Stress Survival Mechanisms Research Articles

Abstract SY39-02: Accessing evolutionary programs to enable therapeutic resistance

Abstract Twenty million people around the world are diagnosed with cancer every year and over ten million people die because their cancer has metastasized and is incurable using current systemic therapies. Metastatic disease remains incurable because tumors evade all forms of programmed cell death. How is this possible? Unicellular and multicellular organisms have evolved multiple strategies to avoid death from internal and external stresses. While the nature of these catastrophes may be varied, their common property is to doom the organism unless it can access a survival mechanism and/or evolve a resistance mechanism rapidly. One such stress survival mechanism that is present across the tree of life in both unicellular and multicellular organisms is the utilization of a program that enables whole genome doubling (polyploidization) and cell cycle arrest. This evolutionary program allows the organism to avoid environmental stresses and protect the integrity of its genomic material by adopting a quiescent phenotype. In addition, by doubling DNA and increasing RNA and protein material, organisms have increased capacity to evolve further resistance mechanisms to stressors. To date, no common genomic signature has been defined that can explain or identify which patients will develop therapeutically resistant and lethal metastatic cancer. In all these incurable patients, therefore, lethal cancer independently evolves capacities for metastasis, avoidance of all cellular death pathways, and resistance. This convergent outcome across all patients demands an explanation beyond the accrual of stochastic mutations. Since the tumor ecosystem is exposed to constantly changing chemical and biologic conditions, it is generally assumed that cancer cell populations must have the ability to evolve rapidly to acquire mutations to adapt and survive. An alternative and complementary theory to explain cancer cell fitness is that a subset of cancer cells access evolutionary stress survival programs that are inherited as development programs. These development programs enable whole genome doubling (polyploidization) and cell cycle arrest and are seen in multiple cell types, including regenerating liver cells, myocytes under physical strain, and megakaryocytes under metabolic stress. In cancer, access to these stress programs result in cancer cell survival though a state of cell cycle arrest that is simultaneously polyploid and aneuploid. Entering the polyaneuploid cancer cell (PACC) state, cancer cells pause their cell cycle and protect the integrity of their DNA from tumor microenvironmental stresses as well as have increased potential to develop further resistance mechanisms to environmental stressors and toxic therapies. After stress is removed, cells in the PACC state undergo depolyploidization and generate resistant progeny. This enables the generation of heritable variation that can be dispensed to their 2N+ aneuploid progeny, providing population rescue in response to therapeutic stress. Targeting cells in the PACC state opens a new avenue for cancer therapy. Citation Format: Kenneth J. Pienta. Accessing evolutionary programs to enable therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY39-02.

The H2O2 inherently released by the mycobacterial minor subpopulation enhances the survival of the major kin subpopulation against rifampicin

Exposure to antibiotics most often generates oxidative stress in bacteria. Oxidative stress survival mechanisms would facilitate the evolution of antibiotic resistance. As part of an effort to understand oxidative stress survival mechanisms in mycobacteria, here we show that the minor subpopulation (SCs; short-sized cells constituting 10% of the population) of Mycobacterium smegmatis significantly increased the survival of its major kin subpopulation (NCs; normal/long-sized cells constituting 90% of the population) in the mid-log-phase (MLP) cultures against the oxidative stress induced by rifampicin and exogenously added H2O2 (positive control). We had earlier shown that the SCs in the MLP cultures inherently and naturally release significantly high levels of H2O2 into the medium. Addition of the SCs' culture supernatant, unlike the supernatant of the dimethylthiourea (H2O2 scavenger) exposed SCs, enhanced the survival of NCs. It indicated that NCs' survival required the H2O2 present in the SCs' supernatant. This H2O2 transcriptionally induced high levels of catalase-peroxidase (KatG) in the NCs. The naturally high KatG levels in the NCs significantly neutralised the endogenous H2O2 formed upon exposure to rifampicin or H2O2, thereby enhancing the survival of NCs against oxidative stress. The absence of such enhanced survival in the furA-katG and katG knockout (KO) mutants of NCs in the presence of wild-type SCs, confirmed the requirement of the H2O2 present in the SCs' supernatant and NCs' KatG for enhanced oxidative stress survival. The presence of SCs:NCs at 1:9 in the pulmonary tuberculosis patients' sputum alludes to the clinical significance of the finding.

