Stimulatory Effect Of Conditioned Medium Research Articles

Diverse effects of Porphyromonas gingivalis on human osteoclast formation

Porphyromonas gingivalis is associated with periodontitis, a chronic inflammatory disease of the tooth-supporting tissues. A major clinical symptom is alveolar bone loss due to excessive resorption by osteoclasts. P. gingivalis may influence osteoclast formation in diverse ways; by interacting directly with osteoclast precursors that likely originate from peripheral blood, or indirectly by activating gingival fibroblasts, cells that can support osteoclast formation. In the present study we investigated these possibilities. Conditioned medium from viable or dead P. gingivalis, or from gingival fibroblasts challenged with viable or dead P. gingivalis were added to human mononuclear osteoclast precursors. After 21 days of culture the number of multinucleated (≥3 nuclei) tartrate resistant acid phosphatase (TRACP)-positive cells was determined as a measure for osteoclast formation. Conditioned medium from viable P. gingivalis, and from fibroblasts with viable P. gingivalis stimulated osteoclast formation (1.6-fold increase p < 0.05). Conditioned medium from dead bacteria had no effect on osteoclast formation, whereas conditioned medium from fibroblasts with dead bacteria stimulated formation (1.4-fold increase, p < 0.05). Inhibition of P. gingivalis LPS activity by Polymyxin B reduced the stimulatory effect of conditioned medium. Interestingly, when RANKL and M-CSF were added to cultures, conditioned media inhibited osteoclast formation (0.6–0.7-fold decrease, p < 0.05). Our results indicate that P. gingivalis influences osteoclast formation in vitro in different ways. Directly, by bacterial factors, likely LPS, or indirectly, by cytokines produced by gingival fibroblasts in response to P. gingivalis. Depending on the presence of RANKL and M-CSF, the effect of P. gingivalis is either stimulatory or inhibitory.

Dihydroorotate dehydrogenase inhibitor A771726 (leflunomide) induces apoptosis and diminishes proliferation of multiple myeloma cells

Multiple myeloma is still an incurable disease; therefore, new therapeutics are urgently needed. A771726 is the active metabolite of the immunosuppressive drug leflunomide, which is currently applied in the treatment of rheumatoid arthritis, BK virus nephropathy, and cytomegaly viremia. Here, we show that dihydroorotate dehydrogenase (DHODH) is commonly expressed in multiple myeloma cell lines and primary multiple myeloma cells. The DHODH inhibitor A771726 inhibits cell growth in common myeloma cell lines at clinically achievable concentrations in a time- and dose-dependent manner. Annexin V-FITC/propidium iodide staining revealed induction of apoptosis of multiple myeloma cell lines and primary multiple myeloma cells. The 5-bromo-2'-deoxyuridine cell proliferation assay showed that inhibition of cell growth was partly due to inhibition of multiple myeloma cell proliferation. A771726 induced G(1) cell cycle arrest via modulation of cyclin D2 and pRb expression. A771726 decreased phosphorylation of protein kinase B (Akt), p70S6K, and eukaryotic translation initiation factor 4E-binding protein-1 as shown by Western blotting experiments. Furthermore, we show that the stimulatory effect of conditioned medium of HS-5 bone marrow stromal cells on multiple myeloma cell growth is completely abrogated by A771726. In addition, synergism studies revealed synergistic and additive activity of A771726 together with the genotoxic agents melphalan, treosulfan, and doxorubicin as well as with dexamethasone and bortezomib. Taken together, we show that inhibition of DHODH by A771726/leflunomide is effective in multiple myeloma. Considering the favorable toxicity profile and the great clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in multiple myeloma.

Fetal/placental regulation of maternal melatonin in rats

We investigated how maternal melatonin is regulated in pregnant rats. To examine the involvement of the conceptus (fetus and placenta) in serum melatonin concentrations, the number of conceptuses was experimentally reduced to one on day 7 of pregnancy (1-conceptus group). Maternal circulating nighttime melatonin levels increased toward day 21 of pregnancy and rapidly decreased to the non-pregnancy levels after parturition, whereas the maternal serum nighttime melatonin levels of the 1-conceptus group on day 21 of pregnancy were significantly lower than normal pregnancy bearing dams more than 10 conceptuses. When the fetuses were removed by fetectomy (all fetuses but not the placentae) on day 12 of pregnancy, serum melatonin concentrations were not decreased. To examine the source of circulating maternal melatonin, mRNA expression of N-acetyltransferase (NAT), which is a late limiting enzyme for melatonin synthesis, was examined in the placenta and fetal pineal. NAT was not expressed in the placenta and was negligible in the pineal gland of the fetus compared with the mother's pineal gland. To examine the effect of placental hormones on maternal melatonin production, a conditioned medium, which was made by incubating placenta of day 20 of pregnancy with medium, was injected into the 1-conceptus dams from day 17 to day 20 of pregnancy. Injection of conditioned medium significantly increased serum melatonin concentrations compared with the control values whereas charcoal treatment abolished the stimulatory effect of conditioned medium. In conclusion, maternal circulating melatonin is from the maternal pineal gland and is increased by placental hormones during pregnancy.

