Cell Death Genes Research Articles

Liver Label Retaining Cancer Cells Are Relatively Resistant to the Reported Anti-Cancer Stem Cell Drug Metformin.

Background & Aims: Recently, we reported that liver Label Retaining Cancer Cells (LRCC) can initiate tumors with only 10 cells and are relatively resistant to the targeted drug Sorafenib, a standard of practice in advanced hepatocellular carcinoma (HCC). LRCC are the only cancer stem cells (CSC) isolated alive according to a stem cell fundamental function, asymmetric cell division. Metformin has been reported to preferentially target many other types of CSC of different organs, including liver. It's important to know if LRCC, a novel class of CSC, are relatively resistant to metformin, unlike other types of CSC. As metformin inhibits the Sorafenib-Target-Protein (STP) PI3K, and LRCC are newly described CSC, we undertook this study to test the effects of Metformin on Sorafenib-treated HCC and HCC-derived-LRCC.Methods: We tested various STP levels and phosphorylation status, associated genes' expression, proliferation, viability, toxicity, and apoptosis profiles, before and after treatment with Sorafenib with/without Metformin.Results: Metformin enhances the effects of Sorafenib on HCC, and significantly decreased viability/proliferation of HCC cells. This insulin-independent effect was associated with inhibition of multiple STPs (PKC, ERK, JNK and AKT). However, Metformin increased the relative proportion of LRCCs. Comparing LRCC vs. non-LRCC, this effect was associated with improved toxicity and apoptosis profiles, down-regulation of cell death genes and up-regulation of cell proliferation and survival genes in LRCC. Concomitantly, Metformin up-regulated pluripotency, Wnt, Notch and SHH pathways genes in LRCC vs. non-LRCC.Conclusions: Metformin and Sorafenib have enhanced anti-cancer effects. However, in contradistinction to reports on other types of CSC, Metformin is less effective against HCC-derived-CSC LRCC. Our results suggest that combining Metformin with Sorafenib may be able to repress the bulk of tumor cells, but as with other anti-cancer drugs, may leave LRCC behind leading to cancer recurrence. Therefore, liver LRCC, unlike other types of CSC, are relatively resistant to the reported anti-cancer stem cell drug metformin. This is the first report that there is a type of CSC that is not relatively resistant to the CSC-targeting drug. Our findings suggest that a drug targeting LRCC may be critically needed to target CSC and prevent cancer recurrence. These may significantly contribute to the understanding of Metformin's anti-cancer effects and the development of novel drugs targeting the relatively resistant LRCC.

Association of two selected polymorphisms with developed endometriosis in women from Slovakia.

To clarify the connection between two selected mononucleotide polymorphisms (rs4957014 and rs3756712) in programmed cell death gene 6 (PDCD6) and endometriosis development risk in patients belonging to the majority population of Slovakia. From all women involved in the research a buccal DNA sample was taken. A genetic analysis of selected polymorphisms was implemented using Real-time PCR method. Variance in allelic and genotype frequencies was statistically evaluated between the controlgroup and the group of patients. The analysed group consisted of 52 women suffering from endometriosis and the control group of 63 women. Variant G allele frequency in the group of patients in case of polymorphism rs3756712 had a value of 0.42 and in the control group 0.29; that represents its statistically and significantly higher occurrence in the group of patients suffering from endometriosis (p = 0.029 and OR = 1.833). Presence of G allele is related to almost 1.9 times higher risk of endometriosis development. Achieved results show that polymorphism rs3756712 is significantly associated with the risk of endometriosis development in Slovak women. Polymorphism rs4957014 did not show any connection with development of endometriosis (Tab. 5, Ref. 10).

Novel axon projection after stress and degeneration in the Dscam mutant retina.

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and in this study we report and describe a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6α Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. In this study we find that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection.

Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death

MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of a series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death.

Feeding on resistant rice leads to enhanced expression of defender against apoptotic cell death (OoDAD1) in the Asian rice gall midge.

