Rationale:
Cancer is a proliferative dis ease that stems from genomic modulations at the cellular level and results in overexpression of characteristic proteins on the cell surface. Targeting oncogene mRNA in vivo with a Tc-99m-labeled specific biomolecule could provide a powerful tool for imaging malignant lesions scintigraphically. Peptide Nucleic Acids (PNAs) display ruggedness and facilitate hybridization better than corresponding phosphorothioate and morpholino phosphoamidate oligonucleotides.
Aims:
Our goals were to 1) synthesize PNAs specific for cMYC and cyclinD-1 specific chimeras with a conjugated chelating moiety, 2) examine their hybridization properties, 3) label them with Tc-99m, and 4) perform feasibility studies to image human breast tumors in nude mice chimeras.
Methods:
Using a solid support and 9-fluorenylmethyloxycarbonyl (Fmoc) coupling technique, PNA dodecamers were extended from the N terminus to a G(D)AGG chelating moiety. This hybrid molecule was further extended from its N terminus with a disulfide bridged peptide. This peptide has a high affinity to insulin-like growth factor (IGF)-1 receptor which is expressed abundantly on breast cancer cells. The hypothesis is that the S-S bridged peptide will enhance PNA specificity for malignant cells. The chimera was purified, characterized using a MALDI mass spectrometer and labeled with Tc-99m. Mismatch PNA chimeras similarly synthesized and characterized served as controls. Hybridization studies were performed using RT-PCR. Tissue distribution and tumor imaging studies at 4 and 24 hrs post-injection were carried out.
Results:
The specific chimera and corresponding mismatch PNA synthesis yields varied from 27.2 to 34%. As compared to the control chimeras, PNA sequences displayed significant gene inhibition as examined by RT-PCR. Radiolabeling efficiencies were >95%. SDS polyacrylamide gel electrophoresis analysis of the radiolabeled product demonstrated a M.W. consistent with the M.W. determined by MALDI analysis. Breast tumors in mice were visualized with a cyclinD-1 chimera but not with the corresponding mismatch PNA chimera or the chimera without the bridged disulfide peptide specific for IGF-1R.
Conclusion:
These results demonstrate that 1) complex PNA-peptide chimeras have been successfully synthesized, 2) These constructs retain their biochemical activity and hybridize with oncogene mRNA, 3) these can be radiolabeled efficiently, and 4) used for scintigraphic localization of malignant tumors in vivo. The approach is worthy of further investigation.