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E Wickstrom

Publications and source records attributed to E Wickstrom.

At least 19 recordsLinked to original sources
PubMed2007

Receptor-mediated internalization of chelator-PNA-peptide hybridization probes for radioimaging or magnetic resonance imaging of oncogene mRNAs in tumours.

Early external detection of cancer gene activity might enable early treatment of cancer and might reduce cancer mortality. We hypothesized that oncogene mRNA overexpressed at thousands of copies per malignant cell in a zone of transformed cells could be imaged externally by scintigraphic imaging, PET (positron emission tomography) or MRI (magnetic resonance imaging) with PNA (peptide nucleic acid) hybridization probes that include chelators for metal cations and a cyclized peptide analogue of IGF-1 (insulin-like growth factor 1), D(Cys-Ser-Lys-Cys), to mediate internalization by IGF1R (IGF-1 receptor) overexpressed on cancer cells. We observed that human MCF7 breast cancer cells that overexpress IGF1R efficiently internalized fluorescein-chelator-PNA-D(Cys-Ser-Lys-Cys) to the cytoplasm, but not with D(Cys-Ala-Ala-Cys). Scintigraphic imaging of MCF7 xenografts in immunocompromised mice revealed that CCND1 and MYC [(99m)Tc]chelator-PNA-D(Cys-Ser-Lys-Cys) probes yielded xenograft. PET imaging with [(64)Cu]chelator-PNA-D(Cys-Ser-Lys-Cys) yielded stronger signals. Scintigraphic imaging of human AsPC1 pancreas cancer xenografts with [(99m)Tc]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys) yielded strong xenograft signals. Stronger xenograft image intensities were obtained by PET imaging of [(64)Cu]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys). MRI required extension of chelator-polydiamidopropanoate dendrimers from the N-termini of the PNA probes to increase the number of contrast paramagnetic gadolinium (III) cations per probe. These results provide a basis for detection of oncogene activity in tissues from outside the body by hybridization with metal-chelator-PNA-peptides that are selectively internalized by cancer cells.

Animals↗
PubMed2005

Tumor-targeting peptide-PNA-peptide chimeras for imaging overexpressed oncogene mRNAs.

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [99sTc] chelator-PNA-D (CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.

Breast Neoplasms↗
PubMed2004

Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42 degrees C-induced reorganization.

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH.

Animals↗
PubMed2004

Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs.

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.

Animals↗
PubMed2003

99mTc-peptide-peptide nucleic acid probes for imaging oncogene mRNAs in tumours.

Imaging oncogene mRNA in tumours would provide a powerful tool for the early detection of occult malignant lesions. The goal was to prepare a chimera consisting of a dodecamer antisense peptide nucleic acid (PNA) specific for c-MYC oncogene overexpressed in human breast cancer cells and a chelating moiety that facilitates quantitative radiolabelling with 99mTc and evaluate it for hybridization and tissue distribution in laboratory animals. The pentapeptide chelator-PNA dodecamer specific for c-MYC mRNA was extended from a solid support by 9-fluorenylmethyloxycarbonyl (Fmoc) coupling. Similarly, a chelator-PNA chimera with four central mismatches was also prepared which served as a control. The chimeras were purified, characterized and evaluated for hybridization to c-MYC mRNA by fluorescent, real-time polymerase chain reaction (RT-PCR). The chimeras were labelled with 99mTc and their tissue distribution was examined in athymic nude mice bearing experimental human breast tumours. 99mTc radiolabelling was quantitative and presented a single peak in reversed phase liquid chromatography. Fluorescent real-time polymerase chain reactions using primer and fluorescent probe sets previously calculated for c-MYC mRNA demonstrated inhibition of reverse transcription by the c-MYC specific chimera as compared to that of the control. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver, and appreciable levels in tumours. These observations suggest that 99mTc-peptide-PNA probes might be useful for imaging gene expression in tumours, and the approach is worthy of further investigation.

