Inhibition of cyclic AMP-triggered aromatase gene expression in human choriocarcinoma cells by antisense oligodeoxynucleotide.

Aromatase, an endomembrane-bound cytochrome P450, is the key enzyme of estrogen biosynthesis. Aromatase inhibitors, therefore, are clinically important tools in the treatment of estrogen-dependent tumor growth. To improve the specificity of these tools, inhibition at the nucleic acid level was examined. An antisense oligodeoxynucleotide complementary to the translation start region of human aromatase transcripts (antisense-arom) was synthesized and used to inhibit cyclic AMP-triggered aromatase gene expression in a human choriocarcinoma cell line (JEG-3), both as occurring in an autocrine fashion by secreted human chorionic gonadotropin or as induced by application of the membrane-permeating dibutyryl cyclic AMP. Significant inhibition was obtained in both cases, reaching 70% and 60%, respectively. In addition, the antisense-arom treatment led to accelerated mRNA degradation. The inhibition at the nucleic acid level was accompanied by a decrease of both the aromatase protein and microsomal aromatase activity. The data appear to indicate the antisense strategy to be a most promising approach for the development of a novel type of specific aromatase inhibitor.

The effects of phosphatase on the components of the cytochrome P-450-dependent microsomal monooxygenase.

Incubation of rabbit liver microsomes with alkaline phosphatase resulted in a marked decrease of NADPH-dependent monooxygenase activities. This decrease was found to be correlated with the decrease of NADPH-cytochrome c reductase activity catalyzed by NADPH-cytochrome P-450 reductase. Neither the content of cytochrome P-450, as determined from its CO difference spectrum, nor the peroxide-supported demethylase activity catalyzed by cytochrome P-450 alone was affected by the phosphatase treatment. NADH-cytochrome b5 reductase and cytochrome b5 were not affected by the phosphatase either. NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes lost its NADPH-dependent cytochrome c reductase activity upon incubation with phosphatase in a way similar to that of microsome-bound reductase. Flavin analysis showed that the phosphatase treatment caused a decrease of FMN with concomitant appearance of riboflavin. Alkaline phosphatase, therefore, inactivates the reductase by attacking its FMN, and the inactivation of the reductase, in turn, leads to a decrease of the microsomal monooxygenase activities.

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.

Rat C alpha catalytic subunit of the cAMP-dependent protein kinase: cDNA sequence and evidence that it is the only isoform expressed in myoblasts.

A full length cDNA clone encoding the C alpha type catalytic subunit of cAMP-dependent protein kinase was isolated from a cDNA library of differentiated rat myoblast L6 cell line. The 2137 bp clone codes for a protein of 351 amino acid residues having more than 90% sequence identity to C alpha subunits of other mammalians. The C alpha isoform was found to be the only isoform of catalytic subunits expressed in myoblast cells as was determined in Northern blot analysis.

Human casein kinase II subunit alpha: sequence of a processed (pseudo)gene and its localization on chromosome 11.

A human 4.3 kb genomic DNA fragment, containing the information of a processed (pseudo)gene of casein kinase II subunit alpha (CKII alpha) was isolated and sequenced. The genomic CKII alpha sequence is 99% homologous to the CKII alpha cDNA, carries several nucleotide exchanges, a poly(A) stretch at its 3' end and is flanked at both ends by a 16 bp repeat. It has a promoter-like region including two TATA boxes and a CAAT box. Although translation of transcripts would be terminated by a stop codon after two third of the coding region, the resulting protein would still contain the catalytic domains. However, so far Northern blots with a 3' specific probe were negative. The 4.3 kb genomic fragment containing the processed CKII alpha (pseudo)gene was mapped by in situ hybridization to chromosome 11p15.

Promoter of the gene encoding the bovine catalytic subunit of cAMP-dependent protein kinase isoform C beta 2.

