Differences in the induction of thymidine kinase isozymes in estrogen-treated immature and adult rats

Thymidine kinase activity in immature and castrated adult rat uterus has been examined in respose to estrogen treatment. Following estrogen administration. it was found that immature uterine thymidine kinase activity was increased 30-fold after 24 h, but almost no effect was produced on castrated or non-castrated adult uterus. Uterine thymidine kinase activity was separated into three peaks (peak 1, 2 and 3) by means of DEAE-cellulose column chromatography. In response to estrogen, the thymidine kinase isozymes differed in adult and immature uteri. In immature uteri, marked and selective increase of the activity was found in peak I, whereas in adult only a slight increase in peak 2 activity was observed. The thymidine kinase activity in peak 1 and peak 2 were found to have different enzymatic properties and molecular weight, as determined by gel filtration of 125 000 for peak 1 and 100 000 for peak 2.From these results, it is suggested that estrogen induces specific thymidine kinase isozyme in immature uterus and that the isozyme may be involved in DNA synthesis. Such a induction mechanism seems to be lost during the development.     

Progestin stimulation of thymidine kinase in the human breast cancer cell line T74D

Our laboratory has previously reported that progestins stimulate growth of the human breast cancer cell line T47D. In an attempt to probe further into this stimulation, we are investigating progestin effects of thymidine kinase (EC 2.7.1.21), an enzyme known to be involved in growth regulation. This report relates our finding that progestins stimulate thymidine kinase activity, at physiological progestin concentrations, in a dose-responsive manner. Estradiol-17β also stimulates, but testosterone, hydrocortisone and aldosterone do not. The antiprogestin RU486 inhibits progestin stimulation, but also stimulates on its own. Maximal by 24 h, the progestin stimulation then falls off with time. Experiments with actinomycin D and cycloheximide suggest that the thymidine kinase stimulation depends on new RNA and protein synthesis. These data shed further light on progestin stimulation of the growth of human breast cancer. To our knowledge, this is the first report of progestin stimulation of thymidine kinase in human breast cancer cells.     

Human mitochondrial thymidine kinase is coded for by a gene on chromosome 16 of the nucleus

The expression of human mitochondrial thymidine kinase (mt TK) was investigated by polyacrylamide electrophoresis in 19 independent human-mouse somatic cell hybrids which allowed all human chromosomes to be analyzed. In 8 hybrid clones the presence of this enzymatic activity could be demonstrated. Human mt TK segregated concordantly with human adenine phosphoribosyltransferase (APRT) and human chromosome 16. Discordant segregation with all other human chromosomes was demonstrated by karyotype and isozyme analyses. These results suggest that human mt TK is coded for by a gene on chromosome 16 of the nucleus. Thus human mt TK is genetically different from human cytosol thymidine kinase which is coded for by a gene on chromosome 17. The appearance of one heteropolymer band after electrophoretic separation of human and murine mt TK supports the notion that both enzymes have dimeric structures.     

Changes in enzyme activities of thymidine kinase and 5'-nucleotidase for dTMP during hormonal regeneration of seminal vesicles of mice.

An increase of thymidine kinase [EC 2.7.1.21] activity and decrease of 5'-nucleotidase [EC 3.1.3.5] activity for dTMP were found during hormonal regeneration of the seminal vesicles by daily or single administration of testosterone propionate into mice castrated 2 weeks previously. Actinomycin D injected on day 0 of testosterone treatment completely inhibited both the increase of thymidine kinase and the decrease of 5'-nucleotidase. When injected on day 2, actinomycin D decreased thymidine kinase activity below the control level and 5'nucleotidase activity was not restored to the normal level. The activity of 5'-nucelotidase in a mixed sample, in which seminal vesicles of castrated mice and those of testosterone-treated mice were homogenized together, was intermediate between the activities determined separately. This indicates the absence of any inhibitor of 5'nucleotidase in the regenerating vesicles. Changes in total activity of 5'nucleotidase and total protein content in extracts during various treatments showed that the decrease in specific activity of 5'-nucleotidase in the first 2 days of testosterone treatment was not due to inhibition of enzyme activity but to dilution of the enzyme with other proteins which increased in content more rapidly than 5'-nucleotidase.     

