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The tumor suppressor p53 is a key regulator of apoptosis induced by various cellular stresses. p53 can induce apoptosis by two mechanisms. First, p53 acts as a transcription factor inducing and repressing pro-apoptotic and anti-apoptotic targets genes, respectively. Second, p53 is able to translocate to the mitochondria, where it interacts with BCL-2 family members to induce membrane permeabilization and cytochrome c release. p53 transcriptional activity is regulated by a set of post-translational modifications that have been well documented. However, how these modifications impact the direct mitochondrial pathway of death remain poorly understood. In this study, we focused on the role of serine 392 phosphorylation in the control of p53-dependent apoptosis. We used CRISPR/Cas9 genome editing to substitute serine 392 by a non-phosphorylatable alanine in HCT-116 colon carcinoma cells. The S392A mutant displayed normal transcriptional activity following genotoxic stress, but markedly impaired ability to localize to mitochondria. The decreased mitochondrial localization of the S392A mutant correlated with a lower ability to induce apoptosis. Confirmatory observations were made following enforced expression of the S392A p53 mutant or a phospho-mimetic S392E mutant in H1299 lung carcinoma cells. Our observations support the premise that serine 392 phosphorylation of p53 influences its mitochondrial translocation and transcription-independent apoptotic function.

Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.

Purpose: Metastasis and drug resistance are the major limitations in the survival and management of patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. Results: We show that a clonal 5F31 cell population, resistant to 1 μmol/L 5FU, express a typical cancer stem cell–like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1 silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, YES1 and YAP transcript levels were higher in liver metastases of patients with colon cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively). Conclusions: We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer. Clin Cancer Res; 20(4); 837–46. ©2013 AACR.

The transcription factor Ets-1 is implicated in various physiological processes and invasive pathologies. We identified a novel variant of ets-1, ets-1Delta(III-VI), resulting from the alternative splicing of exons III to VI. This variant encodes a 27 kDa isoform, named Ets-1 p27. Ets-1 p27 lacks the threonine-38 residue, the Pointed domain and the transactivation domain, all of which are required for the transactivation of Ets-1 target genes. Both inhibitory domains surrounding the DNA-binding domain are conserved, suggesting that Ets-1 p27, like the full-length Ets-1 p51 isoform, is autoinhibited for DNA binding. We showed that Ets-1 p27 binds DNA in the same way as Ets-1 p51 does and that it acts both at a transcriptional and a subcellular localization level, thereby constituting a dual-acting dominant negative of Ets-1 p51. Ets-1 p27 blocks Ets-1 p51-mediated transactivation of target genes and induces the translocation of Ets-1 p51 from the nucleus to the cytoplasm. Furthermore, Ets-1 p27 overexpression represses the tumor properties of MDA-MB-231 mammary carcinoma cells in correlation with the known implication of Ets-1 in various cellular mechanisms. Thus the dual-acting dominant-negative function of Ets-1 p27 gives to the Ets-1 p27/Ets-1 p51 ratio a determining effect on cell fate.

Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which contain a spheroid perinuclear inclusion body, was found in our cellular clone. In conclusion, our data suggest that Huh-7w7.3 cells constitute an excellent model for determining the cellular factor(s) involved in the process of spheroid perinuclear body formation.

The role of tumor-associated macrophages (TAMs) is controversial. Although most studies on different cancer types associate them with a poorer prognosis, interestingly in colon cancer, most articles indicate that TAMs prevent tumor development; patients with high TAMs have better prognosis and survival rate. M1-polarized macrophages produce high level of tumor necrosis factor-alpha, interleukin-1 beta or reactive oxygen species, which can effectively kill susceptible tumor cells. In contrast, M2-polarized macrophages can secrete different factors that promote tumor cell growth and survival or favor angiogenesis and tissue invasion. Considering the beneficial role of TAMs in colon cancer, we speculated that they may not display the M2 polarization commonly observed in tumor microenvironment, but rather develop M1 properties. Therefore, we used an in vitro model to analyze the effects of supernatants from M1-polarized macrophages on DLD-1 colon cancer cells. Our data indicate that the conditioned medium from LPS-activated macrophages (CM-LAM) contains a high level of granulocyte-macrophage colony-stimulating factor, interleukins-1 beta, -6, -8 and tumor necrosis factor-alpha, and that it exerts a marked growth inhibitory activity on DLD-1 cells. Prolonged exposure to CM-LAM results in cell death by apoptosis. Such exposure to CM-LAM leads to the modulation of gal-3 expression: we observed a marked downregulation of gal-3 mRNA and protein expression following CM-LAM treatment. We also describe that the knockdown of gal-3 sensitizes DLD-1 cells to CM-LAM. These data suggest an involvement of gal-3 in the response of colon cancer cells to proinflammatory stimuli, such as the conditioned medium from activated macrophages.

