Ectopic Expression Of Wild-type P53 Research Articles

MiR-125a is upregulated in cancer stem-like cells derived from TW01 and is responsible for maintaining stemness by inhibiting p53.

microRNA (miR)-125a and miR-125b were demonstrated to translationally and transcriptionally inhibit the mRNA level of p53 following the induction of chemo-reagents in our previous report. As a small subpopulation of nasopharyngeal carcinoma (NPC), cancer stem-like cells (CSCs) function critically in multi-malignant behaviors, including tumorigenesis and metastasis; however, the expression pattern and regulatory role of miR-125a, miR-125b and p53 in CSCs derived from NPC remain unclear. In order to investigate the potential regulatory role of miR-125 on p53, firstly CSCs was isolated from TW01 by culturing in serum-free medium. The stemness of isolated CSCs was examined via self-renewal capacity and side population assays. Following this, the miR-125a, miR-125b and p53 mRNA levels were evaluated via reverse-transcription quantitative polymerase chain reaction. Following the transfections of wild-type p53 or p53 without DNA binding activity (p53-mutR248Q) into TW01 or CSCs, Chromatin Immunoprecipitation (ChIP), and cell cycle analyses using flow cytometry or Cell Counting Kit-8 assays were performed. Notably, it was determined that miR-125a was significantly upregulated in CSCs derived from TW01, but not miR-125b, and the mRNA and protein levels of p53 were downregulated. The transfection of p53 significantly decreased the cell viability and stopped cell cycle at the G0/G1 phases in TW01 and CSCs. The ChIP assay confirmed that the ectopic expression of wild-type p53 transcriptionally regulates its downstream gene, p21, but not B-cell lymphoma 2 nor Sco2. Taken together, the results of the present study indicated that p53 regulates CSCs via its DNA binding activity and potentially, in CSCs, miR-125a regulates the expression of p53, maintaining stemness.

Abstract 787: Tumor suppressor p53 inhibits expression of the pro-metastasis protein Toca-1.

Abstract Transducer of Cdc42-dependent actin assembly-1 (Toca-1) is an adaptor protein that recruits key actin regulatory proteins to invadopodia in metastatic breast cancer cells. We recently showed that stable silencing of Toca-1 led to reduced invadopodia formation, cell invasion and tumor metastasis in an orthotopic xenograft model in mice. These results raise the possibility that Toca-1 upregulation may occur during tumor progression to metastatic disease. Here we show that Toca-1 expression is higher in triple negative breast cancers (HER2/ER/PR) compared to other subtypes. Since triple negative breast cancers frequently harbor mutations in the tumor suppressor p53, we tested whether p53 regulates Toca-1 expression. In wild-type (WT) p53-expressing normal breast epithelial and breast cancer cells, stabilization of WT p53 by inducing DNA damage, or treating cells with Mdm2 inhibitor Nutlin-3, led to dose-dependent reductions in Toca-1 mRNA and protein expression. Likewise, ectopic expression of WT p53 in p53-null HCC1806 cells also led to repression of Toca-1 levels. To test if this was a direct effect of p53 on the Toca-1 gene, chromatin immunoprecipitation (ChIP) studies were performed. Indeed, the proximal promoter region and intron 2 of the Toca-1 gene (which contained a predicted p53 binding site) were detected in p53 ChIP assays in WT p53-expressing cells, but not mutant p53 expressing breast cancer cells. Together, these results identify a novel pathway to inhibit Toca-1 expression in cells with WT p53, and imply that the loss of p53 in highly metastatic breast cancer subtypes may lead to elevated Toca-1 expression and progression to metastatic disease. Citation Format: Harish Chander, Colin D. Brien, Peter Truesdell, Doris Germain, Andrew W. B. Craig. Tumor suppressor p53 inhibits expression of the pro-metastasis protein Toca-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 787. doi:10.1158/1538-7445.AM2013-787

Destabilization of CARP mRNAs by aloe‐emodin contributes to caspase‐8‐mediated p53‐independent apoptosis of human carcinoma cells

Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.

