- Email: [email protected]
P33. Vitamin D derivatives, c-myc and chondrocyte differentiation
Abstracts from the Winter Meeting, December 1993
459
The U-Z OS and U-393 OS human osteosarcoma cell lines produce predominantly a truncated mRNA (1.8 kb rather than 2.5 kb) for alkaline phosphatase (ALP). Southern blot analysis revealed no gross DIVA rearrangements in either to account for this abnormality. Enzyme histochemistry revealed each line consisted of three distinct populations relative to ALP activity: (1) constitutive producers of active enzyme (5 and 20% in U-Z OS and U-393 OS, respectively), (2) inducible cells which produce active enzyme after treatment with dexamethasone (30 and 75%, respectively), and (3) cells which were neither. We derived prototype clones from U-2 OS. Northern blot hybridisation revealed that all clonal lines produced the truncated message almost exclusively; dexamethasone treatment enhanced the expression of 1.8 lcb, but not the 2.5 kb, mRNA in the constitutive and inducible clones. The levels of activity detected by histochemical and biochemical assays suggest strongly that the truncated mRNA is translated into active protein. Applying PCR technology to analyse the mRNAs from a constitutive clone and a human osteosarcoma line expressing normal-sized mRNA, it was not possible to identify any mutation, presumably because the clone also expressed low levels, undetectable by northern hybridisation, of the 2.5 kb mRNA. We have prepared a cDNA library from this clone in an attempt to isolate and characterise the truncated message.
P31. Foreign
body
(methacrylate)
macrophages
resorb bone in
Ditf0
R Pandey, J Quinn*, NA Athtiasou Department of Pathology, Nuffield Orthopaedic Centre, Headington, Oxford and *Department of Pathology, University of Oxford, John Radcliffe Hospital, Headington, Oxford Aseptic loosening of cemented arthroplasty components is the major cause of failure in joint replacements. The fibrous membranes around loosened arthroplasty components contain a heavy macrophage reaction to wear debris, which includes particles of polymethyl methacrylate cement (PMMA). We isolated murine blood monocytes and co-cultured them on cortical bone slices and glass coverslips with UMR106 stromal cells in the presence of 1,25 dihydroxyvitamin D3. PMMA particles were added to the co-cultures and were clearly phagocytosed by the mononuclear phagocytes, which were F4/80 antigen positive, non-specific esterase positive and tartrate acid phosphatase (TRAP) negative upon resistant commencement of thle culture. After 14 days in co-culture, extensive lacunar resorption on bone slices was observed (detected by scanning electron microscopy) and TRAP positive cells were noted on coverslips. These results suggest that foreign body macrophages may play a direct role in the pathological bone resorption in aseptic loosening.
P32. The effect of hydrocortisone on the differentiation of adipocytic colonies in ,uitro JH Bennett, ME Owen” Department of Oral Pathology, Institute of Dental Surgery, lMRC Bone Research Laboratory, Nuffield London and Orthopaedic Centre, Oxford Marrow stromal cells plated in vitro form colonies, each derived from a single precursor. The colonies vary in size and morphology accordin to their proliferative and differentiative ability. Marrow stoma1 cells, cultured in the presence of autologous plasma folm colonies which differentiate mainly in an adipocytic direction (Bennett et al, J. Cell Science 99, 131,199l). In this study the effect of hydrocortisone on the formation of colonies was investigaled.
Rabbit marrow strotial cells were cultured in, medium supplemented with 15% autologous plasma with or without IO-7M hydrocortisone (HC) for 11 to 14 days. With HC the total number of colonies increased by about 1%. Colonies were classified either as large (L) colonies (>lOOO cells composed of adipocytic, pre-adipocytic and fibroblastic cells), derived from early stromal precursors with high proliferative potential, or as small (S) colonies (~50 cells of’which almost all were adipccytic), derived from late precursors ivith a low proliferative potential. Sequential observations were made on individual colonies during the culture period. In the presence of HC there was a significant increase in the relative proportion of S compared with L colonies and the ratio of S to L was 0.82. Without HC the ratio was 0.19. Conclusion: The results suggest that HC promotes differentiation rather than proliferation of marrow stromal precursors.
