Antisense RNA Strategy Research Articles

TMIC-74. THERAPEUTICALLY REPROGRAMMING MICROGLIA TO ANTI-TUMORAL ACTIVATION STATES IN DIFFUSE MIDLINE GLIOMA, H3K27 MUTANT

Abstract Diffuse midline glioma (DMG), H3K27M mutant, is one of the most challenging paediatric cancers to date with poor treatment options and dismal survival outcomes. Therefore, new treatment options are desperately needed. Previously we found that activation of the microglial tumour-supportive phenotype by DIPG is associated with a transient H3K27me3 downregulation suggesting a role for this pathway in inducing this activation state. However, it should be noted, that microglia do not carry the H3-K27M mutation when analysed in DIPG patient tissue, otherwise characteristic of the cancer cells. Ezh2 is a histone methyltransferase and the catalytic subunit of the polycomb repressor complex 2 (PRC2), responsible for tri-methylation of histone H3 at lysine 27. We found that repression of Ezh2 in microglia induced an anti-tumour phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumour-related cell death. Antisense RNA strategies were used to target Ezh2 gene expression in BV2 microglia and we have now carried out RNA sequencing on scramble BV2 or siEzh2 microglia co-cultured with DIPG tumour cells to further understand how EZH2, PRC2 and H3K27me3 regulates DIPG-induced microglia activation. Further work has found that activation of the microglial tumour supportive phenotype, in this context, is determined by a complex epigenetic mechanism involving both H3K4me3 and H3K27me3 and their associated methyltransferases. These histone marks are often associated with bivalent regulation of distinct gene transcription programs, and we are currently investigating if this is the case for DIPG-induced activation states using chromatin immunoprecipitation techniques. Overall, our results indicate that microglia, can be reprogrammed epigenetically by EZH2 inhibition to exert anti-tumoral responses and that microglia represent a new therapeutic avenue in DIPG. This future work should provide novel mechanisms governing pro-tumoral microglial activation states and new therapeutic targets that can be combined with EZH2 inhibition to enhance anti-tumoral microglia activation states.

Cellular Computational Logic Using Toehold Switches.

The development of computational logic that carries programmable and predictable features is one of the key requirements for next-generation synthetic biological devices. Despite considerable progress, the construction of synthetic biological arithmetic logic units presents numerous challenges. In this paper, utilizing the unique advantages of RNA molecules in building complex logic circuits in the cellular environment, we demonstrate the RNA-only bitwise logical operation of XOR gates and basic arithmetic operations, including a half adder, a half subtractor, and a Feynman gate, in Escherichia coli. Specifically, de-novo-designed riboregulators, known as toehold switches, were concatenated to enhance the functionality of an OR gate, and a previously utilized antisense RNA strategy was further optimized to construct orthogonal NIMPLY gates. These optimized synthetic logic gates were able to be seamlessly integrated to achieve final arithmetic operations on small molecule inputs in cells. Toehold-switch-based ribocomputing devices may provide a fundamental basis for synthetic RNA-based arithmetic logic units or higher-order systems in cells.

Antisense yycG modulates the susceptibility of Staphylococcus aureus to hydrogen peroxide via the sarA

BackgroundThe infectious pathogen Staphylococcus aureus (S. aureus) is primarily associated with osteomyelitis. Hydrogen peroxide drainage is an effective antimicrobial treatment that has been adopted to combat S. aureus infections. Previous investigations have indicated that the antisense RNA (asRNA) strategy negatively modulates S. aureus YycFG TCS, and it significantly disrupts biofilm formation. However, the effects of the antisense yycG RNA (ASyycG) strategy on the susceptibility of biofilm-producing S. aureus to hydrogen peroxide and the mechanisms underlying this effect have not been elucidated to date.ResultsOverexpression of ASyycG inhibited the transcription of biofilm formation-related genes, including sarA and icaA. Additionally, the CFU counts and the live bacterial ratios of ASyycG biofilm-producing S. aureus treated with H2O2 were notably reduced across the groups. Notably, the predicted promoter regions of the sarA and icaA genes were directly regulated by YycF.ConclusionsASyycG was observed to sensitize biofilm-producing S. aureus to H2O2 intervention synergistically via the sarA and thus may represent a supplementary strategy for managing osteomyelitis. However, future in-depth studies should attempt to replicate our findings in animal models, such as the rat osteomyelitis model.