The regulation of ferroptosis by MESH1 through the activation of the integrative stress response

All organisms exposed to metabolic and environmental stresses have developed various stress adaptive strategies to maintain homeostasis. The main bacterial stress survival mechanism is the stringent response triggered by the accumulation “alarmone” (p)ppGpp, whose level is regulated by RelA and SpoT. While metazoan genomes encode MESH1 (Metazoan SpoT Homolog 1) with ppGpp hydrolase activity, neither ppGpp nor the stringent response is found in metazoa. The deletion of Mesh1 in Drosophila triggers a transcriptional response reminiscent of the bacterial stringent response. However, the function of MESH1 remains unknown until our recent discovery of MESH1 as the first cytosolic NADPH phosphatase that regulates ferroptosis. To further understand whether MESH1 knockdown triggers a similar transcriptional response in mammalian cells, here, we employed RNA-Seq to analyze the transcriptome response to MESH1 knockdown in human cancer cells. We find that MESH1 knockdown induced different genes involving endoplasmic reticulum (ER) stress, especially ATF3, one of the ATF4-regulated genes in the integrative stress responses (ISR). Furthermore, MESH1 knockdown increased ATF4 protein, eIF2a phosphorylation, and induction of ATF3, XBPs, and CHOP mRNA. ATF4 induction contributes to ~30% of the transcriptome induced by MESH1 knockdown. Concurrent ATF4 knockdown re-sensitizes MESH1-depleted RCC4 cells to ferroptosis, suggesting its role in the ferroptosis protection mediated by MESH1 knockdown. ATF3 induction is abolished by the concurrent knockdown of NADK, implicating a role of NADPH accumulation in the integrative stress response. Collectively, these results suggest that MESH1 depletion triggers ER stress and ISR as a part of its overall transcriptome changes to enable stress survival of cancer cells. Therefore, the phenotypic similarity of stress tolerance caused by MESH1 removal and NADPH accumulation is in part achieved by ISR to regulate ferroptosis.

Abstract 42: Treatment-induced embryonic diapause-like adaptation through suppression of Myc activity as mediator of drug persistence in cancer

Abstract Chemo-persistent residual tumors are a major barrier for curative cancer therapy and provide a reservoir of cancer cells for eventual relapse. This clinically critical tumor cell population is poorly understood and lacks faithful in vitro models. We compared the transcriptional profiles of paired samples from patient with solid tumors or hematological neoplasias at the stage of post-treatment residual tumors or minimal residual disease vs. at their respective baselines. For a large proportion of patients, post-treatment tumor cells had a molecular signature that resembled that of embryonic diapause, a dormant stage of suspended development in early mammalian embryos triggered by stress and induced by suppression of Myc activity and overall biosynthesis. This Myc-inactivated molecular program was acquired during treatment and not pre-existing at baseline. Remarkably, baseline tumors with multiple MYC copy numbers maintained their MYC amplification in the post-treatment persistent tumor cells but suppressed Myc activity. We simulated this cancer cell state using preclinical models of breast and prostate cancer and multiple myeloma, including patient-derived cancer organoids and PDX models. Parallel approaches of genome sequencing and DNA barcode-mediated clonal tracking in vitro and in vivo independently indicated that the residual cancer cell subpopulations that persisted after treatment with common chemotherapeutic classes were not driven by rare pre-existing clones or de novo genetic events. Transcriptional analysis of the treatment-persistent cancer cells in 3D organoid cultures and in xenograft/PDX models indicated a diapause-like transcriptional adaptation with inactivated Myc, similar to that in patients. We functionally examined the role of Myc suppression in in vitro models of breast cancer and multiple myeloma using genetic (CRISPR/Cas9 editing and interference) and pharmacological (inhibition of Myc transcriptional co-activator Brd4) approaches. Suppression of Myc in cancer cells induced an embryonic diapause-like molecular profile and attenuated the acute chemotherapeutic cytotoxicity. Myc-inactivated cancer cells had reduced apoptotic priming and maintained low redox stress even in the presence of cytotoxic agents, which may contribute to cell survival during treatment. High-throughput screening of chemo-persistent cells revealed that inhibitors of CDK9 could revert the biosynthetic pause, reactivate Myc, attenuate the diapause-like state, and re-sensitize the residual cancer cells to chemotherapy. Overall, our study shows that cancer cells can co-opt the stress survival mechanism of embryonic diapause by dynamically suppressing Myc to enter transient drug-refractory dormancy. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Dhvanir Kansara, Caroline Vilas, Aziz Al'Khafaji, Juliette Bouyssou, Aedin Culhane, Constantine S. Mitsiades. Treatment-induced embryonic diapause-like adaptation through suppression of Myc activity as mediator of drug persistence in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 42.

