Initial mechanistic studies of antisense targeting in cells.
The continued development of antisense targeting will require a better understanding of the mechanism.


METHODS
We performed initial studies of the mechanism of intracellular antisense targeting through measurements of in situ transcription, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), 32P-labeled uridine-5'-triphosphate (alpha-32P-UTP) incorporation, nuclear accumulations of 99mTc-labeled DNAs, and messenger RNA (mRNA) transcription rate. As reported earlier, an antisense DNA against the mdr1 mRNA coding for P-glycoprotein (Pgp) and its sense DNA control were used in KB-G2 (Pgp++) cells.


RESULTS
Definitive evidence for antisense targeting was obtained by in situ transcription showing complementary DNA elongation in cells exposed to antisense DNA, acting therefore as an intracellular PCR primer of mdr1 mRNA, but not in cells exposed to sense DNA. Immunofluorescence staining showed higher accumulations of antisense versus sense DNAs in KB-G2 cells. Transnuclear migration was confirmed by higher accumulations in the nucleus compared with the cytoplasm in cells incubated with 99mTc-labeled antisense DNA. However, the observed specific accumulations of antisense DNAs of about 10(6) per cell over 10 h could not be explained by a feedback mechanism upregulating transcription in cells exposed to antisense DNA as no increase in mRNA levels was detected by both RT-PCR and 32P-UTP in these cells. To explore an alternative hypothesis, a novel approach using 99mTc-labeled antisense DNA as a probe of total mRNA from cells previously saturated with unlabeled antisense DNA was used to estimate the transcription rate. Compared with controls, mdr1 mRNA levels were found to be initially low after saturation and to recover at about 2,000 copies per minute per cell. If persistent, this transcription rate would provide 10(6) mRNAs in 10 h.


CONCLUSIONS
The results of all studies are consistent with antisense as the mechanism of targeting. Though a feedback mechanism leading to upregulation of mRNA transcription is an unlikely explanation for the high specific accumulations, our results may be explained if antisense DNAs are targeting mdr1 mRNAs produced at high transcription rates. If the target is primarily pre-mRNA in the nucleus rather than mature mRNA in the cytoplasm, this would provide as well an explanation for the observed migration of 99mTc-labeled antisense DNA into the nucleus.
Liu
Xinrong
X
Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Nakamura
Kayoko
K
Wang
Yi
Y
Wang
Yee
Y
Liu
Guozheng
G
He
Jiang
J
Ding
Hongliu
H
Lu
Peiyuan
P
Rusckowski
Mary
M
Kubo
Atsushi
A
Hnatowich
Donald J
DJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000000981:diagnostic imaging* / Q000235:genetics*; D045744:Cell Line, Tumor; D016373:DNA, Antisense / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D011877:Radionuclide Imaging
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016373:DNA, Antisense