Genomic sequences flanking the 5' end of the cDNA encoding isoform C beta 2 of the catalytic subunit of bovine cAMP-dependent protein kinase were cloned, sequenced and analyzed for promoter activity and transcription initiation sites. A region of 913 bp upstream the translation initiator ATG was amplified from genomic DNA by vectorette polymerase chain reaction. In primer extension reactions and RNase protection assays, residues C (at position -91), T (-71) and G (-70) were found to serve as transcription initiation sites of the gene. Amplification products and sub-fragments thereof were ligated upstream of the reporter gene chloramphenicol acetyltransferase to test for promoter activity. Constructs were transiently transfected into a Chinese hamster ovary cell line which was shown to express endogenous C beta 2 mRNA. The genomic sequence upstream the C beta 2 cDNA does have promoter activity. The region from position -51 to -292 proved sufficient to drive efficient transcription of the reporter gene. The promoter is AT rich (68%), does not contain a TATA box within 50 bp upstream of the first initiation site and possesses putative binding sites for several transcription factors such as PEA-3 and a glucocorticoid receptor.

Casein kinase 2 binds and phosphorylates the nucleosome assembly protein-1 (NAP1) in Drosophila melanogaster.

The nucleosome assembly protein-1 (NAP1) was originally identified in HeLa cells as a factor facilitating the in vitro assembly of nucleosomes. However, in yeast cells NAP1 is required in the control of mitotic events induced by the Clb2/p34(CDC28). Here, we show that Drosophila NAP1 is a phosphoprotein that is associated with a kinase able to phosphorylate NAP1. By using an in-gel kinase assay we found that this kinase displays a molecular mass of 38 kDa. Following purification and peptide microsequencing, we identified the kinase phosphorylating NAP1 as the alpha subunit of casein kinase 2 (CK2). With the help of a series of NAP1 segments and synthetic peptides, we assigned the CK2 phosphorylation sites to residues Ser118, Thr120, and Ser284. Interestingly, Ser118 and Thr120 are located within a PEST domain, while Ser284 is adjacent to the nuclear localization signal. Substitution of the identified phosphoresidues by alanine was found to reduce considerably the ability of CK2 to phosphorylate NAP1. The enhanced ability of CK2 to phosphorylate phosphatase-treated NAP1 extracted from Drosophila embryos and the similar tryptic phospho-peptide pattern of in vivo labelled NAP1 and in vitro labelled NAP1 with CK2 indicate that NAP1 is a natural substrate of CK2. Further analysis revealed that both CK2alpha and beta subunits are associated with NAP1 but we found that only the catalytic alpha subunit establishes direct contact with NAP1 on two distinct domains of this protein. The location of CK2 phosphorylation sites in NAP1 suggests that their phosphorylation can contribute to a PEST-mediated protein degradation of NAP1 and the translocation of NAP1 between cytoplasm and nucleus.

Generation of pyrophosphate from extracellular ATP at the surface of HeLa cells.

On incubation of intact HeLa cells with micromolar concentrations of [gamma 32 P]ATP under physiological conditions, the generation of inorganic pyrophophate in the supernatant has been observed. The reaction depends on incubation time and cell number employed; the enzymatic activity is not shed into the supernatant. Isotope dilution data support that the responsible enzyme(s) is located at the cell surface.

Assays of cell surface protein kinase: importance of selecting cytophilic substrates.

The ability of phosvitin and histone to serve as substrates for possible protein kinase(s) at the cell surface was examined with regard to their suitability as indicators of such activity. While phosvitin did not interfere with the membrane barrier of HeLa cells, 3T3 and SV 3T3 cells, histone caused severe damage as indicated by both uptake of viability stains Trypan Blue and diamidino-phenylindol and by release of intracellular compounds such as lactate dehydrogenase, cAMP-dependent protein kinase(s), and metabolically prelabelled proteins. Where intactness of the cell membrane is prerequisite for verification of ecto-protein kinase, histone cannot be used as substrate. In contrast, we found that phosvitin is suitable for assays of cell surface located protein kinase activity.

Serum-stimulated cell cycle entry of fibroblasts requires undisturbed phosphorylation and non-phosphorylation interactions of the catalytic subunits of protein kinase CK2.

