Wild-type P53 mRNA Research Articles

Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer.

The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

62 (PB052) - p53 mRNA rescue of tumor suppressor function prevents tumor growth and restores PARPi sensitivity in p53-deficient cancers in vitro and in vivo

Background: Loss of function in tumor suppressor genes is commonly associated with the onset/progression of cancer and the development of therapeutic resistance. The p53 tumor suppressor mutations resulting in loss of function are common across variety of tumors and control tumor cell proliferation in more than 50% of cancers. P53 null mutations are present in about 10% of p53 driven cancers. However, to date there is no effective treatment for rescuing p53 function in cancers. We have developed a new potent strategy combing p53 mRNA with a tumor selective nanocarrier, to rescue p53 tumor suppressor function as potential therapeutic approach in cancers. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-531 nanoparticles containing full length p53-mRNA were evaluated on 20 different cancer cell lines (osteosarcoma, pancreas, colorectal, ovarian, lung, breast and prostate cancers) harboring different types of p53 mutations (null, deletion, nonsense, missense). Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry, Elisa and Tunel assay. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (0.5 mg/kg) was evaluated on colorectal SW403 (p53 deleted) and osteosarcoma SaOs2 (p53 null) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment in vitro was evaluated on PARPI resistant SUM-149PT and OVCAR-8 cells and on PARPi sensitive MDA-MB436 cells. Results: ADGN-531 NPs markedly delay the growth of a large panel of cancer cells harboring p53-null or nonsense mutations, by inducing cell cycle arrest in G1 due to p21 upregulation and apoptosis following PUMA activation. We demonstrated that ADGN-531 mediated p53 function rescue is directly correlated to the type and level of p53 mutation in the cancer cells. ADGN-531 induces 60% to 80% cell proliferation inhibition in p53 null or nonsense mutated cells and 20 to 50% in p53 missense mutated cells. Intravenous-administration ADGN-531 containing wild type p53-mRNA (0.5 mg/kg) resulted in tumor growth inhibition of 90% and 70% in SaOs2 and SW403 xenografted mouse model, respectively. ADGN-531 NPs treatments are well tolerated, without inducing clinical toxicity or inflammatory response. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to the PARPi veliparib. Conclusions: ADGN-531 nanoparticles are effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by the ADGN-531 NP targeted delivery strategy could be combined together with other therapies for potent combinatorial cancer treatment. No conflict of interest.

Correlation between osteosarcoma and the expression of WWOX and p53.

The objective of this study was to analyze the effect of the expression of WWOX and p53 on the growth of MG-63 osteosarcoma cells and to explore the correlation between osteosarcoma and the expression of WWOX and p53. WWOX and p53-overexpressing MG-63 osteosarcoma cell lines were established by transfection and named the MW and MP cell lines, respectively. Untransfected MG-63 cells (blank control) were used as control. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of WWOX and wild-type p53 mRNA and protein, respectively. The effects of WWOX and p53 (wild-type) on the activity of MG-63 cells were determined by MTT assay and flow cytometry. The expression of mutant p53 protein in 65 cases of osteosarcoma was detected by immunohistochemistry to analyze the correlation between p53 and the development of osteosarcoma. qPCR showed that WWOX and p53 mRNA was overexpressed in MW and MP cells, respectively. Western blot analysis showed that the levels of WWOX and p53 protein in MW and MP cells were higher than in the blank control group. MTT assay showed that the cell proliferation ability of MW and MP cells was significantly lower than in the blank control group. Flow cytometry showed that 78.49% of MW and 66.76% of MP cells were arrested in the G0/G1 phase. Immunohistochemistry showed that mutant p53 was highly expressed in osteosarcoma, with a positive expression rate of 47.7%. The expression rate was positively correlated with the pathological grade of cancer. In conclusion, WWOX can affect the cell cycle of MG-63 osteosarcoma cells to inhibit cell proliferation, which provides new insights into gene therapy for osteosarcoma. The two types of the p53 gene have different functions in the development of osteosarcoma. Wild-type p53 acts as a tumor suppressor, while mutant p53, which is overexpressed in malignant osteosarcoma, has a carcinogenic effect associated with the degree of osteosarcoma.