Induction of the small heat shock protein αB-crystallin by genotoxic stress is mediated by p53 and p73

The small heat shock protein alphaB-crystallin is a molecular chaperone that is induced by stress and protects cells by inhibiting protein aggregation and apoptosis. To identify novel transcriptional regulators of the alphaB-crystallin gene, we examined the alphaB-crystallin promoter for conserved transcription factor DNA-binding elements and identified a putative response element for the p53 tumor suppressor protein. Ectopic expression of wild-type p53 induced alphaB-crystallin mRNA and protein with delayed kinetics compared to p21. Additionally, the induction of alphaB-crystallin by genotoxic stress was inhibited by siRNAs targeting p53. Although the p53-dependent transactivation of an alphaB-crystallin promoter luciferase reporter required the putative p53RE, chromatin immunoprecipitation failed to detect p53 binding to the alphaB-crystallin promoter. These results suggested an indirect mechanism of transactivation involving p53 family members p63 or p73. DeltaNp73 was dramatically induced by p53 in a TAp73-dependent manner, and silencing p73 suppressed the transcriptional activation of alphaB-crystallin by p53. Moreover, ectopic expression of DeltaNp73alpha (but not other p73 isoforms) increased alphaB-crystallin mRNA levels in the absence of p53. Collectively, our results link the molecular chaperone alphaB-crystallin to the cellular genotoxic stress response via a novel mechanism of transcriptional regulation by p53 and p73.

P53 is an important factor for the radiosensitization effect of 2-deoxy-D-glucose

Metabolic change in cancer cells by preferential production of energy through glycolysis is a well-documented characteristic of cancer. However, whether inhibition of glycolysis will enhance the efficacy of radiation therapy is a matter of debate. In this study which uses lung cancer as the model, we demonstrate that the improvement of radiotherapy by 2-deoxy-D-glucose (2DG) is p53-dependent. Based on clonogenic survival data, we show that p53-deficient lung cancer cells (H358) are more sensitive to 2DG treatment when compared to p53 wild-type lung cancer cells (A549). The effective doses of 2DG at 0.5-surviving fraction of A549 and H358 are 17.25 and 4.61 mM, respectively. Importantly, 2DG exhibits a significant radiosensitization effect in A549 cells but not in H358 cells. Treatment with 2DG increases radiation-induced p53 protein levels in A549 cells. siRNA inhibition of p53 in A549 cells reduces the radiosensitization effect of 2DG. Furthermore, ectopic expression of wild-type p53 in H358 cells significantly enhances the radiosensitization effect of 2DG as determined by colony formation assay. In nude mice injected with A549 cells, treatment of 2DG enhances the efficacy of radiation therapy. Together, these results suggest that inhibition of glycolysis may only be beneficial for radiation therapy of cancer expressing wild-type p53.

Role of human HGFIN/nmbin breast cancer

IntroductionHGFIN, previously identified as nmb, and its homolog osteoactivin are single transmembrane proteins that are expressed in differentiated immune cells. These proteins exhibit properties that could potentiate tumorigenesis or decrease invasiveness. These seemingly opposing roles of HGFIN suggest that this protein might be central to malignancies and might also behave as a tumor suppressor. Consistent with the reported roles for HGFIN is the fact that this gene is regulated by p53 through multiple binding sites in the 5' flanking region, and is expressed in osteoblasts.MethodsThis study used siRNA to knock-out HGFIN in non-tumorigenic breast cells and ectopically expressed HGFIN in breast cancer cells. In addition, in situ hybridization studies analyzed primary breast tissues from archived breast surgeries. Reporter gene assays studied the untranslated exon 1 of HGFIN.ResultsHGFIN expression led to reduced cell growth of breast cancer cells and reduced migration. At the molecular level, reporter gene analyses determined the untranslated exon 1 to be a negative regulator of the upstream enhancing effect. Ectopic expression of wild-type p53 in breast cancer cells that expressed endogenous mutant p53 resulted in increased HGFIN reporter gene activities.ConclusionAs the majority of cancer cells have mutations in p53, further studies on the relationship between p53 and HGFIN expression, and its role in tumor genesis and bone invasion, might uncover novel therapy targets for breast and other cancers. The results show a central role for p53 in HGFIN expression, which appears to determine the behavior of the cancer cells.

