Transformation of MC3T3-E1 cells following stress and transfection with pSV2neo plasmid

A continuous cell line, MC3T3-E1 cells, originally derived from murine calvaria bones, loses its osteogenic properties as a result of extended passage number under stress conditions. These aged/stressed MC3T3-S cells, although nontumorigenic, do not display some of the osteogenic properties characteristic of the MC3T3-E1 cells. Altered properties include low expression of alkaline phosphatase, diminished collagen synthesis and inability to form mineralized nodules in vitro. We attempted to reactivate these osteogenic properties by transfections with a pSV2neo plasmid containing the TGFbeta1 gene. During these experiments we found that transfected MC3T3-S cells not only acquired high alkaline phosphatase activity and a potent mineralization potential, but also properties akin to the transformed state, such as ability to grow in soft agar and ability to produce tumors in immunodeficient animals. Further analysis showed that the TGFbeta1 gene is not required and that the changes can be introduced by transfections with pSV2neo alone. In contrast, MC3T3-S cells transfected with pcDNA3 (a plasmid containing only the SV40 origin of replication, early promoter, enhancer and polyadenylation signals) or mock-transfected MC3T3-S cells did not show any transformation traits. The results identify two additional SV40 fragments present in pSV2neo (SV40 virus sequence; Genbank accession number: NC_001669: 4100-4191 and 2668-2774) as functional elements contributing to the transformation of aged/stressed and immortalized osteoblastic cells. These findings are analogous to earlier reports describing the cell modifying potential of pSV2neo. We conclude that stressed and aged MC3T3-S can be transformed by transfection with pSV2neo and that such cells acquire not only the tumorigenic potential but exhibit also some of the osteogenic properties characteristic of the parent MC3T3-E1 cells.