SV-40 Large-T Immortalization of Embryonic Bone Cells: Establishment of Osteoblastic Clonal Cell Lines

Abstract

Cells derived from embryonic rat calvariae were immortalized by retroviral delivery of cDNA for the SV-40 large T antigen and the bacterial neomycin resistance gene. After selection with G418, cells were cloned by limiting dilution and screened for expression of osteoblast characteristics. One clone (RCT-3), derived from cells collected during the third period of enzymatic digestion, showed high constitutive expression of alkaline phosphatase (ALP), synthesized type I collagen in the virtual absence of type III and exhibited a parathyroid hormone (PTH)-responsive adenylate cyclase (EC₅₀, 10 nM). Messenger RNAs for osteonectin and osteopontin were present in RCT-3 cells and osteopontin mRNA was enhanced by 1,25 (OH)₂ vitamin D₃ treatment.

The other cell line (RCT-1). derived from cells released during the first 10 min of digestion, expressed osteoblast features only after 3 d treatment with 1 mm retinoic acid (RA). ALP activity increased from 0.003 to 0.25 m mole/min/mg protein, there was a substantial increase in the steady-state level of type I collagen mRNA and a dose-dependent and saturable response to PTH was induced (EC₅₀, 10 nM). Osteopontin mRNA was induced by 1.25 (OH)₂D₃.

This study has provided two new cell lines which may be useful models for studies of differentiation-related gene expression in bone cells.

Key Words:

• SV-40 Large T
• Immortalization
• Osteoblast
• Retinoic Acid