The soluble-type lectins or galectins constitute a family of proteins defined by shared consensus amino acid sequence and affinity for beta-galactose-containing oligosaccharides. These molecules are widely distributed in the animal kingdom; to date, 15 mammalian galectins have been described but more are likely to be discovered. These proteins are involved in many biological processes including cell-cell and cell-matrix adhesion, growth regulation, signaling, and cytokine secretion. Over the last decade, a vast amount of reports has shown the importance of several galectins in the development and progression of malignancies in the digestive tract, mainly colorectal cancers. More recent data indicate that some of these molecules are also involved in inflammatory bowel diseases. This review focuses on the current knowledge of galectin expression and putative functions in the oesophagus, stomach, small intestine, and colon. It also highlights that the rapid accumulation of research data promises future scenarios in which individual members of the galectin family and/or their ligands will be used as diagnostic and therapeutic modalities for neoplastic as well as inflammatory disorders. However, the concretization of these potential modalities requires substantial improvements in terms of standardization of galectin expression evaluation together with prospective validation of the present data.

Amonafide (1), a naphthalimide which binds to DNA by intercalation and poisons topoisomerase IIalpha, has demonstrated activity in phase II breast cancer trials, but has failed thus far to enter clinical phase III because of dose-limiting bone marrow toxicity. Compound 17 (one of 41 new compounds synthesized) is a novel anticancer naphthalimide with a distinct mechanism of action, notably inducing autophagy and senescence in cancer cells. Compound 17 (2,2,2-trichloro-N-({2-[2-(dimethylamino)ethyl]-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-5-yl}carbamoyl)acetamide (UNBS3157)) was found to have a 3-4-fold higher maximum tolerated dose compared to amonafide and not to provoke hematotoxicity in mice at doses that display significant antitumor effects. Furthermore, 17 has shown itself to be superior to amonafide in vivo in models of (i) L1210 murine leukemia, (ii) MXT-HI murine mammary adenocarcinoma, and (iii) orthotopic models of human A549 NSCLC and BxPC3 pancreatic cancer. Compound 17, therefore, merits further investigation as a potential anticancer agent.

Following a genotoxic stress, the tumor suppressor p53 translocates to mitochondria to take part in direct induction of apoptosis, via interaction with BCL-2 family members such as BAK and BAX. We determined the kinetics of the mitochondrial translocation of p53 in HCT-116 and PA-1 cells exposed to different genotoxic stresses (doxorubicin, camptothecin, UVB). This analysis revealed an early escalation in the amount of mitochondrial p53, followed by a peak amount and a decrease of mitochondrial p53 at later time points. We show that the serine 20 phosphorylated form of p53 is present at the mitochondria and that the decrease of p53 mitochondrial level during late apoptosis correlates with a decrease of Ser-20 phosphorylation. Moreover, the S20A p53 mutant translocates well to mitochondria after a genotoxic stress but its mitochondrial localization is very low during late apoptosis when compared to wt p53. The S20A mutant also appears to be compromised for interaction with BAK. We propose here that the level of serine 20 phosphorylation is influential on p53 mitochondrial localization during late apoptosis. Additionally, we report the presence of a new ≃45 kDa caspase-cleaved fragment of p53 in the cytosolic and mitochondrial fractions of apoptotic cells.

