Biochemical and histological studies on various bone cell preparations

Summary Four different cell populations—designated PF, OB, OC, and PC—were isolated from calvaria of 18-day-old chick embryos for analysis of the effects of hormones on bone tissue. The cell populations were studied with histological and biochemical methods. Apart from the well-known cell types present in calvaria, a new cell type was found in the noncalcified organic matrix between the osteoblastic layer and the calcified matrix. These cells were provisionally called osteocytic osteoblasts. They represent the “transition state” between osteoblasts and osteocytes. On the basis of histological studies with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM), the PF population was considered to originate primarily from the periosteal fibroblasts, the OB population from the osteoblasts and osteocytic osteoblasts. The population of cells still present in calvaria after removal of periosteal fibroblasts and osteoblasts was called the OC population. This cell population was very much enriched with osteocytes. The fourth isolated population (PC) was a mixed population of fibroblasts, osteoblasts, and preosteoblasts. On exposure to parathyroid hormone (PTH), all four cell populations showed increased lactate production, but only the OB and OC populations displayed increased cAMP production. Prostaglandin E₁ (PGE₁) stimulated cAMP production in both OB and PF cells. From the results of this study it was concluded that PTH receptors are present on all of the cell types studied, but that occupancy of the receptor induces adenylate cyclase stimulation only in osteocytes and fully differentiated osteoblasts.     

Comparison of the effects of 1,25-dihydroxycholecalciferol,prostaglandin E2, and osteoclast-activating factor with parathyroid hormone on the ultrastructure of osteoclasts in cultured long bones of fetal rats

Summary The effects of 1,25-dihydroxy vitamin D₃ [1,25(OH)₂D₃], prostaglandin (PGE₂), and osteoclast-activating factor (OAF) on the size of osteoclasts, nuclei, ruffled borders, and clear zones in cultured long bones of fetal rats were quantitated. In addition, the number of osteoclasts in the bones was counted and the release of calcium from the bone into the culture medium was determined. These data were compared with the corresponding effects of parathyroid hormone (PTH). All agents tested increased the size of the ruffled borders significantly after 3 h, the size of the clear zones after 12 h, and the size of the cells after 12–24 h. No important differences in sizes were noticed between the agents tested or between the agents and PTH. The number of osteoclasts was increased after 24 h of treatment with PTH, but not after the other agents. Calcium release was significantly increased for all agents between 12 and 24 h. It is concluded that bone resorption by 1,25(OH)₂D₃, OAF, and PGE₂ is mediated primarily by increased activity of existing osteoclasts similar to PTH activation.     

Characteristics of three human gastric cancer cell lines,NU-GC-2, NU-GC-3 and NU-GC-4

Three human gastric cancer cell lines, NU-GC-2, NU-GC-3 and NU-GC-4 were establishedin vitro from the cancer tissues obtained from 3 patients during surgery. The pathological findings of the gastric tumors of these cases revealed poorly differentiated adenocarcinoma (and partial signet-ring cell carcinoma in the case of NU-GC-4). NU-GC-2 and NU-GC-4 were originally obtained from metastatic paragastric lymph nodes and NU-GC-3 was obtained from a metastatic tumor in the brachial muscle. The cells of NU-GC-2 and NU-GC-3 are polygonal in shape and grow as a monolayer sheet. NU-GC-4 cells, however, are mainly spherical in shape with a few free floating cells. Electron microscopy revealed epithelial characteristics in all 3 cell lines. The average doubling time of NU-GC-2 was 36.1 hours, that of NU-GC-3 was 38.2 hours and that of NU-GC-4 was 29.9 hours. The modal chromosome number of NU-GC-2 was 62, that of NU-GC-3 was 58 and those of NU-GC-4 grown inin vitro andin vivo were 52–54 and 53, respectively.In vitro andin vivo lines of NU-GC-4 were established from the same tumor. These two cell lines are quite similar in morphology, but slightly different in karyotype. Thein vitro sensitivity to anticancer agents was highest in NU-GC-4 and lowest in NU-GC-2. Of the anticancer agents, mitomycin C and adriamycin were most effective on the cells of all 3 cell lines.     

