Radionuclide-peptide nucleic acid in diagnosis and treatment of pancreatic cancer.
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Animals↗
Biomedical subjects
Eric Wickstrom
Publications and source records attributed to Eric Wickstrom.
Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA.
Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells.
Acetylcysteine↗
Correspondence re: S. Heckl et al., Intracellular visualization of prostate cancer using magnetic resonance imaging. Cancer Res 2003;63:4766-72.
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Animals↗
Transposon Tn7 protein TnsD binding to Escherichia coli attTn7 DNA and its eukaryotic orthologs.
Transposon Tn7 inserts itself into the attTn7 target DNA sequence at the 3' end of the Escherichia coli glmS gene with high specificity and efficiency. This site in the E. coli genome displays amino acid conservation and nucleotide similarity with orthologous sequences in Archaebacteria and eukaryotes. On the basis of the high degree of nucleotide similarity, particularly with eukaryotes, we examined the interactions of a set of 20-bp duplex DNA sequences with the Tn7 protein TnsD. The protein was overexpressed in the IPTG-inducible vector pET14b-TnsD in E. coli BL21(DE3)-RIL-Codon-Plus, and purified by nickel chelation and ion exchange chromatography. Changes in the conformation of DNA duplexes upon interaction with TnsD were monitored by circular dichroism (CD) spectroscopy. TnsD binding to and dissociation from immobilized DNA duplexes were monitored by total internal reflectance (TIR). CD and TIR results were analyzed to calculate k(on), k(off), and K(D) values. The 20-bp DNA duplex corresponding to attTn7 at the 3' end of E. coli glmS displayed strong affinity for TnsD protein, with K(D) approximately 20 nM. Eukaryotic orthologs of attTn7 from yeast and mammalian GFPT1 displayed lower affinity, with K(D) approximately 500 nM. Mutant DNA sequences with a single central mismatch did not display any detectable interaction with TnsD. The physical studies validate our biological observation of Tn7 transposition into a plasmid containing the mammalian attTn7 ortholog sequence [Cleaver, S. H., and Wickstrom, E. (2000) Gene 254, 37-44], and suggest that 1-2 amino acid substitutions in TnsD might be sufficient to permit binding to mammalian orthologs that is as strong as wild-type TnsD binding to attTn7.
Animals↗
Downregulation of gene expression with negatively charged peptide nucleic acids (PNAs) in zebrafish embryos.
We found that negatively charged, highly soluble PNA analogs with alternating phosphonates (HypNA-pPNAs) are effective and specific antisense agents in zebrafish embryos, showing comparable potency and greater specificity against chordin, ntl and uroD. In addition, we successfully phenocopied a dharma mutant that had not been found susceptible to MO knockdown. Both MO and HypNA-pPNAs against a tumor suppressor gene induced comparable upregulation of p53, illustrating similar effects on transcription profiles. HypNA-pPNAs are therefore a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes in zebrafish or validating newly identified orthologs, and perhaps for reverse genetic studies in other organisms.
Animals↗
Frank Stinchfield Award. Titanium surface with biologic activity against infection.
Despite immense improvements, periprosthetic infection continues to compromise the result of otherwise successful joint arthroplasty. There are various limitations in the treatment of periprosthetic infection, the most important of which is the inability to deliver antibiotics to the local tissue without the need for intravenous administration. We have developed a novel route to covalently tether vancomycin to a metal (titanium) surface, which showed effective bactericidal activity because of a vancomycin coupling. The chemistry of tethering does not affect the biological activity of the biofactors that are attached to the metal surface. This technology holds great promise for the manufacturing of "smart" implants that can be self protective against periprosthetic infection, or can be used for the treatment of periprosthetic infections when they occur.
Awards and Prizes↗
PET imaging of oncogene overexpression using 64Cu-vasoactive intestinal peptide (VIP) analog: comparison with 99mTc-VIP analog.
