Hexyl-aminolevulinate mediated photodynamic therapy: how to spare normal urothelium. An in vitro approach

Résumé Objectifs : La thérapie photodynamique a pour but la destruction sélective du tissu néoplasique par interaction de lumière, d'oxygène et d'une substance photosensibilisatrice (la Protoporphyrine IX dans notre étude). Malgré une accumulation sélective du photosensibilisateur dans le tissu tumoral, la thérapie photodynamique du carcinome urothélial de la vessie peut endommager les cellules normales de l'épithélium urinaire. La prévention de ces lésions est importante pour la régénération de la muqueuse. Notre étude sur un modèle in vitro d'urothélium porcin étudie l'influence de la concentration du photosensibilisateur, des paramètres d'irradiation et de la production d'intermédiaires réactifs de l'oxygène (ROS) sur les effets photodynamique. Le but était de déterminer les conditions seuil pour épargner l'urothélium sain. Méthode: Dans une chambre de culture transparente à deux compartiments, des muqueuses vésicales de porc maintenues en vie ont été incubées avec une solution d'hexyl-aminolévulinate (HAL), le précurseur de la Protoporphyrine IX. Ces muqueuses ont ensuite été irradiées avec des doses lumineuses croissantes en lumière bleue et en lumière blanche, et les altérations cellulaires ont été évaluées par microscopie électronique à balayage et par un colorant fluorescent, le Sytox green. Nous avons également évalué la production d'intermédiaires réactifs de l'oxygène parla mesure de la fluorescence intracellulaire de Rhodamine 123 (R123), produit de l'oxydation de la Dihydrorhodamine 123 (DHR123) non fluorescente. Ces valeurs ont été corrélées avec celles du photo blanchiment de la PAIX. Résultats : Le taux de mortalité cellulaire était dépendant de la concentration de PAIX. Après 3 heures d'incubation, la valeur seuil de dose lumineuse pour la lumière bleu était de 0.15 et 0.75 J/cm2 (irradiance 30 et 75 mW/cm2, respectivement) et pour la lumière blanche de 0.55 J/cm2 (irradiante 30 mW/cm2). Le taux de photo blanchiment était inversement proportionnel à l'irradiante. Le système de détection des intermédiaires réactifs de l'oxygène DHR123/R123 a démontré une bonne corrélation avec les valeurs seuil pour toutes les conditions d'irradiation utilisées. Conclusions : Nous avons déterminé les doses lumineuses permettant d'épargner 50% des cellules urothéliales saines. L'utilisation d'une faible irradiante associée à des systèmes permettant de mesurer la production d'intermédiaires réactifs de l'oxygène dans les tissus irradiés pourrait améliorer la dosimétrie in vivo et l'efficacité de la thérapie photodynamique. Abstract Background and Objectives: Photodynamic therapy of superficial bladder cancer may cause damages to the normal surrounding bladder wall. Prevention of these is important for bladder healing. We studied the influence of photosensitizes concentration, irradiation parameters and production of reactive oxygen species (ROS) on the photodynamically induced damage in the porcine urothelium in vitro. The aim was to determine the threshold conditions for the cell survival. Methods: Living porcine bladder mucosae were incubated with solution of hexylester of 5-aminolevulinic acid (HAL). The mucosae were irradiated with increasing doses and cell alterations were evaluated by scanning electron microscopy and by Sytox green fluorescence. The urothelial survival score was correlated with Protoporphyrin IX (PpIX) photobleaching and intracellular fluorescence of Rhodamine 123 reflecting the ROS production. Results: The mortality ratio was dependent on PpIX concentration. After 3 hours of incubation, the threshold radiant exposures for blue light were 0.15 and 0.75 J/cm2 (irradiance 30 and 75 mW/cm2, respectively) and for white light 0.55 J/cm2 (irradiance 30 mW/cm2). Photobleaching rate increased with decreasing irradiance. Interestingly, the DHR123/R123 reporter system correlated well with the threshold exposures under all conditions used. Conclusions: we have determined radiant exposures sparing half of normal urothelial cells. We propose that the use of low irradiance combined with systems reporting the ROS production in the irradiated tissue could improve the in vivo dosimetry and optimize the PDT.

