Synthesis and transmethylation of DNA in polyoma-infected cultures.
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Alkylation↗
Biomedical subjects
A M Kaye
Publications and source records attributed to A M Kaye.
Differentiation of cultured mice bone marrow into osteoblast-like cells results in acquisition of sex-specific responsiveness to gonadal steroids.
We have previously demonstrated that mouse skeletal tissue, rat bone as well as rat or human derived bone cells in culture, show a sex-specific response to gonadal steroids in stimulation of the specific activity of the BB isozyme of creatine kinase (CK). This response could be modified by manipulation of the endocrine environment during early postnatal development. Moreover, pretreatment with vitamin D up-regulated the sex-specific responsiveness and sensitivity to gonadal steroids. In the present study we examine the differentiation pattern into osteoblast-like cells using dexamethasone (DEX) and 1,25 dihydroxy vitamin D3 (1,25D) and their effect on the acquisition of responsiveness to gonadal steroids by the differentiated cells. Cultured femoral bone marrow in the presence of DEX or 1,25D or both, were examined for their response to gonadal steroids by measuring the specific activities of alkaline phosphatase (AP) and CK BB. The constitutive level of CK in both male- and female-derived bone cells was decreased by DEX, by 1,25D or by both, whereas the constitutive level of AP was increased by DEX while decreased by 1,25D or by both. Following incubation of the bone marrow cultures with DEX, treatment with estradiol 17beta (E2, 30 nM, 24 h) stimulated CK activity in female derived bone cells, with no effect of treatment with dihydrotestosterone (DHT, 300 nM). In contrast, in male derived bone cells, DHT but not E2 increased CK activity. This sex-specific response was also achieved upon culturing with 1,25D and was significantly augmented by culturing with both. No response to gonadal steroids was seen with undifferentiated bone marrow cells. All cultures responded to IGF-I when cultured with or without DEX and/or 1,25D but with no augmentation by 1,25D. Gonadal steroids increased AP to a much lesser extent; but enzyme activity decreased in the presence of 1,25D. IGF-I stimulated AP slightly with no effect of 1,25D. These findings suggest that manipulation of the hormonal milieu in early stages of differentiation sequence of osteoblast-like cells, determines the subsequent selective responsiveness of the developing bone tissue to gonadal steroids.
Animals↗
The 'estrogen-induced protein': quantitation by autoradiography of polyacrylamide gels.
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Aging↗
Nonhypercalcemic analogs of vitamin D stimulate creatine kinase B activity in osteoblast-like ROS 17/2.8 cells and up-regulate their responsiveness to estrogens.
We have reported that pretreatment with 1 alpha, 25(OH)2D3(1, 25) up-regulates responsiveness and sensitivity to 17 beta estradiol (E2) in osteoblast-like cells, as measured by parallel stimulation of [3H]thymidine incorporation into DNA and the specific activity of creatine kinase BB (CK). Increased responsiveness was correlated with increased E2 receptor concentration. In this study, we have extended these observations to new nonhypercalcemic analogs of 1,25. We compared the analogs hexafluoro vitamin D3 (FL), and the side chain modified derivatives: EB 1089 (EB), CB 1093 (CB) and MC 1288 (MC) with 1,25 and 25 (OH)D3(25 D3). Treatment with 30 nM E2 for 4 h stimulated CK activity in ROS 17/2-8 cells by 40%; there was no further increase after 3 daily additions of E2. Treatment by 3 daily additions, at 1 nM, of all analogs except 25 D3 caused a 2-3-fold increase in CK specific activity. This schedule of treatment also upregulated the response to 4 h exposure to 30 nM E2 by 30-70% above the response to vitamin D analogs alone, and by up to 2 fold compared to E2 without pretreatment. At 1 pM, the analogs doubled CK activity, and, except for 1,25, upregulated the response to E2 to levels characteristic of each analog. Pretreatment with vitamin D analogs also increased the sensitivity to E2 by lowering the dose for a comparable response to E2 by one or two orders of magnitude. Stimulation of specific activity of CK by the analogs was paralleled by increases in the steady state level of mRNA for CKB, but not in its half life. Whereas pretreatment by vehicle followed by E2 for 2 h was unable to increase CKB mRNA, pretreatment with the analogs made possible detection of mRNA responsiveness to E2. These results add to the evidence for the interaction of estrogens and antiestrogens with vitamin D metabolites in regulation of bone growth in vitro. They also strengthen the potential for treatment of bone loss, as occurs in postmenopausal osteoporosis, by a combination of nonhypercalcemic vitamin D analogs and estrogens.
Animals↗
Anabolic effects of estrogen and parathyroid hormone on skeletal tissues: the use of creatine kinase B activity as a response marker.
