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Protein kinase inhibitors as potent inhibitors of bone resorption
Bone Vol. 16, No. 3 March 1995:391-410
Abstracts
33
34
MULTIELEMENT QUANTIFICATION IN CULTURES OF FETAL CHICK CALVARIAE BY TOTAL REFLECTION X-RAY FLUORESCENCE SPECTROMETRY (TXRF) U. Ewald (*/. G. Werner (*). R. Fischer (#). E. Keck (*). (*) Forschungslabor for Osteologie und Rheumatologie Wiesbaden, (#) BKA Wiesbaden, FRGermany
PROTEIN KINASE INHIBITORS AS POTENT I N H I B I T O R S O F B O N E R E S O R P T I O N . J.H.M. Feyen and R.Waelchli. Sandoz Pharma Ltd, 4 0 0 2 Basle, Switzerland p p 6 0 " " is a m e m b e r of a cytoplasmatic non-receptor tyrosine kinase family. These kinases are involved in signal transduction pathways of transmembrane receptors. It has been reported that p p 6 0 .... plays an essential role in osteoclast function. In this report we present the results of several kinase inhibitors on the ability to inhibit bone resorption stimulated by parathyroid h o r m o n e (hPTH 1-34) in vitro. Staurosporine, a widely used nonspecific kinase inhibitor, is an effective inhibitor of bone resorption in the fetal rat long bone assay, with apparent ICso values of 23 nM and 30 nM at day 2 and day 5, respectively. Chelerithrine chloride, a relative specific inhibitor of protein kinase C, was unable to alter the PTH-induced resorption. The inability of chelerithrine chloride to inhibit the bone resorption process indicates that the inhibitory effect of staurosporine can not be attributed to inhibition at the level of protein ldnase C. Herbimycin A and its derivative dihydroherbimycin A were able to inhibit bone resorption in the fetal rat long bone assay with apparent ICso values of 80 nM and 45 nM, respectively. Bone resorption inhibition was complete and persisted during the entire culture period. Similar inhibitory effects were also observed in neonatal mouse calvaria in culture. The inability of genistein and lavendustin A (two additional tyrosine kinase inhibitors) to inhibit bone resorption indicates a certain 'specificity' of herbimycin A towards bone tissue. It has been suggested that calcitonin exerts its inhibitory action on the osteoclast, at least in part, via the inhibition of p p 6 0 "~'' kinase activity. Interestingly, the osteoclast were not able to overcome the herbirnycin A-induced inhibition up to 5 days of culture as would be the case for calcitonin-inhibited resorption . Thus, inhibiting the osteoclast down-stream of the calcitonin receptor could be an approach without an "escape phenomenon" as observed after prolonged culture time in vitro.
A main problem of quantifying elements - playing an improtant role in biomineralization - has been the lack of an analytical approach requiering a minimum of probe preparation together with a high power of detection. The application of the TXRF method to an in vitro tissue culture system has been demonstrated during this investigation. Folded periostal halfes, dissected from 17-day-o|d fetal chick calvariae, were incubated with s-Calcitonin in an serum free medium containing measured ion concentrations of 0.9 mM Ca, 0.7 mM P and 0.02 mM Zn. Additionally it was fortified with vitamins, hormones and Na-glycerophosphate. Cultures were terminated after 2, 4, 6 and 8 days. The organic matrix was digested in one step by suprapure HNO 3 and spiked with a trace amount of gallium for internal standardization. 10/al of the resulting solution were placed on a single quartz-glass support, dried to a thin film by infra-redlight and directly measured for their fluorescence stimulated by the primary X-ray beam of a fine-focus Molybdenum-tube. This technique allows the simultaneous detection of up to 20 elements within the range of atomic numbers 15-40 and 57-92 reaching picomolar concentrations. In our case the quantitative results for Ca, P and Zn significantly documented that s-Calcitonin induces a dose- and time dependent increase during mineralisation as compared to controls. Especially the enrichment of ions like Zn under hormonal control could be clearly demonstrated (i.e. at day 8 for a physiologic s-Calcitonin concentration of 206 vs. 6000 pg/ml [Zn] rose from 0.53 to 0.58 mg/gdw). Thus it migth be concluded that TXRF is a powerful tool to study biomineralization by investigating the influence of varying concentrations of effector hormones as well as relevant ions added to the culture medium. The easy way of preparation together with an automated measuring device allows the assesment of a large number of probes in a given time to gain high statistical safety.
