P53 Antisense Oligonucleotide Research Articles

Determination of cellular uptake and intracellular levels of Cenersen (Aezea®, EL625), a p53 antisense oligonucleotide in acute myeloid leukemia cells

TP53 encodes for tumor protein p53. The suppression of p53 protein results in interruption of DNA repair mechanisms in dividing malignant cells thereby increasing the DNA damage and activating p53-independent mechanisms of apoptosis. This ultimately may translate into enhanced cytotoxic effects of standard chemotherapy. Based on this rationale, Cenersen, a phosphorothioate oligonucleotide antisense to p53-mRNA was synthesized and tested in clinical trials for patients with acute myeloid leukemia (AML). An important component of Cenersen clinical development is to develop a sensitive and specific method to quantify plasma and intracellular levels of Cenersen in different biologic matrices in order to determine tissue and intracellular distribution of the parent compound and its metabolites. Ultimately, this will allow us to determine pharmacokinetic and pharmacodynamic relationship for dose–effect correlation and design effective regimen to be rapidly translate into the clinic. An ELISA-based assay was adapted for assay development and validation of Cenersen in mouse plasma and cell lysate. Cellular uptake of Cenersen was studied in MV4-11 and KASUMI-1 AML cell lines. Real-time RT-PCR was used to measure P53-mRNA expression changes in treated cells. The assay had a limit of quantification of 35pmol/L in mouse plasma. Within-day and between-day precision of <15% and accuracy nearly 100% were observed in a linear range of 10–2000pmol/L (R2=0.99) in AML cell lysate. The selectivity of this assay examined as cross-reactivity with its 3′N-1, 3′N-2-metabolites, was 16.8% and 0.4%, respectively, and with its mismatch and the scramble oligonucleotides was 0.06% and 0.4%, respectively. Cenersen was stable in mouse plasma up to 8h at 37°C. When exposed to 0.1–1μmol/L Cenersen, MV4-11 and KASUMI-1 cells showed intracellular concentration in the range of 9.97–45.34nmol/mg protein and 0.1–2.1nmol/mg protein, respectively. Successful downregulation of p53-mRNA expression was observed in Cenersen treated cells. This ELISA-based assay was applicable to plasma and intracellular concentration measurement of Cenersen. Assessment of achievable concentration of Cenersen in different biologic matrices will be useful to elucidate the biological and clinical activity of this promising drug and define its recommended dose in future clinical trials.

Phase 2 randomized study of p53 antisense oligonucleotide (cenersen) plus idarubicin with or without cytarabine in refractory and relapsed acute myeloid leukemia

The p53 antisense oligonucleotide cenersen has been shown to sensitize acute myeloid leukemia (AML) stem cells to DNA damaging agents. To determine whether cenersen merits testing in larger efficacy studies, an exploratory study of cenersen in combination with idarubicin either alone or with 1 of 2 doses of cytarabine was performed in first-salvage AML patients. Patients who either had failed to respond to a single induction course or had responded to induction but relapsed within 12 months were enrolled. Stopping rules based on an expected 14% complete response (CR) rate were applied to each treatment arm. Fifty-three patients were treated, and none of the arms was terminated for lack of activity. Nearly all patients received a single course unless they responded. Ten of the 53 (19%) patients responded (8 CR and 2 CR with incomplete platelet recovery). There was a positive trend for a better response rate with increasing intensity of chemotherapy in the patients refractory to front-line treatment compared with those who had relapsed previously. One-third (17/53) of the patients received cenersen inhibitors (acetaminophen and/or high dose antioxidants) during treatment, and none of these responded to treatment. No unique toxicity was attributed to cenersen. The results of this study suggested that the combination of cenersen with chemotherapy may have clinical efficacy, and additional studies are warranted to explore its full potential.

