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PubMed2002

Evidence that 2-methoxyestradiol suppresses proliferation and accelerates apoptosis in normal rat growth plate chondrocytes.

PURPOSE: 2-Methoxyestradiol (2ME(2)), a metabolite of 17beta-estradiol, is currently undergoing extensive clinical testing as an antitumor agent. This drug antagonizes both angiogenesis and induces apoptosis in some tumor cell lines. Growth plate alterations as well as an inhibition of longitudinal bone growth have been reported in young rats treated with a pharmacological dose of 2ME(2). However, 2ME(2)'s mechanism of action at the growth plate is unknown. To uncover the mechanism, we examined the dose-response effects of 2ME(2) on growth plate chondrocytes. METHODS: 2ME(2) (4-, 20-, and 75 mg.kg.day) was administered orally to recently ovariectomized 10-week-old rats. The number of cells undergoing DNA synthesis was evaluated with (3)H-thymidine radioautography, apoptosis-induced DNA fragmentation was detected with TUNEL assay, and longitudinal growth rates were determined with fluorochrome labeling. RESULTS: 2ME(2) reduced bone elongation ( P< or =0.05), suppressed chondrocyte proliferation ( P< or =0.05), and induced chondrocyte apoptosis ( P< or =0.05). Furthermore, 1-week treatment with 2ME(2) did not affect the size of hypertrophic chondrocytes. CONCLUSION: Thus, pharmacological doses of 2ME(2) utilized for tumor suppression can inhibit normal bone elongation in growing rats by reducing the number of proliferating chondrocytes in the growth plate and accelerating apoptosis in differentiated chondrocytes. This demonstration identifies another organ in which toxicity should potentially be monitored in ongoing and future clinical trials of 2ME(2).

2-Methoxyestradiol↗

PubMed1996

Relationship of cellular proliferation to expression of osteopontin and bone sialoprotein in regenerating rat periodontium.

Cellular repopulation was studied in a model in which adjacent mineralising and soft connective tissue matrices are regenerated. Window wounds were created through alveolar bone, with either preservation or removal of periodontal ligament, in 30 male Wistar rats. Three animals per time period were killed on days 1, 3, 7, 14, and 28 after surgery for each wound type. Cellular proliferation in alveolar bone and periodontal ligament was assessed by 3H-thymidine labelling 1 h before death, followed by radioautographic analysis. Cellular differentiation was determined by the temporal expression of the bone-related markers osteopontin and bone sialoprotein, using immunohistochemical methods. In regenerating periodontium, osteopontin was expressed earlier than bone sialoprotein, which was restricted to alveolar bone. After wounding, transient expression of osteopontin was detected in the periodontal ligament at days 1 and 3. In general, wounding induced fivefold higher proliferation and clonal growth of periodontal ligament cells compared to the unwounded (control) side. Combined immunostaining and radioautography demonstrated colocalisation of osteopontin in sites with high numbers of labelled cells in both nascent periodontal ligament and regenerating alveolar bone at days 3 and 7. In contrast, bone sialoprotein, which appeared in regenerating alveolar bone on days 14-28 after wounding, was expressed much later than the peak of cellular proliferation. We conclude that (1) the intact periodontal ligament influences cell proliferation and osteopontin expression; (2) osteopontin is an early marker of periodontal tissue regeneration that is temporally and spatially associated with intensive cell proliferation and migration in osteogenic and periodontal ligament cell populations; and (3) bone sialoprotein is expressed after proliferation at sites of mineralising bone formation.

Animals↗

PubMed1998

Osteogenic inhibition by rat periodontal ligament cells: modulation of bone morphogenic protein-7 activity in vivo.

Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by 3H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of alpha-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P<0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P>0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P<0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for alpha-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis.

Administration, Topical↗

PubMed2000

Etidronate (HEBP) promotes osteoblast differentiation and wound closure in rat calvaria.