BackgroundThe Asian rice gall midge (Orseolia oryzae) is a destructive insect pest of rice. Gall midge infestation in rice triggers either compatible or incompatible interactions leading to survival or mortality of the feeding maggots, respectively. In incompatible interactions, generation of plant allelochemicals/defense molecules and/or inability of the maggots to continue feeding on the host initiate(s) apoptosis within the maggots. Unraveling these molecular events, triggered within the maggots as a response to feeding on resistant hosts, will enable us to obtain a better understanding of host resistance. The present study points towards the likely involvement of a defender against apoptotic cell death gene (DAD1) in the insect in response to the host defense.ResultsThe cDNA coding for the DAD1 orthologue in the rice gall midge (OoDAD1) consisted of 339 nucleotides with one intron of 85 bp and two exons of 208 and 131 nucleotides. The deduced amino acid sequence of OoDAD1 showed a high degree of homology (94.6 %) with DAD1 orthologue from the Hessian fly (Mayetiola destructor) —a major dipteran pest of wheat. Southern hybridization analysis indicated that OoDAD1 was present as a single copy in the genomes of the Asian rice gall midge biotypes (GMB) 1, 4 and 4 M. In the interactions involving GMB4 with Jaya (susceptible rice host) the expression level of OoDAD1 in feeding maggots gradually increased to 3-fold at 96hai (hours after infestation) and peaked to 3.5-fold at 96hai when compared to that at 24 hai. In contrast, expression in maggots feeding on RP2068 (resistant host) showed a steep increase of more than 8-fold at 24hai and this level was sustained at 48, 72 and 96hai when compared with the level in maggots feeding on Jaya at 24hai. Recombinant OoDAD1, expressed in E. coli cells, when injected into rice seedlings induced a hypersensitive response (HR) in the resistant rice host, RP2068, but not in the susceptible rice variety, Jaya.ConclusionsThe results indicate that the expression of OoDAD1 is triggered in the feeding maggots probably due to the host resistance response and therefore, is likely an important molecule in the initial stages of the interaction between the midge and its rice host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0618-y) contains supplementary material, which is available to authorized users.

A cerato-platanin-like protein HaCPL2 from Heterobasidion annosum sensu stricto induces cell death in Nicotiana tabacum and Pinus sylvestris

The cerato-platanin family is a group of small secreted cysteine-rich proteins exclusive for filamentous fungi. They have been shown to be involved in the interactions between fungi and plants. Functional characterization of members from this family has been performed mainly in Ascomycota, except Moniliophthora perniciosa. Our previous phylogenetic analysis revealed that recent gene duplication of cerato-platanins has occurred in Basidiomycota but not in Ascomycota, suggesting higher functional diversification of this protein family in Basidiomycota than in Ascomycota. In this study, we identified three cerato-platanin homologues from the basidiomycete conifer pathogen Heterobasidion annosum sensu stricto. Expression of the homologues under various conditions as well as their roles in the H. annosum s.s.–Pinus sylvestris (Scots pine) pathosystem was investigated. Results showed that HaCPL2 (cerato-platanin-like protein 2) had the highest sequence similarity to cerato-platanin from Ceratocystis platani and hacpl2 was significantly induced during nutrient starvation and necrotrophic growth. The treatment with recombinant HaCPL2 induced cell death, phytoalexin production and defense gene expression in Nicotiana tabacum. Eliciting and cell death-inducing ability accompanied by retardation of apical root growth was also demonstrated in Scots pine seedlings. Our results suggest that HaCPL2 might contribute to the virulence of H. annosum s.s. by promoting plant cell death.

