Tissue Culture Lines Research Articles

Early nodulin 93 protein gene: essential for induction of somatic embryogenesis in oil palm.

Transcript profiling during the early induction phase of oil palm tissue culture and RNAi studies in a model somatic embryogenesis system showed that EgENOD93 expression is essential for somatic embryogenesis. Micropropagation of oil palm through tissue culture is vital for the generation of superior and uniform elite planting materials. Studies were carried out to identify genes to distinguish between leaf explants with the potential to develop into embryogenic or non-embryogenic callus. Oil palm cDNA microarrays were co-hybridized with cDNA probes of reference tissue, separately with embryo forming (media T527) and non-embryo (media T694) forming leaf explants sampled at Day 7, Day 14 and Day 21. Analysis of the normalized datasets has identified 77, 115 and 127 significantly differentially expressed genes at Day 7, Day 14, and Day 21, respectively. An early nodulin 93 protein gene (ENOD93), was highly expressed at Day 7, Day 14, and Day 21 and in callus (media T527), as assessed by RT-qPCR. Validation of EgENOD93 across tissue culture lines of different genetic background and media composition showed the potential of this gene as an embryogenic marker. In situ RNA hybridization and functional characterization in Medicago truncatula provided additional evidence that ENOD93 is essential for somatic embryogenesis. This study supports the suitability of EgENOD93 as a marker to predict the potential of leaf explants to produce embryogenic callus. Crosstalk among stresses, auxin, and Nod-factor like signalling molecules likely induces the expression of EgENOD93 for embryogenic callus formation.

Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.)

BackgroundSwitchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration.ResultsRegenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety ‘Performer’ tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines.ConclusionThe new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6–7 months from culture establishment.

Optimization of DamID for use in primary cultures of mouse hepatocytes.

DamID, a method to identify DNA associating with a particular protein, was originally developed for use in immortalized tissue culture lines. The power of this technique has led to its adaptation for a number of additional systems. Here we report adaptations for its use in primary cells isolated from rodents with emphasis on the challenges this presents. Specifically, we present several modifications that allow the method to be performed in mouse acutely isolated primary hepatocytes while seemingly maintaining tissue genome architecture. We also describe the downstream bioinformatic analysis necessary to identify LADs and discuss some of the parameters and their effects with regards to the sensitivity of the method.

Heritable Epigenomic Changes to the Maize Methylome Resulting from Tissue Culture.

DNA methylation can contribute to the maintenance of genome integrity and regulation of gene expression. In most situations, DNA methylation patterns are inherited quite stably. However, changes in DNA methylation can occur at some loci as a result of tissue culture resulting in somaclonal variation. To investigate heritable epigenetic changes as a consequence of tissue culture, a sequence-capture bisulfite sequencing approach was implemented to monitor context-specific DNA methylation patterns in ∼15Mb of the maize genome for a population of plants that had been regenerated from tissue culture. Plants that have been regenerated from tissue culture exhibit gains and losses of DNA methylation at a subset of genomic regions. There was evidence for a high rate of homozygous changes to DNA methylation levels that occur consistently in multiple independent tissue culture lines, suggesting that some loci are either targeted or hotspots for epigenetic variation. The consistent changes inherited following tissue culture include both gains and losses of DNA methylation and can affect CG, CHG, or both contexts within a region. Only a subset of the tissue culture changes observed in callus plants are observed in the primary regenerants, but the majority of DNA methylation changes present in primary regenerants are passed onto offspring. This study provides insights into the susceptibility of some loci and potential mechanisms that could contribute to altered DNA methylation and epigenetic state that occur during tissue culture in plant species.

Cryopreservation of the Norway spruce tissue culture line able to produce extracellular lignin