International audience; The transcription factor E-twenty-six version 5 (ETV5) has been linked with obesity in genome-wide association studies. Moreover, ETV5-deficient mice (knockout; KO) have reduced body weight, lower fat mass, and are resistant to diet-induced obesity, directly linking ETV5 to the regulation of energy balance and metabolism. ETV5 is expressed in hypothalamic brain regions that regulate both metabolism and HPA axis activity, suggesting that ETV5 may also modulate HPA axis function. In order to test this possibility, plasma corticosterone levels were measured in ETV5 KO and wildtype (WT) mice before (pre-stress) and after (post-stress) a mild stressor (intraperitoneal injection). ETV5 deficiency increased both pre- and post-stress plasma corticosterone, suggesting that loss of ETV5 elevated glucocorticoid tone. Consistent with this idea, ETV5 KO mice have reduced thymus weight, suggestive of increased glucocorticoid-induced thymic involution. ETV5 deficiency also decreased the mRNA expression of glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and vasopressin receptor 1A in the hypothalamus, without altering vasopressin, corticotropin-releasing hormone, or oxytocin mRNA expression. In order to test whether reduced MR and GR expression affected glucocorticoid negative feedback, a dexamethasone suppression test was performed. Dexamethasone reduced plasma corticosterone in both ETV5 KO and WT mice, suggesting that glucocorticoid negative feedback was unaltered by ETV5 deficiency. In summary, these data suggest that the obesity-associated transcription factor ETV5 normally acts to diminish circulating glucocorticoids. This might occur directly via ETV5 actions on HPA-regulatory brain circuitry, and/or indirectly via ETV5-induced alterations in metabolic factors that then influence the HPA axis.

Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural polyphenol synthesized by various plants such as grape vine. Resveratrol (RSV) is a widely studied molecule, largely for its chemopreventive effect in different mouse cancer models. We propose a mechanism underlying the cytotoxic activity of RSV on colon cancer cells. Our data show that resveratrol induces apoptosis, as observed by the cleavage of PARP-1 and chromatin condensation. We show that the tumor suppressor p53 is activated in response to RSV and participates to the apoptotic process. Additionally, we show that HCT-116 p53 wt colon carcinoma cells are significantly more sensitive than HCT-116 p53-/- cells to RSV. RSV induces DNA damage including double strand breaks, as evidenced by the presence of multiple γ-H2AX foci in 50% of cells after a 24. h treatment with 25. μM RSV. The formation of DNA damage does not appear to rely on a pro-oxidant effect of the molecule, inhibition of topoisomerase I, or DNA intercalation. Rather, we show that DNA damage is the consequence of type II topoisomerase poisoning. Exposure of HCT-116 cells to RSV leads to activation of the Ataxia Telangiectasia Mutated (ATM) kinase, and ATM is required to activate p53.

Non-small cell lung cancers (NSCLCs) are the leading cause of cancer deaths in most developed countries. Targeting heat shock protein 70 (Hsp70) expression and function, together with the induction of lysosomal membrane permeabilization (LMP), could overcome the multiple anti-cell death mechanisms evidenced in NSCLCs that are responsible for the failure of currently used chemotherapeutic drugs. Because cardenolides bind to the sodium pump, they affect multiple signaling pathways and thus have a number of marked effects on tumor cell behavior. The aim of the present study was to characterize in vitro and in vivo the antitumor effects of a new cardenolide (UNBS1450) on experimental human NSCLCs. UNBS1450 is a potent source of in vivo antitumor activity in the case of paclitaxel-and oxaliplatin-resistant subcutaneous human NCI-H727 and orthotopic A549 xenografts in nude mice. In vitro UNBS1450-mediated antitumor activity results from the induction of nonapoptotic cell death. UNBS1450 mediates the decrease of Hsp70 at both mRNA and protein levels, and this is at least partly due to UNBS1450-induced downregulation of NFAT5/TonEBP (a factor responsible for the transcriptional control of Hsp70). These effects were paralleled by the induction of LMP, as evidenced by acridine orange staining and immunofluorescence analysis for cathepsin B accumulation., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The incidence of prostate cancer is increasing in western countries because of population aging. Prostate cancer begins as an androgen-dependent disease, but it can become androgen independent at a later stage or in tumors recurring after an antihormonal treatment. Although many genetic events have been described to be involved in androgen-dependent and/or -independent prostate cancer growth, little is known about the contribution of epigenetic events. Here we have examined the possibility that the methyl-CpG-binding protein MECP2 might play a role in controlling the growth of prostate cancer cells. Inhibition of MECP2 expression by stable short hairpin RNA stopped the growth of both normal and cancer human prostate cells. In addition, ectopic expression of the MECP2 conferred a growth advantage to human prostate cancer cells. More importantly, this expression allowed androgen-dependent cells to grow independently of androgen stimulation and to retain tumorigenic properties in androgen-depleted conditions. Analysis of signaling pathways showed that this effect is independent of androgen receptor signaling. Instead, MECP2 appears to act by maintaining a constant c-myc level during antihormonal treatment. We further show that MECP2-expressing cells possess a functional p53 pathway and are still responsive to chemotherapeutic drugs.

