increase the number of PML-NBs, to increase the levels of PML protein and to inhibit PML polyubiquitylation in nasopharyngeal carcinoma cells. This effect of USP7 was independent of p53 as PML loss was observed in p53-null cells. PML-NBs disruption was induced by USP7 overexpression independently of its catalytic activity and was induced by either of the protein interaction domains of USP7, each of which localized to PML-NBs. USP7 also disrupted NBs formed from some single PML isoforms, most notably isoforms I and IV. CK2α and RNF4, which are known regulators of PML, were dispensable for USP7-associated PML-NB disruption. The results are consistent with a novel model of PML regulation where a deubiquitylase disrupts PML-NBs through recruitment of another cellular protein(s) to PML NBs, independently of its catalytic activity.