nutlin-3a and Cell-Transformation--Neoplastic

Emerging evidence indicates that lncRNAs play important roles in cancer tumourigenesis and could be used as potential diagnostic biomarkers or therapeutic targets. However, the clinical significance and molecular mechanism of lncRNAs in gastric cancer (GC) is still unclear. The aim of this study was to explore the expression and role of lncRNAs in GC. The relative expression level of lncRNAs in GC samples was examined by an lncRNA microarray analysis, northern blot analysis and qRT-PCR analysis. A Kaplan-Meier survival analysis and univariate and multivariate Cox proportional hazards models were performed to evaluate the clinical and prognostic significance of PANDAR (promoter of CDKN1A antisense DNA damage activated RNA) in GC patients. The binding activity of PANDAR with the p53 protein was analysed by an RNA immunoprecipitation analysis and RNA pull-down analysis. The depletion of PANDAR was conducted using the CRISPR/Cas9 system for PANDAR. The biological functions of PANDAR in GC cells were determined both in vitro and in vivo. Upregulated PANDAR in GC patients was positively correlated with increased tumour size, advanced TNM classification and a poor survival rate in GC patients. The ROC curves identified that the PANDAR level was a marker for discriminating the early-stage tumour group from the healthy group, the metastasis group from the non-metastasis group and the chemoresistance group from the chemosensitive group in GC patients. As a target, the CDKN1A gene was successfully downregulated by PANDAR. PANDAR controlled the transcription of the CDKN1A gene by competitively binding with p53 protein. In combination with a p53 activator (nutlin3), the knockout of PANDAR by CRISPR/Cas9 technology synergistically inhibited GC tumour growth in vivo. Our results suggest that the PANDAR is a powerful diagnostic and therapeutic marker for patients with GC and, combined with other chemotherapeutics, may have distinct antitumour effects.

Topics: Apoptosis; Base Sequence; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Piperazines; Prognosis; Promoter Regions, Genetic; Protein Binding; RNA, Long Noncoding; Stomach Neoplasms; Transcription, Genetic; Tumor Suppressor Protein p53

Alcohol consumption is associated with increased breast cancer risk; however, the underlying mechanisms that contribute to mammary tumor initiation and progression are unclear. Alcohol is known to induce oxidative stress and DNA damage; likewise, p53 is a critical modulator of the DNA repair pathway and ensures genomic integrity. p53 mutations are frequently detected in breast and other tumors. The impact of alcohol on p53 is recognized, yet the role of p53 in alcohol-induced mammary carcinogenesis remains poorly defined. In our study, we measured alcohol-mediated oxidative DNA damage in MCF-7 cells using 8-OHdG and p-H2AX foci formation assays. p53 activity and target gene expression after alcohol exposure were determined using p53 luciferase reporter assay, qPCR, and Western blotting. A mechanistic study delineating the role of p53 in DNA damage response and cell cycle arrest was based on isogenic MCF-7 cells stably transfected with control (MCF-7/Con) or p53-targeting siRNA (MCF-7/sip53), and MCF-7 cells that were pretreated with Nutlin-3 (Mdm2 inhibitor) to stabilize p53. Alcohol treatment resulted in significant DNA damage in MCF-7 cells, as indicated by increased levels of 8-OHdG and p-H2AX foci number. A p53-dependent signaling cascade was stimulated by alcohol-induced DNA damage. Moderate to high concentrations of alcohol (0.1-0.8% v/v) induced p53 activation, as indicated by increased p53 phosphorylation, reporter gene activity, and p21/Bax gene expression, which led to G0/G1 cell cycle arrest. Importantly, compared to MCF-7/Con cells, alcohol-induced DNA damage was significantly enhanced, while alcohol-induced p21/Bax expression and cell cycle arrest were attenuated in MCF-7/sip53 cells. In contrast, inhibition of p53 degradation via Nutlin-3 reinforced G0/G1 cell cycle arrest in MCF-7 control cells. Our study suggests that functional p53 plays a critical role in cellular responses to alcohol-induced DNA damage, which protects the cells from DNA damage associated with breast cancer risk.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Alcohol Drinking; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Deoxyguanosine; DNA Damage; DNA Repair; Ethanol; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Histones; Humans; Imidazoles; MCF-7 Cells; Oxidative Stress; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Diethylnitrosamine; Enzyme Activation; Hepatocytes; Imidazoles; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred C57BL; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

TP53 mutations occur in half of all human tumours. Mutagen-induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock-in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen-treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2-5 months) and much effort is expended maintaining TP53-WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin-3a, an MDM2 inhibitor that leads to stabilisation and activation of wild-type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin-3a to examine the effect on cell growth and p53 activation. Nutlin-3a induced the p53 pathway in TP53-WT HUFs and inhibited cell growth, whereas most TP53-mutated HUFs were resistant to Nutlin-3a. We then assessed whether Nutlin-3a treatment could discriminate between TP53-WT and TP53-mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin-3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin-3a-resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin-3a-sensitive clones were TP53-WT. These data suggest that including a Nutlin-3a counter-screen significantly improves the specificity and efficiency of the HIMA, whereby TP53-mutated clones are selected prior to sequencing and TP53-WT clones can be discarded.