Therapeutic potential of antisense oligodeoxynucleotides in downregulating p53 oncogenic mutations in cancers

mutation of the p53 gene is the most common genetic alteration in human cancers. Our study proposes to rationally design a p53 antisense oligonucleotide (ASO) repository, which contains a series of ASOs containing single nucleotide differences to discriminate between each mutant and wild type (WT) p53. The Sfold software was used to predict target-accessibility and we designed an initial series of antisense oligonucleotides (ASO) that target the p53 mutants A161T, R175H and R249S. Western-blot analysis indicated that ASOs strongly inhibited the expression of p53 mutants in a panel of human tumor cell lines (SNU-449, SK-BR-3 and PLC/PRF/5) while having little effect on the expression of WT p53 (HepG2 cells). In three cancer lines harboring each of the p53 mutations, mutant-specific ASO treatment led to a dose-dependent inhibition of cell growth, cell viability, colony formation and invasion, and expression of mutant p53-dependent survival proteins. Our preliminary results indicate that a single nucleotide difference in ASOs can discriminate between mutant and WT p53. These observations support the hypothesis that a p53 ASO repository can be a potentially valuable tool to knock down oncogenic mutant p53 and warrant the testing of a p53 ASO repository in in vivo settings.

Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

Apigenin, a plant flavone, potentially activates wild-type p53 and induces apoptosis in cancer cells. We conducted detailed studies to understand its mechanism of action. Exposure of human prostate cancer 22Rv1 cells, harboring wild-type p53, to growth-suppressive concentrations (10–80 μM) of apigenin resulted in the stabilization of p53 by phosphorylation on critical serine sites, p14 ARF-mediated downregulation of MDM2 protein, inhibition of NF-κB/p65 transcriptional activity, and induction of p21/WAF-1 in a dose- and time-dependent manner. Apigenin at these doses resulted in ROS generation, which was accompanied by rapid glutathione depletion, disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. Interestingly, we observed accumulation of a p53 fraction to the mitochondria, which was rapid and occurred between 1 and 3 h after apigenin treatment. All these effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine, p53 inhibitor pifithrin-α, and enzyme catalase. Apigenin-mediated p53 activation and apoptosis were further attenuated by p53 antisense oligonucleotide treatment. Exposure of cells to apigenin led to a decrease in the levels of Bcl- XL and Bcl-2 and increase in Bax, triggering caspase activation. Treatment with the caspase inhibitors Z-VAD-FMK and DEVD-CHO partially rescued these cells from apigenin-induced apoptosis. In vivo, apigenin administration demonstrated p53-mediated induction of apoptosis in 22Rv1 tumors. These results indicate that apigenin-induced apoptosis in 22Rv1 cells is initiated by a ROS-dependent disruption of the mitochondrial membrane potential through transcriptional-dependent and -independent p53 pathways.

Calpain counteracts mechanosensitive apoptosis of vascular smooth muscle cellsin vitroandin vivo

Mechanical forces contribute to vascular remodeling processes. Elevated mechanical stress causes apoptosis of vascular smooth muscle cells (VSMCs) within the media. This study examined the role of the cystein protease calpain in force-induced vascular cell apoptosis and its effect on injury-induced vascular remodeling processes. VSMCs were exposed to cyclic tensile force in vitro, which resulted in increased p53 protein expression and transcriptional activity as well as a significant increase of apoptotic VSMCs. Apoptosis was prevented by the p53 inhibitor pifithrin and by p53 antisense oligonucleotides, indicating dependency of force-induced apoptosis on p53. Simultaneously, calpain activity increased by mechanical stress. Prevention of calpain activation by calpeptin or antisense oligonucleotides augmented strain-induced p53 expression and transcriptional activity, resulting in a further increase of apoptotic rate. p53 protein was directly disintegrated by activated calpain. The in vivo relevance of the findings was tested: pharmacologic inhibition of initial calpain activation augmented early apoptosis of medial VSMCs 24 h after balloon injury in a p53-dependent manner but resulted in a marked increase in late neointima formation. We conclude that calpain counteracts mechanically induced excessive VSMC apoptosis through its p53-degrading properties, which identifies calpain as a key regulator of mechanosensitive remodeling processes of the vascular wall.

