Proffered Papers— Pediatric Oncology

INTRODUCTION OF CHEMORESISTANCE TO DOXORUBICIN IN CELLS CARRYING A P53 GERMLINE MUTATION DETECTED IN A LI-FRAUMENI FAMILY

Sangiorgi, L.¹, Cerone, M.A.², Soddu, S.², Gobbi, G.1, Lucarelli, E 1, Brach del Prever A³, Picci, P.¹, Helman, L.J.^{4 1}Rizzoli Orthopedic Institute, Bologna, ²Regina Elena Cancer Institute, Rome, ³University of Turin, Turin, Italy and ⁴NCI, NIH, Bethesda, MD, USA

A phenotypic Li-Fraumeni family (mother died of breast carcinoma at the age of 35, brother died of rabdomyosarcoma at the age of 3, 2 sisters died of osteosarcoma at the age of 10 and 14) was investigated for the presence of germline p53 mutations. SSCP for exons 4 through 11 of the gene on tumor DNA from the two sisters revealed an abnormal conformer in exon 6. DNA sequence analysis showed a transversion from adenine to cytosine at codon 220 (amino acid change from tyrosine to serine). The same pattern was observed on microdissected paraffin-embedded tissue of both the mother and the brother. We generated by site directed mutagenesis a plasmid encoding the p^53SER220 mutation (pLp53-S220). We transfected fibroblasts from p53 -/- mice (F10) with pLp-S220 and pLp53-H175 (a plasmid encoding the p^53His175 mutant). The presence of the 2 mutations did not increase the proliferation rate of F10 fibroblast. Drug sensitivity (assessed by IC₅₀ value) of the fibroblasts carrying the mutations was evaluated for doxorubicin, cisplatin and 5-florouracil. Fibroblasts carrying the p^53SER220 and p^53His175 mutations showed a selective resistance for doxorubicin with, respectively, a 3.5 and a 2.9 fold increase. These data were confirmed by the evaluation of the clonogenic ability of the fibroblasts carrying the different transfectants. In conclusion, the p^53SER220 germ-line mutation seems to induce a gain of function in term of chemoresistency for the mutated p53 protein.