Caterina Tanzarella Antonio Antoccia Antonella Sgura Francesco Berardinelli

1. Status Report: “Characterization of a panel of cell lines to be used in in the framework of a Treatment Planning System” Dipartimento di Biologia, Università “Roma Tre” INFN – Sezione di “Roma Tre” Caterina Tanzarella Antonio Antoccia Antonella Sgura Francesco Berardinelli

2. ENDPOINTS TO BE ANALYZED • Characterization of cell lines • 1) Nuclear area • 2) Nuclear thickness InV79, AG01522D, CCD18Lu, HSG, T98G b. First priority measures in collaboration with the LNL group 1) Cell growth curves 2) Cell inactivation to evaluate the surviving fraction in IR-exposed cells c. Additional measures 1) -H2AX assay for quantitation of tracks at t=30 min 2) Cytogenetic analysis to evaluate the clastogen effect in IR-exposed cells.

3. Measure of nuclear area • Cells were seeded at a final concentration of 12000/cm2 in chamber slides successively fixed in absolute methanol and stained with 0,5µg/ml DAPI • - Images were captured at 63X magnification and nuclear area evaluated using the ISIS sofware by Metasystems

4. Measure of nuclear thickness Analysis performed in vivo using DRAQ5 dye in confocal microscopy ~ 0,38 µm Each cell was analysed indipendently and at least 100 cells were scored for each cell line BF 1 2 3 4 5 6 7 BF BF 8 9 10 11 12 13 14 Thickness (µm) = (number of slices -1) * thickness of a single slice (µm)

5. Nuclear area and thickness in AG01522 D

6. Nuclear area and thickness in V79

7. Cell growth of AG01522 D Cells were seeded at a final concentration of 16*104 in plastic Petri dishes and counted in triplicate every 24 hours for 5 days. T =tlog2 / (log N -log N0) Doubling time: ~ 22 hrs