Abstract A17: p53 missense mutants R172H and R270H exhibit differential effects on tumorigenesis

Abstract p53 is a well-characterized transcription factor that is mutated in about 50% of human cancers. The majority of p53 genomic alterations are missense mutations which result in expression of p53 protein isoforms with deficient functionality. Such missense mutations cluster in several hotspots, with the most frequent alterations located at amino acids 175, 248 and 273 of human p53 (mouse amino acids 172, 245 and 270). p53 missense mutants disrupt the function of p53 tetramers, thereby behaving similarly to loss of p53. However, there is evidence that p53 missense mutants have additional tumor-promoting functions that differ from p53 deletions: p53 missense mutants can transform p53 null cells in vitro and in mouse models, and the p53 missense mutants induce a different tumor spectrum from that induced by p53 loss or other p53 missense mutants. The information obtained from the engineered murine models featuring p53 missense mutations has greatly elucidated the role of p53 mutagenesis in cancer. However, the currently-available p53 models bear several drawbacks that make them sub-optimal for preclinical studies. These models contain a global p53 haploinsufficiency, potentially impacting the biology of tumor-modifying structures like stroma, the immune system, and the vasculature. Furthermore, activation of the p53 missense mutant alleles results in a transition from a single functional wild-type allele to a combination of a wild-type and a mutant allele. These features substantially deviate from those of observed in human tumorigenesis, where somatic p53 mutagenesis in cells undergoing transformation results in a transition from two wild-type alleles to a combination of a wild-type and a mutant alleles, while tumor-modifying structures retain an unaltered p53 biallelic configuration. Here we describe the molecular and phenotypic characterization of a new allelic series of conditional p53 missense mutant mouse lines in which Cre-mediated recombination converts p53 from a wild-type to a missense mutant (R172H, R270H or R270C) configuration. To characterize the gradual molecular changes induced by expression of R172H and R270H mutants, we derived MEF lines harboring the alleles in a heterozygous conditional (p53-R172H fl/+ or p53-R270H fl/+) configuration. We observed that the wild-type p53 mRNA was lost but p53 protein abundance was increased after Cre-mediated activation of the mutant allele. Interestingly, we found that the R270H mutant allele provided a greater proliferative advantage, a distinct growth pattern and a greater ability to grow in vitro under starvation conditions than a similarly engineered R172H mutant allele. Consistent with published evidence, these observations suggest that the acquired functional changes are dissimilar between different p53 missense mutants. We further characterized the transcriptome profiles in MEF lines harboring R172H and R270H alleles and identified a number of unique and common transcriptional changes that could be causal for gain-of-function phenotypes and informative for uncovering additional mechanisms for carcinogenesis driven by p53 missense mutants. We used the newly-derived p53 mutant alleles to establish improved autochthonous and orthotopic pancreatic cancer models suitable for preclinical efficacy studies and we are currently developing lung cancer, ovarian cancer, and medullary thyroid carcinoma models. Citation Format: Tomas Vilimas, Keith Collins, Theresa Guerin, Roackie Awasthi, Lionel Feigenbaum, Guillermina Lozano, Terry Van Dyke, Serguei Kozlov. p53 missense mutants R172H and R270H exhibit differential effects on tumorigenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A17.

MicroRNA Hsa-miR-125a-3p Activates p53 and Induces Apoptosis in Lung Cancer Cells

The mature microRNA hsa-miR-125a-3p is derived from the 3′ end of pre-miR-125a. Here, we reported that hsa-miR-125a-3p suppressed proliferation and induced apoptosis in A549 cells. In addition, wild-type p53 mRNA and protein expression was increased by hsa-miR-125a-3p over-expression. Moreover, blocking wild-type p53 attenuated the effect of hsa-miR-125a-3p on apoptosis but could not restore completely. In p53-deficient cell line H1299, hsa-miR-125a-3p still induced apoptosis. Taken together, these data suggest that hsa-miR-125a-3p induces apoptosis not only via the p53 pathway in human lung cancer cells. These results provide new insight into the roles of the miR-125a family in lung cancer.

