Alvin M. Kaye

Kinetic of DNA synthesis in immature rat uterus: Age dependence and estradiol stimulation

Abstract 1. 1. The stimulation of DNA synthesis by estradiol-17β was studied in surving uteri of 1- to 30-day-old rats injected 24 h previously with estradiol-17β. 2. 2. Uteri from untreated immature rats showed an age-dependent decrease in incorporation of [Me−3H]thymidine into DNA during the first month after birth; incorporation decreased by a factor of 10 over this period. 3. 3. In rats less than 15 days old, a single estradiol-17β injection did not result in an increase in the rate of DNA synthesis; uterine wet weight, RNA content and protein content also showed no increase. At 15 days, estradiol caused an increase in uterine wet weight, but only slight increases in the other parameters measured. In uteri of 20-day-old rats, estradiol-17β caused a significant increase in the rate of DNA synthesis and in RNA and protein content. 4. 4. Amounts of estradiol-17β as low as 50 pg per 20-day-old rat (33 g in weight) significantly increased the rate of DNA synthesis. Dibutyryl cyclic AMP, administered in doses of 0.5 mg into the uterine lumen or 5 mg intraperitoneally, showed no effect on uterine nucleic acid or protein synthesis. 5. 5. In the 20-day-old rat uterus, the epithelial, stromal and myometrial tissues all showed a wave of cell division with a peak mitotic activity between 24 and 28 h after estradiol injection. In the epithelium, the increase was from 3 mitoses per 1000 cells to 164 per 1000 cells 26 h after hormone treatment.

The transduction of mechanical force into biochemical events in bone cells may involve activation of phospholipase A2

SummaryMechanical forces applied to cultured bone cells induce the production of cAMP via stimulation of the formation of prostaglandin E2 (PGE2) and its release into the medium, resulting in stimulation of adenylate cyclase. In this paper we show that either the antibiotic gentamycin (100 μg/ml) or antiphospholipid antibodies (0.1%) which bind to membrane phospholipids abolish cAMP formation induced by mechanical forces; exogenously added arachidonic acid or PGE2 stimulates cAMP formation, even in the presence of these agents. Addition of exogenous phospholipase A2 (but not phospholipase C) causes an increase in the formation of cAMP in bone cells, a response that is also inhibited by gentamycin or antiphospholipase antibodies. These observations suggest that mechanical forces exert their effect on bone cells via the following chain of events: (1) activation of phospholipase A2, (2) release of arachidonic acid, (3) increased PGE synthesis, (4) augmented cAMP production.

Mouse nuclear satellite dna, 5-methylcytosine content, pyrimidine isoplith distribution and electron microscopic appearance.

Abstract Nuclear satellite DNA of high specific activity labelled with [ 3 H- methyl ]- l -methionine and 32 P was isolated from newborn mice and from cultured mouse embryo cells. This DNA was found to have more than twice the molar concentration of 5-methylcytosine than the main band of nuclear DNA. Labelled pyrimidine isopliths were separated by column chromatography on DEAE cellulose. The distribution of radioactivity in such isopliths did not reveal any feature to distinguish nuclear satellite DNA from the main band DNA. Visualization of renatured satellite DNA by electron microscopy revealed a population of linear molecules of up to 4.0 μ in length, distributed into four groups with average lengths of 0.9, 1.8, 2.7 and 3.6 μ. Loops of approximately 0.2 μ in circumference were seen at intervals along the molecules.

Stimulation by estrogens of ornithine and S-adenosylmethionine decarboxylases in the immature rat uterus

Abstract 1. 1. Injection of 0.5 μg of estradiol-17β into 20-day-old rats caused a 14–25 fold increase in the specific activity of ornithine decarboxylase (EC 4.1.1.17) in the 38000 × gmax supernatant fraction of uterine homogenates but not in liver homogenates. This peak value occurred 4 h after administration of the hormone. 2. 2. Putrescine-dependent S-adenosylmethionine decarboxylase specific activity doubled within 4 h after estradiol treatment and remained constant thereafter. 3. 3. The increase in ornithine decarboxylase activity was suppressed completely by treatment with the inhibitors of protein synthesis, cycloheximide (6 μg/g) or puromycin (0.2 mg/g). 4. 4. Non-steroidal estrogens (diethylstilbestrol, 50 μg; coumestrol, 80 μg; and genistein, 1250 μg) also increased the specific activity of ornithine decarboxylase. Estradiol-17α showed activity at 0.5 μg but not at 0.05 μg, a dose at which estradiol-17β was still active. Testosterone (1 mg) was completely inactive.

