External Imaging of CCND1 Cancer Gene Activity in Experimental Human Breast Cancer Xenografts with ^99mTc-Peptide-Peptide Nucleic Acid-Peptide Chimeras

Analysis of ^99mTc-AcGlyd(Ala)GlyGlyAba-CTGGTGTTCCAT-AEEA-d(CysSerLysCys), WT4185. (A) A sample from the labeling reaction mixture was analyzed by reversed-phase HPLC on a Microbond C₁₈ column (10 × 250 mm) eluted with a gradient from 10% to 100% CH₃CN in aqueous 0.1% CF₃CO₂H at 1 mL/min over 28 min at 25°C. %B (CH₃CN) is shown on right axis, and NaI (Tl) radiometric γ-emission is shown on left axis. The single labeled peak eluted at 9.3 min. (B) Denaturing gel electrophoresis on polyacrylamide (10%–20%)–Tris–Tricine–SDS gels. Left panel is an autoradiogram; right panel is stained with Coomassie blue. Lanes 1 and 5: ^99mTc labeling reaction; lanes 2 and 6: mock reaction without ^99mTc; lanes 3 and 7: purified WT4185; lanes 4 and 8: 9.3-min ^99mTc peak from A; lane 9: peptide mass standards.

MCF7:IGF1R cell uptake of CCND1 fluoresceinyl-PNA-mismatch peptide probe, WT4361 (A–C), and CCND1 fluoresceinyl-PNA-IGF1 peptide probe, WT4433 (D–F). Cells were incubated with fluoresceinyl-PNA-peptide at 1 μmol/L for 8 h at 37°C in PRF-SFM, fixed, and examined by confocal microscopy. (Left) Phase contrast. (Middle) Fluorescence. (Right) Overlay.

BT474 (left) and MCF7:IGF1R (right) breast cancer cell mRNAs (10, 1, and 0.1 ng) were analyzed by QRT–PCR with a Prism 7700 (Applied Biosystems) to determine the levels of expression of HER2 (blue), CCND1 (green), IGF1R (red), MYC (pink), and TATA-box binding protein (yellow). ΔRn = relative difference in fluorescence at cycle n.

Western blots of 100 μg of protein extracted from MCF7:IGF1R estrogen receptor-positive breast tumor cell xenografts at 24 h after direct injection of the PNA mismatch chimera, WT4172, the peptide mismatch chimera, WT4113, and the PNA antisense chimera, WT4185. CD1 = cyclin D1; B-actin = β-actin.

Scintigraphic images of γ-rays emitted by decaying ^99mTc in nude mice carrying human MCF7:IGF1R estrogen receptor-positive breast tumor cell xenografts at 4, 12, and 24 h after injection of the PNA-free control probe, WT990, the PNA mismatch control probe, WT4172, the peptide mismatch control probe, WT4113, and the PNA antisense probe, WT4185.