In vitro investigations of tumor targeting with 99(m)Tc-labeled antisense DNA

Ym. Zhang et al., In vitro investigations of tumor targeting with 99(m)Tc-labeled antisense DNA, J NUCL MED, 42(11), 2001, pp. 1660-1669
Citations number
Categorie Soggetti
Radiology ,Nuclear Medicine & Imaging","Medical Research Diagnosis & Treatment
Journal title
ISSN journal
0161-5505 → ACNP
Year of publication
1660 - 1669
SICI code
One objective of this investigation was to determine whether chemical modif ications of oligonucleotices to permit radiolabeling with gamma- or positro n emitters interferes with hybridization and target cell accumulation. A se cond objective was to establish to a reasonable extent whether cellular acc umulation 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 a of cyclic adenosin e monophosphate-dependent protein kinase A (RI alpha) was conjugated with t he N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MA G3) through a primary amine/linker and investigated in vitro in cell cultur e. Results: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were iden tical. Melting temperatures were also identical for native DNA, amine/linke r-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modificati ons had no detectable influence on hybridization. However, cellular accumul ation of Tc-99m-MAG3-DNA was lower than that of S-35-MAG3-DNA, suggesting t hat chemical modifications can have an important influence on cellular accu mulation. In tissue culture studies of ACHN tumor cells (a human renal aden ocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of S-35- or Tc-99m-labeled antisense versus sense DN A, an increased accumulation of Tc-99m-antisense DNA in another RI alpha -p ositive tumor cell line (LS174T) but not in a murine transfected control ce ll line (HC-2), and the disappearance of the increased cellular accumulatio n of Tc-99m-antisense DNA with increasing dosage of antisense DNA. Higher t han expected cellular accumulations of about 10(5) antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA produc tion by the presence of the antisense DNA. In support of this suggestion, w e observed, first, an increased incorporation of uridine-5'-triphosphate in to 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 ant isense DNA but not to the control DNA. Conclusion: This evidence suggests t umor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcrip tion rate may be an important determinant of tumor counting rates.