Interactions between ipriflavone and the estrogen receptor

Estrogen replacement therapy is effective in the prevention of postmenopausal osteoporosis, and a direct action of 17--estradiol (17E₂) on osteoblastic and osteoclastic cells has been demonstrated. The inhibition of bone resorption by ipriflavone (IP), an isoflavone derivative devoid of estrogenic properties but active in potentiating the effects of estroge on bone tissue, has been shown in in vitro and in vivo studies and confirmed by clinical data. To investigate the molecular mechanisms that underlie IP effect, we studied the possible interactions of IP and its four main in vivo metabolites (I, II, III, and V) with the estrogen receptor (ER) in the human preosteoclastic cell line FLG 29.1, whose growth and function are modulated by the compound. In parallel experiments, the human breast cancer cell line MCF7 was also analyzed. IP binding sites were demonstrated in the nuclear fraction of FLG 29.1 cells. 17E₂ and other steroid compounds failed to displace IP binding to intact FLG 29.1 cells. Similarly, IP and metabolites I, III, and V were not able to displace 17E₂ binding to intact MCF7 cells, whereas metabolite II showed an IC₅₀ of 61 nM. 17E₂ binding to FLG 29.1 cells was increased after preincubation with metabolites I, III, and V. IP and its metabolites did not induce FR-dependent gene expression in FLG 29.1 and MCF7 cells transfected with a reporter gene and an estrogen response element (ERE). These results suggest that IP effects on osteoclast precursors are not mediated by a direct interaction with the ER, even if a crosstalk between the mechanisms of action of IP and 17E₂ cannot be excluded.     

A Human Homolog of the 150 kD Avian Osteoclast Membrane Antigen Related to Superoxide Dismutase and Essential for Bone Resorption is Induced by Developmental Agents and Opposed by Estrogen in FLG 29.1 Cells

Osteoclast development from hematopoietic bone marrow precursors is associated with the expression of various enzymes, receptors, adhesion molecules, and other specialized components. Among these is a novel 150 kD superoxide dismutase-related membrane glycoprotein, originally identified by its reaction with the anti-osteoclast monoclonal antibody 121F. This antigen is uniquely restricted to osteoclasts in bone, universally present on osteoclasts from multiple species, induced during osteoclast differentiation in vitro and in ovo, and required at high levels for avian osteoclastic bone pit resorption. Expression of a comparable human antigen was investigated using human leukemic FLG 29.1 cells capable of differentiating towards an osteoclast-like phenotype. Phorbol ester, 1,25 (OH)₂ vitamin D₃, and osteoblast-derived soluble factors elicited dose and time-dependent inductions of this antigen as measured by enzyme-linked immunosorbent assay (ELISA) and immunocytochemical staining, coincident with their display of multiple other osteoclastic features. Synergistic interactions of these modulators led to further elevations in the ultimate expression levels of this antigen, although not to the full extent associated with in vivo-formed avian osteoclasts. The potent antiresorptive hormone 17β-estradiol, but not its inactive α isomer, partially suppressed the phorbol ester-induced elevation of the 121F antibody-reactive antigen in FLG 29.1 cells as it does in avian osteoclast-like cells. Characterization of the human antigen isolated from FLG 29.1 cells by 121F immunoaffinity purification demonstrated that this regulated membrane component was synthesized by these human cells, more abundant following their differentiation into osteoclast-like cells, and similar biochemically and immunologically to the 150 kD integral membrane glycoprotein previously described from avian osteoclasts. Therefore, this report is the first documentation that human osteoclast-like FLG 29.1 cells express, in a developmentally regulated fashion, a homolog of the specific 150 kD avian osteoclast surface antigen that is related to superoxide dismutase, a protective free radical scavenging enzyme and is essential for osteoclastic bone resorption. Received: 1 April 1996 / Accepted: 19 July 1996     

Effects of transforming growth factor-β1 on formation and activation of osteoclasts

