Blood thymidine level and iododeoxyuridine incorporation and reutilization in DNA in mice given long-acting thymidine pellets.
A long-acting thymidine pellet consisting of 190 mg of cholesterol and 60 mg of thymidine has been developed for the study of thymidine metabolism and reutilization in vivo. Implantation of such a pellet s.c. in adult mice will maintain the blood plasma concentration of thymidine at levels between 40 and 8 X 10(-6) M, which are from 36 to 7 times those of normal mice, for periods up to 48 hr. During this period, in vivo uptake and reutilization of [125I]iododeoxyuridine, a thymidine analog, into intestinal and tumor DNA were almost completely suppressed. While iododeoxyuridine reutilization is not large in normal proliferative tissue even in the absence of pellet implants, reutilization of over 30% was measured in large, rapidly growing ascites tumors. The inhibition of iododeoxyuridine incorporation by elevated thymidine blood levels is directly proportional to serum concentration. This appears to be due to a thymidine pool in rapid equilibrium with blood thymidine. This pool is at least 10 times larger than the 4-nmole pool of extracellular thymidine. (+info)
The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.
We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites. (+info)
The effects of glucocorticoids and progesterone on hormone-responsive human breast cancer in long-term tissue culture.
Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporation in three lines of human breast cancer maintained in long-term tissue culture. At steroid concentrations sufficient to inhibit thymidine incorporation 50%, little or no effect is seen on protein synthesis 48 hr after hormone addition. All three of these lines are shown to have glucocorticoid receptors demonstrable by competitive protein binding assays. Receptors are extensively characterized in one line by sucrose density gradient analysis and binding specificity studies. Good correlation between receptor-binding specificity and biological activity is found except for progesterone, which binds to glucocorticoid receptor but is noninhibitory. Cross-competition and quantification studies demonstrate a separate receptor for progesterone. This receptor has limited binding specificities restricted largely to progestational agents, whereas the glucocorticoid receptor bound both glucocorticoids and progesterone. Two other human breast cancer lines neither contain glucocorticoid receptor nor are inhibited by glucocorticoids. It is concluded that in some cases glucocorticoids can directly limit growth in human breast cancer in vitro without requiring alterations in other trophic hormones. (+info)
The effects of androgens and antiandrogens on hormone-responsive human breast cancer in long-term tissue culture.
We have examined five human breast cancer cell lines in continuous tissue culture for androgen responsiveness. One of these cell lines shows a 2- to 4-fold stimulation of thymidine incorporation into DNA, apparent as early as 10 hr following androgen addition to cells incubated in serum-free medium. This stimulation is accompanied by an acceleration in cell replication. Antiandrogens [cyproterone acetate (6-chloro-17alpha-acetate-1,2alpha-methylene-4,6-pregnadiene-3,20-dione) and R2956 (17beta-hydroxy-2,2,17alpha-trimethoxyestra-4,9,11-triene-1-one)] inhibit both protein and DNA synthesis below control levels and block androgen-mediated stimulation. Prolonged incubation (greater than 72 hr) in antiandrogen is lethal. The MCF- cell line contains high-affinity receptors for androgenic steroids demonstrable by sucrose density gradients and competitive protein binding analysis. By cross-competition studies, androgen receptors are distinguishable from estrogen receptors also found in this cell line. Concentrations of steroid that saturate androgen receptor sites in vitro are about 1000 times lower than concentrations that maximally stimulate the cells. Changes in quantity and affinity of androgen binding to intact cells at 37 degrees as compared with usual binding techniques using cytosol preparation at 0 degrees do not explain this difference between dissociation of binding and effect. However, this difference can be explained by conversion of [3H]-5alpha-dihydrotestosterone to 5alpha-androstanediol and more polar metabolites at 37 degrees. An examination of incubation media, cytoplasmic extracts and crude nuclear pellets reveals probable conversion of [3H]testosterone to [3H]-5alpha-dihydrotestosterone. Our data provide compelling evidence that some human breast cancer, at least in vitro, may be androgen dependent. (+info)
Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.
The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly. (+info)
The histone acetylase PCAF is a phorbol-ester-inducible coactivator of the IRF family that confers enhanced interferon responsiveness.
