Sophie North

ΔN-p53, a natural isoform of p53 lacking the first transactivation domain, counteracts growth suppression by wild-type p53

The tumor suppressor protein p53 is ubiquitously expressed as a major isoform of 53 kD, but several forms of lower molecular weight have been observed. Here, we describe a new isoform, ΔN-p53, produced by internal initiation of translation at codon 40 and lacking the N-terminal first transactivation domain. This isoform has impaired transcriptional activation capacity, and does not complex with the p53 regulatory protein Mdm2. Furthermore, ΔN-p53 oligomerizes with full-length p53 (FL-p53) and negatively regulates its transcriptional and growth-suppressive activities. Consistent with the lack of Mdm2 binding, ΔN-p53 does not accumulate in response to DNA-damage, suggesting that this isoform is not involved in the response to genotoxic stress. However, in serum-starved cells expressing wild-type p53, ΔN-p53 becomes the predominant p53 form during the synchronous progression into S phase after serum stimulation. These results suggest that ΔN-p53 may play a role as a transient, negative regulator of p53 during cell cycle progression.

Hypoxia-inducible Factor-1α, a Key Factor in the Keratinocyte Response to UVB Exposure

Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor sensitive to oxygen levels, which responds to stress factors under both hypoxic and nonhypoxic conditions. UV irradiation being a common stressor of skin, we looked at the effect of UVB on HIF-1α expression in keratinocytes. We found that UVB induces a biphasic HIF-1α variation through reactive oxygen species (ROS) generation. Whereas rapid production of cytoplasmic ROS down-regulates HIF-1α expression, delayed mitochondrial ROS generation results in its up-regulation. Indeed, activation of p38 MAPK and JNK1 mediated by mitochondrial ROS were required for HIF-1α phosphorylation and accumulation after UVB irradiation. Our experiments also revealed a key role of HIF-1α in mediating UVB-induced apoptosis. We conclude that the broad impact of the HIF-1 transcription factor on gene expression could make it a key regulator of UV-responsive genes and photocarcinogenesis.

Restoration of wild‐type conformation and activity of a temperature‐sensitive mutant of p53 (p53V272M) by the cytoprotective aminothiol WR1065 in the esophageal cancer cell line TE‐1

The aminothiol WR1065, the active metabolite of the cytoprotector amifostine, exerts its antimutagenic effects through free‐radical scavenging and other unknown mechanisms. In an earlier report, we showed that WR1065 activates wild‐type p53 in MCF‐7 cells, leading to p53‐dependent arrest in the G1 phase of the cell cycle. To determine whether WR1065 activates p53 by modulating protein conformation, we analyzed its effects on p53 conformation and activity in the esophageal cancer cell line TE‐1. This cell line contains a mutation in codon 272 of p53 (p53V272M, with methionine instead of a valine), conferring temperature‐sensitive properties to the p53 protein. At the nonpermissive temperature (37°C), p53V272M adopts the mutant p53 conformation (nonreactive with the antibody PAb1620), does not bind specifically to DNA, and is not activated in response to DNA‐damaging treatment. However, treatment with 0.5–4 mM WR1065 partially restored wild‐type conformation at 37°C, stimulated DNA binding activity, and increased the expression of p53 target genes WAF‐1, GADD45, and MDM2, leading to cell‐cycle arrest in G1. These results suggest that WR1065 activates p53 through a mechanism distinct from DNA‐damage signaling, which involves modulation of p53 protein conformation.

Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation

High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice.

The cytoprotective aminothiol WR1065 activates p21waf-1 and down regulates cell cycle progression through a p53-dependent pathway.

The phosphoaminothiol WR1065, the active metabolite of the pro-drug amifostine (WR2721), protects cultured cells and tissues against cytotoxic exposure to radiation or chemotherapeutic agents. We show here that WR1065 and the pro-drug WR2721 activate the p53 tumor suppressor protein and induce the expression of the cyclin-dependent kinase inhibitor p21waf-1 in the breast cancer cell line MCF-7, and in the mouse fibroblast cell line balb/c 3T3. Using two MCF-7 derived cell lines, MN1 and MDD2, we show that induction of p21waf-1 is detectable in MN1 (expressing a functional p53) but not in MDD2 (p53 disabled). These effects are observed at concentrations of WR1065 (0.5 to 1 mM) identical to those required to protect against cytotoxicity by hydrogen peroxide. Induction of p53 is not prevented by addition of aminoguanidine, an inhibitor of Cu-dependent amine-oxidases which blocks the extra-cellular degradation of WR1065 into toxic metabolites. Moreover, spermidine, a natural polyamine structurally related to amifostine, does not activate p53. Induction of p53 by WR1065 results in a delay in the G1/S transition in MCF-7 and MN-1 cells, but not in the p53 disabled cells MDD2. These data indicate that WR1065, a polyamine analog with thiol anti-oxidant properties, activates a cell cycle check-point involving p53.

