Growth Arrest In Phase Research Articles

Diverse intrinsic properties shape transcript stability and stabilization in Mycolicibacterium smegmatis.

Mycobacteria regulate transcript degradation to facilitate adaptation to environmental stress. However, the mechanisms underlying this regulation are unknown. Here we sought to gain understanding of the mechanisms controlling mRNA stability by investigating the transcript properties associated with variance in transcript stability and stress-induced transcript stabilization. We measured mRNA half-lives transcriptome-wide in Mycolicibacterium smegmatis in log phase growth and hypoxia-induced growth arrest. The transcriptome was globally stabilized in response to hypoxia, but transcripts of essential genes were generally stabilized more than those of non-essential genes. We then developed machine learning models that enabled us to identify the non-linear collective effect of a compendium of transcript properties on transcript stability and stabilization. We identified properties that were more predictive of half-life in log phase as well as properties that were more predictive in hypoxia, and many of these varied between leadered and leaderless transcripts. In summary, we found that transcript properties are differentially associated with transcript stability depending on both the transcript type and the growth condition. Our results reveal the complex interplay between transcript features and microenvironment that shapes transcript stability in mycobacteria.

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

Understanding how MSCs reverse radiation-induced fibrosis: HGF vs. TGF-B1

Understanding how MSCs reverse radiation-induced fibrosis: HGF <i>vs.</i> TGF-B1

Lipidomics analysis reveals the effect of Sirex noctilio infestation on the lipid metabolism in Pinus radiata needles

The Sirex noctilio’s climatic adaption and rapid proliferation have caused Pinus mortality worldwide. The infestation combines the early effect of female S. noctilio gland secretion and the spreading symbiotic fungus Amylostereum areolatum. 'Lipidomics' is the study of all non-water-soluble components of the metabolome. Most of these non-water-soluble compounds correspond to lipids which can provide information about a biological activity, an organelle, an organism, or a disease. Using HPLC-MS/MS based lipidomics, 122 lipids were identified in P. radiata needles during S. noctilio infestation. Phosphatidic acids, N-acylethanolamines, and phosphatidylinositol-ceramides accumulated in infested trees could suggest a high level of phospholipases activities. The phosphatidylcholines were the most down-regulated species during infection, which could also suggest that they may be used as a substrate for up-regulated lipids. The accumulation of very long-chain fatty acids and long-chain fatty acids during the infestation could imply the tree defense response to create a barrier in the drilled zone to avoid larvae development and fungus proliferation. Also, the growth arrest phase of the trees during the prolonged infestation suggests a resistance response, regulated by the accumulation of NAE, which potentially shifts the tree energy to respond to the infestation.

Implication of Adipogenesis-Coupled CRMP2 Functional Profile in Metabolic Homeostasis and Imbalance

Our previous studies demonstrated that collapsin response mediator protein 2 (CRMP2) is associated with obesity and, in addition, that hyperglycemia-suppressed CRMP2 augments malignant traits of colorectal cancer and is associated with advanced tumor stage. Regulation of CRMP2 profile was further explored in this study using 3T3-L1 pre-adipocyte adipogenesis as a study model for illustrating the roles of CRMP2 in metabolic homeostasis. Hyperglycemia inhibited expression of CRMP2, adipogenic machinery and adipocyte markers. CRMP2 displayed f-CRMP2 (62~66 kDa) and s-CMRP2 (58 kDa) isoforms at the growth arrest phase. Expression of s-CRMP2 was coupled with the mitotic clonal expansion (MCE) phase to direct cell proliferation and rapidly down-regulated in post-mitotic cells. In the late differentiation phase, f-CRMP2 was co-localized with tubulin in the cortical area. Insulin-enhanced CRMP2-glucose transporter 4 (GLUT4) co-localization and CRMP2 puncta on lipid droplets (LDs) suggested participation of CRMP2 in GLUT4 translocation and LD fusion. Collectively, the CRMP2 functional profile must be finely controlled to adjust cytoskeletal stability for meeting dynamic cellular needs. Manipulating the s-CRMP2/f-CRMP2 ratio and thus the cytoskeleton dynamics is anticipated to improve glucose uptake and insulin sensitivity. In summary, our data provide molecular evidence explaining the functions of CRMP2 in physiological, pathological and disease progression in metabolic homeostasis and disorders related to metabolic abnormalities, including cancer.

