A tridentate single amino acid chelate (SAAC) derived from N-alpha-Fmoc-l-lysine was incorporated within a short peptide sequence using an automated peptide synthesizer. Novel derivatives of the chemotactic peptide fMLF were prepared such that the SAAC and its Re complex were selectively placed between a terminal glycine amino acid and the targeting fMLF sequence. The products, which were synthesized in parallel, were characterized by mass spectrometry and multi-NMR spectroscopy. The latter technique demonstrated that the structures of the targeting portions of the peptides are the same in the SAAC and Re-SAAC derivatives. The affinities of the reported compounds for the formyl peptide receptor were subsequently determined using flow cytometry and were found to be comparable to that of the parent peptide. The results of this work demonstrate the feasibility and numerous benefits of using the SAAC system to prepare peptide-targeted Tc(I) and Re(I) radiopharmaceuticals.

We examined bystander cell death produced in T98G cells by exposure to irradiated cell conditioned medium (ICCM) produced by high-energy 20 MeV electrons at a dose rate of 10 Gy min(-1) and doses up to 20 Gy. ICCM induced a bystander response in T98G glioma cells, reducing recipient cell survival by more than 25% below controls at 5 and 10 Gy. Higher doses increased survival to near control levels. ICCM was analyzed for the presence of transforming growth factor alpha (TGF-alpha) and transforming growth factor beta1 (TGF-beta1). Monoclonal antibodies for TGF-alpha (mAb TGF-alpha) and TGF-beta1 (mAb TGF-beta1) were added to the ICCM to neutralize any potential effect of the cytokines. The results indicate that TGF-alpha was not present in the ICCM and addition of mAb TGF-alpha to the ICCM had no effect on bystander cell survival. No active TGF-beta1 was present in the ICCM; however, addition of mAb TGF-beta1 completely abolished bystander death of reporter cells at all doses. These results indicate that bystander cell death can be induced in T98G glioma if a large enough radiation stress is applied and that TGF-beta1 plays a downstream role in this response.

While nontargeted and low-dose effects such as the bystander effect are now accepted, the mechanisms underlying the response have yet to be elucidated. It has been shown that the transfer of irradiated cell conditioned medium (ICCM) can kill cells that are not directly irradiated; however, to date the effect of ICCM concentration on cell killing has not been reported. The occurrence of a bystander effect was determined by measuring cell survival after exposure to various ICCM dilutions, using the colony-forming assay, in cells of six human cell lines with varied bystander responses and tumor/ p53 status. Autologous ICCM transfer for these cell lines induced a bystander effect as reported previously. ICCM from these cell lines was transferred to cells of a common reporter cell line (HPV-G) to investigate whether the lack of an induced bystander effect was due to their inability to generate or to respond to a bystander signal(s). ICCM from cells of four cell lines induced a bystander effect in HPV-G reporter cells, confirming that signal production is a critical factor. A saturation response was observed when ICCM was diluted. Survival was found to increase linearly until a plateau was reached and the bystander effect was abolished at 2x dilution. The effect of ICCM from the different cell lines reached a plateau at different dilutions, which were found to correlate with the cell line's radiosensitivity.