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The TLS-CHOP oncoprotein, found in the majority of human myxoid liposarcomas, consists of a fusion between the transcription factor CHOP/GADD153 and the N terminus of an RNA-binding protein TLS/FUS. Clinical correlation and in vitro transformation assays indicate that the N terminus of TLS plays an important role in oncogenesis by TLS-CHOP. Until now, however, the only activity attributed to the oncoprotein is that of inhibiting the binding of transcription factors of the C/EBP class to certain adipogenic target genes, a function that TLS-CHOP shares with the nononcogenic CHOP protein. Here we report the isolation of a gene, DOL54, that is activated in primary fibroblasts by the expression of TLS-CHOP. DOL54 is expressed in the neoplastic component of human myxoid liposarcomas and increases the tumorigenicity of cells injected in nude mice. Activation of DOL54 requires an intact DNA-binding and dimerization domain in TLS-CHOP, a suitable cellular dimerization partner, and depends on the TLS N terminus. Normal adipocytic differentiation is associated with an early and transient expression of DOL54, and the gene encodes a secreted protein that is tightly associated with the cell surface or extracellular matrix. TLS-CHOP thus leads to the unscheduled expression of a gene that is normally associated with adipocytic differentiation.

CHOP (also called GADD153) is a stress-inducible nuclear protein that dimerizes with members of the C/EBP family of transcription factors and was initially identified as an inhibitor of C/EBP binding to classic C/EBP target genes. Subsequent experiments suggested a role for CHOP-C/EBP heterodimers in positively regulating gene expression; however, direct evidence that this is the case has so far not been uncovered. Here we describe the identification of a positively regulated direct CHOP-C/EBP target gene, that encoding murine carbonic anhydrase VI (CA-VI). The stress-inducible form of the gene is expressed from an internal promoter and encodes a novel intracellular form of what is normally a secreted protein. Stress-induced expression of CA-VI is both CHOP and C/EBPbeta dependent in that it does not occur in cells deficient in either gene. A CHOP-responsive element was mapped to the inducible CA-VI promoter, and in vitro footprinting revealed binding of CHOP-C/EBP heterodimers to that site. Rescue of CA-VI expression in c/ebpbeta-/- cells by exogenous C/EBPbeta and a shorter, normally inhibitory isoform of the protein known as LIP suggests that the role of the C/EBP partner is limited to targeting the CHOP-containing heterodimer to the response element and points to a preeminent role for CHOP in CA-VI induction during stress.

CHOP (GADD153) is a small nuclear protein that dimerizes avidly with members of the C/EBP family of transcription factors. Normally undetectable, it is expressed at high levels in cells exposed to conditions that perturb protein folding in the endoplasmic reticulum and induce an endoplasmic reticulum stress response. CHOP expression in stressed cells is linked to the development of programmed cell death and, in some instances, cellular regeneration. In this study, representational difference analysis was used to compare the complement of genes expressed in stressed wild-type mouse embryonic fibroblasts with those expressed in cells nullizygous for chop. CHOP expression, in concert with a second signal, was found to be absolutely required for the activation by stress of a set of previously undescribed genes referred to as DOCs (for downstream of CHOP). DOC4 is a mammalian ortholog of a Drosophila gene, Tenm/Odz, implicated in patterning of the early fly embryo, whereas DOC6 encodes a newly recognized homolog of the actin-binding proteins villin and gelsolin. These results reveal the existence of a novel CHOP-dependent signaling pathway, distinct from the known endoplasmic reticulum unfolded protein response, which may mediate changes in cell phenotype in response to stress.

