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Initial Mechanistic Studies of Antisense Targeting in Cells

Xinrong Liu, Kayoko Nakamura, Yi Wang, Yee Wang, Guozheng Liu, Jiang He, Hongliu Ding, Peiyuan Lu, Mary Rusckowski, Atsushi Kubo and Donald J. Hnatowich
Journal of Nuclear Medicine February 2006, 47 (2) 360-368;

Article Figures & Data

Figures

  • FIGURE 1. 
    FIGURE 1. 

    Hybridization of AS to mdr1 mRNA in KB-G2 and KB-31 cells detected using in situ reverse transcription. Cells growing on glass coverslips were incubated with 100 nmol/L DNA in the presence of TransFast Reagent and then cells were permeabilized, fixed, and subjected to in situ reverse transcription using DIG-11-dUTP. Incorporated label was detected using antidigoxigenin antibodies linked to alkaline phosphatase. (A and B) Cells incubated with mdr1 AS or S in KB-G2 cells. (C and D) Cells incubated with mdr1 AS DNA or S DNA in KB-31 cells. (E and F) Cells incubated with β-actin AS DNA or no DNA in KB-G2 cells. (G and H) Cells incubated with β-actin AS DNA or no DNA in KB-31 cells. (×400)

  • FIGURE 2. 
    FIGURE 2. 

    32P-UTP incorporation during RNA synthesis in KB-G2 cells. Cells were cultured with 37 kBq (1 μCi) α-32P–UTP per well in the presence of AS DNA, S DNA, or no DNA (100 nmol/L) in DMEM with 1% FCS for 24 h. Total RNA was extracted and counted from each sample. Error bars signify 1 SD. *P < 0.05 AS vs. S.

  • FIGURE 3. 
    FIGURE 3. 

    Results of RT-PCR on KB-G2 cells treated with AS DNA, S DNA, or no DNA. (A) Gels with ethidium bromide staining show correct molecular weights (M) for RT-PCR products of mdr1 mRNA and reference β-actin mRNA. (B) Histograms show similar relative abundances of mRNA when KB-G2 cells are treated with AS DNA compared with S DNA or no DNA. bp = base pairs.

  • FIGURE 4. 
    FIGURE 4. 

    Cellular accumulation and distribution of mdr1 AS by immunofluorescence microscopy in KB-G2 cells incubated with biotinylated AS DNA (A) and S DNA (B) and detected with antibiotin-Cy3 antibody. (×400)

  • FIGURE 5. 
    FIGURE 5. 

    Accumulation of 99mTc in nucleus (N) compared with cytoplasm (C) for cells incubated with AS DNA and S DNA for up to 9 h.

  • FIGURE 6. 
    FIGURE 6. 

    The relative concentration of mdr1 mRNA as shown by 99mTc–AS DNA probing of KB-G2 cells incubated at saturating concentrations with unlabeled AS DNAs (•) compared with cells incubated with unlabeled S DNA (○). As further controls, cells incubated with unlabeled AS DNA (▴) and unlabeled S DNA (▵) were also probed with 99mTc–S DNA. *P < 0.05, cells saturated with AS DNA vs. cells “saturated” with S DNA when probed with 99mTc–AS DNA. P < 0.05, cells saturated with AS DNA and probed with 99mTc–AS DNA vs. 99mTc–S DNA. P < 0.05, also for cells saturated with AS DNA and probed with 99mTc–AS DNA vs. cells “saturated” with S DNA and probed with 99mTc–S DNA.

  • FIGURE 7. 
    FIGURE 7. 

    Plot of counting rate of total RNA from Figure 6 divided by number of cells for the earliest 4 time points. Regression line through data points provides a slope of 1.95 × 103 copies per minute per cell (R2 = 0.961).

Tables

  • TABLE 1

    RT-PCR Primer Sequences

    PrimerSequenceTm (°C)
    mdr1 sense5′-GCTCCTGACTATGCCAAAGC59
    mdr1 antisense5′-CTTCACCTCCAGGCTCAGTC59
    β-Actin sense5′-GATCATTGCTCCTCCTGAGC59
    β-Actin antisense5′-AAAGCCATGCCAATCTCATC60

    • Tm = melting temperature.

  • TABLE 2

    Results of In Situ Reverse Transcription in KB-G2 and KB-31 Cells

    KB-G2KB-31
    GroupPositive cells (no.)Total cells (no.)Positive cells (%)Positive cells (no.)Total cells (no.)Positive cells (%)
    AS*4,382 ± 1,1548,747 ± 42450.1020 ± 78,299 ± 9960.24
    S309 ± 806,386 ± 1,4964.8433 ± 1110,817 ± 2,2570.31
    AS+TF†4,695 ± 1,0298,423 ± 1,76755.7416 ± 48,485 ± 1690.19
    S+TF39 ± 810,718 ± 3,3750.3620 ± 18,736 ± 2,8580.23

    • ↵* P < 0.01, AS vs. S in KB-G2 cells only.

    • ↵† P < 0.01, AS+TF vs. S+TF in KB-G2 cells only.

    • AS = antisense DNA; S = sense DNA; AS+TF = AS DNA complexed withTransFast Reagent; S+TF = S DNA complexed with TransFast Reagent.

    • Values are mean ± SD for number of positive cells and total number of cells.

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