The mechanisms that regulate the migration, proliferation and differentiation of osteogenic cell populations in vivo are poorly understood. Elucidation of these mechanisms is essential for an understanding of the basic processes that determine mineralized connective tissue homeostasis and regeneration. Bisphosphonates are known to regulate bone metabolism, in part through effects on osteoclastic resorption. Given previous data from other in vitro and in vivo investigations, we considered that they could also affect the proliferation and differentiation of osteoblasts in vivo. We tested this hypothesis using a bisphosphonate (ethane-1-hydroxy-1,1-bisphosphonate, HEBP, etidronate) and a calvarial wound model in which osteogenic differentiation and bone formation are coordinately induced by the wounding stimulus. Wounds through the calvarial bone were created in 20 male Wistar rats. After surgery, animals were treated every day for 1 or 2 weeks with HEBP or saline (controls) and five rats in each group were killed at 1 or 2 weeks following surgery. Cellular proliferation and clonal growth were assessed by 3H-thymidine labeling 1 h before death followed by radioautography. Cellular differentiation of osteogenic cell populations was determined by immunohistochemical staining for osteopontin and bone sialoprotein. Von Kossa and toluidine blue staining were used for the assessment of mineralization and osteoid formation, and for morphometric analysis of wound closure. At 1 and 2 weeks after surgery HEBP promoted wound closure (> twofold greater than controls, P < 0.001) and mineralized/osteoid tissue formation in the bony compartment of the wound (> 50% higher than saline controls, P < 0.05). In HEBP-treated animals there was a > 50% increase in intracellular staining for osteopontin in the endosteum-lined spaces adjacent to the wound (P < 0.05) and increased staining for osteopontin in the nascent bone at the wound margin (> 50% greater than controls, P < 0.05). However, there were reduced cell counts and labeling indices at stromal precursor sites (65% reduction compared to controls; P < 0.01). As HEBP increased osteopontin expression and osteoid/mineralized tissue formation but reduced the proliferation of precursor cells, we conclude that in addition to blockade of bone resorption and mineralization, this drug, at doses which also reversibly inhibit mineralization, may promote osteoblast differentiation as well.

Animals↗

PubMed2003

Electron microscopic radioautographic study on nucleic acid synthesis in amitotic hepatocytes of the aging mouse.

Twenty groups of aging mice, each consisting of three individuals from fetal day 19 to postnatal month 24, were injected with either (3)H-thymidine or (3)H-uridine. The mice were killed 1 h and the livers processed for light and electron microscopic radioautography. On radioautograms obtained from each animal, amitotic nuclear divisions and resulting binucleate hepatocytes were detected and compared to mononucleate hepatocytes. From the results, it was demonstrated that only a few hepatocytes showing amitotic nuclear division were found labeled with the two precursors demonstrating DNA and RNA synthesis. However, there were very few silver grains showing incorporation of labeled precursors in respective amitotic cells. It was clarified that the amitotic cells did not synthesize such macromolecules as mononucleate hepatocytes did. On the other hand, the binucleate cells were found more often than the amitotic cells. DNA synthesis of binucleate hepatocyte nuclei was observed at the perinatal stage but disappeared at the adult stage. RNA syntheses in karyoplasm and cytoplasm in both mononucleate and binucleate cells significantly increased from the perinatal stage, reaching the maxima at the adult stage, then significantly decreased to the senescent stage. Grain counts revealed that RNA was synthesized significantly more in binucleate cells than in mononucleate cells at the respective aging stages.

Aging↗

PubMed2004

Electron microscopic radioautographic study on protein synthesis in amitotic hepatocytes of the aging mouse.

Ten groups of aging mice, each consisting of three individuals, from fetal day 19 to postnatal month 24, were injected with (3)H-leucine and killed 1 h later; the livers were processed for light and electron microscopic radioautography. On radioautograms obtained from each animal, amitotic nuclear divisions and resulting binucleate hepatocytes were detected and compared to mononucleate hepatocytes. From the results, it was demonstrated that only a few hepatocytes showing amitotic nuclear divisions were found labeled with the precursor demonstrating protein synthesis. However, the numbers of silver grains showing incorporations of labeled precursors in respective amitotic cells were very few. It was clarified that the amitotic cells did not synthesize such macromolecules as mononucleate hepatocytes did. On the other hand, more binucleate cells were found than amitotic cells. Protein synthesis in karyoplasm and cytoplasm in both mononucleate and binucleate cells increased from the perinatal stage, reaching the maxima at adult stage, then decreased to the senescent stage. Grain counts revealed that synthesized proteins were more abundant in binucleate cells than in mononucleate cells at the respective aging stages.

Aging↗

PubMed2006

Radioautographic study of palatine tonsillar cells.

Proliferative and functional (protein producing) activities of palatine tonsillar cells were studied by light radioautography (semithin sections). 3H-Thymidine incorporation demonstrated the dynamics of renewal of palatine tonsillar cell populations in various pathological states. 3H-Uridine labeling objectively evaluated functional activity of different cells and more accurately showed distribution of destructive processes in the organ.

Adult↗

PubMed1986

Epithelial regulation of prolactin effect on amnionic permeability.