Phase II study of EL625, a p53 antisense oligonucleotide, and chemotherapy in refractory and relapsed acute myelogenous leukemia (AML)

6617 EL625, an antisense phosphorothioate, induces RNase H dependent cleavage of p53 transcripts resulting in loss of p53 production. In vitro, EL625 combined with low doses of p53-inducing genome damaging agents, e.g. idarubicin, eliminates self-renewal of AML blast stem cells without adversely affecting normal cells. To determine the safety & efficacy of EL625 in combination with chemotherapy we are conducting a phase II study in patients (pts) with AML who were refractory to induction chemotherapy or had a 1st remission duration of <12 months, & were receiving their first salvage. As previously published, the expected complete remission rate with salvage chemotherapy for pts with these characteristics is 10–20% (Estey et al. Blood 1996; 88: 756). Pts were randomized to 1 of 3 groups. All pts received EL625 (0.1 mg/kg/hr continuous infusion over 4 days [d]) & Idarubicin (12 mg/m2/d x 3 d) starting 24 hrs after the start of EL625. Ara-C was administered starting 24 hrs after the start of EL625 infusion as follows: Arm 1, no Ara-C; Arm 2, 100 mg/m2/d over 7d; Arm 3, 1 g/m2/d x4d (3d if 60 yrs or older). Pts could receive up to 2 courses of induction therapy. We have treated 11 pts (4 in arm 1; 3 in arm 2; 4 in arm 3). Median age is 56 yrs (range, 25–73 yrs). 7 were refractory to induction & 6 had relapsed after median 1st complete remission (CR) duration of 30 wks (range, 4–46). 4 pts had diploid karyotype & 7 had cytogenetic abnormalities (complex -5/-7 in 2, misc 5). 9 pts are evaluable for efficacy & toxicity (2 too early). 5 pts have received 2 courses of therapy. 4 pts (44%) achieved CR (1 arm 1, 2 arm 2, 1 arm 3). CR occurred in 2 pts with refractory disease & 2 with relapse (1st CR 32 & 46 wks, respectively). All 4 pts continue in CR after 6, 5, 4 & 1 month, respectively; 1 received stem cell transplant in CR. 2 other pts had a morphologic leukemia free state as per the International Working Group (Cheson et al, JCO 2003; 21:4642). 1 pt died on course 1, day 15 of pneumonia. Toxicity was as expected with myelosuppression as the most common adverse event and no grade 3–4 toxicity related to EL625. Although preliminary, these results suggest that EL625 in combination with chemotherapy is well tolerated. The early efficacy data is encouraging. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Eleos Eleos

P53 may positively regulate hepatocyte proliferation in rats

p53, known as a tumor suppressor gene, is a transcription factor that regulates various cellular functions. Recently, several growth factor gene promoters, including that of transforming growth factor α (TGF-α), were shown to be direct targets of p53-mediated transcription. Hepatic p53 mRNA is up-regulated during liver regeneration in rats. The aim of this study is to examine the role of p53 in hepatocyte proliferation. p53 protein levels were examined in rat hepatocytes cultured in the medium containing hepatocyte growth factor (HGF). p53 levels began to increase after 6 hours of incubation, reached a maximum at 18 hours, and decreased thereafter. DNA synthesis increased at 12 hours and peaked at 30 hours. When hepatocytes were incubated with p53 antisense oligonucleotide in addition to HGF, increases of p53 and TGF-α levels were suppressed, and DNA synthesis was reduced. The increases of TGF-α levels and DNA synthesis were also suppressed by a chemical inhibitor of p53, pifithrin-α. In rats after two-thirds partial hepatectomy, hepatic p53 increased and reached maximal levels around 16 hours when hepatic HGF levels have been shown to reach a maximum followed by an increase in hepatic TGF-α levels or hepatocyte proliferation. In contrast, sham-operated rats showed minor elevations of hepatic p53 levels. In conclusion, p53 production is stimulated by HGF and may contribute to the proliferation of rat hepatocytes. Considering previous findings indicating the importance of endogenous TGF-α for the proliferation of hepatocytes stimulated by HGF, TGF-α might play a role in HGF-p53 mediated hepatocyte proliferation. (H EPATOLOGY 2002;36:336-344.)