Human breast-cancer cells stimulate the fusion, migration and resorptive activity of osteoclasts in bone explants.

A central event in bone resorption is the recruitment of osteoclasts to future resorption sites. Breast-cancer cells invariably metastasise to the skeleton and induce extensive bone destruction by osteoclasts. However, our understanding of the mechanisms by which cancer cells interact with osteoclasts remains unclear. Consequently, we compared the effects of conditioned medium (CM) from 2 human breast-cancer cell lines, MB-MDA-231 and MCF-7, with those of a normal human breast epithelial cell line, HME, on osteoclastic fusion, resorptive activity and migration from the periosteum to the developing marrow cavity of fetal mouse metatarsals in culture. Osteoclastic resorptive activity was assessed by pre-labelling 17-day-old fetal metatarsal explants with 45Ca, whilst fusion and migration were monitored by histomorphometry and osteoclasts were identified by their tartrate-resistant acid phosphatase activity. CM from TPA-stimulated breast-cancer cell lines produced a significant increase in osteoclastic resorptive activity, whilst the normal breast cell line produced a minimal increase. The breast-cancer cell lines also stimulated osteoclastic fusion and migration in the metatarsal explants, but the normal breast cell line was without effect. The stimulatory effect of CM from MDA-MB-231 cells on osteoclastic fusion, but not migration, was partially inhibited by preventing prostaglandin and leukotriene synthesis by cells within the bone explants. In contrast, a synthetic matrix metalloproteinase (MMP) inhibitor, but not a cysteine proteinase inhibitor, prevented the migration of osteoclasts to the calcified centre of the metatarsal explants in response to CM from MDA-MB-231 cells. MDA-MB-231 cells also induced an increase in the expression of MMP-9 by migrating osteoclasts. Fractionation of the TPA-stimulated breast cancer cell CM established that the resorptive activity was associated with factors of m.w. >3 kDa. We determined by immuno-assay that human breast-cancer cells secrete parathyroid hormone-related protein (PTH-rP), tumour necrosis factor-alpha (TNF-alpha) and interleukins (ILs) 6 and 11. Neutralizing experiments with human antibodies to these cytokines established that PTH-rP and TNF-alpha production by MDA-MB-231 cells were responsible for mediating their effects on osteoclastic migration and ultimately bone resorption in the metatarsal explants.

4.P.248 PFGF-AA is responsible for the stimulatory effect of conditioned medium from colon carcinoma cell line (SW480) on LDL receptor activity or proliferation in cultured skin fibroblasts

4.P.248 PFGF-AA is responsible for the stimulatory effect of conditioned medium from colon carcinoma cell line (SW480) on LDL receptor activity or proliferation in cultured skin fibroblasts

Suppression of tyrosine kinase activity inhibits [3H]thymidine uptake in cultured human pituitary tumor cells.

Tyrosine kinases are involved in the phosphorylation of proteins that regulate cell growth and proliferation. The mitogenic effect of several growth factors requires tyrosine kinase activity of their receptors. The effect of inhibition of tyrosine kinase activity on thymidine uptake into cultured human pituitary adenoma cells was studied using two inhibitors, genestein and methyl-2,3-dihydroxycinnamate (MDHC). Of 33 pituitary adenomas, 7 incorporated sufficient [3H]thymidine to be investigated in the experiments. Genestein and MDHC both potently inhibited thymidine uptake into these tumors, with a mean inhibition by 74 mumol/L genestein of 61.96 +/- 18.96% (+/- SD inhibition of basal), by 740 mumol/L genestein of 92.65 +/- 8.59%, and by 100 mumol/L MDHC of 93.84 +/- 3.85%. The 7 pituitary adenomas were all large with suprasellar extension and secreted interleukin-6 in vitro. They included 2 prolactinomas, 1 somatotropinoma, 1 mammosomatropinoma, and 3 clinically nonfunctioning adenomas. Epidermal growth factor stimulated thymidine uptake in 2 of the 3 clinically nonfunctioning adenomas studied, and this stimulation was inhibited by genestein. Both of these tumors released FSH in cell culture and are probably silent gonadotropinomas. The growth stimulatory effect of conditioned medium from human pituitary cell culture on GH3 cells was inhibited by both genestein and MDHC. We conclude that tyrosine kinase activity is crucial for the integrity and growth of pituitary adenomas in culture. Growth factors released by pituitary adenomas potentially may maintain and promote tumor growth by stimulating tyrosine kinase activity.