Regulation of proliferation of rat cartilage and bone by sex steroid hormones.

We have demonstrated previously that 17 beta-estradiol (E2) stimulates proliferation of skeletal tissues, both in vivo and in vitro, as measured by increased DNA synthesis and creatine kinase (CK) specific activity. The effect of E2 on bone is sex specific. E2 is active only in females and androgens only in males. By contrast, in cartilage of both sexes, dihydrotestosterone (DHT) as well as E2 stimulates CK specific activity and DNA synthesis. In bone, we find that sex steroids stimulate skeletal cell proliferation in gonadectomized as well as in immature rats. Ovariectomized (OVX) rats, between 1 and 4 weeks after surgery, show stimulation of CK by E2. The basal activity and response of CK changes with the varying endogenous levels of E2 in cycling rats, in which the highest basal activity is at proestrus and estrus and the highest response is in diestrus. In rats of all ages tested, both the basal and stimulated specific activity of CK is higher in diaphysis and epiphysis than in the uterus, or in the adipose tissue adjacent to the uterus, which has a response similar to that of the uterus itself. The effect of E2 in vivo, and in chrondroblasts and osteoblasts in vitro, is inhibited by high levels of the antiestrogen tamoxifen which, by itself, in similar high concentrations, shows stimulatory effects. In addition to the sex steroids, skeletal cells are also stimulated by secosteroid and peptide calciotrophic hormones. The interactions of the sex steroids with these hormones modulate the response of cartilage and bone cells to both sex steroids and the other calciotrophic hormones. These results provide the first steps towards understanding the regulation of bone cell proliferation and growth by the concerted action of a variety of hormones and growth factors.

Methylation of RNA by mouse organs and tumors: Ionic stimulation in vitro

Abstract 1. 1. In experiments designed to investigate possible organ- and tumor-specific differences in methylation of soluble RNA reported in the literature, RNA methylase activity from dialyzed high-speed supernatant extracts of adult and newborn mouse organs and from mouse tumors was measured. Soluble RNA from Escherichia coli B was used as acceptor, and tests were made over a range of concentrations of ammonium acetate and/or magnesium chloride. 2. 2. It was found that the rate of methylation by organ and tumor extracts was stimulated 4- to 8-fold, and the extent of methylation 2- to 4-fold by 0.36 M ammonium acetate. 3. 3. The products of methylation in vitro were separated electrophoretically. The pattern of incorporation into nucleotides of C, A, G and U was measured in the presence and absence of additional ions. Addition of ions increased the methylation of each major nucleotide from an average of 2-fold in guanylic acid to almost 9-fold in cytidylic acid. 4. 4. Neither the rate, the extent nor the pattern of methylation served to distinguish unequivocally extracts from tumors or from normal organs, or to characterize extracts of several normal organs.

Hormonal stimulation of bone cell proliferation.

The recent demonstration of estrogen receptors in bone derived cells has stimulated the study of direct effects of sex steroids on bone. We have shown direct stimulation of proliferation by 17 beta-estradiol (E2) of ROS 17/2.8 rat osteogenic osteosarcoma cells, and other bone-derived cells in culture, as well as sex-specific stimulation of diaphyseal bone in vivo by estrogen and testosterone, using [3H]thymidine incorporation into DNA and stimulation of the specific activity of creatine kinase as markers. ROS 17/2.8 cells were used as models of osteoblast-like cells to study the reciprocal modulation of stimulation of bone cell proliferation by sequential treatment by sex steroid and calciotrophic hormones. Pretreatment with 1,25(OH)2D3 and PTH augmented stimulation by E2, while pretreatment with PGE2 followed by E2 resulted in no additional stimulation. Reciprocally, pretreatment with E2 significantly reduced the response to PGE2 while showing an insignificant effect on the response to the other hormones. Gonadectomized Wistar-derived rats provided a useful model system for study of postmenopausal osteoporosis. In diaphyseal bone, [3H]thymidine incorporation and creatine kinase activity decreased 4 weeks after gonadectomy. At that time, a single i.p. injection of E2 in females, and testosterone in males, resulted in a highly significant increase in both these parameters within 24 h.