Transforming growth factor-₁ (TGF-₁) has biological functions in various types of cells. However, its roles in the regulation of osteoclast formation and function are unclear. To examine them, we employed a culture system in which unfractionated cells obtained from long bones of 13-day-old mice were cultured on a dentine slice. We found that TGF-₁ has a potent inhibitory effect on osteoclastic bone resorption at a dose of 0.2–5 ng/ml. By electron microscopy the osteoclasts appeared to have fewer mitochondria and ruffled borders than those in control cultures. But in the presence of 1,25-dihydroxyvitamin D₃, [1,25-(OH)₂D₃], TGF-₁ at a dose of 0.2–1 ng/ml stimulated the formation of osteoclasts from unfractionated bone cell cultures in which preexistent osteoclasts had degenerated. Thus, using stromal cell-free he-mopoietic blast cells, we examined the direct action of TGF-₁ on osteoclast precursors. Although TGF-₁ inhibited tartrate-resistant acid phosphatase-positive (TRAP) multinucleate cell (MNC) formation induced by 1,25-(OH)₂D₃, the conditioned medium (CM) of TGF-₁-treated MC3T3-E1 cells stimulated such formation. These results suggest that TGF-₁ inhibits osteoclastic bone resorption but stimulates osteoclast formation via the action of factor(s) produced by TGF-₁-treated osteoblasts in the presence of 1,25-(OH)₂D₃.     

Effects of lead on osteoclast-like cell formation in mouse bone marrow cell cultures

To examine an effect of lead (Pb) on the process of osteoclast-like cell formation from its progenitors, we used a mouse bone marrow culture system in which osteoclast-like multinucleated cells (MNCs) were formed in response to bone-resorbing agents. In a 9-day culture period, Pb dose-dependently stimulated MNC formation over the concentration range 2–10 M, whereas at 40 M Pb, MNC formation declined. In an 11-day culture period, MNC formation reached a maximum at 5 M Pb and decreased with increasing concentration of Pb at 10–40 M. Pb-stimulated MNC formation was inhibited by both indomethacin and SC19220, an antagonist of prostaglandin E₂ (PGE₂) receptor. Pb stimulated the production of PGE₂ in marrow cell cultures, suggesting that Pb-stimulated MNC formation is dependent on the production of PGE₂. 3-Isobutyl-1-methylxanthine potentiated Pb-stimulated MNC formation and 2,5-dideoxyadenosine, an inhibitor of adenylate cyclase, inhibited it. A calcium ionophore A23187 increased Pb-induced MNC formation and verapamil, a calcium channel blocker, depressed it. It is possible that a PGE₂-induced increase in the levels of cyclic adenosine 3,5-monophosphate (cAMP) and calcium ions in marrow cells is involved in Pb-induced MNC formation. Pb and parathyroid hormone showed a synergistic stimulation on MNC formation. From these results, Pb is thought to induce osteoclast-like cell formation by a mechanism involving PGE₂ which increases the intracellular levels of cAMP and calcium ions.     

Effect of 1,25-dihydroxycholecalciferol on adenosine 3′,5′-cyclic monophosphate production in calvaria of mice

Summary 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃), added at doses which stimulate bone resorption in the system used, did not increase adenosine 3,5-cyclic monophosphate (cAMP) production in vitro in mouse calvaria incubated for up to 48 h. Thus 1,25-(OH)₂D₃ does not appear to stimulate bone resorption through involvement of an increased cAMP production.     

Testosterone metabolism in primary cultures of epithelial cells and stroma from benign prostatic hyperplasia

We studied the metabolism of testosterone in primary cultures of prostate epithelial cells and fibroblasts obtained from patients with benign prostatic hyperplasia (BPH). The conversion of ³H-testosterone in both cell cultures was predominantly to the oxidative pathway, with the formation of ³H-androstenedione increasing with cell number and time of incubation. Although we also detected some 5-reductase activity in these cells, the activity in the stroma component (0.00688 pmol/mg protein/min) was nonetheless insignificant when compared to the 5-reductase activity in the tissue of origin (0.0616 pmol/mg protein/min) and well below the 17-hydroxysteroid dehydrogenase activity of the same cells (0.0518 pmol/mg protein/min). The aromatase activity in our cells was also measured by two separate techniques, but neither the deuterium procedure nor the production of oestrone from androgen precursors yielded any positive results, suggesting that under these experimental conditions there was no aromatase activity within the cells. The shift from the reductive to the oxidative pathways in these primary cell cultures was reminiscent of the androgen-metabolizing enzyme profiles seen in poorly differentiated prostate cancer. Whether this fransition is an obligatory step in the development of hormone refractiveness remains to be elucidated.     

