Inhibition of Potentially Anti-Apoptotic Proteins by Antisense Protein Kinase C-α (Isis 3521) and Antisense bcl-2 (G3139) Phosphorothioate Oligodeoxynucleotides: Relationship to the Decreased Viability of T24 Bladder and PC3 Prostate Cancer Cells

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Abstract
Isis 3521 and G3139 are 20- and 18-mer phosphorothioate oligonucleotides, respectively, targeted to the protein kinase C (PKC)-α and bcl-2 mRNAs. Treatment of T24 bladder and PC3 prostate carcinoma cells with full-length and 3′-truncation mutants of Isis 3521 causes down-regulation of PKC-α protein and mRNA. However, at the level of a 15-mer and shorter, down-regulation of mRNA expression is no longer observed. Further, no diminution in cellular viability, as measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay, in response to increasing concentrations of paclitaxel, can be observed for these shorter oligomers. These observations not only indicate that PKC-α protein expression can be down-regulated by both RNase H-dependent and -independent mechanisms but also that down-regulation of PKC-α is insufficient by itself to “chemosensitize” cells. G3139, which down-regulates bcl-2 protein and mRNA expression, also down-regulates PKC-α protein and mRNA expression but not that of PKC-βI, -ε, or -ζ. However, the down-regulation of PKC-α and bcl-2 are not linked. When the carrier Eufectin 5 is employed, only bcl-2 is down-regulated in both T24 and PC3 cells at 50 nM oligonucleotide concentration. At 100 nM, both bcl-2 and PKC-α expression are down-regulated, and only at this concentration can “chemosensitization” to paclitaxel and carboplatin be observed. In contrast, the down-regulation of bcl-2 seems to be linked with that of RelA (p65). However, this too is also not sufficient for chemosensitization, even though it leads to the loss of expression of genes under the putative control of nuclear factor-κB and to detachment of the cells from plastic surfaces. These results underscore the complexity of the intracellular requirements for the initiation of chemosensitization to anti-neoplastic agents.

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