Growth Of Suspension Cultures Research Articles

Source of nitrogen as a factor limiting saponin production by hairy root and suspension cultures of Calendula officinalis L.

Triterpenic saponins represented in Calendula officinalis L. by oleanolic acid (OA) glycosides are pentacyclic triterpene compounds with a wide range of biological and medicinal properties. This report demonstrates nitrogen source impact on growth, saponin accumulation, and secretion in hairy root and suspension cultures of marigold. Hairy roots preferred nitrate as a mineral source of nitrogen, but its impact on growth, OA glycosides accumulation, and secretion were line-dependent. The best productivity of OA glycosides was found in CC16 line (74.86 mg flask−1) in ½ MS medium modified by 2.5× KNO3 and ammonium elimination with 2.5 g l−1 peptone. Organic nitrogen source at 27.5-g l−1 impairs the growth rate of hairy roots. Its effect on saponin accumulation and secretion to the surrounding medium depended on line and media composition. Nitrate:ammonium ratio of 4:2 for CC16 resulted in 5.7-fold increment of saponin secretion comparing to the standard medium. Embryo roots, apical bud, and hypocotyls explants were crucial for induction of suspension culture synthesizing saponins; however, effect of mineral form of nitrogen in cultivating medium had to be considered. The highest OA glycosides level (171.97 μg g−1 of dry weight) was recorded in the root derived culture with nitrate as a sole mineral form of nitrogen. Peptone from lactalbumin decidedly inhibited the saponin formation; however, it was essential for culture initiation, proliferation, and organ differentiation.

Optimization of cell growth and bacoside-A production in suspension cultures of Bacopa monnieri (L.) Wettst. using response surface methodology

Plant secondary metabolites have emerged as potential raw materials, which are used in the pharmaceutical, food, chemical, and cosmetic industries. Bacoside-A, a secondary metabolite produced by Bacopa monnieri, is known for its memory-facilitating properties. In recent years, various strategies have been developed to enhance biomass accumulation and synthesis of secondary compounds in cultures. In the present investigation, various factors affecting the production of biomass and bacoside-A in the cell suspension cultures of B. monnieri were optimized using the statistical experimental design approach. Preliminary screening by Plackett–Burman’s design revealed that among the tested factors, glucose, KNO3, KH2PO4, and inoculum density significantly influenced cell growth and bacoside-A production. Furthermore, using response surface methodology (RSM), glucose, KNO3, and KH2PO4 at a concentration of 5.67, 0.313, and 0.29%, respectively, and an inoculum density of 0.66% in basal MS medium were found to be optimal for cell growth and bacoside-A production. After optimization, the biomass yield increased about twofold (from 5.52 to 12.58 g L−1 fresh cell weight) and bacoside-A production about 1.7-fold (5.56 to 9.84 mg g−1 dry weight). The present study results show the successful application of RSM to enhance the production of biomass and accumulation of bacoside-A content in cell suspension cultures of B. monnieri.

Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3–4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.

Vanilla (Vanilla planifolia Jacks.) cell suspension cultures: establishment, characterization, and applications

The establishment and characterization of cell suspension cultures are an in vitro culture technique very useful for various plant biotechnological applications (production of secondary metabolites, mass micropropagation, protoplast isolation and fusion, gene transfer and the investigation of cell pathways). The objective of this study was to establish and characterization of cell suspension cultures of V. planifolia by inducing friable calluses. For that, friable calluses were obtained from immature seeds cultivated in MS medium supplemented with 0.45µM thidiazuron (TDZ). The effect of benzyladenine (BA) in different concentrations was evaluated. Cultures were incubated under photoperiod at continuous stirring at 120rpm on an orbital shaker. The optimal condition found for biomass growth in suspension cultures was 0.5g of inoculum density (fresh weight) in MS liquid, supplemented with 8.88µM BA. The growth kinetics of the cell suspension culture revealed a maximum cell growth (exponential growth phase) at day 16 and an 80% cell viability. The establishment and characterization of cell suspension cultures of V. planifolia constitute the bases of future studies and above all a better biotechnological use of this crop.