Abstract 3199: Ceramide restores the expression of wild-type p53 tumor suppressor in mRNA splicing through PP1 and ASF/SF2.

Abstract p53 mutants that have been detected in approximately 50% of tumors loss the activity in stabilizing the genome and tumor suppression, and further exhibit oncogenic function in cancer cells. Most tumors that exhibit disrupted p53 signaling pathways remain addicted to p53 mutants, and p53 mutants have emerged as perhaps the most important target to improve cancer treatment. Most works in restoring p53 activity focus on either altering protein conformations of the mutants or introducing foreign p53 gene into cells. Using gene silencing strategy and mass spectrometry, here we demonstrated that ceramide modulates the RNA splicing to restore the expression of wild-type p53 in p53 deletion-mutant ovarian cancer cells. Enhanced endogenous long-carbon chain ceramide species (C16- to C24-ceramides) and exogenous C6-ceramide, but not other sphingolipids, restored wild-type p53 mRNA (intact exon-5), phosphorylated p53 protein (Ser15 in exon-5) and p53-responsive proteins (p21, Bax) in cells that dominantly expressed a deleted p53-mRNA and protein in exon-5. Thus, the restored p53 sensitized the cells to induced apoptosis. Furthermore, ceramide activated protein phosphatase 1 (PP1), rather than PP2a, increased the translocation of alternative splicing factor ASF/SF2 from cytoplasm to nucleus and thus determined the pre-mRNA splicing for wild-type p53. Alternative pre-mRNA splicing not only generates more than one protein isoform from single gene, it also results in gene mutants in cancer and other diseases. Our study elucidates a previous unrecognized mechanism in regulating the expression of wild-type versus mutant protein of p53 and indicates mutants can be restored in posttranscriptional levels by using an epigenetic approach. Citation Format: Yong-Yu Liu, Gauri A. Patwardhan. Ceramide restores the expression of wild-type p53 tumor suppressor in mRNA splicing through PP1 and ASF/SF2. [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 3199. doi:10.1158/1538-7445.AM2013-3199

Prognostic Role of p53 Messenger Ribonucleic Acid Expression in Patients after Curative Resection for Stage I to III Colorectal Cancer: Association with Colon Cancer Stem Cell Markers

The current study was performed to examine the prognostic role of 53 messenger ribonucleic acid (mRNA) expression in patients with colorectal cancer and analyze its relationship with the expression of CD44 and CD133 mRNA levels. We retrospectively reviewed 137 consecutive patients who underwent curative surgery for stage I to III colorectal cancer in 2006. Prognostic factors, including wild-type (wt) p53, cyclooxygenase-2, CD44, and CD133 mRNA levels, were determined using reverse transcriptase polymerase chain reaction and clinical outcomes were analyzed. Wild-type p53 mRNA expression was correlated with the expression of CD44 and CD133 mRNA (p= 0.005 and p= 0.013, respectively). With a median follow-up period of 64months, the 5-year disease-free survival rate of patients with elevated wt-p53 mRNA expression was significantly higher than that of those patients with low levels of wt-p53 mRNA expression (84.9% and 67.6%, respectively; p= 0.014). A multivariate analysis identified 3 independent factors that substantially affected the disease-free survival: depth of tumor invasion, lymph node metastasis, and wt-p53 mRNA expression. The 5-year disease-free survival rate in patients with stage III or rectal tumors differed significantly between the low and high wt-p53 expression groups. In stage III cancers, high wt-p53 expression was associated with better survival than low wt-p53 expression in patients treated with adjuvant chemotherapy (p= 0.005). A significant association between combined p53/CD44 expression and survival was evident (p= 0.006). Expression of p53 mRNA is a useful predictor of survival in patients with stage III or rectal cancers, with a significant association with CD44 mRNA expression.

Novel luminescent silica nanoparticles (LSN): p53 gene delivery system in breast cancer in vitro and in vivo.

