Regulation of AUF1 expression via conserved alternatively spliced elements in the 3' untranslated region.

The A+U-rich RNA-binding factor AUF1 exhibits characteristics of a trans-acting factor contributing to the rapid turnover of many cellular mRNAs. Structural mapping of the AUF1 gene and its transcribed mRNA has revealed alternative splicing events within the 3' untranslated region (3'-UTR). In K562 erythroleukemia cells, we have identified four alternatively spliced AUF1 3'-UTR variants, including a population of AUF1 mRNA containing a highly conserved 107-nucleotide (nt) 3'-UTR exon (exon 9) and the adjacent downstream intron (intron 9). Functional analyses using luciferase-AUF1 3'-UTR chimeric transcripts demonstrated that the presence of either a spliceable or an unspliceable intron 9 in the 3'-UTR repressed luciferase expression in cis, indicating that intron 9 sequences may down-regulate gene expression by two distinct mechanisms. In the case of the unspliceable intron, repression of luciferase expression likely involved two AUF1-binding sequences, since luciferase expression was increased by deletion of these sites. However, inclusion of the spliceable intron in the luciferase 3'-UTR down-regulated expression independent of the AUF1-binding sequences. This is likely due to nonsense-mediated mRNA decay (NMD) owing to the generation of exon-exon junctions more than 50 nt downstream of the luciferase termination codon. AUF1 mRNA splice variants generated by selective excision of intron 9 are thus also likely to be subject to NMD since intron 9 is always positioned >137 nt downstream of the stop codon. The distribution of alternatively spliced AUF1 transcripts in K562 cells is consistent with this model of regulated AUF1 expression.

Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover.

Prostaglandin A(2) (PGA(2)), an experimental chemotherapeutic agent, causes growth arrest associated with decreased cyclin D1 expression in several cancer cell lines. Here, using human non-small-cell lung carcinoma H1299 cells, we investigated the mechanisms whereby PGA(2) down-regulates cyclin D1 expression. Transcription rates of the cyclin D1 gene, studied using a cyclin D1 promoter-luciferase construct and nuclear run-on assays, were not affected by PGA(2) treatment. Instead, the cyclin D1 mRNA was rendered unstable after exposure to PGA(2). Since the stability of labile mRNA is modulated through binding of proteins to specific mRNA sequences, we sought to identify protein(s) recognizing the cyclin D1 mRNA. In electrophoretic mobility-shift assays using radiolabeled RNA probes derived from different regions of cyclin D1 mRNA, we observed that (i) lysates prepared from PGA(2)-treated cells exhibited enhanced protein-cyclin D1 RNA complex formation; (ii) the kinetics of complex formation correlated closely with that of cyclin D1 mRNA loss; and (iii) binding occurred within a 390-base cyclin D1 3' untranslated region (UTR) (K12). This binding activity could be cross-linked, revealing proteins ranging from 30 to 47 kDa. The RNA-binding protein AUF1, previously associated with the degradation of target mRNAs, bound cyclin D1 mRNA, because anti-AUF1 antibodies were capable of supershifting or immunoprecipitating cyclin D1 mRNA-protein complexes. Finally, insertion of K12 in the 3'UTR of reporter genes markedly reduced the expression and half-life of the resulting chimeric mRNAs in transfected, PGA(2)-treated cells. Our data demonstrate that PGA(2) down-regulates cyclin D1 expression by decreasing cyclin D1 mRNA stability and implicates a 390-base element in the 3'UTR in this regulation.

Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe.

The effects of the expression of the human Bcl-2 family proteins Bax, Bak, Bcl-2, and Bcl-XL were examined in the fission yeast Schizosaccharomyces pombe and compared with Bax-induced cell death in mammalian cells. Expression of the proapoptotic proteins Bax and Bak conferred a lethal phenotype in this yeast, which was strongly suppressed by coexpression of the anti-apoptotic protein Bcl-XL. Bcl-2 also partially abrogated Bax-mediated cytotoxicity in S. pombe, whereas a mutant of Bcl-2 (Gly145Ala) that fails to heterodimerize with Bax or block apoptosis in mammalian cells was inactive. However, other features distinguished Bax- and Bak-induced death in S. pombe from animal cell apoptosis. Electron microscopic analysis of S. pombe cells dying in response to Bax or Bak expression demonstrated massive cytosolic vacuolization and multifocal nuclear chromatin condensation, thus distinguishing this form of cell death from the classical morphological features of apoptosis seen in animal cells. Unlike Bax-induced apoptosis in 293 cells that led to the induction of interleukin-1 beta-converting enzyme (ICE)/CED-3-like protease activity, Bax- and Bak-induced cell death in S. pombe was accompanied neither by internucleosomal DNA fragmentation nor by activation of proteases with specificities similar to the ICE/CED-3 family. In addition, the baculovirus protease inhibitor p35, which is a potent inhibitor of ICE/CED-3 family proteases and a blocker of apoptosis in animal cells, failed to prevent cell death induction by Bax or Bak in fission yeast, whereas p35 inhibited Bax-induced cell death in mammalian cells. Taken together, these findings suggest that Bcl-2 family proteins may retain an evolutionarily conserved ability to regulate cell survival and death but also indicate differences in the downstream events that are activated by overexpression of Bax or Bak in divergent cell types.

