Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6-to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H 2 O 2 -sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.The complex relationships between programmed cell death (PCD) and natural senescence observed during leaf development are far from understood. However, one clear distinction is that senescence in leaves is essentially reversible, but PCD is not (Thomas et al., 2003). The genetically programmed cell suicide events that comprise PCD are triggered by enhanced levels of reactive oxygen species (ROS; Chen and Dickman, 2004;Laloi et al., 2004;Wagner et al., 2004). However, senescence-enhanced genes are also expressed in response to ROS (Navabpour et al., 2003).While the chemical nature of ROS dictates that they are potentially harmful to cells, plants use ROS as second messengers in signal transduction cascades regulating diverse processes such as mitosis, tropisms, and cell death. It is now well accepted that ROS accumulation is crucial to plant development as well as defense (Foyer and Noctor, 2005a). ROS signal transduction will ensue only if ROS escape destruction by cellular antioxidants that determine the lifetime and specificity of the signal. Ascorbic acid (AA) and glutathione are the major redox buffers of the plant cells, and they themselves are also signal-transducing molecules that can either signal independently or further transmit ROS signals (Fig. 1). They are thus intrinsic to redox homeostasis and redox-signaling events (Foyer and Noctor, 2005b).ROS production is often genetically programmed, for example, during the hypersen...

Accumulation of free L-proline (Pro) is a typical stress response incited by osmotic injuries in plants and microorganisms. Although the protective role of Pro in osmotic stress is not well understood, it is thought to function as compatible osmolyte or as a scavenger of reactive oxygen species (ROS). Here we show that, in Arabidopsis thaliana, Pro biosynthesis can be activated by incompatible plant-pathogen interactions triggering a hypersensitive response (HR). Pro accumulates in leaf tissues treated with Pseudomonas syringae pv. tomato avirulent strains (avrRpt2 and avrRpm1) but remains unchanged in leaves infected with isogenic virulent bacteria. Incompatible interactions lead to transcriptional activation of AtP5CS2, but not AtP5CS1, encoding the rate limiting enzyme in Pro biosynthesis pyrroline-5-carboxylate synthase (P5CS). AtP5CS2:GUS and AtP5CS2:LUC transgenes were induced inside and around the HR lesions produced by avirulent Pseudomonas spp. in transgenic plants. Pro accumulation was faster and stronger when stimulated by avrRpm1 than by avrRpt2, and was compromised in the low-salicylic acid plants NahG and eds5 when signaled through the RPS2-dependent pathway. In addition, Pro content and AtP5CS2 expression were enhanced by ROS in wild-type plants, suggesting that ROS may function as an intermediate signal in AtP5CS2-mediated Pro accumulation.

Plant tissues display major alterations upon the perception of microbial pathogens. Changes of cytoplasmic and apoplastic components that sense and transduce plant defenses have been extensively characterized. In contrast, less information is available about modifications affecting the plant nuclear genome under these circumstances. Here, we investigated whether the Arabidopsis thaliana DNA methylation status is altered in tissues responding to the attack of Pseudomonas syringae pv. tomato DC3000. We applied amplified fragment length polymorphism analysis to monitor cytosine methylation at anonymous 5'-CCGG-3' and 5'-GATC-3' sites in naive and infected samples. Plant genomic fragments reducing methylation upon infection, including peri/centromeric repeats such as the 180-bp unit, Athila retrotansposon, and a portion of the nuclear insertion of mitochondrial DNA, were isolated and characterized. P. syringae pv. tomato-induced hypomethylation was detected by high-performance liquid chromatography assays and at the molecular level it did not seem to equally affect all 5-methyl cytosine (5-mC) residues. Nuclei from challenged tissues displayed structural chromatin alterations, including loosening of chromocenters, which also were stimulated by avirulent P. syringae pv. tomato, but not by the P. syringae pv. tomato hrpL- mutant. Finally, P. syringae pv. tomato-induced hypomethylation was found to occur in the absence of DNA replication, suggesting that it involves an active demethylation mechanism. All these responses occurred at 1 day postinfection, largely preceding massive plant cell death generated by pathogen attack.

Ongoing clinical trials are exploring anticancer approaches based on signaling by TRAIL, a ligand for the cell death receptors DR4 and DR5. In this study, we report on the selective apoptotic effects of multivalent DR5 binding peptides (TRAIL mim/DR5 ) on cancer cells in vitro and in vivo. Surface plasmon resonance revealed up to several thousand-fold increased affinities of TRAIL mim/DR5 -receptor complexes on generation of divalent and trivalent molecules, the latter of which was achieved with a conformationally restricted adamantane core. Notably, only multivalent molecules triggered a substantial DR5-dependent apoptotic response in vitro. In tumor models derived from human embryonic kidney cells or primary foreskin fibroblasts, TRAIL mim/DR5peptides exerted a cancer cell-selective action that could synergize with resveratrol in a manner independent of p53. In a xenograft model of human colon cancer, a divalent TRAIL mim/DR5 peptide inhibited tumor growth.Our results offer a proof-of-principle for the development of synthetic small molecules to trigger the TRAIL apoptosis pathway for cancer therapy.

