Evaluating the Specificity of Antisense Oligonucleotide Conjugates: A DNA ARRAY ANALYSIS

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (approximately 2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution

Analysis of Aurora kinase A expression in CD34+ blast cells isolated from patients with myelodysplastic syndromes and acute myeloid leukemia

Aurora kinase A, also known as aurora A, is a serine/threonine kinase that plays critical roles in mitosis entry, chromosome alignment, segregation, and cytokinesis. Overexpression of aurora A has been observed in many solid tumors and some hematopoietic neoplasms, but little is known about its expression in myeloid diseases. Because cytogenetic abnormalities play an essential role in the pathogenesis of myeloid malignancies, we hypothesized that aurora A deregulation may be involved in myelodysplastic syndromes and acute myeloid leukemia and contribute to the chromosomal instability observed in these diseases. We assessed aurora A mRNA levels in CD34+ bone marrow blasts from nine patients with acute myeloid leukemia, 20 patients with myelodysplastic syndromes, and five normal patients serving as controls. CD34+ blasts were isolated from bone marrow aspirate specimens using magnetic activated cell separation technology. RNA was extracted from purified CD34+ cells, and quantitative real-time reverse transcriptase polymerase chain reaction for aurora A was performed. Immunocytochemical analyses for total aurora A, phosphorylated aurora A, Ki-67, and activated caspase 3 were performed on cytospin slides made from purified CD34+ cells in myelodysplastic syndrome patients using standard methods. Aurora A mRNA and protein levels were correlated, as was aurora A mRNA level, with blast counts, cytogenetic abnormalities, and International Prognostic Scoring System score. We found that CD34+ cells in myelodysplastic syndromes and acute myeloid leukemia expressed aurora A at significantly higher levels (P = 0.01 and P = 0.01, respectively) than normal CD34+ cells. Aurora A mRNA levels correlated with total and phosphorylated protein levels (P = 0.0002 and P = 0.02, respectively). No significant correlation was found between aurora A mRNA level and blast count, blast viability, cytogenetic abnormalities, or the International Prognostic Scoring System score in patients with myelodysplastic syndromes. We conclude that aurora A is up-regulated in CD34+ blasts from myeloid neoplasms