Antisense targeting of p-glycoprotein expression in tissue culture.
Radiolabeled DNA and other oligomers are now under investigation for antisense targeting of a variety of messenger RNA (mRNA). Multidrug resistance (MDR) is detectable as P-glycoprotein (Pgp) expression in most cells and is often elevated in tumor cells, especially those exposed to chemotherapeutic drugs. Radiolabeled antisense DNA has not previously been considered for the targeting of mdr1 mRNA, the product of the mdr1 gene controlling Pgp expression of MDR.


METHODS
A 20-mer uniform phosphorothioate DNA, described elsewhere as targeting the AUG start codon of mdr1 mRNA, was used naked along with the sense phosphorothioate DNA control. The 3 cell lines were KB-G2, an epidermal carcinoma cell line that had been transfected to overexpress mdr1 mRNA (i.e., Pgp++) compared with its parent (Pgp+) KB-31, and TCO-1, a thyroid carcinoma cell line also reported to be Pgp++. The relative expression of mdr1 mRNA in these 3 cell lines was confirmed elsewhere by reverse transcriptase polymerase chain reaction. As a marker of Pgp expression, the uptake of (99m)Tc-sestamibi was measured in the 3 cell lines after 20 h of incubation with different concentrations of both antisense and sense DNA. Both DNAs were radiolabeled with (99m)Tc via mercaptoacetyltriglycine, and cellular uptake was measured after 24 h of incubation.


RESULTS
In the case of the sense DNA, the ratio of sestamibi uptake in cells incubated with the DNA to those not exposed to the DNA was unaffected regardless of cell line and regardless of DNA concentration. In contrast, this ratio was significantly higher in both the KB-G2 and TCO-1 cells when incubated with antisense DNA at concentrations greater than about 25 nmol/L (i.e., 150 ng/mL). Only in the KB-31 cells was the sestamibi accumulation unaffected by incubation with the antisense DNA. Thus, the antisense DNA was interfering with Pgp expression to a measurable extent in both Pgp++ cells, but not the Pgp+ cells. This behavior is almost certainly due to antisense targeting of mdr1 mRNA by the antisense DNA since the sense control DNA had no effect. A significant increased accumulation of (99m)Tc-antisense versus (99m)Tc-sense DNA was observed in all 3 cell lines. In all cases, this difference was greatest at the lowest DNA concentrations and decreased with increasing concentration as expected for specific binding. In the KB-G2 cells, cellular accumulation of (99m)Tc-antisense DNA was strikingly high at the lowest concentration at 54%, compared with 22% for (99m)Tc-sense DNA. These accumulations therefore probably reflect the higher mRNA target concentration in the MDR++ cells than in the MDR+ cells and the higher specific binding of (99m)Tc-antisense DNA than nonspecific binding of (99m)Tc-sense DNA.


CONCLUSIONS
Further evidence was obtained suggesting that an antisense mechanism is responsible for the accumulation of (99m)Tc-oligomers in cells in culture. Finally, whereas evidence of in vitro targeting is not necessarily evidence of in vivo targeting, our results do suggest that radiolabeled antisense DNA against the mdr1 mRNA may potentially be useful for antisense imaging of MDR in cancer.
Nakamura
Kayoko
K
Department of Radiology, Keio University School of Medicine, Tokyo, Japan. kumako@sc.itc.keio.ac.jp
Kubo
Atsushi
A
Hnatowich
Donald J
DJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism*; D002277:Carcinoma / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage / Q000493:pharmacokinetics*; D020869:Gene Expression Profiling / Q000379:methods; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D011877:Radionuclide Imaging; D013667:Technetium / Q000493:pharmacokinetics*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D013667:Technetium; D004247:DNA