Carbon nanoparticle induced cytotoxicity in human mesenchymal stem cells through upregulation of TNF3, NFKBIA and BCL2L1 genes

Carbon based nanomaterials, including carbon nanotubes, graphene, nanodiamond and carbon nanoparticles, have emerged as potential candidates for a wide variety of applications because of their unusual electrical, mechanical, thermal and optical properties. However, our understanding of how increased usage of carbon based nanomaterials could lead to harmful effects in humans and other biological systems is inadequate. Our present investigation is focused on the cellular toxicity of carbon nanoparticles (CNPs) on human mesenchymal stem cells (hMSCs). Following exposure to CNPs, cell viability, nuclear morphological changes, apoptosis and cell cycle progression were monitored. Furthermore, the expression of genes involved in both cell death (e.g., P53, TNF3, CDKN1A, TNFRSF1A, TNFSF10, NFKBIA, BCL2L1) and cell cycle regulation (e.g., PCNA, EGR1, E2F1, CCNG1, CCND1, CCNC, CYCD3) were assessed using qPCR. Our results indicated that CNPs reduce cell viability and cause chromatin condensation and DNA fragmentation. Cell cycle analysis indicated that CNPs affect the cell cycle progression. However, the gene expression measurements confirmed that CNPs significantly upregulated the P53, TNF3, CDKNIA, and NFKBIA genes and downregulated the EGR1 gene in hMSCs. Our findings suggest that CNPs reduce cell viability by disrupting the expression of cell death genes in human mesenchymal stem cell (hMSC). The results of this investigation revealed that CNPs exhibited moderate toxicity on hMSCs.

Abstract 2207: Elucidating mechanisms employed by NFIL3 to impact cancer cell survival

Abstract NFIL3 was recently identified as a novel modulator of FOXO1 output on the PI3K pathway. NFIL3 bound to DNA to physically block FOXO1 recruitment to genes that induce apoptosis. The diminishment in NFIL3 expression via RNA interference led to FOXO recruitment to cell death genes, an increase in Histone H3 acetylation on these genes, and increased expression of these genes. However, a number of observations suggest that FOXO1 is not the main target of NFIL3 in these cells. First, the NFIL3 and FOXO1 DNA consensus sites have almost no similarity. Second, the loss of FOXO1 or all three FOXO factors failed to suppress the cell death phenotype upon the diminishment of NFIL3 in MEFs. The goal of this project is to identify the factor(s) that NFIL3 directly blocks from DNA recruitment to promote cellular survival. In previous work, the NFIL3 and FOXO1 sites were mapped as being adjacent on the DNA. The mapped NFIL3 site was required for NFIL3 to associate with the DNA in biochemical experiments and for NFIL3 to regulate the promoter sequence in reporter assays. The mapped NFIL3 site is a perfect match for the bZIP transcription factor CCAAT/enhancer binding protein beta (CEBPB) consensus sequence. NFIL3 has been shown to block the ability of CEBPB to activate transcription by competing for access to the same DNA consensus site in numerous contexts including neuronal regeneration, PKA pathway transcriptional programs and the regulation of hepatitis C viral genes. Aside from DBP in circadian rhythm, CEBPB is the most commonly antagonized factor by NFIL3 in the literature. We are investigating if NFIL3 hinders FOXO1 recruitment to cell death genes by directly antagonizing CEBPB recruitment to these genes using cell biological and bioinformatics approaches. We have found that CEBPB is required for efficient induction of a subset of NFIL3 repressed genes. Citation Format: Megan Keniry, Omar Caballero, Itzel Flores, Andrea Salinas. Elucidating mechanisms employed by NFIL3 to impact cancer cell survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2207. doi:10.1158/1538-7445.AM2015-2207

Abstract 4558: Using reduction mammoplasty tissues from healthy young women living in USA's high breast cancer (BC) risk posing environment to open up to study the “black box” of latent stages of breast carcinogenesis; a key to develop strategies for BC