Transcription factors of the interferon regulatory factor (IRF) family bind to the type I interferon (IFN)-responsive element (ISRE) and activate transcription from IFN-inducible genes. To identify cofactors that associate with IRF proteins, DNA affinity binding assays were performed with nuclear extracts prepared from tissue culture cells. The results demonstrated that the endogenous IRFs bound to the ISRE are complexed with the histone acetylases, PCAF, GCN5, and p300/CREB binding protein and that histone acetylase activities are accumulated on the IRF-ISRE complexes. By testing recombinant proteins, we show that PCAF directly binds to some but not all members of the IRF family through distinct domains of the two proteins. This interaction was functionally significant, since transfection of PCAF strongly enhanced IRF-1- and IRF-2-dependent promoter activities. Further studies showed that expression of PCAF and other histone acetylases was markedly induced in U937 cells upon phorbol ester treatment, which led to increased recruitment of PCAF to the IRF-ISRE complexes. Coinciding with the induction of histone acetylases, phorbol ester markedly enhanced IFN-alpha-stimulated gene expression in U937 cells. Supporting the role for PCAF in conferring IFN responsiveness, transfection of PCAF into U937 cells led to a large increase in IFN-alpha-inducible promoter activity. These results demonstrate that PCAF is a phorbol ester-inducible coactivator of the IRF proteins which contributes to the establishment of type I IFN responsiveness. (+info)
The 3' end CCA of mature tRNA is an antideterminant for eukaryotic 3'-tRNase.
Cytoplasmic tRNAs undergo posttranscriptional 5' and 3' end processing in the eukaryotic nucleus, and CCA (which forms the mature 3' end of all tRNAs) must be added by tRNA nucleotidyl transferase before tRNA can be aminoacylated and utilized in translation. Eukaryotic 3'-tRNase can endonucleolytically remove a 3' end trailer by cleaving on the 3' side of the discriminator base (the unpaired nucleotide 3' of the last base pair of the acceptor stem). This reaction proceeds despite a wide range in length and sequence of the 3' end trailer, except that mature tRNA containing the 3' terminal CCA is not a substrate for mouse 3'-tRNase (Nashimoto, 1997, Nucleic Acids Res 25:1148-1154). Herein, we extend this result with Drosophila and pig 3'-tRNase, using Drosophila melanogaster tRNAHis as substrate. Mature tRNA is thus prevented from recycling through 3' end processing. We also tested a series of tRNAs ending at the discriminator base (-), with one C added (+C), two Cs added (+CC), and CCA added (+CCA) as 3'-tRNase inhibitors. Inhibition was competitive with both Drosophila and pig 3'-tRNase. The product of the 3'-tRNase reaction (-) is a good 3'-tRNase inhibitor, with a KI approximately two times KM for the normal 3'-tRNase substrate. KI increases with each nucleotide added beyond the discriminator base, until when tRNA+CCA is used as inhibitor, KI is approximately forty times the substrate KM. The 3'-tRNase can thus remain free to process precursors with 3' end trailers because it is barely inhibited by tRNA+CCA, ensuring that tRNA can progress to aminoacylation. The active site of 3'-tRNase may have evolved to make an especially poor fit with tRNA+CCA. (+info)
Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentrations.
Daidzein and genistein glucuronides (DG and GG), major isoflavone metabolites, may be partly responsible for biological effects of isoflavones, such as estrogen receptor binding and natural killer cell (NK) activation or inhibition. DG and GG were synthesized using 3-methylcholanthrene-induced rat liver microsomes. The Km and Vmax for daidzein and genistein were 9.0 and 7.7 micromol/L, and 0.7 and 1.6 micromol/(mg protein. min), respectively. The absence of ultraviolet absorbance maxima shifts in the presence of sodium acetate confirmed that the synthesized products were 7-O-glucuronides. DG and GG were further purified by a Sephadex LH-20 column. DG and GG competed with the binding of 17beta-(3H) estradiol to estrogen receptors of B6D2F1 mouse uterine cytosol. The concentrations required for 50% displacement of 17beta-(3H) estradiol (CB50) were: 17beta-estradiol, 1.34 nmol/L; diethylstilbestrol, 1.46 nmol/L; daidzein, 1.6 micromol/L; DG, 14.7 micromol/L; genistein, 0.154 micromol/L; GG, 7.27 micromol/L. In human peripheral blood NK cells, genistein at <0.5 micromol/L and DG and GG at 0.1-10 micromol/L enhanced NK cell-mediated K562 cancer cell killing significantly (P < 0.05). At > 0.5 micromol/L, genistein inhibited NK cytotoxicity significantly (P < 0.05). The glucuronides only inhibited NK cytotoxicity at 50 micromol/L. Isoflavones, and especially the isoflavone glucuronides, enhanced activation of NK cells by interleukin-2 (IL-2), additively. At physiological concentrations, DG and GG were weakly estrogenic, and they activated human NK cells in nutritionally relevant concentrations in vitro, probably at a site different from IL-2 action. (+info)