Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027651 (tumor)
 685,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reactivity of recombinant and tumor-derived preparations of oncomodulin toward 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent) and dansylaziridine was investigated. In contrast to previously published data (Mutus, B., Palmer, E. J., and MacManus, J. P. (1988) Biochemistry 27, 5615-5622), the apoprotein was observed to react far more rapidly than the calcium-bound form with Ellman's reagent. Attempts to quantitatively label the native protein with dansylaziridine met with little success, either with the metal-free or calcium-bound forms. In neither case did the extent of modification approach the level observed with the sodium dodecyl sulfate-denatured form of the protein. These results suggest that access to the sulfhydryl group of Cys-18 is severely restricted in the native protein, particularly when the high affinity ion-binding sites are occupied. Consistent with these observations, prolonged incubation of native oncomodulin at room temperature in the absence of reductant did not result in the generation of disulfide-linked dimers, either in the presence or absence of Ca2+. Interestingly, however, Cu2+ ion was observed to facilitate the apparent dimerization of oncomodulin. This reaction, which occurs more rapidly with the Ca2(+)-free form of the protein, affords material with the expected electrophoretic mobility. However, in contrast with the results of Mutus et al., dimeric oncomodulin prepared in this manner fails to stimulate bovine heart cAMP phosphodiesterase.
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PMID:Reactivity of cysteine 18 in oncomodulin. 215 44

Retinoblastoma is a malignant intraocular tumor that primarily affects small children. These tumors are primitive neuroectodermal malignancies, however some of them show morphologic evidence of differentiation into photoreceptors. Phototransduction cascades are a series of biochemical reactions that convert a photon of light into a neural impulse in rods and cones. The components of these cascades are uniquely expressed in photoreceptors and, although functionally similar, distinct components of these cascades are expressed in rods and cones. Using HPLC anion exchange chromatography, Western blot analysis, and specific monoclonal and polyclonal antibodies, we found that the cone but not the rod cGMP phosphodiesterase is functionally expressed in all six primary retinoblastomas examined and in three continuous retinoblastoma cell lines. Morphologic evidence of differentiation did not correlate with the expression of the enzyme. Furthermore, GTP analogues could activate the phosphodiesterase activity suggesting that an intact phototransduction cascade is present in the tumors. The presence of the cone phototransduction cascade in retinoblastoma confirms that this tumor has biochemically differentiated along the cone cell lineage.
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PMID:Expression of the functional cone phototransduction cascade in retinoblastoma. 216 31

The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was cloned and a consensus sequence for a glucocorticoid response element (GRE) was found at -513 bp, 5' to the transcriptional start site. In order to define the function of this element, fusion genes containing the PNMT promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were constructed. These constructs did not express after transfection into any of 7 continuous cell lines, none of which endogenously produce PNMT. A system for transfecting chromaffin cells in primary culture was therefore devised using constructs containing 200 bp of the proenkephalin (ENK) promoter, whose expression characteristics are well known. pENK beta GAL-1, containing the ENK promoter with a lac Z reporter, was introduced into these cells and beta-galactosidase activity was visualized in situ. Approximately 90% of cells transfected were chromaffin; transfection efficiency was 5%. High levels of CAT activity were measured in chromaffin cells transfected with pENKAT12, possessing a CAT reporter. In contrast to tumor cell lines, pENKAT12 induction in these cells by forskolin and phorbol esters did not require a phosphodiesterase inhibitor. In this chromaffin system, both basal and regulated expression of the PNMT fusion genes were detected. Dexamethasone (dex) induced expression of pPNMT3000 and pPNMT900, containing the putative GRE and 3000 bp or 863 bp of PNMT promoter sequence, 4- to 10-fold. Expression of pPNMT300 and pPNMT100, which lack the GRE and contain 273 bp or 99 bp of PNMT promoter sequence, was unaffected by dex. Addition of the PNMT region spanning -490 to -863 bp conferred full dex responsiveness to a thymidine kinase promoter. Deletion of the putative GRE sequence by site-directed mutagenesis abolished the dex response. These data identify the sequence at -513 bp in the rat PNMT gene as a functional, positively acting GRE. Primary cultures of bovine chromaffin cells provide a biologically relevant expression system for transcriptional studies of catecholamine genes and their related neuropeptides.
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PMID:Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture. 230 57