Abstract Knowledge of latent stages of breast carcinogenesis is limited to when histological changes are already present due to a lack of; a) cytochemical marker(s) needed to identify loci in histologically normal breast tissues where procarcinogenic conditions exist and; b) microscopic systems needed to geno- and phenotype cells in such loci. In 2 recent publications we presented evidence that protein adducts of 4-hydroxy-2-nonenal (4HNE), markers of a procarcinogenic state of chronic oxidative stress (OxS), might provide the needed fiducial marker. Specifically, a) we documented the presence of 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells in terminal ductal units (TDLUs) in breast tissues of healthy teenagers and young women and their association with altered expression of OxS-related mRNAs and; b) presented Fourier-transform infrared microspectroscopic evidence of differences in biomolecular profile of ME cells and associated intra- and interlobular stromal cells in 4HNE+ vs. 4HNE- TDLUs, i.e., of cells of origin of BCs and cells in their supportive stromal microenvironment. Here we present further evidence of molecular changes in breast tissues of young women (ages 17-30 y) associated with 4HNE adducts. A comparison of the transcriptome of breast tissues with many 4HNE+ vs. few 4HNE+ TDLUs (controls) by Illumina 24,000 gene platform and SABioscience 84 gene Cell Death PCR Array revealed reprogramming of many homeostatic mechanism in 4HNE+ TDLUs. Notably, a) DASL platform identified transcripts for 395 genes that differed (p = <.05) between 4HNE+ vs. 4HNE- tissues, transcripts that were assigned by GO bioinformatics tool to 21 molecular mechanism and; b) Cell Death array identified 30 genes that differed >2 fold between 4HNE+ vs. 4HNE- tissues. The findings support using 4HNE adducts to identify in histologically normal breast tissues loci harboring cells that might initiate a cancer. Our findings fit an evolutionary model of carcinogenesis and reflect adaptive responses to stressors that serve to preserve tissue integrity, e.g., by developing resistance to OxS and to apoptosis, responses however that over time can lead to the emergence of a cancer. Our recently acquired Toponome Imaging System (TIS) enables identifying such responses to chronic OxS implied by the presence of 4HNE adducts. TIS is the first high throughput fluorescence robotic microscopic system that can provide a map, a toponome, of up to 100 proteins at 40nm functional resolution in individual cells in a tissue section. In ongoing experiments we are using TIS and antibodies against up to 21 proteins to test the postulate that 4HNE+ TDLUs are enriched with cells with an apoptosis resistant phenotype, a postulate supported by our transcriptional data and predicted by the evolutionary model of carcinogenesis. Citation Format: Judith Weisz, Debra Shearer, Anna C. Salzberg. Using reduction mammoplasty tissues from healthy young women living in USA's high breast cancer (BC) risk posing environment to open up to study the “black box” of latent stages of breast carcinogenesis; a key to develop strategies for BC [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4558. doi:10.1158/1538-7445.AM2015-4558

Involvement of Kruppel-like factor 9 in bleomycin-induced pulmonary toxicity

Oxidative stress or the production of reactive oxygen species (ROS) has been implicated as an important factor in the development of bleomycin (BLM)-induced pulmonary toxicity; however, the mechanism behind the toxicity remains to be elucidated. The present study aimed to investigate the key factor involved in BLM‑induced toxicity. The study was conducted in human pulmonary fibroblast (HPF) cells and in a mouse model. The ROS level, cell death assay, protein and gene expression levels of Kruppel‑like factor 9 (Klf9) and other associated factors were assessed. A dose‑dependent increase in ROS, lipid peroxidation, cell death, and protein and mRNA expression levels of NF‑E2‑related transcription factor 2 (Nrf2) and Klf9 were observed in BLM‑treated cells. However, the expression levels of the other antioxidant proteins assessed, including catalase, super oxide dismutase, glutathione reductase and thioredoxin reductase 2, were decreased. The expression levels of Nrf2 were decreased in cells treated with a higher concentration (>200 µM) of BLM. These results suggested that in response to increased intracellular levels of ROS, above a critical threshold, Nrf2 stimulates the expression of Klf9, resulting in a further increase in Klf9‑mediated ROS production and subsequent cell death. Furthermore, the data suggested that Klf9 may be considered as an adjunctive therapeutic target for BLM-induced pulmonary toxicity.

Analyzing the Response of RNAi-Treated Drosophila Cells to Death Stimuli by Quantitative Real-Time Polymerase Chain Reaction.