The search for trans-acting factors controlling messenger RNA decay.

Control of mRNA turnover is an integral component of regulated gene expression. Individual mRNAs display a wide range of stabilities, which in many cases have been linked to discrete sequence elements. The most extensively characterized determinants of rapid constitutive mRNA turnover in mammalian systems are A + U-rich elements (AREs), first identified in the 3' untranslated regions of many cytokine/lymphokine and protooncogene mRNAs. In this article, we describe recent advances in the characterization of ARE-directed mRNA turnover, including links to deadenylation kinetics and functional heterogeneity among AREs from different mRNAs. We then describe strategies employed in the search for trans-acting factors interacting with these elements. Using such techniques, an ARE-binding activity capable of accelerating c-myc mRNA turnover in vitro was identified, and named AUF1. Subsequent cloning and characterization revealed that AUF1 exists as a family of four proteins formed by alternative splicing of a common pre-mRNA and appears to function as part of a multisubunit trans-acting complex to promote ARE-directed mRNA turnover. Investigations using several systems have demonstrated that AUF1 expression and/or activity correlate with rapid decay of ARE-containing mRNAs, and that both expression and activity of AUF1 are regulated by developmental and signal transduction mechanisms.

Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells.

We have previously identified and characterized a novel member of the ATP-binding cassette superfamily of transport proteins, multidrug resistance protein (MRP), and subsequently demonstrated that its overexpression is sufficient to confer multidrug resistance on previously sensitive cells (Cole et al., Science (Washington DC), 258: 1650-1654, 1992; Grant et al., Cancer Res. 54: 357-361, 1994). In the present study, we have transfected two different eukaryotic expression vectors containing MRP complementary DNA into HeLa cells to study the pharmacological phenotype produced exclusively by overexpression of human MRP. The drug resistance patterns of the two MRP-transfected cell populations were similar. They were characterized by a moderate (5- to 15-fold) level of resistance to doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide, and a low (< or = 3-fold) level of resistance to taxol, vinblastine, and colchicine. The transfectants were not resistant to 9-alkyl anthracyclines, mitoxantrone, or cisplatin. The MRP-transfected cells were also resistant to some heavy metal anions including arsenite, arsenate, and trivalent and pentavalent antimonials but were not resistant to cadmium chloride. Accumulation of radiolabeled vincristine was reduced by 45% in the MRP-transfected cells and could be restored to the levels found in sensitive cells by depletion of ATP. Rates of vincristine efflux did not differ greatly in the sensitive and resistant cells. The cytotoxic effects of vincristine and doxorubicin could be enhanced in a dose-dependent fashion by coadministration of verapamil. Cyclosporin A also increased vincristine toxicity but had less effect on doxorubicin toxicity. The degree of chemosensitization by verapamil and cyclosporin A was similar in MRP-transfected cells and in cells transfected with the vector alone, suggesting that sensitization involved mechanisms independent of MRP expression. Verapamil and cyclosporin A caused a modest increase in vincristine accumulation in the resistant cells but did not restore levels to those of the sensitive cells. Taken together, these data indicate that drug-resistant cell lines generated by transfection with MRP complementary DNA display some but not all of the characteristics of MRP-overexpressing cell lines produced by drug selection in vitro. They further demonstrate that the multidrug resistance phenotype conferred by MRP is similar but not identical to that conferred by P-glycoprotein and includes resistance to arsenical and antimonial oxyanions.

The effect of E1 mutations on biochemical transformation by an adenovirus carrying the herpes simplex virus thymidine kinase gene in region E3.