35 M U L T I - H O R M O N A L E F F E C T S ON O S T E O C L A S T FORMATION: ROLE OF IL-6. C, Schiller. K. Redlicb. P. Pietschmann and M. Peterlik. Department of General and Experimental Pathology, University of Vienna, Waehdnger Guerte118-20, A-1090 Vienna, Austria Apart from estrogen deficiency, other hormonal imbalances such as mild vitamin D deficiency or slightly elevated thyroid hormone production are likely to contribute to the development of osteoporosis. We therefore addressed the question whether the increased rate of bone loss, which is the hallmark of this diseasae, could be due to a respective multi-hormonal effect on osteoclast formation from undifferentiated haematopoietic cells. Bone marrow was isolated from 8-10 week old mice, and adherent cells were grown in c~-MEM for 6-8 days. Osteoclast-like cell formation was monitored by counting multi-nucleated tartrate-resistant acid phosphatase-positive (TRAP + ) cells. ": ,25-dihydroxyvitamin D 3 (1,25(OH)2D3) was essential for the generation of osteoclasts and c a u s e d a dose-dependent increase in TRAP + cell numbers between 10-10-10-8 M. The effect of 10 -8 M vitamin D was inhibited by 17f~-estradiol (Eg) at and above 10 -12 M, with maximal inhibition of 66% at 10 -`9 M. Triiodothyronine (T3), which per se (10-10-10 -7 M) was ineffective, augmented induction of TRAP + cells by 10 -e M 1,25(OH)2D 3. The synergistic action of T 3 and 1,25(OH)2D 3 was however completely blocked by 10-UM E2. Since estrogen defiency is considered a cause of elevated circulating IL-6 levels, which, in turn, stimulate osteoclast formation, we sought to elucidate the role of i'L-6 in hormonal modulation of 1,25(OH)2D3-dependent osteoclast formation. When a monoclonal anti-lL-6 antibody (lmg/ml) was added to the bone marrow cell cultures, osteoclastinduction by 1,25(OH)2D3 alone or by 1,25(OH)2D3 and T3 was markedly inhibited (50% of control). The majority of bone marrow cells expressed receptors for the three hormones on days 1 and 8 of culture. Therefore a receptor-mediated action of either hormone in modulation of IL-6-mediated osteoclast-like cell formation can be assumed.
36 THE EFFECTS OF 1,25(OH)2-16,23-DIENECHOLECALCIFEROL ON PROLIFERATION AND DIFFERENTIATION OF CACO-2 CELLS: RELATION TO IN VITRO BONE RESORPTION. Martin G. Bischof, Kurt Redlich. Meinrad Peterlik, Heide S. Cross, Institute of General and Experimental Pathology, University of Vienna Medical School, Austria. Although 1,25-dihydroxycholecalciferol (1,25-CC) is a potent antimitogen in many cell systems, its use in effective doses in cancer therapy is limited by the occurrence of hypercalcemia. This has led to the development of synthetic, so-called non-hypercalcemic, analogs. We have tested the effects of 1,25dihydroxy-16,23-dienecholecalciferol (Ro 24-2201) on proliferation and differentiation of human colon adenocarcinoma derived Caco-2 cells as well as on calcium release from cultured mouse calvariae. Caco-2 cells were seeded at a density of 7500 cells/cm2 in DMEM Proliferation was assessed by measuring incorporation of [3H]thymidine into cellular DNA after 14 days of culture. Calvariae were dissected from 4-6 day old mice, precultured for 6 h in DMEM containing 0.5 laM indomethacin and then washed and cultured for additional 72 h in indomethacin-frec medium. Ro 24-2201 and 1,25oCC both significantly decreased proliferation of Caco-2 cells to 80% of control at a concentration of 10s M. Interestingly, alkaline phosphatase activity was significantly more increased by Ro 24-2201 than by 1,25-CC (202% vs 176% of control). In contrast to these results, Ro 24-2201 stimulated bone resorption to only 164% of control which was significantly less than calcium release induced by 1,25-CC (202%). We conclude that, in comparison to 1,25-CC, the analog Ro 242201 combines a similarly high antimitogenic and differentiating potency with a significantly reduced hypercalcemic activity. As stimulation of bone resorption seems to be the most important cause of hypercalcemia in vivo, Ro 24-2201 constitutes a promising new approach to the therapy of 1,25-dihydroxycholecalciferol responsive cancers.