UNLABELLED: The purpose of this study was to assess the feasibility of PET imaging of oncogene VPAC1 receptors overexpressed in human breast cancer cells. METHODS: Vasoactive intestinal peptide (VIP) analog (TP3982) was synthesized to harbor a carboxy-terminus lysine (Lys) residue separated from VIP-asparagine (Asn(28)) by 4-aminobutyric acid (Aba) as a spacer. Lys was derivatized with diaminopropionic acid coupled to a pair of dibenzoylthioglycolic acid residues as protecting groups. The analog was labeled with (64)Cu at pH 9 ((64)Cu-TP3982) and (99m)Tc at pH 12 ((99m)Tc-TP3982). (99m)Tc-TP3982 and VIP derivatized with Aba-GAGG and labeled with (99m)Tc ((99m)Tc-TP3654) were used as reference agents. Smooth muscle relaxivity assays performed with each derivative and compared with unaltered VIP(28) demonstrated functional integrity. In vitro stability of (64)Cu-TP3982 was determined by challenging the complex with 100-mol excess of diethylenetriaminepentaacetic acid (DTPA), human serum albumin (HSA), and cysteine. In vivo stability was determined in urine and serum for up to 24 h. The mass of the Cu-TP3982 complex was determined by mass spectrometry. Human T47D breast tumor xenografts were grown in athymic nude mice. Planar scintigraphic imaging was performed at 4 and 24 h after the intravenous administration of (99m)Tc-TP3982 and (99m)Tc-TP3654 and PET imaging was performed using a small animal MOSAIC PET scanner, also at 4 and 24 h after injection of (64)Cu-TP3982. Tissue-distribution studies were also performed. In a separate experiment, receptors were blocked by intravenous injection of authentic VIP(28) 30 min before the administration of (64)Cu-TP3982 and tissue distribution was examined. RESULTS: (64)Cu-TP3982 labeling yields were 98% +/- 1.2% and those for (99m)Tc-TP3982 and (99m)Tc-TP3654 were 98.2% +/- 1.1% and 97% +/- 1.6%, respectively. The biologic activity of both VIP analogs was uncompromised. When (64)Cu-TP3982 was challenged with 100-mol excess of DTPA, HSA, or cysteine, >98% radioactivity remained as (64)Cu-TP3982. In vivo, >98% of (64)Cu circulating in plasma remained as (64)Cu-TP3982. Of the (64)Cu excreted in urine 4, 20, and 24 h after injection, >98%, 89.9% +/- 0.9%, and 85% +/- 3%, respectively, were bound to TP3982. The mass of Cu-TP3982 as determined by surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) was 4,049.7 Da. Four hours after receptor blocking with VIP(28), there was a significant reduction in uptake of all tissues except in the liver. With (64)Cu-TP3982, the 4-h postinjection tumor uptake was 10.8 +/- 2.1 %ID/g versus 0.5 +/- 0.02 %ID/g and 0.24 +/- 0.08 %ID/g for (99m)Tc-TP3982 and (99m)Tc-TP3654, respectively. Twenty-four hours after injection, the corresponding numbers were 17 +/- 0.7 %ID/g, 0.77 +/- 0.1 %ID/g, and 0.23 +/- 0.1 %ID/g. The severalfold greater uptake (21.2-74) of (64)Cu-TP3982 is attributable to the in vivo stability of the agent. CONCLUSION: The results suggest that the uncompromised biologic activity and the significantly greater tumor uptake of (64)Cu-TP3982, combined with the high sensitivity and enhanced resolution of PET imaging, make (64)Cu-TP3982 highly desirable for further studies in PET imaging of oncogene receptors overexpressed in breast and other types of cancers.
Animals↗
External imaging of CCND1 cancer gene activity in experimental human breast cancer xenografts with 99mTc-peptide-peptide nucleic acid-peptide chimeras.
UNLABELLED: Detection of a new or recurrent breast cancer lesion relies on physical examination and imaging studies, primarily mammography, followed by histopathologic evaluation of biopsy tissue for morphologic confirmation. Approximately 66%-85% of detected lesions are not malignant. Therefore, biopsies are unnecessary for at least two thirds of patients. Human estrogen receptor-positive breast cancer cells typically display an elevated level of cyclin D1 protein because of the overexpression of CCND1 messenger RNA (mRNA) and an elevated level of insulin-like growth factor 1 (IGF1) receptor (IGF1R) because of the overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of CCND1 peptide nucleic acid (PNA) hybridization probes with a (99m)Tc-chelating peptide on the N terminus and an IGF1 peptide loop on the C terminus could detect CCND1 mRNA in human MCF7 breast cancer xenografts in nude mice from outside the body. METHODS: We prepared the CCND1 probes as well as mismatched controls by solid-phase synthesis. We used fluorescence microscopy to detect the cellular uptake of fluoresceinyl probes and quantitative reverse transcription-polymerase chain reaction to detect the hybridization of probes to mRNA. We imaged (99m)Tc-probes in MCF7 xenografts scintigraphically and measured distribution by scintillation counting of dissected tissues. RESULTS: IGF1R-overexpressing MCF7 cells internalized the fluorescein-chelator-CCND1 PNA-IGF1 peptide but not the mismatched control peptide. The chelator-CCND1 PNA-IGF1 peptide but not the control peptide lowered the level of cyclin D1 protein in IGF1R-overexpressing MCF7 xenografts in nude mice after intratumoral injection. IGF1R-overexpressing MCF7 xenografts in nude mice were visualized at 4, 12, and 24 h after tail vein administration of the (99m)Tc-CCND1 antisense probe but not the control probe. (99m)Tc-chimeras were distributed normally in the kidneys, liver, tumors, and other tissues. CONCLUSION: Cancer gene activity can be detected from outside the body by probing with radionuclide-chelator-PNA-peptide chimeras.
Animals↗
Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction.