Urethane produces threefold more skin papillomas when administered orally than dermally in SENCAR mice, a strain susceptible to tumorigenesis. To better understand the relation of distribution to the initiation stage, [14C]urethane (0.10 mg/kg, 2.5 muCi/25 g) was administered orally and dermally to male SENCAR and BALB/c mice. Absorption of urethane was greater in the first hour in SENCAR mice by both routes, as indicated by more label in the liver, lung, and stomach than found in these tissues in BALB/c mice. These differences were not observed at later time periods after oral administration. Following dermal application, higher levels were maintained in the liver, lungs, and stomach through 48 h in the SENCAR mice when compared to BALB/c mice. Binding of [14C]urethane (0.062 mg/g body weight, 20 microCi/20 g body weight) to DNA, RNA, and protein 6 h after oral administration varied with tissue (liver greater than stomach greater than skin = lung) but did not differ with strain. Binding to DNA in skin, lung, and stomach, RNA in stomach, and protein in stomach and liver after 48 h were significantly higher in SENCAR mice than in BALB/c mice. Dermal application of [14C]urethane resulted in severalfold higher binding to liver DNA of SENCAR mice than BALB/c mice, but DNA binding was comparable in other tissues after 6 h. At 48 h after dermal application, significantly higher levels of [14C]urethane remained bound to skin DNA, RNA, and protein in BALB/c mice, although all values were lower than at 6 h after treatment. Differences in the distribution and binding of urethane probably do not account for the discrepancies in tumor sensitivity. Liver DNA hydrolysates were examined after 48 h. Thin-layer chromatography showed little incorporation of the 14C into the normal deoxyribonucleotide or deoxyribonucleoside bases, and no modified bases were apparent. Radioactivity was present in the fraction that remained at the origin and was consistent with a dinucleotide fragment resistant to phosphodiesterase cleavage, such as a phosphotriester.
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PMID:Distribution of urethane and its binding to DNA, RNA, and protein in SENCAR and BALB/c mice following oral and dermal administration. 241 23

The effect of isobutyl 1-methyl xanthine (IBMX) upon cyclic AMP level and DNA synthesis was determined in vivo in Ehrlich ascites tumor (EAT) cells. Cyclic AMP was found to be elevated by IBMX while 3H-thymidine incorporation into the cells was inhibited when the phosphodiesterase inhibitor was injected in the mice bearing the tumor cells. The effect upon cyclic AMP level and H thymidine incorporation was more pronounced in cells obtained 4 days after the implantation compared to the effect found 8 days after cells implantation. Following 12 days of implantation, a weaker effect was found upon cyclic AMP level and no effect was found on 3H-thymidine incorporation.
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PMID:Effect of in vivo administration of 3-isobutyl 1-methyl xanthine (IBMX) on cyclic AMP levels, and DNA synthesis in Ehrlich ascites tumor cells. 243 34

The diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS, have been previously shown to act as agonists and antagonists, respectively, in the activation of several mammalian cAMP-dependent protein kinases. In an effort to characterize further the involvement of cAMP in the activation of Leydig cell steroidogenesis by lutropin/choriogonadotropin (LH/CG), we examined the effects of these cyclic nucleotide analogues on a clonal strain of cultured murine Leydig tumor cells (designated MA-10). Our results show that (i) (Sp)-cAMPS activates and (Rp)-cAMPS inhibits the isolated cAMP-dependent protein kinase of the MA-10 cells; (ii) both analogues inhibit the isolated cAMP phosphodiesterase(s); (iii) (Sp)-cAMPS activates steroid biosynthesis in intact cells, but (Rp)-cAMPS does not; and (iv) (Rp)-cAMPS is a competitive inhibitor of the activation of steroidogenesis by (Sp)-cAMPS, 8-bromo-cAMP, human CG, cholera toxin, and forskolin. However, (Rp)-cAMPS is a more effective inhibitor when steroidogenesis is activated by (Sp)-cAMPS or 8-bromo-cAMP than when it is activated by human CG, cholera toxin, or forskolin. This difference appears to be related to the combined effects of (Rp)-cAMPS on the cAMP-dependent protein kinases and cAMP phosphodiesterase(s). We conclude that cAMP is a quantitatively important mediator of the activation of steroidogenesis by LH/CG even at low concentrations of hormone where an increase in steroid biosynthesis cannot be easily correlated with increased cAMP accumulation. Thus, our data indicate that if other second messengers are involved in the activation of steroidogenesis by LH/CG, they must do so by acting together with, rather than independently of, cAMP.
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PMID:Inhibition of choriogonadotropin-activated steroidogenesis in cultured Leydig tumor cells by the Rp diastereoisomer of adenosine 3',5'-cyclic phosphorothioate. 243 13

Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]orthophosphate under conditions where 2-chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (pheochromocytoma) cells. When NCB-20 cells were exposed to activators [5-hydroxytryptamine (5-HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP-dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5-HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa protein. [D-Ala2,D-Leu5]-enkephalin, which decreased cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa protein in NCB-20 cells. Pretreatment of NCB-20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90- and 130-kDa proteins, but phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80-kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid-dependent kinase with tumor promoting agent. Neither the 90-kDa nor the 80-kDa protein showed any immunological cross-reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase, but not calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique proteins can be phosphorylated by cyclic AMP-dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80-kDa protein is the primary substrate for calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP-dependent protein kinase.
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PMID:Neuromodulator-mediated phosphorylation of specific proteins in a neurotumor hybrid cell line (NCB-20). 245 Jan 74

The expression of the microsomal (M) antigen on the surface and in the cytoplasm of a strain of rat thyroid cells (FRTL-5) is under the regulation of TSH. In the present report the mechanism by which TSH induces such expression was investigated with the use of human microsomal antibody-positive serum and an indirect immunofluorescence technique. Studies were also performed to ascertain whether the M antigen of FRTL-5 cells could be identified with thyroid peroxidase (TPO), as suggested by recent data obtained in human thyroid tissue. Preabsorption experiments showed that, like solubilized human thyroid microsomes, purified human TPO completely abolished the binding of microsomal antibody to FRTL-5 cells. No inhibition was obtained by preabsorption with control human tissues (placenta, liver, and spleen) or human thyroglobulin, indicating that the antigen recognized by microsomal antibody in FRTL-5 cells was TPO. After 72 h of TSH withdrawal from the culture medium the M/TPO antigen disappeared from the surface and the cytoplasm of FRTL-5 cells. Readdition of TSH (250 microU/ml) to the culture medium of cells lacking the M/TPO antigen elicited its reappearance within 24-48 h. This effect of TSH was prevented by 10 microM cycloheximide or 0.5-5 micrograms/ml actinomycin D. Two well known stimulators of the adenylate cyclase-cAMP system, cholera toxin and forskolin, mimicked TSH in inducing the reappearance of the M/TPO antigen. A similar effect was observed with use of the phosphodiesterase inhibitor isobutylmethylxanthine. Reappearance of M/TPO antigen was also produced by the cAMP analog 8-bromo-cAMP. The tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate, which stimulates thyroid cell growth through a cAMP-independent pathway, was ineffective in inducing the M/TPO antigen in FRTL-5 cells. The present data indicate that 1) thyroid peroxidase accounts for most, if not all, of the microsomal antigen of FRTL-5 cells; and 2) TSH modulates the expression of the M/TPO antigen in FRTL-5 cells by a mechanism that involves cAMP production and requires mRNA formation and subsequent protein synthesis.
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PMID:Studies on the mechanism responsible for thyrotropin-induced expression of microsomal/peroxidase antigen in FRTL-5 cells. 245