Induction of thymidine kinase synthesis by 5-androstene-3 beta,17 beta-diol in the uterus of the immature rat

In uteri from immature female rats, thymidine kinase activity was largely increased by administration of 5-androstene-3 beta, 17 beta-diol. Kinetic studies showed that the enzyme activity reached its maximum level 30 h after hormone administration. That increase in thymidine kinase activity was dose-dependent and could be related to the synthesis of new molecules of enzyme. Moreover, it was exclusively observed in target-organs for estrogens. It was concluded that 5-androstene-3 beta, 17 beta-diol which results from the metabolism of dehydroepiandrosterone had estrogen-like properties with regard to the induction of thymidine kinase.     

Metabolic alterations of liver regeneration. XV. Cadmium-mediated depression of thymidine and thymidylate kinase induction in rats.

Cadmium administered shortly before or after partial hepatectomy blocks in a dose-dependent manner the increase of thymidine and thymidylate kinase activities in regenerating rat livers. The effect of cadmium can be partially antagonized by simultaneous zinc administration. The intraperitoneal injection of cadmium is more effective than its subcutaneous administration. While there are in vitro differences in the sensitivity of thymidine kinase and thymidylate kinase towards Cd2+-, Zn2%- and Cu2+-ions, both enzymes are equally depressed following their in vivo administration. Cadmium displays the highest inhibitory activity and resembles in this respect beryllium [1].     

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.     

Selective expression of type I and type II cyclic AMP-dependent protein kinases in subcellular fractions of concanavalin A-stimulated rat thymocytes

Total protein kinase activity and the expression of the type I and type II cyclic adenosine 3′:5′-monophosphate-dependent protein kinases were studied in subcellular fractions of rat thymocytes and the effect of concanavalin A treatment on protein kinase activity was assessed. At a concentration of 100 μ/ml of concanavalin A a marked decline of total nuclear protein kinase activity occurred which lasted approximately 20 to 90 min. Concomitantly, a twofold increase of total protein kinase activity in the 900g supernatant fraction was observed which lasted from 5 to 30 min. Studies using the heat-stable protein kinase inhibitor revealed that the concanavalin A-mediated activity changes were primarily due to changes of cAMP-dependent protein kinase activity, whereas cAMP-independent protein kinase activity remained unchanged. Analysis of the type I and type II cAMP-dependent protein kinase isozyme pattern before and after concanavalin A treatment revealed a selective change of the relative expression of isozyme activities. Whereas type I protein kinase was the major nuclear isozyme before concanavalin A treatment, nuclear type II cAMP-dependent protein kinase increased markedly with a concomitant loss of type I isozyme expression. In the 900g supernatant fraction, containing primarily the type II isozyme in unstimulated cells, concanavalin A treatment caused an increase of the expression of the type I isozyme. The concanavalin A-mediated relative changes of cAMP-dependent protein kinase isozyme expression were confirmed by photoaffinity labeling of the regulatory subunits R^I and R^II before and after concanavalin A stimulation. The intracellular concanavalin A-mediated isozyme changes were time dependent, exhibiting maximal effects about 20 min after concanavalin A addition. These results indicate that selective regulation of intracellular cAMP-dependent protein kinase isozyme expression may be a mechanism related to isozyme-specific phosphorylation of specific intracellular substrates in concanavalin A-activated thymocytes.     

The effects of the administration of testosterone propionate alone or with phenobarbitone and of testosterone metabolites to neonatal female rats

The effects of graded doses of testosterone propionate administered to female rats on Day 4 of postnatal life have been determined. The incidence of failure of ovulation as adults was related to the dose. With increasing dosage over the range 1–100 μg no significant evidence of a progressive decrease in immunoassayable luteinizing hormone concentration in the plasma or anterior pituitary was obtained. The reported protective action of sodium phenobarbitone when administered with testosterone propionate could not be confirmed. Single injections of 100 μg of testosterone, dihydrotestosterone, 5 α-androstanediol, or a combination of the two latter compounds had no masculinizing effect. When the 17-β propionate derivatives of these compounds were administered at the same dose level only testosterone propionate had a masculinizing effect.     

Isoproterenol injection alters protein kinase DEAE-cellulose profiles in selected rat tissues.