A cryopreservation method was developed for a Norway spruce (Picea abies L. Karst.) cell line characterised by highly vacuolated cells and ability to produce natural-like extracellular lignin in a cell suspension culture. Spruce callus cultured in a photoperiod of 16 h light, 8 h dark contained two types of callus morphologies. Soft callus was composed of loosely bound cells that dispersed into single cells and small cell aggregates when transferred into liquid medium. The callus with hard morphology had also cells that were more tightly attached to each other; this callus formed bigger cell aggregates in liquid medium in addition to single cells and small cell aggregates. The hard callus contained higher concentration of intracellular phenolic compounds as compared to soft callus. For cryopreservation, a vitrification method with plant vitrification solution 2 (PVS2) was used. To reduce cellular water content, spruce calli were pre-cultured on a culture medium with increasing sucrose concentration (0.2 and 0.4 M; one day on each). The cryopreservation survival rate of callus with hard morphology was significantly higher than that with soft morphology (45 ± 8% and 5 ± 5%, respectively). Pre-culturing in continuous light for several weeks led exclusively to formation of a hard-type callus, which had a survival rate of 48 ± 16% in cryopreservation. Expression of candidate genes of the monolignol biosynthesis pathway, Fourier transform infrared spectra and pyrolysis breakdown products of extracellular lignin were similar in control cultures and those originating from cryopreserved cells suggesting that cryopreservation is a feasible method for long-term storage of the lignin-forming cell line.

Abstract 4034: Immunotherapy generates selective pressure to create an escape tumor with increased susceptibility to treatment with T cell based therapies

Abstract Host immune surveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. Less is known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application of a T cell-dependent selection pressure in the form of a whole tumor vaccine would alter the immunogenic architecture in our transplantable murine melanoma model SB-3123. We generated an escape variant to test this hypothesis. We performed whole-exome and RNA-sequencing of the tumor line SB-3123 and two fresh tumors generated by subcutaneous implantation of SB-3123 to identify mutations and to assess levels of expression. There were 349 mutations shared between the tumor line and fresh tumors and less than 5 mutations were unique to each sample. We then investigated whether tumors that had undergone an additional round of host immunoediting would acquire different mutations. To test this, we generated a re-derived tumor cell line from a fresh SB-3123 tumor. Remarkably, the re-derived tissue culture line retained 333 mutations in common with the prior samples and produced only 2 unique mutations. To test the immunogenicity of SB-3123, we vaccinated mice with irradiated SB-3123 and administered a live tumor challenge two weeks after vaccination. This resulted in a profound vaccination response with mice being completely protected from tumor for over 200 days. However, one vaccinated mouse developed a recrudesced tumor at the site of implantation 40 days after challenge and a tumor line was created (SB-3123-esc). We asked whether SB-3123-esc represented an escape variant by immunizing mice with SB-3123 and then challenging with either SB-3123 or SB-3123-esc. Vaccination with SB-3123 did not protect against SB-3123-esc. We also performed sequencing on SB-3123-esc and discovered that 441 nonsynonymous mutations were common to both tumor lines while 80 were unique to SB-3123 and 40 to SB-3123-esc. Furthermore, we were able to determine that SB-3123-esc lost 2 immunogenic neoantigens but had acquired 5. In addition, we identified a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. Our results demonstrate that the expressed mutations of SB-3123 tumor did not change, regardless of whether we sequenced the tumor line, fresh tumors, or a re-derived tumor cell line. However, the escape tumor had new unique mutations and at least one of these was demonstrably immunogenic; we were able to develop a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. In conclusion, we posit that these observations offer a proof of concept that the process of cancer immune evasion can create new opportunities to treat escape tumors with T cell based therapies. Citation Format: Jenny H. Pan, Suman Vodnala, Robert L. Eil, Zhiya Yu, Jared Gartner, David Clever, Rahul Roychoudhuri, Shashank J. Patel, Christopher A. Klebanoff, Madhu Sukumar, Tori Yamamoto, Nicholas P. Restifo. Immunotherapy generates selective pressure to create an escape tumor with increased susceptibility to treatment with T cell based therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4034.

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

BackgroundThe somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels.ResultsIn this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization.ConclusionsSystematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

The use of a tissue culture line to determine the estrogen-active substances in municipal water of Potchefstroom in the North West Province

The use of a tissue culture line to determine the estrogen-active substances in municipal water of Potchefstroom in the North West Province. Substances called hormone disruptors are released in fresh water systems by industrial and agricultural run-off as well as unprocessed sewage. These substances influence the reproductive, immune and nervous systems of humans, as well as the thyroid function. The present study focuses on the incidence of estrogen-active substances in the aquatic environment and the ability of sewage works to purify water from these substances.

Mechano-growth factor peptide, the COOH terminus of unprocessed insulin-like growth factor 1, has no apparent effect on myoblasts or primary muscle stem cells

A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed "mechano-growth factor" (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.

Abstract 2769: DSRCT: Establishing xenograft and tissue culture cell lines and assessing chemosensitivity.

