Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.     

C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wt p53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (m p53) cells transfected with a vector carrying the m p53 gene (SAS/m p53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/m p53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/m p53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/m p53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/m p53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53 -mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in m p53 cancer cells. This novel tool for enhancement of apoptosis induction in m p53 cells might be useful for p53-targeted radio-cancer therapy.     

ErbB2 overexpression in p53-inactivated mammary epithelial cells

Functional loss of p53 and ErbB2 overexpression are the frequent genetic alterations in human breast carcinomas. Here, we found that ErbB2 expression was upregulated in primary cultured mammary epithelial cells (MECs) isolated from mice with a defect in exons 5 and 6 of the p53 gene (p53(Delta5,6)). The reporter gene activity in the p53(Delta5,6) MECs transfected with the -756bp flanking region of the hErbB2 gene was higher than the wild type MECs. p53 inactivation selectively increased the level of AP-2alpha, but not AP-2beta and AP-2gamma and a mutation of the two AP-2 binding sites completely inhibited the reporter activity.     

Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5 mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (?1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5 mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (?1% oxygen) and hypoxia mimetic CoCl₂. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5 mM glucose after 48 h of physical hypoxia. However, combining the latter with 100 μM CoCl₂ was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl₂ in 5 mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.     

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.     

Rearrangement and overexpression of the gene coding for tumor antigen p53 in a rat histiocytoma AK-5

The gene coding for the cellular tumor antigen p53 is rearranged and overexpressed in a rat histiocytoma, AK-5. The protein coded by the gene was detected by immunofluorescence and its full size was confirmed by immunoprecipitation using monoclonal antibodies against p53. Southern hybridizations with a full length cDNA probe specific for p53 indicated rearrangement of the gene. Alterations in the upstream region, which probably disrupt the normal regulatory control are suggested by the pattern obtained using a 5'-specific p53 probe in Southern hybridization.     

Immune responses to p53 in patients with cancer:enrichment in tetramer+ p53 peptide-specific T cells and regulatory T cells at tumor sites

Objective: A majority of human cancers, including head and neck cancer (HNC), overexpress p53. Although T cells specific for wild-type (wt) sequence p53 peptides are detectable in the peripheral blood of patients with HNC, it is unknown whether such T cells accumulate in tumor-involved tissues. Also, the localization of regulatory T cells (Treg) to tumor sites in HNC has not been investigated to date. Methods: Tumor infiltrating lymphocytes (TIL), tumor-involved or non-involved lymph node lymphocytes (LNL) and peripheral blood mononuclear cells (PBMC) were obtained from 24 HLA-A2.1+ patients with HNC. Using tetramers and four-color flow cytometry, the frequency of Treg and CD3+CD8+ T cells specific for wt p53 epitopes as well as their functional attributes were determined. Results: The CD3+CD8+ tetramer+ cell frequency was significantly higher (P<0.001) in TIL than autologous PBMC as was the percentage of CD4+CD25+ T cells (P<0.003). TIL were enriched in FOXp3+, GITR+ and CTLA-4+ Treg. CD8+ TIL had low expression and produced little IFN- after ex vivo stimulation relative to autologous PBMC or PBMC from NC. Conclusions: Anti-wt p53 epitope-specific T cells and Treg preferentially localize to tumor sites in patients with HNC. However, despite enrichment in tumor peptide-specific T cells, the effector cell population (CD3+CD8+) in TIL or PBMC was unresponsive to activation in the tumor microenvironment enriched in Treg.     

ADP-ribosylation of p53 tumor suppressor protein: Mutant but not wild-type p53 is modified

Poly(ADP-ribosyl)ation of mutant and wild-type p53 was studied in transformed and nontransformed rat cell lines constitutively expressing the temperature-sensitive p53^135val. It was found that in both cell types at 37.5°C, where overexpressed p53 exhibits mutant conformation and cytoplasmic localization, a considerable part of the protein was poly(ADP-ribosyl)ated. Using densitometric scanning, the molecular mass of the modified protein was estimated as 64 kD. Immunofluorescence studies with affinity purified anti-poly(ADP-ribose) transferase (pADPRT) antibodies revealed that, contrary to predictions, the active enzyme was located in the cytoplasm, while in nuclei chromatin was depleted of pADPRT. A distinct intracellular localization and action of pADPRT was found in the cell lines cultivated at 37.5°C, where p53 adopts wild-type form. Despite nuclear coexistence of both proteins no significant modification of p53 was found. Since the strikingly shared compartmentalization of p53 and pADPRT was indicative of possible complex formation between the two proteins, reciprocal immunoprecipitation and immunoblotting were performed with anti-p53 and anti-pADPRT antibodies. A poly(ADP-ribosyl)ated protein of 116 kD constantly precipitated at stringent conditions was identified as the automodified enzyme. It is concluded that mutant cytoplasmic p53 is tighly complexed to pADPRT and becomes modified. At 32.5°C binding to DNA of p53 or its temperature-dependent conformational alteration might prevent an analogous modification of the tumor suppressor protein. © 1996 Wiley-Liss, Inc.     

5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 M 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 M 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 M 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.     