Diol- and Triol-Type Ginseng Saponins Potentiate the Apoptosis of NIH3T3 Cells Exposed to Methyl Methanesulfonate

In this study we investigated the effect of ginseng saponins on the p53-dependent apoptosis in NIH3T3 cells exposed to methyl methanesulfonate (MMS), an alkylating agent. Trypan blue exclusion assay, cell morphology studies, and apoptotic index determined by acridine orange staining showed that the postincubation of MMS-exposed cells in medium containing diol- (PD) or triol-type (PT) ginseng saponins potentiate the apoptotic cell death. FACS analysis indicated that the increased apoptotic cell population in the saponin-postincubation group was accompanied by the accumulation of cells in G0/G1 phase. By Western blot analyses it was demonstrated that postincubation of saponins increases the expression of p53 and p21 in MMS-exposed cells but decreased that of CDK2, cyclin E and D1, and PCNA. The upregulation of p53 and p21 and downregulation of CDK2 was shown to be p53-dependent in experiments using the p53 antisense oligonucleotide. These results suggest that ginseng saponins contain components potentiating the apoptosis of MMS-exposed NIH3T3 cells via p53 and p21 activation, accompanied with by downregulation of cell cycle-related protein expression.

P53 mediates ceramide-induced apoptosis in SKN-SH cells.

Ceramide induces apoptotic cell death in a dose- and time-dependent manner in neuroblastoma SKN-SH cells. Pretreatment with caspase inhibitors blocks cell death, suggesting that a set of caspase activities including caspase 1, as well as caspase 3, are involved in ceramide-induced apoptosis in SKN-SH cells. Treatment with a caspase inhibitor 3 h after ceramide addition did not inhibit cell death, although caspase activity was substantially reduced. Ceramide-induced apoptosis is accompanied by accumulation of p53 followed by an increase of Bax and decrease of Bcl-2 levels. Inhibition of p53 expression with p53 antisense oligonucleotides inhibits apoptosis and prevents the increase in Bax and decrease in Bcl-2. Furthermore, pretreatment with p53 antisense oligonucleotides markedly inhibits the induction of caspase activity. These results suggest that p53 regulates the ratio Bcl-2/Bax and the expression/activation of caspases during ceramide-induced apoptosis in SKN-SH cells. Caspase inhibition did not alter the expression of p53, Bcl-2 and Bax. Thus ceramide-induced reduction in the Bcl-2/Bax ratio, increase in caspase activity, and apoptosis is dependent upon increases in cellular p53 levels which play a critical role in the regulation of apoptotic cell death.

Resveratrol induces apoptosis in thyroid cancer cell lines via a MAPK- and p53-dependent mechanism.

Two papillary thyroid carcinoma (PTC) and two follicular thyroid carcinoma (FTC) cell lines treated with resveratrol (RV), 1-10 microM, showed activation and nuclear translocation of MAPK (extracellular signal-regulated kinase 1/2). Cellular abundance of the oncogene suppressor protein p53, serine phosphorylation of p53, and abundance of c-fos, c-jun, and p21 mRNAs were also increased by RV. Inhibition of the MAPK pathway by either H-ras antisense transfection or PD 98059, an MAPK kinase inhibitor, blocked these RV-induced effects. Addition of pifithrin-alpha, a specific inhibitor of p53, or transfection of p53 antisense oligonucleotides caused decreased RV-induced p53 and p21 expression in PTC and FTC cells. Studies of nucleosome levels estimated by ELISA and of DNA fragmentation showed that RV induced apoptosis in both papillary and follicular thyroid cancer cell lines; these effects were inhibited by pifithrin-alpha and by p53 antisense oligonucleotide transfection. PD 98059 and H-ras antisense transfection also blocked induction of apoptosis by RV. Thus, RV acts via a Ras-MAPK kinase-MAPK signal transduction pathway to increase p53 expression, serine phosphorylation of p53, and p53-dependent apoptosis in PTC and FTC cell lines.

Resveratrol Induces Apoptosis in Thyroid Cancer Cell Lines via a MAPK- and p53-Dependent Mechanism

Two papillary thyroid carcinoma (PTC) and two follicular thyroid carcinoma (FTC) cell lines treated with resveratrol (RV), 1–10 μm, showed activation and nuclear translocation of MAPK (extracellular signal-regulated kinase 1/2). Cellular abundance of the oncogene suppressor protein p53, serine phosphorylation of p53, and abundance of c-fos, c-jun, and p21 mRNAs were also increased by RV. Inhibition of the MAPK pathway by either H-ras antisense transfection or PD 98059, an MAPK kinase inhibitor, blocked these RV-induced effects. Addition of pifithrin-α, a specific inhibitor of p53, or transfection of p53 antisense oligonucleotides caused decreased RV-induced p53 and p21 expression in PTC and FTC cells. Studies of nucleosome levels estimated by ELISA and of DNA fragmentation showed that RV induced apoptosis in both papillary and follicular thyroid cancer cell lines; these effects were inhibited by pifithrin-α and by p53 antisense oligonucleotide transfection. PD 98059 and H-ras antisense transfection also blocked induction of apoptosis by RV. Thus, RV acts via a Ras-MAPK kinase-MAPK signal transduction pathway to increase p53 expression, serine phosphorylation of p53, and p53-dependent apoptosis in PTC and FTC cell lines.