Construction and analysis of a new conditional ferritin H knockout mouse strain/

Résumé Le fer joue un rôle important dans la plupart des fonctions biologiques mais sa présence excessive provoque la production de molécules réactives d'oxygène (ROS) qui peuvent contribuer à diverses maladies. La protéine de stockage du fer, la ferritine H, capte l'excès en fer et le stocke sous forme non-toxique, ce qui empêche des dommages potentiels. La délétion de la ferritine H dans des souris knock-out a été essayée antérieurement, mais ces souris mouraient au stade précoce du développement embryonnaire. Pour étudier l'importance du fer, et en particulier son stockage dans la ferritine, et pour pouvoir mieux comprendre les fonctions de la ferritine H, nous avons créé un modèle de souris knock-out conditionnelles de la ferritine H, selon le système classique de Cre-LoxP. Le premier exon et la région du promoteur du gène de la ferritine H ont été entourés de sites loxP. La mortalité embryonnaire provoquée par la délétion constitutive du gène de la ferritine H a été confirmée en croisant nos souris avec des souris exprimant nestin-Cre1. En croisant nos souris avec des souris transgéniques Mx-Cre, nous avons observé que l'induction de Cre par injection de polyI-polyC provoque la délétion presque complète de la ferritine H dans le foie (> 99%) et la rate (> 88%). Ces tissus ont également perdu une grande partie de leur réserve de fer. Cette observation apporte pour la première fois la preuve in vivo que la ferritine H est indispensable pour le stockage du fer, que les fonctions de la ferritine H et de la ferritine L ne sont pas équivalentes, et que la ferritine L ne peut pas assumer seule la fonction de stockage du fer. Dans le foie des souris knock-out, l'expression de l'ARN messager de l'hepcidine a été induite après 10 jours. En même temps, l'expression de l'ARN messager des gènes codant pour des protéines de l'absorption de fer (DMT1, ferroportin, Dcytb1 et hephaestin) a été réprimée mais dans le duodénum seulement. L'expression d'hepcidine est inversément corrélée avec celle des gènes liés à l'absorption de fer. Cette observation corrobore des études antérieures. Mais, en plus, elle montre également que cette répression se produit seulement dans l'intestin. Nous pouvons ainsi tirer la conclusion suivante : ou bien l'hepcidine a un récepteur spécifique dans le duodénum ou bien les gènes liés à l'absorption de fer dans le duodénum ont un facteur spécifique de transcription sensible à l'hepcidine. Aucune répression de DMT1 et de ferroportin n'a été observée dans les macrophages de la rate après l'induction d'hepcidine. La délétion de ferritine H a entraîné une augmentation du taux de mortalité des cellules hépatiques, ainsi que des altérations dans l'architecture normale du tissu de la rate. Vu par l'immunohistologie, le nombre de lymphocytes B et T était réduit dans la rate, tendant à démontrer que la ferritine H et l'homéostase du fer jouent un rôle dans l'immunité. En conclusion, le modèle de souris knock-out conditionnelles de la ferritine H nous fournit un outil précieux pour l'étude in vivo du rôle joué par la ferritine dans l'homéostase du fer, dans les dommages créés par les ROS, ainsi que dans l'apoptose et l'immunité. Summary Iron plays an important role in most biological functions. However, excess of iron results in production of reactive oxygen species (ROS) which could substantially contribute to pathology of various diseases. Ferritin H scavenges excess of iron and stores it in non-toxic form and potentially prevents the damage. Fenitin H targeting in mice has been attempted before, however, straight knockout was lethal in early embryonic stage. To study the role of iron and its storage protein ferritin and to further elucidate ferritin H functions, we aimed at creating a conditional ferritin H knockout mouse model by classical Cre-LoxP system. First exon along with promoter region of the ferritin H gene was foxed. Embryonic lethality of the constitutive ferritin H deletion was confirmed by crossing the foxed mice with mice expressing nestin Cre-1 as transgene. Almost complete deletion was observed in liver (> 99%) and spleen (>88%) upon induction of Cre by injecting polyI-polyC in Fth Lox/Lox; MxCre mice. These tissues also lost substantial fraction of their iron stores. This provides first in vivo evidence that ferritin H is required for iron storage, ferritin H and L functions are not redundant and that ferritin L cannot perform iron storage function alone. Hepcidin mRNA expression was induced after 10 days in the livers of deleted mice and, simultaneously, mRNA expression of iron absorption related genes (DMT 1, ferroportin, Dcytb1 and hephaestin) was repressed in duodenum only. Hepcidin expression is inversely correlated with that of duodenal iron absorption related genes. This is in agreement with previous studies. However, we also show that this repression happens only in intestine. This leads to the conclusion that either hepcidin has a specific receptor in duodenum or the iron absorption related genes have duodenum specific transcription factor that is responsive to hepcidin. No repression of DMT1 and ferroportin was observed in spleen macrophages upon hepcidin induction. Ferritin H deletion showed increased cell death in liver and disruption of normal architecture of spleen. B lymphocytes were reduced in spleen on immunohistology which point towards a role of ferritin H and iron homeostasis in immunity. In conclusion, ferritin H conditional knockout mouse model provides us with an invaluable tool to study the in vivo role of ferritin H in iron homeostasis, ROS mediated damage, apoptosis and immunity.