Cellular Memory of HipA-Induced Growth Arrest: The Length of Cell Growth Arrest Becomes Shorter for Each Successive Induction.

Toxin–antitoxin (TA) systems are genetic modules found commonly in bacterial genomes. HipA is a toxin protein encoded from the hipBA TA system in the genome of Escherichia coli. Ectopic expression of hipA induces cell growth arrest. Unlike the cell growth arrest caused by other TA toxins, cells resume growth from the HipA-induced cell growth arrest phase after a defined period of time. In this article, we describe the change in the length of growth arrest while cells undergo repeated cycles of hipA induction, growth arrest and regrowth phases. In the multiple conditions tested, we observed that the length of growth arrest became successively shorter for each round of induction. We verified that this was not due to the appearance of HipA-resistant mutants. Additionally, we identified conditions, such as the growth phase of the starting culture and growth vessels, that alter the length of growth arrest. Our results showed that the length of HipA-induced growth arrest was dependent on environmental factors—in particular, the past growth environment of cells, such as a previous hipA induction. These effects lasted even after multiple rounds of cell divisions, indicating the presence of cellular “memory” that impacts cells’ response to HipA-induced toxicity.

Identification of Novel Targets Based on Splicing Alterations for Undruggable RAS/CDK Signaling Cascade in Multiple Myeloma

Background: RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis. Targeting these pathways represents a novel therapeutic strategy in MM. Our ongoing studies (>420 patients) demonstrate that aberrantly spliced transcript expressions can predict MM patient survival outcomes better than gene expression alone, indicating a significant role of splicing mechanism in MM pathophysiology. These studies also identified intron retentions as the predominant recurrent alterations (~32% of spliced genes were retained introns) in MM. We evaluated splicing alterations associated with pathway-level responses after RAS/CDK inhibition in order to identify and validate novel molecular targets.Methods/results: MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to inhibit RAS and CDK pathways. Our studies demonstrated strong synergistic (IC<0.5) MM cytotoxicity triggered by this combination treatment, which triggered dose-dependent manner G0/G1 phase growth arrest. We assessed early death cascade in MM cells after Ei+Ci treatment, and demonstrated significant priming to selective peptides BIM, BAD, and MS1 or HRK, suggesting dependency on BCL2 and MCL1 or on BCL-XL proteins. Our studies showed that Ei+Ci treatment induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling including c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of Ei and Ci. Patient MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that this combination can overcome the protective effects of the MM BM milieu. Moreover, our in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. We evaluated the effect of Ei+Ci treatment on target gene expression in BM cells isolated from flushed femurs of treated animals with Ei, Ci or Ei+Ci, and observed downregulation of Erk1/2-CDK4/6-dependent gene signature. Therefore, we suggest that these inhibitors selectively target Erk1/2, CDK4/6 and their downstream substrates both in vitro/vivo.We next evaluated aberrantly spliced transcript expression in MM cells, with/without Erk1/2 knockdown (KD) or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. This observation was further supported by principal component analyses: upregulation in response to Erk1/2 KD and downregulation due to treatment with Ei+Ci were considered spliced gene-signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complex and are involved in intron retention processes, a marker of malignant transformation. We compared signature-gene expressions from 558 MM patient samples to the signature-genes in plasma cells from normal donors and observed significant (p<2e-11) upregulation of genes with progression from MGUS to sMM, and, also to overt MM . SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 is linked with poor outcomes. SNRPB proteins are also overexpressed in MM cells. Our studies evaluating SNRPB effects on RNA splicing showed both upregulation of transcripts with full intron retention and transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. We evaluated RAVER1 and SNRPB expression in BM cells from animals treated with Ei and Ci alone or in combination. We observed significant downregulation of RAVER1/SNRPB (p=0.001) in BM samples obtained from animals treated with Ei+Ci. We observed decreased intron retention events in genes in treated samples, consistent with our in vitro analyses in MM cell lines and patient samples. Thus, RAVER1/SNRPB overexpression contributes to the aberrant transcriptome splicing associated with RAS/CDK cascade in MM.Conclusions: Our studies 1) show an association between RNA processing and RAS-CDK pathways in MM, 2) identify a core splicing protein, SNRPB/RAVER1, as a novel target for modulating this cascade, and 3) suggest that targeting spliceosome complexes represents a promising therapy in MM. DisclosuresLetai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Anderson: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Anticancer Activity of a Novel High Phenolic Sorghum Bran in Human Colon Cancer Cells.