Inhibition of microglial EZH2 leads to anti-tumoral effects in pediatric diffuse midline gliomas.

BackgroundDiffuse intrinsic pontine gliomas (DIPG), within diffuse midline gliomas are aggressive pediatric brain tumors characterized by histone H3-K27M mutation. Small-molecule inhibitors for the EZH2-H3K27 histone methyltransferase have shown promise in preclinical animal models of DIPG, despite having little effect on DIPG cells in vitro. Therefore, we hypothesized that the effect of EZH2 inhibition could be mediated through targeting of this histone modifying enzyme in tumor-associated microglia.MethodsPrimary DIPG tissues, and cocultures between microglia and patient-derived DIPG or -pediatric high-grade glioma (pHGG) cell lines, were used to establish the H3-K27M status of each cell type. Antisense RNA strategies were used to target EZH2 gene expression in both microglia and glioma cells. Microglia anti-tumoral properties were assessed by gene expression profile, tumor cell invasion capacity, microglial phagocytic activity, and associated tumor cell death.ResultsIn primary DIPG tissues, microglia do not carry the H3-K27M mutation, otherwise characteristic of the cancer cells. Activation of a microglial tumor-supportive phenotype by pHGG, independently of their H3-K27M status, is associated with a transient H3K27me3 downregulation. Repression of EZH2 in DIPG cells has no impact on tumor cell survival or their ability to activate microglia. However, repression of EZH2 in microglia induces an anti-tumor phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumor-related cell death.ConclusionsThese results indicate that microglia, beyond the tumor cells, contribute to the observed response of DIPG to EZH2 inhibition. Results highlight the potential importance of microglia as a new therapeutic avenue in DIPG.

Antisense RNA-Based Strategy for Enhancing Surfactin Production in Bacillus subtilis TS1726 via Overexpression of the Unconventional Biotin Carboxylase II To Enhance ACCase Activity.

The antisense RNA (asRNA) strategy is commonly used to block protein expression and downregulate the contents of metabolites in several microorganisms. Here, we show that the asRNA strategy can also be used to block gfp expression in Bacillus subtilis TS1726, which could further be utilized in the identification of new genes and functions. Via application of this strategy, biotin carboxylase II encoded by yngH (GeneID 939474) was identified to play a more significant role in maintaining acetyl-CoA carboxylase (ACCase) activity and enhancing surfactin synthesis compared to those of other ACCase subunits. The yngH gene was then overexpressed in the engineered strain B. subtilis TS1726(yngH). The surfactin titer of TS1726(yngH) increased to 13.37 g/L in a flask culture, representing a 43% increase compared to that of parental strain TS1726. This strategy opens the door to achieving large-scale production and broad application of surfactin.

Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach.

CRISPR interference (CRISPRi) using dCas9-sgRNA is a powerful tool for the exploration and manipulation of gene functions. Here we quantify the reversible switching of a central process of the bacterial cell cycle by CRISPRi and an antisense RNA mechanism. Reversible induction of filamentous growth in E. coli has been recently demonstrated by controlling the expression levels of the bacterial cell division proteins FtsZ/FtsA via CRISPRi. If FtsZ falls below a critical level, cells cannot divide. However, the cells remain metabolically active and continue with DNA replication. We surmised that this makes them amenable to an inducible antisense RNA strategy to counteract FtsZ inhibition. We show that both static and inducible thresholds can adjust the characteristics of the switching process. Combining bulk data with single cell measurements, we characterize the efficiency of the switching process. Successful restoration of division is found to occur faster in the presence of antisense sgRNAs than upon simple termination of CRISPRi induction.

Joint antisense RNA strategies for regulating isoprene production in Escherichia coli

Isoprene (C5H8) is a key chemical ingredient for the production of synthetic rubber and plastic.

1-Butanol production from glycerol by engineered Klebsiella pneumoniae

To utilize the by-product of biodiesel production, Klebsiella pneumoniae, a well-known glycerol-fermenting microorganism, was engineered to produce 1-butanol. The modified CoA-dependent and 2-keto acid pathways were established by expressing the genes ter-bdhB-bdhA and kivd, respectively. The 1-butanol titer and specific BuOH yield were 15.03 mg L−1 and 27.79 mg-BuOH per g cell in KpTBB (K. pneumoniae overexpressing the genes ter-bdhB-bdhA), and 28.7 mg L−1 and 51.58 mg-BuOH per g cell in Kp-kivd (K. pneumoniae overexpressing the gene kivd), respectively. Moreover, the native products in K. pneumoniae fermentation were down regulated using the antisense RNA strategy. The resulting yield of 1,3-propanediol and 2,3-butanediol was reduced by 81% and 15%, respectively. This work reports a new strain, K. pneumoniae, for 1-butanol production and the application of an antisense RNA strategy as an effective method for reducing the main by-products.