Mutations in the p53 tumor suppressor gene are one among the most common genetic abnormalities to be described in breast cancer. However, there are a few recant reports on non-viral vector-mediated p53 gene delivery in breast cancer. A new formulation of luminescent silica nanoparticles (LSNs) for gene delivery was produced by the two-step method with slight modification. The pp53 plasmid constructs (p53-EGFP)/LSNs complexes were transfected into human breast cancer cell (MCF-7) and transfection efficiency was determined by FACS analysis. The gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis respectively. Further the growth inhibition through induced apoptosis with pp53-EGFP/LSNs complex were assessed by trypan blue exclusion assay and annexin V staining, respectively. Interestingly the in vivo biodistribution of plasmid DNA study revealed the occurrence was investigated by PCR and RT-PCR. The transfection efficiency of LSNs showed the highest transfection efficiency among the LSN formulation was higher than that of commercially available Lipofectin®. The LSNs-mediated transfection of the p53 gene resulted in efficient high level of wild-type p53 mRNA and protein expression levels in MCF-7 cells. Selected tissues were analyzed for any potential toxicity by histological analysis the efficient reestablishment of wild-type p53 function in breast cancer cells restored the p53 dependent apoptotic pathway. Taken together, our results reveal that cationic LSN-mediated p53 gene delivery may have potential application as a non-viral vector-mediated breast cancer gene therapy due to its effective induction of apoptosis and tumor growth inhibition.

103P P53 Gene Therapy of Human Breast Carcinoma Using a Cationic Non - Viral Vector, Luminescent Silica Nanoparticles

ABSTRACT Mutations in the p53 tumor suppressor gene are the most common genetic abnormalities to be described in breast cancer. A new formulation of luminescent silica nanoparticles (LSNs) for gene delivery was produced by the two-step method with slight modification. Plasmid DNA (pp53-EGFP)/LSNs complexes were transfected into human breast cancer cells (MCF-7) and transfection efficiency was determined by FACS analysis. The gene expression was determined by RT-PCR and Western blot analysis. The cellular growth inhibition and apoptosis of treated cells were assessed by trypan blue exclusion assay and annexin V staining, respectively. In vivo biodistribution of plasmid DNA was investigated by PCR and RT-PCR. The transfection efficiency of LSNs showed the highest transfection efficiency among the LSN formulation was higher than that of commercially available Lipofectin®. The LSNs-mediated transfection of the p53 gene resulted in efficient high level of wild-type p53 mRNA and protein expression levels in MCF-7 cells. The efficient reestablishment of wild-type p53 function in breast cancer cells restored the apoptotic pathway. Taken together, our results reveal that cationic LSN-mediated p53 gene delivery may have potential for clinical application as a nonviral vector-mediated breast cancer therapy due to its effective induction of apoptosis and tumor growth inhibition. Disclosure All authors have declared no conflicts of interest.

MicroRNA HSA-miR-125a-5p induces apoptosis by activating p53 in lung cancer cells

ABSTRACTThe mature microRNA hsa-miR-125a-5p is derived from the 5′ end of pre-miR-125a. Although hsa-miR-125a-5p is dysregulated in some tumors, its specific roles in lung cancer cell apoptosis is still unknown. To study its function, the authors examined the effects of hsa-miR-125a-5p on apoptosis in lung cancer cells and investigated its probable regulatory mechanism. The authors showed that hsa-miR-125a-5p expression was lower in different lung cancer cell lines than in Human bronchial epithelial (HBE) cells by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). In gain-of-function experiments, the authors found that hsa-miR-125a-5p suppressed proliferation and induced apoptosis in A549 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and flow cytometry, respectively. In addition, wild-type p53 mRNA and protein expression was increased by hsa-miR-125a-5p overexpression. Moreover, blocking wild-type p53 attenuated the effect of hsa-miR-125a-5p on apoptosis. In loss-of-function experiments, wild-type p53 mRNA and protein expression was decreased by blocking hsa-miR-125a-5p. The effect of hsa-miR-125a-5p inhibitor on apoptosis was also weakened by blocking wild-type p53. Taken together, these data suggest that hsa-miR-125a-5p induces apoptosis via a p53-dependent pathway in human lung cancer cells. These results provide new insight into the roles of the miR-125a family in lung cancer.

