Exista la ora actuala tot mai multe dovezi in ceea ce priveste faptul ca mutatiile genei supresoare pentru proteina p53 sunt printre cele mai frecvente alterari genetice din neoplaziile umane. Studiul de fata a urmarit determinarea semnificatiei expresiei accentuate a proteinei p53 in melanomul malign cunoscut fiind faptul ca radiatiile ultraviolete pot induce mutatii specifice pentru p53. De asemenea s-a incercat determinarea modului in care aceste modificari pot influenta tratamentul specific.

S-au analizat 6 linii celulare de melanom atat pentru expresia genei p53 cat si pentru expresia proteinei p53. Exonii 4-9 ai genei p53 au fost amplificati folosind un protocol multiplex/nested PCR (Berg et al. 1995). Produsul obtinut prin polimerizarea in lant a fost analizat baza cu baza prin secventiere ciclica, marcand diferit A, C, T. G (Big Dye Terminators, Perking Elmer, Norwalk, Connecticut, USA). Secventa obtinuta a fost identificata si comparata cu secventa normala a genei p53 obtinuta din baza de date BLASTIN, NCBI, folosind un program de analiza a secventei DNA (Gene Codes). Expresia proteinei p53 a fost analizata atat prin metoda Western blott cat si imunohistochimic.

Rezultate: p53 este implicata in aparitia melanomului malign si este posibila utilizarea genei p53 ca tinta terapeutica.

There is increasing evidence that mutation in the p53 suppresser genes are among the most common genetic alterations in human malignancies. Because ultraviolet light can induce specific p53 mutation and is linked to the development of skin cancer, this stdy was done to determinate the significance of p53 protein overexpresion in melanoma, and how could be use this modification for treatment.

Six-melanoma cell line was analysed for either p53 gene expression or p53 protein expression: Exon 4-9 of the human p53 was amplified from celular DNA using a multiple/nested PCR protocol (Berg et al., 1995). The outer multiplex amplification was performed with 14 primers annealing the intronic sequences that span exon 4 to exon 9 of the p53 gene. The inner exon-specific PCR was done using 100-fold to 25-fold dilution of the outer PCR template. PCR products were directly sequnced by cycle sequncing with dye labeled terminators (big Dye Terminators, Perking Elmer, Norwalk, Connecticut, USA), and analyzed on a DNA sequencer ABI PRISM 377XL (PE Applied Biosystems, Foster City, California, USA). Primers used in the PCR amplification were used as sequencing primers. The sequences obtained were identified and aligned together with the wild type sequence (from BLASTIN, NCBI) using the DNA analyzer program sequencer (Gene Codes). Expression of p53 protein was analyzed using both western blotting and IHC. Our result strongly suggests implication of p53 in melanoma pathway, and possibility for use p53 gene as a therapeutic target.