C-Abl-independent p73 stabilization during gemcitabine- or 4′-thio-β-d-arabinofuranosylcytosine–induced apoptosis in wild-type and p53-null colorectal cancer cells

Nucleoside anticancer drugs like gemcitabine (2'-deoxy-2',2'-difluorocytidine) are potent inducers of p53, and ectopic expression of wild-type p53 sensitizes cells to these agents. However, it is also known that nucleosides are efficient activators of apoptosis in tumor cells that do not express a functional p53. To clarify this issue, we examined the effects of gemcitabine and 4'-thio-beta-d-arabinofuranosylcytosine (T-ara-C) on p73, a structural and functional homologue of p53, whose activation could also account for nucleoside-induced apoptosis because no functionally significant mutations of p73 have been reported in cancers. Acute treatment of HCT 116 colon carcinoma cells with gemcitabine or T-ara-C induced marked cytotoxicity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. T-ara-C and gemcitabine markedly induced p53 accumulation as well as increased levels of phospho-p53 (Ser15/Ser20/Ser46) and induced its binding to a consensus p53 response element. Despite robust activation of p53 by T-ara-C and gemcitabine, we found that wild-type and p53-/- HCT 116 cells exhibited almost equivalent sensitivity towards these nucleosides. Examination of p73 revealed that T-ara-C and gemcitabine markedly increased p73 protein levels and p73 DNA-binding activities in both p53-/- and wild-type cells. Furthermore, T-ara-C- and gemcitabine-induced increases in p73 levels occur due to a decrease in p73 protein turnover. RNA interference studies show that nucleoside-induced p73 increases are independent of c-Abl, a nucleoside-activated kinase recently implicated in p73 stabilization. HCT 116 lines, wherein the downstream p53/p73 targets Bax and PUMA (p53 up-regulated modulator of apoptosis) were deleted, were less sensitive to T-ara-C and gemcitabine. Together, these studies indicate that c-Abl-independent p73 stabilization pathways could account for the p53-independent mechanisms in nucleoside-induced apoptosis.

Induction of p53-regulated genes in lung cancer cells: implications of the mechanism for adenoviral p53-mediated apoptosis.

The p53 gene is the most frequently mutated gene in human cancers. Loss of functional p53 leads to impaired responses of cancer cells to apoptosis induction and to poor prognosis in patients with certain types of cancer. Cancer gene therapies using ectopic expression of wild-type p53 to force cancer cells through the apoptotic pathway have been tested extensively preclinically and clinically, and genes in various cell lines have been reported to be regulated upon ectopic p53 overexpression. However, the effect of p53 on many other p53-dependent and apoptosis-related genes remains unclear, as does the mechanism of p53-induced apoptosis in human cancers. In this study, we used real-time reverse transcription polymerase chain reaction to evaluate the changes in expression of various p53-dependent and apoptosis-related genes in five human non-small-cell lung cancer cell lines with varying p53 statuses after adenoviral p53 treatment. We found that Ad/p53 induced the expression of the proapoptotic genes PUMA, Bak, Bax, and Fas in a cell type- and time-dependent manner. Among these genes, PUMA was upregulated the most dramatically and broadly. However, when a specific siRNA construct against PUMA was employed, we observed no attenuation of apoptosis in H1299 cells. Our data suggest that Ad-p53 induces the expression of a variety of proapoptotic genes and that lack of induction in one of these genes does not block Ad/p53-mediated cell killing in human lung cancer cells.