The rapid stimulation of the specific activity of the brain type isozyme of creatine kinase (CK BB) is an almost universal marker of cell stimulation. We have studied its stimulation in skeletal-derived cells and shown that the increase in its activity is closely correlated with the biochemical parameter of cell proliferation - [(3)thymidine incorporation into DNA - and with the morphological parameters of bone growth, increase in thickness of cortical bone and of the number of cells in the proliferating zone of the epiphyseal growth plate. We have used the increase in CK activity to demonstrate sex specific stimulation of diaphyseal bone, exclusively by estrogens in females and by androgens in males, and the dependence of sex steroid stimulation on an adequate level of vitamin D. After finding that parathyroid hormone can act as a mitogen via a phospholipase-C-phosphoinositide turnover pathway, we collaborated with colleagues at the GBF in Braunschweig to find that mid-region fragments of PTH could act exclusively as mitogens, without stimulating cAMP production leading to bone resorption. hPTH (28-48) variants designed to be resistant to proteolysis were efficient in stimulating CK specific activity in vitro and in vivo and increased cortical bone thickness and the number of proliferating epiphyseal cartilage cells in rat long bones. These results are put into an historical context and compared with recent studies, in this short, selective review.
Journal Article↗
"Non-hypercalcemic" analogs of 1alpha,25 dihydroxy vitamin D augment the induction of creatine kinase B by estrogen and selective estrogen receptor modulators (SERMS) in osteoblast-like cells and rat skeletal organs.
We have demonstrated previously that daily treatments for 3 days with the so-called "non-hypercalcemic" analogs of 1alpha,25 dihydroxy vitamin D in ROS 17/2.8 osteoblast-like cells, stimulate the specific activity of creatine kinase BB (CK), and that such treatment with these analogs followed by a single treatment with gonadal steroids, upregulates responsiveness and sensitivity to estradiol 17beta (E(2)) for the induction of CK. This study was designed to determine if these same "non-hypercalcemic" vitamin D analogs could upregulate in vivo the response to E(2) and whether substitution of selective estrogen receptor modulators (SERMS) for E(2) would result in the same upregulation. We found that one week or 2 weeks pretreatment of prepubertal rats with vitamin D analogs led to increased induction of CK by E(2) and by the SERMS tamoxifen, tamoxifen methiodide and raloxifene, in epiphysis and diaphysis of the femur but not in the uterus. However, in contrast to their antiestrogenic activity in the uterus, there was no inhibition of E(2) action by the SERMS in skeletal tissues. The induction of mRNA for ckb in ROS 17/2.8 cells by E(2) or SERMS was demonstrated only after vitamin D pretreatment; there was no inhibition of E(2) induction by SERMS. Antagonists of vitamin D dependent calcium transport (transcaltachia) did not inhibit stimulation by vitamin D analogs. These results support the involvement of a nuclear mechanism in the upregulation of induction of CK by E(2), which may be due, in part, to the ability of vitamin D to increase estrogen receptor(s).
Animals↗
Paradoxical interactions among estrogen receptors, estrogens and SERMS: mutual annihilation and synergy.
The phenomenon of mutual annihilation of action between 17beta estradiol (E(2)) and a selective estrogen receptor modulator (SERM), previously described in prepubertal rat diaphysis, epiphysis and uterus, has been investigated in ROS 17/2.8 rat osteoblastic cells and in transiently co-transfected cells in culture. In ROS 17/2.8 cells, the estrogen-induced marker enzyme creatine kinase B (CKB) was stimulated by raloxifene, tamoxifen and tamoxifen methiodide to a specific activity equal to or greater than that induced by 10 nM E(2). However, when a fully inhibitory dose of any of these SERMS was given simultaneously with E(2), no stimulation of CK activity resulted. Therefore, SERMS can be full agonists when acting alone, but complete antagonists to a super-physiological dose of estrogen. It is expected that excess tamoxifen would prevent the action of a SERM, but that the agonist activity of a SERM is abolished by 1000-fold less estrogen is a phenomenon without obvious explanation by classical pharmacology of competitive inhibition. To probe the mechanism of this interaction further, a ckb-CAT reporter plasmid, plus the human receptor expression plasmid, HEO, was transfected transiently into several cell types. In MCF-7 cells, a 1:10 ratio of E(2) to tamoxifen produced mutual annihilation, but the same ratio in ROS 17/2.8 or HeLa cells led to synergistic stimulation. In HeLa cells, co-transfected with the more efficient wild-type estrogen receptor plasmid, HEGO, synergy was demonstrated only at sub-saturation levels of HEGO. We speculate that, in the presence of estradiol and a SERM, not only active homodimers would be formed, but also hetero-dimers of estrogen-liganded and tamoxifen-liganded receptor monomers, depending on the molar ratio of their ligands and their relative affinities. The resulting hetero-dimer conformation would change the specific receptor surface for interactions with the growing number of co-activators and co-repressors, structural changes which could help to explain the mutual annihilation and synergy phenomena and their cell selectivity.
Antineoplastic Agents, Hormonal↗