Topics: Animals; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Drug Resistance; Fibroblasts; Gene Knock-In Techniques; Genes, p53; Humans; Imidazoles; Mice; Mutation; Oxygen; Piperazines; Proto-Oncogene Proteins c-mdm2; Selection, Genetic; Signal Transduction

Nutlin-3a increases p53 levels after UVB radiation, which could result in a decrease in DNA damage and thus lead to a lower risk of non-melanoma skin cancer. Especially, organ transplant recipients might derive benefit from such a topical formulation with an active ingredient to prevent DNA damage.. To investigate whether topical nutlin-3a can decrease photocarcinogenesis induced by simulated solar radiation.. 72 hairless C3.Cg/TifBomTac mice were treated 3 days/week topically with 100 μl nutlin-3a (9 mM) [Groups 1 and 3 (120 days)) or 100 μl vehicle (Group 2). Three hours later, all mice were exposed to simulated solar radiation (a radiometric equivalent of three standard erythema dose units).. The median time to tumours did not differ between the mice treated with nutlin-3a and with the vehicle. The median time to the first and second tumours did not differ between 'nutlin-3a-120 days' and vehicle-treated mice, but there was a small significant difference in the median time to the third tumour (211 vs. 196 days, P = 0.043). However, after Bonferroni correction, there was no difference at all.. Nutlin-3a had no reductive effect on photocarcinogenesis and we do not believe in nutlin-3a as a potential drug against DNA damage in a topical formulation for organ transplant patients.

Topics: Administration, Topical; Animals; Cell Transformation, Neoplastic; DNA Damage; Female; Humans; Imidazoles; Mice; Mice, Hairless; Piperazines; Skin Neoplasms; Sunlight; Ultraviolet Rays

In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.. We made use of an in vitro transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential.. Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors in vivo. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines.. Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.

Topics: Animals; Cell Adhesion; Cell Cycle Proteins; Cell Proliferation; Cell Shape; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chorioallantoic Membrane; Fibroblasts; Gene Expression; Gene Expression Profiling; Humans; Imidazoles; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Neoplasm Transplantation; Nuclear Proteins; Oncogenes; Piperazines; Primary Cell Culture; Proto-Oncogene Proteins; Recombinant Proteins; Retina; Retinoblastoma; Tumor Suppressor Protein p53

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cellular Senescence; Cyclin D; Cyclin-Dependent Kinases; Cyclins; DNA Damage; Eye Neoplasms; Genes, p16; Genes, p53; Genes, Retinoblastoma; Humans; Imidazoles; Mice; Models, Biological; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Retinoblastoma; Retinoblastoma Protein; Topotecan; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Interaction of cyclin D1 with cyclin-dependent kinases (CDK) results in the hyperphosphorylation of the RB family of proteins, thereby inactivating the tumor-suppressive function of RB. Our previous findings suggest that constitutive cyclin D1/CDK activity inhibits p53-mediated gene repression by preventing the appropriate regulation of CDK activity by the CDK inhibitor p21, a transcriptional target of p53. To study the role of cyclin D1 in driving human mammary cell transformation, we expressed a constitutively active cyclin D1-CDK fusion protein (D1/CDK) in immortalized human mammary epithelial cells. D1/CDK-expressing human mammary epithelial cells grew anchorage-independently in the presence of wild-type p53, consistent with the idea that D1/CDK disrupts downstream p53 signaling. Using this transformation model, we examined the sensitivity of the D1/CDK-expressing cells to Nutlin-3, an HDM2 antagonist that activates p53. Surprisingly, treatment of D1/CDK-transformed cells with Nutlin-3 prevented their anchorage-independent growth. The Nutlin-3-induced growth arrest was enforced in D1/CDK-expressing cells despite the presence of hyperphosphorylated RB implicating a p53-dependent, RB-independent mechanism for growth suppression. Further analysis identified that CDC2 and cyclin B1, key cell cycle regulators, were stably down-regulated following p53 stabilization by Nutlin-3, consistent with direct interaction between p53 and the CDC2 and cyclin B1 promoters, leading to the repression of transcription by methylation. In contrast to D1/CDK expression, direct inactivation of p53 resulted in no repression of CDC2 and no cell cycle arrest. We conclude that induction of p53 by Nutlin-3 is a viable therapeutic strategy in cancers with constitutive CDK signaling due to the direct repression of specific p53 target genes.

Topics: Cell Growth Processes; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Epithelial Cells; HCT116 Cells; Humans; Imidazoles; Mammary Glands, Human; Piperazines; Recombinant Fusion Proteins; Retinoblastoma Protein; Tumor Suppressor Protein p53

Hdm2 and HdmX coordinately regulate the stability and function of p53. Each is overexpressed in subsets of many different types of malignancy, and most of these subsets maintain wild-type p53. Nutlins, newly discovered small-molecule inhibitors of the Hdm2-p53 interaction, offer a novel strategy for therapy of tumors with wild-type p53. We now show that Nutlin-3 efficiently induces apoptosis and diminishes long-term survival of human fibroblasts transformed in vitro by Hdm2 but not HdmX. The resistance of cells overexpressing HdmX to Nutlin-3 is due to its inability to disrupt the p53-HdmX interaction, resulting in continued suppression of p53 activity. Although HdmX overexpression yielded cells resistant to Nutlin-3, ablation of HdmX expression by short hairpin RNA sensitized tumor cells to Nutlin-3-mediated cell death or arrest. Furthermore, deletion of the COOH-terminal RING finger domain of HdmX completely reversed the resistance to Nutlin-3, probably reflecting the requirement of the RING finger for interaction with Hdm2. Thus, the relative abundance of Hdm2 and HdmX and the specificity of Nutlin-3 for Hdm2 influence the sensitivity of cells to p53-dependent apoptosis or arrest in response to Nutlin-3. Our findings establish Hdm2 and HdmX as independent therapeutic targets with respect to reactivating wild-type p53 as a means for cancer therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Proteins; Cell Transformation, Neoplastic; Colonic Neoplasms; Doxorubicin; Drug Screening Assays, Antitumor; Drug Synergism; Fibroblasts; HCT116 Cells; Humans; Imidazoles; Nuclear Proteins; Piperazines; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Tumor Suppressor Protein p53