Human colon cancer is the third leading cause of mortality in the United States and worldwide. Chemoprevention using diet is widely accepted as a promising approach for cancer management. Numerous population studies indicate a negative correlation between the incidence of colon cancer and consumption of whole grains with a high content of bioactive phenolic compounds. In the current study, we evaluated the anticancer properties of a high phenolic sorghum bran extract prepared using 70% ethanol with 5% citric acid solvent at room temperature. A significant dose-dependent suppression of cell proliferation was observed in human colon cancer cells treated with the high phenolic sorghum bran extract. Apoptosis and S phase growth arrest were induced, while cell migration and invasion were inhibited by this treatment; these effects were accompanied by altered expression of apoptosis, cell cycle, and metastasis-regulating genes. We also found that the high phenolic sorghum bran extract stimulated DNA damage in association with induction of extracellular signal-regulated kinase (ERK) and c-Jun-NH2-terminal kinase (JNK) and subsequent expression of activating transcription factor 3 (ATF3). The present study expands our understanding of the potential use of high phenolic sorghum bran to prevent human colon cancer.

Synthesis and biological evaluation of N-Alkylamide derivatives as anti-tumor agents

N-Alkylamides (NAAs), derived from Anacyclus pyrethrum (L.) DC, have potential anti-tumor effects. To explore the molecular mechanism and chemo-preventive capacity of NAAs, we synthesized an NAA (H-10) and evaluated whether it could inhibit the proliferation of B16, HepG2, HeLa, and HCT116 cancer cells in 2D culture. To evaluate the antiproliferative activity of H-10 in 2D and 3D culture of BD, HepG2, HeLa, and HCT116 cells, multicellular tumor spheroids were constructed to more accurately reflect the cell tumor environment. To visualize nuclear changes related to apoptosis, Hoechst 33258 staining and propidium iodide-Annexin V double staining were performed. Compound H-10 strongly inhibited the growth of all tested cell lines. Hoechst 33258 staining and propidium iodide-Annexin V double staining revealed that H-10 did not cause morphological alterations in the nuclei and moderately induced late apoptosis only when treated at 180 μM. The strongest inhibitory effect was observed in HCT116 cells. Flow cytometry analysis indicated that treatment of HCT116 cells with compound H-10 resulted in robust cell growth arrest in G2 phase in 2D and 3D cell culture; in 3D-cultured HCT116 cells, growth arrest occurred in G1 phase. Treatment with compound H-10 also significantly suppressed angiogenesis of chick chorioallantoic membrane in vivo . Treatment with compound H-10 strongly affected clonogenic survival (in the long-term) and migration of HCT116 cells. Therefore, H-10, a compound of NAAs may be useful for treating cancer because of its anti-neoplastic effect and easy synthesis.

Regulatory role of transcription factor HBP1 in anticancer efficacy of EGFR inhibitor erlotinib in HNSCC.

Epidermal growth factor receptor (EGFR) is often hyperactivated in head and neck squamous cell carcinoma (HNSCC); however, its downstream mediators are not fully identified. Here, we investigate the role of transcription factor HBP1 in the anticancer efficacy of EGFR inhibitor erlotinib in HNSCC. The effect of erlotinib and HBP1 on cell proliferation and invasion was examined by flow cytometric analysis and a Matrigel invasion assay, respectively. Oral tumor specimens were used to evaluate the association between the expression level of EGFR and HBP1, and metastatic potential. Erlotinib caused cell growth arrest in the G1 phase and sluggish invasion with a concomitant increase in HBP1 and p27 expression. The erlotinib effect was attenuated upon HBP1 knockdown. Analysis of oral tumor specimens revealed that the low HBP1/high EGFR status can predict metastatic potential. Our data support HBP1 as a crucial mediator of EGFR-targeting inhibitors in HNSCC.

The use of catechins in Chinese hamster ovary cell media for the improvement of monoclonal antibody yields and a reduction of acidic species.