Down Regulation of ackA-pta Pathway in Escherichia coli BL21 (DE3): A Step Toward Optimized Recombinant Protein Expression System.

Background:One of the most important problems in production of recombinant protein is to attain over-expression of the target gene and high cell density. In such conditions, the secondary metabolites of bacteria become toxic for the medium and cause cells to die. One of these aforementioned metabolites is acetate, which enormously accumulated in the medium, so that both cell and protein yields are affected.Objectives:To overcome this problem, several strategies applied. In this research we used antisense RNA strategy, where the transcription of phosphotransacetylase (PTA) and acetate kinase (ACK), two acetate pathway key enzymes, could be controlled, which led to reduced acetate production.Materials and Methods:In order to achieve this, recombinant plasmid harboring antisense sequences targeting both of pta and ackA was assembled, after transfecting to the cells, its effects on the cell growth and acetate accumulation in the minimal media was assessed and compared with the control, the plasmid without antisense cassette, in presence and absence of IPTG in Escherichia coli BL21 (DE3).Results:It was observed that the mentioned strategy partially affect the growth and amount of excreted acetate in comparison with the control. In addition it was found that high down-regulation of the acetate production pathway reduces the growth rate of E. coli BL21 (DE3).Conclusions:The study principally proved the importance of this strategy in acetate excretion control.

English

The enzyme taxoid 14β-hydroxylase (14OH) directs a side-route of taxol pathway to 14β-hydroxy taxoids. Suppression of this side-route could increase the production of taxol. To suppress taxoid 14β-hydroxylase gene (14OH) expression in the Taxus× media TM3 cell line, antisense RNA inhibition approach was used in this study. Following the construction of an antisense RNA expression vector of 14OH fromTaxus chinensis, the antisense 14OH cDNA (as14OH) was introduced into TM3 cells by Agrobacterium tumefaciens-mediated transformation. Southern blot analysis of hygromycin phosphotransferase gene (HYG) revealed that this selection gene was integrated successfully into the genome of Taxus × media cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the 14OHmRNA level in transgenic cells dropped dramatically, suggesting that the expression of endogenous14OH gene was significantly suppressed by the exogenous as14OH gene. Correspondingly, the total yield of three major C-14 oxygenated taxoids (yunnanxane, taxuyunnanine C, sinenxan C) was markedly reduced in the silenced cell lines when compared with those of the non-transgenic controls. These results indicated that the antisense RNA strategy is a useful tool in suppressing the expression of genes in Taxus and this method could be used to silence other important genes that divert Taxol pathway to side-route metabolites.   Key words: Taxus × media, taxoid 14β-hydroxylase, antisense, gene suppression.

In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

AbstractAbstract 815Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3‘ splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3‘ splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs.To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures:No relevant conflicts of interest to declare.

Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium

SummaryCobalamin (vitamin B12) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome‐based overexpression of genes involved in vitamin B12 biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysGAcbiYbtuR,and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B12 riboswitch upstream of the cbiXJCDETLFGAcysGAcbiYbtuRoperon and the recombinant production of three different vitamin B12 binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B12‐dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B12 concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.

Expression and location of endo-β-mannanase during the ripening of tomato fruit, and the relationship between its activity and softening

Endo-beta-mannanase is thought to play a role in tomato fruit ripening by participating in the degradation of cell walls. Its spatial and temporal expression during ripening was examined, as was the relationship between its activity and softening of the fruit using a large number of tomato lines, and by suppression of transcription of the endo-beta-mannanase (LeMan4a) gene. Immunolocalization studies showed that the enzyme is expressed in the fruit cell wall at all ripening stages, but it is not active during the initial green stage; this is not due to the presence of inhibitors of its activity, nor due to changes in its mRNA sequence. Transient expression in onion epidermal cells of endo-beta-mannanase transcripts fused to green fluorescent protein resulted in the expressed enzyme being localized to the cell walls. Transgenic tomato plants expressing a GUS gene attached to the LeMan4a promoter showed that this occurs initially during ripening in the skin and outer pericarp of the fruit, and later in the skin and throughout the pericarp. Fruit firmness and activity of endo-beta-mannanase were not strongly correlated during ripening of many lines of tomato. Several plants of cv. Micro-Tom were transformed using RNA interference (mRNAi) and antisense RNA strategies to suppress transcription of the LeMan4a gene. When endo-beta-mannanase activity was much reduced in the transgenic fruits, their firmness was higher compared to those of control fruits at the turning and orange-color stages, but at the red-ripe stage firmness was similar between the two fruit types. We suggest that while the enzyme does participate in fruit ripening it alone is not sufficient to cause hydrolysis of the cell walls which results in their weakening; it likely plays a cooperative role with other known wall-modifying enzymes, and/or is involved in cell wall rearrangement.