BACKGROUND: The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH), in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2. RESULTS: Using polony technology, Panc-1 was determined to possess only one copy of p53, which possessed a mutation in codon 273, and two copies of K-ras2, one wildtype and one with a mutation in codon 12. To further demonstrate the general approach of this method, polonies were also used to detect K-ras2 mutations in the pancreatic cell lines, AsPc-1 and CAPAN-1. CONCLUSIONS: In conclusion, we have developed an assay that can detect mutations in hotspots of p53 and K-ras2 as well as diagnose LOH in these same genes.
Cell Line, Tumor↗
Targeted gene knockdown in zebrafish using negatively charged peptide nucleic acid mimics.
Negatively charged homo-oligomers of alternating trans-4-hydroxy-L-proline/phosphonate polyamides with DNA bases (HypNA-pPNA) display excellent hybridization properties toward DNA and RNA, while preserving the mismatch discrimination, nuclease resistance, and protease resistance of peptide nucleic acids (PNAs). Similar properties are associated with morpholino phosphorodiamidate (MO) DNA mimics, which have been used in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. We evaluated mixed sequence HypNA-pPNAs as an alternative to MOs, and found that even a single central DNA mismatch lowered the HypNA-pPNA melting temperature by 16 degrees C. We then observed that the melting temperatures of HypNA-pPNA 18-mers hybridized to RNA 25-mers were comparable to the melting temperatures of MO 25-mers, and that two HypNA-pPNA mismatches lowered the melting temperature with RNA by 18 degrees C. 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. Finally we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. HypNA-pPNAs designed to inhibit translation of specific zebrafish RNA targets thus demonstrated stringent hybridization properties, relative to DNA and MO oligomers, and present a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes.
Animals↗
Imaging oncogene expression.
In 2003, approximately 39,800 women in the US will die from breast cancer. Mammography and physical examination miss up to 40% of early breast cancers. Moreover, if an abnormality is found, an invasive diagnostic procedure must still be performed to determine if the breast contains atypia or cancer, even though approximately 85% of abnormalities are benign. Scintigraphic imaging of gene expression in vivo by noninvasive means could direct physicians to appropriate targets for intervention at the onset of disease and thereby significantly impact patient management. Until now, no method has been available to image specific overexpressed oncogene mRNAs in vivo by scintigraphic imaging. We hypothesize that gamma-emitting Tc-99m-PNA-peptides can be taken up by human ER+ and ER- breast cancer xenografts, hybridize to complementary mRNA targets in those cells, and concentrate sufficiently in tumor tissue to allow noninvasive imaging of oncogene overexpression. To prepare the probes, peptide analogs of insulin-like growth factor 1 (IGF1) were extended from a solid support by Fmoc coupling. Peptide nucleic acid (PNA) dodecamers antisense to CCND1 and MYC mRNAs were then extended from the N-terminus of IGF1, followed by a chelator peptide, using Fmoc coupling for all residues. The cysteine thiols were cyclized on the solid support, either before or after PNA extension. This simplified synthetic approach allows preparation of a variety of multipeptide disulfide-bridged PNA chimeras. A chelating peptide-PNA chimera antisense to MYC mRNA was then labeled efficiently with Tc-99m, yielding a single product. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver and kidneys, with appreciable levels in tumors. This result enables testing of Tc-99m-peptide-PNA probes to image gene expression in tumors.
Animals↗
Continuous solid-phase synthesis and disulfide cyclization of peptide-PNA-peptide chimeras.
Chelator peptides were extended from the N-terminus of peptide nucleic acid (PNA) dodecamers, which in turn were extended from the N-termini of disulfide-bridged peptide ligand analogues, using Fmoc coupling for all residues. The cysteine thiols were cyclized on a solid support, either before or after PNA extension. This simplified synthetic approach might allow preparation of a variety of multipeptide disulfide-bridged PNA chimeras. [structure: see text]
Amino Acid Sequence↗
Stereospecific Grignard-Activated Solid Phase Synthesis of DNA Methylphosphonate Dimers.
Stereoregular R(p) or S(p) DNA methylphosphonate dimers have been synthesized on a solid phase support. A deprotected 5'-hydroxyl-N(2)-isobutanoyldeoxyguanosine 3'-O-succinate coupled to high-loaded polyethylene glycol (PEG) coated polystyrene beads (HLP) was activated with a Grignard reagent, t-BuMgCl. After activation was complete, a pure diastereoisomer of 5'-(dimethoxytrityl) N-benzoyldeoxynucleoside 3'-(p-nitrophenyl methylphosphonate) p-nitrophenyl ester (R(p) or S(p)) was added. Coupling of the activated 5'-hydroxyl to the 3'-methylphosphonate ensued, releasing nitrophenol, yielding the R(p) or S(p) dimer, respectively. The dimers were then cleaved from the solid support, deprotected, and purified, yielding methylphosphonate DNA dimers of defined stereochemistry.
Journal Article↗
Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates.
Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.
Binding Sites↗