Catechin compounds have potential benefits for recombinant monoclonal antibody (Mab) production as chemical additives in cell culture media. In this study, four catechin compounds catechin (Cat), epicatechin (EC), gallocatechin-gallate (GCG), and epigallocatechin-gallate (EGCG) were added to cell culture media (at 50 μM) and their effects on the recombinant Chinese hamster ovary (CHO) cell culture, specific productivity, and Mab quality were assessed. The results indicate that the improvement of specific productivity was linked to cell growth inhibition. All catechins caused cell phase growth arrest by lowering the number of cells in the G1/G0 phase and increasing the cells in the S and G2/M phases. Late addition of the catechin resulted in a significantly higher final IgG concentration. Cat and EC caused an improvement in the final antibody titer of 1.5 ± 0.1 and 1.3 ± 0.1 fold, respectively. Catechins with a galloyl group (GCG and EGCG) arrested cell growth and reduced cell specific productivity at the concentrations tested. The Cat-treated IgG was found to have reduced acidic species with a corresponding increase in the main peak.

Deciphering the Molecular Mechanism of Spontaneous Senescence in Primary Epithelial Ovarian Cancer Cells.

Spontaneous senescence of cancer cells remains a puzzling and poorly understood phenomenon. Here we comprehensively characterize this process in primary epithelial ovarian cancer cells (pEOCs). Analysis of tumors from ovarian cancer patients showed an abundance of senescent cells in vivo. Further, serially passaged pEOCs become senescent after a few divisions. These senescent cultures display trace proliferation, high expression of senescence biomarkers (SA-β-Gal, γ-H2A.X), growth-arrest in the G1 phase, increased level of cyclins D1, D2, decreased cyclin B1, up-regulated p16, p21, and p53 proteins, eroded telomeres, reduced activity of telomerase, predominantly non-telomeric DNA damage, activated AKT, AP-1, and ERK1/2 signaling, diminished JNK, NF-κB, and STAT3 pathways, increased formation of reactive oxygen species, unchanged activity of antioxidants, increased oxidative damage to DNA and proteins, and dysfunctional mitochondria. Moreover, pEOC senescence is inducible by normal peritoneal mesothelium, fibroblasts, and malignant ascites via the paracrine activity of GRO-1, HGF, and TGF-β1. Collectively, pEOCs undergo spontaneous senescence in a mosaic, telomere-dependent and telomere-independent manner, plausibly in an oxidative stress-dependent mechanism. The process may also be activated by extracellular stimuli. The biological and clinical significance of pEOC senescence remains to be explored.

Ribosome Reconstruction during Recovery from High-Hydrostatic-Pressure-Induced Injury in Bacillus subtilis.

Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf, whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn2+ or Zn2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn2+ or Zn2+ Moreover, Zn2+, but not Mn2+, promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn2+-dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn2+ and Zn2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells.IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn2+ and Zn2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells.

New synthetic chitosan hybrids bearing some heterocyclic moieties with potential activity as anticancer and apoptosis inducers

Wastes of the freshwater crayfish Procambarus clarkii were used as precursor for the extraction of chitin, which was deacetylation with alkali giving chitosan. Chitosan was transformed into the nanoparticles using sodium tripolyphosphate. Chitosan Cs and its nanoparticles CNP were characterized through FTIR, 1H NMR and surface morphology. Moreover, it was converted into some chitosan Schiff bases; CSB-1,2,3, containing different five membered heterocyclic moieties and also they were characterized. All of the synthesized compounds were evaluated against three cell lines, such as HepG-2, HCT-116 and MCF-7, whereas compounds CSB-2 and CSB-3 showed the highest cytotoxic activities. The higher activities for the latter compounds were confirmed through apoptosis studies (Caspase-3 and Bax/Bcl-2 ratio) against HePG-2 cells. Cell cycle analysis and apoptosis induction showed that compound CSB-2 induces apoptosis at pre G1 phase and cell growth arrest at G2/M phase.