Abstract 2569: Disruption of ceramide glycosylation restores p53-dependent apoptosis in p53 mutant drug-resistant cancer cells: Alternative splicing determines the feature of p53 activity

Abstract Mutation of tumor suppressor p53 is frequently detected in many different types of cancers. p53 mutants acts as oncogenic proteins preventing programmed cell death and promoting cancer progression. Restoration of p53 that is to increase the expression of wild-type p53 and to eliminate mutant expression can substantially increase the efficacy of chemotherapy and stop malignant progression. However, incomplete understanding the molecular mechanism underlying p53 gene regulation limits the success in p53 restoration. We, for the first time, report that disruption of ceramide glycosylation restores p53 dependent-apoptosis in p53 mutant drug-resistant cancer cells. A lose of 21 base-pair in the mRNA of exon-5 can result in the accumulation of mutant p53 and cells insensitiveness to induced-apoptosis in human NCI/ADR-RE ovarian cancer cells. We found that treatment of mixed-backbone oligonucleotide against human glucosylceramide synthase (MBO-asGCS) increased the levels of wild-type p53 mRNA (containing the 21 bp in exon-5) and protein of wild-type p53 in dose-dependent manner (50-200 nM). In response to doxorubicin-induced DNA damage, phosphorylated p53 (Ser15 in the exon-5) was significantly increased, that in turn increased the protein levels of p21 and Bax, and apoptosis (52% vs. 3%) after MBO-asGCS treatment compared to control in NCI/ADR-RE cells. Silencing of GCS using MBO-asGCS (1 mg/kg) restored wild-type p53 expression and substantially sensitizes NCI/ADR-RE tumors to doxorubicin-induced apoptosis in vivo (10 mice per group), as compared to MBO-scramble control or other treatments. In contrast, introduction of GCS gene in NCI-ADR-RE cells reduces wild-type p53 expression and p53-depedent apoptosis. Further mechanic studies indicated that increased ceramide after GCS silencing modulates the function of SRp30a (ASF/SF2) regulating alternative pre-mRNA splicing. Increasing endogenous ceramide by inhibition of its glycosylation using PDMP or enhanced its production using sphingomylinase promoted wild-type p53 expression at the levels of mRNA, protein and phosphorylation; decreasing ceramide by inhibition of ceramide synthesis using fumonisin (FB1) repressed wild-type p53 expression. Present study indicates that cancer cells marked with mutant p53 phenotype are capable of expressing functional p53; ceramide glycosylation is one molecular mechanism that shifts expression to wild-type form of p53 at alternative splicing of post-transcription processing. MBO-asGCS, a specific agent silencing GCS restores p53-dependent apoptosis in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2569.

Selective Inhibition of p53 Dominant Negative Mutation by shRNA Resulting in Partial Restoration of p53 Activity

The tumor suppressor gene p53 is the most frequently mutated gene found in human cancer. The majority of p53 mutations are missense mutations occurring within the DNA binding domain. Some of these mutations exhibit dominant negative effects (e.g., p53 R273H) that disrupt the normal function of wild-type p53. It is of therapeutic interest to determine if the normal function of p53 can be restored when the dominant negative effect is selectively inhibited. In this study, we tested this possibility using small hairpin RNA (shRNA) to specifically reduce the level of p53 R273H. The antisense strand of shRNA is fully complementary to mRNA of p53 R273H, but leaves a mismatch base in the middle of the duplex to wild-type p53 mRNA. Both wild-type p53 and mutant p53 R273H were transiently expressed in p53-null H1299 lung cancer cells. The shRNA we designed selectively reduced the mRNA level of p53 R273H, but had no RNA interference or antisense effects on wild-type mRNA. As a result, the transactivation activity of p53 was partially restored. In this report, we provide a new strategy for studying functional alterations for point mutation or single nucleotide polymorphism, and treating some dominant mutant-derived diseases.