Abstract Desmoplastic small round cell tumor (DSRCT) is an extremely rare and aggressive neoplasm which mainly affects young males and generally presents as a widely disseminated tumor within the peritoneal cavity. Due to the rarity of the tumor, a younger and overall healthier patient population, and because it lacks definitive histologic and immunohistologic features, it is possible that DSRCT is frequently delayed in diagnosis or is entirely misdiagnosed as a different kind of abdominal sarcoma. This study seeks to rectify the dearth of models that exist for this rare neoplasm through the development of several DSRCT tissue culture and xenograft lines. Samples were received from surgeries and biopsies from patients around the world and were immediately processed for xenograft development in nude mice. Tumor tissues were minced and fragments were injected onto the dorsal flank of nude mice. Of the 15 samples received, 9 were established into xenograft lines and 6 of those into tissue culture lines. Xenografts displayed the microscopic histology of their parent tumors and two different growth rates among established xenograft lines. Chemosensitivity of tumors was assessed against a variety of chemotherapeutic agents and IC50 values established for each drug and cell line. Interestingly, the cell lines were split into two distinct groups in terms of morphology, growth rate and chemosensitivity. Overall, the establishment of these xenograft and tissue culture lines gives researchers tools to evaluate DSRCT response to chemotherapy and investigate DSRCT-specific signaling pathways or mechanisms. Citation Format: Constantine SA Markides, Dana M. Vardeman, Linh Luong, Douglas R. Coil, Anthony Kozielski, John Mendoza, Beppino C. Giovanella. DSRCT: Establishing xenograft and tissue culture cell lines and assessing chemosensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2769. doi:10.1158/1538-7445.AM2013-2769

The role of the transcription factor - Prospero homeobox 1 (Prox1) in the biology of differentiated thyroid cancer - the effect of the expression of Prox1 on migration and invasiveness of thyroid cancer cells

The feature of malignancy is its ability to infiltrate and metastasis. The differentiated thyroid carcinoma (DTC), which constitutes of more than 80% of all thyroid cancers is spread by two major paths: papillary carcinoma (PTC) usually by the lymphatic vessels, and follicular cancer (FTC) mainly through the circulatory system. It was proposed that the cancers microenvironment induced by lymphangiogenesis could be an important factor affecting the expansion of primary tumour cells to lymph nodes. The aim of our work was to investigate signaling pathways associated with migration and invasiveness of thyroid cancer cells. We were especially interested in the role of Prospero homeobox 1 (Prox1) protein, an important element of the lymphangiogenesis. The studies were performed in a series of thyroid cancer FTC and PTC cell lines. The protein expression level and intracellular localization of the Prox1 gene was determined using molecular biology methods including: Q-RT-PCR, Western Blot and immunocytochemistry. The tumour cells' dynamics and invasiveness were monitored by microscopic observation assay and using invasion test, respectively. Additionally, the cells phenotype under Prox1 gene overproduction and silencing conditions was investigated. We observed significant differences in the level of expression Prox1 between tested thyroid cancer cell lines. Additionally, protein subcellular localization was also variable and correlated with the tumour type. The protein concentration was not directly combined with mRNA level and even under small mRNA concentration in some tissue culture lines, the Prox1 protein was cumulated in the cells over the time. We noticed that the increased expression level of Prox1 gene resulted in a lower rate of cell migration. Interestingly, a similar effect was demonstrated under overexpression conditions of the Prox1 gene, but uniquely, the lower dynamic rate of cells was additionally accompanied by a significantly higher invasive potential of the tested cells. The differences in the expression level and localization of Prox1 protein in various DTC lines may suggest an important role of it in the spreading of follicular and papillary carcinomas. We showed that Prox1 is a factor that has a significant effect on the migration and invasiveness of thyroid cancer cells, and thus affects the spread of the tumour. We propose that Prox1 may lead to changes in the signaling pathway controlling cytoskeleton dynamics in the tumour cells and the turnover of invasiveness. Further on-going studies will precisely determine the role of Prox1 lymphangiogenesis factor in DTC propagation.

