Alterations in the INK4a/ARF locus and their effects on the growth of human osteosarcoma cell lines

Two different proteins, p16INK4a and p14ARF, encoded by the INK4a/ARF locus play important roles in the RB and p53 pathways, respectively. This study was performed to determine genetic and epigenetic alterations in the INK4a/ARF locus and their effects on the growth of osteosarcoma. Among six cell lines examined, both p16INK4a and p14ARF exons were homozygously deleted in two cell lines, MG63 and HOS, and both p16INK4a and p14ARF promoters were methylated in one cell line, U2OS. Wild-type mRNA and proteins for p16INK4a and p14ARF were expressed in three other cell lines, SaOS2, HuO9, and G292. Transfection studies were performed using two cell lines, U2OS and MG63. Both the RB and p53 genes were wild types in U2OS, whereas p53 but not RB was mutated in MG63. Both p16INK4a and p14ARF suppressed the growth of U2OS, whereas p16INK4a but not p14ARF suppressed the growth of MG63. p53 only did not suppress the growth of MG63 either; however, coexpression of p14ARF with p53 increased the fraction of the G0/G1 phase in MG63 cells. The data presented here demonstrate the importance of genetic and epigenetic alterations in the INK4a/ARF locus for the growth of osteosarcoma and thus will be useful to further understand the biologic behavior of osteosarcoma in association with the defects in the p53 and RB pathways.