Identification of Cellular Genes Affecting the Infectivity of Foot-and-Mouth Disease Virus

Foot-and-mouth disease virus (FMDV) produces one of the most infectious of all livestock diseases, causing extensive economic loss in areas of breakout. Like other viral pathogens, FMDV recruits proteins encoded by host cell genes to accomplish the entry, replication, and release of infectious viral particles. To identify such host-encoded proteins, we employed an antisense RNA strategy and a lentivirus-based library containing approximately 40,000 human expressed sequence tags (ESTs) to randomly inactivate chromosomal genes in a bovine kidney cell line (LF-BK) that is highly susceptible to FMDV infection and then isolated clones that survived multiple rounds of exposure to the virus. Here, we report the identification of ESTs whose expression in antisense orientation limited host cell killing by FMDV and restricted viral propagation. The role of one such EST, that of ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase6; also known as CD39L2), a membrane-associated ectonucleoside triphosphate diphosphohydrolase that previously was not suspected of involvement in the propagation of viral pathogens and which we now show is required for normal synthesis of FMDV RNA and proteins, is described in this report.

Metabolic engineering for solvent productivity by downregulation of the hydrogenase gene cluster hupCBA in Clostridium saccharoperbutylacetonicum strain N1-4

The selective production of acetone and butanol is highly desirable from the viewpoint of biofuel production. We have manipulated the activity level of a hydrogenase for this purpose because hydrogen and solvent production are closely correlated with each other. First, we cloned the hydrogenase gene cluster from Clostridium saccharoperbutylacetonicum strain N1-4 and downregulated its expression using an antisense RNA strategy. The cloned hydrogenase gene cluster contained three adjacent open reading frames, designated hupC, hupB, and hupA. Sequence analysis revealed that HupA could accommodate an H-cluster, which is the catalytic domain of the Fe-hydrogenase. HupB and HupC contained no H-cluster but could accommodate several Fe-S clusters. The hupCBA genes were co-transcribed, and the level of the transcript was maximized in the solventogenic phase. When the antisense RNA of the hupC upstream region (180 bp) was expressed under the bdh (encoding butanol dehydrogenase) promoter, significant reduction of hupC translation was observed, indicating that this antisense RNA is effective in strain N1-4. Production of hydrogen in the antisense transformant increased 3.1-fold. Hydrogen-evolving activity was comparable in both the control and antisense strains, but hydrogen uptake activity significantly decreased in the antisense strain (13% remaining). These results indicate that the HupCBA proteins are involved in hydrogen uptake. Importantly, the level of acetone in the antisense transformant increased 1.6-fold, and butanol production decreased to 75.6% compared to the control strain. Thus, we successfully altered solvent productivity by controlling electron flow in an acetone/butanol-producing Clostridium species.

A high-affinity molybdate transporter in eukaryotes

Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.

The Cytoskeleton-associated PDZ-LIM Protein, ALP, Acts on Serum Response Factor Activity to Regulate Muscle Differentiation

In this report, an antisense RNA strategy has allowed us to show that disruption of ALP expression affects the expression of the muscle transcription factors myogenin and MyoD, resulting in the inhibition of muscle differentiation. Introduction of a MyoD expression construct into ALP-antisense cells is sufficient to restore the capacity of the cells to differentiate, illustrating that ALP function occurs upstream of MyoD. It is known that MyoD is under the control of serum response factor (SRF), a transcriptional regulator whose activity is modulated by actin dynamics. A dramatic reduction of actin filament bundles is observed in ALP-antisense cells and treatment of these cells with the actin-stabilizing drug jasplakinolide stimulates SRF activity and restores the capacity of the cells to differentiate. Furthermore, we show that modulation of ALP expression influences SRF activity, the level of its coactivator, MAL, and muscle differentiation. Collectively, these results suggest a critical role of ALP on muscle differentiation, likely via cytoskeletal regulation of SRF.

