TY - JOUR T1 - In Vitro Investigations of Tumor Targeting with <sup>99m</sup>Tc-Labeled Antisense DNA JF - Journal of Nuclear Medicine JO - J Nucl Med SP - 1660 LP - 1669 VL - 42 IS - 11 AU - Yu-Min Zhang AU - Yi Wang AU - Ning Liu AU - Zhi-Hong Zhu AU - Mary Rusckowski AU - Donald J. Hnatowich Y1 - 2001/11/01 UR - http://jnm.snmjournals.org/content/42/11/1660.abstract N2 - One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with γ- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an antisense mechanism. Methods: An 18mer uniform phosphorothioate DNA antisense to the messenger RNA (mRNA) of the type I regulatory subunit α of cyclic adenosine monophosphate-dependent protein kinase A (RIα) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. Results: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of 99mTc-MAG3-DNA was lower than that of 35S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of 35S- or 99mTc-labeled antisense versus sense DNA, an increased accumulation of 99mTc-antisense DNA in another RIα-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of 99mTc-antisense DNA with increasing dosage of antisense DNA. Higher than expected cellular accumulations of about 105 antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5′-triphosphate into RNA in cells exposed to the antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the antisense DNA but not to the control DNA. Conclusion: This evidence suggests tumor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates. ER -