The gene TP53, encoding p53, has a common sequence polymorphism that results in either proline or arginine at amino-acid position 72. This polymorphism occurs in the proline-rich domain of p53, which is necessary for the protein to fully induce apoptosis. We found that in cell lines containing inducible versions of alleles encoding the Pro72 and Arg72 variants, and in cells with endogenous p53, the Arg72 variant induces apoptosis markedly better than does the Pro72 variant. Our data indicate that at least one source of this enhanced apoptotic potential is the greater ability of the Arg72 variant to localize to the mitochondria; this localization is accompanied by release of cytochrome c into the cytosol. These data indicate that the two polymorphic variants of p53 are functionally distinct, and these differences may influence cancer risk or treatment.

No consensus exists so far on the definition of cellular senescence. The narrowest definition of senescence is irreversible growth arrest triggered by telomere shortening counting cell generations (definition 1). Other authors gave an enlarged functional definition encompassing any kind of irreversible arrest of proliferative cell types induced by damaging agents or cell cycle deregulations after overexpression of proto-oncogenes (definition 2). As stress increases, the proportion of cells in “stress-induced premature senescence-like phenotype” according to definition 1 or “stress-induced premature senescence,” according to definition 2, should increase when a culture reaches growth arrest, and the proportion of cells that reached telomere-dependent replicative senescence due to the end-replication problem should decrease. Stress-induced premature senescence-like phenotype and telomere-dependent replicatively senescent cells share basic similarities such as irreversible growth arrest and resistance to apoptosis, which may appear through different pathways. Irreversible growth arrest after exposure to oxidative stress and generation of DNA damage could be as efficient in avoiding immortalisation as “telomere-dependent” replicative senescence. Probabilities are higher that the senescent cells (according to definition 2) appearingin vivoare in stress-induced premature senescence rather than in telomere-dependent replicative senescence. Examples are given suggesting these cells affectin vivotissue (patho)physiology and aging.

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

WI-38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 microM tert-butylhydroperoxide (t-BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 microM H2O2 stress or five repeated 30 microM t-BHP stresses along the same PD, respectively a 322 +/- 55 and 380 +/- 129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress.

No alternative in vitro method exists fordetecting the potential long-term genotoxic effects ofmolecules at subcytotoxic concentrations, in terms ofdays and weeks after exposure(s) to the moleculetested. A theoretical model of cellular senescence ledto the concept that subcytotoxic stresses under anymolecules at subcytotoxic doses, such as moleculesunder development in the pharmaceutical, cosmetics andfood industry, might lead human fibroblasts into a stateclosely related to in vitro senescence. Thisconcept was then experimentally confirmed invitro: many biomarkers of replicative senescence ofhuman fibroblasts were found 72 h after theirexposure to various kinds of stressors used at non-cytotoxic concentrations. This phenomenon has beentermed stress-induced premature senescence (SIPS).Moreover, proteomics studies have revealed that,besides their effects on the appearance of thebiomarkers of senescence, sublethal stresses under avariety of stressors also lead to long-term specificchanges in the expression level of proteins which arestress-specific. These changes have been coined themolecular scars of stress. The proteins correspondingto these molecular scars may be identified using thelatest developments in mass spectrometry. This modelof stress-induced premature senescence may be appliedto the toxicological sciences when testing for thepotential irreversible long-term effects of moleculeson the cell fate.

The Ets family transcription factor Pea3 (ETV4) is involved in tumorigenesis especially during the metastatic process. Pea3 is known to induce migration and invasion in mammary epithelial cell model systems. However, the molecular pathways regulated by Pea3 are still misunderstood. In the current study, using in vivo and in vitro assays, Pea3 increased the morphogenetic and tumorigenic capacity of mammary epithelial cells by modulating their cell morphology, proliferation, and migration potential. In addition, Pea3 overexpression favored an epithelial–mesenchymal transition (EMT) triggered by TGF-β1. During investigation for molecular events downstream of Pea3, Cyclin D2 (CCND2) was identified as a new Pea3 target gene involved in the control of cellular proliferation and migration, a finding that highlights a new negative regulatory loop between Pea3 and Cyclin D2. Furthermore, Cyclin D2 expression was lost during TGF-β1–induced EMT and Pea3-induced tumorigenesis. Finally, restored Cyclin D2 expression in Pea3-dependent mammary tumorigenic cells decreased cell migration in an opposite manner to Pea3. As such, these data demonstrate that loss of the negative feedback loop between Cyclin D2 and Pea3 contributes to Pea3-induced tumorigenesis. Implications: This study reveals molecular insight into how the Ets family transcription factor Pea3 favors EMT and contributes to tumorigenesis via a negative regulatory loop with Cyclin D2, a new Pea3 target gene. Mol Cancer Res; 11(11); 1412–24. ©2013 AACR.