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.

Novel cationic solid lipid nanoparticles enhanced p53 gene transfer to lung cancer cells

Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in lung cancer. However, there have been few reports of nonviral vector-mediated p53 gene delivery in lung cancer. A new formulation of cationic solid lipid nanoparticles (SLNs) for gene delivery was produced by the melt homogenization method with slight modification, and the SLNs were formulated by mixing tricaprin (TC) as a core, 3β[ N-( N′, N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), dioleoylphosphatidylethanolamine (DOPE) and Tween 80 in various ratios. Plasmid DNA (pp53-EGFP)/SLNs complexes were transfected into human non-small cell lung cancer cells (H1299 cells) and transfection efficiency was determined by FACS analysis. The gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The cellular growth inhibition and apoptosis of treated cells with pp53-EGFP/SLNs complexes were assessed by trypan blue exclusion assay and annexin V staining, respectively. In vivo biodistribution of plasmid DNA was investigated by PCR and RT-PCR. The transfection efficiency of SLN1 (TC:DC-Chol:DOPE:Tween 80 = 0.3:0.3:0.3:1), which showed the highest transfection efficiency among the SLN formulations, was higher than that of commercially available Lipofectin ®. The SLNs-mediated transfection of the p53 gene resulted in efficient high levels of wild-type p53 mRNA and protein expression levels in H1299 cells. The efficient reestablishment of wild-type p53 function in lung cancer cells restored the apoptotic pathway. Taken together, our results reveal that cationic SLN-mediated p53 gene delivery may have potential for clinical application as a nonviral vector-mediated lung cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.

Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro

DNA damage is a critical event preceding cellular apoptosis or necrosis. This study was carried out to investigate the effect of aristolochic acid I (AAI) on DNA damage and cell cycle in porcine proximal tubular epithelial cell lines (LLC-PK1 cells). LLC-PK1 cells were stimulated with AAI at the concentrations of 80, 320, and 1,280 ng/ml for 24 h. DNA damage was examined by comet assay and the cell cycle was assayed by flow cytometry (FCM), cellular apoptosis and lysis were examined simultaneously. Cellular nuclear changes were observed by electron microscopy and the expression of wild-type p53 protein and mRNA were measured by FCM and RT-PCR. We found that AAI-induced DNA damage prior to apoptosis and lysis in LLC-PK1 cells in a dose-dependent manner (P<0.01). The percentage of cells in the G2/M phase that were treated with AAI (320 and 1,280 ng/ml) for 24 h increased significantly (P<0.01). Electron micrographs showed the nuclear abnormalities in AAI-treated cells. The expression of p53 protein and mRNA did not change in the AAI-treated cells. AAI may cause DNA damage and cell cycle arrest in LLC-PK1 cells through a wild-type p53-independent pathway, prior to apoptosis or necrosis. This study on the molecular mechanism of AAI-induced toxicity may explain why tubular epithelial cells present limited proliferation and regeneration abilities in the clinical presentation of AAI-associated nephrotoxicity.

Enhanced p53 gene transfer to human ovarian cancer cells using the cationic nonviral vector, DDC

Objective Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo. Method DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription–polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed. Results The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group. Conclusion The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.

Restoration of p53 gene function in 12-O-tetradecanoylphorbor 13-acetate-resistant human leukemia K562/TPA cells

The human leukemia K562 cell line does not express wild-type p53 protein. Due to the loss of one p53 allele and an insertion mutation in exon 5 of the other allele resulting in a frameshift mutation, K562 cells express a truncated p53 protein of 148 amino acids. A human leukemia phorbol ester-resistant subline, K562/TPA, is cross-resistant to some anticancer agents. A remarkable difference in cell cycle progression at G1/S phase was observed in the synchronised K562/TPA cells as compared with K562 cells. Southern blot and DNA sequence analysis revealed no mutation in exon 5 of the p53 gene in K562/TPA cells. p21Cip1 expression was also restored in K562/TPA cells confirming that the reversal of this p53 gene mutation restored wild-type p53 function in these cells. This is a unique report describing reversal of p53 gene mutation by drugs. This was associated with the expression of wild-type p53 mRNA and protein in K562/TPA cells. The K562/TPA cell line may provide a very useful tool for the investigation of the relationship between p53 status and chemosensitization.