Production of biomass and useful compounds through elicitation in adventitious root cultures of Fagonia indica

Fagonia indica is an important medicinal plant highly traded for its multitude of potentials against breast cancer. The natural availability of this high-valued herb is not fulfilling the ever-increasing demand. Alternative options should be adopted to balance the supply with demand. Adventitious root culture provides a preferred platform to produce commercially important secondary metabolites. In the present report, adventitious roots (AR) were induced from callus explants in F. indica for the first time. Higher adventitious root induction frequency (88%) and biomass accumulation (6.9 DW g/L) were observed when callus explants were treated with 0.5mg/L Methyl Jasmonate (Me-J) for 2h and subsequently incubated on solid Murashige and Skoog (MS) media containing 1.0mg/L Naphthalene acetic acid (NAA). Variations in growth of roots in suspension cultures were analyzed to estimate the impact of elicitors on adventitious rooting in F. indica. In response to 1.0mg/L NAA, a lag phase of 6days, logarithmic (log) phase of 15days and stationary phase of 6days were observed in the growth curve of AR in suspension culture. Maximum biomass (9.5g DW g/L) was recorded on the 27th day in log phase of culture. Analysis of AR samples derived from different growth stages showed that at 0.5mg/L Me-J, maximum Total Phenolic Content (TPC; 6.0mg GAE/g of DW) and Total Flavonoid Content (TFC; 5.0mg QE/g of DW) were produced, respectively in the lag phase compared to other growth lines. Quantification of vital plant secondary metabolites through High-Performance Liquid Chromatography (HPLC) revealed a higher amount of Gallic acid (148.0±4.8μg/mg of DW) detected in Control (CTR) samples raised in vitro at 1.0mg/L NAA. This was followed by Rutin (122.3± 3.8μg/mg of DW) in LOG phase and Myricetin (32.3±0.7μg/mg of DW) in LAG phase during growth of AR suspension cultures. In response to 0.5mg/L Me-J, more Apigenin (25.3±0.6μg/mg of DW), Caffeic acid (25.3±0.6μg/mg of DW) and Catechin (9.4±0.07μg/mg DW) were accumulated in the LOG phase in comparison to other growth stages. Thus, a feasible protocol was developed for induction and proliferation of adventitious roots with sustainable metabolite profiles through elicitation.

Growth, lipid peroxidation, organic solutes, and anti-oxidative enzyme content in drought-stressed date palm embryogenic callus suspension induced by polyethylene glycol

Polyethylene glycol (PEG) can be used in somatic embryogenesis to enhance embryogenic development and improve the success of in vitro culture but PEG also causes osmotic stress in developing embryos. The effects of PEG on embryo growth and development in date palm cell suspension culture and associated antioxidant enzyme activities were evaluated. Callus maintained on MS basal media was transferred to regeneration liquid media supplemented with increasing levels (0–20%) of polyethylene glycol 6000 (PEG) to induce osmotic stress. The degree of embryogenic callus formation, its fresh weight, and the percentage of normal embryo callus shapes were increased with an increase in the level of PEG up to 10%. Total soluble protein (TSP), proline, glycine betaine (GB), total soluble phenol (TSPh), total sugars (TS), and total soluble organic acids (TOA) also increased whereas superoxide dismutase (SOD) activity decreased in response to PEG supplementation. Raising the PEG level increased malondialdehyde (MDA) concentration up to 10% PEG and thereafter decreased. Glutathione reductase (GR) and catalase (CAT) activities decreased at the highest levels of PEG. The proportion of normal embryo developmental shapes were about 50% compared with 20% abnormal shapes at optimum levels of PEG. Proliferation of somatic embryos was influenced by their developmental shapes. Cv. Samani accumulated more organic solutes compared with cv. Sewi in both control and stress inducing media. In contrast, lipid peroxidation, GR, SOD, and CAT activities were significantly higher in cv. Sewi than in cv. Samani indicating that the cv. Samani had the ability to tolerate a higher level of osmotic stress compared to cv. Sewi due to the enhanced osmotic re-balancing within its tissues.

Effect of gold and silver nanoparticles on the growth of the Arabidopsis thaliana cell suspension culture

The addition of 20-nm gold and silver nanoparticles to a growth medium has a positive effect on the growth of biomass of a cell suspension culture of Arabidopsis thaliana (L.) Heynh. Growing the cells with silver and gold nanoparticles causes different changes in the pH of the medium: the culture medium is made markedly acidic by the addition of silver nanoparticles and alkaline by the addition of gold nanoparticles. As is found by the MTT assay, metallic nanoparticles are responsible for a slight but steady decrease in the specific respiratory activity of cells of the A. thaliana suspension culture. Growth with the nanoparticles is accompanied by an increase in the intracellular free amino acid pool (alanine, γ-aminobutyric acid, and valine), which is characteristic of responses to abiogenic stresses. Furthermore, the addition of the nanoparticles changes the composition of extracellular proteins in the A. thaliana cell culture.