P33. Vitamin D derivatives, c-myc and chondrocyte differentiation C Farquharson. CC Whitehead, AL Kwan, JS Rennie, N Loveridge Roslin Institute (Edinburgh), Roslin EH25 9PS, University of Manchester, Manchester, and Rowetf Research Institute, Aberdeen AB2 9SB previous studies have shown that expression of c-myc increases during chondrocyte differentiation and decreases in tibia1 dyschondroplasia (TD) where the differentiation process is interrupted. As 1,25(OH)zD3 prevents TD and is known to regulate c-myc, we have investigated the in viw role of vitamin D derivatives in chondrocyte differentiation and the expression of this proto-oncogene. the tibiotarsi from chicks fed a normal diet or given vitamin D derivatives were chilled, sectioned and examined for alkaline phosphatase (ALP) activity, type X collagen and c-myc immunoreactivity and quantified by microdensitometry. Feeding 1,25(OH)zD3 for 3 weeks prior to death .increased the rate of differentiation as evidenced by the earlier appearance of both ALP activity and type X collagen while 1,25(OH)2-22-ene-24- hydroxymethyl-cholecalciferol (RO 23-6474) delayed chondrocyte differentiation, as assessed by the distance of maximum activity from top of growth plate. (ALP: 1,25(OH)zD3, 3OOym; RO 23-6474, 48Ocm; UnsupRlemented, 360pm. Type X: 1,25(OH)2D3,420pm; RO 23-6474, 540rm; unsupplemented, 480rm). C-myc levels (MIA) in the transitional chondrocytes were higher in the 1,25(OH)zD3 treated animals and decreased in the 1,25(OH)2-22-ene-24-hydroxymethyl-cholecalciferol treated chicks (1,25(OH)zD3 41.3 f 2.3; RO 23-6474, 31.4 f 1.6; unsupplemented, 37.3 f 1.7). These studies confirm the importance of vitamin D derivatives in differentiation and give further support to the involvement of c-myc in the cascade of events initiated by them.
P34. Detection of cytokines in human osteoblast-like cells cultured from normal and osteoporotic bone AF Cinty, MA Birch, CA Walsh, J Robinson*, WD Fraser’, JA Gallagher Department of Human Anatomy and Cell Biology and ‘Clinical Chemistry, University of Liverpool, Liverpool In the adult skeleton continual bone remodellmg is regulated by a complex interaction of systemic and local factors which ensures that skeletal mass is maintained. Osteoporosis is characterised by a breakdown in the regulation of remodelling which leads to bone loss. We have investigated the cytokines expressed by osteoblasts derived from explants of trabecular bone. Samples were obtained at trans-iliac crest biopsy from patients with proven osteoporosis and X-ray evidence of vertebral fractures. Non-osteoporotic samples were obtained from patients, with no X-ray evidence of osteopenia, undergoing total hip replacement.
459
The U-Z OS and U-393 OS human osteosarcoma cell lines produce predominantly a truncated mRNA (1.8 kb rather than 2.5 kb) for alkaline phosphatase (ALP). Southern blot analysis revealed no gross DIVA rearrangements in either to account for this abnormality. Enzyme histochemistry revealed each line consisted of three distinct populations relative to ALP activity: (1) constitutive producers of active enzyme (5 and 20% in U-Z OS and U-393 OS, respectively), (2) inducible cells which produce active enzyme after treatment with dexamethasone (30 and 75%, respectively), and (3) cells which were neither. We derived prototype clones from U-2 OS. Northern blot hybridisation revealed that all clonal lines produced the truncated message almost exclusively; dexamethasone treatment enhanced the expression of 1.8 lcb, but not the 2.5 kb, mRNA in the constitutive and inducible clones. The levels of activity detected by histochemical and biochemical assays suggest strongly that the truncated mRNA is translated into active protein. Applying PCR technology to analyse the mRNAs from a constitutive clone and a human osteosarcoma line expressing normal-sized mRNA, it was not possible to identify any mutation, presumably because the clone also expressed low levels, undetectable by northern hybridisation, of the 2.5 kb mRNA. We have prepared a cDNA library from this clone in an attempt to isolate and characterise the truncated message.
P31. Foreign
body
(methacrylate)
macrophages
resorb bone in
Ditf0
R Pandey, J Quinn*, NA Athtiasou Department of Pathology, Nuffield Orthopaedic Centre, Headington, Oxford and *Department of Pathology, University of Oxford, John Radcliffe Hospital, Headington, Oxford Aseptic loosening of cemented arthroplasty components is the major cause of failure in joint replacements. The fibrous membranes around loosened arthroplasty components contain a heavy macrophage reaction to wear debris, which includes particles of polymethyl methacrylate cement (PMMA). We isolated murine blood monocytes and co-cultured them on cortical bone slices and glass coverslips with UMR106 stromal cells in the presence of 1,25 dihydroxyvitamin D3. PMMA particles were added to the co-cultures and were clearly phagocytosed by the mononuclear phagocytes, which were F4/80 antigen positive, non-specific esterase positive and tartrate acid phosphatase (TRAP) negative upon resistant commencement of thle culture. After 14 days in co-culture, extensive lacunar resorption on bone slices was observed (detected by scanning electron microscopy) and TRAP positive cells were noted on coverslips. These results suggest that foreign body macrophages may play a direct role in the pathological bone resorption in aseptic loosening.