Prostaglandin E2 stimulates osteoclast formation via endogenous IL-1 beta expressed through protein kinase A.

The present study was conducted to determine whether endogenous IL-1 is involved as a potent mediator of PGE2-stimulated osteoclast formation in 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3)-primed calvarial cells from mouse embryos. PGE2 induced IL-1 beta gene expression in the primed calvarial cells. IL-1 beta gene expression was also induced in a dose-dependent manner by forskolin and dibutyryl cAMP. PGE2-induced IL-1 beta gene expression was markedly inhibited by H-89, a potent inhibitor of protein kinase A. On the other hand, osteoclast formation in 1 alpha,25-(OH)2D3-primed calvarial cells was also stimulated by forskolin and dibutyryl cAMP, and their stimulatory effects were dose dependent. H-89 also inhibited PGE2-stimulated osteoclast formation. The presence of the IL-1 beta gene product in the conditioned medium of 1 alpha,25-(OH)2D3-primed calvarial cells treated with PGE2 was proved by the results of an immunoprecipitation assay using anti-mouse IL-1 beta Ab. The addition of anti-mouse IL-1 beta Ab to 1 alpha,25-(OH)2D3 primed calvarial cell cultures markedly inhibited PGE2-stimulated osteoclast formation. The stimulatory effect of conditioned medium of primed calvarial cells treated with PGE2 on osteoclast formation was also inhibited by anti-IL-1 beta Ab pretreatment. Furthermore, we found that endogenous IL-6 is partially involved in PGE2-stimulated osteoclast formation.

Glomerular endothelial cells promote mesangial cell growth via a PDGF-Like substance.

We conducted these experiments to determine whether glomerular endothelial cells (GEN) may play a role in the regulation of glomerular mesangial cells (GMC) growth. When GMC were separately co-cultured with GEN for 24 h, [3H]thymidine incorporation into GMC increased by 22%. In addition, when GMC were co-cultured with GEN for 4 days, the GMC count increased statistically. Conditioned medium (CM) was obtained from confluent GEN monolayers and tested for its effect on GMC growth. When CM was mixed with RPMI 1640 medium containing fetal calf serum (final concentration: 10%), GMC growth was potentiated in a dose-dependent manner. This stimulatory effect of CM from GEN cultures was inhibited by the addition of anti-human platelet-derived growth factor (PDGF) antibody. However, antibodies against interleukin 1-beta, tumor necrosis factor-alpha and endothelin-1 did not affect the stimulatory effect of CM. Moreover, the modulator contained in CM was stable at 100 degrees C, and pH 2.5 at 22 degrees C for 30 min. When treated with 2 mM mercaptoethanol, this activity was almost completely lost. These findings suggest that GEN promote GMC growth in co-culture systems, without direct contact, by means of a PDGF-like substance.

Glomerular endothelial cells promote mesangial cell growth via a PDGF-like substance

We conducted these experiments to determine whether glomerular endothelial cells (GEN) may play a role in the regulation of glomerular mesengial cells (GMC) growth. When GMC were separately co-cultured with GEN for 24 h, [ 3H]thymidine incorporation into GMC increased by 22%. In addition, when GMC were co-cultured with GEN for 4 days, the GMC count increased statistically. Conditioned medium (CM) was obtained from confluent GEN monolayers and tested for its effect on GMC growth. When CM was mixed with RPMI 1640 medium containing fetal calf serum (final concentration: 10%), GMC growth was potentiated in a dose-dependent manner. This stimulatory effect of CM from GEN cultures was inhibited by the addition of anti-human platelet-derived growth factor (PDGF) antibody. However, antibodies against interleukin 1-β, tumor necrosis factor-α and endothelin-1 did not affect the stimulatory effect of CM. Moreover, the modulator contained in CM was stable at 100°C, and pH 2.5 at 22°C for 30 min. When treated with 2 mM mercaptoethanol, this activity was almost completely lost. These findings suggest that GEN promote GMC growth in co-culture systems, without direct contact, by means of a PDGF-like substance.