Piriformospora indica enhances the production of pentacyclic triterpenoids in Lantana camara L. suspension cultures

The stimulation of secondary metabolite synthesis by the endophytic fungus, Piriformospora indica, was investigated for the production of triterpenoids—ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA)—in the suspension cultures of Lantana camara. Growth and product formation profiles of the cell suspension cultures of L. camara were established for optimum elicitation strategy. The elicitation potential of P. indica was compared with typical elicitors like methyl jasmonate (MJ), chitin and chitosan. MJ at 62.5 µM and 2.5 % (v/v) filter-sterilized culture filtrate of P. indica enhanced the triterpenoids production to similar level. Filter-sterilized culture filtrate of P. indica at 2.5 % (v/v) increased the concentration of UA by 3.5-fold (3869.3 µg/g DW), OA by 5.6-fold (1425.7 µg/g DW) and BA by 7.8-fold (117.02 µg/g DW) in the cell cultures of L. camara.

BET Protein Bromodomain Antagonist-Based Combinations Exert Synergistic Pre-Clinical Activity Against Ibrutinib-Sensitive or -Resistant Human Mantle Cell Lymphoma (MCL) Cells

Mantle Cell Lymphoma (MCL) exhibits genetic alterations in p53, CDK4, CDKN2A, MYC, BCL2, B cell receptor (BCR) and NFkB signaling, which confer a cell autonomous, pro-growth and pro-survival advantage on MCL cells. Treatment with the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib inhibits BCR and NFκB activity, mediating growth inhibition and apoptosis of MCL cells. Although ibrutinib induces impressive clinical responses in patients with MCL, long-term durable remissions and cure remain elusive. Ibrutinib-resistant MCL cells exhibit mutations in TRAF2/3 and MAP3K14 (NIK), activating the alternative NFκB signaling. Additionally, a cysteine-to-serine C481S mutation in BTK at the binding site of ibrutinib, which confers resistance to ibrutinib, has also been documented in MCL that relapsed while on ibrutinib. These observations suggest that ibrutinib-resistant MCL cells remain dependent for growth and survival on the NFκB-activated gene-expressions. In the ibrutinib-refractory primary MCL cells, mutations in MLL2, CREBBP, PIM1 and ERB4 were also detected. Our findings have shown that the BET (bromodomain and extra-terminal) protein BRD4 antagonists (BA), e.g., JQ1, transcriptionally repress the levels of MCL-relevant oncogenes BCL-2, c-Myc, cyclin D1, CDK6 and PIM1, while inducing the levels of HEXIM1, p21 and p27 in the MCL cells. Consistent with the reported findings that BRD4 is essential for the transcriptional activity of NFkB, JQ1 treatment also attenuated the nuclear RelA and inhibited the expression of several NFkB target genes, including XIAP, IkBα, cFLIP, cIAP2, as well as BTK. Collectively, this resulted in growth arrest and apoptosis of the cultured (MO2058, Mino, Z138 and JeKo-1) and primary MCL (pMCL) cells. We also demonstrate that compared to ibrutinib alone, co-treatment with JQ1 and ibrutinib markedly inhibits pBTK, BTK, pPLCγ2, pAKT, PIM1 and nuclear RelA levels, as well as synergistically induced apoptosis of the cultured and pMCL cells with combination indices (CI) of less than 1.0 by the median dose effect isobologram analysis. Following tail-vein infusion and engraftment of Mino cells in the bone marrow and spleen, co-treatment with JQ1 and ibrutinib, versus each agent alone, significantly improved the survival of NOD/SCID mice. By exposing parental MCL Mino or MO2058 cells to escalating levels of ibrutinib, we isolated Ibrutinib-resistant Mino/IR (>13-fold resistant) and MO2058/IR cells (>8-fold resistant), respectively. Sequencing of the BTK gene did not reveal any mutation in the BTK gene (e.g. C481S) in the ibrutinib-resistant cells. Notably, compared to Mino, Mino/IR cells expressed higher protein levels of BTK, AKT, CDK6, PIM1, XIAP, Bcl-xL and BCL2 levels, while simultaneously displaying lower NOXA, PUMA and p21 levels. Importantly, treatment with JQ1 inhibited the suspension culture growth of Mino/IR cells. JQ1 treatment also induced similar level of apoptosis in Mino/IR, as compared to Mino cells. JQ1-induced apoptosis of Mino/IR cells was associated with a reduction in the nuclear levels of RelA, as well as inhibition of the mRNA levels of BTK, CDK6, and BCL2. In contrast, JQ1 treatment induced the mRNA expression of BCL2L11 and HEXIM1. JQ1 treatment also markedly attenuated the protein levels of BTK, c-Myc, PIM and CDK6 in Mino/IR cells; however, a more modest inhibition of Bcl-xL, BCL2, and XIAP protein levels, and a lack of effect on the protein levels of AKT was observed. Furthermore, pre-treatment of Mino/IR cells with JQ1 enhanced their sensitivity to ibrutinib. Next, we determined that co-treatment with JQ1 and the BCL2 antagonist ABT-199, palbociclib (a CDK4 and CDK6 inhibitor) or AZD1208 (a PIM1 & 2 kinase inhibitor) was synergistically lethal against Mino and Mino/IR cells (CI < 1.0). Co-treatment with JQ1 and ABT199, AZD1208 or palbociclib was also synergistically lethal against pMCL cells, but not against the normal B or hematopoietic cells. These findings demonstrate that treatment with the BA JQ1 sensitizes MCL cells to not only ibrutinib but also to BCL2, PIM1 & 2 and CDK4/6 antagonists. Furthermore, co-treatment with BA and BCL2, PIM1 & 2 or CDK4/6 antagonist are promising combination therapies for testing against ibrutinib-sensitive or ibrutinib-resistant MCL cells. DisclosuresWang:Pharmacyclics, Janssen, Celgene, Oncopep, Kite, Juno: Research Funding; Janssen: Honoraria.

