Synergistic effect of estrogen and androgen on induction of uterine thymidine kinase activities in immature rats

Estrdiol-17β induced a significant increase in the uterine thymidine kinase activity with a characteristic isozyme pattern 30 h after injection into immature rats. Testosterone propionate also revealed a similar increase. Following combined injection of estradiol-17β and testosterone propionate, the overall and separate isozyme activities of thymidine kinase increased to nearly the total amount of those when each hormone was injected separately.     

Arabinosylcytosine downregulates thymidine kinase and induces cross-resistance to zidovudine in T-lymphoid cells

The aim of this study was to determine molecular mechanism(s) responsible for the reduced thymidine kinase activity (TK) observed earlier in an arabinosylcytosine (araC) resistant lymphoid cell line (H9-araC cells), which was obtained following continuous cultivation of H9 cells in the presence of 0.5 microM araC. Compared to H9 cells, in H9-araC cells TK1 and TK2 gene expressions were reduced to 17.7% and 2.5%, respectively, and the cellular AZT accumulation was diminished to 35.8%. These cells were also found cross-resistant to azidothymidine (>42-fold). There was no significant difference in the expression of MDR1, MRP4 or TK protein. The lack of correlation between the expressions of TK protein and TK1 and TK2 suggests that post-translational factors may also play a role in the reduced TK activity in H9-araC cells. These findings suggest that araC affects TK expression at the genetic level.     

Cloning and sequence analysis of thymidine kinase from the oral bacterium Streptococcus gordonii

Abstract Thymidine kinase is an important enzyme in the pyrimidine nucleotide salvage pathway and catalyzes the formation of thymidylate from thymidine using ATP as a phosphate donor. The gene encoding thymidine kinase of the oral bacterium Streptococcus gordonii was cloned and the nucleptide sequence determined. The inferred amino acid sequence of thymidine kinase (191 amino acids) exhibited 43% identity with type H thymidine kinase from Escherichia coli . The S. gordonii thymidine kinase expressed in Escherichia coli KY895 ( tdk⁻ ) was inhibited by thymidline triphosphate, a feature typical of type II thymidine kinases. Immediately 3' to the tdk gene, and possibly co-transcribed with it, was the gene encoding release factor 1 ( prfA ).     

Dynamics of estrogen induction of glandular kallikrein in the rat anterior pituitary

Glandular kallikrein has recently been identified as an estrogen-induced protein of the rat anterior pituitary. This study examined the dynamics of the estrogen induction of anterior pituitary glandular kallikrein in the ovariectomized rat. The estrogen induction of uterine dry weight was also examined for purposed of comparison. 17β-Estradiol (0.1–100 μg/day) produced dose-dependent increases in anterior pituitary glandular kallikrein, with the highest dose producing a 60-fold increase. Time-course studies demonstrated that a lag phase of 2–3 days was required before these estrogen effects on glandular kallikrein became evident, and levels were still rising between 7 and 10 days of treatment. The dynamics of the estrogen induction of glandular kallikrein resembled the estrogen induction of uterine dry weight with regard to estrogen sensitivity and the presence of a lag phase before estrogen-induced increases. However, uterine dry weight responde more rapidly to estrogen than did anterior pituitary glandular kallikrein, and reached a plateau after 5 days of estrogen treatment.     

Preferential alkaline phosphatase isoenzyme induction by sodium butyrate

SW-620, a continuous cell line derived from a poorly differentiated human colon carcinoma, produces two alkaline phosphatases. Under basal conditions the heat-stable, term-placental is the major isoenzyme and the heat-labile, liver/bone/kidney form represents a minor component. Exposing SW-620 cells to sodium butyrate causes induction of increased levels of activity accompanied by a striking shift in isoenzyme distribution not observed heretofore. The activity increase is accounted for entirely by augmentation of the liver/bone/kidney isoenzyme, with the term-placental form not being affected. Two other known alkaline phosphatase inducers, prednisolone and hyperosmolality, do not influence specific activity and isoenzyme distribution. The preferential induction of the liver/bone/kidney form of alkaline phosphatase in SW-620 cells may reflect a butyrate-elicited expression of a more differentiated state.     

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.     

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.     