Desmoplastic small round cell tumor (DSRCT) xenografts and tissue culture lines: Establishment and initial characterization

Desmoplastic small round cell tumor (DSRCT) is an extremely rare and aggressive neoplasm, which mainly affects young males and generally presents as a widely disseminated tumor within the peritoneal cavity. Due to the rarity of the tumor, its younger and overall healthier patient population (compared with other tumor types) and the fact that it lacks definitive histological and immunohistological features, the diagnosis of DSRCT may be frequently delayed or the tumor may be entirely misdiagnosed as a different type of abdominal sarcoma. The present study aimed to rectify the lack of models that exist for this rare neoplasm, through the development of several DSRCT tissue cultures and xenograft lines. Samples were received from surgeries and biopsies from patients worldwide and were immediately processed for xenograft development in nude mice. Tumor tissues were minced and fragments were injected into the dorsal flanks of nude mice. Of the 14 samples received, nine were established into xenograft lines and five into tissue culture lines. Xenografts displayed the microscopic histology of their parent tumors and demonstrated two different growth rates among the established xenograft lines. Overall, the establishment of these xenograft and tissue culture lines provides researchers with tools to evaluate DSRCT responses to chemotherapy and to investigate DSRCT-specific signaling pathways or mechanisms.

Genome Sequence of Mycoplasma hyorhinis Strain GDL-1

Mycoplasma hyorhinis impacts swine health and production in many countries, either as a primary pathogen or as a component of a polymicrobial infection. Isolates of this species are also common contaminants of tissue culture lines. The genome sequence of the cell culture isolate M. hyorhinis GDL-1 is presented herein.

A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense.

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

TCUP: A Novel hAT Transposon Active in Maize Tissue Culture

Transposable elements (TEs) are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced TEs that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP), which is transcribed at high levels in long-term maize black Mexican sweet (BMS) tissue culture and is transcribed in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected novel TCUP insertions in two long-term tissue-cultured cell lines of the genotype Hi-II A × B and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

Abstract 3818: CTC profiling: Integration of biomarker analysis with current Veridex CellSearch technology

Abstract The presence and number of circulating tumor cells (CTCs) in the blood of cancer patients is predictive of clinical outcome. To date only the CellSearch system by Veridex has been FDA approved for use in breast, prostate and colon cancer (cc). However, a limited number of biomarkers are examined in the CellSearch system. In the case of colorectal cancer, cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) expression is used to identify cells as CTCs. Additional analysis of other biomarkers by Cheshire technology is provided as fee for service and only uses the current fluorescent channels utilized by the CellSearch Analyzer. Our aim is to greatly enhance the number of biomarkers that can be tested in an economic and efficient manner and integrating the analysis into the existing CellSearch isolation and analytic platform. We combined Nuance multispectral imaging with quantum dot technology to expand the biomarkers analysis. Quantum dots (QD) have the advantage that they can be excited in the UV wavelengths while emitting across the visible spectrum. Only DAPI in the original CellSearch panel of reagents is excited in this range and its emission does not overlap with the QDs. This allows us to visualize additional biomarkers without interfering with CellSearch CTC analysis. Antibodies against a panel of known biomarkers were labeled with quantum dots. Each antibody was verified by standard immunofluorescence in appropriate tissue culture lines before quantum dot labeling. Antibody labeling and titration was confirmed on tissue culture cell lines before adding it to the panel of CTC staining reagents. To model multiplexing assays, blood is spiked with a known number of cells from appropriate cancer cell lines. Following standard CTC isolation and enumeration, cells are retained in the MagNest and allowed to dry. Cells are then stained with our biomarker panel. Currently 8 quantum dots are commercially available for simultaneous analysis. Our panel of biomarkers is under revision, but includes phospho-AKT, Ki-67, CD26, CD133 and EGFR. We are expanding the panel to include markers for death pathway signaling including Mcl-1, Bcl-XL, Flip, TRAIL, and death receptors. Separate panels are also being developed for breast cancer. Additional panels can include markers for sensitivity to chemotherapeutic reagents (TS, DPD, PTHFR, GSTP1, ERCC1, ERCC2, UGT1A1, BRAF), cell proliferation, known tumor susceptibility genes or tumor markers (APC, p53, HER2, PSA, IGF1-R) or probes for specific signal transduction pathways (NFκB, Wnt). Analysis of samples from patients undergoing chemotherapy -/+ targeted therapy is underway to evaluate the status of the stem cell, prognostic markers and cell death determinants in individual CTCs as a function of time. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3818. doi:10.1158/1538-7445.AM2011-3818