The forgotten face of regular physical exercise: a 'natural' anti-atherogenic activity.

Humans are not programmed to be inactive. The combination of both accelerated sedentary lifestyle and constant food availability disturbs ancient metabolic processes leading to excessive storage of energy in tissue, dyslipidaemia and insulin resistance. As a consequence, the prevalence of Type 2 diabetes, obesity and the metabolic syndrome has increased significantly over the last 30 years. A low level of physical activity and decreased daily energy expenditure contribute to the increased risk of cardiovascular morbidity and mortality following atherosclerotic vascular damage. Physical inactivity leads to the accumulation of visceral fat and consequently the activation of the oxidative stress/inflammation cascade, which promotes the development of atherosclerosis. Considering physical activity as a 'natural' programmed state, it is assumed that it possesses atheroprotective properties. Exercise prevents plaque development and induces the regression of coronary stenosis. Furthermore, experimental studies have revealed that exercise prevents the conversion of plaques into a vulnerable phenotype, thus preventing the appearance of fatal lesions. Exercise promotes atheroprotection possibly by reducing or preventing oxidative stress and inflammation through at least two distinct pathways. Exercise, through laminar shear stress activation, down-regulates endothelial AT1R (angiotensin II type 1 receptor) expression, leading to decreases in NADPH oxidase activity and superoxide anion production, which in turn decreases ROS (reactive oxygen species) generation, and preserves endothelial NO bioavailability and its protective anti-atherogenic effects. Contracting skeletal muscle now emerges as a new organ that releases anti-inflammatory cytokines, such as IL-6 (interleukin-6). IL-6 inhibits TNF-α (tumour necrosis factor-α) production in adipose tissue and macrophages. The down-regulation of TNF-α induced by skeletal-muscle-derived IL-6 may also participate in mediating the atheroprotective effect of physical activity.

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

The NLRP3 inflammasome: a sensor for metabolic danger?

Interleukin-1beta (IL-1beta), reactive oxygen species (ROS), and thioredoxin-interacting protein (TXNIP) are all implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Here we review mechanisms directing IL-1beta production and its pathogenic role in islet dysfunction during chronic hyperglycemia. In doing so, we integrate previously disparate disease-driving mechanisms for IL-1beta, ROS, and TXNIP in T2DM into one unifying model in which the NLRP3 inflammasome plays a central role. The NLRP3 inflammasome also drives IL-1beta maturation and secretion in another disease of metabolic dysregulation, gout. Thus, we propose that the NLRP3 inflammasome contributes to the pathogenesis of T2DM and gout by functioning as a sensor for metabolic stress.

Deletion of Sirt3 does not affect atherosclerosis but accelerates weight gain and impairs rapid metabolic adaptation in LDL receptor knockout mice: implications for cardiovascular risk factor development.

Sirt3 is a mitochondrial NAD(+)-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR(-/-)) and LDLR/Sirt3 double-knockout (Sirt3(-/-)LDLR(-/-)) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR(-/-) mice. Sirt3 deficiency does not affect atherosclerosis in LDLR(-/-) mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.

Mechanisms of Photoaging and Cutaneous Photocarcinogenesis, and Photoprotective Strategies with Phytochemicals.