Abstract GS1-07: Treatment persistence of residual breast tumors through an embryonic diapause-like cancer cell state with suppressed myc activity

Abstract Systemic breast cancer treatments often fail to achieve complete and sustained responses due to drug-persistent residual tumor foci, the “seed” for eventual relapse. Recent clinical studies have revealed that the chemo-persistent tumor cells undergo extensive transcriptional reprogramming in response to neo-adjuvant treatment; however, the impact of this acquired molecular signature on the ability of residual cancer cells to survive during therapy is not clear. To further study the molecular hallmarks of chemo-persistent cancer cells, we analyzed the transcriptional signatures of post-treatment residual tumors in a large number of breast cancer patients from several neoadjuvant clinical studies. We observed that the treatment-persistent tumor cells had a distinct cellular state which molecularly resembles that of the embryonic diapause, a dormant stage of transiently suspended development in undifferentiated epiblasts triggered by stress and induced by suppression of Myc activity and overall biosynthesis. Remarkably, the propensity for residual tumors with an embryonic diapause-like (EDL) molecular signature was significantly associated with worse outcome in breast cancer patients. To dissect this distinct cancer cell state, we developed novel in vitro models using 3D breast cancer organoids which responded to cytotoxic treatment by generating longitudinally-persistent residual organoid fractions that phenotypically and molecularly simulated the in vivo emergence of post-treatment residual tumors in preclinical and clinical settings. The treatment-persistent tumor fractions in cancer organoids and in the respective in vivo xenografts did not exhibit significant genetic changes compared to baseline tumor cells, but had reduced apoptotic priming and an EDL transcriptional reprograming similar to that of residual tumors in patients. Similarly to embryonic diapause, residual persistent fractions in cancer organoids, xenografts and patient tumors had markedly suppressed Myc transcriptional output and biosynthetic levels. Ectopic induction of MYC expression enhanced acute chemotherapeutic cytotoxicity in breast cancer organoids. Conversely, suppression of MYC or pharmacological inhibition of Myc transcriptional co-activators, BET bromodomains, abrogated chemotherapeutic cytotoxicity and induced in breast cancer cells an EDL molecular signature characterized by below-baseline redox stress levels which were maintained during drug exposure. High-throughput interrogation of residual persistent breast cancer organoids indicated broad refractoriness to specific anticancer drug classes thought to operate through induction of cellular stress (e.g. agents targeting DNA or DNA-repair). However, maintaining the residual cells in dormancy after completion of cytotoxic chemotherapy via inhibition of Brd4/Myc axis or pharmacologically interfering with the diapause-like transcriptional reprogramming of treatment-persistent cancer cells represent potential therapeutic strategies to target chemo-persistent tumor cells. Overall, our study shows that breast tumors dynamically co-opt the stress survival mechanism of embryonic diapause to persist during treatment, and reveals an unexpected role of Myc as regulator of cancer cell entry into transient drug-refractory dormancy. The diapause-like persister organoid cancer models provide ex vivo tractability for studying the otherwise elusive, dormant, drug-refractory residual tumors, with potential implications in personalized medicine and drug discovery. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Dhvanir Kansara, Juliette Bouyssou, Jennifer Roth, Michal Sheffer, Rinath Jeselsohn, Nathanael Gray, Ulrich Steidl, Boris Bartholdy, Myles Brown, Aedin Culhane, Constantine Mitsiades. Treatment persistence of residual breast tumors through an embryonic diapause-like cancer cell state with suppressed myc activity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS1-07.

Increased mitochondrial turnover in the skeletal muscle of fasted, castrated mice is related to the magnitude of autophagy activation and muscle atrophy.

Androgen-deficiency promotes muscle atrophy in part by increasing autophagy-mediated muscle protein breakdown during the fasted state, but factors contributing to this remain undefined. To identify novel factors, mice were subjected to sham or castration surgery. Seven-weeks post-surgery, mice were fasted overnight, refed for 30 min, and fasted another 4.5 h before sacrifice. BNIP3-mediated turnover of mitochondria was increased within the atrophied tibialis anterior (TA) of castrated mice and related to the magnitude of muscle atrophy and autophagy activation (i.e. decreased p62 protein content), thus linking turnover of potentially dysfunctional mitochondria with autophagy-mediated atrophy. Autophagy induction was likely facilitated by AMPK activation as a stress survival mechanism since phosphorylation of AMPK (Thr172), as well as the pro survival kinases Akt (Thr308) and (ERK1/2 Thr202/Tyr204), were increased by castration. Together, these data identify a novel relationship between mitochondrial turnover in the fasted state with autophagy activation and muscle atrophy following androgen depletion.