International audience; The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells.

Caspases play a key role in the apoptotic pathway by virtue of their ability to cleave key protein substrates within the dying cell. Caspases are produced as inactive zymogens, and need to become proteolytically processed in order to become active. A key executioner caspase, caspase-3, has previously been found to exist in both the cytosol and the mitochondria. At the mitochondria, caspase-3 is associated with both the inner and outer mitochondrial membranes, where it interacts with heat shock proteins Hsp60 and Hsp10. Like caspase-3, a small portion of the p53 tumor suppressor protein is localized to mitochondria, particularly after genotoxic stress. p53 interacts with various members of the Bcl2 family at the mitochondria, and this interaction is key to its ability to induce apoptosis. In this study, we sought to determine the identity of other mitochondrial p53-interacting proteins. Using immunoprecipitation from purified mitochondria followed by mass spectrometry we identified caspase-3 as a mitochondrial p53-interacting protein. Interestingly, we find that tumor-derived mutant forms of p53 retain the ability to interact with mitochondrial caspase-3. Further, we find evidence that these mutant forms of p53 may interfere with the ability of procaspase-3 to become proteolytically activated by caspase-9. The combined data suggest that tumor-derived mutants of p53 may be selected for in tumor cells due to their ability to bind and inhibit the activation of caspase-3.

Pea3 and Erm are transcription factors expressed in normal developing branching organs such as the mammary gland. Deregulation of their expression is generally associated with tumorigenesis and particularly breast cancer. By using RNA interference (RNAi) to downregulate the expression of Pea3 and/or Erm in a mammary cancer cell line, we present evidence for a role of these factors in proliferation, migration and invasion capacity of cancer cells. We have used different small interfering RNAs (siRNAs) targeting pea3 and erm transcripts in transiently or stably transfected cells, and assessed the physiological behavior of these cells in in vitro assays. We also identified an in vivo alteration of tumor progression after injection of cells that overexpress pea3 and/or erm short hairpin RNAs (shRNAs) in immunodeficient mice. Using transcriptome profiling in Pea3- or Erm-targeted cells, two largely independent gene expression programs were identified on the basis of their shared phenotypic modifications. A statistically highly significant part of both sets of target genes had previously been already associated with the cellular signaling pathways of the ;proliferation, migration, invasion' class. These data provide the first evidence, by using endogenous knockdown, for pivotal and complementary roles of Pea3 and Erm transcription factors in events crucial to mammary tumorigenesis, and identify sets of downstream target genes whose expression during tumorigenesis is regulated by these transcription factors., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

From the conditioned medium of the human colon carcinoma cells, HT-29 5M21 (CM-5M21), expressing a spontaneous invasive phenotype, tumor-associated trypsin inhibitor (TATI) was identified and characterized by proteomics, cDNA microarray approaches and functional analyses. Both CM-5M21 and recombinant TATI, but not the K18Y-TATI mutant at the protease inhibitor site, trigger collagen type I invasion by several human adenoma and carcinoma cells of the colon and breast, through phosphoinositide-3-kinase, protein kinase C and Rho-GTPases/Rho kinase-dependent pathways. Conversely, the proinvasive action of TATI in parental HT29 cells was alleviated by the TATI antibody PSKAN2 and the K18Y-TATI mutant. Stable expression of K18Y-TATI in HT-29 5M21 cells downregulated tumor growth, angiogenesis and the expression of several metastasis-related genes, including CSPG4 (13.8-fold), BMP-7 (9.7-fold), the BMP antagonist CHORDIN (5.2-fold), IGFBP-2 and IGF2 (9.6- and 4.6-fold). Accordingly, ectopic expression of KY-TATI inhibited the development of lung metastases from HT-29 5M21 tumor xenografts in immunodeficient mice. These findings identify TATI as an autocrine transforming factor potentially involved in early and late events of colon cancer progression, including local invasion of the primary tumor and its metastatic spread. Targeting TATI, its molecular partners and effectors may bring novel therapeutic applications for high-grade human solid tumors in the digestive and urogenital systems.

