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BMC Cancer Jun 2010Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of...
BACKGROUND
Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells.
METHODS
We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT).
RESULTS
We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406) targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512) targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping) and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone.
CONCLUSION
We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.
Topics: Aminacrine; Blotting, Western; Cell Proliferation; Exons; Humans; Introns; Mutagens; Peptide Nucleic Acids; Proto-Oncogene Proteins c-mdm2; RNA Precursors; RNA Splicing; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 20591158
DOI: 10.1186/1471-2407-10-342 -
Journal of Lipid Research Sep 2010A method of direct lipid analysis by MALDI mass spectrometry in intact membranes, without prior extraction/separation steps, is described. The purple membrane isolated...
A method of direct lipid analysis by MALDI mass spectrometry in intact membranes, without prior extraction/separation steps, is described. The purple membrane isolated from the extremely halophilic archaeon Halobacterium salinarum was selected as model membrane. Lyophilized purple membrane were grinded with 9-aminoacridine (9-AA) as dry matrix, and the powder mixture was crushed in a mechanical die press to form a thin pellet. Small pieces of the pellet were then attached to the MALDI target and directly analyzed. In parallel, individual archaebacterial phospholipids and glycolipids, together with the total lipid extract of the purple membrane, were analyzed by MALDI-TOF/MS using 9-AA as the matrix in solution. Results show that 9-AA represents a suitable matrix for the conventional MALDI-TOF/MS analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized archaebacterial membranes. This method might be of general application, offering the advantage of quickly gaining information about lipid components without disrupting or altering the membrane matrix.
Topics: Aminacrine; Archaea; Fluorescent Dyes; Freeze Drying; Halobacterium salinarum; Lipids; Molecular Structure; Purple Membrane; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 20538644
DOI: 10.1194/jlr.D007328 -
Journal of Lipid Research Jun 2010Recently, we used the favorable properties of 9-aminoacridine (9-AA) as matrix for the quantitative analysis of acidic metabolites and glycerophospholipids from extracts...
Recently, we used the favorable properties of 9-aminoacridine (9-AA) as matrix for the quantitative analysis of acidic metabolites and glycerophospholipids from extracts of biological materials [Sun, G., Yang, K., Zhao, Z., Guan, S., Han, X., and Gross, R.W. (2007) A shotgun metabolomics approach for rapid analysis of negatively-charged water-soluble cellular metabolites from mouse heart tissue. Anal. Chem. 79: 6629-6640; Sun, G., Yang, K., Zhao, Z., Guan, S., Han, X., and Gross, R.W. (2008) Matrix-assisted laser desorption/ionization-time of flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions. Anal. Chem. 80: 7576-7585.] by MALDI-MS. Herein, we extend this discovery and identified the selective desorption/ionization of sulfatides over other examined anionic lipids present in lipid extracts of biological samples by MALDI-MS using 9-AA as matrix. Through this approach, a high throughput method for the quantitative analysis of low to very low abundance sulfatide molecular species directly from crude lipid extracts has been developed. This method possessed a linear dynamic range of over 1,000-fold, a detection limit at the high attomole level, and a reproducibility of approximately 10% deviation. Many potential factors that might affect the quantitation of sulfatide species employing the method were examined and their effects were found to be negligible within experimental error. Collectively, these results demonstrate a powerful high throughput method for the measurement of sulfatides directly from extracts of biological samples, facilitating the study of sulfatide metabolism, trafficking, and homeostasis in health and disease.
Topics: Aminacrine; Animals; Cattle; Linear Models; Male; Mice; Phospholipids; Reference Standards; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfoglycosphingolipids
PubMed: 20124011
DOI: 10.1194/jlr.D004077 -
Virology Journal Jul 2009As part of a continued search for more efficient anti-HIV-1 drugs, we are focusing on the possibility that small molecules could efficiently inhibit HIV-1 replication...
As part of a continued search for more efficient anti-HIV-1 drugs, we are focusing on the possibility that small molecules could efficiently inhibit HIV-1 replication through the restoration of p53 and p21WAF1 functions, which are inactivated by HIV-1 infection. Here we describe the molecular mechanism of 9-aminoacridine (9AA) mediated HIV-1 inhibition. 9AA treatment resulted in inhibition of HIV LTR transcription in a specific manner that was highly dependent on the presence and location of the amino moiety. Importantly, virus replication was found to be inhibited in HIV-1 infected cell lines by 9AA in a dose-dependent manner without inhibiting cellular proliferation or inducing cell death. 9AA inhibited viral replication in both p53 wildtype and p53 mutant cells, indicating that there is another p53 independent factor that was critical for HIV inhibition. p21WAF1 is an ideal candidate as p21WAF1 levels were increased in both p53 wildtype and p53 mutant cells, and p21WAF1 was found to be phosphorylated at S146, an event previously shown to increase its stability. Furthermore, we observed p21WAF1 in complex with cyclin T1 and cdk9 in vitro, suggesting a direct role of p21WAF1 in HIV transcription inhibition. Finally, 9AA treatment resulted in loss of cdk9 from the viral promoter, providing one possible mechanism of transcriptional inhibition. Thus, 9AA treatment was highly efficient at reactivating the p53 - p21WAF1 pathway and consequently inhibiting HIV replication and transcription.
