GENERATION OF MOUSE OSTEOCLASTOGENIC CELL-LINES IMMORTALIZED WITH SV40 LARGE T-ANTIGEN

Progress in the field of osteoclast gene regulation has been hampered significantly by the lack of such cell lines. In this study, mouse ost eoclast precursor cells were elicited in an osteoclast-inductive cocul ture system and immortalized using SV40 large T antigen. One of the os teoclast precursor cell lines (MOCP-5) forms 95% tartrate-resistant ac id phosphatase positive (TRAP(+)) multinuclear osteoclast-like cells ( OCLs) in the coculture system. The yield of TRAP(+) OCLs was 4.5-7 x 1 0(4) cells per 10 cm(2) dish. Expression of SV40 large T antigen was v isualized in the nucleus of MOCP-5 cells and OCLs by immunohistochemis try. MOCP-5 cells were positive for MoMa-2 antigen and nonspecific est erase but negative for F4/80 antigen. OCLs derived from MOCP-5 cells w ere able to form extensive resorption bone pits on bone slices. The re sorbing activity of the OCLs was comparable to that of authentic mouse osteoclasts. Pit formation was inhibited by salmon calcitonin (CT). A cid production by OCLs was demonstrated by vital staining with acridin e orange. The OCLs expressed cathepsin K and CT receptors, MOCP-5 tell s could be transfected by a construct that carries the beta-galactosid ase gene. Transfected MOCP-5 cells expressing beta-galactosidase retai n the ability to differentiate into OCLs, indicating a useful model fo r osteoclast gene regulation. To date, the MOCP-5 cell line has been m aintained in continuous culture for 23 months and has maintained the c apacity to differentiate into osteoclasts throughout this time. In sum mary, these data show that a stable immortalized osteoclast precursor cell line has beers established and that the immortalization with SV40 large T oncogene does not prevent osteoclast precursor cell different iation.