Assessing factors that affect the growth of Corylus avellana cell suspension cultures: a statistical approach

The detection of taxol and related taxanes in Corylus avellana has generated considerable interest, particularly for in vitro cell cultures. Cell suspensions are a sustainable and rational option for obtaining a continuous and reliable source of secondary metabolites in large-scale processes. We therefore focused our study on the main factors that affect the growth of C. avellana cell suspensions as a key approach to improving culture productivity. In this work, calli were successfully induced from C. avellana seeds, leaves, and stems, and the efficiency of different sterilization methods was analyzed. The effects of the basal medium, carbon source, and the type and quantity of plant growth regulators on culture growth were studied. A fractional factorial design allowed us to reduce the number of experiments and analyze all the combinations in one run, thereby reducing time, variability, and costs. Statistical analysis (ANOVA) revealed that 1-naphthaleneacetic acid (NAA) and sucrose are mandatory for the growth of C. avellana cell suspension cultures, with no interactions detected between the parameters analyzed, while growth did not depend on the addition of cytokinins. The secondary metabolism was not inhibited, detecting 1175.45 ng/L of baccatin III and traces of taxol, deacetyltaxol, and cephalomannine. Additionally, prompted by the high growth rate of the C. avellana calli, we assayed a new cold-temperature-based method to maintain a stock of calli using half-strength Murashige and Skoog solid medium, concluding that up to 5 mo at 4°C is optimal to ensure white friable calli upon regrowth at 25°C.

Effect of methyl jasmonate on growth characteristics and accumulation of phenolic compounds in suspension culture of tartary buckwheat

The effect of methyl jasmonate (MeJA) was studied at concentrations of 0.01, 0.1, 1, and 10 μM on the following physiological and biochemical characteristics of the suspension culture of tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.): growth, morphogenesis, the content of phenolic compounds (PC), and antioxidant activity (AOA). Qualitative and quantitative HPLC analysis of PC revealed that the dark-grown suspension culture of tartary buckwheat synthesizes rutin, quercitrin, epicatechin, ferulic and p-coumaric acids, with rutin being the most abundant component. It was shown that MeJA had no inhibitory effect on growth of suspension culture and stimulated the formation of PC, especially in media supplemented with 1 and 10 μM MeJA. At increasing concentrations of MeJA, the accumulation of PC was accompanied by the decline in AOA, which was probably due to the development of oxidative stress in the suspension culture. MeJA at a concentration of 10 μM stimulated slightly the growth of biomass; however, after the passage in the medium with 10 μM MeJA, the suspension culture almost lost its ability to form embryoids on the hormone-free medium. Cultivation of cells in the medium supplemented with 10 μM MeJA not only elevated the total content of PC but also modified their qualitative composition and the content of individual PC. The epicatechin content was higher than in the control culture throughout the whole passage; the increase in the content of p-coumaric acid and rutin was noted between the 4th and 8th days, and quercitrin content increased on the 8–14th day. Synthesis of these compounds was activated not by the end of the passage, as it occurred in the control culture, but at earlier stages of culture growth. The intracellular level of ferulic acid increased sharply on the 4th day of cultivation, while control cultures contained only trace quantities of this substance. It was found that MeJA activated biosynthesis of individual PC to different extents: the accumulation of epicatechin, p-coumaric and ferulic acids was more significant than that of rutin and quercitrin. In addition to the peaks of identified PC, MeJA stimulated the appearance of new compounds that were absent or weakly evident in the control culture.