A useful complement to animal studies is the use of Drosophila cell lines to analyze cell-death responses. There are numerous Drosophila cell lines available, such as S2 cells, which possess the advantages of being semi-adherent, fast growing, relatively robust, and useful for transfection and knockdown studies, whereas other lines, such as mbn2, are more suitable for analyzing hormone-induced cell death and gene expression. Drosophila cell lines are very amenable to knockdown studies as the cells take up double-stranded RNA (dsRNA) from the medium, initiating gene silencing and resulting in a high level of gene knockdown. This means that the cell lines are useful for investigating the response to death stimuli, following gene knockdown, by examining the expression of cell-death genes. This protocol describes the synthesis of dsRNA for treatment of Drosophila cells and the subsequent analysis of cell-death gene expression by quantitative real-time polymerase chain reaction (qPCR).

Network-based survival-associated module biomarker and its crosstalk with cell death genes in ovarian cancer

Ovarian cancer remains a dismal disease with diagnosing in the late, metastatic stages, therefore, there is a growing realization of the critical need to develop effective biomarkers for understanding underlying mechanisms. Although existing evidences demonstrate the important role of the single genetic abnormality in pathogenesis, the perturbations of interactors in the complex network are often ignored. Moreover, ovarian cancer diagnosis and treatment still exist a large gap that need to be bridged. In this work, we adopted a network-based survival-associated approach to capture a 12-gene network module based on differential co-expression PPI network in the advanced-stage, high-grade ovarian serous cystadenocarcinoma. Then, regulatory genes (protein-coding genes and non-coding genes) direct interacting with the module were found to be significantly overlapped with cell death genes. More importantly, these overlapping genes tightly clustered together pointing to the module, deciphering the crosstalk between network-based survival-associated module and cell death in ovarian cancer.

Osmotic stress affects polyamine homeostasis and phenolic content in proembryogenic liquid cell cultures of Scots pine

Polyamines (PAs) are ubiquitous polycations involved in many physiological processes in plants, including somatic embryogenesis, cellular growth and stress reactions. In the present study, we focus on the consequences in PA metabolism caused by polyethylene glycol (PEG) in proembryogenic Scots pine (Pinus syl- vestris L.) liquid cultures. The growth and viability of the cell masses and changes in PA concentrations and phenolic secondary metabolites were investigated under control, 5 and 10 % PEG treatments. The effect of osmotic stress responses was investigated at the gene expression level including stress, cell division, programmed cell death and PA-related genes and PA metabolites. Moreover, the expression of ethylene and proline biosynthesis genes and phenylalanine ammonia lyase and stilbene synthase (psSTS) was analyzed. Under osmotic stress conditions, we found a consistent pattern of endogenous PAs in Scots pine proembryogenic cells. However, accumulation of free spermine (Spm) and methyl putrescine under osmotic stress might indicate their specific role in stress protection. Expression of polyamine oxidase was down-regulated under osmotic stress, suggesting the role of PA catabolism in regulation of Spm levels. Scots pine proliferating proembryogenic cells are in a developmentally undiffer- entiated stage where the content of secondary metabolites is generally low. However, in the present study the total content of phenolic compounds increased but the biosyn- thesis of phenylpropanoids and stilbenes, generally con- sidered as stress-protective molecules, was not affected by osmotic stress.

Studies on the characteristics and mechanisms of testicular toxicity induced by Hydroxyurea

Objective: Apoptosis plays a dominant role in both spontaneous spermatogenesis and germ cell death. This study was aimed to investigate the functions of related genes in testicular germ cell death induced by Hydroxyurea (HU).Method: Wild-type (WT) and FasL transgenic (TG) DBA/C57BL mice were intraperitoneal injected with 400 mg/kg HU. Twelve hours later, testes were collected. Histomorphology of testis was observed by staining with Periodic Acid Schiff (PAS). Apoptosis was assessed by TUNEL assay. mRNA and protein levels of related genes were evaluated by quantitative RT-PCR and Western blot, respectively.Results: The 2 × 2 factorial design comparative experiments between the WT and TG mice showed that the TG mice exhibited a higher basal apoptotic index. The basal mRNA levels of Fas and FasL and protein levels of Fas, FasL, Caspase-3, Caspase-8 and Caspase-9 in the TG mice were also higher than that in the WT mice. Twelve hours after injection of HU, the testicular tubules exhibited no significantly morphological changes but apoptosis index remarkably increased in both the WT and TG mice, with the latter having the higher amplitude. Although, HU up-regulated the mRNA of apoptosis-related genes, such as Fas and FasL, in both the TG and WT mice, the increased amplitude was more obvious in the TG mice. By Western blot analysis, apoptosis-related proteins Fas, FasL Caspase-3, Caspase-8 and Caspase-9 were significantly increased in both the WT and TG mice, with the TG mice exhibiting a greater up-regulation.Conclusion: Germ cell apoptosis induced by the HU treatment may be related to the FasL-mediated signal transduction pathway.

