Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.

Harvest protocol to reduce variability of soluble enzyme yield from cultured cells.

Many aspects of physiology and gene regulation can be studied by examining the levels of enzymes harvested from cultured cells. We found that the yield from cultured cells of two different cytosolic enzymes, creatine kinase and the common reporter gene product chloramphenicol acetyltransferase (CAT), could be highly variable despite superficially identical harvest procedures. Analysis of multiple harvest and assay parameters disclosed that fluctuations in enzyme yield were correlated with the time cells that were allowed to remain in an EDTA-containing buffered saline solution prior to scraping from the dishes with a rubber policeman. The highest and most consistent yields were obtained when the cells were allowed to remain in the solution for 6-10 min before scraping: this protocol has cut variability approximately by a factor of three.

Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.

We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.

MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer.

MyoD is a skeletal muscle-specific protein that is able to induce myogenesis in a wide variety of cell types. In this report, we show that MyoD is a DNA binding protein capable of specific interaction with two regions of the mouse muscle creatine kinase gene upstream enhancer, both of which are required for full muscle-specific enhancer activity. MyoD shares antigenicity and DNA binding specificity with MEF1, a myocyte-specific DNA binding factor. The contiguous basic and myc homology regions of MyoD that are necessary and sufficient for specific DNA interaction are the same regions of the protein required to convert 10T1/2 fibroblasts into muscle. These findings suggest that the biological activity of MyoD is mediated via its capacity for specific DNA interaction.

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence.

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer.

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. We have examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.

Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts.

The muscle-specific form of creatine kinase (MCK) is induced in differentiating myoblast cultures, and a dramatic increase in mRNA levels precedes and parallels the increase in MCK protein. To study this induction, the complete MCK gene was cloned and characterized. The transcription unit was shown to span 11 kilobases and to contain seven introns. The splice junctions were identified and shown to conform to the appropriate consensus sequences. Close homology with branchpoint consensuses was found upstream of the 3' splice sites in six of seven cases. Transcriptional regulation of the gene in differentiating myoblast cultures was demonstrated by nuclear run-on experiments; increases in transcription accounted for a major part of the increased mRNA levels. Regulated expression of a transfected MCK gene containing the entire transcription unit with 3.3 kilobases of 5'-flanking sequence was also demonstrated during differentiation of the MM14 mouse myoblast cell line. The MCK 5'-flanking region was sufficient to confer transcriptional regulation to a heterologous structural gene, since chloramphenicol acetyl transferase activity was induced during differentiation of cultures transfected with an MCK-chloramphenicol acetyl transferase fusion construct. Examination of the DNA sequence immediately upstream of the transcription start site revealed a 17-nucleotide element which occurred three times. Comparisons with other muscle-specific genes which are also transcriptionally regulated during myogenesis revealed upstream homologies in the alpha-actin and myosin heavy chain genes, but not in the myosin light-chain genes, with the regions containing these repeats. We suggest that coordinate control of a subset of muscle genes may occur via recognition of these common sequences.

The mouse muscle creatine kinase cDNA and deduced amino acid sequences: comparison to evolutionarily related enzymes.

The nucleotide sequence of cloned DNA corresponding to full-length mouse muscle creatine kinase mRNA has been determined. This 1415 base pair DNA sequence and the deduced 381 amino acid sequence of the protein have been compared to creatine kinase sequences from other vertebrate species and to invertebrate guanidino kinase sequences. These comparisons show that the vertebrate muscle creatine kinases constitute a remarkably conserved protein family with a unit evolutionary period of 30. The creatine kinases also retain marked sequence similarity with the more distantly related invertebrate guanidino kinases. A portion of the sequence, presumably part of the ATP binding site, shows similarity to other nucleotide binding proteins with diverse functions. Comparisons of the untranslated regions of the creatine kinase cDNA sequences show that the 5' untranslated regions are more highly conserved than are the 3' untranslated regions; this may point to some regulatory function in the 5' region.

Single dose pharmacokinetics of mabuterol in man.

The pharmacokinetics of 1-(4-amino-3-chloro-5-trifluoromethyl-phenyl)-2-tert.-butylamino-ethanol hydrochloride (mabuterol), a new bronchodilator in the series of phenyl-ethanolamine derivatives, was investigated in six healthy young male volunteers. Use of tritiated compound allowed characterization of the plasma concentration-time profile of unchanged drug after a single oral dose of 40 micrograms in the form of a drinking solution. Peak concentrations of 96-160 pg/ml were reached between 1.5 and 3.2 h and drug concentrations declined thereafter in a biphasic pattern. Elimination of unchanged drug was slow (t 1/2 beta 20-30 h) due to a low clearance (CL/F 3.0 ml/min/kg) and a large volume of distribution (V beta/F 5.8 l/kg). Absorption of the drug from the gastrointestinal tract was complete and availability of unchanged drug to the systemic circulation could be estimated (F greater than 0.91) using flow model concepts. At peak concentrations mabuterol accounted for only 46% of total radioactivity in plasma but no major conjugated or unconjugated metabolite has been identified. The active tert.-butanol-amino-metabolite was present in negligible amounts (less than 10 pg/ml). The time profile of total radioactivity in plasma closely resembled that reported for structurally related compounds. Excretion of total radioactivity was mainly renal (80% of dose) but the fraction of the dose eliminated unchanged in urine was only 24%. Fecal excretion was low with a cumulative amount recovered over the entire study period of 3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of probenecid on ketoprofen kinetics.