Mechanistic Role of HEXIM1 Induction in BRD4-Antagonist Mediated Growth Inhibition, Differentiation and in Vivo Lethal Activity Against Human AML Blast Progenitor Cells

The BET (bromodomain and extra terminal) protein family, including BRD4 are chromatin reader proteins that bind to acetylated lysines on histone proteins and recruit transcriptional regulatory complexes to gene promoters, thereby coupling histone acetylation to RNA polymerase II (RNAP2)-mediated transcript elongation. Preferential binding of BRD4 to clustered enhancers or ‘super enhancers’ enables BRD4, through the mediator complex, to regulate the transcription of specific oncogenes, including c-MYC, BCL2 and CDK4/6 in AML blast progenitor cells (BPCs). Through its C-terminal domain (CTD), BRD4 recruits pTEFb (positive transcription elongation factor b), the heterodimer composed of cyclin T and CDK9, which phosphorylates the heptad repeats on the CTD of RNAP2 at serine 2. The kinase activity of CDK9 in pTEFb is also required for the phosphorylation of DSIF and NELF, which abrogates their inhibitory hold on the pause-release of RNAP2 and productive transcript elongation of specific oncogenes and their target genes. pTEFb exists in two forms, the catalytically active form, which is associated with BRD4 and RNAP2, and a catalytically inactive form associated with 7SK snRNP (small nuclear ribonucleoprotein) and the inhibitory protein HEXIM1 (hexamethylene bis-acetamide or HMBA-inducible protein 1). The transition of pTEFb between the inactive and active complex is dynamic and regulated by cell growth and stress signaling. HEXIM1 binds to and sequesters pTEFb, thereby inhibiting its kinase activity for RNAP II. We recently reported (Mol Cancer Ther, 2014) that the BRD4 antagonist JQ1 and I-BET151 alone, and synergistically with the pan-histone deacetylase inhibitor panobinostat, inhibits the growth and induces apoptosis in cultured (OCI-AML3 and MOLM13) and patient-derived primary AML (pAML) BPCs. JQ1 treatment also improved the survival of the immune-depleted mice engrafted with MOLM13 or pAML BPCs. Notably, the lethal activity of JQ1 was associated with marked induction of HEXIM1. Therefore, we hypothesized that HEXIM1 induction, by sequestering pTEFb and inhibiting RNAP2, contributes to the growth inhibitory and lethal effects of JQ1. We additionally hypothesized that co-treatment with those agents that further augment HEXIM1 induction would synergize with JQ1 against AML BPCs. To validate these hypotheses, we achieved a stable lentivirus mediated knockdown (KD) of the mRNA and protein expressions of HEXIM1 by approximately 90% in OCI-AML3 and MOLM13 cells, as well as transient KD in pAML BPCs. Compared to the parental control cells, OCI-AML3/HKD and MOLM13/HKD cells showing HEXIM1 knockdown (HKD) exhibited reduced binding of HEXIM1 with cyclin T (as revealed by cyclin T pull-down and immunofluorescence microscopy), but showed augmented suspension culture growth and increased protein levels (by Western blot) of cyclin T, pRNAP2 and cMYC levels. However, in contrast to the parental control cells, JQ1 (1000 nM) treatment-mediated HEXIM1 induction and binding to cyclin T was markedly inhibited in the HKD AML BPCs, which was associated with significant inhibition of JQ1-induced pRNAP2, as well as differentiation and apoptosis of HKD BPCs (p < 0.01). Notably, whereas treatment with JQ1 alone improved the median survival of NOD/SCID mice engrafted with MOLM13 BPCs, this survival improvement was significantly impaired in the mice engrafted with MOLM13/HKD AML BPCs (p < 0.02). This indicated that HEXIM1-induction mechanistically contributes to JQ1-induced growth inhibition, differentiation and apoptosis of AML BPCs. Further, the synergistic apoptosis observed in MOLM13, OCI-AML3 and primary AML cells induced by co-treatment with JQ1 (250 to 1000 nM) and PS (5 to 10 nM) or HMBA (1 to 2 mM) was also associated with greater induction of HEXIM1. However, this synergy was impaired in the cultured and pAML BPCs with HEXIM1 knockdown. These findings confirm that HEXIM1 induction is a biomarker and plays a mechanistic role in mediating growth inhibition, differentiation and apoptosis induced by BRD4 antagonist in human AML BPCs. These findings support, and we have embarked upon, studies involving future pre-clinical development of those agents that in combination significantly augment BRD4 antagonist-induced HEXIM1 in human AML BPCs. DisclosuresNo relevant conflicts of interest to declare.