In addition to a well-defined transcriptional activity that is necessary for efficient apoptosis induction, the p53 tumor suppressor also has a direct apoptogenic role at the mitochondria. This direct role in cell death is mediated at least in part by interaction of p53 with BCL2 family members, including the pro-apoptotic protein BAK. Whereas it is currently accepted that the mitochondrial function of p53 contributes to its tumor suppressive role, the regulation of p53 function at this organelle is poorly understood. In this manuscript we examine the role of p53 oligomerization in the regulation of its pro-apoptotic function at the mitochondria, specifically in regard to its ability to induce BAK oligomerization. We find that deletion or mutation of p53's oligomerization domain markedly impairs the ability of this protein to oligomerize BAK. Along these lines, cross-linking studies indicate that the majority of p53 localized to mitochondria is in dimeric or higher-order oligomeric form. In support of the importance of the p53-BAK interaction in the localization of p53 to mitochondria, we find that mouse embryo fibroblasts from the BAK null mouse have greatly reduced mitochondrial p53 compared to wild type fibroblasts. These data indicate that pro-apoptotic BAK, unlike other BCL2 family members, may serve as a major receptor for p53 on the mitochondria.

Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosismediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation.

In addition to a common polymorphism at codon 72, the p53 tumor suppressor gene also contains a rare single nucleotide polymorphism at amino acid 47. Wild type p53 encodes proline at this residue, but in

The tumour suppressor activity of the p53 protein has been explained by its ability to induce apoptosis in response to a variety of cellular stresses1,2. Thus, understanding the mechanism by which p53 functions in the execution of cell death pathways is of considerable importance in cancer biology. Recent studies have indicated that p53 has a direct signalling role at mitochondria in the induction of apoptosis3,4,5,6, although the mechanisms involved are not completely understood. Here we show that, after cell stress, p53 interacts with the pro-apoptotic mitochondrial membrane protein Bak. Interaction of p53 with Bak causes oligomerization of Bak and release of cytochrome c from mitochondria. Notably, we show that formation of the p53–Bak complex coincides with loss of an interaction between Bak and the anti-apoptotic Bcl2-family member Mcl1. These results are consistent with a model in which p53 and Mcl1 have opposing effects on mitochondrial apoptosis by interacting with, and modulating the activity of, the death effector Bak.

This study investigates the involvement of the c- cbl proto-oncogene during the first stages of the apoptotic process. We have already shown that a c-Cbl aptotosis-related protein of 90kDa (CARP 90) is detected very rapidly in the cytoplasm as well as in the nucleus of murine thymocytes after hydrocortisone (HC) treatment. We report here that this protein appeared as well after in vivo treatment of mice by γ-irradiation or injection of anti-CD3 monoclonal antibody, two potent thymic apoptosis inductors, providing a close relationship between the occurrence of apoptosis and the appearance of CARP 90. We showed that CARP 90 and p120 cbl share numerous epitopes strikingly suggesting that CARP 90 is coded by c- cbl . In addition, KO mice do not sustain CARP 90 appearance. We finally showed that CARP 90 contains N- and C-terminal end epitopes of p120 cbl , which suggests that CARP 90 is an alternative spliced form of c- cbl . This protein was also observed under γ-irradiation in tissues of different origin, which enlarges the physiological significance of this phenomenon. The very rapid CARP 90 appearance under apoptotic conditions in the nucleus of cells originating in different tissues makes this protein if not a possible new actor of the apoptotic process, at least an interesting marker of this process.

Replicative senescence is a process in which normal proliferative cells lose replicative potential and acquire biomarkers of cellular senescence as a result of serial passages in culture instead of chronological time. Oxidative stress-induced premature senescence is the long-term appearance of the biomarkers of replicative senescence after exposure of human diploid cells to sublethal stress. Cytokine-induced premature senescence is the long-term appearance of biomarkers of replicative senescence after repeated exposure of human diploid fibroblasts to proinflammatory cytokines. Keywords: stress-induced premature senescence; oxidative stress; fibroblasts; cytokines; toxicology

Sublethal oxidative stresses increase the proportions of human fibroblasts positive for senescence associated beta-galactosidase activity and accelerate the transition in the fibroblast morphotypes characterising fibroblast ageing. Stimulation of fibroblasts with TNF-alpha or IL-1alpha transiently increases the production of reactive oxygen species (ROS) in human fibroblasts. Here we propose that repeated stimulation of WI-38 fibroblasts with TNF-alpha or IL-1alpha can generate enough ROS to accelerate the transition in the fibroblast morphotypes and increase the proportion of cells positive for senescence associated beta-galactosidase activity. The involvement of ROS is suggested by experiments where the stimulation of fibroblasts with TNF-alpha or IL-1alpha are performed in the presence of N-acetylcysteine which increases the intracellular antioxidant potential. It is proposed that the decrease in the proportions of morphotypes I and II, and the increase in the proportions of morphotypes III to VI observed after successive stimulation with TNF-alpha or IL1-alpha is attributed to an increased ROS production occurring during the stimulation.

