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Biomedical subjects

A P Smyth

Publications and source records attributed to A P Smyth.

7 recordsLinked to original sources
PubMed1997

Sensitization of multidrug-resistant human leukemia cells with MDR1-targeted antisense and inhibition of drug-mediated MDR1 induction.

Increased expression of MDR1 is strongly implicated in the appearance of chemotherapeutic drug resistance in cancer, especially hematological malignancies. We therefore examined the potential of antisense oligonucleotides to inhibit MDR1 and restore sensitivity to drug-resistant human lymphoblastic cells (CCRF-CEM). Treatment with two different phosphorothioate-modified antisense sequences as well as a DNA-RNA hybrid sequence resulted in a 30 to 45% decrease in MDR1 expression as determined by staining with the monoclonal antibody MRK16 followed by flowcytometry (FCM) analysis. Further, inhibition of MDR1 expression persisted for 3 days after removal of oligonucleotides. Increased accumulation of rhodamine 123 and nearly a three-fold sensitization of cells to vincristine paralleled the reduction in staining with MRK16. Reversed or scrambled control sequences had no effect in any of the assays. During the course of these studies, we observed a 25 to 75% increase in MRK16 staining of cells treated with the chemotherapeutic agents daunorubicin and vincristine as well as by the resistance reversal agents verapamil and cyclosporin. Treatment of cells with antisense oligonucleotides prior to exposure to daunorubicin or cyclosporin reduced the increase in MRK16 staining. These results indicate that antisense targeted to MDR1 can sensitize drug-resistant leukemia cells and suggest that antisense treatment may prevent the emergence of MDR1-mediated drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Antisense oligonucleotides inhibit vascular endothelial growth factor/vascular permeability factor expression in normal human epidermal keratinocytes.

In psoriatic lesions, epidermal keratinocytes overexpress vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and transforming growth factor alpha (TGF-alpha). TGF-alpha has been shown to induce VEGF/VPF in normal human epidermal keratinocytes in vitro. By using a 19-mer antisense phosphorothioate oligodeoxynucleotide (PS-ODN) complementary to bases 6-24 relative to the translational start site of the VEGF/VPF mRNA, the control sense and mismatched PS-ODNs, we examined modulation of VEGF/VPF induction by TGF-alpha in vitro. Normal human epidermal keratinocytes were treated with PS-ODNs and Lipofectin for 8 h prior to the addition of TGF-alpha. Inhibition was assayed at the level of secreted protein by capture ELISA and mRNA expression was assayed by Northern blot analysis. The anti-sense PS-ODN was capable of inhibiting VEGF/VPF RNA and protein to near-basal levels. This inhibition was concentration dependent. No effect was observed with the sense or mismatch control PS-ODNs. These studies suggest that antisense oligonucleotide technology may be a potential therapy for the inhibition of angiogenesis associated with certain skin disorders such as psoriasis.

Down-Regulation↗
PubMed1993

A human genome YAC library in a selectable high-copy-number vector.

An experimental yeast artificial chromosome (YAC) library consisting of one genome equivalent of human DNA was prepared in a selectable high-copy-number (hcn) YAC vector. Screening for unique loci was accomplished by PCR of successively smaller DNA pools and by hybridization to high-density microcolony blots. Inserts averaged 200 kb in size, but several YACs with inserts averaging about 650 kb were obtained when polyamines were added prior to yeast transformation. YACs were identified for 17 out of 29 sequence-tagged sites (STS) screened by a PCR-based approach. All YACs in the size range of 100-600 kb that were examined could be obtained at significantly elevated copy numbers following growth of the clones in methotrexate/sulfanilamide/thymidine-supplemented medium. The hcn YACs could also be selected during growth in microtiter dishes, and the resulting clones were used to prepare high-density microcolony DNA blots for hybridization with radiolabeled PCR products. DNA pools for the PCR-based screening of this experimental library are available to investigators interested in applications of hcn YACs.

Base Sequence↗
PubMed1993

Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination.

We have developed a pair of vectors for exchanging yeast artificial chromosome (YAC) arms by targeted homologous recombination. These conversion vectors allow the introduction of copy-number control elements into YACs constructed with pYAC4 or related vectors. YACs modified in this way provide an enriched source of DNA for genetic or biochemical studies. A LYS2 gene on the conversion vector provides a genetic selection for the modified YACs after transformation with appropriately prepared vector. A background of Lys+ clones that do not contain modified YACs is also present. However, clones with converted YACs can be distinguished from this background by counter-screening for loss of the original p YAC4 TRP1 arm (Trp- phenotype). The elimination of yeast replication origins (ARS elements) from the conversion vectors increased the frequency of Lys+ Trp- clones, but resulted in weaker amplification. Several YACs have been converted with these vectors, and the fate of the transformed DNA and of the resident YAC DNA has been systematically investigated.

Chromosomes, Fungal↗
PubMed1991

Mutations affecting the activity of urokinase-type plasminogen activator.

Mutagenesis throughout the single-chain urokinase-type plasminogen activator (scu-PA) cDNA molecule, followed by expression of the mutant genes and secretion of the resulting mutant proteins from yeast, has been used to determine the amino acid residues important for activity of scu-PA molecules. Twelve out of 13 colonies secreting variant scu-PA molecules with decreased ability to form a zone of fibrinolysis had mutant genes with a single codon alteration in the serine protease encoding domain (B-chain). Many of these changes are of highly conserved residues in the serine proteases and are consequently of considerable interest. A model three-dimensional structure of the protease domain of urokinase was used to explain the basis for the effects of these down mutations. The model showed that the strongest down mutations result from either interference of the mutated side chain with substrate binding at the active site or the introduction of bulky or charged groups at structurally sensitive internal positions in the molecule. Attempts to find second site revertants of five down mutants, altered either at the plasmin activation site or near the serine at the active site, only resulted in same-site revertants, with the original or closely related amino acids restored.

Base Sequence↗
PubMed1990

Amplification of large artificial chromosomes.

Yeast artificial chromosome cloning is an attractive technology for genomic mapping studies because very large DNA segments can be readily propagated. However, detailed analyses often require the extensive application of blotting-hybridization techniques because artificial chromosomes are normally present at only one copy per haploid genome. We have developed a cloning vector and host strain that alleviate this problem by permitting copy number amplification of artificial chromosomes. The vector includes a conditional centromere that can be turned on or off by changing the carbon source. Strong selective pressure for extra copies of the artificial chromosome can be applied by selecting for the expression of a heterologous thymidine kinase gene. When this system was used, artificial chromosomes ranging from about 100 to 600 kilobases in size were readily amplified 10- to 20-fold. The selective conditions did not induce obvious rearrangements in any of the clones tested. Reactivation of the centromere in amplified artificial chromosome clones resulted in stable maintenance of an elevated copy number for 20 generations. Applications of copy number control to various aspects of artificial chromosome analysis are addressed.

Base Sequence↗