Zur Bedeutung von β-hCG im serum als tumormarker fur das magenkarzinom

Zusammenfassung Einleitung: Nach neueren Untersuchungen ist davon auszugehen, daß bei des Hälfte von Patienten mit einem Magenkarzinom -hCG-positive Zellen im Tumor immunhistochemisch gefunden werden können. Ziel war daher, systematisch zu untersuchen, inwieweit -hCG-immunreaktive Magenkarzinome von einem Anstieg des Serum--hCG begleitet werden and dieses damit als Verlaufsparameter zur Verfügung steht. Methode: Bei 54 Patienten mit einem Magenkarzinom wurde zur immunhistochemischen Darstellung ein gegen -hCG gerichteter monoklonaler Antikörper (Fa. Sigma, 1:100) im APAAP-System verwendet. Die Auswertung wurde nach positiver and negatives Reaktion graduiert. Parallel wurde im Serum des Patienten -hCG präoperativ mit einem Enzymimmunoassay (MEIA, Fa. Abbot) bestimmt. Tumor-stadium, Grading and Tumor-lokalisation werden in die Auswertung mit einbezogen. Ergebnisse: Es wird bestätigt, daß 41% (22 von 54) des Karzinome, unabhängig von ihrer Lokalisation im Magen, eine positive immunhistochemische Reaktion gegen -hCG auslösen. Es zeigte sich in Abhängigkeit vom Tumorstadium eine positive -hCG-Immunreaktivität in 27% (6 von 22) des Tumoren ohne Lymphknoten- and Fernmetastasierung (T_1–4 N₀ M₀), in 54% (7 von 13) des Tumoren mit Lymphknotenaber ohne Fernmetastasen (T_1–4 N₁ M₀) und in 47% (9 von 35) des Tumoren mit Fernmetastasierung. Schlecht differenzierte Tumoren (G3–4) waren zu 42% (15 von 36) und gut differenzierte Tumoren (G_1–2) nur zu 39% (7 von 18) positiv. Aber lediglich bei einer Patientin war der -hCG-Spiegel im Serum erhöht. Zusammenfassung: Immunhistochemisch -hCG-positive Magenkarzinome werden vermehrt bei fortgeschrittenem Tumorstadium und Schlecht differenzierten Karzinomen gefunden. Diese Kar zinome scheinen aber nicht in ausreichender Menge -hCG ins Serum abzugeben, was zu serologisch meßbar erhöh-ten Werten führt. -hCG im Serum kann daher nicht als Prognosefaktor bzw. zur Verlaufskontrolle herangezogen werden. Abzuwarten bleibt, inwieweit die -hCG-Expression von Tumorzellen u. U. Einfluß auf die Propose der Patienten besitzt.

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Significance of -hCG in the serum as a tumour marker for gastric cancer