Protein kinase CK2 is a pleiotropic Ser/Thr kinase occurring as alpha2beta2, alpha'2beta2, or alphaalpha'beta2 tetramers. A requirement in serum-stimulated cell cycle entry in both the cytoplasm and the nucleus of human fibroblasts for phosphorylation(s) by CK2 has been concluded from stimulation inhibition by microinjected antibodies against the regulatory subunit (beta). We have now examined this idea more directly by microinjection-mediated perturbation of phosphorylation and non-phosphorylation interactions of the catalytic subunits (alpha and alpha'), and by verifying the supposed matching of the cellular partition of CK2 subunits in the fibroblasts employed. While immunostaining and cell fractionation indicate that the partitions of subunits indeed match each other (with their predominant location in the nucleus in both quiescent and serum-stimulated cells), microinjection of substrate or pseudosubstrate peptides competing for the CK2-mediated phosphorylation in vitro resulted in significant inhibition of serum stimulation when placed into the nucleus but not when placed into the cytoplasm. Also inhibitory were nuclear but not cytoplasmic injections of antibodies against alpha and alpha' that affect neither their kinase activity in vitro nor their complexing to beta. The data indicate that the role played by CK2 in serum-stimulated cell cycle entry is predominantly nuclear and more complex than previously assumed, involving not only phosphorylation but also experimentally separable non-phosphorylation interactions by the catalytic subunits.

Tumour initiation in mouse skin by 7,12-dimethylbenz[a]anthracene: irrelevance of systemic activation.

The effect was investigated of 7,8-benzoflavone (BF) on tumour initiation by 7,12-dimethylbenz[a]anthracene (DMBA) applied either intragastrically or topically. A single topical application of BF drastically decreased tumour initiation via both routes. This decrease was in both cases inversely proportional to the interval between administration of DMBA and BF. The data suggest that the decisive step of metabolic activation of DMBA is mediated by cytochrome P-448 dependent monooxygenase(s) located within the epidermis.

Extent of skin tumour initiation in mice by 7,12-dimethylbenz[alpha]anthracene and induction of arylhydrocarbon monooxygenase are not causally related.

In order to clarify further the relation between extent of arylhydrocarbon monooxygenase (AHM) induction and tumour initiation by polycyclic aromatic hydrocarbons, AHM activity and concentration of 7,12-dimethyl-benz[alpha]anthracene (DMBA) equivalents in dorsal epidermis were determined after both intragastric and topical administration of DMBA. Although in each case the dose applied caused comparable tumor incidences, both the extent of AHM induction and the concentration of DMBA equivalents were remarkably different: while topically applied DMBA stimulated AHM several-fold, intragastric instillation led to only a slight increase, if any. The concentration of DMBA equivalents in epidermis was about 100-fold higher after topical DMBA application than after intragastric administration. These data provide further evidence that extent of tumour initiation by DMBA and of AHM induction are not causally related; positive correlations found in certain cases appear to be fortuitous. Induction of AHM seems to be rather a function of DMBA concentration adjustment in epidermis and to indicate, if anything, elevated detoxification.

Conversion of hepatic microsomal cytochrome P-450 to P-420 upon phosphorylation by cyclic AMP dependent protein kinase.

Cytochrome P-450, purified from liver microsomes of phenobarbital-induced rabbits, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Upon phosphorylation P-450 was found to be converted to its denatured form, P-420, as verified spectroscopically from the CO-bound form of the reduced cytochrome. The conversion was dependent on both kinase and ATP. Thus, cyclic AMP may regulate the biotransformation system through the control of the degradation rate of microsomal P-450 in vivo.

Phospholipid bilayer membranes play decisive roles in the cytochrome P-450-dependent monooxygenase system.

Hepatic microsomal monooxygenase was reconstituted by incorporating cytochrome P-450 and NADPH-cytochrome P-450 reductase, which had been purified from phenobarbital-pretreated rabbit liver microsomes, into phospholipid liposomal membranes. The NADPH-dependent monooxygenase activity of the reconstituted system was found to be dependent on the phospholipid-to-protein ratio, i.e., the two-dimensional concentration of the two proteins on the plane of the membranes. A similar concentration dependence was also observed in the cytochrome b5 and NADH-cytochrome b5 system, which had been incorporated into liposomal membranes. The diffusion process of the proteins in the membrane, therefore, plays an important role in the monooxygenase system. When the fluidity of the membrane was changed by utilizing a synthetic dimyristoylphosphatidylcholine, which shows a well-defined gel to liquid crystalline phase transition, the activation energy of the monooxygenase reaction was changed at around the phase transition temperature, suggesting a conformational change of cytochrome P-450 caused by the fluidity change of the membrane. The incorporation of P-450 into liposomes was also found to affect the binding of substrates to cytochrome P-450. The decrease in the apparent dissociation constant of substrates upon incorporation into membranes suggests that the lipid membrane acts as a pool for hydrophobic substrates, which are concentrated in the lipid phase, and that cytochrome P-450 takes substrates directly from the membrane phase. Phospholipid membranes, therefore, play very important roles in various phases of the reaction of cytochrome P-450-dependent monooxygenase.

