RESUMO
The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.
Assuntos
Regulação Neoplásica da Expressão Gênica , Oligonucleotídeos Antissenso/metabolismo , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Neoplasias da Próstata/genética , Protaminas/metabolismo , Neoplasias da Bexiga Urinária/genética , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Western Blotting , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cloroquina/farmacologia , Regulação para Baixo , Humanos , Masculino , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/genética , Oligopeptídeos/genética , Paclitaxel/farmacologia , Peptídeos/química , Neoplasias da Próstata/enzimologia , Protaminas/química , Ligação Proteica , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Espectrometria de Fluorescência , Neoplasias da Bexiga Urinária/enzimologiaRESUMO
Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.
Assuntos
Endorribonucleases/metabolismo , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Western Blotting , Regulação para Baixo , Ativação Enzimática , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , Oligorribonucleotídeos/genética , Fosfatidiletanolaminas/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/genética , Processamento Pós-Transcricional do RNA/genética , RNA Antissenso/química , RNA Antissenso/genética , RNA Antissenso/fisiologia , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonuclease H/metabolismo , Ribonuclease P , Especificidade por Substrato , Transfecção/métodos , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Proteína bcl-X , Pequeno RNA não TraduzidoRESUMO
The early gene expression changes mediating carcinogen enhancement of viral transformation (CET) remain to be elucidated. A model cell culture system has been developed that is now permitting a molecular analysis of CET. Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with the cold-sensitive host-range type 5 adenovirus mutant, H5hr1, results in a dose-dependent increase in viral transformation. The present study investigates the role of immediate-early response genes, specifically c-fos, in the CET process. MMS pretreatment, alone or in combination with infection with H5hr1 temporally and differentially increases c-fos, c-jun, jun-B, jun-D and c-myc steady-state mRNA levels. Maximum induction occurs with c-fos and c-jun 8 to 12 h posttreatment and the magnitude of response is generally greatest in CREF cells pretreated with MMS and then infected with H5hr1. Enhancement in RNA levels is observed in the presence of cycloheximide indicating that ongoing protein synthesis is not required for induction of c-fos, c-jun, jun-B or c-myc expression. Nuclear run-on analysis indicates an enhancement in transcriptional rates for c-fos, c-jun, jun-B and c-myc in CREF cells treated with MMS or MMS plus infection with H5hr1. A requirement for elevated c-fos in the early stages of CET is indicated by the ability of c-fos antisense oligonucleotides to prevent the CET process. Direct evidence implicating early increases in c-fos as a mediator of the CET process is demonstrated by stably expressing mouse mammary tumor virus promoter-regulated human sense and antisense c-fos genes in CREF cells. Induction of c-fos sense expression by dexamethasone (DEX) in the absence of MMS treatment results in enhanced c-fos mRNA, Fos protein, AP-1 DNA-binding activity and H5hr1-induced transformation and CET. Induction of c-fos expression by DEX in stable c-fos-sense CREF constructs also results in elevated levels of c-jun, jun-B and c-myc mRNA and protein. Conversely, induction of c-fos antisense expression prevents the increase in c-fos mRNA, Fos protein and AP-1 DNA-binding activity and eliminates CET. In the antisense-c-fos constructs, increases in c-jun, jun-B and c-myc mRNA and protein normally induced by MMS also are not apparent. Thus, induction or inhibition in c-fos expression affects the level of expression of additional immediate-early response genes, including c-jun, jun-B and c-myc.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Adenoviridae , Transformação Celular Viral , Genes fos/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Transformação Celular Viral/efeitos dos fármacos , Transformação Celular Viral/genética , DNA/metabolismo , Fibroblastos , Expressão Gênica/efeitos dos fármacos , Genes fos/efeitos dos fármacos , Genes jun/efeitos dos fármacos , Genes jun/fisiologia , Genes myc/efeitos dos fármacos , Genes myc/fisiologia , Metanossulfonato de Metila/farmacologia , Dados de Sequência Molecular , Podofilina/análogos & derivados , Podofilina/metabolismo , Podofilotoxina/análogos & derivados , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Ratos , Fator de Transcrição AP-1/metabolismoRESUMO
The mechanism of the antineoplastic effects of suramin may involve interference with signal transduction, but in general is not well understood. We examined several polyanions to determine their effects on the kinase activity of the protein kinase C (PKC) beta1 and other PKC isoforms. Similar to suramin, a phosphorothioate oligodeoxynucleotide 28-mer homopolymer of cytidine (SdC28) inhibited the phosphatidylserine and Ca2+-dependent phosphorylation of an epidermal growth factor receptor octapeptide substrate. The inhibition by suramin was mixed competitive/noncompetitive with respect to ATP, but uncompetitive with respect to substrate. In contrast, the inhibition by SdC28 was competitive with respect to substrate (Ki = 5.4 microM) and not competitive with respect to ATP. The PKC alpha and beta1 isoforms were inhibited to the same extent with SdC28, while PKC epsilon was not inhibited. SdC28, in the absence of lipid cofactor, stimulated substrate phosphorylation, and in the absence of substrate induced PKC beta1 autophosphorylation. Similar behavior was seen with another polyanion, the polysulfated carbohydrate pentosan polysulfate (polyxylyl hydrogen sulfate). H4, a bis-naphthalene disulfonate tetraanion structurally related to suramin, also inhibited kinase activity but was not competitive with respect to ATP. Dianions closely related to H4 failed to inhibit PKC beta1, suggesting that multiple (>2) negative charges are required. The interactions of polyanions with PKC are complex, and are dependent on the molecular structure of the polyanion, the presence of cofactors, and the PKC isoform.