Induction of TNF-sensitive cellular phenotype by c-Myc involves p53 and impaired NF-kappaB activation.

Normal fibroblasts are resistant to the cytotoxic action of tumor necrosis factor (TNF), but are rendered TNF-sensitive upon deregulation of c-Myc. To assess if oncoproteins induce the cytotoxic TNF activity by modulating TNF signaling, we investigated the TNF-elicited signaling responses in fibroblasts containing a conditionally active c-Myc protein. In association with cell death, c-Myc impaired TNF-induced activation of phospholipase A2, JNK protein kinase and cell survival-signaling-associated NF-kappaB transcription factor complex. The TNF-induced death of mouse primary fibroblasts expressing deregulated c-Myc was inhibited by transient overexpression of the p65 subunit of NF-kappaB, which increased NF-kappaB activity in the cells. Unlike other TNF-induced signals, TNF-induced accumulation of the wild-type p53 mRNA and protein was not inhibited by c-Myc. TNF, with c-Myc, induced apoptosis in mouse primary fibroblasts but only weakly in p53-deficient primary fibroblasts. The C-terminal domain of p53, which is a transacting dominant inhibitor of wild-type p53, failed to inhibit apoptosis by c-Myc and TNF, suggesting that the cell death was not dependent on the transcription-activating function of p53. Taken together, the present findings show that the cytotoxic activity of TNF towards oncoprotein-expressing cells involves p53 and an impaired signaling for survival in such cells.

Mutations of the p53 Gene in Myelodysplastic Syndrome (MDS) and MDS-Derived Leukemia

The p53 gene is currently thought to be a tumor suppressor gene, and its alterations have been suggested to be involved in the pathogenesis of several human malignancies, including some leukemias and lymphomas. We present here evidence for the possible involvement of p53 gene mutations in the myelodysplastic syndrome (MDS), although the incidence is relatively low. Forty-four patients with MDS and six patients with overt leukemias that developed from MDS were studied for p53 gene alterations using reverse transcriptase-polymerase chain reaction, single-strand conformation polymorphism analysis, and nucleotide sequencing. Three patients with MDS (2 RAEB and 1 RAEB in T) had missense point mutations in the conserved regions of the p53 coding sequence. Furthermore, expression of the wild-type p53 mRNA was not detected in these three patients. The probable absence of normal p53 function in the three cases studied here suggests that alterations in the p53 gene may occasionally play a role in MDS. These three MDS patients with p53 gene mutations and an MDS-derived erythroleukemia cell line that we had previously reported to carry a p53 gene mutation showed no N-ras gene mutations, suggesting heterogeneity in the oncogenic mechanism of MDS.

Mutations of the p53 gene in myelodysplastic syndrome (MDS) and MDS- derived leukemia

The p53 gene is currently thought to be a tumor suppressor gene, and its alterations have been suggested to be involved in the pathogenesis of several human malignancies, including some leukemias and lymphomas. We present here evidence for the possible involvement of p53 gene mutations in the myelodysplastic syndrome (MDS), although the incidence is relatively low. Forty-four patients with MDS and six patients with overt leukemias that developed from MDS were studied for p53 gene alterations using reverse transcriptase-polymerase chain reaction, single-strand conformation polymorphism analysis, and nucleotide sequencing. Three patients with MDS (2 RAEB and 1 RAEB in T) had missense point mutations in the conserved regions of the p53 coding sequence. Furthermore, expression of the wild-type p53 mRNA was not detected in these three patients. The probable absence of normal p53 function in the three cases studied here suggests that alterations in the p53 gene may occasionally play a role in MDS. These three MDS patients with p53 gene mutations and an MDS-derived erythroleukemia cell line that we had previously reported to carry a p53 gene mutation showed no N-ras gene mutations, suggesting heterogeneity in the oncogenic mechanism of MDS.