P38 Kinase Regulates Nitric Oxide-induced Apoptosis of Articular Chondrocytes by Accumulating p53 via NFκB-dependent Transcription and Stabilization by Serine 15 Phosphorylation

Nitric oxide (NO) during primary culture of articular chondrocytes causes apoptosis via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation, and differentiation status. In this study, we characterized the molecular mechanism by which p38 kinase induces apoptosis through activation of p53. We report here that NO-induced activation of p38 kinase leads to activation of NFkappaB, which in turn induces transcription of the p53 gene. Activated p38 kinase also physically associates and phosphorylates the serine 15 residue of p53, which results in accumulation of p53 protein during NO-induced apoptosis. Ectopic expression of wild-type p53 enhanced NO-induced apoptosis, whereas expression of a dominant negative p53 blocked it, indicating that p53 plays an essential role in NO-induced apoptosis of chondrocytes. The increased accumulation of p53 caused expression of Bax, a pro-apoptotic member of the Bcl-2 family that is known to cause apoptosis via release of cytochrome c and caspase activation. These results suggest that NO-activated p38 kinase activates p53 function in two different ways, transcriptional activation by NFkappaB and direct phosphorylation of p53 protein, leading to apoptosis of articular chondrocytes.

Boswellic acids and malignant glioma: induction of apoptosis but no modulation of drug sensitivity.

Steroids are essential for the control of oedema in human malignant glioma patients but may interfere with the efficacy of chemotherapy. Boswellic acids are phytotherapeutic anti-inflammatory agents that may be alternative drugs to corticosteroids in the treatment of cerebral oedema. Here, we report that boswellic acids are cytotoxic to malignant glioma cells at low micromolar concentrations. In-situ DNA end labelling and electron microscopy reveal that boswellic acids induce apoptosis. Boswellic acid-induced apoptosis requires protein, but not RNA synthesis, and is neither associated with free radical formation nor blocked by free radical scavengers. The levels of BAX and BCL-2 proteins remain unaltered during boswellic acid-induced apoptosis. p21 expression is induced by boswellic acids via a p53-independent pathway. Ectopic expression of wild-type p53 also induces p21, and facilitates boswellic acid-induced apoptosis. However, targeted disruption of the p21 genes in colon carcinoma cells enhances rather than decreases boswellic acid toxicity. Ectopic expression of neither BCL-2 nor the caspase inhibitor, CRM-A, is protective. In contrast to steroids, subtoxic concentrations of boswellic acids do not interfere with cancer drug toxicity of glioma cells in acute cytotoxicity or clonogenic cell death assays. Also, in contrast to steroids, boswellic acids synergize with the cytotoxic cytokine, CD95 ligand, in inducing glioma cell apoptosis. This effect is probably mediated by inhibition of RNA synthesis and is not associated with changes of CD95 expression at the cell surface. Further studies in laboratory animals and in human patients are required to determine whether boswellic acids may be a useful adjunct to the medical management of human malignant glioma. © 1999 Cancer Research Campaign

Roles of the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in receptor-mediated apoptosis of WEHI 231 B lymphoma cells.

Treatment of WEHI 231 immature B lymphoma cells with an antibody against their surface immunoglobulin M (anti-IgM) induces apoptosis and has been studied extensively as a model of self-induced B cell tolerance. Since the tumor suppressor protein p53 has been implicated in apoptosis in a large number of cell types and has been found to be mutated in a variety of B cell tumors, here we sought to determine whether p53 and the p53 target gene cyclin-dependent kinase inhibitor p21(WAF1/CIP1) were involved in anti-IgM-induced cell death. Anti-IgM treatment of WEHI 231 cells increased expression of p53 and p21 protein levels. Ectopic expression of wild-type p53 in WEHI 231 cells induced both p21 expression and apoptosis. Ectopic expression of p21 similarly induced apoptosis. Rescue of WEHI 231 cells from apoptosis by costimulation with CD40 ligand ablated the increase in p21 expression. Lastly, a significant decrease in anti-IgM-mediated apoptosis was seen upon downregulation of endogenous p53 activity by expression of a dominant-negative p53 protein or upon microinjection of an antisense p21 expression vector or antibody. Taken together, the above data demonstrate important roles for p53 and p21 proteins in receptor-mediated apoptosis of WEHI 231 B cells.