Effect of glucose on the induction of δ-aminolevulinic acid synthase and ferrochelatase in isolated rat hepatocytes by allylisopropylacetamide

The present work shows that allylisopropylacetamide exerts an inducing effect on δ-aminolevulinic acid synthase and ferrochelatase activities in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the inducing effect produced in both enzymes. Glucose inhibits the induction of δ-aminolevulinic acid synthase and ferrochelatase in this in vitro system. A similar effect was observed with fructose and 2-deoxyglucose. No glucose effect was observed with galactose, mannose, glycerol, pyruvate and lactate. The glucose effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. The simple in vitro method used in the present work promises to be a very useful tool for studies of regulatory mechanisms of porphyrin and heme biosynthesis in hepatocytes under normal and pathological conditions (hepatic porphyrias).     

Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin-binding protein

Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTK deltaC36, DdTK deltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTK deltaN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113+/-0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2+/-0.8%) and W13 (96.6+/-0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein.     

Down-regulation of mitochondrial thymidine kinase 2 and deoxyguanosine kinase by didanosine: Implication for mitochondrial toxicities of anti-HIV nucleoside analogs

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2′,3′-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs.     

The induction of liver microsomal proteins by 3-methylcholanthrene in rats of different age,sex and strain

The treatment of male and female adult and male and female immature rats with 3-methylcholanthrene results in increases in the intensity of two liver microsomal protein bands separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The induced protein species have estimated molecular weights of about 55 000 and 52 500. The time course of the induction of these species has been followed in a semi-quantitative manner. In immature rats an additional band, corresponding to a protein species with a molecular weight of about 43 000, is also increased by 3-methylcholanthrene treatment. In male adults rats exclusively, treatment with 3-methylcholanthrene also results in a decrease in the intensity of a band corresponding to a protein species of about 51 000. The appearance of this band in the liver microsomal fraction of adult male rats occurs following sexual maturation and its presence represents a significant difference in the microsomal proteins between male and female rats, this protein is not detected in younger male rats or in female rats at any age. These findings are discussed in terms of their possible relevance to the cytochrome P-450 content of the endoplasmic reticulum of rat hepatocytes.     

Gene expression and localization of diacylglycerol kinase isozymes in the rat spinal cord and dorsal root ganglia

The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKζ and DGKι in the DRG. By immunohistochemical analysis, DGKζ immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKι immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKζ in the nucleus and DGKι in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKζ between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.The work was supported by grants-in-aid from the Ministry of Education, Science, Culture, and Sports of Japan (M.T., K.G.) and from the Ono Medical Research Foundation, Kato Memorial Bioscience Foundation, and Janssen Pharmaceutical (K.G.) and by the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Gene expression, cellular localization, and enzymatic activity of diacylglycerol kinase isozymes in rat ovary and placenta

Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGK, -, -, and - was detected in the ovary and placenta. Intense expression signals for DGK and - were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGK were seen more intensely in granulosa cells. In the placenta, signals for DGK and - were observed in the junctional zone, whereas those for DGK were detected in the labyrinthine zone. At higher magnification, the signals for DGK were mainly discerned in giant cytotrophoblasts, and those for DGK were found in small cytotrophoblasts of the junctional zone. DGK signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGK signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.This work was supported by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, the Uehara Memorial Foundation, the ONO Medical Research Foundation, the Ciba-Geigy Foundation (Japan) for the Promotion of Science, the Kato Memorial Bioscience Foundation, and the Yamagata Health Support Foundation (to K.G.).     

Effect of glucose on induction of δ-aminolevulinic acid synthase,ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead

In the present work we have been able to demonstrate the phenobarbital and lead exert an inducing effect on the biosynthesis of δ-aminovulenic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the induction effect produced by phenobarbital in this in vitro system. Glucose inhibits the induction of δ-aminolevulinic acid synthase and ferrochelatase. This repression effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. No glucose effect was observed on the phenobarbital- and lead-mediated inductions of cytochrome P-450. The present results add more experimental evidence to support the concept that the last enzyme of the heme pathway is inducible, and as such may have a significant role in regulatory mechanisms of porphyrin and heme biosynthesis.     