The permeability of human amnion to tritiated water is reduced in the presence of both human and ovine prolactin. The cellular composition of amnion is such that the action of prolactin on this membrane probably occurs by way of the epithelium lining the fetal surface. The present study sought to confirm an epithelial site of action of prolactin on the permeability of amnionic membrane to tritiated water. In addition, radioautography and competition experiments were conducted to determine a possible receptor-mediated mechanism for prolactin action. Membrane permeability to tritiated water was found to be equivalent for both intact membranes and membranes enzymatically stripped of the lining epithelial cells. However, when ovine prolactin was presented to the fetal surface of amnion, only intact membrane displayed decreased permeability to tritiated water. Although localization of iodine 125-labeled prolactin to the light cell population of amniotic epithelium was observed, positive evidence of a receptor-mediated mechanism could not be established. The results indicate that the permeability of human amnion to water is influenced principally by cells of the epithelium in response to prolactin.

Autoradiography↗

PubMed1983

A rapid method for detecting specific RNA transcripts by hybridization to DNA probes in solution.

A method is described for detecting specific transcripts in crude mixtures of RNA. The method employs hybridization of single-stranded or double-stranded radioactive DNA probes in solution, followed by electrophoretic separation of the hybrid and probe on agarose and visualization by radioautography. The procedure offers the advantages of decreased preparation time and increased sensitivity over currently used methods.

Autoradiography↗

PubMed1983

Determination of levels of glycolytic intermediates and nucleotides in platelets by pulse-labeling with [32P]orthophosphate.

A method is presented for the separation and quantification of 32P-labeled carbohydrates and nucleotides in blood platelets which have been pulse-labeled with [32P]orthophosphate. The procedure is based on two-dimensional paper chromatography, identification of the spots by radioautography and enzymatic methods, and quantitation of 32P radioactivity by liquid scintillation counting. The data show that 32P is homogeneously distributed among the compounds studied so that the total radioactivity is proportional to the levels of these compounds in the metabolic compartment of the cells. Thus, this method provides a sensitive and accurate means to evaluate phosphorylated intermediates in glycolysis and nucleotide metabolism and to assess the transfer of energy-rich phosphate groups between these pathways in particular.

Autoradiography↗

PubMed1983

Subpicogram analysis of sweat proteins using two-dimensional polyacrylamide gel electrophoresis.

Sweat collected from six normal volunteers was analyzed to determine if reproducible protein patterns could be obtained using two-dimensional polyacrylamide gel electrophoresis of 125I-labeled sweat proteins. This method has the capability of easily detecting picogram quantities of protein. Once the methods of collection of the sweat had been standardized, reproducible patterns were obtained from these volunteers. Over 100 discrete spots were revealed by a combination of fluorography and rare earth screen radioautography of dried two-dimensional gels. This method will allow analysis of sweat for qualitative and quantitative variations in protein content in pathologic conditions such as cystic fibrosis, renal failure, and diabetes.

Electrophoresis, Polyacrylamide Gel↗

PubMed1983

An assay for the rates of cleavage of specific sites in DNA by restriction endonucleases: its use to study the cleavage of phage lambda DNA by EcoRI and phage P22 DNA containing thymine or 5-bromouracil by HindIII.

A method to measure the rates of cleavage of specific sites in DNAs by restriction endonucleases is described. Partial digests are prepared by incubating DNAs with limiting amounts of endonuclease. The termini generated by cleavage are labeled with 32P by the polynucleotide kinase-exchange reaction. The labeled termini are then identified by completing the digestion with the same endonuclease and separating the products by gel electrophoresis. As the products of complete digestion of DNA are often easily separated and can be unequivocally identified, this procedure permits comparison of the rates of cleavage of specific sites in DNAs; furthermore, because detection of the products of cleavage utilizes radioautography and does not depend upon their size, or amount, only small amounts of DNA need to be utilized. This method has been used to examine the cleavage of phage lambda DNA by EcoRI endonuclease, and to demonstrate that 5-bromouracil substitution in phage P22 DNA reduces the rate of cleavage of most sites by HindIII endonuclease approximately threefold and the rate of cleavage of one site approximately tenfold.

Bacteriophage lambda↗

PubMed1986

A dot-blot assay for heparin-binding proteins.

A method for the detection and quantitation of picomole amounts of heparin-binding proteins is described. Proteins are first spotted on nitrocellulose and then incubated with 125I-heparin. Binding of heparin to the proteins is detected by radioautography and quantitated by scanning densitometry; proteins are quantitated by densitometric analysis of the amido black stained nitrocellulose. Heparin-binding was time-dependent and sensitive to the presence of metal ions, urea, and detergents (anionic, nonionic, and zwitterionic). The divalent cations Ca2+ and Mg2+ and the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased heparin binding whereas NaCl, urea, sodium dodecylsulfate, and La3+ decreased binding. This assay is applicable to the identification and characterization of a variety of heparin-binding proteins.

Animals↗

PubMed1988

Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation.

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.

Alkylation↗

PubMed1985

Radioautographic analysis of [3H]-fucose utilization by mouse odontoblasts with emphasis on intracytoplasmic and plasma membrane glycoproteins.