Introduction: Recent investigations indicate that in 50% of patients with gastric cancer, -hCG-posiitive cells can be found in the tumour by immunohistochemical investigations. The objective of this study was to investigate how often -hCG-immunoreactive gastric carcinomas were accompanied by an elevation in serum -hCG, that could have been used as a course control variable. Methods: In 54 patients with gastric carcinoma a monoclonal antibody directed against -hCG was used for immunohistochemical marking in the APAAP system. The evaluation was graded positive or negative. In parallel, serum -hCG was determined preoperatively using an enzyme immunoassay (MEIA). Tumour stage, grading and tumour locallization were determinants in the evaluation. Results: We found that 41% (22 of 54) of the carcinomas induced a :positive immunohistochemical response to -hCG, regardless of their location in the stomach. In relation to tumour stage, a positive -hCG immunoreactivity was apparent in 27% (6/22) of tumours without lymph node or distant metastases (TI -4N0M0), in 54% (7/13) of tumours with lymph node and without distant metastases (T1–4N1 M0) and in 47% (9/35) of tumours with distant metastases. Poorly differentiated tumours (G3–4) were positive in 42% (15/36) and well-differentiated tumors (G1–2) in 39% (7/18) of cases. In only 1 patient was the -hCG, level in serum elevated, however. Conclusions: -hCG-Positive gastric carcinomas are found more frequently in advanced tumour stages and poorly differentiated carcinomas. These carcinomas, however, seem not to excrete -hCG in sufficient amounts to produce measurable serum values. Therefore, -hCG cannot be used a prognostic factor or for course control. The relevance of -hCG expression of tumour cells to the patients' prognosis remains obscure.

     

Bone-resorbing activities of 24-epi-1α-hydroxyvitamin D2 and 24-epi-1α,25-dihydroxyvitamin D2

Bone-resorbing activities of 24-epi-1-hydroxyvitamin D₂ [24-epi-1(OH)D₂], 24-epi-1,25-dihydroxyvitamin D₂ [24-epi-1,25(OH)₂D₂], and 1,24S,25-trihydroxyvitamin D₂ [1,24S,25(OH)₃D₂], which might be a metabolite of 24-epi-1,25(OH)₂D₂, were investigated. In an in vitro bone resorption test, the activity of 24-epi-1(OH)D₂ was similar to that of 1-hydroxyvitamin D₃ [1(OH)D₃] at 10^-9 M-10^-6 M. The activity of 24-epi-1,25(OH)₂D₂ was weaker than that of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] at 10^-11 M-10^-8 M. On the other hand, the activity of 1,24S,25(OH)₃D₂ was similar to that of 24-epi-1,25(OH)₂D₂ at 10^-11 M-10^-9 M. In the formation assay of osteoclast-like cells, the activity of 24-epi-1(OH)D₂ was weaker than that of 1(OH)D₃ at 10^-7 M. The activity of 24-epi-1,25(OH)₂D₂ was almost similar to that of 1,25(OH)₂D₃ at 10^-11 M-10^-7 M. The activity of 1,24S,25(OH)₃D₂ was significantly weaker than that of 24-epi-1,25(OH)₂D₂ at 10^-11 M-10^-9 M. In the two experiments, the potencies of 24-epi-1,25(OH)₂D₂ were about 100 times higher than those of 24-epi-1(OH)D₂. In an in vivo/in vitro bone resorption test, the activity of 24-epi-1(OH)D₂ was almost similar to those of 1(OH)D₃ and 1,25(OH)₂D₃ and higher than those of 24-epi-1,25(OH)₂D₂ and 1,24S,25(OH)₃D₂. 24-epi-1-(OH)D₂ and 1(OH)D₃ were longer lasting than 24-epi-1,25(OH)₂D₂ and 1,25(OH)₂D₃ in this experiment. These results suggested that 24-epi-1(OH)D₂ as well as 1(OH)D₃ was converted into dihydroxy form in vivo.     

Expression of interferon-γ receptors on bladder cancer cells: does it correlate with biological response?

Summary Previously we have shown a differential biological response of three human bladder cancer cell lines (RT4, RT112 and MGH-U1) to gamma interferon (IFN-). The present study examines the relationship between the biological response and the expression of the interferon- receptor on the tumour cell surface. Using a competitive radioligand binding assay and Scatchard analysis, we measured the number and affinity of the IFN- receptors on each of the above cell lines. Individual cells from each line expressed large numbers (29,100–41,800) of high-affinity receptors (k _d =2.4–3.9×10¹⁰M). There was no statistically significant difference in either of these parameters between the three lines. We therfore conclude that the biological response of these bladder lines to IFN- does not relate to the number or affinity of its receptor on the plasma membrane of these tumour cells.     