Estrogenic activity of glabridin and glabrene from licorice roots on human osteoblasts and prepubertal rat skeletal tissues

Data from both in vivo and in vitro experiments demonstrated that glabridin and glabrene are similar to estradiol-17beta in their stimulation of the specific activity of creatine kinase, although at higher concentrations, but differ in their extent of action and interaction with other drugs. In pre-menopausal human bone cells, the response to estradiol-17beta and glabridin (at higher concentration) was higher than in post-menopausal cells; whereas, glabrene (at higher concentration) was more effective in post-menopausal cells. At both ages, the response to estradiol-17beta and glabridin was enhanced by pretreatment with the less-calcemic Vitamin D analog CB 1093 (CB) and the demonstrably non-calcemic analog JK 1624 F(2)-2 (JKF). The response to glabrene was reduced by this pretreatment. Both glabridin and glabrene stimulated creatine kinase specific activity in diaphyseal bone and epiphyseal cartilage of prepubertal female rats. Daily feeding (3-14 days) of prepubertal female rats with glabridin, estradiol-17beta or their combination, also stimulated creatine kinase specific activity. Glabridine, similarly to estradiol-17beta, also stimulated creatine kinase specific activity in ovariectomized female rats. Raloxifene, in combination with glabridin or estradiol-17beta, demonstrated the phenomenon of mutual annihilation of stimulation of creatine kinase specific activity in both epiphysis and diaphysis. Glabrene activity was not inhibited by raloxifene. Therefore, glabridin shows greater similarity to estradiol-17beta and thus greater potential, with or without Vitamin D, to modulate bone disorders in post-menopausal women.

Sex-specific response of bone cells to gonadal steroids : modulation in perinatally androgenized females and in testicular feminized male rats

We have found previously that rat diaphyseal bone in vivo, as well as rat embryo calvaria cells in culture, show a sex-specific response to gonadal steroids in stimulation of creatine kinase (CK)-specific activity, and the rate of [3H]thymidine incorporation into DNA; male-derived cells responded only to testosterone or to dihydrotestosterone (DHT), whereas female-derived cells were stimulated exclusively by estradiol (E2). In this study, we tested whether developmental hormone manipulation could alter this sex specificity. We showed that diaphyseal bone of prenatally or neonatally androgenized female rats responds to a single injection of either E2 (5 micrograms/rat) or DHT (50 micrograms/rat) at 3-4 weeks postandrogenization. This response of androgenized female diaphyseal bone to androgen gradually declines; 3 months posttreatment, diaphyseal bone no longer responds to DHT and reverts to its original sex specificity. Rat embryo calvaria cell cultures prepared from female fetuses androgenized in utero showed the same lack of hormonal specificity, that is, the cells responded to both E2 (30 nM) or DHT (300 nM). Cells derived from the male siblings of the prenatally androgenized rats were not affected and responded only to DHT. In contrast to experiments in utero, in vitro administration of testosterone (1 microM) or E2 (1 microM) to calvaria cells from female embryos failed to cause the cells to respond to DHT. Androgen receptor-deficient (Tfm) male rats, which have approximately 10% of the normal response to androgens, also showed a response to both testosterone and E2 in comparison to their normal male siblings, whose bones responded only to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)

24R,25-Dihydroxyvitamin D stimulates creatine kinase BB activity in chick cartilage cells in culture

In chick limb‐bud cartilage cell cultures 24R,25‐dihydroxycholecalciferol (24R,25(OH)2D3), but not 24S,25(OH)2D3, 1α,25(OH)2D3 or 25(OH)D3, stimulates the activity of the brain type (BB) isozyme of creatine kinase (EC 2.7.3.2), the ‘estrogen‐induced protein’ first identified in rat uterus. Cultures treated with bromodeoxyuridine, in which cartilage formation in inhibited, show no stimulation of creatine kinase BB by 24R,25(OH)2D3.