No abstract

ABSTRACTcAMP regulates transcription of the gene encoding the a-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the a-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the a-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element.The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the a-subunit gene.Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone expressed in the placenta. Both the a-subunit and the P-subunit are required for biological activity (1). While a physiological regulator of hCG production has not been identified, the synthesis of both subunits can be stimulated by cAMP in placental explants and in human choriocarcinoma cells (2, 3). Recent reports from several laboratories have shown that cAMP regulates expression of the chorionic gonadotropin a-and ,-subunit genes, at least in part, at the level of transcription (refs. 4 and 5; A.M., R. Cox, and J.H.N., unpublished data).In the human a-subunit gene, the first 140 base pairs (bp) of 5' flanking sequence are sufficient to confer cAMP regulation to a heterologous gene after transfection and transient expression in choriocarcinoma cells (4). This suggests that a cAMP response element lies within this region. In the present study, we have constructed several expression vectors and have used a transient expression assay to localize this element to an 18-bp sequence that is repeated between positions -146 and -111 in the 5' flanking region of the a-subunit gene. A single copy ofthis cAMP response element is sufficient to confer the same degree of cAMP regulation as a 1500-bp fragment containing the a-subunit promoter. This response element functions independently of other promoter regulatory elements. PROCEDURES Construction of Vectors. Construction of the expression vector pHaCAT (Fig. 1A) was initiated by isolating a 1500-bp DNA fragment from the genomic clone of the human asubunit gene provided by J. Fiddes (6). ...

Modified nucleosides are present in mRNA of all eukaryotes, albeit at much lower levels than in other RNA moieties such as rRNA, tRNA, and snRNA. Modification by methylation occurs on the terminal guanosine of the cap (N 7 -methylguanosine), and the first two encoded nucleosides (2'-O-methylnuculeosides) in most higher eukaryotes. Additional modifications of cap nucleosides occur in special cases where the cap is derived by trans-splicing in nematodes and kinetoplastids. Modification by methylation also occurs at internal adenosine residues in many species (N 6 -methyladenosine). Modification by deamination occurs at specific adenosine residues (forming inosine) and cytidine residues (forming uridine) in very specific cases leading to post-transcriptional editing. Numerous studies have shown the importance of the cap N 7 -methylguanosine in translation, splicing, transport, and mRNA stability. The role of the 2'-O-methylnucleosides is not as well understood, but there is evidence that these modifications play some role in translation efficiency. The role of internal N 6 -methyladenosine residues is least known, and is the focus of this review. The formation of N 6 -methyladenosine is catalyzed by a complex enzyme containing a subunit (MT-A70) that co-localizes with nuclear speckles and appears to be widely expressed in all higher eukaryotes. Loss of this enzyme leads to a sporulation defect in yeast and to apoptosis in mammalian cells, although the exact mechanism by which the effects occur remains obscure.

There were no objective responses seen with single-agent fosbretabulin as administered in this trial, and we did not observe a doubling of survival as our primary endpoint. This is among the largest prospective trials ever conducted for ATC. Fosbretabulin has an acceptable safety profile in patients with advanced ATC, and one-third survived more than 6 months. Despite a small sample size, low baseline sICAM levels were predictive of event-free survival. Further prospective validation of sICAM as a therapeutic biomarker and exploring combination regimens with fosbretabulin are warranted.