Chromatographic separation of two acid phosphatases from rat bone

Summary Extracts of tibiae of suckling rats were prepared with 0.3 M KCl containing 0.1% Triton X-100 and were chromatographed with CM-52 cellulose. Most of the acid phosphatase activity determined with p-nitrophenylphosphate (p-NPP) was bound to the cellulose and could be eluted with a sodium acetate buffer gradient in 2 distinct peaks. The major peak, E₂, was bound strongly to the cellulose and showed high activity with p-NPP and inorganic pyrophosphate (P-P_i), but only slight activity with-glycerophosphate (-GP) and was unaffected by tartrate. The minor peak, E₁, was weakly bound to the adsorbent, showed equal activity with p-NPP and-GP, but negligible activity with P-P_i and was completely inhibited by tartrate. These results support earlier evidence suggesting that bone contains at least 2 different acid phosphatases and that the more abundant enzyme may function as a pyrophosphatase.     

β2-microglobulin in Paget's bone disease

On the basis of earlier findings of increased serum ₂-microglobulin concentration in women with postmenopausal osteoporosis, we decided to study serum ₂-microglobulin concentration in other bone diseases. In 28 patients with untreated Paget's bone disease, serum ₂-microglobulin concentration was normal (1.49±0.41 mg/liter versus 1.36±0.21 mg/liter in 42 control subjects, P= ns), a finding that contradicts reports in the literature. We found that serum ₂-microglobulin concentration was related negatively and significantly (r²=–0.154, P=0.0354) with serum total alkaline phosphatase concentration, but not with serum tartrate-resistant acid phosphatase concentration (p =ns). Urinary elimination of ₂-microglobulin was lower in the patients with Paget's disease than in the controls (34±28 versus 120±21 mg/liter, P<0.001). These findings suggest that ₂-microglobulin behaves similarly to osteocalcin (BGP) in Paget's bone disease and that its concentration remains within normal levels perhaps because of the rate of reuptake of ₂-microglobulin in bone neoformation.     

Changes in plasma catecholamine levels following injection of prostaglandin F2α into the basal cistern in rabbits

We measured plasma epinephrine and norepinephrine concentrations in a rabbit model simulating subarachnoid hemorrhage (SAH), following the injection of prostaglandin F₂ (PGF₂) into the basal cistern. In this model, plasma epinephrine values increased significantly (to 4.2-fold those before injection), substantially more than norepinephrine (which increased 1.3-fold) at 5 minutes (min) after PGF₂ injection. Dissection of autonomic outflow from the cervical spinal cord or ligation of the suprarenal veins reduced the changes in plasma catecholamine concentrations associated with PGF₂ injection. These results suggest that the sympathetic discharge seen after PGF₂ injection into the basal cistern in rabbits occurred through the sympatho-adrenal pathways.(Yokoyama Y, Uchida M, Matsumoto S, et al.: Changes in plasma catecholamine levels following injection of prostaglandin F₂ into the basal cistern in rabbits. J Anesth 6: 161–166, 1992)     

β2-Microglobulin in postmenopausal osteoporosis

Summary The so-called bone-derived growth factor, or ₂-microglobulin, has a regulatory function in bone metabolism, stimulating osteoclastic activity. Osteoclastic activity is enhanced in postmenopausal osteoporosis, suggesting that ₂-microglobulin concentration may also be increased in this disease. ₂-microglobulin concentration was found to be raised (P < 0.001) in 30 women with postmenopausal osteoporosis as compared with 30 normal women of similar age; tartrate-resistant acid phosphatase concentration also was raised (P < 0.001), and total body bone mineral content was decreased (P < 0.001). Linear regression analysis revealed a highly negative correlation result between total body bone mineral content and ₂-microglobulin (r = 0.577,P < 0.001), and a positive correlation result between ₂-microglobulin and tartrate-resistant acid phosphatase concentration (r² = 0.806,P < 0.001). These findings, and the stimulatory effect of ₂-microglobulin on osteoclastic and osteoblastic activity, suggest that ₂-microglobulin may play an important role as a local regulatory factor in the pathogenesis of postmenopausal osteoporosis.     