A series of human adenovirus type 5 (Ad5) vectors has been constructed in which a vector containing the human herpes simplex virus thymidine kinase (TK) gene has been recombined with several Ad5 early region 1 (E1) mutants. The resulting viruses were used to study host-virus interactions in TK- rat cells and to examine the importance of E1 functions in a biochemical transformation assay. One of the most important parameters affecting transformation efficiency in this system was the cytotoxicity of the transforming virus. Ad5 viruses expressing the E1a 289 amino acid protein were all highly cytotoxic and induced significantly fewer colonies than did less cytotoxic mutants which were defective in expression of the 289 amino acid product. When correction was made for differential cell viability the variation in transformation efficiencies was considerably reduced although some E1a mutants still demonstrated an enhanced ability to transform in comparison to wt virus. The significance of these results to morphological transformation by adenoviruses is discussed.

Diuretics: mechanism of action and clinical application.

Despite the bewildering number of diuretics available to the physician, these drugs can be divided into 4 main groups, characterised by their site of action on sodium reabsorption in the kidney. Drugs acting on the ascending limb of the loop of Henle have a powerful but short acting diuretic effect; they include frusemide, ethacrynic acid and bumetanide. The benzothiadiazines and related compounds have a moderate diuretic action spread over a longer period, whilst the potassium-sparing diuretics, triamterene, amiloride and spironolactone, have only a weak diuretic effect but a marked ability to diminish urinary potassium excretion. The fourth group is made up of miscellaneous substances which function as vasodilator or osmotic agents. The pathogenesis of oedema formation in heart failure is outlined and a logical approach to treatment suggested. Duiretics are being increasingly used in the treatment of non-oedematous states, in particular hypertension, diabetes insipidus and hypercalciuria; their exact role in pregnancy and acute renal failure remains controversial. Side-effects can be related to their effect on electrolyte excretion and include hypokalaemia, hyponatraemia, hyperkalaemia and hyperuricaemia. The incidence of disturbed carbohydrate tolerance in previously normal individuals is low. Other less common side-effects are also discussed.

Coincidence of true and false left ventricular aneurysm.

Coincidence of true and false left ventricular aneurysm is very rare. To date 6 cases have been reported in the world literature. We present a case of false aneurysm emanating from a posterior true aneurysm of the left ventricle. These findings were demonstrated preoperatively by transesophageal echocardiography and were confirmed at operation. The aneurysms were successfully resected and the ventricle repaired.

The clinical utility of automatic boundary detection for the determination of left ventricular volume: a comparison with conventional off-line echocardiographic quantification.

The aim of this study was to compare measurements of echocardiographic volume with an on-line automatic boundary detection imaging system with those of a conventional off-line method for routine clinical studies. Automatic boundary detection imaging shows promise as a rapid, on-line method for quantitating left ventricular volumes by echocardiography. However, there is little information about the role of automatic boundary detection for routine clinical studies. Ninety-seven patients with a variety of clinical diseases who were referred for clinical transthoracic echocardiographic evaluation were studied in apical four-chamber and two-chamber imaging planes. End-diastolic volume, end-systolic volume, and ejection fraction obtained with automatic boundary detection images were compared with those of conventional off-line analysis. Segmental endocardial definition and border tracking were evaluated on all automatic boundary detection images. Left ventricular end-diastolic volumes obtained by automatic boundary detection correlated well but were systematically under-estimated compared with off-line analysis for the apical two-chamber (r = 0.83; underestimation = 42 +/- 33 ml; p < 0.05) and four-chamber views (r = 0.83; underestimation = 43 +/- 31 ml; p < 0.05). Left ventricular end-systolic volumes also correlated well but were underestimated by automatic boundary detection for the apical two-chamber (r = 0.83; underestimation = 14 +/- 26 ml; p < 0.05) and four-chamber views (r = 0.83; underestimation = 18 +/- 24 ml; p < 0.05). Ejection fraction was not predicted accurately for the entire study population (n = 97). However, for patients with complete endocardial definition (n = 32), automatic boundary detection accurately predicted ejection fraction with no systematic error compared with manually traced images for both the apical two-chamber (r = 0.86; p < 0.05) and four-chamber (r = 0.82; p < 0.05) views. Segmental analysis of endocardial tracking revealed significantly better tracking of the septal and lateral walls compared with other regions (p < 0.05). End-diastolic and end-systolic volumes determined by automatic boundary detection correlate well but underestimate volume compared with conventional off-line analysis. However, ejection fraction compares favorably for the two methods when there is complete endocardial definition.