Isoproterenol injection produces cardiac hypertrophy and salivary gland enlargement in rats. After subcutaneous injection (5 mg/kg) twice daily for 10 days, the soluble extracts from these and other tissues were subjected to DEAE-cellulose chromatography and the activity of isozymes I and II of cyclic AMP-dependent protein kinase was measured. The activity of isozyme I is decreased 80% in salivary gland and 40% in liver. No significant changes were observed in kidney, brain or skeletal muscle. In contrast to a previous report, no change was observed in the amount of isozyme I in hypertrophied heart. Furthermore, no changes were observed in the activity of isozyme II in response to isoproterenol injection in any of these tissues.     

TGF-beta-exposed plasmacytoid dendritic cells participate in Th17 commitment

TGF-β is required for both Foxp3(+) regulatory T cell (Treg) and Th17 commitment. Plasmacytoid DCs (pDC) have been shown to participate to both Treg and Th17 commitment as well. However, few studies have evaluated the direct effect of TGF-β on pDC, and to our knowledge, no study has assessed the capacity of TGF-β-exposed pDC to polarize naive CD4(+) T cells. In this paper, we show that TGF-β-treated pDC favor Th17 but not Treg commitment. This process involves a TGF-β/Smad signal, because TGF-β treatment induced Smad2 phosphorylation in pDC and blockade of TGF-β signaling with the SD208 TGF-βRI kinase inhibitor abrogated Th17 commitment induced by TGF-β-treated pDC. Moreover, TGF-β mRNA synthesis and active TGF-β release were induced in TGF-β-treated pDC and anti-TGF-β Ab blocked Th17 commitment. Unexpectedly, TGF-β treatment also induced increased IL-6 production by pDC, which serves as the other arm for Th17 commitment driven by TGF-β-exposed pDC, because elimination of IL-6-mediated signal with either IL-6- or IL-6Rα-specific Abs prevented Th17 commitment. The in vivo pathogenic role of TGF-β-treated pDC was further confirmed in the Th17-dependent collagen-induced arthritis model in which TGF-β-treated pDC injection significantly increased arthritis severity and pathogenic Th17 cell accumulation in the draining lymph nodes. Thus, our data reveal a previously unrecognized effect of TGF-β-rich environment on pDC ability to trigger Th17 commitment. Such findings have implications in the pathogenesis of autoimmune diseases or immune responses against mucosal extracellular pathogens.     

Inhibitory effects of Celiptium on thymidine kinase synthesis induced in the uterus by 17 beta-estradiol

Inhibitory effects of Celiptium on the thymidine kinase synthesis induced by oestradiol-17 beta in the rat uterus. In the rat uterus, the synthesis of thymidine kinase specifically induced by oestradiol-17 beta was inhibited by Celiptium. The synthesis was totally inhibited when the drug was administered before the oestrogen and partially when it was administered after. These facts suggested that Celiptium was competitive to the acceptor sites for oestradiol-receptors and inhibited the expression of the thymidine kinase gene.     

Genetic homology between man and the chimpanzee: syntenic relationships of genes for galactokinase and thymidine kinase and adenovirus-12-induced gaps using chimpanzee-mouse somatic cell hybrids.

The induction by adenovirus-12 of a site-specific gap and assignment of the chimpanzee genes for thymidine kinase and galactokinase were studied by utilizing chimpanzee-mouse hybrid cells. It has been shown that adenovirus-12 induces a specific gap in the long arm of human chromosome 17 (HS 17); with chimpanzee-mouse hybrid cells the specific gap appears on the short arm of the chimpanzee homolog [PTR 19 (HS 17)] of HS 17. This result supports the proposed relationship of HS 17 to PTR 19 (HS 17) by means of a pericentric inversion. The chimpanzee thymidine kinase and galactokinase genes were assigned to PTR 19 (HS 17), further confirming the homology to HS 17. Other syntenic relationships and gene assignments were consistent with proposed homologies between chimpanzee and human chromosomes.     