The effect of sodium salicylate on the osteoclast-like cell formation and bone resorption in a mouse bone marrow culture

Salicylates are reported to have an inhibitory effect on bone resorption in vivo and in vitro. The present study examined the effect of sodium salicylate on the formation of osteoclast-like cells in vitro. When mouse bone marrow cells were cultured for 8 days with 10^-8 M 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), numerous clusters of mononuclear and multinucleated cells (MNCs) formed, which stained positive for tartrate-resistant acid phosphatase (TRAP-positive). In similar cultures using sodium salicylate, the number of both TRAP-positive mononuclear and TRAP-positive MNCs were found to diminish in proportion to the concentration of sodium salicylate. A time-course experimental model showed that the number of TRAP-positive MNCs decreased slightly when sodium salicylate was given early in the culture period, and decreased markedly when the drugs were given later in the culture period. Pit formation and bone-resorption area on the bone slices were also inhibited by adding sodium salicylate continuously with 1,25(OH)₂D₃. The sodium salicylate showed no cytotoxic effect because the total number of adherent cells, including both TRAP-positive and TRAP-negative cells, was independent of the presence of sodium salicylate. These results suggest that sodium salicylate has an inhibitory effect on the recruitment of osteoclast-like MNCs and that this inhibition is greater during the later stage of mouse bone marrow culture.     

Blockade of the pore-forming P2X7 receptor inhibits formation of multinucleated human osteoclasts in vitro

Osteoclasts are large, multinucleated, terminally differentiated cells formed by the fusion of mononuclear hemopoietic precursors. Their function is the resorption of bone, which is an essential part of the growth, modeling and remodeling of the skeleton. Though some osteoclast differentiation factors have recently been identified, the molecular basis for the fusion process that leads to multinucleation is poorly understood. The ATP-gated P2X₇ receptor is a plasma membrane receptor belonging to the family of P2X purinergic receptors. It is known to be expressed by cells of hemopoietic origin where its activation leads to multiple downstream events including cytokine release, cell permeabilization and apoptosis. More recently this receptor has been implicated in the generation of multinucleated giant cells and polykaryons. Here we show that human osteoclasts express P2X₇ receptors in vitro and in vivo, and that these receptors are functional in vitro, as assessed by pore-formation studies. More importantly, blockade of the P2X₇ receptor with the antagonist oxidized ATP or a blocking monoclonal antibody significantly inhibits the fusion of osteoclast precursors to form multinucleated osteoclasts. Taken in combination with previous results from our laboratory demonstrating P2X₇ receptor-mediated apoptosis and inhibition of bone resorption in vitro, these data suggest an important role for the P2X₇ receptor in the regulation of the osteoclast population. The P2X₇ receptor provides a significant new target for modulating osteoclast function in diseases characterized by increased osteoclast number and excessive bone turnover. Abbreviations: TRAP, tartrate-resistant acid phosphatase; RANKL, receptor activator of nuclear factor-B ligand; MCSF, macrophage colony-stimulating factor; BzATP, 2,3-(4-benzoyl)-benzoyl-ATP; oATP, oxidized ATP; APBMC, adherent peripheral blood mononuclear cells; OC, osteoclast.     

Calcium regulating activity of 26,27-dialkyl analogs of 1α,25-dihydroxyvitamin D3