5‐Aminolevulinic acid promotes callus growth and paclitaxel production in light‐grown Taxus cuspidata suspension cultures

Cultured plant cells generally produce low levels of secondary metabolites, and elicitors of secondary metabolites usually inhibit callus growth. The aim of this study was to determine the effect of 5‐aminolevulinic acid (ALA), a chlorophyll precursor that promotes plant growth, on callus induction from leaves of Taxus cuspidata, and on callus growth on solid medium. ALA at 0.76, 7.6, and 76 μM had similar effects on callus induction and growth, while ALA at 760 μM had negative effects. Next, the effects of ALA concentrations on callus growth and paclitaxel production in suspension cultures in the dark were evaluated. The results showed that 0.76 and 7.6 μM ALA stimulated growth and paclitaxel production, while 76 μM ALA had negative effects. ALA is thought to promote cellular activity under light conditions. Therefore, the effects of light intensity on callus growth and paclitaxel production in the presence of ALA were evaluated. Our results showed that the best conditions for callus growth and paclitaxel production were 7.6 μM ALA under photosynthetically active radiation of 12 μmol photons m−2 s−1. Callus growth and paclitaxel production were inhibited under stronger light (24 μmol photons m−2 s−1). Together, these results show that ALA promoted callus growth and the production of paclitaxel by light‐grown cultured T. cuspidata cells.

Abstract 4435: Deubiquitinases Usp9x and Usp5 control tumorigenicity and apoptotic responsiveness in malignant melanoma

Abstract Melanoma is an aggressive skin cancer characterized by gene mutations, high metastatic potential and few clinically effective systemic therapies. BRAF, MEK1 kinase inhibitors and CTLA4 antagonist have improved patient survival, but only a subset of patients respond or derived long-lasting clinical benefit, illustrating the need for discovery of additional targets and pathways that may be inhibited with small molecules. Given the clinical impact of inhibitors of the ubiquitin-proteasome system in other cancers, we explored the potential for aberrant deubiquitinase (DUB) activity to play a role in the tumorigenic properties of melanoma. We also assessed whether specific DUBs play a role in apoptotic responsiveness and resistance to kinase inhibitors. Initially, we noted that the activity of several DUBs, including Usp9x and Usp5, was elevated in melanoma compared to normal melanocytes. Further, Usp9x and Usp5 expression and activity were up-regulated by expression of BRAFV600E in 293T cells and kinase inhibition partially suppressed both Usp9x and Usp5 activity in vemurafenib sensitive, but not resistant cells. Usp9x knockdown (KD) in BRAF mutant or wild-type melanoma did not affect 2-dimensional cell growth or survival but significantly amplified the apoptotic activity of BRAF or MEK1 inhibitors, respectively. Usp5 KD in melanoma suppressed cell growth by reinforced the S/G2-M checkpoint, enhanced extrinsic caspase activation through modulation of p53 and FAS levels and amplified the apoptotic activity of kinase inhibitors (and other chemotherapeutic agents) and overcame vemurafenib resistance in A375R cells. To further assess the clinical relevance of activated DUBs in melanoma, primary human melanoma explants characterized for their metastatic efficiency in patients and NSG mice demonstrated that Usp9x and Usp5 expression and activity were elevated in efficient metastasizers, but only a subset of inefficient metastasizers, suggesting a potential role for DUBs in the regulation of metastatic potential. To investigate that possibly, control and Usp9x KD cells were grown in Matrigel or suspension culture and colony growth and survival, as well as Usp9x activity were examined. Usp9x KD significantly suppressed melanoma colony growth and fully reduced survival of highly metastatic SK-Mel147 cells in 3D culture. Growth in 3D conditions resulted in activation of Usp9x, but also increased their sensitivity (IC50 &amp;lt;400 nM) to our partially selective Usp9x inhibitor, EOAI3402143 (G9). G9 inhibits both Usp9x and Usp5 activity and phenocopied the activities of both Usp9x and Usp5 KD cells. G9 was safely administered to NSG mice, with complete block of melanoma growth. We conclude that targeted inhibition of specific DUBs will suppress metastatic melanoma and amplify existing melanoma therapies. Citation Format: Harish Potu, Anupama Pal, Hanshi Sun, Luke Peterson, Moshe Talpaz, Monique Verhaegen, Juxiang Cao, Ugur Eskiocak, Sean Morrison, Nicholas J. Donato. Deubiquitinases Usp9x and Usp5 control tumorigenicity and apoptotic responsiveness in malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2014-4435

