Olivier Pluquet

Genotoxic and non-genotoxic pathways of p53 induction

Since the initial concept of p53 as a sensor of DNA-damage, the picture of the role of p53 has widened to include the sensing of much more diverse forms of stress, including hypoxia and constitutive activation of growth-promoting cascades. The pathways by which these processes regulate p53 are partially overlapping, but imply different patterns of post-translational modifications. In this review, we summarize current knowledge on post-translational modifications of p53, and we discuss how hypoxia and oncogene activation stresses may induce p53 independently of DNA damage.

The eIF2α Kinases PERK and PKR Activate Glycogen Synthase Kinase 3 to Promote the Proteasomal Degradation of p53

Phosphorylation of eukaryotic initiation factor 2α (eIF2α) is mediated by a family of kinases that respond to various forms of environmental stress. The eIF2α kinases are critical for mRNA translation, cell proliferation, and apoptosis. Activation of the tumor suppressor p53 results in cell cycle arrest and apoptosis in response to various types of stress. We previously showed that, unlike the majority of stress responses that stabilize and activate p53, induction of endoplasmic reticulum stress leads to p53 degradation through an Mdm2-dependent mechanism. Here, we demonstrate that the endoplasmic reticulum-resident eIF2α kinase PERK mediates the proteasomal degradation of p53 independently of translational control. This role is not specific for PERK, because the eIF2α kinase PKR also promotes p53 degradation in response to double-stranded RNA. We further establish that the eIF2α kinases induce glycogen synthase kinase 3 to promote the nuclear export and proteasomal degradation of p53. Our findings reveal a novel cross-talk between the eIF2α kinases and p53 with implications in cell proliferation and tumorigenesis.

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.

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.

Calnexin Phosphorylation Attenuates the Release of Partially Misfolded α1-Antitrypsin to the Secretory Pathway

Calnexin is a type I integral membrane phosphoprotein resident of the endoplasmic reticulum. Its intraluminal domain has been deduced to function as a lectin chaperone coordinating the timing of folding of newly synthesized N-linked glycoproteins of the secretory pathway. Its C-terminal cytosolic oriented extension has an ERK1 phosphorylation site at Ser563 affecting calnexin association with the translocon. Here we find an additional function for calnexin phosphorylation at Ser563 in endoplasmic reticulum quality control. A low dose of the misfolding agent l-azetidine 2-carboxylic acid slows glycoprotein maturation and diminishes the extent and rate of secretion of newly synthesized secretory α1-antitrypsin. Under these conditions the phosphorylation of calnexin is enhanced at Ser563. Inhibition of this phosphorylation by the MEK1 inhibitor PD98059 enhanced the extent and rate of α1-antitrypsin secretion comparable with that achieved by inhibiting α-mannosidase activity with kifunensine. This is the first report in which the phosphorylation of calnexin is linked to the efficiency of secretion of a cargo glycoprotein.

Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells

The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis.

Activation of p53 by the cytoprotective aminothiol WR1065: DNA-damage-independent pathway and redox-dependent modulation of p53 DNA-binding activity

WR1065 is an aminothiol with selective cytoprotective effects in normal compared to cancer cells, which is used to protect tissues against the damaging effect of radiation and chemotherapeutic drugs. WR1065 has been shown to induce wild-type p53 accumulation and activation in cultured cells, suggesting a role of p53 in cytoprotection. However, the molecular mechanisms by which WR1065 activates p53 remain unclear. Here, we demonstrated that p53 accumulation by WR1065 in MCF-7 cells did not result from the formation of DNA-damage as measured by DNA fragmentation and Comet assay, nor from oxidative stress as detected by measurement of glutathione levels, lipid peroxidation and reactive oxygen species production. p53 activation by WR1065 was not prevented by inhibition of PI-3 kinases, and was still detectable in MCF-7 cells stably transfected with the oncoprotein E6, which repressed p53 induction by DNA damage. These data provided evidence that WR1065 induces p53 by a pathway different than the one elicited by DNA-damage. Direct reduction by WR1065 of key cysteines in p53 may play an important role in this alternative pathway, as shown by the fact that WR1065 activated the redox-dependent, DNA-binding activity of p53 in vitro.

Poor intercellular transport and absence of enhanced antiproliferative activity after non-viral gene transfer of VP22-P53 or P53-VP22 fusions into p53 null cell lines in vitro or in vivo.

The herpes simplex virus type 1 (HSV‐1) VP22 protein has the property to mediate intercellular trafficking of heterologous proteins fused to its C‐ or N‐terminus. We have previously shown improved delivery and enhanced therapeutic effect in vitro and in vivo with a P27‐VP22 fusion protein. In this report, we were interested in studying the spread and biological activity of VP22 fused to the P53 tumor suppressor.

Role of the unfolded protein response in tumor cell characteristics and cancer outcome

Purpose of review In the present review, we discuss the possible role of the unfolded protein response (UPR) in the acquisition of tumor cell characteristics and in the prognosis of cancer outcome, which could assist and contribute to the development of more promising therapeutic strategies. Recent findings Accumulating evidence supports the idea that alteration of endoplasmic reticulum proteostasis is a key player in cancer development and aggressiveness. Some UPR components were reported as independent prognostic biomarker. Recent evidence supports a relationship between the UPR activation status and prognosis of tumors. This may represent an interesting avenue for better characterization of carcinogenesis and tumor type. Summary The contribution of the UPR to the characteristics of malignant tumors is complex and dependent on both intrinsic (e.g. oncogene addiction) and extrinsic (e.g. hypoxia) contexts. Through adaptation to severe microenvironmental conditions, UPR branches are generally a survival strategy for cancer cells, which are able to cope with this challenging context. We address the question of whether the activation status of the UPR is related to tumor properties and discuss the role of the UPR in the clinical context.

Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?

Senescence is a cell state occurring in vitro and in vivo after successive replication cycles and/or upon exposition to various stressors. It is characterized by a strong cell cycle arrest associated with several molecular, metabolic and morphologic changes. The accumulation of senescent cells in tissues and organs with time plays a role in organismal aging and in several age-associated disorders and pathologies. Moreover, several therapeutic interventions are able to prematurely induce senescence. It is, therefore, tremendously important to characterize in-depth, the mechanisms by which senescence is induced, as well as the precise properties of senescent cells. For historical reasons, senescence is often studied with fibroblast models. Other cell types, however, much more relevant regarding the structure and function of vital organs and/or regarding pathologies, are regrettably often neglected. In this article, we will clarify what is known on senescence of epithelial cells and highlight what distinguishes it from, and what makes it like, replicative senescence of fibroblasts taken as a standard.