Natural history of a Balaustium sp. (Parasitengona: Erythraeidae) from eastern North America, with emphasis on moisture and temperature requirements

ABSTRACTXeric-suitability and heat tolerance by having a low water loss rate (3%/h) and high upper lethal temperature (48°C) are reported for adults of a Balaustium sp., which match typical hot, dry Balaustium habitat where pollen and arthropod prey on landscape plants are available. They do not have the capacity for water vapour absorption; i.e. the critical equilibrium humidity ≥100% relative humidity with body water losses in unsaturated air. They drink water from droplets, occurring only on occasion and by chance. Prey is their primary source of water. This mite has several features: (1) the inability to recover from heat shock, (2) little heat shock resistance, and (3) no acclimatizing effects by conditioning at a sublethal temperature. In absence of a heat stress-survival mechanism, cooler, shady refuges are sought and used more often that simultaneously help to minimize desiccation risks. This selection for shady spots permits niche differentiation from the bright-red B. murorum, a more desiccation- and heat-resistant species, that coexists in the same habitat and is active in well-lit, sunny locations.

Acute Myeloid Leukemia Cells Resist Chemotherapy through a Reversible Senescence-like State Maintaining Repopulation Potential

Cancer cells can undergo a senescence-like phenotype in response to genotoxic stress from chemotherapy. Relapse of acute myeloid leukemia (AML) frequently occurs months or years after initial chemotherapy and the functional consequence of chemotherapy-induced senescence (CIS) has not been elucidated. We propose that CIS acts as a stress-survival mechanism in AML, allowing cells to persist in a dormant state with potential to repopulate leukemia post-treatment.To quantify senescence-associated-β-galactosidase (SA-β-gal) activity, we used a fluorogenic substrate (C12-FDG) that enabled sensitive quantification of the senescence marker in viable cells by flow cytometry. We found increased SA-β-Gal activity after exposure to the chemotherapeutic agent cytarabine (AraC) in p53-deficient myeloid leukemia cell lines (K-562 and KG-1), indicating that p53 is not essential for CIS in AML. Using an ex vivo culturing model, we found that patient-derived AML cells demonstrate a greater SA-β-gal response to increasing dosage or duration of AraC treatment until cells undergo apoptosis and exhibit diminished C12-FDG levels. This suggested that CIS is not a distinct condition, but rather a continuous response to the degree of genotoxic stress. ATR kinase activation mediates senescence by enforcing cell cycle arrest in response to genotoxic stress during replication. Treatment of AML specimens with an inhibitor of ATR (VE-821) reduced the initiation of senescence and markedly impaired cell survival after exposure to a moderate dose of AraC. This implied that ATR mediates CIS, independent of p53, to increase stress survival following AraC treatment.In order to characterize the response to genotoxic stress, we treated patient-derived AML cells with AraC, analyzed RNA expression, and performed gene set enrichment analysis (GSEA) with senescence-associated genes compiled from literature. Inflammatory mediators and extracellular matrix proteins are strongly induced after AraC treatment; these are partly related to the senescence-associated secretory phenotype (SASP) and senescence-messaging secretome (SMS) (p<0.001). Thus, primary AML cells also display a senescent gene expression signature after AraC exposure.To model the latent dormant state after chemotherapy, we treated patient-derived AML cells with high doses of AraC (1000 and 10 000 nM for 3 days) that killed the majority of the leukemia population in vitro. Cells surviving initial AraC treatment with 1000 nM persisted in a senescent-like dormant state for 3-4 weeks before initiating leukemia repopulation, while cells treated with 10 000 nM AraC continued to be in a dormant state. To recapitulate the relapse in vivo, we established an AML relapse model using primary human specimens engrafted in NSG mice. Administration of AraC, using a physiologically relevant dose and schedule (60 mg/kg/day x 5 days), to AML-engrafted NSG mice reduced peripheral blood leukemic cells and total body leukemic burden 8 days after initiation of therapy to a small residual leukemia fraction (nadir). This reduction in leukemic burden was reversed after 4 weeks. Gene expression analysis of purified human AML cells at nadir (day 8) were significantly enriched for senescence signatures (p<0.001). This enrichment was reduced by day 29 post-treatment as expression was partially reversed to untreated levels. Notably, GSEA revealed reduction of hematopoietic and leukemia stem cell signatures at nadir, which partially recovered in some AML cases at day 29.Finally, to demonstrate that senescent-like cells maintain leukemia-repopulating potential, we sorted for low (untreated control cells), moderate, and high levels of SA-β-gal activity in C12-FDG+ AML cells after AraC treatment. Transplantation of the sorted cells into NSG mice demonstrated repopulation of leukemia from senescent-like cells. Notably, mice transplanted with equal numbers of high C12-FDG+ senescent-like cells had shorter overall survival compared to mice with moderate or low C12-FDG+ cells.Altogether, our results show that AML cells after chemotherapy can persist in a reversible senescent-like dormant state with leukemia repopulation capacity and reveal a novel mechanism of chemotherapy resistance with therapeutic potential in AML. DisclosuresDuy:GlaxoSmithKline: Research Funding. Melnick:Janssen: Research Funding.