Adriamycin inhibits PTH-mediated but not PGE2-mediated stimulation of cyclic AMP formation in isolated bone cells

Summary We have examined the effect of adriamycin, an anthracycline antibiotic which modifies plasma membrane functions, on the cyclic AMP response to PTH and PGE₂ in isolated osteoblastlike cells. Adriamycin blunted the increment in bone cell cyclic AMP caused by exposure to PTH. This effect appeared rapidly (within 3 min after bone cells were exposed to adriamycin) and disappeared soon after exposure of adriamycin-treated cells to adriamycin-free incubation medium. Inhibition was evident over the entire time course of PTH action, at low as well as high PTH concentrations, and was one-half maximal at 31 μM adriamycin. It could not be attributed to alterations in cyclic AMP exodus, degradation or interference with the cyclic AMP assay, nor to impaired cell viability. Adriamycin also reduced the stimulatory effect of PTH on adenylate cyclase activity in a crude plasma membrane preparation. By contrast, adriamycin failed to modify the effects of PGE₂ on cyclic AMP generation in intact bone cells, and on adenylate cyclase activity in broken cells. Moreover, concentrations of adriamycin that blunted the effect of PTH on adenylate cyclase activity did not inhibit the stimulatory effects of sodium fluoride or of GppNHp. These results suggest that adriamycin selectively alters the interaction between PTH and its receptor or impairs the transmission of information from hormone-receptor complex to adenylate cyclase (or both), perhaps by binding to specific lipid domains in the plasma membrane. Structural analogues of adriamycin, which vary in their lipophilic properties, also varied in their capacity to perturb the cyclic AMP response. One such analogue in fact inhibited the response to PGE₂, and several appeared to augment the PGE₂ effect. These substances may well be useful in probing the membrane properties required for selectivity in hormone action. Supported by NIH grant AM 19855-07     

In vitro evidence that local and systemic skeletal effectors can regulate3[H]-thymidine incorporation in chick calvarial cell cultures and modulate the stimulatory actions(s) of embryonic chick bone extract

Summary These investigations were intended to determine whether local and systemic skeletal effectors—3′5′-cyclic adenosine monophosphate (cAMP), prostaglandin E₂ (PGE₂), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)₂D), calcitonin, and NaF—could regulate³[H]-thymidine incorporation (i.e., into DNA) in serum-free, monolayer cultures of embryonic chick calvarial cells, and/or modulate the activity of embryonic chick bone extracts to increase³[H]-thymidine incorporation. In the absence of added bone extract, we found that calcitonin (0.1 U/ml), NaF (100 μM) and low-dose PTH (0.1 nM) stimulated³[H]-thymidine incorporation,P<.05 for each; isobutylmethylxanthine (IBMX-1 mM), 1,25OHD (10 nM), and high-dose PTH (10 nM) decreased³[H]-thymidine incorporation; and PGE₂ (1 μM) had no effect. The stimulatory actions of calcitonin, fluoride, and low-dose PTH were inductive, and the inhibitory actions of IBMX and 1,25(OH)₂D were acute. PTH had complex time-dependent actions on³[H]-thymidine incorporation, being inhibitory after 4–8 hours of exposure and stimulatory after 20–24 hours (P<.001 for each). The effects of calcitonin, fluoride, and low-dose PTH to increase³[H]-thymidine incorporation were greater in calvarial cell cultures enriched for undifferentiated osteoprogenitor cells than in cultures enriched for differentiated osteoblastlike cells. PTH inhibited³[H]-thymidine incorporation in the latter (i.e., osteoblastlike) cultures (P<.005). The inhibitory actions of IBMX and 1,25(OH)₂D were independent of cell differentiation. Additional studies further revealed that these local and systemic skeletal effectors could also modulate the activity of embryonic chick bone extracts to increase³[H]-thymidine incorporation in calvarial cell cultures. We found that calcitonin, fluoride, and low-dose PTH enhanced the effect of the extracts to increase³[H]-thymidine incorporation (P<.001 for each). These activations were noncompetitive, indicating (1) mechanistic differences between the stimulatory actions of the effectors and the chick bone extract (i.e., different rate-limiting steps for the effects of each on³[H]-thymidine incorporation); and (2) that neither calcitonin, fluoride, nor 0.1 nM PTH altered the apparent affinity of the cells for stimulatory activity(s) in the extract. High-dose PTH was a noncompetitive inhibitor with respect to bone extract activity, indicating that the effect of 10 nM PTH to decrease³[H]-thymidine incorporation was mechanistically distinct from the effect of the bone extract to increase³[H]-thymidine incorporation. Both IBMX and PGE₂ were competitive inhibitors of bone extract-stimulated³[H]-thymidine incorporation (P<.001 for each), implying that these effectors (IBMX, PGE₂, and embryonic chick bone extract) shared a common (or coincidentally equal) rate-limiting step. The effects of 1,25(OH)₂D on bone extract-stimulated³[H]-thymidine incorporation were different at high and low doses. At a low concentration (1 nM), 1,25(OH)₂D enhanced the effect of bone extract to increase³[H]-thymidine incorporation, but higher concentrations (e.g., 100 nM) were inhibitory (P<.01 for each). Together, these data demonstrate that local and systemic skeletal effectors can have direct effects on embryonic chick calvarial cells,in vitro, to regulate the basal rate of³[H]-thymidine incorporation, and to modulate the stimulatory action of an embryonic chick bone extract.     