[3H]-fucose utilization by odontoblasts was studied by light and electron microscopic radioautography. At 10 min after injection, fucose label was concentrated in the Golgi area. By 20-30 min, there was a progressive decline in Golgi labelling with label present at the plasma membrane, terminal web, odontoblast process and predentine matrix. At 4 h, the predentine and the predentine-dentine junction were heavily labelled. At the ultrastructural level, Golgi labelling at 10 min was mostly localized to cisternal elements and at 20 and 30 min secretory granules and dense bodies were also labelled. Most of the silver grains observed in the terminal web were associated with microfilaments near the plasma membrane. In the predentine, the matrix itself accounted for 23.0 per cent of the label at 4 h and the plasma membrane of the odontoblast process accounted for 19 per cent. The results indicate that odontoblasts, in addition to secreting glycoproteins into the dentinal matrix, also continuously manufacture glycoproteins for incorporation into the cell surface, the lysosomal system and the terminal web.

Animals↗

PubMed1986

Penetration of various molecular-weight proteins into the enamel organ and enamel of the rat incisor.

During enamel maturation, most of the organic matrix is removed as the mineral content increases; it is postulated that proteolytic enzymes within enamel break down large proteins into more mobile fragments. To predict how such fragments might leave the enamel, the entry and penetration of various proteins into it was examined. Rats (100 g) were injected via the external jugular vein with 125I-iodinated calcitonin (3600), insulin (5700), epidermal growth factor (EGF; 6100) and albumin (68,000). They were killed after 10 min and radioautographs made to visualize these molecules in the incisor enamel organ and enamel. In addition, dissected incisors were wiped free of their enamel organs, dipped in the iodinated protein solutions for 10 min, and processed for radioautography. In all dipped teeth, except those exposed to albumin, there was a gradient of silver-grain density over the entire thickness of enamel in both the secretion and maturation zones. In all injected animals, enamel labelling in the secretion zone was only slightly above background. In the maturation zone of animals injected with calcitonin and insulin, many grains were over enamel adjacent to smooth-ended ameloblasts but not ruffle-ended ones. Animals injected with EGF and albumin had no labelled enamel in the maturation zone. Thus dipped rat incisor enamel was permeable to proteins with molecular weights as high as 6100. Localization of injected proteins indicates that the enamel organ restricts their passage into enamel, but proteins with molecular weights as high as 5700 may pass into enamel through or between smooth-ended ameloblasts. As exogenous proteins readily diffused into the enamel, it seems likely that enamel proteins of similar size can leave enamel by a similar route.

Animals↗

PubMed1988

The effects of cerebral hemidecortication on the eruption rate and uptake of [3H]-glycine by the periodontal ligament of the rat incisor.

Hemidecortication, which consists of removing one cerebral hemisphere, leaving intact the thalamus and hypothalamus, affects the hypothalamus-hypophyseal axis producing hypothyroidism. Hemidecorticate rats showed a significant decrease in the weekly eruption rate of the upper incisors and partial recovery after the administration of thyrotrophin-releasing hormone (TRH). The uptake of [3H]-glycine, 1 and 4 h after a single injection, shown by radioautography, was 25 to 50 per cent higher in the periodontal ligament of the experimental animals. Most of the labelled material was non-collagenous proteins because only 20 to 30 per cent was removed by collagenase.

Animals↗

PubMed1994

The effect of vinblastine on the incorporation of [3H]-glycine into proteins of the periodontal ligament of impeded and unimpeded mouse incisors.

The effect of vinblastine on the protein metabolism of the periodontal ligament of impeded and unimpeded mouse incisors was studied by [3H]-glycine labelling and radioautography. The silver-grain concentration was determined in areas adjacent to the tooth, areas adjacent to bone and, as an internal control, in the dentine matrix. From 1 to 12 h there was no difference between treated and control animals; thus the drug did not alter protein biosynthesis. Later, the silver-grain concentration was significantly higher in areas adjacent to both bone and tooth in vinblastine-treated animals, suggesting a longer half-life of the labelled proteins. No significant differences between normal or unimpeded erupting incisors of both groups were detected. Dentine matrix showed a possibly higher re-utilization of the labelled amino acid in vinblastine-treated animals. The amount of labelled protein removed by collagenase was similar in both groups, while the concentration of grains due to collagenase-resistant proteins was significantly higher in treated animals, particularly at 96 h after the injection of labelled glycine. The relation between the increased amount of non-collagenous proteins in the periodontal ligament and the decrease in the rate of eruption caused by vinblastine was not established. However, among these proteins, fibronectin and proteoglycans are thought to be important factors in tooth eruption.

Alveolar Process↗