P32. The effect of hydrocortisone on the differentiation of adipocytic colonies in ,uitro JH Bennett, ME Owen” Department of Oral Pathology, Institute of Dental Surgery, lMRC Bone Research Laboratory, Nuffield London and Orthopaedic Centre, Oxford Marrow stromal cells plated in vitro form colonies, each derived from a single precursor. The colonies vary in size and morphology accordin to their proliferative and differentiative ability. Marrow stoma1 cells, cultured in the presence of autologous plasma folm colonies which differentiate mainly in an adipocytic direction (Bennett et al, J. Cell Science 99, 131,199l). In this study the effect of hydrocortisone on the formation of colonies was investigaled.
Rabbit marrow strotial cells were cultured in, medium supplemented with 15% autologous plasma with or without IO-7M hydrocortisone (HC) for 11 to 14 days. With HC the total number of colonies increased by about 1%. Colonies were classified either as large (L) colonies (>lOOO cells composed of adipocytic, pre-adipocytic and fibroblastic cells), derived from early stromal precursors with high proliferative potential, or as small (S) colonies (~50 cells of’which almost all were adipccytic), derived from late precursors ivith a low proliferative potential. Sequential observations were made on individual colonies during the culture period. In the presence of HC there was a significant increase in the relative proportion of S compared with L colonies and the ratio of S to L was 0.82. Without HC the ratio was 0.19. Conclusion: The results suggest that HC promotes differentiation rather than proliferation of marrow stromal precursors.
P33. Vitamin D derivatives, c-myc and chondrocyte differentiation C Farquharson. CC Whitehead, AL Kwan, JS Rennie, N Loveridge Roslin Institute (Edinburgh), Roslin EH25 9PS, University of Manchester, Manchester, and Rowetf Research Institute, Aberdeen AB2 9SB previous studies have shown that expression of c-myc increases during chondrocyte differentiation and decreases in tibia1 dyschondroplasia (TD) where the differentiation process is interrupted. As 1,25(OH)zD3 prevents TD and is known to regulate c-myc, we have investigated the in viw role of vitamin D derivatives in chondrocyte differentiation and the expression of this proto-oncogene. the tibiotarsi from chicks fed a normal diet or given vitamin D derivatives were chilled, sectioned and examined for alkaline phosphatase (ALP) activity, type X collagen and c-myc immunoreactivity and quantified by microdensitometry. Feeding 1,25(OH)zD3 for 3 weeks prior to death .increased the rate of differentiation as evidenced by the earlier appearance of both ALP activity and type X collagen while 1,25(OH)2-22-ene-24- hydroxymethyl-cholecalciferol (RO 23-6474) delayed chondrocyte differentiation, as assessed by the distance of maximum activity from top of growth plate. (ALP: 1,25(OH)zD3, 3OOym; RO 23-6474, 48Ocm; UnsupRlemented, 360pm. Type X: 1,25(OH)2D3,420pm; RO 23-6474, 540rm; unsupplemented, 480rm). C-myc levels (MIA) in the transitional chondrocytes were higher in the 1,25(OH)zD3 treated animals and decreased in the 1,25(OH)2-22-ene-24-hydroxymethyl-cholecalciferol treated chicks (1,25(OH)zD3 41.3 f 2.3; RO 23-6474, 31.4 f 1.6; unsupplemented, 37.3 f 1.7). These studies confirm the importance of vitamin D derivatives in differentiation and give further support to the involvement of c-myc in the cascade of events initiated by them.
P34. Detection of cytokines in human osteoblast-like cells cultured from normal and osteoporotic bone AF Cinty, MA Birch, CA Walsh, J Robinson*, WD Fraser’, JA Gallagher Department of Human Anatomy and Cell Biology and ‘Clinical Chemistry, University of Liverpool, Liverpool In the adult skeleton continual bone remodellmg is regulated by a complex interaction of systemic and local factors which ensures that skeletal mass is maintained. Osteoporosis is characterised by a breakdown in the regulation of remodelling which leads to bone loss. We have investigated the cytokines expressed by osteoblasts derived from explants of trabecular bone. Samples were obtained at trans-iliac crest biopsy from patients with proven osteoporosis and X-ray evidence of vertebral fractures. Non-osteoporotic samples were obtained from patients, with no X-ray evidence of osteopenia, undergoing total hip replacement.