The effect of strain on bone cell prostaglandin E2 release: A new experimental method

Summary A new method of investigating the mechanisms of strain-induced bone remodeling has been developed. Bone cells were subjected to cyclical strainsin vitro by computer-controlled stretching of the plastic substrate on which they were cultured, enabling both physiological and pathological strains to be investigated. Physiological strains have not previously been investigatedin vitro. The prostaglandin E₂ (PGE₂) released by the cells was found to depend on the strain magnitude. It was independent of cycle time, and 5 hours after straining had ceased, it had returned to control levels. These results are similar to thein vivo findings that bone remodeling is dependent on strain magnitude and not strain frequency, indicating that PGE₂ may play an important role in strain-induced bone remodeling. The relationship between PGE₂ release and strain magnitude was biphasic, with particularly high levels being released at strains that would be associated with either abnormally strenuous activity or microstructural bone damage. It is therefore possible that PGE₂ stimulates the osteogenesis caused by increased functional demands, and initiates the remodeling caused by bone damage. This new method of investigating strain-induce remodeling is useful, as any cell type, any mediator, and any strain pattern or parameter can be individually studied.     

The hemodynamic and metabolic changes in prostaglandin E1-induced hypotension in dogs

The hemodynamic and metabolic changes in hypotensive state induced with prostaglandin E₁ (PGE₁) or trimetaphan (TMT) infusion were investigated in dogs. Mean arterial pressure was decreased by about 50% with 1.58µg/kg/min of PGE₁ or 45µg/kg/min of TMT. Heart rate, pulmonary capillary wedge pressure and central venous pressure remained virtually unchanged in the two groups. Cardiac output was well maintained in PGE₁ group, whereas cardiac output showed the tendency to decline in TMT group.Greater reduction in systemic vascular resistance was seen in PGE₁ group than in TMT group. Pulmonary vascular resistance showed no significant change in PGE₁ group, whereas it increased significantly in TMT group. Gradual decreases in arterial pH, Pa_{O 2} and base excess and slight but significant increase in Pa_{CO 2} was observed in PGE₁ group, and these abnormalities recovered 30min after hypotension. Abnormalities in blood gases and acid-base balance were considerably more severe and prolonged in TMT group compared with those in PGE₁ group. Blood lactate and pyruvate concentrations showed no significant changes in PGE₁ group, whereas substantial elevation was seen in L/P ratio especially 30min after induction of hypotension in TMT group. Oxygen consumption showed minimal changes in PGE₁ group, whereas a significant decrease was observed in TMT group. The conclusions derived from these results are as follows;1) PGE₁ maintained cardiac output better than TMT, probably because of its direct inotropic action on the heart, and of its greater reduction of systemic vascular resistance than TMT.2) PGE₁ seemed to provide the better blood perfusion throughout the body than TMT.3) PGE₁ showed less possibility to produce the metabolic derangement compared with TMT.(Nam YT, Takahashi S, Tominaga M et al.: The hemodynamic and metabolic changes in prostaglandin E₁-induced hypotension. J Anesth 3: 210–217, 1989)     

Keloid-derived fibroblasts have a diminished capacity to produce prostaglandin E2