Amifostine (WR2721) restores transcriptional activity of specific p53 mutant proteins in a yeast functional assay

Many p53 mutants found in human cancer have an altered ability to bind DNA and transactivate gene expression. Re-expression of functional p53 in cells in which the endogenous TP53 gene is inactivated has been demonstrated to restore a non-tumorigenic phenotype. Pharmacological modulation of p53 mutant conformation may therefore represent a mechanism to reactivate p53 function and consequently improve response to radio- and chemotherapy. We have recently reported that the radio- and chemoprotector Amifostine (WR2721, Ethyol®) activates wild-type p53 in cultured mammalian cells. In the present study, we have used a yeast functional assay to investigate the effect of WR2721 on the transcriptional activity of p53. WR2721 restored this activity in a temperature-sensitive mutant V272M (valine to methionine at codon 272) expressed at the non-permissive temperature and it also partially restored the transcriptional activity of several other conformationally flexible p53 mutants. The results indicate that the yeast functional assay may be used to identify compounds that modulate p53 activity, with potential therapeutic implications.

Acute L-glutamine deprivation compromises VEGF-a upregulation in A549/8 human carcinoma cells.

Tumor ischemia participates in angiogenesis and cancer progression through cellular responses to hypoxia and nutrient deprivation. However, the contribution of amino acids limitation to this process remains poorly understood. Using serum‐free cell culture conditions, we tested the impact of L‐glutamine deprivation on metabolic and angiogenic responses in A549/8 carcinoma cells. In these cells, lowering glutamine concentration modified the cell cycle distribution and significantly induced apoptosis/necrosis. Although glutamine deprivation led to a HIF‐independent increase in VEGF‐A mRNA, the corresponding protein level remained low and correlated with the inhibition of protein synthesis and activation of the GCN2/eIF2α pathway. Limitation of glutamine availability also hampers hypoxia‐ and hypoglycemia‐induced VEGF‐A protein upregulation. Thus, glutamine deprivation may have no direct effect on VEGF‐dependent angiogenesis, compared to hypoxia or to glucose deprivation, and may instead be detrimental to cancer progression by antagonizing ischemia‐induced stresses. J. Cell. Physiol. 212: 463–472, 2007.

High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor

BackgroundEpidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma.MethodsExpression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown.ResultsEREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α.ConclusionEREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the growth factor through ErbB1.

The Cytoprotective Aminothiol WR1065 Activates p53 through a Non-genotoxic Signaling Pathway Involving c-Jun N-terminal Kinase

WR1065 is an aminothiol with selective cytoprotective effects in normal cells compared with cancer cells. In a previous study (North, S., El-Ghissassi, F., Pluquet, O., Verhaegh, G., and Hainaut, P. (2000) Oncogene 19, 1206–1214), we have shown that WR1065 activates wild-type p53 in cultured cells. Here we show that WR1065 induces p53 to accumulate through escape from proteasome-dependent degradation. This accumulation is not prevented by inhibitors of phosphatidylinositol 3-kinases and is not accompanied by phosphorylation of Ser-15, -20, or -37, which are common targets of the kinases activated in response to DNA damage. Furthermore, WR1065 activates the JNK (c-Jun N-terminal kinase), decreases complex formation between p53 and inactive JNK, and phosphorylates p53 at Thr-81, a known site of phosphorylation by JNK. A dominant negative form of JNK (JNK-APF) reduces by 50% the activation of p53 by WR1065. Thus, WR1065 activates p53 through a JNK-dependent signaling pathway. This pathway may prove useful for pharmacological modulation of p53 activity through non-genotoxic mechanisms.

Inhibition of angiogenesis and the angiogenesis/invasion shift.

Angiogenesis has become a major target in cancer therapy. However, current therapeutic strategies have their limitations and raise several problems. In most tumours, anti-angiogenesis treatment targeting VEGF (vascular endothelial growth factor) has only limited overall survival benefit compared with conventional chemotherapy alone, and reveals several specific forms of resistance to anti-VEGF treatment. There is growing evidence that anti-VEGF treatment may induce tumour cell invasion by selecting highly invasive tumour cells or hypoxia-resistant cells, or by up-regulating angiogenic alternative pathways such as FGFs (fibroblast growth factors) or genes triggering new invasive programmes. We have identified new genes up-regulated during glioma growth on the chick CAM (chorioallantoic membrane). Our results indicate that anti-angiogenesis treatment in the experimental glioma model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. We have identified a molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme. Furthermore, we are focusing our research on alternative inhibitors that act, in part, independently of VEGF. These are endogenous molecules that play a role in the control of tumour growth and may constitute a starting point for further development of novel therapeutic or diagnostic tools.