Properties of acetate kinase isozymes and a branched-chain fatty acid kinase from a spirochete

Spirochete MA-2, which is anaerobic, ferments glucose, forming acetate as a major product. The spirochete also ferments (but does not utilize as growth substrates) small amounts of l-leucine, l-isoleucine, and l-valine, forming the branched-chain fatty acids isovalerate, 2-methylbutyrate, and isobutyrate, respectively, as end products. Energy generated through the fermentation of these amino acids is utilized to prolong cell survival under conditions of growth substrate starvation. A branched-chain fatty acid kinase and two acetate kinase isozymes were resolved from spirochete MA-2 cell extracts. Kinase activity was followed by measuring the formation of acyl phosphate from fatty acid and ATP. The branched-chain fatty acid kinase was active with isobutyrate, 2-methylbutyrate, isovalerate, butyrate, valerate, or propionate as a substrate but not with acetate as a substrate. The acetate kinase isozymes were active with acetate and propionate as substrates but not with longer-chain fatty acids as substrates. The acetate kinase isozymes and the branched-chain fatty acid kinase differed in nucleoside triphosphate and cation specificities. Each acetate kinase isozyme had an apparent molecular weight of approximately 125,000, whereas the branched-chain fatty acid kinase had a molecular weight of approximately 76,000. These results show that spirochete MA-2 synthesizes a branched-chain fatty acid kinase specific for leucine, isoleucine, and valine fermentation. It is likely that a phosphate branched-chain amino acids is also synthesized by spirochete MA-2. Thus, in spirochete MA-2, physiological mechanisms have evolved which serve specifically to generate maintenance energy from branched-chain amino acids.     

Adenylate deaminase deficiency in a mutant murine T cell lymphoma cell line

From a population of wild type S49 cells, a clone, DTB6, was isolated in a single step from selective medium containing thymidine and dibutyryl cyclic AMP that exhibited a 60% deficiency in AMP deaminase (AMP-D) activity. The AMP-D deficiency conferred to the DTB6 cells a striking susceptibility to killing by low concentrations of either adenine or adenosine, the latter in the presence of an inhibitor of adenosine deaminase activity. This growth supersensitivity of DTB6 cells toward adenine could be ameliorated by the addition of hypoxanthine to the culture medium. Immunoprecipitation of AMP-D from wild type and mutant cells revealed that the DTB6 cell line contained markedly diminished amounts of the AMP-D isozyme which reacts with antisera to the predominant isoform expressed in adult kidney. The quantities of the AMP-D isozyme immunoprecipitated by antisera raised to the predominant isoform prepared from adult heart were equivalent in the two cell lines. Although Northern blot analyses revealed no alterations in mRNA sizes or levels encoded by either of the AMP-D genes, Southern blots of genomic DNA hybridized to a cDNA specific for the ampd2 gene revealed the presence of a new BamHI restriction fragment in the DNA of DTB6 cells. These data suggested that a point mutation has occurred in the ampd2 gene of DTB6 cells which encodes the AMP-D isozyme recognized by the kidney antisera. The DTB6 cells also possessed a virtual complete deficiency in thymidine kinase activity. The two enzyme deficiencies were distinguishable. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism.     

Induction of fetal thymidine kinase by phyto-estrogens in the immature rat uterus

Administration of phytooestrogens to immature female rats leads to a large increase in uterine thymidine kinase activity. That increase concerns to a large extent the fetal isoenzyme of thymidine kinase. These results confirm the estrogenic properties of phytoestrogens and allow to specify their physiological effects.     

Evolution of isozyme loci and their differential tissue expression

Summary The phylogeny of the creatine kinase (CK, EC 2.7.3.2) isozyme loci and their differential tissue expressions were determined for representatives of 65 families of vertebrates, with emphasis on the fishes. The transition from the single creatine kinase locus, characteristic of certain echinoderms, to the two creatine kinase loci which are orthologous to those present in all vertebrates, occurred early in the chordate line. The majority of pre-teleostean fishes possesses only these two CK loci (A and C). These loci are relatively generalized in their tissue expressions which are variable among species of primitive fishes. The third and fourth creatine kinase loci (B and D) arose separately in the ancestors of the bony fishes and appear to be the result of regional genome duplications. Concomitant with the increase in the number of isozyme loci has been an increase in the specificity of their tissue expression. In the advanced teleost fishes the four CK loci are differentially expressed in a characteristic manner. The A₂ isozyme predominates in skeletal muscle, the B₂ isozyme in eye and brain, the C₂ isozyme in stomach muscle, and the D₂ isozyme is found exclusively in testis. We propose a phylogeny of the creatine kinase genes in the lower chordates based on the time of appearance of new CK loci, the sequence in which the loci achieve a tissue restricted expression, and the immunochemical relatedness of the orthologous and paralogous gene products.