Keloids result from pathological wound healing responses. However, the pathogenesis of keloids is still poorly understood. PGE2 was shown to decrease fibroblast proliferation, inhibit collagen synthesis and enhance the expression of matrix-metalloproteinases (MMPs). This study sought to delineate the production of PGE2 by normal and keloid-derived dermal fibroblasts. Human normal and keloid dermal fibroblasts were cultured in vitro. Cell proliferation and viability were determined based on WST-1 assay. IL-1beta-induced PGE2 production and effects of PGE2 on the synthesis of procollagen by culture-derived fibroblasts were determined by using enzyme-linked immunosorbant assay (ELISA) kits. IL-1beta-induced MMP-1 production by culture-derived fibroblasts was determined with an MMP-1 immunoassay kit. Our results showed that normal and keloid-derived fibroblasts exhibited a statistically significant increase (p<0.05) in cell proliferation when the cells were cultured in media with an increase in the concentrations (0%, 2% and 10%) of fetal bovine serum (FBS). In culture medium without FBS, an increase in cell proliferation of keloid-derived fibroblasts was detectable when compared with those of control fibroblasts. IL-1beta (1 ng/ml and 10 ng/ml) stimulated statistically significant production (p<0.01) of PGE2 by both normal and keloid-derived fibroblasts. However, lower levels of PGE2 produced by keloid-derived fibroblasts were detectable compared with those produced by normal-derived fibroblasts (p<0.05). In this study, although not statistically significant, inhibition of procollagen production by PGE2 in a dose-dependent manner was found. In addition, decreased production of MMP-1 by keloid-derived fibroblasts compared with those of control fibroblasts was also observed. In conclusion, keloid-derived fibroblasts produced less PGE2 than those produced by control fibroblasts. The role of diminished capacity of PGE2 production in keloid formation is presently unknown and needs further study.     

Early manifestations of vitamin D effects in rat osteogenic sarcoma cells

Summary We have recently demonstrated that 48 hour exposure of ROS 17/2 cells to low concentrations of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) (1.0 pg/ml) stimulated the cellular accumulation of⁴⁵Ca, and exposure to high concentrations (160 pg/ml) inhibited such accumulation. In the present study, short-term (15 min) effects of the sterol on⁴⁵Ca accumulation in ROS 17/2 cells were compared with the long-term (48 hours) effects in order to clarify mechanisms responsible for 1,25(OH)₂D₃ control of calcium metabolisms in ROS 17/2 cells. ROS 17/2 cells were grown for 48 hours in the presence and absence of 1,25(OH)₂D₃ and then incubated for an additional 15 min in the presence and absence of 1,25(OH)₂D₃ immediately before measuring⁴⁵Ca accumulation. Cellular⁴⁵Ca was measured after incubating the cells in the medium containing 0.5 μCi/ml of⁴⁵CaCl₂ for 4 min at 25°C. The effect of actinomycin D was determined by preincubating the cells in 0.1 μg/ml of actinomycin D for 45 min at 25°C. Exposure to low concentrations (1.0 pg/ml) of 1,25(OH)₂D₃ for either 48 hours or 15 min increased⁴⁵Ca in the cells by 10–20%. An additional 15 min exposure following 48 hour exposure yielded an increase in the cellular⁴⁵Ca similar to that after 48 hours or 15 min exposure. Exposure to high concentrations (160 pg/ml) for either 48 hour or 15 min decreased cell⁴⁵Ca by approximately 20%.An additional 15 min exposure to the high concentrations did not change the 48 hour effect. Actinomycin D reversed early inhibitory effects of high concentrations, but had no effect on the early stimulatory effects of low concentrations. These results suggest that the mechanisms underlying the 15 min and 48 hour effects of 1,25(OH)₂D₃ are the same. Moreover, the mechanism responsible for the inhibitory effect of high concentrations is dependent onde novo protein synthesis whereas that for the stimulatory effects of low concentrations is not.     