STAMP alters the growth of transformed and ovarian cancer cells

BackgroundSteroid receptors play major roles in the development, differentiation, and homeostasis of normal and malignant tissue. STAMP is a novel coregulator that not only enhances the ability of p160 coactivator family members TIF2 and SRC-1 to increase gene induction by many of the classical steroid receptors but also modulates the potency (or EC50) of agonists and the partial agonist activity of antisteroids. These modulatory activities of STAMP are not limited to gene induction but are also observed for receptor-mediated gene repression. However, a physiological role for STAMP remains unclear.MethodsThe growth rate of HEK293 cells stably transfected with STAMP plasmid and overexpressing STAMP protein is found to be decreased. We therefore asked whether different STAMP levels might also contribute to the abnormal growth rates of cancer cells. Panels of different stage human cancers were screened for altered levels of STAMP mRNA. Those cancers with the greatest apparent changes in STAMP mRNA were pursued in cultured cancer cell lines.ResultsHigher levels of STAMP are shown to have the physiologically relevant function of reducing the growth of HEK293 cells but, unexpectedly, in a steroid-independent manner. STAMP expression was examined in eight human cancer panels. More extensive studies of ovarian cancers suggested the presence of higher levels of STAMP mRNA. Lowering STAMP mRNA levels with siRNAs alters the proliferation of several ovarian cancer tissue culture lines in a cell line-specific manner. This cell line-specific effect of STAMP is not unique and is also seen for the conventional effects of STAMP on glucocorticoid receptor-regulated gene transactivation.ConclusionsThis study indicates that a physiological function of STAMP in several settings is to modify cell growth rates in a manner that can be independent of steroid hormones. Studies with eleven tissue culture cell lines of ovarian cancer revealed a cell line-dependent effect of reduced STAMP mRNA on cell growth rates. This cell-line dependency is also seen for STAMP effects on glucocorticoid receptor-mediated transactivation. These preliminary findings suggest that further studies of STAMP in ovarian cancer may yield insight into ovarian cancer proliferation and may be useful in the development of biomarker panels.

Cell Wall Lignin is Polymerised by Class III Secretable Plant Peroxidases in Norway Spruce

Class III secretable plant peroxidases occur as a large family of genes in plants with many functions and probable redundancy. In this review we are concentrating on the evidence we have on the catalysis of lignin polymerization by class III plant peroxidases present in the apoplastic space in the xylem of trees. Some evidence exists on the specificity of peroxidase isozymes in lignin polymerization through substrate specificity studies, from antisense mutants in tobacco and poplar and from tissue and cell culture lines of Norway spruce (Picea abies) and Zinnia elegans. In addition, real time (RT-)PCR results have pointed out that many peroxidases have tissue specific expression patterns in Norway spruce. Through combining information on catalytic properties of the enzymes, on the expression patterns of the corresponding genes, and on the presence of monolignols and hydrogen peroxide in the apoplastic space, we can show that specific peroxidases catalyze lignin polymerization in the apoplastic space of Norway spruce xylem.

The effect of the crosstalk between photoperiod and temperature on the heading-date in rice.

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions.

The role of xylem class III peroxidases in lignification

Lignification is a cell wall fortifying process which occurs in xylem tissue in a scheduled manner during tissue differentiation. In this review, enzymes and the genes responsible for lignin biosynthesis have been studied with an emphasis on lignin polymerizing class III secretable plant peroxidases. Our aim is to understand the cell and molecular biology of the polymerization of lignin especially in tracheids and vessels of woody species but much of the experimental evidence comes from herbaceous plants. Class III peroxidases pose many problems for empirical work as their encoding genes are variable, their substrate specificities are wide and the half-life of many of the isozymes is very long. However, there is some evidence for the role of specific peroxidases in lignin polymerization through antisense mutants in tobacco and poplar and from tissue and cell culture lines of Picea abies and Zinnia elegans. Peroxidase enzyme action has been shown by substrate specificity studies and, for example, RT-PCR results have pointed out that many peroxidases have tissue-specific expression patterns. Tissue-level location of gene expression of some peroxidases has been studied by in situ hybridization and their cellular localization with antibodies and using EGFP-fusion genes. From these, it can be concluded that, although many of the xylem class III peroxidases have the potential for functioning in the synthesis of the lignin polymer, the combined information of catalytic properties, expression, and localization can reveal differences in the significance of different peroxidases in the lignification process.