Photoaging and photocarcinogenesis are primarily due to solar ultraviolet (UV) radiation, which alters DNA, cellular antioxidant balance, signal transduction pathways, immunology, and the extracellular matrix (ECM). The DNA alterations include UV radiation induced thymine-thymine dimers and loss of tumor suppressor gene p53. UV radiation reduces cellular antioxidant status by generating reactive oxygen species (ROS), and the resultant oxidative stress alters signal transduction pathways such as the mitogen-activated protein kinase (MAPK), the nuclear factor-kappa beta (NF-κB)/p65, the janus kinase (JAK), signal transduction and activation of transcription (STAT) and the nuclear factor erythroid 2-related factor 2 (Nrf2). UV radiation induces pro-inflammatory genes and causes immunosuppression by depleting the number and activity of the epidermal Langerhans cells. Further, UV radiation remodels the ECM by increasing matrixmetalloproteinases (MMP) and reducing structural collagen and elastin. The photoprotective strategies to prevent/treat photoaging and photocarcinogenesis include oral or topical agents that act as sunscreens or counteract the effects of UV radiation on DNA, cellular antioxidant balance, signal transduction pathways, immunology and the ECM. Many of these agents are phytochemical derivatives and include polyphenols and non-polyphenols. The flavonoids are polyphenols and include catechins, isoflavones, proanthocyanidins, and anthocyanins, whereas the non-flavonoids comprise mono phenolic acids and stilbenes. The natural sources of polyphenols include tea, cocoa, grape/wine, soy, pomegranate, and Polypodium leucotomos. The non-phenolic phytochemicals include carotenoids, caffeine and sulphoraphance (SFN). In addition, there are other phytochemical derivatives or whole extracts such as baicalin, flavangenol, raspberry extract, and Photomorphe umbellata with photoprotective activity against UVB radiation, and thereby carcinogenesis.

NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production?

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a multiprotein complex that activates caspase 1, leading to the processing and secretion of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and IL-18. The NLRP3 inflammasome is activated by a wide range of danger signals that derive not only from microorganisms but also from metabolic dysregulation. It is unclear how these highly varied stress signals can be detected by a single inflammasome. In this Opinion article, we review the different signalling pathways that have been proposed to engage the NLRP3 inflammasome and suggest a model in which one of the crucial elements for NLRP3 activation is the generation of reactive oxygen species (ROS).

Assessment of particulate exposure and surface characteristics in association with urinary levels of 8-hydroxy-2'-deoxyguanosine, considered as marker of oxidative stress

Epidemiological studies have demonstrated that exposure to fine particles is associated to adverse health effects, including cancer, respiratory and cardiovascular diseases. However, mechanisms by which particles induce health effects remain unclear. According to one of the most investigated hypotheses, particles cause adverse effects through the production of reactive oxygen species (ROS), which are very hazardous compounds able to attack directly biological structures, including the DNA strand or the lipid bilayer of the cells. If the defense mechanisms, constituted of antioxidants, are not able to counter ROS, then these compounds will cause in the body a range of oxidation reactions called "oxidative stress". The aim of the present research project was to better understand mechanisms by which exposure to fine particles induces oxidative stress. The first point of this project was to check whether exposure to high levels of fine particles is directly linked to oxidative stress, and whether this oxidative stress is accompanied by the activation of the defense mechanisms (antioxidants). The second point was to study the role played by the particle surface characteristics in the oxidative stress process. For that purpose, a study was conducted in bus depots with the participation of 40 mechanics. First, occupational exposure to particles (PM4) and to other pollutants (NOx, O3) was measured over a two-day period. Then, urine samples of mechanics were collected in order to measure levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and antioxidants. 8OHdG is a molecule formed by the oxidation of DNA and allowing to assess the oxidative stress status of the mechanics. Finally, particles were collected on filters, and functional groups located on the particle surface were analyzed in the laboratory using a Knudsen flow reactor. This technique allows not only to quantify functional groups on the particle surface, but also to measure the reaction kinetics. Results obtained during the field campaign in bus depots showed that mechanics were exposed to rather low levels of PM4 (20-85 μg/m3) and of pollutants (NOx: 100-1000 ppb; O3: <15 ppb). However, despite this low exposure, urinary levels of the oxidative stress biomarker (8OHdG) increased significantly for non-smoking workers over a two-day period of shift. This oxidative stress was accompanied by an increase of antioxidants, indicating the activation of defense mechanisms. On the other hand, the analysis of functional groups on the particle surface showed important differences, depending on the workplace, the date and the activities of workers. The particle surface contained simultaneously antagonistic functional groups which did not undergo internal reactions (such as acids and bases), and was usually characterized by a high density of carbonyl functions and a low density of acidic sites. Reaction kinetics measured using the Knudsen flow reactor pointed out fast reactions of oxidizable groups and slow reactions of acidic sites. Several exposure parameters were significantly correlated with the increase of the oxidative stress status: the presence of acidic sites, carbonyl functions and oxidizable groups on the particle surface; reaction kinetics of functional groups on the particle surface; particulate iron and copper concentrations; and NOx concentration.