Abstract 2897: Phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy

Abstract Autophagy is a cellular stress survival mechanism in which newly formed double-membrane vesicles target parts of the cytosol and organelles for lysosomal degradation to maintain cellular energy production. The source of the autophagosome membrane in starvation has been subject to much debate, but has not been investigated in the more clinically relevant setting of drug-induced autophagy where nutrients are available. By 1H-magnetic resonance spectroscopy (MRS) we show that autophagy, induced in human colorectal carcinoma cells HCT116 wt, HCT116 Bax-ko and HT29 by dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, by PI-103, a dual PI3K/mTOR inhibitor, or by starvation in medium lacking amino acids and growth factors, is associated with significant increases in membrane phospholipid phosphatidylcholine (PtdCho) (p≤0.02). 13-C labeled choline 13C-MRS revealed that these increases are due to de novo synthesis of PtdCho (p≤0.02) and are linked with increased expression of the rate-limiting enzyme, CDP:phosphocholine cytidylyltransferase (CCTα) in its active, membrane-bound form. Autophagic cells labeled with propargylcholine, a choline analog, had significant increase in propargylcholine phospholipid synthesis (p≤0.02) and vesicular structures consistent with autophagosomes when imaged by fluorescence confocal microscopy. Electron microscopy with immunogold labeling reveals the incorporation of newly synthesized propargylcholine phospholipids into the membranes of autophagic vacuoles and the digested material in autophagolysosomes, as well as endoplasmic reticulum, mitochondrial membranes and nuclear envelope as in control cells. To assess the role of PtdCho synthesis and CCTα in autophagy, we used Chinese hamster ovarian cells CHO-K1 MT58, with a temperature-sensitive mutation in the CCTα gene, resulting in reduced PtdCho synthesis at 40°C. PI-103 induced autophagy in wild-type CHO-K1 cells at both 33°C and 40°C. MT58 cells had normal autophagy induction with PI-103 at 33°C, but the autophagy marker LC3B-II was lower at 40°C than in the CHO-K1 cells. Loss of CCTα activity prevented PI-103-induced increase in PtdCho levels (p≤0.0003) and resulted in a reduced volume of autophagosomes in MT58 cells at 40°C (p≤0.01). Our evidence suggests that increased PtdCho synthesis in drug-induced autophagy provides membrane phospholipids for the growing autophagosomes and replaces phospholipids consumed from other organelles during autophagosome formation and degradation. Expression of CCTα, the rate-limiting enzyme of PtdCho synthesis, is increased in autophagy and its loss results in the inability of cells to maintain autophagosome formation. PtdCho synthesis might provide a new target for autophagy inhibition. Also, fluorescence imaging of propargylcholine labeling may offer a novel way to visualize newly formed autophagosome membranes in vitro. Citation Format: Gabriela Andrejeva, Gigin Lin, Harry G. Parkes, James Mui, Anne-Christine LF Wong Te Fong, Gema Vizcay-Barrena, Roland Fleck, Martin O. Leach, Yuen-Li Chung. Phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy. [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 2897. doi:10.1158/1538-7445.AM2015-2897

Earthworms as agents for ecotoxicity in roxarsone-contaminated soil ecosystem: a modeling study of ultrastructure and proteomics.