When six normal men took probenecid with ketoprofen in a two-treatment crossover study, steady-state plasma concentrations of ketoprofen and ketoprofen conjugates rose, but plasma protein binding of ketoprofen and urinary excretion of ketoprofen conjugates decreased. Probenecid decreased protein binding of ketoprofen by 28 +/- 7%, total ketoprofen clearance by 67 +/- 11%, clearance of unbound ketoprofen by 74 +/- 10%, clearance of unbound ketoprofen by conjugation by 91 +/- 5%, and renal clearance of ketoprofen conjugates by 93 +/- 4%. An apparent decrease (22 +/- 29%) in unbound ketoprofen clearance by mechanisms other than conjugation might have been established in a study of more than six subjects. Probenecid, which reaches plasma concentrations that approach 100 times those of ketoprofen or its conjugates, appears to inhibit both the conjugation of ketoprofen and the renal excretion of ketoprofen conjugates. An alternative explanation to inhibition of conjugation involves cumulation and subsequent hydrolysis of ketoprofen conjugates as a result of the renal action of probenecid. In addition to the advantages of obtaining simultaneous uricosuric and anti-inflammatory effects, there may be clinical kinetic advantages of administration of probenecid with ketoprofen, because the large interdose concentration swings of ketoprofen are then substantially reduced.

Improved liquid-chromatography of aspirin, salicylate, and salicyluric acid in plasma, with a modification for determining aspirin metabolites in urine.

Detailed performance specifications are given for a specific and sensitive liquid-chromatographic assay for aspirin, salicylic acid, and salicyluric acid in plasma. The sensitivity of this method for aspirin (50 micrograms/L) is 10-fold that of previous methods, so that concentrations of aspirin in plasma can now be followed for about four or five half-lives after the peak plasma concentration arising from a single 650-mg dose. In addition, this assay is as sensitive for salicylic and salicyluric acid in plasma as any hitherto. A modification permits measurement of gentisic acid, salicylic acid, and salicyluric acid in urine; further modifications allow indirect measurement of conjugated gentisate and salicylate in urine.

Ketoprofen-aspirin interactions.

To assess an interaction between ketoprofen and aspirin we gave both drugs orally, to steady state, to healthy men. Although ketoprofen did not alter salicylate absorption and disposition, we observed that concurrent administration of aspirin decreased ketoprofen protein binding and increased its plasma clearance. Salicylate also appeared to reduce metabolic ketoprofen conversion to conjugates and their renal elimination. The data suggested that salicylate also enhanced the metabolic conversion of ketoprofen to nonconjugate metabolites (p = 0.091). Our findings indicate that the drug-drug interaction between aspirin and ketoprofen is complex.

Ketoprofen pharmacokinetics and bioavailability based on an improved sensitive and specific assay.

A commercial capsule containing 50 mg of ketoprofen (Orudis), a simple capsule containing 50 mg of ketoprofen alone and 50 mg of ketoprofen in an aqueous solution were given as separate doses in a randomized sequence to 12 normal adult males. The areas under the resulting plasma concentration-time curves (AUC) were remarkably consistent for each volunteer. The bioavailability from the commercial capsule relative to that from the solution was 99.7% +/- 10.5% and that from the simple capsule was 102% +/- 10%. After 6 of the volunteers had taken the commercial capsule 6 hourly for thirteen doses, their AUC extrapolated to infinity was significantly higher (by 22%) than that after the single dose indicating, contrary to previous reports, accumulation upon multiple dosing. The interdose AUC after the thirteenth dose was, however, statistically indistinguishable from the AUC-to-infinity after the single dose as might be expected from linear kinetics. The ketoprofen solution generated peak plasma concentrations in only one-third the time (21 +/- 7 min) required for the capsules (commercial, 72 +/- 45; simple, 61 +/- 39 min). Despite plasma concentrations being tracked over a 200-fold range, log linearity was not established within 12 h in any of the 42 profiles obtained. A two-compartment open model was fitted to the solution data giving excellent prediction of the time-to-peak and clearance (Cl/F = 5.2 +/- 1.1l/h) as determined by eye and by log-trapezoidal rule, respectively.

Negligible excretion of unchanged ketoprofen, naproxen, and probenecid in urine.

On the average, 0.6% of a dose of ketoprofen or naproxen or 1.2% of a dose of probenecid was found in the urine of normal male volunteers assayed immediately after its collection. Between approximately 60 and 85% of the dose of these drugs can be excreted in the urine as conjugates, which rapidly hydrolyze at body temperature, at room temperature, and even during frozen storage, thereby regenerating the parent drug. Since urine collections involved sample retention in the bladder at 37 degrees for collection intervals as long as 2--3 hr, the given percentages excreted unchanged probably are overestimates. It is possible that no unchanged ketoprofen, naproxen, or probenecid is excreted in urine. This study contrasts with previous reports of up to 50% of a dose of ketoprofen and 15--17% of doses of naproxen and probenecid being excreted in urine as the parent compound. Those reports probably reflect primarily the duration of frozen sample storage between collection and assay along with the urine collection schedules employed the speed of the clinical procedures, and the analytical procedures used. Attention should be given to potential conjugate hydrolysis whenever the pharmacokinetics of carboxylic acids are studied.

Convenient and sensitive high-performance liquid chromatography assay for ketoprofen, naproxen and other allied drugs in plasma or urine.

A new high-performance liquid chromatography technique enables convenient and rapid assay of ketoprofen and naproxen in biological samples at a sensitivity (10 and 2 ng/ml, respectively in plasma; 20 and 50 ng/ml in urine) far greater than previously available. Superior sensitivity is attributable to the buffered neutral eluent employed, which yields improved separation from material of biological origin. There is no interference from the major ketoprofen and naproxen metabolites tested and excellent reproducibility and accuracy can be maintained. Moreover, the same system can be used to assay probenecid and also shows promise of applicability to ibuprofen, fenoprofen and other members of the aryl-alkanoic acid class of non-steroidal anti-inflammatory agents.