Altered Local Beta and Gamma Synchronization in Prefrontal Cortex of Mice with Redox Dysregulation or Maternal Immune Activation

Background: A developmental dysregulation of glutathione (GSH) synthesis leading to oxidative stress, when combined with environmental risk factors (viral infections) generating reactive oxygen species, can play a critical role in inducing schizophrenia phenotypes. GSH deficit induces morphological, physiological and behavioral anomalies analogous to those reported in schizophrenic patients, including disrupted parvalbumine (PV) inhibitory interneuron's integrity and neuronal synchrony (β/γ-oscillations). Methods: We assessed PV immunoreactivity (PV-IR) and local synchronization in prefrontal cortex of two mouse models: (1) mice with a genetic deficit in GSH (GCLM-/-) and (2) mice with prenatal immune activation at embryonic day17 (PolyI:C). Results: Adults from both mice models display reduced PV-IR in prefrontal cortex. In anterior cingulate (ACC) of GCLM-/-, appearance and maturation of PVI are delayed and worsened with peribubertal stress but not in adult one. This effect is reversed by treatment with the GSH precursor N-acetyl-cysteine. The power of beta and gamma oscillations are decreased in ACC of GCLM-/- while they increased in prelimbic cortex of PolyI:C mice. Conclusions: Despite reduced PV-IR in both models, alteration of the synchronization was different, indicating that the structural/functional disruption of the cortical circuitry was partly different in both models. Novel therapeutic strategies are proposed, based on interference with oxidative stress and inflammatory processes.

CD44 attenuates activation of the hippo signaling pathway and is a prime therapeutic target for glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor that, by virtue of its resistance to chemotherapy and radiotherapy, is currently incurable. Identification of molecules whose targeting may eliminate GBM cells and/or sensitize glioblastoma cells to cytotoxic drugs is therefore urgently needed. CD44 is a major cell surface hyaluronan receptor and cancer stem cell marker that has been implicated in the progression of a variety of cancer types. However, the major downstream signaling pathways that mediate its protumor effects and the role of CD44 in the progression and chemoresponse of GBM have not been established. Here we show that CD44 is upregulated in GBM and that its depletion blocks GBM growth and sensitizes GBM cells to cytotoxic drugs in vivo. Consistent with this observation, CD44 antagonists potently inhibit glioma growth in preclinical mouse models. We provide the first evidence that CD44 functions upstream of the mammalian Hippo signaling pathway and that CD44 promotes tumor cell resistance to reactive oxygen species-induced and cytotoxic agent-induced stress by attenuating activation of the Hippo signaling pathway. Together, our results identify CD44 as a prime therapeutic target for GBM, establish potent antiglioma efficacy of CD44 antagonists, uncover a novel CD44 signaling pathway, and provide a first mechanistic explanation as to how upregulation of CD44 may constitute a key event in leading to cancer cell resistance to stresses of different origins. Finally, our results provide a rational explanation for the observation that functional inhibition of CD44 augments the efficacy of chemotherapy and radiation therapy.

Oviposition by pierid butterflies triggers defense responses in Arabidopsis.

Insect eggs represent a threat for the plant as hatching larvae rapidly start with their feeding activity. Using a whole-genome microarray, we studied the expression profile of Arabidopsis (Arabidopsis thaliana) leaves after oviposition by two pierid butterflies. For Pieris brassicae, the deposition of egg batches changed the expression of hundreds of genes over a period of 3 d after oviposition. The transcript signature was similar to that observed during a hypersensitive response or in lesion-mimic mutants, including the induction of defense and stress-related genes and the repression of genes involved in growth and photosynthesis. Deposition of single eggs by Pieris rapae caused a similar although much weaker transcriptional response. Analysis of the jasmonic acid and salicylic acid mutants coi1-1 and sid2-1 indicated that the response to egg deposition is mostly independent of these signaling pathways. Histochemical analyses showed that egg deposition is causing a localized cell death, accompanied by the accumulation of callose, and the production of reactive oxygen species. In addition, activation of the pathogenesis-related1::beta-glucuronidase reporter gene correlated precisely with the site of egg deposition and was also triggered by crude egg extract. This study provides molecular evidence for the detection of egg deposition by Arabidopsis plants and suggests that oviposition causes a localized response with strong similarity to a hypersensitive response.