P53 Protects renal inner medullary cells from hypertonic stress by restricting DNA replication.

We previously found that p53 upregulation by hypertonicity protected renal inner medullary collecting duct (mIMCD3) cells from apoptosis. The purpose of the present study was to investigate the mechanism by which p53 protects the cells. We now find that hypertonicity (NaCl added to a total of 500 mosmol) inhibits DNA replication and delays G(1)-S transition as concluded from analysis of cell cycle distributions and bromodeoxyuridine (BrDU) incorporation rates. Lowering of p53 with p53 antisense oligonucleotide attenuated such effects of hypertonicity, resulting in an increased number of apoptotic cells in S phase and cells with >4 N DNA. Results with synchronized cells are similar, showing that cells in the early S phase are more sensitive to hypertonicity. Immunocytochemistry revealed that p53 becomes phosphorylated on Ser(15) and translocates to the nucleus in S both in isotonic and hypertonic conditions. Caffeine (2 mM) greatly reduces the p53 level and Ser(15) phosphorylation, followed by a remarkable increase of DNA replication rate, by failure of hypertonicity to inhibit it, and by reduction of cell number during hypertonicity. Finally, inhibition of DNA replication by the DNA polymerase inhibitor aphidicolin significantly improves cell survival, confirming that keeping cells in G(1) and decreasing the rate of DNA replication is protective and that these actions of p53 most likely are what normally help protect cells against hypertonicity.

Neurons Are Protected from Excitotoxic Death by p53 Antisense Oligonucleotides Delivered in Anionic Liposomes

The potential of anionic liposomes for oligonucleotide delivery was explored because the requirement for a net-positive charge on transfection-competent cationic liposome-DNA complexes is ambiguous. Liposomes composed of phosphatidylglycerol and phosphatidylcholine were monodisperse and encapsulated oligonucleotides with 40-60% efficiency. Ionic strength, bilayer charge density, and oligonucleotide chemistry influenced encapsulation. To demonstrate the biological efficacy of this vector, antisense oligonucleotides to p53 delivered in anionic liposomes were tested in an in vitro model of excitotoxicity. Exposure of hippocampal neurons to glutamate increased p53 protein expression 4-fold and decreased neuronal survival to approximately 35%. Treatment with 1 microm p53 antisense oligonucleotides in anionic liposomes prevented glutamate-induced up-regulation of p53 and increased neuronal survival to approximately 75%. Encapsulated phosphorothioate p53 antisense oligonucleotides were neuroprotective at 5-10-fold lower concentrations than when unencapsulated. Replacing the anionic lipid with phosphatidylserine significantly decreased neuroprotection. p53 antisense oligonucleotides complexed with cationic liposomes were ineffective. Neuroprotection by p53 antisense oligonucleotides in anionic liposomes was comparable with that by glutamate receptor antagonists and a chemical inhibitor of p53. Anionic liposomes were also capable of delivering plasmids and inducing transgene expression in neurons. Anionic liposome-mediated internalization of Cy3-labeled oligonucleotides by neurons and several other cell lines demonstrated the universal applicability of this vector.

Ionizing radiation enhances matrix metalloproteinase-2 production in human lung epithelial cells.

Radiation pneumonitis is a major complication of radiation therapy. However, the detailed cellular mechanisms have not been clearly defined. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase (MMP)-2 can degrade type IV collagen, one of the major components of the basement membrane, we hypothesized that ionizing radiation would modulate MMP-2 production in human lung epithelial cells. To evaluate this, the modulation of MMP-2 with irradiation was investigated in normal human bronchial epithelial cells as well as in A549 cells. We measured the activity of MMP-2 in the conditioned medium with zymography and the MMP-2 mRNA level with RT-PCR. Both of these cells constitutively expressed 72-kDa gelatinolytic activity, corresponding to MMP-2, and exposure to radiation increased this activity. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. This radiation-induced increase in MMP-2 expression was mediated via p53 because the p53 antisense oligonucleotide abolished the increase in MMP-2 activity as well as the accumulation of p53 after irradiation in A549 cells. These results indicate that MMP-2 expression by human lung epithelial cells is involved in radiation-induced lung injury.