401
Abstracts
33
34
MULTIELEMENT QUANTIFICATION IN CULTURES OF FETAL CHICK CALVARIAE BY TOTAL REFLECTION X-RAY FLUORESCENCE SPECTROMETRY (TXRF) U. Ewald (*/. G. Werner (*). R. Fischer (#). E. Keck (*). (*) Forschungslabor for Osteologie und Rheumatologie Wiesbaden, (#) BKA Wiesbaden, FRGermany
PROTEIN KINASE INHIBITORS AS POTENT I N H I B I T O R S O F B O N E R E S O R P T I O N . J.H.M. Feyen and R.Waelchli. Sandoz Pharma Ltd, 4 0 0 2 Basle, Switzerland p p 6 0 " " is a m e m b e r of a cytoplasmatic non-receptor tyrosine kinase family. These kinases are involved in signal transduction pathways of transmembrane receptors. It has been reported that p p 6 0 .... plays an essential role in osteoclast function. In this report we present the results of several kinase inhibitors on the ability to inhibit bone resorption stimulated by parathyroid h o r m o n e (hPTH 1-34) in vitro. Staurosporine, a widely used nonspecific kinase inhibitor, is an effective inhibitor of bone resorption in the fetal rat long bone assay, with apparent ICso values of 23 nM and 30 nM at day 2 and day 5, respectively. Chelerithrine chloride, a relative specific inhibitor of protein kinase C, was unable to alter the PTH-induced resorption. The inability of chelerithrine chloride to inhibit the bone resorption process indicates that the inhibitory effect of staurosporine can not be attributed to inhibition at the level of protein ldnase C. Herbimycin A and its derivative dihydroherbimycin A were able to inhibit bone resorption in the fetal rat long bone assay with apparent ICso values of 80 nM and 45 nM, respectively. Bone resorption inhibition was complete and persisted during the entire culture period. Similar inhibitory effects were also observed in neonatal mouse calvaria in culture. The inability of genistein and lavendustin A (two additional tyrosine kinase inhibitors) to inhibit bone resorption indicates a certain 'specificity' of herbimycin A towards bone tissue. It has been suggested that calcitonin exerts its inhibitory action on the osteoclast, at least in part, via the inhibition of p p 6 0 "~'' kinase activity. Interestingly, the osteoclast were not able to overcome the herbirnycin A-induced inhibition up to 5 days of culture as would be the case for calcitonin-inhibited resorption . Thus, inhibiting the osteoclast down-stream of the calcitonin receptor could be an approach without an "escape phenomenon" as observed after prolonged culture time in vitro.
A main problem of quantifying elements - playing an improtant role in biomineralization - has been the lack of an analytical approach requiering a minimum of probe preparation together with a high power of detection. The application of the TXRF method to an in vitro tissue culture system has been demonstrated during this investigation. Folded periostal halfes, dissected from 17-day-o|d fetal chick calvariae, were incubated with s-Calcitonin in an serum free medium containing measured ion concentrations of 0.9 mM Ca, 0.7 mM P and 0.02 mM Zn. Additionally it was fortified with vitamins, hormones and Na-glycerophosphate. Cultures were terminated after 2, 4, 6 and 8 days. The organic matrix was digested in one step by suprapure HNO 3 and spiked with a trace amount of gallium for internal standardization. 10/al of the resulting solution were placed on a single quartz-glass support, dried to a thin film by infra-redlight and directly measured for their fluorescence stimulated by the primary X-ray beam of a fine-focus Molybdenum-tube. This technique allows the simultaneous detection of up to 20 elements within the range of atomic numbers 15-40 and 57-92 reaching picomolar concentrations. In our case the quantitative results for Ca, P and Zn significantly documented that s-Calcitonin induces a dose- and time dependent increase during mineralisation as compared to controls. Especially the enrichment of ions like Zn under hormonal control could be clearly demonstrated (i.e. at day 8 for a physiologic s-Calcitonin concentration of 206 vs. 6000 pg/ml [Zn] rose from 0.53 to 0.58 mg/gdw). Thus it migth be concluded that TXRF is a powerful tool to study biomineralization by investigating the influence of varying concentrations of effector hormones as well as relevant ions added to the culture medium. The easy way of preparation together with an automated measuring device allows the assesment of a large number of probes in a given time to gain high statistical safety.