International audience; In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg) during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.

The experiments aimed at evaluating the optimal parameters in the chemo-immunotherapeutic treatment of the L1210 lymphoid leukaemia grafted to [female BALB/c (H2d) X male DBA/2 (H2d)]F1 hybrid mice, hereafter referred to as CDF1 mice. In vitro irradiation of leukaemic ascites cells by X- or gamma-rays and subsequent inoculation in mice showed that optimum immunogenicity is radiation dose-dependent. Grafting mice with 10(7) leukaemic ascites cells irradiated at optimum dose (80 GyX- or gamma-rays) delays mortality of the animals when challenged later with untreated L1210 cells, but is unable to cure mice. By contrast, specific immunoprophylaxis induced by Micrococcus, complement-triggering polysaccharides or BCG and irradiated leukaemic cells was able to protect mice against grafts of 10(4) L1210 cells. The i.p. route was notably superior to the i.v. route. When mice bearing advanced L1210 tumour were treated by chemotherapy (12 mg/kg of BCNU) on Day 6.5 after grafting 10(4) L1210 cells and subsequently treated by immunotherapy, a very high percentage (up to 90%) of mice with 10(8) leukaemic cells could be cured by repeated 1mg injections of bacterium or polysaccharide, and challenge with irradiated leukaemic cells was unnecessary. Because of the high cure rate obtained, the very regular response pattern and the non-pathogenicity, the bacterium Micrococcus lysodeikticus would seem a promising new candidate for chemo-immunotherapeutic antitumour strategies.

In the subrenal capsule assay, the immuno-competent mice used have a host immune response to the graft which may introduce a bias in the determination of tumour size. A study was therefore conducted to verify the quality of the correlation that could exist between "macroscopical" and "microscopical" evaluation. A histological study of 12 different tumours treated with cis-Pt, L-PAM, DTIC, vindesine, BCNU or TGU demonstrated that a cellular host immune response was observed in 11 out of the 12 control groups and was uneven in the treated groups. The "microscopical" and "macroscopical" evaluations of tumour-take were 100 and 95% respectively. Although there was a fair correlation between the "microscopic" and "macroscopic" parameters, tumour variations in absence of host reaction--which reflect pure chemosensitivity of the treated tissue--could be measured in 50% of the cases. It was possible to determine a base-line for rejection of the test results when the control group showed a decrease in mean tumour size exceeding 20% of its initial size on day 0. If the decrease is less than 20%, then the histological evaluation appears to be of considerable additional value.

The 6 day subrenal capsule (SRC) assay was performed in normal mice using 20 fresh human non-small cell lung cancers and nine fresh ovarian cancers. Different multi-agent chemotherapy regimens administered intravenously on days 2 + 3 of the assay were evaluated for activity against these two tumor types. Macroscopic results measured via an ocular microscope showed high activity for some combinations as well as for single agents. However, when subjected to microscopic examination, the SRC implants in the untreated control animals did not show viable tumor growth on day 6. Detailed histologic evaluation of over 800 SRC grafts reveals an intense inflammatory and fibrotic reaction in the majority of the grafts. These results indicate that drug sensitivity patterns obtained with this assay using only macroscopic criteria do not correlate with actual tumor regression. Microscopic analysis of the grafts prior to transplant shows absence or only minimal presence of tumor in many cases which also contributes to the poor growth observed in these two tumors.