Arsenic trioxide induces apoptosis in human gastric cancer cells through up-regulation of p53 and activation of caspase-3.

Arsenic trioxide (As(2)O(3)) can induce clinical remission in patients suffering from acute promyelocytic leukemia, through induction of apoptosis and activation of caspases. We investigated the potential use of As(2)O(3) in human gastric cancer and its possible mechanisms. Human gastric cancer cell lines AGS and MKN-28 were treated with various concentrations (0.1 to 100 microM) of As(2)O(3) for 24 to 72 hr. Apoptosis was determined by acridine orange staining, flow cytometry and DNA fragmentation. Protein levels of p53, p21(waf1/cip1), c-myc, bcl-2 and bax were detected by Western blotting. Effects of As(2)O(3) on caspase-3 protease activity, its protein concentration and cleavage of poly(ADP)-ribose polymerase (PARP) were also studied. As(2)O(3) inhibited cell growth and induced apoptosis in both cell lines, though AGS cells were more sensitive. As(2)O(3) induced apoptosis in AGS cells in a concentration- and time-dependent manner. Treatment resulted in a marked increase in p53 protein levels as early as 4 hr. Co-incubation with p53 anti-sense oligo-nucleotide suppressed As(2)O(3)-induced intracellular p53 over-expression and apoptosis. As(2)O(3) increased the activity of caspase-3, with appearance of its 17 kDa peptide fragment, and cleavage of PARP, with appearance of the 85 kDa cleavage product, both in parallel with the induction of apoptosis. Both the tripeptide caspase inhibitor zVAD-fmk and the specific caspase-3 inhibitor DEVD-fmk partially suppressed As(2)O(3)-induced caspase-3 activation and apoptosis. As(2)O(3) inhibits cell growth and induces apoptosis in gastric cancer cells, involving p53 over-expression and activation of caspase-3. The potential use of this compound in the treatment of gastric cancer is worth further investigation.

Mediation of Nerve Growth Factor-driven Cell Cycle Arrest in PC12 Cells by p53: SIMULTANEOUS DIFFERENTIATION AND PROLIFERATION SUBSEQUENT TO p53 FUNCTIONAL INACTIVATION

Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G(1)/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G(1) arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells.

Induction of p53 in the glutamate-induced cell death program.

Fifteen minute exposure of primary cultures of cerebellar granule cells to micromolar concentrations of glutamate results in apoptotic cell death. Among the intracellular events triggered by glutamate, we identified two transcriptional factors, i.e. the p50 member of the NF-kappaB family and the tumor suppressor phosphoprotein p53, that are apparently linked by a sequential trascriptional program. We found that pretreatment of the cultures with aspirin (ASA), which inhibits NF-kappaB activation, resulted in a complete prevention of glutamate-induced p53 immunoreactivity. The same results were obtained pretreating the cells with a specific p53 antisense oligonucleotide. Both ASA and p53 antisense abolished glutamate-induced apoptosis. We also found that two other proteins, the cyclin dependent kinase inhibitor p21 and DNA mismatches repair MSH2, whose encoding genes are well known target of p53, were upregulated by glutamate. On these bases, we propose NF-kappaB, p53, p21 and MSH2 as relevant contributors of the glutamate-induced pro-apoptotic pathway.

