Etsuko Abe Bo Abrahamsen Yousef Abu-Amer Silvano Adami Sherrill Adams Douglas Adams John S. Adams Sudha Agarwal Thomas Aigner Takuhiko Akatsu Kristina Akesson Mohammed P. Akhter Richard Alexander Luisa Alvarez Shreyasee Amin Norio Amizuka Michael Amling Patrick Ammann H. Clarke Anderson John J.B. Anderson Troels T. Andreassen Dennis Andress Frattini Annalisa Sara B. Arnaud Timothy R. Arnett James Aronson Brian Ashton Per Aspenberg Gerald James Atkins Jane E. Aubin Maurice Audran Peter Augat Itai A. Bab Laura K. Bachrach Donald A. Bailey Kathleen Elise Bainbridge Jameela Banu John Baron Zvi Bar-Shavit Julia Barsony Shona Bass Douglas Bauer David J. Baylink Wesley G. Beamer Thomas J. Beck Norman H. Bell Teresita M. Bellido Dafna Benayahu Claude Laurent Benhamou Douglas Benson Jon N. Beresford Suzanne M. Bernier Paolo Bianco Daniel D. Bikle Neil Binkley Stanley J. Birge Alessandro Bisello Nicholas Bishop Alison Black Dennis Black Harry C. Blair Rosemary Bland Robert D. Blank Michael Bliziotes Aubrey Blumsohn Scott D. Boden Peter V.N. Bodine Georges Boivin Mark E. Bolander Herbert H. Bolotin Lynda Bonewald Sydney Bonnick Johannes Boonstra Adele L. Boskey Mathias Bostrom Roger Bouillon Mary L. Bouxsein Barbara Boyan Brendan F. Boyce Alan Boyde Richard Brand Rolf E. Brenner F. Richard Bringhurst Kim Brixen Robert Brommage Dieter Bromme Matthew Brown Robert E. Brown Edward M. Brown Alex Jay Brown Matthew Brown Henry Burger David B. Burr David A. Bushinsky William T. Butler Kenneth Byrd Suzanne Cadarette Mona S. Calvo Nancy Camacho Ernesto Canalis M. Leonor Cancela Christopher Cann Georges Carle Thomas O. Carpenter Antonio Carrascosa Jean-Paul Casez Jane A. Cauley Joseph Caverzasio Anthony J. Celeste Timothy J. Chambers Daniel Chappard Arthur B. Chausmer Su-Li Cheng Michael Cher Russell W. Chesney Charles H. Chesnut III Thierry Chevalley Kit Chiu Je-Yong Choi Ung-il Chung Roberto Civitelli Bart L. Clarke Gregory Adam Clines Denis Clohisy Jack W. Coburn Augusto Cogoli William G. Cole Ada Cole Judith A. Cole Patricia Collin-Osdoby Ricki Jean Colman Cathleen Colon-Emeric Juliet E. Compston Arthur Conigrave Cheryl A. Conover Cyrus Cooper Glinda S. Cooper Jillian Cornish Felicia Cosman Gilbert J. Cote Veronique Coxam Thomas Crenshaw Peter Ian Croucher Diane M. Cullen Robert G. Cumming Steven R. Cummings Evis Daci Anastasia Daifotis Gail Dalsky Patricia Dargent U. N. Das K. Michael Davies Chris E.D.H. de Laet Marie Christine de Vernejoul Paul C. Dechow Leonard J. Deftos Miriam Frances Delaney Anne M. Delany Leigh Delbridge

Many osteoblastic cell lines are currently in use, but these have limitations either in terms of their relevance to adult human biology and disease or in terms of their suitability for biochemical and molecular analyses. Consequently, we undertook the development of conditionally transformed adult human osteoblastic cell lines. Osteoblasts were obtained from a normal explant cancellous bone chip culture. These cells were infected with adenovirus-ori-SV40 tsA 209, which encodes a temperature-sensitive large T-antigen mutant. Cells immortalized with this virus express a transformed phenotype at the permissive temperature of 34 degrees C but revert to a normal phenotype at the nonpermissive temperature of 40 degrees C. Using this approach, we have isolated several cell clones and describe the characterization of one that was designated HOB-02-C1. Immunocytochemistry revealed that > 95% of the cells express the large T-antigen at both temperatures. These cells exponentially proliferate at 34 degrees C with a doubling time of approximately 2 days but irreversibly stop dividing at 40 degrees C. However, cell volume increases > 2-fold when the cells are maintained for 6 days at the higher temperature. This clone expresses alpha 1 type (I) procollagen mRNA and secretes type I procollagen C-peptide at both temperatures, although the levels were slightly elevated at 40 degrees C. The cell line expresses alkaline phosphatase activity at 34 degrees C, and the basal level of this enzyme increases 2- to 6-fold at 40 degrees C. Alkaline phosphatase activity is induced 4- to 8-fold by 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor-beta 1 (TGF-beta 1) suppresses enzyme expression > 90% at 40 degrees C. Vitamin D3 also induces a 10-fold increase in osteocalcin secretion when the clone is maintained at 34 degrees C, and this induction is enhanced > 8-fold at 40 degrees C. Parathyroid hormone and forskolin stimulate a 4- to 6-fold increase in the production of intracellular cyclic AMP (cAMP) by the cells at 34 degrees C, and this stimulation is enhanced 2- to 4-fold at 40 degrees C. In contrast, prostaglandin E2 stimulates a 7- to 8-fold increase in cAMP only when the cells are maintained at 34 degrees C. This cell line secretes TGF-beta 1 and interleukin-6 (IL-6) at 34 degrees C, but only the basal secretion of IL-6 increases 70% at 40 degrees C. Finally, alizarin red-S histochemical staining demonstrates that these cells produce mineralized nodules at both temperatures. In summary, the results of this study indicate that the HOB-02-C1 cells have a mature osteoblastic phenotype. Consequently, this new cell line and others obtained in a similar fashion should be valuable in vitro tools for cellular, biochemical, and molecular studies of adult human osteoblast biology.