Topics: Aminacrine; Anti-HIV Agents; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; HIV-1; Humans; T-Lymphocytes; Transcription, Genetic; Tumor Suppressor Protein p53; Virus Replication; tat Gene Products, Human Immunodeficiency Virus
PubMed: 19630958
DOI: 10.1186/1743-422X-6-114 -
Bioorganic & Medicinal Chemistry Letters Aug 2009The cytotoxicity and mechanism of action of a series of substituted 9-aminoacridines is evaluated using topoisomerase I and cancer cell growth inhibition assays. In...
The cytotoxicity and mechanism of action of a series of substituted 9-aminoacridines is evaluated using topoisomerase I and cancer cell growth inhibition assays. In previous work, compounds of this type were shown to catalytically inhibit topoisomerase II, leading to a G1-S phase arrest of the cell cycle and apoptosis in pancreatic cancer cells in vitro and in vivo. The present study expands the potential utility of these compounds in the development of cancer therapeutics by showing that these compounds inhibit proliferation of cell lines derived from the nine most common human cancers. Further results show that at least one of the compounds effectively stabilizes topoisomerase I-DNA adduct formation in intact cells. RNA interference experiments, however, indicate that this interaction does not contribute to the drug-induced killing of cancer cells indicating the compounds may be non-lethal poisons of topoisomerase I.
Topics: Acridines; Aminacrine; Antineoplastic Agents; Cell Line; Chemistry, Pharmaceutical; DNA Topoisomerases, Type I; Dose-Response Relationship, Drug; Doxorubicin; Drug Design; Drug Screening Assays, Antitumor; Etoposide; Humans; Models, Chemical; Neoplasms; RNA, Small Interfering
PubMed: 19501511
DOI: 10.1016/j.bmcl.2009.05.037 -
Nucleic Acids Research Jul 2009While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA...
While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes--mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na(+)). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally located mismatches was more than 150-fold. Together the data support the use of homopyrimidine PNAs as efficient and sequence selective tools in triplex targeting strategies under physiological relevant conditions.
Topics: Aminacrine; Base Sequence; Cytosine; DNA; Hydrogen-Ion Concentration; Intercalating Agents; Peptide Nucleic Acids
PubMed: 19474349
DOI: 10.1093/nar/gkp437 -
Analytical Chemistry Oct 2008A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based approach was developed for the rapid analyses of cellular...
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions.
A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based approach was developed for the rapid analyses of cellular glycerophospholipids. Through multiplexed solvent-enabled optimization of analyte-matrix interactions during the crystallization process, over a 30-fold increase in S/N was achieved using 9-aminoacridine as the matrix. The linearity of response (r(2) = 0.99) and dynamic range of this method (over 2 orders of magnitude) were excellent. Moreover, through multiplexing ionization conditions by generating suites of different analyte-matrix interactions in the absence or presence of different alkali metal cations in the matrix, discrete lipid classes were highly and selectively ionized under different conditions resulting in the de facto resolution of lipid classes without chromatography. The resultant decreases in spectral complexity facilitated tandem mass spectrometric analysis through high energy fragmentation of lithiated molecular ions that typically resulted in informative fragment ions. Anionic phospholipids were also detected as singly negatively charged species that could be fragmented using MALDI tandem mass spectrometry leading to structural assignments. Collectively, these results identify a rapid, sensitive, and highly informative MALDI-TOF MS approach for analysis of cellular glycerophospholipids directly from extracts of mammalian tissues without the need for prior chromatographic separation.
Topics: Aminacrine; Animals; Cardiolipins; Crystallization; Glycerophospholipids; Male; Mice; Mice, Inbred C57BL; Myocardium; Phosphatidylcholines; Solvents; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tissue Extracts; Triglycerides
PubMed: 18767869
DOI: 10.1021/ac801200w -
Journal of Virology Sep 2008Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the...
Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the HTLV-1-encoded Tax protein inhibits the function of tumor suppressor p53 through a Tax-induced NF-kappaB pathway. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kappaB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is "druggable" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kappaB suggests a promising strategy for the treatment of HTLV-1-transformed cells.
Topics: Aminacrine; Anticarcinogenic Agents; Apoptosis; Cell Cycle; Cell Death; Cell Line, Transformed; Cell Survival; Cell Transformation, Viral; Dose-Response Relationship, Drug; G1 Phase; Genes, Reporter; Human T-lymphotropic virus 1; Humans; In Situ Nick-End Labeling; Luciferases; NF-kappa B; Plasmids; RNA, Small Interfering; Time Factors; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53
PubMed: 18550670
DOI: 10.1128/JVI.00690-08 -
Molecular Cancer Jun 2008Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of...
BACKGROUND
Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis.
RESULTS
To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice.
CONCLUSION
Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.
Topics: Aminacrine; Animals; Breast Neoplasms; Cell Line, Transformed; Cell Line, Tumor; Chromosomal Instability; Female; Frameshift Mutation; Genes, Tumor Suppressor; Humans; Mammary Neoplasms, Experimental; Mice; Models, Biological; Mutagenesis; Neoplasm Transplantation; Nitrogen Mustard Compounds; Nucleic Acid Hybridization; RNA Stability; Spectral Karyotyping
PubMed: 18534021
DOI: 10.1186/1476-4598-7-51 -
Biophysical Chemistry Jun 2008We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and...
We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.
Topics: Aminacrine; Antimutagenic Agents; Binding, Competitive; Chemoprevention; Chlorophyllides; DNA; DNA Damage; HL-60 Cells; Humans; Macromolecular Substances; Mutagens; Nitrogen Mustard Compounds
PubMed: 18423964
DOI: 10.1016/j.bpc.2008.03.004