35 M U L T I - H O R M O N A L E F F E C T S ON O S T E O C L A S T FORMATION: ROLE OF IL-6. C, Schiller. K. Redlicb. P. Pietschmann and M. Peterlik. Department of General and Experimental Pathology, University of Vienna, Waehdnger Guerte118-20, A-1090 Vienna, Austria Apart from estrogen deficiency, other hormonal imbalances such as mild vitamin D deficiency or slightly elevated thyroid hormone production are likely to contribute to the development of osteoporosis. We therefore addressed the question whether the increased rate of bone loss, which is the hallmark of this diseasae, could be due to a respective multi-hormonal effect on osteoclast formation from undifferentiated haematopoietic cells. Bone marrow was isolated from 8-10 week old mice, and adherent cells were grown in c~-MEM for 6-8 days. Osteoclast-like cell formation was monitored by counting multi-nucleated tartrate-resistant acid phosphatase-positive (TRAP + ) cells. ": ,25-dihydroxyvitamin D 3 (1,25(OH)2D3) was essential for the generation of osteoclasts and c a u s e d a dose-dependent increase in TRAP + cell numbers between 10-10-10-8 M. The effect of 10 -8 M vitamin D was inhibited by 17f~-estradiol (Eg) at and above 10 -12 M, with maximal inhibition of 66% at 10 -`9 M. Triiodothyronine (T3), which per se (10-10-10 -7 M) was ineffective, augmented induction of TRAP + cells by 10 -e M 1,25(OH)2D 3. The synergistic action of T 3 and 1,25(OH)2D 3 was however completely blocked by 10-UM E2. Since estrogen defiency is considered a cause of elevated circulating IL-6 levels, which, in turn, stimulate osteoclast formation, we sought to elucidate the role of i'L-6 in hormonal modulation of 1,25(OH)2D3-dependent osteoclast formation. When a monoclonal anti-lL-6 antibody (lmg/ml) was added to the bone marrow cell cultures, osteoclastinduction by 1,25(OH)2D3 alone or by 1,25(OH)2D3 and T3 was markedly inhibited (50% of control). The majority of bone marrow cells expressed receptors for the three hormones on days 1 and 8 of culture. Therefore a receptor-mediated action of either hormone in modulation of IL-6-mediated osteoclast-like cell formation can be assumed.
36 THE EFFECTS OF 1,25(OH)2-16,23-DIENECHOLECALCIFEROL ON PROLIFERATION AND DIFFERENTIATION OF CACO-2 CELLS: RELATION TO IN VITRO BONE RESORPTION. Martin G. Bischof, Kurt Redlich. Meinrad Peterlik, Heide S. Cross, Institute of General and Experimental Pathology, University of Vienna Medical School, Austria. Although 1,25-dihydroxycholecalciferol (1,25-CC) is a potent antimitogen in many cell systems, its use in effective doses in cancer therapy is limited by the occurrence of hypercalcemia. This has led to the development of synthetic, so-called non-hypercalcemic, analogs. We have tested the effects of 1,25dihydroxy-16,23-dienecholecalciferol (Ro 24-2201) on proliferation and differentiation of human colon adenocarcinoma derived Caco-2 cells as well as on calcium release from cultured mouse calvariae. Caco-2 cells were seeded at a density of 7500 cells/cm2 in DMEM Proliferation was assessed by measuring incorporation of [3H]thymidine into cellular DNA after 14 days of culture. Calvariae were dissected from 4-6 day old mice, precultured for 6 h in DMEM containing 0.5 laM indomethacin and then washed and cultured for additional 72 h in indomethacin-frec medium. Ro 24-2201 and 1,25oCC both significantly decreased proliferation of Caco-2 cells to 80% of control at a concentration of 10s M. Interestingly, alkaline phosphatase activity was significantly more increased by Ro 24-2201 than by 1,25-CC (202% vs 176% of control). In contrast to these results, Ro 24-2201 stimulated bone resorption to only 164% of control which was significantly less than calcium release induced by 1,25-CC (202%). We conclude that, in comparison to 1,25-CC, the analog Ro 242201 combines a similarly high antimitogenic and differentiating potency with a significantly reduced hypercalcemic activity. As stimulation of bone resorption seems to be the most important cause of hypercalcemia in vivo, Ro 24-2201 constitutes a promising new approach to the therapy of 1,25-dihydroxycholecalciferol responsive cancers.
401