APO2 ligand: a novel lethal weapon against malignant glioma?

APO2L (TRAIL) is a novel CD95L (Fas/APO-1-L) homologous cytotoxic cytokine that interacts with various receptors which transmit (DR4, DR5) or inhibit (DcR1, DcR2) an apoptotic signal. Here, we report that human glioma cell lines preferentially express mRNAs for agonistic death receptors DR4 (8/12) and DR5 (11/12) rather than the death-inhibitory decoy receptors DcR1 (4/12) and DcR2 (2/12). Ten of 12 cell lines are susceptible to APO2L-induced apoptosis. The resistant cell lines, U138MG and U373MG, are cross-resistant to CD95L-induced apoptosis. Similar to CD95L-induced apoptosis, APO2L-induced apoptosis is inhibited by ectopic expression of the caspase inhibitor, crm-A, or of bcl-2, or by coexposure to the corticosteroid, dexamethasone, or the lipoxygenase inhibitor, nordihydroguaretic acid. There is no correlation between p53 genetic status of the cell lines and their susceptibility to APO2L-induced apoptosis, but the latter is moderately enhanced by ectopic expression of wild-type p53. APO2L targeting may be a promising approach for selectively targeting apoptosis to human malignant glioma cells.

Tumorigenic conversion of p53-deficient colon epithelial cells by an activated Ki-ras gene.

Distinct genetic abnormalities (loss-of-function mutations of APC and p53 and oncogenic activation of Ki-ras) are associated with specific stages of the sporadic, most common types of colorectal tumors. However, the inability to maintain primary colon epithelial cells in culture has hindered the analysis of the pathogenetic role of these abnormalities in colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of p53-deficient mice; these cells are nontumorigenic as indicated by their failure to form colonies in soft agar and to grow as tumors in immunodeficient SCID mice and in immunocompetent syngeneic hosts. Upon ectopic expression of an activated Ki-ras gene, p53-deficient colon epithelial cells form colonies in soft agar and highly invasive subcutaneous tumors in both immunodeficient and immunocompetent mice. Ectopic expression of wild-type p53, but not of a DNA-binding-deficient mutant, markedly suppressed the colony-forming ability of the Ki-ras-transformed p53-deficient epithelial cells. Together, these findings establish a functional synergism in colorectal tumorigenesis dependent on the effects of an oncogenic Ki-ras in a p53-deficient background. This model of tumorigenic conversion of colon epithelial cells might be useful to identify genetic changes associated with disease progression and to evaluate the therapeutic response to conventional and novel anticancer drugs.

Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1.

Deregulated expression of the c-myc proto-oncogene can lead to apoptosis under certain physiological conditions. By introducing a conditionally active Myc allele into primary embryo fibroblasts null for p53, and into fibroblasts without endogenous p53 expression but ectopically expressing a temperature-sensitive p53 allele, we show that expression of wild-type p53 is required for susceptibility to Myc-mediated apoptosis. Although ectopic expression of wild-type p53 blocked cells in the G1 phase of the cell cycle, G1 arrest by isoleucine starvation, in a manner independent of p53, did not confer susceptibility to apoptosis. Thus, growth arrest per se is not sufficient to induce Myc-mediated apoptosis; instead, a property intrinsic to p53 is specifically required. Moreover, apoptosis did not require induction of p53 target proteins, including the cyclin-dependent kinase inhibitor p21waf1/cip1. Therefore, the role of p53 in apoptosis may be distinct from its role in cell cycle arrest.