调控水稻异倍性杂交种子败育的相关基因分析

The MADS29 gene regulates the plumpness of rice seeds and affects seed development by regulating the degradation of maternal tissues and maintaining the plants hormone balance. Rice seeds produced by heteroploid crosses exhibit greater rate of sterility than that by seeds of other plants. To determine whether MADS29 and its related genes regulate seed development, we used four rice cultivars (two diploid and two tetraploid) to produce four self-crossed combinations as the control and eight hybridized combinations as reciprocal crosses. Then, pollen grain fertility, pollen germination, and seed development were observed, and the expressions of MADS29 and related genes were quantified using RT-PCR. The results showed the following: (1) heteroploid crosses showed 100% successful fertilization, with non-negligible rate of seed abortion. (2) Based on examinations of paraffin sections of seeds at 3 days after pollination (3 DAP) in a 4 n ×2 n cross, the endosperm had entered the cellularization stage, but the number of cells was lower than that in the control. However, in the endosperm of seeds of 2 n ×4 n crosses, the cell walls did not form around the nucleus. In addition, some vacuolation was noted in the endosperm, and cellularization was delayed. (3) qRT-PCR results showed that the relative expression levels of MADS29 , IAA , and programmed cell death (PCD) genes associated with maternal tissues in hybrid seeds at 6 DAP and 8 DAP were significantly higher than those of the control. On the contrary, the relative expression of starch synthesis genes in hybrid seeds was significantly lower than that of the control. This suggests that MADS29 is more highly expressed in the seeds of heteroploid crosses. In addition, it suggested that the expressions of IAA and other genes involved in the PCD of maternal tissues was upregulated in the same seeds. However, starch synthesis genes showed downregulated expression levels, leading to the abnormal development of the endosperm, followed by seed abortion. Thus, high expression of MADS29 leads to hybrid seed abortion, a novel regulatory pattern that is not observed in diploid rice seeds.

Transcriptional profiling provides insights into metronomic cyclophosphamide-activated, innate immune-dependent regression of brain tumor xenografts.

BackgroundCyclophosphamide treatment on a six-day repeating metronomic schedule induces a dramatic, innate immune cell-dependent regression of implanted gliomas. However, little is known about the underlying mechanisms whereby metronomic cyclophosphamide induces innate immune cell mobilization and recruitment, or about the role of DNA damage and cell stress response pathways in eliciting the immune responses linked to tumor regression.MethodsUntreated and metronomic cyclophosphamide-treated human U251 glioblastoma xenografts were analyzed on human microarrays at two treatment time points to identify responsive tumor cell-specific factors and their upstream regulators. Mouse microarray analysis across two glioma models (human U251, rat 9L) was used to identify host factors and gene networks that contribute to the observed immune and tumor regression responses.ResultsMetronomic cyclophosphamide increased expression of tumor cell-derived DNA damage, cell stress, and cell death genes, which may facilitate innate immune activation. Increased expression of many host (mouse) immune networks was also seen in both tumor models, including complement components, toll-like receptors, interferons, and cytolysis pathways. Key upstream regulators activated by metronomic cyclophosphamide include members of the interferon, toll-like receptor, inflammatory response, and PPAR signaling pathways, whose activation may contribute to anti-tumor immunity. Many upstream regulators inhibited by metronomic cyclophosphamide, including hypoxia-inducible factors and MAP kinases, have glioma-promoting activity; their inhibition may contribute to the therapeutic effectiveness of the six-day repeating metronomic cyclophosphamide schedule.ConclusionsLarge numbers of responsive cytokines, chemokines and immune regulatory genes linked to innate immune cell recruitment and tumor regression were identified, as were several immunosuppressive factors that may contribute to the observed escape of some tumors from metronomic CPA-induced, immune-based regression. These factors may include useful biomarkers that facilitate discovery of clinically effective immunogenic metronomic drugs and treatment schedules, and the selection of patients most likely to be responsive to immunogenic drug scheduling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1358-y) contains supplementary material, which is available to authorized users.