Cancer is a complex progressive multistep disorder that results from the accumulation of genetic and epigenetic abnormalities, which lead to the transformation of normal cells into malignant derivatives. Despite enormous progress in the understanding of cancer biology including the decryption of multiple regulatory networks governing cell growth and death, and despite the possibility of analyzing (epi)genetic deregulation at the genome-wide scale, cancertargeted therapy is still the exception. In fact, to date there are still far too few examples of therapies leading to cure; treatment-derived toxicity is a major issue, and cancer remains to be one of the largest causes of death worldwide. The purpose of this review is to discuss the state of the art of cancer therapy with respect to the key issue of any treatment, namely its target selectivity. Therefore, we recapitulate and discuss current concepts and therapies targeting tumorspecific features, including oncofusion proteins, aberrant kinase activities and epigenetic tumor makeup. We analyze strategies designed to induce tumor-selective death such as the use of oncolytic virus, tumoricidal proteins (NS1, Eorf4, apoptin, HAMLET (human a-lactalbumin made lethal to tumor cells)) and activation of signaling pathways involved in tumor surveillance. We emphasize the potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway, an essential component of the evolutionary developed defense systems that eradicate malignant cells. Finally, we discuss the necessity of targeting tumor-initiating cells (TICs) to avoid relapse and increase the chances of complete remission, and describe emerging concepts that might provide novel avenues for cancer therapy.

SUMMARYCells can change identity during normal development, in response to tissue damage or defined artificial treatments, or during disease processes such as cancer. Strikingly, not only the reprogramming of tissue cells to an embryonic stem cell-like state, but also the direct conversion from one cell type to another have been described. Direct cell type conversion could represent an alternative strategy for cellular therapies. However, little is known about the actual cellular steps undertaken by a cell as it changes its identity and their possible consequences for the organism. Using an in vivo single-cell system of natural direct reprogramming, in which a C. elegans rectal cell transforms into a motoneuron, we present an in-depth analysis of the cellular transformations involved. We found that the reprogrammed cell transits through intermediate states during direct in vivo reprogramming. We identified and characterised a mutant in the conserved COE transcription factor UNC-3 in which this cellular transformation is blocked. We determined that complete erasure of initial identity first takes place, followed by stepwise, unc-3-dependent, redifferentiation into a motoneuron. Furthermore, unlike in vitro induced reprogramming, reversion to a dedifferentiated identity does not lead to an increase in cellular potential in a natural, in vivo context. Our findings suggest that direct cell type conversion occurs via successive steps, and that dedifferentiation can occur in the absence of cell division. Furthermore, our results suggest that mechanisms are in place in vivo to restrict cell potential during reprogramming, a finding with important implications for regenerative medicine.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10/Apo2L) holds promise for cancer therapy as it induces apoptosis in a large variety of cancer cells while exerting negligible toxicity in normal ones. However, TRAIL can also induce proliferative and migratory signaling in cancer cells resistant to apoptosis induced by this cytokine. In that regard, the molecular mechanisms underlying the tumor selectivity of TRAIL and those balancing apoptosis versus survival remain largely elusive. We show here that high mRNA levels of PLAU, which encodes urokinase plasminogen activator (uPA), are characteristic of cancer cells with functional TRAIL signaling. Notably, decreasing uPA levels sensitized cancer cells to TRAIL, leading to markedly increased apoptosis. Mechanistic analyses revealed three molecular events taking place in uPA-depleted cells: reduced basal ERK1/2 prosurvival signaling, decreased preligand decoy receptor 2 (DcR2)-death receptor 5 (DR5) interaction and attenuated recruitment of DcR2 to the death-inducing signaling complex upon TRAIL challenge. These phenomena were accompanied by increased FADD and procaspase-8 recruitment and processing, thus guiding cells toward a caspase-dependent cell death that is largely independent of the intrinsic apoptosis pathway. Collectively, our results unveil PLAU mRNA levels as marker for the identification of TRAIL-responsive tumor cells and highlight a key role of uPA signaling in ‘apoptosis versus survival' decision-making processes upon TRAIL challenge.

Recent evidence has suggested the existence of sense-antisense transcription in mammals, but the existence of double-stranded RNAs endowed with biological function has remained elusive. Herein we show that hundreds of putative natural double-stranded RNAs (ndsRNAs) are expressed from interspersed genomic locations and respond to cellular cues. We demonstrate that a subset of ndsRNAs localize in the nucleus and, in their double-stranded form, interact with nuclear proteins. Detailed characterization of an ndsRNA (nds-2a) revealed that this molecule displays differential localization throughout the cell cycle and directly interacts with RCC1 and RAN and, through the latter, with the mitotic RANGAP1-SUMO1-RANBP2 complex. Notably, altering nds-2a levels led to postmitotic abnormalities, mitotic catastrophe and cell death, thus supporting a mitosis-related role. Altogether, our study reveals a hitherto-unrecognized class of RNAs that potentially participate in major biological processes in human cells.