软骨Ⅱ号对骨关节炎兔不同部位PGE2选择性抑制作用的实验研究

目的 观察以中医学柔肝为治法的中药软骨方及非甾体类抗炎药扶他林对实验性骨关节炎家兔正常效应部位（胃粘膜,肾皮质）PGE2和发病部位（关节滑液）PGE2的影响。方法 按Hulth法建立家兔关节炎模型,并随机分为正常组、模型组、西药组（扶他林）、中药组（软骨Ⅱ号方）。抽取家兔患膝关节滑液并对相应的胃粘膜及肾皮质组织匀浆,采用放射免疫法测定滑液及匀浆液内PGE2含量。结果 持他林对实验性骨关节炎家兔肾皮质部PGE2无明显影响（P＞0．05）,对胃粘膜和关节滑液中的PGE2有明显抑制作用（P＜0．05）。软骨Ⅱ号方只对关节滑液中的PGE2有一定抑制作用,但其作用强度不及扶他林（P＜0．05）,而对正常效应部位（胃粘膜、肾皮质）的PGE2不抑制。结论 在骨关节炎的治疗中,软骨Ⅱ号方只抑制发病部位关节滑液的PGE2。     

Inhibitory effects of prostaglandin D2 against the proliferation of human colon cancer cell lines and hepatic metastasis from colorectal cancer

The inhibitory action of prostaglandin D₂ (PGD₂) and its effect on the cell cycle were examined in cell lines SW480 and LS174T of human colon cancer. The growth of the cell lines were assessed 24h and 48h after the addition of 1.0μg/ml and 10.0μg/ml PGD₂. The growth of SW480 cells and 24h and 48h after the addition of 10.0μ/ml, while that of LS174T was inhibited by both doses after 24h and 48h. S-Phase DNA synthesis in the SW480 cells was significantly blocked 24h after the addition of 10.0μg/ml PGD₂. The cell cycle of LS174T cells was arrested at the G₀+G₁ phase 24h after the addition of 1.0μg/ml and 10.0μg/ml PGD₂. The correlation between hepatic metastasis and PGD₂ concentration in human cancer tissue was examined. The mean value of PGD₂ concentrations in the primary cancer tissue was significantly lower in the hepatic metastasis group than that in the group without hepatic metastasis. These findings suggest that measuring the PGD₂ in cancer tissue may be useful for detecting and predicting the hepatic metastasis from human colorectal cancer.     

Congenital lack of COX-2 affects mechanical and geometric properties of bone in mice

We compared the mechanical properties of bones from mice lacking either a functional cycloxygenase-1 (C57BL6/DBA COX-1–/–; n = 9) or COX-2 (C57BL6/DBA COX-2–/–; n = 9) gene and wild type mice (C57BL6/DBA; n = 10). Twenty-eight right femora from 3-month-old male mice were used to determine bulk structural and material properties of bone by three-point bending. Bone matrix properties were also measured by nanoindentation to access the changes in bulk mechanical properties due to changes in bone matrix or bone geometry. The bulk material properties (elastic modulus, P < 0.05; ultimate stress, P < 0.01) of COX-2–/– bones were lower than those of wild-type mice whereas the bulk structural properties (stiffness, P > 0.2; breaking force, P > 0.1) were similar to those of the wild-type mice. COX-2–/– mice had a longer moment of inertia but their cortical bones were thinner and contained many more intra-cortical pores compared with the bones of the other two groups. Finally, the bone matrix properties of COX-1–/– mice, COX-2–/– mice and their heterozygous littermates were similar to those of C57BL6/DBA wild-type mice. Deseased on December 30, 2002     

Actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on citrate decarboxylation in osteoblast-like bone cells differ in calcium requirement and in sensitivity to trifluoperazine

Summary The actions of PTH in OB bone cells appear to involve both calcium and cAMP. At present little information exists regarding the relationship, if any, between these two putative second messengers of hormone action in bone cells. In this report the molecular role of calcium in the actions of PTH and 1,25(OH)₂D₃ has been compared, since like PTH, the steroid 1,25(OH)₂D₃ is a potent bone resorbing hormone that exerts inhibition of citrate decarboxylation in OB cells, but unlike PTH does not activate adenylate cyclase. It was found that 1,25(OH)₂D₃ could initiate near maximum inhibition of citrate decarboxylation at extracellular calcium levels as low as 0.05 mM, whereas PTH effects began to be apparent only at 0.1 mM calcium, and maximum inhibition of citrate decarboxylation by PTH required 0.5 mM Ca. In addition, PTH-induced decrease in citrate decarboxylation was inhibited by low doses of TFP, an inhibitor of calmodulin and calcium-dependent, phospholipid-sensitive protein kinases, in contrast to 1,25(OH)₂D₃, whose effects were not reduced by this agent. These results suggest that: (a) the actions of 1,25(OH)₂D₃ may not be directly dependent on calcium influx; (b) in OB cell response to PTH a relationship probably exists between cAMP and calcium; and (c) this relationship may involve calmodulin, or calcium-dependent protein kinases that can be inhibited by TFP.     