PKM2 Subcellular Localization Is Involved in Oxaliplatin Resistance Acquisition in HT29 Human Colorectal Cancer Cell Lines.

Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells.

Characterization of a vacuolar processing enzyme expressed in Arachis diogoi in resistance responses against late leaf spot pathogen, Phaeoisariopsis personata.

Vacuolar processing enzymes are cysteine proteases responsible for maturation of vacuolar proteins. They have been shown to possess caspase-1-like activity, mediate cell death and display increased activity during pathogen infections. A transcript derived fragment corresponding to VPE was found to be up-regulated in a cDNA-AFLP analysis of host responses of a wild peanut, Arachis diogoi upon challenge from the late leaf spot pathogen Phaeoisariopsis personata, which was subsequently validated by q-PCR in a time course analysis, where susceptible peanut did not show its upregulation. In transient conditional and constitutive expression studies in tobacco leaves using agroinfiltration, we have observed that expression of AdVPE was associated with hypersensitive response (HR) like cell death. AdVPE expression was found to be high at 24h post estradiol application and this was associated with the enhanced co-expression of molecular markers of HR cell death genes and genes for pathogenesis related proteins indicating that AdVPE positively regulates defense responses and its estradiol induced expression is sufficient for HR-like cell death in tobacco. We found that AdVPE expression was very strongly induced in response to sodium nitroprusside, which indicates its involvement in stress signaling. Induced expression of AdVPE in response to jasmonic acid and ethylene also indicates its involvement in an interconnected network of signaling. Transgenic tobacco plants ectopically expressing AdVPE exhibited enhanced resistance against Phytophthora parasitica var. nicotianae, Alternaria alternata var. nicotianae and Rhizoctonia solani. To our knowledge, this is the first report on the heterologous expression of a pathogen induced VPE enhancing resistance to fungal pathogens with cell death phenomenon under transient expression.

Forebrain neuronal specific ablation of p53 gene provides protection in a cortical ischemic stroke model

Cerebral ischemic injury involves death of multiple cell types at the ischemic sites. As a key regulator of cell death, the p53 gene has been implicated in the regulation of cell loss in stroke. Less focal damage is found in stroke animals pre-treated with a p53 inhibitor or in traditional p53 knockout (ko) mice. However, whether the p53 gene plays a direct role in regulating neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 ko mice, we utilized a neuronal specific conditional ko mouse line (CamcreTRP53loxP/loxP) to achieve forebrain neuronal specific deletion of p53 and examined the role of the p53 gene in ischemia-induced cell death in neurons. Expression of p53 after stroke is examined using the immunohistochemical method and the outcome of stroke is examined by analysis of infarction size and behavioral deficits caused by stroke. Our data showed that p53 expression is upregulated in the ischemic region in neuronal cells in wildtype (wt) mice but not in CamcreTRP53loxP/loxP ko mice. Deletion of the p53 gene in forebrain neurons results in a decreased infarction area in ko mice. Locomotor behavior, measured in automated activity chambers, showed that CamcreTRP53loxP/loxP ko mice have less locomotor deficits compared to wt mice after middle cerebral artery occlusion (MCAo). We conclude that manipulation of p53 expression in neurons may lead to unique therapeutic development in stroke.

Structure guided design of potent and selective ponatinib-based hybrid inhibitors for RIPK1.

RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective "hybrid" RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.