Animals↗
PubMed2001

Oligonucleotide treatment of ras-induced tumors in nude mice.

Oligonucleotides have shown an ability to target specific oncogene transcripts and inhibit their expression in cells, but the degree to which sustained treatment can suppress the levels of an oncogenic protein enough to benefit a patient remains to be determined. This question has been studied in several ways. First, the relationship of antisense DNA inhibition to the predicted secondary structure of human H-RAS oncogene mRNA was examined in transformed mouse cells that form solid tumors. Inhibition of H-Ras expression was sequence-specific, dose-dependent, and correlated with inhibition of focus formation. The efficacy of the first intron antisense sequence in reducing H-Ras expression was greater than that of the initiation codon target. Second, H-RAS transformed solid tumor cells were pretreated in vitro with normal oligonucleotides, after which tumor growth from the treated cells was tested in nude mice. The three days of treatment with the first intron antisense DNA reduced H-Ras cellular levels by more than 90% whereas a nonspecific control DNA reduced H-Ras levels by approx 20%. Tumor growth of cells treated with H-RAS antisense oligonucleotide was significantly reduced for up to 14 d following the end of treatment and implantation into the mice, whereas the nonspecific control DNA had no significant effect. Third, H-RAS transformed bladder cancer cells were implanted into nude mice, after which the mice were treated for 31 d with oligonucleotide phosphorothioates. Tumor growth in mice treated with H-RAS 12th codon antisense oligonucleotide was reduced by about 80% throughout the treatment period, reiterating the sustained effect seen in pretreated tumor cells. However, the scrambled phosphorothioate control inhibited tumor growth by about 60%, illustrating some nonspecific inhibition. Fourth, K-RAS transformed pancreatic cancer cells were treated in culture and in nude mice. Inhibition of K-Ras expression with a phosphorothioate oligonucleotide directed against a 5'-UTR sequence was sequence-specific and dose-dependent. K-RAS transformed pancreatic cancer cells were implanted into nude mice, after which the mice were treated for 14 d with oligonucleotide phosphorothioates. Tumor growth in mice treated with K-RAS 5'-UTR antisense oligonucleotide was reduced by about 50% throughout the treatment period, reiterating the sustained effect seen with H-RAS transformed cells. In this case, the sense phosphorothioate control did not inhibit tumor growth, demonstrating that nonspecific inhibition is not a characteristic of all phosphorothioate sequences. The next logical steps include testing oligonucleotide efficacy against other tumor types, toxicological testing in higher species, and clinical trials in human subjects.

3T3 Cells↗
PubMed2000

Transposon Tn7 gene insertion into an evolutionarily conserved human homolog of Escherichia coli attTn7.

Escherichia coli transposon Tn7 can integrate into its target DNA sequence, attTn7 at the 3' end of glmS, with high specificity and efficiency. Remarkably, the insertional recognition sequence in the E. coli genome displays a high degree of identity with the corresponding region at the 3' end of the corresponding human gene for glutamine-fructose-6-phosphate transaminase (GFPT), located at 2p13. It was therefore of interest to determine whether Tn7 could recognize the corresponding human sequence, and transpose at that site. Strains of E. coli DH5alpha were prepared carrying the tnsA-E genes on one plasmid, and attTn7 or the human equivalent on a second recipient plasmid within the alpha-complementation fragment of the lacZ gene. Each strain was transformed with a donor plasmid carrying a gentamycin resistance gene within the Tn7L and Tn7R cassettes. Restriction mapping and sequence analysis of recipient plasmids isolated from white colonies demonstrated that Tn7 inserted the gentamycin resistance gene both into the E. coli attTn7 sequence, and into its human counterpart. No nonspecific insertion was observed in a control plasmid containing only the lacZ fragment. These results provide a basis to investigate whether TnsA-D proteins can mediate gene insertion into comparably conserved sites in eukaryotic chromosomes.

Bacterial Proteins↗
PubMed1998

Antisense c-myc and immunostimulatory oligonucleotide inhibition of tumorigenesis in a murine B-cell lymphoma transplant model.