Contamination of roxarsone has been recognized as a potential environmental hazard. In this study, Eisenia fetida samples were collected after roxarsone exposures to analyze their intestinal epithelium ultrastructure, expression levels of stress-related genes, and proteomics. Our results showed that mitochondria and endoplasmic reticulum in roxarsone-treated earthworms demonstrated variety of damages. Furthermore, 149 proteins were displayed in 2-DE, and 36 of them were identified by MALDI-TOF/TOF-MS. Those identified proteins are involved in several important processes including cell immunity, cell stress responses, and cell genetic behaviors. Our study demonstrates the toxicity responses of earthworms toward arsenic-based animal drug roxarsone with practical usefulness and demonstrates a proteomic profile change that may be critical for the roxarsone stress survival mechanisms of E. fetida. Graphical Abstract Inspiration of this referred to the form of Fig. 4 in the article "Proteomic analysis of a high aluminum tolerant yeast Rhodotorula taiwanensis RS1 in response to aluminum stress" of Chao, W et al.

Deciphering the response of Mycobacterium smegmatis to nitrogen stress using bipartite active modules

BackgroundThe ability to adapt to environments with fluctuating nutrient availability is vital for bacterial survival. Although essential for growth, few nitrogen metabolism genes have been identified or fully characterised in mycobacteria and nitrogen stress survival mechanisms are unknown.ResultsA global transcriptional analysis of the mycobacterial response to nitrogen stress, showed a significant change in the differential expression of 16% of the Mycobacterium smegmatis genome. Gene expression changes were mapped onto the metabolic network using Active Modules for Bipartite Networks (AMBIENT) to identify metabolic pathways showing coordinated transcriptional responses to the stress. AMBIENT revealed several key features of the metabolic response not identified by KEGG enrichment alone. Down regulated reactions were associated with the general reduction in cellular metabolism as a consequence of reduced growth rate. Up-regulated modules highlighted metabolic changes in nitrogen assimilation and scavenging, as well as reactions involved in hydrogen peroxide metabolism, carbon scavenging and energy generation.ConclusionsApplication of an Active Modules algorithm to transcriptomic data identified key metabolic reactions and pathways altered in response to nitrogen stress, which are central to survival under nitrogen limiting environments.

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states.

Nitric Oxide Modulates the Expression of Proteins and Promotes Epiphyllous Bud Differentiation in Kalanchoe pinnata

Nitric oxide (NO), a recent addition to the signaling molecules in plants, plays an important role in mediating both biotic and abiotic stress responses. The occurrence of reproductive/vegetative structures, known as epiphylly, on the surface of leaves is a stress survival mechanism exhibited by some plants, including Kalanchoe pinnata. In the present study the role of NO during epiphyllous bud differentiation was investigated. NO donors l-arginine, NaNO2, and SNP promoted epiphyllous bud differentiation in a dose-dependent manner, whereas NO inhibitors reversed the effect, suggesting the involvement of NO in the process. Albeit numerous physiological processes are reported to be modulated by NO, but their correlation with NO-responsive genes and proteins needs to be established. To address this issue, NO-responsive proteins (NORPs) were identified using 2D-PAGE and MS analysis. Major NORPs that were identified belonged to photosynthesis, oxidative phosphorylation, signaling, and stress metabolism, aptly indicating their probable role in this stress-induced growth process. The present study clearly indicates the role of NO in this stress survival mechanism, thus strongly affirming its role in stress responses.

Compatible solutes

Recently we reported a role for compatible solute uptake in mediating bile tolerance and increased gastrointestinal persistence in the foodborne pathogen Listeria monocytogenes.1 Herein, we review the evolution in our understanding of how these low molecular weight molecules contribute to growth and survival of the pathogen both inside and outside the body, and how this stress survival mechanism may ultimately be used to target and kill the pathogen.

Compatible solutes: the key to Listeria's success as a versatile gastrointestinal pathogen?

Recently we reported a role for compatible solute uptake in mediating bile tolerance and increased gastrointestinal persistence in the foodborne pathogen Listeria monocytogenes[1]. Herein, we review the evolution in our understanding of how these low molecular weight molecules contribute to growth and survival of the pathogen both inside and outside the body, and how this stress survival mechanism may ultimately be used to target and kill the pathogen.

Patho-Biotechnology - Making New Friends of Old Foes

The rationale for choosing pathogens over commensal species as a source of potential stress survival mechanisms is based on a number of factors; not least of which is the fact that certain pathogenic bacteria, with lifecycles bridging the host and external milieu, have evolved sophisticated stress management strategies enabling them to succeed in more diverse ecological niches than those normally occupied by most non-pathogenic species [7]. Pathogens thus represent a rich source of genes which could potentially improve the physiological vigor of less versatile probiotic strains, both external to and within the host.