Assuntos
Antineoplásicos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Suramina/análogos & derivados , Suramina/farmacologia , Animais , Linhagem Celular , Isoenzimas/antagonistas & inibidores , Cinética , Camundongos , Oligodesoxirribonucleotídeos/farmacologia , Poliéster Sulfúrico de Pentosana/farmacologia , Fosforilação , Proteína Quinase C beta , Proteína Quinase C-alfa , Proteína Quinase C-delta , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , TionucleotídeosRESUMO
We established a continuous cell line, NCI-H295, from an invasive primary adrenocortical carcinoma. The cell line was established in a fully defined medium (HITES) and later could be adapted for growth in a simple medium supplemented only with selenium, insulin, and transferrin and devoid of serum, steroids, fibroblast growth factor, and a source of exogenous cholesterol. NCI-H295 cells had a relatively long population doubling time and were tumorigenic when inoculated s.c. into athymic nude mice. The cultured cells had ultrastructural features of steroid-secreting cells and contained complex cytogenetic abnormalities including the presence of multiple marker chromosomes. Steroid analyses (radioimmunoassays and mass spectrometry), performed 7 to 9 years after culture initiation, demonstrated secretion of more than 30 steroids characteristic of adrenocortical cells. Total unconjugated steroid secretion in serum-supplemented medium was 2.83 micrograms/10(6) cells/24 h and about 4-fold less in serum-free medium. The major pathway of pregnenolone metabolism in NCI-H295 cells is androgen synthesis, with formation of dehydroepiandrosterone, androstenedione, testotesterone, and at least three sulfated androgens, as well as estrogens. In addition, formation of cortisol, corticosterone, aldosterone, and 11 beta-hydroxyandrostenidione indicated the presence of 11 beta-hydroxylase. Thus, multiple pathways of steroidogenesis are expressed by NCI-H295 cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens. Our findings indicate the presence in NCI-H295 cells of all of the major adrenocortical enzyme systems, including 11 beta-hydroxylase, desmolase, 21 alpha-hydroxylase, 17 alpha-hydroxylase, 18-hydroxylase, lyase, sulfokinase, and aromatase. The NCI-H295 cell line should prove of value in studying the regulation, metabolic pathways, and enzymes involved in steroid formation and secretion. In addition, it may provide insights into the biology and treatment of adrenocortical carcinoma.
Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Esteroides/metabolismo , Células Tumorais Cultivadas/citologia , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/ultraestrutura , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Bandeamento Cromossômico , Técnicas de Cultura/métodos , Humanos , Cariotipagem , Espectrometria de Massas , Camundongos , Camundongos Nus , Microscopia Eletrônica , Esteroides/biossíntese , Transplante HeterólogoRESUMO
Oligodeoxynucleotides with a phosphorus atom in which one of the non-bridging oxygen atoms is substituted by selenium were prepared and investigated with respect to their antisense properties. A general synthesis of phosphoroselenoate analogs of oligonucleotides is described using potassium selenocyanate as the selenium donor. The compounds, characterized by 31P NMR, were shown to decompose to phosphate with a half-life of ca. 30 days. Melting temperatures of duplexes between poly(rA) or poly(rI) with oligo(dT) and oligo(dC), respectively, indicate diminished hybridization capability of phosphoroselenoate oligomers relative to both the unmodified phosphodiester oligomers and the phosphorothioate congeners. A phosphoroselenoate 17-mer is a sequence specific inhibitor of rabbit beta-globin synthesis in wheat germ extract and in injected Xenopus oocytes. In contrast phosphoroselenoate analogs are potent non-sequence specific inhibitors in rabbit reticulocyte lysate. In vitro HIV assays were carried out on a phosphoroselenoate sequence and compared with a phosphorothioate analogue that has previously been shown to exhibit anti-HIV activity (Matsukura et al., Proc. Natl. Acad. Sci. (1987) 84, 7706-7710). The phosphoroselenoate was somewhat less active, and was much more toxic to the cells.