BACKGROUND: Because the development of drug-resistant cells can lead to relapses in patients with lymphoma treated with chemotherapy, new approaches are needed for effective disease management, such as those targeting the c-MYC proto-oncogene with antisense oligonucleotides. Our goal was to investigate whether antisense c-myc oligonucleotides could prevent tumorigenesis in a B-cell lymphoma model. METHODS: Immunocompetent mice received subcutaneous injections of tumor cells from a transgenic mouse model of Burkitt's lymphoma. For 7 consecutive days, beginning 1 day after tumor cell transplantation, the mice were given either a DNA phosphorothioate oligonucleotide complementary to c-myc codons 1-5 (myc6) or other c-myc-related oligonucleotides at a dose of 0.76 mg per day subcutaneously. Myc protein expression, normalized to beta-actin expression, was measured by western blotting of tumor and splenic proteins. To determine whether tumor inhibition by myc6 could be a result of B-cell activation, we compared the activity of myc6 with that of an immunostimulatory oligonucleotide, mcg. RESULTS: In comparison with control treatments (saline vehicle, scrambled-sequence oligonucleotide, or double-mismatch oligonucleotide), treatment with myc6 delayed tumor onset by 3 days, decreased total tumor mass at sacrifice (i.e., 17 days after tumor cell transplantation) by 40% +/- 16% (mean +/- standard error), and decreased the splenic Myc-to-actin ratio. Inhibition of tumors by myc6 and mcg (both of which share a dACGTT motif) was comparable. Administration of an oligonucleotide sequence complementary to c-myc codons 384-388 (myc55) delayed tumor onset by 5-6 days, decreased total tumor mass at sacrifice by 65% +/- 6%, and reduced the splenic Myc-to-actin ratio to below that produced by myc6. A 14-day treatment regimen of myc55 alternating with mcg completely inhibited tumor formation during the therapeutic schedule. CONCLUSIONS: A combined oligonucleotide regimen, based on antisense c-MYC and immunostimulatory oligonucleotides, should be investigated to increase the number and duration of complete remissions obtained after standard chemotherapy for B-cell lymphoma.

Adjuvants, Immunologic↗
PubMed1997

Transformed and immortalized cellular uptake of oligodeoxynucleoside phosphorothioates, 3'-alkylamino oligodeoxynucleotides, 2'-O-methyl oligoribonucleotides, oligodeoxynucleoside methylphosphonates, and peptide nucleic acids.

Direct quantitative comparisons of cellular uptake across a wide variety of analogs and cell types are necessary for the design of oligonucleotide diagnostic and therapeutic applications. This work reports quantitative cellular uptake and nuclear localization of [14C]oligodeoxynucleoside phosphorothioates (PS), 3'-alkylamino oligodeoxynucleoside phosphodiesters (PO-NH2), 2'-O-methyl oligoribonucleoside phosphodiesters (2OM), peptide nucleic acids (PNA), and oligodeoxynucleoside methylphosphonates (MP) in several transformed or immortalized cell lines. All analogs demonstrated active cellular uptake in that intracellular concentrations greatly exceeded the extracellular 1 microM concentration within 1-3 hr. However, by 9-24 hr, cellular accumulations of PS exceeded those of PO-NH2 and 2OM by 3- to 5-fold, PNA by 6- to 7-fold, and MP by 8- to 10-fold. Similar results were observed in two transformed cell lines, HL-60 leukocytes and H-ras transformed fibroblasts, using three different heterogeneous sequences. H-ras and IGF-1R transformed fibroblasts had a 2- to 5-fold higher uptake of all analogs than non-transformed immortalized fibroblasts. Nuclear levels of the PO-NH2, PS, and MP analogs were approximately 25% of total cellular uptake, while nuclear percentages of 2OM and PNA were less than 20%, suggesting some differences in nuclear localization among the analogs. These observations provide a direct quantitative comparison of cellular uptake as a function of oligonucleotide modification, and imply that transformation enhances cellular uptake. From the perspective of therapy and diagnosis, clear trade-offs were apparent between efficiency of uptake on the one hand, and nuclease resistance and hybridization strength on the other.