P1.14-41 The Combination of the PARP Inhibitor Olaparib and the ATR Inhibitor VE-821 Selectively Targets ATM-Deficient Lung Cancer Cells

The driving principle behind precision medicine is to specifically target genetic variations that arise in tumorigenesis while leaving normal cells unaffected. Mutations in Ataxia Telangiectasia Mutated (ATM) may offer such a therapeutic target. ATM is mutated in approximately 12% of lung cancers and up to 40% of lung adenocarcinoma have been reported to lack ATM protein expression. ATM is an apex signaling kinase that responds to DNA-double strand breaks, playing a direct role in DNA repair as well as the initiation of signaling cascades that can lead to cell cycle arrest and apoptosis. We asked whether ATM-deficient human lung cancer cells are sensitive to the poly-ADP ribose polymerase (PARP) inhibitor olaparib, and investigated the mechanism of action of olaparib in these cells. We analyzed drug sensitivity for 61 lung adenocarcinoma cell lines from the Genomics of Drug Sensitivity in Cancer (GDSC) project and deleted ATM from lung adenocarcinoma A549 cells using CRISPR/Cas9. We determined the effects on cell viability using trypan blue exclusion and clonogenic survival assays. To investigate the mechanism of sensitivity of ATM-deficient cells to PARP and ATR inhibitors we used flow cytometry and cell viability assays as above. We observed a positive correlation between olaparib IC50 values and ATM mRNA expression. ATM mutant cell lines were more sensitive to olaparib compared to ATM wild-type cell lines or cell lines with amplified ATM. Additionally, ATM-deficient lung cancer cells were sensitive to olaparib, as are lung cancer cells (A549) with CRISPR/Cas9 deletion of ATM. Mechanistically, olaparib caused the temporary and reversible accumulation of G2 phase cells in ATM-deficient cells which manifested as a decrease in proliferation in both the trypan blue exclusion assay and clonogenic survival assay. Olaparib did not induce cell death in ATM-deficient cells, however cell death was induced when olaparib was used in combination with the ATR inhibitor VE-821. We show that olaparib acts as a cytostatic agent in ATM-deficient lung cancer cells, inducing a reversible and temporary growth arrest in G2 phase. Only when combined with the ATR inhibitor VE-821 was cell death observed and only in ATM-deficient cells. Our data suggest that patients with ATM-deficient lung cancer could benefit from combinatorial treatment with PARP and ATR inhibitors.

Abscisic acid-dependent histone demethylation during postgermination growth arrest in Arabidopsis.

After germination, seedlings undergo growth arrest in response to unfavourable conditions, a critical adaptation enabling plants to survive harsh environments. The plant hormone abscisic acid (ABA) plays a key role in this arrest. To arrest growth, ABA-dependent transcription factors change gene expression patterns in a flexible and reversible manner. Although the control of gene expression has important roles in growth arrest, the epigenetic mechanisms in the response to ABA are not fully understood. Here, we show that the histone demethylases JUMONJI-C domain-containing protein 30 (JMJ30) and JMJ32 control ABA-mediated growth arrest in Arabidopsis thaliana. During the postgermination stage (2-3days after germination), the ABA-dependent transcription factor ABA-insensitive3 (ABI3) activates the expression of JMJ30 in response to ABA. JMJ30 then removes a repressive histone mark, H3 lysine 27 trimethylation (H3K27me3), from the SNF1-related protein kinase 2.8 (SnRK2.8) promoter, and hence activates SnRK2.8 expression. SnRK2.8 encodes a kinase that activates ABI3 and is responsible for JMJ30- and JMJ32-mediated growth arrest. A feed-forward loop involving the ABI3 transcription factor, JMJ histone demethylases, and the SnRK2.8 kinase fine-tunes ABA-dependent growth arrest in the postgermination phase. Our findings highlight the importance of the histone demethylases in mediating adaptation of plants to the environment.