Recent studies conducted in our laboratory have demonstrated that plasminogen activator (PA) is present in granulosa cells collected from the largest preovulatory follicle in the ovary of the domestic hen, and that its activity can be modulated by a variety of hormones in vitro. The present study was conducted to evaluate the intracellular mechanisms involved in the control of hen granulosa cell PA activity through the use of physiological and pharmacological agents. Treatment of granulosa cells with increasing doses (1, 10, and 50 ng/tube) of ovine LH resulted in a significant reduction of PA activity, which was accompanied by an increase in intracellular levels of cAMP. Furthermore, the effects of LH were potentiated by cotreatment with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM). Exposure of cells to increasing concentrations of the adenylyl cyclase activator forskolin (0.005, 0.01, 0.05, and 0.1 mM) resulted in a significant reduction in PA activity at all doses given. Similarly, the presence of the cAMP analog 8-bromo-cAMP (0.005, 0.01, 0.05, 0.1, 0.5, 1.5, and 10 mM) caused a dose-dependent inhibition of PA activity from 0.005 to 1.0 mM, further suggesting the involvement of cAMP in the inhibitory regulation of hen granulosa cell PA activity. The induction of intracellular calcium mobilization through the use of the calcium ionophore A23187 (0.1, 0.5, 1, and 2 microM) resulted in a dose-dependent suppression of PA activity. By contrast, treatment of granulosa cells with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA; 0.5, 5, 10, 25, and 50 micrograms/tube), a compound that activates protein kinase-C, stimulated PA activity in a dose-dependent fashion; a non-tumor-promoting phorbol ester (phorbol 13-monoacetate; 0.5, 10, and 50 ng/tube) was without effect. Coincubation of granulosa cells with a submaximal dose of PMA (5 ng/tube) and low concentrations of A23187 (0.001, 0.005, 0.01, and 0.05 microM) could not significantly enhance the stimulatory effects of PMA on PA activity; however, higher concentrations of the ionophore (0.1, 0.5, and 1.0 microM) completely abolished PMA-stimulated PA activity. The stimulatory effects of PMA could also be eliminated by cotreatment with a protein kinase-C inhibitor (H-7; 100 microM), a mRNA transcription blocker (actinomycin-D; 5 micrograms/tube), or a protein synthesis inhibitor (cycloheximide; 50 micrograms/tube).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of a phorbol ester, a calcium ionophore, and 3',5'-adenosine monophosphate production on hen granulosa cell plasminogen activator activity. 245 14

Cloned cell lines have proven to be useful models in understanding the regulation of endocrine cells and steroid synthesis. In this study we report the isolation and characterization of a subclone of the MA-10 Leydig tumor cell line. Whereas there was no difference in basal steroid production between the clone (MA-10 LP) and the parent stock (MA-10), MA-10 LP produces very low levels of progesterone after stimulation by hCG or (Bu)2cAMP. In both cell populations, hCG stimulation resulted in the accumulation of comparable amounts of cAMP in the presence of a phosphodiesterase inhibitor, and similar levels of cAMP were measured at 30 min without inhibitor. Measurement of cholesterol side-chain cleavage activity using two separate methods demonstrated that the low steroid production in MA-10 LP could not be accounted for by a decrease in the activity of this enzyme complex. Additionally, no difference in 3 beta-hydroxysteroid dehydrogenase activity could be demonstrated between the two cell populations. Since the lesion that attenuates the ability of MA-10 to synthesize progesterone is somewhere after the production of cAMP and before cholesterol side-chain cleavage activity, this system may provide a useful model for understanding the regulatory mechanisms controlling steroid biosynthesis in Leydig cells.
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PMID:Initial characterization of a subclone of the MA-10 mouse Leydig tumor cell line. 246 36


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