3T3 Cells↗
PubMed1997

Temperature and salt dependence of higher order structure formation by antisense c-myc and c-myb phosphorothioate oligodeoxyribonucleotides containing tetraguanylate tracts.

The use of complementary RNA or DNA sequences to selectively interfere with the utilization of mRNA of a target gene is an attractive therapeutic strategy. Two well-studied targets for oligonucleotide therapy are the c-mycand c-mybproto-oncogenes. It has been reported that sequences which contain four contiguous Gs can elicit a non-antisense response, due to the formation of a homotetrameric G quartet structure. Therefore, it was of interest to determine whether anti-c-mycand anti-c-mybphosphorothioate DNAs including tetraguanylate form higher order structures under physiologically relevant salt conditions and temperature. First, the identity of the higher order structure was established and was found to be a tetraplex. Employing intracellular (high K+), extracellular (low K+) and normal saline (no K+) salt mixtures, native gel electrophoresis revealed no tetraplex formation at 37 degrees C, the physiologically relevant temperature. On the other hand, tetraplex structure formation was observed at 4 and 23 degrees C. Hence, the potential for these sequences to form tetraplex structures at lower temperatures may not be relevant for their activity in cells and animals at physiological temperature.

Animals↗
PubMed1997

Differential oligonucleotide activity in cell culture versus mouse models.

The usual course of drug discovery begins with the demonstration of compound activity in cells and, usually, a lower level of activity in animals. Successive rounds of drug design may result in a compound with sufficient activity in animals to justify clinical trials. The basic endpoints of therapeutic oligonucleotide experiments include target antigen reduction, target messenger reduction and inhibition of transformed cell proliferation or viral replication. However, one should expect oligonucleotides to exhibit pleiotropic behaviour, as do all other drugs. In an animal oligonucleotides will necessarily bind to and dissociate from all macromolecules encountered in the blood, in tissues, on cell surfaces and within cellular compartments. Contrary to expectations, oligonucleotides designed to be complementary to certain transcripts have sometimes been found moderately effective in cell-free extracts, more effective in cell culture and most effective in animal models. If greater potency against standard endpoints is reported in mouse models than was observed in cell culture, critical examination must consider alternate modes of action in animals that may not apply in cell culture. This counterintuitive paradox will be examined, based on studies of Ha-ras expression in bladder cancer, Ki-ras expression in pancreatic cancer, erbB2 expression in ovarian cancer and c-myc expression in B cell lymphoma.

Animals↗
PubMed1997

Hepatic distribution and clearance of antisense oligonucleotides in the isolated perfused rat liver.

PURPOSE: This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides. METHODS: The disposition of backbone-modified antisense oligonucleotides [phosphorothioate (PS) and methylphosphonate (MP)] of the same base-length and sequence (5'-TAC-GCC-AAC-AGC-TCC-3'), complementary to the codon 12 activating mutation of Ki-ras, was investigated in the isolated perfused rat liver. Livers were perfused for 2 hr: perfusate and bile concentrations were analyzed by HPLC. Hepatocellular distribution was examined by measuring the amount of radiolabeled PS oligonucleotide associated with hepatocytes and Kupffer cells. Protein binding of the PS and MP oligonucleotides was determined in rat serum by ultrafiltration. RESULTS: MP oligonucleotide perfusate concentrations remained constant during the 2-hour perfusion. In contrast, PS oligonucleotide was eliminated slowly by the isolated perfused liver [CI = 1.05 +/- 0.21 mL/min; extraction ratio = 0.06 +/- 0.01]. Uptake of PS oligonucleotide by Kupffer cells appeared to exceed uptake by hepatocytes, based on standard cell separation techniques as well as confocal microscopy. The degree of protein binding in rat serum was greater for the PS oligonucleotide (79.9 +/- 2.2%) than for the MP oligonucleotide (53.0 +/- 4.7%). CONCLUSIONS: Backbone modifications significantly-influence the hepatic clearance of oligonucleotides. Uncharged MP oligonucleotides are not extracted by the isolated perfused rat liver, whereas the charged PS oligonucleotide is processed more readily.

