BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19).

Translocation t(15;19)(q13;p13.1) defines a lethal midline carcinoma arising adjacent to respiratory tract in young people. To characterize molecular alterations responsible for the distinctly aggressive biological behavior of this cancer, we mapped the chromosome 15 and 19 translocation breakpoints by fluorescence in situ hybridization (FISH) and Southern blotting. To evaluate preliminarily the frequency, anatomical distribution, and histological features of t(15;19) cancer, we developed a FISH assay for paraffin sections. Our findings reveal a novel oncogenic mechanism in which the chromosome 19 translocation breakpoint interrupts the coding sequence of a bromodomain gene, BRD4. These studies implicate BRD4 as a potential partner in a t(15;19)-associated fusion oncogene. In addition, we localized the chromosome 15 breakpoint to a 9-kb region in each of two cases, thereby identifying several candidate oncogenes which might represent the BRD4 fusion partner. FISH evaluation of 13 pediatric carcinomas revealed t(15;19) in one of four sinonasal carcinomas, whereas this translocation was not detected in thymic (n = 3), mucoepidermoid (n = 3), laryngeal (n = 2), or nasopharyngeal (n = 1) carcinomas. Our studies shed light on the oncogenic mechanism underlying t(15;19) and provide further evidence that this highly lethal cancer arises from respiratory mucosa.

Transcriptional repression and developmental functions of the atypical vertebrate GATA protein TRPS1.

Known vertebrate GATA proteins contain two zinc fingers and are required in development, whereas invertebrates express a class of essential proteins containing one GATA-type zinc finger. We isolated the gene encoding TRPS1, a vertebrate protein with a single GATA-type zinc finger. TRPS1 is highly conserved between Xenopus and mammals, and the human gene is implicated in dominantly inherited tricho-rhino-phalangeal (TRP) syndromes. TRPS1 is a nuclear protein that binds GATA sequences but fails to transactivate a GATA-dependent reporter. Instead, TRPS1 potently and specifically represses transcriptional activation mediated by other GATA factors. Repression does not occur from competition for DNA binding and depends on a C-terminal region related to repressive domains found in Ikaros proteins. During mouse development, TRPS1 expression is prominent in sites showing pathology in TRP syndromes, which are thought to result from TRPS1 haploinsufficiency. We show instead that truncating mutations identified in patients encode dominant inhibitors of wild-type TRPS1 function, suggesting an alternative mechanism for the disease. TRPS1 is the first example of a GATA protein with intrinsic transcriptional repression activity and possibly a negative regulator of GATA-dependent processes in vertebrate development.

PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma [corrected].

Chromosomal translocations that encode fusion oncoproteins have been observed consistently in leukemias/lymphomas and sarcomas but not in carcinomas, the most common human cancers. Here, we report that t(2;3)(q13;p25), a translocation identified in a subset of human thyroid follicular carcinomas, results in fusion of the DNA binding domains of the thyroid transcription factor PAX8 to domains A to F of the peroxisome proliferator-activated receptor (PPAR) gamma1. PAX8-PPARgamma1 mRNA and protein were detected in 5 of 8 thyroid follicular carcinomas but not in 20 follicular adenomas, 10 papillary carcinomas, or 10 multinodular hyperplasias. PAX8-PPARgamma1 inhibited thiazolidinedione-induced transactivation by PPARgamma1 in a dominant negative manner. The experiments demonstrate an oncogenic role for PPARgamma and suggest that PAX8-PPARgamma1 may be useful in the diagnosis and treatment of thyroid carcinoma.

Expression of laminin chains during myogenic differentiation.

Expression of two Ae-related chains of the extracellular matrix glycoprotein laminin was induced as multipotent C3H10T1/2 mouse embryo fibroblasts differentiated into myoblasts and myofibers. C3H10T1/2 fibroblasts expressed the B1e (M(r) = 215,000) and B2e (M(r) = 205,000) laminin chains based on metabolic radiolabeling, immunoprecipitation, peptide mapping, and mRNA analysis. In contrast, myoblasts derived from C3H10T1/2 fibroblasts treated with DNA demethylating agents or transfected with the cDNA encoding MyoD expressed the Ae (M(r) = 400,000) and a novel Ae-related laminin chain (designated Ac3h, M(r) = 350,000) in addition to the B1e and B2e chains. Expression of the Ae and Ac3h chains paralleled the capacity for myofiber formation in six additional C3H10T1/2 myoblast clones with varied potentials for terminal differentiation and coincided with a switch in laminin isoforms from those of M(r) = 850,000 synthesized by C3H10T1/2 fibroblasts to those of M(r) = 900,000-950,000 synthesized by C3H10T1/2 myoblasts and myofibers. Cultures of mouse C2C12, mouse BC3H1, rat L6, and primary mouse myoblasts also synthesized the Ae, Ac3h, B1e, and B2e laminin chains. The results demonstrate that expression of the Ae and Ac3h laminin chains is associated with expression of MyoD and the mammalian myogenic differentiation program.

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells.

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.

Differential occurrence of free beta and free alpha subunits of human chorionic gonadotropin (hCG) in pregnancy sera.

Levels of hCG alpha beta dimer, free alpha-subunit and free beta-subunit were measured in pregnancy sera. Dimer and free alpha were quantitated by radioimmunoassays (RIAs) using specific polyclonal antisera. Free beta was quantitated both by monoclonal anti-beta RIA and by polyclonal anti-beta RIA following the complete adsorption of cross-reacting hCG by immobilized alpha-antisera. Consistent with the findings of other laboratories, hCG levels in pregnancy sera peaked at around 10 weeks after the last menstrual period (post LMP), and declined thereafter. Free alpha levels rose as hCG levels declined and accounted for 30-40% of total serum alpha in the third trimester. Although free beta accounted for only a small proportion of the total beta-subunit at the time of the hCG peak and thereafter (2.4-3.6%), in early pregnancy serum samples, 4-6 weeks post LMP, when hCG was generally first detected, an average of 16% free beta was detected. At this time, the higher the hCG level (20-2000 ng/ml), the lower the percent free beta (54-3%). Thus, the free beta portion started high and declined prior to the hCG peak; the free alpha portion increased thereafter. To explain these findings, we propose a two phase regulation of hCG dimer formation. Up to the time of the hCG peak, supplies of alpha-subunit are limiting (hence the presence of free beta). Thereafter, beta-subunit levels drop, restricting dimer formation and leaving uncombined alpha.