The influence of the micrococcal vaccine on the proliferation of normal immunocompetent and neoplastic plasma (MOPC 173 ) and of carcinoma (Ehrlich ascites) cells is studied. BALBc mice and CDF 1 mice, hypervaccinated with Micrococcus Lysodeikticus , developed significantly ( 0.001 0.005 ) lower primary immune responses against sheep erythrocytes and also displayed impaired secondary immune responses. Administration of Bacillus Calmette-Guerin could partially restore the immunologic potential of vaccinated mice as measured by the capability of mice to answer with primary or secondary immune responses to sheep erythrocytes but did not increase immunoresponsiveness of normal mice sensitized at nearly optimal antigenic dose. Switching on a secondary antimicrococcus immune response after grafting 200,000 Ehrlich carcinoma cells in the peritoneum of BALBc mice resulted in a 30% increase in mean life span over control mice. However, mice hyperimmunized for months with Micrococcus lysodeikticus were offered the strongest immunoprotection and we found 67% of mice surviving to day 90 whereas the other vaccinated but tumour bearing animals showed a 29% increase in life span over control mice. Similarly, we demonstrated that 44% of BALBc mice, hyperimmunized for months, were able to reject a tumour challenge of 200,000 grafted MOPC 173 plasmacytoma cells, whereas the other micrococcus vaccinated but tumour bearing animals died with a 26% increase in mean survival time over control mice.

When testing in vitro, we often observed that erythrocytes taken from Ehrlich ascites tumor bearing mice displayed enhanced agglutinability by the lectin Concanavalin A, suggesting that protease activity operates in vivo. In several studies, we were able to inhibit Ehrlich ascites tumor growth by the repeated administration of soya bean trypsin inhibitor. Studies of dosages and schedules of treatment showed that for 20,000 initially grafted cells, treatment resulted in 0–70% of long-term survivors and induced a significant increase in mean survival time of treated mice over control mice. For 200,000 grafted tumor cells, 0–40% of long-term survivors were recorded. In a comparative study, we found that different inhibitors of urokinase displayed a similar chemotherapeutic effect against 200,000 inoculated cells. Our results corroborate the idea that a plasminogen activator monitored chain of fibrinolytic and proteolytic activity controls tumor growth and metastasis enzymatically.

A comparative study of the effects of BCG, Micrococcus lysodeikticus, and a series of structurally related polysaccharides (complement triggers) on the non-specific and specific immune resistance against L1210 lymphoid leukaemia was carried out and commented on. In contrast with authors of earlier reports, we were unable to generate any effective non-specific or specific immunotherapy after the graft of 10(4) leukaemic cells to 8--10-week-old CDF1 mice. However, when mice were prevaccinated with irradiated (8 krad X-rays) cultured cells combined with 1 mg of bacterium or polysaccharide one month before grafting 10(4) cells, they were given an immunoprotection that was more pronounced with the i.p. than with the i.v. route. Prevaccinated mice were afforded a stronger immunoprotection when boosted repeatedly with 1mg injections of bacterium or polysaccharide after tumour challenge.

An immunization schedule that leads to the production of antimicrococcus antibodies of restricted electrophoretic heterogeneity in (female BALB/c × male DBA/2) F1 mice (CDF1) is described and commented on. Although mouse antimicrococcus sera agglutinate cultured L1210 cells in vitro, micrococcus vaccinated mice never showed any anti-leukaemia immunoprotection. However, vaccinated mice, given booster doses of 1 mg heat-killed Micrococcus lysodeikticus on days 4, 8 and 12 after a tumour challenge of 104 cells, were considerably protected (P Our results underline the idea that, after establishment of tumour growth, the humoral antitumour immunity requires an adequate trigger of immunocompetent effector cells to be operationally effective.

By analyzing, at different times after grafting 300,000 Ehrlich ascites cells to BALB/c mice, the primary immune response to 2.10 8 sheep erythrocytes, the carcinoma monitored immunosuppression was outlined. The fact that murine ascites fluid contains less immunoglobulins and less pre-albumin migrating components than serum of tumour bearing mice suggests that the tumour site may be the consuming focus. When different treatment schedules were assayed, we found that the intraperitoneal route (intratumoral) was better than other treatment routes: for 30,000 initially grafted tumour cells, i.p. treatment with 0.08 and 0.4 mg heat-killed Micrococcus on day 1, 3, 5, 7 and 9 resulted respectively in a 120 and 148% increase in mean life span over control mice and 50 and 20% of long-term survivors were recorded. However, the administration of 1 mg Micrococcus on days 1, 5, 9 and 12 after grafting 300,000 Ehrlich carcinoma cells considerably enhanced tumour growth. The administration of 1 mg of Micrococcus, cell wall, cell wall conjugated chitin, chitin and zymosan A on days 1, 2, 3, 4 and 5 resulted in a 71, 45, 29.5, 68 and 82% increase in mean life span over control mice and 30, 20 and 30% of long-term survivors were recorded for chitin, cell wall conjugated chitin and zymosan A respectively.