Regulation of creatine kinase activity in rat osteogenic sarcoma cell clones by parathyroid hormone,prostaglandin E2, and vitamin D metabolites

Summary We have previously shown that both parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂) stimulate the activity of creatine kinase BB (CKBB) in rat bone cells in culture. Therefore, morphologically distinct rat osteogenic sarcoma cells in culture were tested for stimulation of CKBB activity by hormones that regulate skeletal tissues. PTH stimulated CKBB in the osteoblast-like clone ROS 17/2; 1α,25(OH)₂D₃ inhibited this activity while PGE₂, CT and 24R,25(OH)₂D₃ had no significant effect. PGE₂ stimulated CKBB activity in the fibroblast-like clone ROS 24/1, which was unresponsive to PTH, CT and Vitamin D metabolites. 24R,25(OH)₂D₃ as well as PGE₂ (but not PTH, CT or 1α25(OH)₂D₃) stimulated CKBB in clone ROS 25/1, suggesting that this fibroblast-like clone has some chondroblast-like character. Both PTH and PGE₂ stimulated the brain type isoenzyme of CK (CKBB), although the osteogenic sarcoma cell clones contain a significant proportion of the muscle type of CK (CKMM). Thus, increased CKBB activity can serve as an additional characteristic marker for the action of steroid and polypeptide hormones and for prostaglandins.     

Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

Summary We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)₂D₃ and for glucocorticoids, and have examined the modulation of the 1,25(OH)₂D₃ receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)₂D₃ receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)₂D₃; this phenomenon occurred in a dosedependent fashion for triamcinolone (10⁻⁹ to 10⁻⁷ M) with a maximum increase of 1,25(OH)₂D₃ receptor concentration of ⋍twofold. During the culture period, the 1,25(OH)₂D₃ receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)₂D₃ receptor concentration was higher in the triamcinolone-treated cells.     

Proteomic analysis of dunning prostate cancer cell lines with variable metastatic potential using SELDI-TOF

BACKGROUND: Surface enhanced laser desorption and ionization-time-of-flight (SELDI-TOF) is an evolving proteomic technology for improving biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures generated from body fluids, cells, and/or tissues. SELDI--based profiling identifies unique, differentially expressed proteins relating to specific cancer-related disease states. We utilized SELDI-TOF following pre-processing with molecular separation and chemical fractionation of cell membrane extracts from three Dunning rat prostate cancer cell lines of varying metastatic potential to search novel proteins that are differentially expressed. METHODS: Dunning rat cell sublines of variable (%) metastatic potential; G (0%), AT-1 (20%), and Mat-Ly-Lu (100%) were cultured in two different laboratories. Cell lysis was performed in a homogenation buffer (320 mM sucrose/50 mM Tris/0.5 mM PSMF) using Dounce homogenation. After centrifugation, the membrane pellet was washed 2x and then solublized in 2% CHAPS/8 M urea. This sample was further processed using positive pressure molecular ultrafiltration at 30 kDa or precipitation with 50% ammonium sulfate. Next, each sample was applied to an IMAC3-Ni ProteinChip (Ciphergen Biosystems, Freemont, CA) and analyzed using Ciphergen's Protein Biology System with protein peak analysis software. RESULTS: SELDI-TOF analysis differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20,000 Da. The preparations from the three cells lines showed clear differences when the extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kDa (decrease), 12 kDa (significant decrease), 14 kDa (decrease), and 17 kDa (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate laboratory, and using different lots of SELDI ProteinChips. CONCLUSIONS: The application of SELDI-TOF to a series of Dunning rat prostate cancer cell lines illustrated apparent changes in protein profiles among the three cell lines with known differences in metastatic biologic activity. SELDI-TOF identified four reproducible changes in protein expression in the AT1 and MLL metastatic cell sublines. Three of the expression changes were manifested as decreases, but one protein (17 kDa) was over-expressed in the AT1 and MLL cell lines. Emphasis will be placed on the isolation, purification, and characterization of the 17 kDa over-expressed protein and its potential role in PCa metastasis.     