Posttranslational modifications of the cytochrome P-450 monooxygenase system.

Two forms of enzymatic posttranslational modifications of the monooxygenase system are described: modification by phosphatase and modification by protein kinase. Phosphatase treatment of microsomes isolated from phenobarbital-pretreated rabbits and rats caused a marked decrease of monooxygenase activity which was paralleled by a comparable decrease of NADPH-cytochrome P-450 reductase activity while the second essential component of the system, cytochrome P-450, remained unaltered. Thus phosphatase attacks monooxygenase via reductase. Protein kinases showed the opposite preference; while cytochrome P-450 was phosphorylated, NADPH-cytochrome P-450 reductase was not. Thus the kinase affects monooxygenase via cytochrome P-450. The phosphorylation of cytochrome P-450 turned out to be a specific reaction observed only with certain cytochrome P-450 isoenzymes and certain protein kinases.

Monooxygenase activity of human epithelial cells (HeLa): comparison of the response to treatment by arylhydrocarbons and diterpene esters.

Arylhydrocarbons are inducers of monooxygenase activity in epithelial cells in culture (Hela) whereas diterpene esters are not. This is one more example of a specific biochemical difference - here in gene activation - exhibited by tumor initiators and tumor promoters of the classes of compounds assayed.

Human casein kinase II: structures, genes, expression and requirement in cell growth stimulation.

Casein kinase II (CKII) is an ubiquitous Ser/Thr protein phosphotransferase in control of a variety of crucial cellular functions including metabolism, signal transduction, transcription, translation and replication. CKII levels are consistently higher in neoplastic tissues. The human CKII is composed of subunits alpha, alpha', and beta with molecular masses of 43, 38 and 28 kDa, respectively, that form heterotetrameric holoenzymes (alpha 2 beta 2; alpha alpha' beta 2, alpha'2 beta 2) showing autophosphorylation particularly at subunit beta and hence suspected to play a regulatory role. The amino acid sequences of subunits indicate high evolutionary conservation. Employing the complete set of tissue-derived (placenta) and recombinant (expressed in E. coli) subunits and CKII holoenzymes, the catalytic function of alpha and alpha' and the several-fold stimulation by beta is shown to occur comparably in tissue-derived and recombinant CKII and the autophosphorylation of beta is shown by site-directed mutagenesis to be not decisive for the tuning of CKII activity. The human genome contains two genes encoding CKII alpha. First, there is a processed (pseudo)gene which is 99% homologous to the CKII alpha cDNA and which possesses a promoter-like region adjacently upstream with TATA and CAAT boxes so that transcription cannot be excluded. Second, there is an active gene of which we have characterized so far a 18.9 kb long central fragment which contains 8 exons comprising bases 102-824 of the CKII alpha coding region. The gene fragment contains repetitive elements, most prominently 16 Alu repeats. The genome further contains one as yet uncharacterized CKII alpha' gene and one gene encoding CKII beta. The CKII beta gene has been characterized as a 4.2 kb spanning gene composed of seven exons which possesses three transcription start sites and the translation start site in the second exon. The first intron harbors an Alu repeat also. The promoter region of the CKII beta gene contains elements such as multiple GC boxes, a CpG island, and nonstandard-positioned CAAT boxes but lacks a TATA box thus characterizing the gene as a housekeeping gene. The CKII genes are not clustered at a certain chromosome but rather are distributed over the whole human genome. Using the genomic clones as the probes for in situ hybridization, the active CKII alpha gene was mapped to chromosome 20p13, the processed CKII alpha (pseudo)gene to chromosome 11p15, and the CKII beta gene to chromosome 6p21. (The CKII alpha' gene has been localized on chromosome 16 with a cDNA probe.).(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin.

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.