1. RESPONSE OF A RESISTANT HUMAN MELANOMA CELL LINE TO A THERAPEUTIC PROTON BEAM A. Ristić-Fira 1, I Petrović 1, D. Todorović 1, L. Korićanac 1, L. Valastro 2 and G. Cuttone 2 1 Vinča Institute of Nuclear Sciences, Belgrade, Serbia and Montenegro 2 Istituto Nazionale di Fisica Nucleare, LNS, Catania, Italy

2. Malignant melanomas: –highly metastatic tumours, –poor curing prognosis. Phenotypic heterogeneity of melanoma cells: –differences in degree and type of pigmentation, –different cell morphology, –different growth rate and metastatic capacity. Therapeutic approaches: –surgery, –chemotherapy, –radiotherapy, but uneven effectiveness. For certain melanoma (uveal melanoma and other eye tumours - conjunctival melanoma, iris melanoma, choroidal melanoma or retinoblastoma) rather good results obtained with proton beams.

3. Individual response of malignant melanoma to radiotherapy is variable, reflecting different radiobilogical characteristics of tumour, especially the ability to repair radiation damage. Radiosensitivity of melanomas is related to intrinsic characteristic of the melanocyte - specific metabolic activity, i.e., melanogenesis. Types of melanin: eumelanin (black, dark brown, efficient radioprotector) pheomelanin (red, Cys rich) mixed-type pigmentation cells

4. Aim To investigate effects of protons at four positions within the spread-out Bragg peak (SOBP), thus simulating corresponding therapeutic effects along the tumour volume. Parameters of analyses:  level of cell inactivation,  quality of cell inactivation.

5. Cell culture conditions Irradiation of exponentially growing HTB140 human melanoma cells. Plating efficiency (PE) for HTB140 cells - approximately 60 - 70%. Doubling time (Td) for HTB140 cells - 24±2,7 h.

6. Irradiation conditions Irradiations at 6.6, 16.3, 25.0 and 26.0 mm in Perspex (Polymethyl methacrylate - PMMA) within the SOBP of the 62 MeV proton beam (produced by the superconducting cyclotron at the CATANA treatment facility, INFN, LNS – Catania). Reference dosimetry - plane-parallel PTW 34045 Markus ionization chamber calibrated according to IAEA code of practice (IAEA-TRS-398 2000). Single doses delivered to the cells: 2, 4, 8, 12 and 16 Gy, at dose rate of 15 Gy/min. Irradiations with γ-rays, at the same dose levels, were performed using 60 Co source at the Vinca Institute of Nuclear Sciences in Belgrade, at average dose rate of 1 Gy/min. All cell irradiations were carried out in air at room temperature.

7. Figure 1. Depth dose distribution of the spread-out Bragg peak in Perspex of the 62 MeV proton beam produced at the CATANA treatment facility in the INFN-LNS, Catania. Arrows correspond to irradiation positions at 6.6 mm (A), 16.3 mm (B), 25 mm (C) and 26 mm (D). SOBP

8. Table 1. Irradiation position parameters in SOBP for HTB140 cells Irradiation Depth in Dose Ē * position Perspex (mm) (%) (MeV) A 6.6 87.24±2.61 50.90±4.33 B 16.3 99.42±0.58 34.88±2.15 C 25.0 102.21±3.43 11.74±1.23 D 26.0 32.12±4.27 5.99±1.36 * mean energy

9. Biological assays Cell viability: –microtetrasolium (MTT) assay, –sulforhodamine B (SRB) assay, –clonogenic assay (CA). Cell proliferation: –incorporation of 5-bromo-2`-deoxyuridine (BrdU ) during DNA synthesis. Cell cycle redistribution: –fluorescence activated cell sorter (FACS) with propidium iodide (PI) staining. DNA ladder fragmentation on 2 % agarose gel electrophoresis.

10. Results

11. A Figure 2. Dose dependent surviving fractions, estimated by microtetrasolium, sulforhodamine B and clonogenic assay, of HTB140 melanoma cells irradiated with  - rays and protons. Irradiation position within the proton spread-out Bragg peak corresponds to 6.6 mm depth in Perspex (A).

12. A B CD Figure 3.

13. BrdU Figure 4. Cell proliferation of HTB140 melanoma cells irradiated at 2, 4, 8, 12 and 16 Gy as a function of depth, estimated by 5-bromo-2`-deoxyuridine assay. Irradiation position within the proton spread-out Bragg peak correspond to 6.6 mm (A), 16.3 mm (B), 25 mm (C) and 26 mm (D) depth in Perspex.

14. AB C D FACS Figure 5.

15. 1 2 3 4 5 6 7 AB Figure 6. DNA gel electrophoresis of HTB140 cells irradiated with  -rays 6 h (panel A) and 48 h (panel B) post-irradiation. Lane 2 non-irradiated melanoma cells, lanes 3 – 7 cells irradiated with 8, 12, 16, 20 and 24 Gy respectively. Lane 1 molecular weight marker, 100 bp DNA Ladder (Gibco BRL).

16. 1 2 3 4 5 6 7 Figure 7. Proton induced DNA fragmentation in HTB140 cells 6 h (panel C) and 48 h (panel D) post-irradiation. Lane 2 non-irradiated melanoma cells, lanes 3 – 7 cells irradiated with 8, 12, 16, 20 and 24 Gy respectively. Lane 1 molecular weight marker, 100 bp DNA Ladder (Gibco BRL). CD

17. Figure 8. Phase-contrast photomicrographs of HTB140 melanoma cells irradiated with  –rays and protons at 8, 12, 16, 20 and 24 Gy (original magnification, x 100).

18. Conclusions  The number of viable cells estimated by microtetrasolium (MTT), sulforhodamine B (SRB) and clonogenic (CA) assays revealed cell inactivation, showing an increase when approaching the end of the SOBP at lower doses.  With increase of the doses applied (8 to 16 Gy) and position within the SOBP, the level of cell elimination, although increasing, had a less important descent than for smaller doses (2 and 4 Gy), suggesting that these cells are very radio- resistant.

19. Conclusions  Cell cycle phase distribution exhibited major accumulation of irradiated HTB140 cells in G1/S phase, expressing mainly high metabolic activity of melanoma cells.  The level of G2/M cell population was relatively low, thus confirming the very pronounced radio-resistant nature of these cells.  With the increase of dose and position within the SOBP, this tendency was kept in general, indicating that even 7 days after proton irradiation cells that survived were still rather active.  BrdU assay has shown considerable proliferative activity of irradiated cells with the increase of depth within the SOBP. Inside the same irradiation position, with the increase of dose cell proliferative capacity, although still very high, was significantly reduced.

20. From our previous results, including this study, it seems that protons eliminate HTB140 cells both by apoptosis and irreparable DNA damage, including genomic instability generation, while γ-rays, almost only by the irreparable DNA damage. Time course, extent and qualitative features of various lesions are reported to be different after irradiation with γ- rays and protons, indicating higher effectiveness of proton irradiation.