Repression ofOsEXPA3Expression Leads to Root System Growth Suppression in Rice

ABSTRACTExpansins are cell‐wall–loosening proteins known to be involved in a wide range of physical processes during plant development.OsEXPA3, a rice α‐expansin gene, was found to be preferentially expressed in the root and highly induced by NaCl, while suppressed by nitrate (N) and phosphate (Pi) starvation. Repression ofOsEXPA3expression by RNA interference (RNAi) in rice (Oryza sativaL.) severely impaired the growth of the root system. A significant reduction in the primary root length and lateral root density was observed in 1‐wk‐oldOsEXPA3‐RNAilines at seedling stage, and the total size and density of the root system was clearly decreased in 1‐mo‐old plants. Besides, the root hair of RNAi seedlings was clearly shorter than that of wild‐type plants. Moreover, RNAi plants exhibited clearly inhibited growth, which is mostly attributed to the poorer root system architecture. Further study indicated that the cell length of root vascular bundles and cell growth in suspension cultures inOsEXPA3‐RNAilines decreased significantly, which revealed the cellular basis forOsEXPA3‐mediated rice root growth. Further, the cell‐wall mechanical properties of suspension cells probed by atomic force microscopy (AFM) showed that repression ofOsEXPA3led to a significant increase in cell‐wall stiffness, and cell‐wall polymer compositions revealed by Fourier transform infrared (FTIR) spectra were also strongly changed. Taken together, the results suggest thatOsEXPA3is required for root system development in rice and involved in mediating root growth by mediating cell‐wall loosening.

Changes in auxin level in the course of growth of a sunflower crown-gall suspension culture

The auxin level in the cell mass and culture medium was determined by means of the &lt;em&gt;Avena&lt;/em&gt; straight caleoptile test in various periods of the suspension culture cycle of the sunflower crown-gall tumour. The investigations were performed in the course of the zero passage (PO) and first one (Pl), differing in their time of duration of maximum growth and its intensity. In both passages the intra- and extra-cellular auxin levels reach values of the same order. At the beginning of the maximal growth phase the activity corresponding to IAA in the cells prevails over that of the other auxin-like compounds. This disproportion diminishes with further development of the culture, and with the beginning of the stationary phase the cellular IAA level is lower than that of the remaining auxin-like compounds. The short phase of maximal growth (PO) occurs with an auxin level decreasing in the cell mass and increasing in the medium, and towards the end of the cycle these levels become equal. During the long phase of maximal growth (Pl) the total amount of auxins in the cells increases and is 2-3 times higher than in the medium, whereas IAA in the cells remains at a constant level. These results suggest that the participation of IAA in the intracellular pool of auxin-like substances is decisive for the mitotic activity of the cells and maintenance of growth in the culture.

Effects of several factors on cell growth and ginsenoside accumulation of Panax ginseng suspension culture

To improve cell suspension culture system of Panax ginseng, the dynamic of cell growth and medium consumption were studied, and the effects of filter on the culture vessel, revolution number, and inoculation density on cell growth and ginsenoside accumulation were also investigated. The maximum cell growth and ginsenoside accumulation was found on the 20th days of suspension culture, therefore, 20 days were confirmed as a suitable culture period for mass production of ginsenoside. Cell growth and ginsenoside content were promoted when the culture vessel had a ventilated filter. Revolution speed during suspension culture affected cell growth, but not ginsenoside content, a peak of ginsenoside productivity was found in the treatment of 120 r x min(-1). Inoculation density also influenced cell growth and ginsenoside accumulation, inoculation density of 6 g was better than other inoculation densities, the ginsenoside content and productivity were up to 12.8 mg x g(-1) DW and 146.6 mg x L(-1), respectively.