A Novel Ras Antagonist Regulates Both Oncogenic Ras and the Tumor Suppressor p53 in Colon Cancer Cells

In colon cancer, K-Ras oncogenes, which appear to be linked to chemoresistance and poor prognosis, are activated in more than 50% of cases, whereas the tumor suppressor gene p53 is mutationally altered in about 70% of all cases. The transcription factor p53, which is frequently mutated at codon 273, maintains wild-type configuration and possibly carries out residual functions. Although blocking of activated K-Ras may constitute a rational therapeutic concept for this treatment-resistant malignancy, a strategy influencing both oncogenic Ras and the tumor suppressor p53 may be even more promising. We evaluated the effects of S-trans, trans-farnesyl-thiosalicylic acid (FTS), a novel Ras antagonist on human SW480 and HT-29 colon cancer cells, which both harbor a p53 His273 mutation but express activated K-Ras and wild-type, but overexpressed, H-Ras, respectively. Besides cell growth and morphology, levels of cellular Ras proteins, regulation of p53 and p21(waf1/cip1) expression were analyzed by immunoblotting. The cell cycle arresting potential of FTS was quantified by flow cytometry. We demonstrate that FTS treatment alters the morphology and blocks the growth of SW480 and HT-29 colon cancer cells by both reducing the total amount of Ras and up-regulating the tumor suppressor p53. Furthermore, FTS caused an upregulation of the cyclin-cyclin-dependent kinase (CDK) inhibitor p21(waf1/cip1) and blocked the cell cycle. p53 antisense oligonucleotides not only reduced the level of p53 proteins but correspondingly also blocked the expression of p21(waf1/cip1) in FTS-treated colon cancer cells. FTS, a unique compound capable of regulating both oncogenic Ras and the tumor suppressor p53 may prove particularly useful for the therapy of colon cancer and other treatment-resistant malignancies where Ras is altered and p53 is either wild-type or mutated in positions that allow residual p53 functions.

Protection of Renal Inner Medullary Epithelial Cells from Apoptosis by Hypertonic Stress-induced p53 Activation

Acute hypertonicity causes cell cycle delay and apoptosis in mouse renal inner medullary collecting duct cells (mIMCD3) and increases GADD45 expression. Because the tumor suppressor protein p53 may be involved in these effects, we have investigated the role of p53 in mIMCD3 response to hyperosmotic stress. Acute elevation of osmolality with NaCl addition from the control level of 320 mosmol/kg to 500-600 mosmol/kg greatly increased the levels of total and Ser(15)-phosphorylated p53 within 15 min. However, similar elevation of osmolality with urea did not increase p53 levels. Our studies indicate that induced p53 is transcriptionally active because NaCl addition to 500-600 mosmol/kg stimulated transcription of a luciferase reporter containing a p53 consensus element and appropriately altered mRNA levels of known transcriptional targets of p53, i.e. increased MDM-2 and decreased BCL-2 levels. Elevating NaCl further to 700-800 mosmol/kg rapidly killed most of the cells by apoptosis. At these higher NaCl concentrations, p53 levels were further increased although Ser(15) phosphorylation and transcriptional activity were significantly lower than levels at 500-600 mosmol/kg. At NaCl-induced 500 mosmol/kg, apoptosis was rare in the presence of control, nonspecific oligonucleotide but highly prevalent upon addition of p53 antisense oligonucleotide that substantially reduced p53 levels. We conclude that induction of active p53 in mIMCD3 cells by hypertonic stress contributes to cell survival.

P53-independent regulation of cyclin B1 in normal human fibroblasts during UV-induced G2-arrest

Recently we demonstrated, using normal human fibroblasts (NHFs), that UVc radiation induces a G2 M arrest which was even more pronounced when p53 expression was inhibited. So, the aim of this study was to evaluate in NHFs the relationship between UV-induced G2 M arrest and cyclin B1 regulation and to investigate if p53 could contribute to the cyclin B1 regulation in these conditions. Following exposure of asynchronous NHFs to UV light, we showed that the induced G2 M arrest was accompanied by a dose-dependent down-regulation of cyclin B1 mRNA as evaluated by RT-PCR. Concomitantly, using flow cytometric analysis, we observed a strong accumulation of cyclin B1 protein which was correlated to the apparition of the G2 M arrest. In order to study the contribution of p53 to the cyclin B1 accumulation in response to UV exposure, we inhibited p53 induction using p53 antisense oligonucleotides. We found that the inhibition of p53 protein induction after UV exposure had no effect on the level of cyclin B1 mRNA. Moreover, although inhibition of p53 protein induction increased the number of the cells in the G2-M phase, the mean content of cyclin B1 protein was not augmented in these cells. These results indicate clearly that the induction of p53 protein following UV exposure does not regulate the level of cyclin B1 mRNA or protein in normal cells.