PO-503 HDAC inhibitor resistance in colorectal cancer: RAS and AMP; MYC – the partners in crime

IntroductionOncogenic KRAS is widely acknowledged as a critical determinant of the therapeutic response of colorectal cancer (CRC) – a fact that to date is pivotal for defining an appropriate treatment strategy. Whether this also extends beyond RTK- and MAPK- targeted molecular cancer therapies, to a novel and promising class of anti-cancer drugs, namely HDAC inhibitors (HDACi), is an aspect that until now has remained largely undefined. Our aim is, therefore, to establish whether RAS is an effective predictor of response to HDACi in CRC. Ultimately, we intend to shed light on the cause underlying the limited clinical benefits of HDACi as a treatment option for solid tumours, including CRC and thereby identify opportunities to improve the prospects of HDACi treatment.Material and methodsWe investigated the presence of an oncogenic-RAS dependency against a wide range of different HDAC inhibitors using model systems with intrinsic and conditionally active RAS oncogenes.Results and discussionsWe uncovered an oncogenic RAS-dependent ‘safeguard’ mechanism imposed in order to evade the cytotoxic effect of HDACi and thereby apoptosis. Cells harbouring oncogenic RAS were observed to undergo a reversible senescence-like growth arrest in G2 phase, allowing for re-entry into cell cycle following a withdrawal of the inhibitor. This mechanism is implemented as a consequence of the inhibition of the RAS deacetylase, namely HDAC2, which in turn result in the generation of (hyper)acetylated RAS with increased binding affinity to BRAF and CRAF. This translates to a further amplification in MAPK-signalling and thus an increase in the priming of c-MYC for ubiquitin-mediated proteasomal degradation, thereby enabling the cells to exit the cell cycle and enter the defined protective state of G2 arrest. The prospect of HDACi treatment was effectively improved using current MAPK-targeted therapy and senolytic drugs by effectively preventing the observed pro-oncogenic effect of the HDACi treatment alone.ConclusionOur study reveals an oncogenic RAS-dependent resistance mechanism, enabling cells harbouring oncogenic RAS, to establish a favourable cellular state of prolonged pharmacological hideout - a phenomenon that is replicated in patient-derived 3D cell culture models of CRC. This highlights the potential clinical relevance of our findings and thus the importance of a rational mechanism-based combinatorial therapeutic design in order to realise the true therapeutic potential of HDACi.

A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway.

Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53mt), MiaPaCa-2 (p53mt), and Capan-2 (p53wt) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

A Stress Response that Monitors and Regulates mRNA Structure Is Central to Cold Shock Adaptation

Temperature influences the structural and functional properties of cellular components, necessitating stress responses to restore homeostasis following temperature shift. Whereas the circuitry controlling the heat shock response is well understood, that controlling the E.coli cold shock adaptation program is not. We found that during the growth arrest phase (acclimation) that follows shift to low temperature, protein synthesis increases, and open reading frame (ORF)-wide mRNA secondary structure decreases. To identify the regulatory system controlling this process, we screened for players required for increased translation. We identified a two-member mRNA surveillance system that enables recovery of translation during acclimation: RNase R assures appropriate mRNA degradation and the Csps dynamically adjust mRNA secondary structure to globally modulate protein expression level. An autoregulatory switch in which Csps tune their own expression to cellular demand enables dynamic control of global translation. The universality of Csps in bacteria suggests broad utilization of this control mechanism.

The two novel DLL4-targeting antibody-drug conjugates MvM03 and MGD03 show potent anti-tumour activity in breast cancer xenograft models

The anti-human Delta-like 4 (DLL4) monoclonal antibody MMGZ01 has a high affinity to hrDLL4 and arrests the DLL4-mediated human umbilical vein endothelial cell (HUVEC) phenotype, promotes immature vessels, and effectively reduces breast cancer cell growth in vivo. To develop a much more effective therapy, we conjugated MMGZ01 with two small-molecule cytotoxic agents, i.e., monomethyl auristatin E (MMAE) and doxorubicin (DOX), with different linkers to generate antibody–drug conjugates (ADCs), i.e., MMGZ01-vc-MMAE (named MvM03) and MMGZ01-GMBS-DOX (named MGD03), that are more potent therapeutic agents than naked antibody therapeutic agents. The produced anti-DLL4 ADCs can be effectively directed against DLL4 and internalized. Then, the release of MMAE or DOX into the cytosol can induce G2/M or G0/G1 phase growth arrest and cell death through the induction of apoptosis. In vitro, MvM03 was highly potent and selective against DLL4 cell lines. The anti-DLL4 ADCs, particularly MvM03, showed more potent anti-tumour activity than Docetaxel, which is an inhibitor of the depolymerisation of microtubules, in two xenograft breast cancer tumour models. Our findings indicate that anti-DLL4 ADCs have promising potential as an effective therapy for breast cancer.