CD4+ T cell responses to HLA-DP5-restricted wild-type sequence p53 peptides in patients with head and neck cancer

Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Evidence has been accumulating which indicates that CD4+ Th cells have an important role in generating and maintaining antitumor immune responses. To elucidate the nature of CD4+ Th responses to wt p53 epitopes in patients with squamous cell carcinoma of the head and neck (SCCHN), peripheral blood mononuclear cells (PBMCs) from HLA-DP5+ patients were stimulated with HLA-DP5-restricted wt p53 peptides, p53_108–122 or p53_153–166, and tested for the release of IFN-γ and IL-5 in ELISPOT assays. Immunohistochemistry for p53 accumulation in tumors, and ELISA for serum antibodies to p53 were also performed. Eleven (57.9%) of 19 HLA-DP5+ patients but none of 5 healthy donors had detectable Th1 and/or Th2 responses to wt p53 peptides by ELISPOT assay. Among these 11 responding patients, 9 (81.8%) and all 11 (100%) patients had a tumor burden and p53 accumulation, respectively. On the other hand, two responding patients were in post-operative condition. Interestingly, among nine patients with a tumor burden, four patients with early disease showed either Th1-polarized or mixed Th1/Th2 responses, while five patients with advanced disease showed either Th2-polarized or mixed Th1/Th2 responses. Our results suggest that wt p53_108–122 and p53_153–166 peptides stimulate both Th1- and Th2-type CD4+ T cell responses in patients with SCCHN, and anti-p53 Th responses may persist even after surgical resection of the tumor; however, the presence of a tumor and its progression may affect the nature of immune responses to wt p53 peptides.     

Antitumor effect of miR-197 targeting in p53 wild-type lung cancer

Lung cancer is the leading cause of tumor-related death. The lack of effective treatments urges the development of new therapeutic approaches able to selectively kill cancer cells. The connection between aberrant microRNA (miRNA – miR) expression and tumor progression suggests a new strategy to fight cancer by interfering with miRNA function. In this regard, LNAs (locked nucleic acids) have proven to be very promising candidates for miRNA neutralization. Here, we employed an LNA-based anti-miR library in a functional screening to identify putative oncogenic miRNAs in non-small-cell lung cancer (NSCLC). By screening NIH-H460 and A549 cells, miR-197 was identified as a new functional oncomiR, whose downregulation induces p53-dependent lung cancer cell apoptosis and impairs the capacity to establish tumor xenografts in immunodeficient mice. We further identified the two BH3-only proteins NOXA and BMF as new miR-197 targets responsible for induction of apoptosis in p53 wild-type cells, delineating miR-197 as a key survival factor in NSCLC. Thus, we propose the inhibition of miR-197 as a novel therapeutic approach against lung cancer.     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

Establishment and characterization of a human cell line derived from a uterine papillary serous carcinoma with wild-type p53 function

Uterine papillary serous carcinoma is an uncommon histologic subtype of endometrial cancer that behaves aggressively and has a poor prognosis. We successfully established a uterine papillary serous carcinoma cell line. The population-doubling time was approximately 16 h. Although loss of p53 function is considered critical for the molecular pathogenesis of uterine papillary serous carcinoma, p53 was not only mutated but functionally active in this cell line. This newly established cell line should be useful for investigating the characteristics of uterine papillary serous carcinoma.     

Regulation of p53 in embryonic stem cells

Despite an increasing interest in the role of the p53 tumour suppressor protein in embryonic stem cells, not much is known about its regulation in this cell type.We show that the relatively high amount of p53 protein correlates with a higher amount of p53 RNA in ES cells compared to differentiated cells. Moreover, p53 RNA is more stable in embryonic stem cells and the p53 protein is more often transcribed. This is at least partly due to decreased expression of miRNA-125a and 125b in embryonic stem cells. Despite its cytoplasmic localisation, p53 is degraded in 26S proteasomes in embryonic stem cells. This process is controlled by Mdm2, the deubiquitinating enzyme Hausp and Ubc13. In contrast, the E3 ligase PirH2 appears to be less important for the control of p53 in embryonic stem cells. During differentiation, p53 protein and RNA levels are decreased which corresponds to increased expression of miRNA-125a and miRNA-125b.     

Serine 312 phosphorylation is dispensable for wild-type p53 functions in vivo

Cellular stimulation results in phosphorylation of the tumor suppressor p53 on multiple residues, though the functional relevance is not always clear. It is noteworthy that the serine (S) 315 residue is unique, as it has been suggested to be phosphorylated not only by genotoxic signals, but also during cell-cycle progression and by endoplasmic-reticulum stress. However, in vitro data have been conflicting as phosphorylation at this site was shown to both positively and negatively regulate p53 functions. We have thus generated knock-in mice expressing an unphosphorylable S312 (equivalent to human S315), by substitution with an alanine (A) residue, to clarify the conflicting observations and to evaluate its functional relevance in vivo. Born at Mendelian ratios, the p53^S312A/S312A mice show no anomalies during development and adulthood. p53 activation, stability, localization and ability to induce apoptosis, cell-cycle arrest and prevent centrosome amplification are not compromised in p53^S312A/S312A cells. p53^S312A/S312A mice are unable to rescue mdm2^−/− lethality, and tumorigenesis – both spontaneous and irradiation/oncogene-induced – is not accentuated. Taken together, the results show that the S312 phosphorylation site is not in itself necessary for efficient p53 function, and advocates the possibility that it is neither relevant in the mouse context nor important for p53 functions in vivo.