Overexpression of OsEXPA8, a Root-Specific Gene, Improves Rice Growth and Root System Architecture by Facilitating Cell Extension

Expansins are unique plant cell wall proteins that are involved in cell wall modifications underlying many plant developmental processes. In this work, we investigated the possible biological role of the root-specific α-expansin gene OsEXPA8 in rice growth and development by generating transgenic plants. Overexpression of OsEXPA8 in rice plants yielded pleiotropic phenotypes of improved root system architecture (longer primary roots, more lateral roots and root hairs), increased plant height, enhanced leaf number and enlarged leaf size. Further study indicated that the average cell length in both leaf and root vascular bundles was enhanced, and the cell growth in suspension cultures was increased, which revealed the cellular basis for OsEXPA8-mediated rice plant growth acceleration. Expansins are thought to be a key factor required for cell enlargement and wall loosening. Atomic force microscopy (AFM) technology revealed that average wall stiffness values for 35S::OsEXPA8 transgenic suspension-cultured cells decreased over six-fold compared to wild-type counterparts during different growth phases. Moreover, a prominent change in the wall polymer composition of suspension cells was observed, and Fourier-transform infrared (FTIR) spectra revealed a relative increase in the ratios of the polysaccharide/lignin content in cell wall compositions of OsEXPA8 overexpressors. These results support a role for expansins in cell expansion and plant growth.

Availability of public goods shapes the evolution of competing metabolic strategies

Tradeoffs provide a rationale for the outcome of natural selection. A prominent example is the negative correlation between the growth rate and the biomass yield in unicellular organisms. This tradeoff leads to a dilemma, where the optimization of growth rate is advantageous for an individual, whereas the optimization of the biomass yield would be advantageous for a population. High-rate strategies are observed in a broad variety of organisms such as Escherichia coli, yeast, and cancer cells. Growth in suspension cultures favors fast-growing organisms, whereas spatial structure is of importance for the evolution of high-yield strategies. Despite this realization, experimental methods to directly select for increased yield are lacking. We here show that the serial propagation of a microbial population in a water-in-oil emulsion allows selection of strains with increased biomass yield. The propagation in emulsion creates a spatially structured environment where the growth-limiting substrate is privatized for populations founded by individual cells. Experimental evolution of several isogenic Lactococcus lactis strains demonstrated the existence of a tradeoff between growth rate and biomass yield as an apparent Pareto front. The underlying mutations altered glucose transport and led to major shifts between homofermentative and heterofermentative metabolism, accounting for the changes in metabolic efficiency. The results demonstrated the impact of privatizing a public good on the evolutionary outcome between competing metabolic strategies. The presented approach allows the investigation of fundamental questions in biology such as the evolution of cooperation, cell-cell interactions, and the relationships between environmental and metabolic constraints.

Mda-7/IL-24 Expression Inhibits Breast Cancer through Upregulation of Growth Arrest-Specific Gene 3 (gas3) and Disruption of β1 Integrin Function

Melanoma differentiation-associated gene (MDA)-7)/interleukin (IL)-24, a member of the IL-10 family of cytokines, inhibits growth of various human cancer cells, yet the underlying mechanism is largely unknown. Here, we report that mda-7/IL-24 efficiently suppresses the development of rat mammary tumors in vivo. Microarray analysis for genes differentially expressed in rat mammary tumor cells overexpressing MDA-7/IL-24 compared with those that do not express this cytokine identified growth arrest-specific gene-3 (gas3) as a target for mda-7/IL-24. Upregulation of gas3 by mda-7/IL-24 was STAT3 dependent. Induction of gas3 inhibited attachment and proliferation of tumor cells in vitro and in vivo by inhibiting the interaction of β1 integrin with fibronectin. A mutated GAS3, which is unable to bind β1 integrin, was also unable to inhibit fibronectin-mediated attachment and cell growth both in adherent and suspension cultures, suggesting that GAS3 exerts its effects through interaction with and regulation of β1 integrin. Thus, mda-7/IL-24 inhibits breast cancer growth, at least in part, through upregulation of GAS3 and disruption of β1 integrin function. Importantly, the expression of the mda-7/IL-24 receptor, IL-20R1, is highly correlated with GAS3 expression in human breast cancer (P = 1.02 × 10(-9)), and the incidence of metastases is significantly reduced in patients with HER2(+) breast cancer expressing high-levels of IL-20R1. Together, our results identify a novel MDA-7/IL-24-GAS3-β1integrin-fibronectin signaling pathway that suppresses breast cancer growth and can be targeted for therapy.