Animals↗
PubMed1997

Rapid measurement of modified oligonucleotide levels in plasma samples with a fluorophore specific for single-stranded DNA.

Animal studies of therapeutic oligonucleotides require measurement of circulating levels of oligonucleotides by multistep, time-consuming methods. In contrast, addition of a single-stranded DNA binding fluorophore, OliGreen, to oligonucleotides in plasma samples allowed rapid quantitation. Dose-response curves were measured for five different oligonucleotide analogs added to plasma or serum. Phosphorothioate or 3'-amino phosphodiester oligodeoxynucleotides in calf serum reliably exhibited linear, dose-dependent fluorescence at 15-500 nM. The assay was equally sensitive in human and mouse plasma, with a heterogeneous variety of sequences. Oligonucleotides shorter than 10 nucleotides yielded substantially reduced fluorescence. In contrast, 2'-O-methyl oligoribonucleotides, DNA methylphosphonates, and peptide nucleic acids demonstrated little or no fluorescence with OliGreen. Following intravenous injection of a phosphorothioate pentadecamer into mice, fluorescence measurements of plasma phosphorothioate levels displayed a dose-dependent, biexponential decline over a 90 min period. Chronic infusion at 2.5 nmol/hour into mice yielded plasma oligonucleotide values equivalent to 0.1 microM, a value reflecting the contributions of intact and partially degraded strands. Tumor-bearing mouse plasma evidenced high fluorescence values in the absence of oligonucleotide administration, presumably because of elevated intrinsic plasma DNA fragments. Although limited in its ability to differentiate intact from partially degraded strands, OliGreen fluorescence provides a simple, rapid, and sensitive method for measuring circulating levels of phosphorothioate or phosphodiester oligonucleotides in healthy animals or humans.

Animals↗
PubMed1997

Comparative pharmacokinetics, tissue distribution, and tumor accumulation of phosphorothioate, phosphorodithioate, and methylphosphonate oligonucleotides in nude mice.

The goals of this study were to systematically compare the pharmacokinetics and tissue distribution of phosphorothioate (PS), methylphosphonate (MP), and phosphorodithioate (PS2) oligonucleotide analogs; 15-mers of sequence d-TAC GCC AAC AGC TCC (5'-3') complementary to the AUG region of K-ras were radiolabeled with carbon-14. Oligomers were administered as a single dose in the tail vein of nude mice harboring a K-ras-dependent human pancreatic tumor (CFPAC1). The kinetics of PS, PS2, and MP oligomer availability in the bloodstream was followed. Concentration versus time profiles for all oligomers were biphasic, indicative of a two-compartment model. A rapid distribution phase with t1/2 alpha values of 1 minute or less and an elimination phase with average t1/2 beta values of 24-35 minutes were observed. Volumes of distribution (Vd) were 3.2, 4.8, and 6.3 ml for PS2, MP, and PS, respectively, in comparison to 3.6 ml for sucrose, a fluid-phase marker. Relative tissue drug levels obtained at 1 and 24 hours after administration were kidney > liver > spleen > tumor > muscle. Total kidney and liver oligonucleotide accumulation was approximately 7%-15% of the initial dose, with tumor accumulating 2%-3%. Intact compound was recovered from all tissues, including tumor, as assessed by high-pressure reversed-phase HPLC coupled to radiometric detection. Integrity of the oligonucleotides ranged from 73% in blood to 43%-46% in kidney and liver. Kidney and liver appear to be the primary sites of metabolism. These results demonstrate widespread tissue availability of these compounds and suggest their development as potential antitumor agents.

Animals↗