The effects of glucocorticoids on thymidine kinase and nucleoside phosphotransferase during development of chicken embryo retina

Thymidine kinase in chick embryo retina reaches its highest values on the 8–10th day of development, then declines reaching the lowest value at hatching. The rate of DNA synthesis essentially follows this activity while, in contrast, nucleoside phosphotransferase increases progressively during development. Glucocorticoids at 5 × 10^?6M lower the level of thymidine kinase in isolated retinas of chick embryo. The most effective steroid was hydrocortisone. The effect was observed in retinas from 8–18-day-old chick embryo and, except on the 18th day, was always of the same magnitude. We suggest that a glucocorticoid can be the natural factor responsible for the marked fall in thymidine kinase during development. Brief periods of exposure to steriods increase nucleoside phosphotransferase activity in isolated chick embryo retinas. When the exposure was longer than 3 h this activity was also clearly decreased. We conclude that other factors are responsible for the natural increment which occurs for this activity during development.     

Expression and activation of Akt/protein kinase B in sexually immature and mature rat uterus

This study investigated the expression and activation of Akt/PKB in developing and adult rat uterus. Expression of Akt was observed in uteri from adult ovariectomized and 7–35-day-old rats and no changes were observed in response to in vivo estradiol treatment (1–100 μg/100 g b.w.). To examine the mechanisms of PKB/Akt activation, phosphorylation at Thr³⁰⁸ and Ser⁴⁷³ regulatory sites were studied in uteri. Akt was constitutively phosphorylated on Ser⁴⁷³ residue in the untreated, control uteri, while phosphorylation of Thr³⁰⁸ was observed only after estradiol 17β (E2) treatment. The effects of E2 treatment were age dependent, no response was induced in 11-day-old uteri, while in 28 days and older rats the activation of Akt at both regulatory sites, Ser⁴⁷³ and Thr³⁰⁸, increased, the first response was detected 2 h after treatment, reaching the highest rate at 6 h. The rate of phosphorylation was stronger at Ser⁴⁷³ residue. The results suggest that the regulation of Akt activation at two regulatory sites in rat uteri are different, phosphorylation of Thr³⁰⁸ seems to be entirely estrogen dependent, while the phosphorylation of Ser⁴⁷³ is regulated by other factors as well as estrogen.     

Induction of chemosensitivity in nasopharyngeal carcinoma cells using a human papillomavirus regulatory sequence and the thymidine kinase gene

Nasopharyngeal carcinoma (NPC) is a human cancer of epithelial cell origin. Infection by Epstein-Barr virus has been shown to be closely associated with this tumor. Recent studies have indicated that another common epitheliotropic virus, human papillomavirus (HPV), is also found in a significant number of NPC cases. In this study, we evaluated the feasibility of using the HPV regulatory long control region (LCR) to drive the expression of the thymidine kinase (tk) gene to achieve chemosensitivity for gene therapeutic treatment of NPC. Testing HPV-11-LCR-tk constructs in NPC cell lines in the presence of ganciclovir (GCV) led to 50-60% cell death of transfected cells. The therapeutic efficacy was further tested in an in vivo model using nude mice transplanted with tumors derived from transfected NPC cells. Injection of 50 mg/kg body weight GCV twice daily for 14 days resulted in visually complete regression of the transplanted NPC tumor loads within 20 days after GCV treatment. Taken together, results from this pilot study indicate the feasibility of the development of a gene therapeutic protocol based on the chemosensitive gene constructs described in this paper.     

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae

Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.     

Synthesis and characterization of a C6 nucleoside analogue for the in vivo imaging of the gene expression of herpes simplex virus type-1 thymidine kinase (HSV1 TK)

The synthesis and biological evaluation of '6-(1,3-dihydroxyisobutyl)thymine' (DHBT; 1), which corresponds to 6-[3-hydroxy-2-(hydroxymethyl)propyl]-5-methylpyrimidine-2,4(1H,3H)-dione, is reported. DHBT (1) was designed as a new substrate for herpes simplex virus type-1 thymidine kinase (HSV1 TK). The compound was found to be exclusively phosphorylated by HSV1 TK, and to exhibit good binding affinity (Ki = 35.3+/-1.3 microM). Cell-proliferation assays with HSV1-TK-transduced human osteosarcoma cells (143B-TK+-HSV1-WT) and with both human-thymidine-kinase-1-negative (143B-TK-) and non-transduced parental (MG-63) cells indicate that 1 is less cytotoxic than the standard drug Ganciclovir. Thus, DHBT (1) represents a promising precursor of a nontoxic reporter probe for the monitoring of HSV1 TK gene expression by means of positron-emission tomography (PET).