Antisense RNA downregulation of bcl-xL protein expression.

摘要

Chapter 1 provides an overview of antisense theory. One of the goals of this work was to determine the properties of the anti-apoptotic bcl-xL protein. That was done by RNA antisense downregulation of the level of expression of bcl-xL protein in the DU145 and LNCaP prostate cancer cells as described in Chapter 2. It was hypothesized that since bcl-xL is an antiapoptotic protein, decrease of its levels of expression would result in an induction of apoptosis as well as an increase in chemosensitivity of the transfected cells. In this work, the 433 base pair 5′-terminus of the bcl-xL cDNA was cloned in the antisense orientation and stably transfected into prostate cancer cells. Phenotypic changes included an increase in doubling time and appearance of polyploid phenotype. The downregulation of bcl-xL also led to desensitization of the cells to a number of cytotoxic agents. Re-inducing the bcl-xL protein in these transfectants reversed the observed phenotypes. Also, significant upregulation of a member of another antiapoptotic family, cIAP-1 protein, was observed.; The other goals of this thesis were to optimize the intracellular delivery, and to reduce the non-sequence specificity of antisense oligonucleotides. The first part of Chapter 3 describes the successful use of a porphyrin as delivery vehicle, which is expected to form stable complexes with oligodeoxynucleotides. To evaluate delivery, an antisense 20-mer phosphorothioate oligonucleotide targeted to the 3′-UTR of the PKC-α mRNA, was complexed with porphyrin. The levels of expression of PKC-α protein and its mRNA were reduced by approximately 80% in T24 bladder carcinoma cells.; The formation of higher order structures by an 18-mer oligonucleotide that contain four continuous guanosine bases was demonstrated in the second part of Chapter 3. A phosphorothioate antisense c-myb oligodeoxyribonucleotide was employed in the study. Although not observable by conventionally employed techniques such as PAGE and dimethyl sulfate protection, the formation of higher order structures was revealed by several other techniques. These included capillary gel electrophoresis, which demonstrated the presence of molecules with increased retention time compared to the monomer; magnetic circular dichroism spectroscopy, which demonstrated a band at 290 nm, a characteristic of antiparallel tetraplexes; and fluorescence energy transfer measurements.