Reduced expression of top1 gene induces programmed cell death and alters ascorbate metabolism in Daucus carota cultured cells

Topoisomerase I (topo I) is a nuclear enzyme which plays a fundamental role in several pathways involving changes in DNA topology. The topo I-mediated reaction is accomplished by the transient covalent binding of the enzyme to DNA (topo I-DNA complex). Stabilization of the topo I-DNA complex, leading to irreversible double-strand breaks, has been reported to occur in animal cells under oxidative stress conditions and during apoptosis. In order to study the existence of a putative link between the topo I-mediated DNA damage and ascorbate (ASC) metabolism, also involved in the responses against oxidative stress and in the apoptotic process in plants, Daucus carota cells showing reduced expression of the top1beta gene encoding the topo Ibeta isoform were produced, using an antisense RNA strategy. Two independent transgenic lines (AT1-beta/22 and beta/36), characterized by a slow growth phenotype, resistance to camptothecin, a specific inhibitor of topo I, but sensitivity to etoposide, an inhibitor of topo II, were investigated in this study. In the absence of external stimuli, AT1-beta/22 and beta/36 cells underwent programmed cell death (PCD) in a precocious phase of the growth curve. ASC metabolism showed remarkable differences in AT1-beta/22 and beta/36 cells, compared with control, and the observed alterations were similar to those occurring in tobacco Yellow Bright-2 cells induced to enter PCD by exogenous stimuli. However, differently from other studied examples of PCD, overproduction of reactive oxygen species was not detected in AT1-beta/22 and beta/36 cells. The relevance of these findings in relation to the signalling pathways leading to PCD is discussed.

Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro

Small interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limited because of in vivo instability. As a first step to overcome delivery issues, a series of graft copolymers of polyethylene glycol and polyethylenimine (PEI-g-PEG) were synthesized and investigated as nontoxic carriers for delivery of siRNA targeting the signaling peptide of secretory clusterin (sCLU), a prosurvival factor that protects cells from ionizing radiation (IR) injury, as well as chemotherapeutic agents. Three copolymers with different PEG grafting densities were tested for their abilities to bind and form nanocomplexes with siRNA. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2k10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and had the smallest nanocomplex size (115 +/- 13 nm diameter). In human breast cancer MCF-7 cells, 2k10-siRNA-sCLU nanocomplexes suppressed both basal as well as IR-induced sCLU protein expression, which led to an over 3-fold increase in IR-induced lethality over 2k10-siRNA scrambled controls. In summary, this study demonstrates the proof-of-principle in using nanoparticle-mediated delivery of specific siRNAs to enhance the lethality of IR exposure in vitro, opening the door for siRNA-mediated knockdown of specific cytoprotective factors, such as DNA repair, anti-apoptotic, free radical scavenging, and many other proteins.

Combined effects of GSTP1 and MRP1 in melanoma drug resistance

Glutathione-S-transferase Pi1 (GSTP1) and multidrug resistance protein 1 (MRP1) are overexpressed in melanoma, a skin cancer notoriously resistant to all current modalities of cancer therapy. To investigate the involvement of these detoxifying enzymes in the drug resistance of melanoma, an inducible (Tet-On™ system) antisense (AS) RNA strategy was used to specifically inhibit GSTP1 expression in A375 cells, a human melanoma cell line expressing high levels of GSTP1 and MRP1. Stable transfectant clones were established and analysed for GSTP1 inhibition by AS RNA. The clone A375-ASPi1, presenting a specific 40% inhibition of GSTP1 expression in the presence of doxycycline, was selected. Lowering the GSTP1 level significantly increased (about 3.3-fold) the sensitivity of A375-ASPi1 cells to etoposide. Inhibitors of glutathione synthesis (BSO), GSTs (curcumin, ethacrynic acid), and also of MRPs (MK571, sulphinpyrazone) improved the sensitising effect of GSTP1 AS RNA. All these inhibitors had stronger sensitising effects in control cells expressing high GSTP1 level (A375-ASPi1 cells in the absence of doxycycline). In conclusion, GSTP1 can act in a combined fashion with MRP1 to protect melanoma cells from toxic effects of etoposide.