Role of prostaglandins in the pathogenesis of X-linked hypophosphatemia

X-linked hypophosphatemia is an X-linked dominant disorder resulting from a mutation in the PHEX gene. PHEX stands for phosphate-regulating gene with endopeptidase activity, which is located on the X chromosome. Patients with X-linked hypophosphatemia have hypophosphatemia due to renal phosphate wasting and low or inappropriately normal levels of 1,25-dihydroxyvitamin D. The renal phosphate wasting is not intrinsic to the kidney but likely due to an increase in serum levels of fibroblast growth factor-23 (FGF-23), and perhaps other phosphate-wasting peptides previously known as phosphatonins. Patients with X-linked hypophosphatemia have short stature, rickets, bone pain and dental abscesses. Current therapy is oral phosphate and vitamin D which effectively treats the rickets and bone pain but does not adequately improve short stature. In this review, we describe recent observations using Hyp mice; mice with the same mutation as patients with X-linked hypophosphatemia. We have recently found that Hyp mice have abnormal renal prostaglandin production, which may be an important factor in the pathogenesis of this disorder. Administration of FGF-23 in vivo results in phosphaturia and an increase in prostaglandin excretion, and FGF-23 increases proximal tubule prostaglandin production in vitro. In Hyp mice, indomethacin improves the phosphate transport defect in vitro and in vivo. Whether indomethacin has the same effect in patients with X-linked hypophosphatemia is unknown.     

The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells

Summary Controversy exists over a direct effect of 1,25(OH)₂D₃ on PTH secretion. To investigate the possibility that the suppressive effect of 1,25(OH)₂D₃ on PTH secretion may be demonstrable in 1,25(OH)₂D₃-depleted tissue and/or after prolonged periods of exposure to 1,25(OH)₂D₃, primary monolayer cultures of bovine parathyroid cells were established in 1∶1 DMEM/Ham's F-12 media supplemented with 2% calf serum but not 1,25(OH)₂D₃. Ionized calcium was maintained at 1.0 mM. Experiments were performed on 4-day-old culture cells. PTH concentration was measured using both a mid-region/carboxyl and an amino-terminal PTH antisera. 1,25(OH)₂D₃ at a concentration of 0.1 ng/ml suppressed PTH secretion by 32±7% after 48 hours. High calcium concentration (2.0 mM) suppressed PTH secretion by 37±10% and this effect was not additive over that of 1,25(OH)₂D₃. PTH secretion rate recovered fully 48 hours after normalization of the external calcium concentration but not after the removal of 1,25(OH)₂D₃. It is concluded that 1,25(OH)₂D₃ directly suppresses PTH secretion by monolayer culture of bovine parathyroid cells.     

Functional angiotensin II type 2 receptors inhibit growth factor signaling in LNCaP and PC3 prostate cancer cell lines

BACKGROUND: There is clear evidence of a tissue-based renin-angiotensin system in the prostate and studies to date suggest that AT(1)-receptor blocking drugs inhibit the growth of some prostate cancer cell lines and delay the development of prostate cancer. The present studies examine the action of Ang II in two prostate cancer cell lines and report the presence of functional AT(2)-receptors that regulate the actions of growth factors. METHODS: Immunohistochemistry was used to identify the presence of Ang II and QPCR techniques to examine AT(1)- and AT(2)-receptor mRNA expression in androgen-dependent (LNCaP) and independent (PC3) cell lines. The effects of AT(1)- and AT(2)-receptor activation upon EGF-induced DNA synthesis and ERK2 phosphorylation in these cells were also examined. RESULTS: Functional AT(2)-receptors together with Ang II were identified in both cell lines and stimulation of these receptors inhibited EGF-induced DNA synthesis and ERK2 phosphorylation. AT(1)-receptors, although present in both cell lines, were only functional in LNCaP cells where activation stimulated DNA synthesis. CONCLUSIONS: Functional AT(2)-receptors are present and have the capacity to inhibit EGF-induced prostate cancer cell growth in LNCaP and fast growing androgen-independent PC3 cell lines, whereas functional AT(1)-receptors are found only in LNCaP cells where their activation stimulates DNA synthesis.