Summary A series of analogs of 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] with alkyl substitutions in 26- and 27-positions were tested for calcium (Ca) regulating activity. The potencies of dialkyl analogs in stimulating bone resorption in neonatal mouse calvaria cultures were the highest in 1,25-dihydroxy-26,27-dimethylvitamin D₃[1,25(OH)₂-(Me)₂D₃], followed by 1,25(OH)₂D₃, 1,25-dihydroxy-26,27-diethylvitamin D₃[1,25(OH)₂(Et)₂D₃], and 1,25-dihydroxy-26,27-dipropylvitamin D₃[1,25(OH)₂(Pr)₂D₃] in that order. A similar order of potential regarding formation of osteoclast-like cells in mouse bone marrow cell cultures and on bone Ca mobilization with long-term vitamin D-deficient rats was observed in the same series. The relative potencies of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 1,25(OH)₂(Et)₂D₃, and 1,25(OH)₂(Pr)₂D₃ in competing with 1,25(OH)₂D₃ for binding to chick intestinal cytosol receptors were 1:1:0.16:0.036. A similar order of potential in case of intestinal Ca transport in situ was observed in the same series. The potencies of dialkyl analogs in competing with 25-hydroxy-vitamin D₃ for binding to rat serum vitamin D binding protein were much lower than that of 1,25(OH)₂D₃. Effect of 1,25(OH)₂(Me)₂D₃ on osteopenia in rats induced by ovariectomy and right sciatic neurotomy was higher than that of 1,25(OH)₂D₃. From these results, the lengthening by one carbon at 26- and 27-positions was shown to maintain the Ca regulatory activity of 1,25(OH)₂D₃.     

Differentiation of pancreatic ductal carcinoma cells associated with selective expression of protein kinase C isoforms

Background: The signal transduction pathways important in regulating the growth and differentiation of malignant cells are poorly understood. Recent evidence has implicated activation of the protein kinase C (PKC) family of signaling proteins in pancreatic carcinoma during cytokine-induced cytostasis and differentiation. Methods: A human pancreatic adenocarcinoma (HPAC) cell line was exposed to tumor necrosis factor- (TNF-; 40 ng/ml) for 6 days. Cytostasis and viability were confirmed by daily MTT [(3(4,5)-dimethyl-thiazol-2-yl) 2,5-diphenyl-tetrazolium bromide] and trypan exclusion assay. Protein fractions were isolated daily and subjected to immunoblot analysis for the normal (terminally differentiated) pancreatic ductal cell marker carbonic anhydrase II (CA II) as well as specific PKC isoforms (, , , , and). Results: Growth arrest occurred in HPAC cells after exposure to TNF- for 48 h, with viability maintained above 90% throughout the 6-day time course. CA II immunoreactivity was not detected in untreated controls but appeared after 2 days of TNF- exposure, peaking on day 6. Concurrently, TNF- induced the selective downregulation of PKC-, whereas PKC- levels increased. PKC- and PKC- immunoreactivity did not change. The atypical PKC- isoform developed a doublet banding pattern in response to TNF-, although overall PKC- levels did not change. Conclusions: TNF--induced growth arrest and differentiation in HPAC cells is associated with the selective downregulation of PKC- and upregulation of PKC-.Presented at the 48th Annual Cancer Symposium of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

Effects of 1α-hydroxyvitamin D3 on lumbar bone mineral density and vertebral fractures in patients with postmenopausal osteoporosis

The effects of 1-hydroxyvitamin D₃ [1(OH)D₃] on bone mineral density, fracture incidence, and bone metabolism were evaluated by a double-blind, placebo-controlled study. Eighty postmenopausal osteoporotic Japanese women (71.9±7.3 years, mean±SD) were randomly assigned to 1 g of 1(OH)D₃ daily or inactive placebo for 1 year. All patients were given supplemental calcium (300 mg of elemental calcium daily). Lumbar (L2–L4) bone mineral density (BMD) determined by dual energy X-ray absorptiometry increased 0.65% with 1(OH)D₃ treatment and decreased 1.14% with placebo (P=0.037). BMD in both the femoral neck and Ward's triangle did not yield any significant differences between the two groups, whereas trochanter BMD in the 1(OH)D₃-treated group increased 4.20% and decreased 2.37% with placebo (P=0.055). X-ray analysis demonstrated that new vertebral fractures occurred in two patients with 1(OH)D₃ and in seven patients with placebo. The vertebral fracture rate in the treated group was significantly less (75/1000 patient years) than in the control group (277/1000 patient years; P=0.029). Hypercalcemia (12.1 mg/100 ml) occurred in one patient receiving 1(OH)D₃; however, the serum calcium level in this patient promptly decreased to the reference range after cessation of the treatment. There were no significant changes in serum creatinine level in either group. A significant increase in urinary excretion of calcium was found but there was no significant change in urinary excretion of hydroxyproline in the treated group. The serum level of bone-derived alkaline phosphatase activity significantly decreased by–26±26 (mU/ml) after the treatment (P=0.003). These results indicate that 1(OH)D₃ treatment is effective for maintaining trabecular bone mass and prevents further vertebral fractures without any serious adverse effects in postmenopausal osteoporosis.     

Involvement of phosphatidylcholine hydrolysis by phospholipase C in prostaglandin F2Α-induced 1,2-diacylglycerol formation in osteoblast-like MC3T3-E1 cells

We previously demonstrated that a prostaglandin F2 (PGF2)-induced, sustained increase in 1,2-diacylglycerol (DAG) production was important for proliferation in osteoblast-like MC3T3-E1 cells. The 1,2-DAG formation is mediated by various enzymes, such as phos-phoinositide (PI)-specific phospholipase C (PLC), phospholipase D (PLD), and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). In the present study, to elucidate the mechanism of the 1,2-DAG formation, we have examined the PGF2-induced production of [³H]phosphorylcholine, a product of PC-PLC activity, in [³H]choline-labeled MC3T3-E1 cells. The PGF2-induced [³H]phosphorylcholine production was inhibited by genistein, a potent protein tyrosine kinase inhibitor, and increased by vanadate, a potent protein tyrosine phosphatase inhibitor. However, there were no effects after treatment with protein kinase C (PKC) inhibitors, the guanosine triphosphate (GTP) binding protein activator, NaF/AlCl₃, a Ca²⁺-ionophore, or the potent activator of PKC, phorbol 12-myristate 13-acetate (PMA), suggesting that a tyrosine kinase(s) was involved in the PGF2-induced [³H]phosphorylcholine formation. Furthermore, a PGF2 analogue, 16-(3-trifluoromethylphenoxy)--tetranor-trans-² PGF2 methyl ester (ONO-995), stimulated the proliferation of MC3T3-E1 cells to a level similar to that seen with PGF2, and also caused phosphorylcholine and 1,2-DAG generation. However, neither an increase in intracellular free calcium ion ([Ca²⁺]i) levels by PI-PLC, nor phosphatidylethanol formation (and choline production) by PC-PLD were observed. From these results, we conclude that PGF2-induced 1,2-DAG accumulation was mediated mainly via tyrosine kinase(s)-dependent PC hydrolysis by PLC activity in osteoblast-like MC3T3-E1 cells.     

Identification of α1-adrenoceptor subtypes in the dog prostate

Summary The ₁-adrenoceptor subtypes of dog prostate were characterized in binding and functional experiments. In saturation experiments, [³H]prazosin bound to ₁-adrenoceptors with high affinity. In the displacement experiments, unlabelled prazosin and WB4101 biphasically inhibited the binding of 400 pM [³H]prazosin, suggesting the presence of at least two distinct affinity sites for prazosin or WB4101. The proportion of high-affinity sites was approximately 10%. HV723 also recognized two distinct affinity sites but the proportion of high-affinity sites was approximately 20%. From these results the presence of three distinct ₁-adrenoceptor subtypes was suggested: presumably subtypes _1A (high affinity for prazosin and WB4101), _1N (high affinity for only HV723) and _1L (low affinity for the three antagonists) according to the recently proposed ₁-adrenoceptor subclassification. The density of subtype _1L was much higher than that of subtypes _1A and _1N subtypes. In the functional experiments, prazosin, WB4101 and HV723 competitively antagonized the contractile response to noradrenaline with low affinities close to those estimated for the _1L subtypes. These results suggest that the contractile response to noradrenaline in the dog prostate is mediated predominantly through _1L subtype -adrenoceptors.