TLS, the product of a gene commonly translocated in liposarcomas (TLS), is prototypical of a newly identified class of nuclear proteins that contain a C-terminal domain with a distinct RNA recognition motif (RRM) surrounded by Arg-Gly-Gly (RGG) repeats. Its unique N terminus serves as an essential transforming domain for a number of fusion oncoproteins in human sarcomas and leukemias. In this study we use an in vivo UV crosslinking procedure to probe the interactions of TLS with RNA. TLS is found to bind RNA in vivo and the association of TLS with RNA is rapidly diminished by treating cells with transcriptional inhibitors. This suggests that the species bound by TLS turns over rapidly. Surprisingly, the RRM was found to be dispensable for RNA binding by TLS in vivo, suggesting that at any one time most of the interactions between TLS and RNA in the cell are not sequence specific. Analysis of inter specific heterokaryons formed between human and mouse or Xenopus cells revealed that TLS engages in rapid nucleocytoplasmic shuttling, a finding confirmed by the ability of anti-TLS antibodies to trap TLS when injected into the cytoplasm of HeLa cells. Cellular fractionation experiments suggest that TLS binds to RNA in both the nucleus and cytoplasm and support the hypothesis that TLS functions as a heterogeneous ribonuclear protein (hnRNP)-like chaperone of RNA. These findings are discussed in the context of the role altered forms of TLS play in cellular transformation.

A 35-YEAR-OLD WOMAN PRESENTED WITH AN 8-monthhistoryofprogressiverighteyeproptosis without nasal congestion, nasal discharge, or epistaxis. For several weeks, she had experienced horizontal diplopia along with intermittent pain in the right eye. Her medical and social histories were otherwise unremarkable. Nasal endoscopy revealed prominence of the right agger nasi, with a normal-appearing infundibulum and bulla ethmoidalis. A computed tomographic scan of the paranasal sinus showed a homogeneous soft tissue mass obliterating the frontal sinus and extending into the right orbit, with bony erosion (Figure1). The mass was pushing the right globe laterally without extension into the anterior cranial fossa or fovea ethmoidalis. The mass demonstrated high signal intensity on T2-weighted magnetic resonance images (MRIs) and low signal intensity on T1-weighted MRIs, with rim enhancement after contrast injection (Figure 2). The presumptive diagnosis of a mucocele was made, and endonasal endoscopy revealed a solid, soft tissue mass. A right eyebrow incision was used to reexplore and then completely resect the mass. Intraoperatively, it was noted that the mass was not encapsulated and that the bony wall between the frontal sinus and orbit had been eroded. Grossly, the mass was soft, grayish white, and gelatinous, similar in appearance to an edematous nasal polyp. Hematoxylineosin–stained sections revealed sparse, spindle and stellate cells surrounded by a loose, pale-pink myxoid stroma (Figure 3 and Figure 4). There were no mitotic or pleomorphic cells. Mucicarmine stains demonstrated prominent mucin deposition in the tumor stroma. Moreover, immunostaining of the specimen was positive for vimentin and negative for desmin, S100 protein, CD34, keratin, and cytokeratins (AE1/AE3). What is your diagnosis?

Objectives To identify distinct gene expression profiles of human head and neck squamous cell carcinomas (HNSCCAs) using complementary DNA (cDNA) microarray analysis and to create a preliminary, comprehensive database of HNSCCA gene expression. Patients and Methods Nine patients with histologically confirmed HNSCCAs, staged according to the American Joint Committee on Cancer, were enrolled. The HNSCCA tumor tissue and normal mucosal tissue were harvested at the time of surgery. A cDNA library was constructed from the paired fresh-frozen human surgical specimens of HNSCCAs and nonmalignant epithelial tissues. Biotinylated RNA was transcribed from the cDNA library and hybridized to high-density microarrays containing approximately 12 000 human genes. Altered gene expression of HNSCCAs was identified by comparison to corresponding normal mucosal tissues after a bayesian statistical analysis of variance. Results were analyzed using the gene database of the National Institutes of Health. Hierarchical clustering of the genomic data sets was determined by similarity metrics based on Pearson correlation. Results Hierarchical clustering analysis revealed that the gene expression profiles obtained from the nonselected panel of 12 000 genes could distinguish the tumors from nonmalignant tissues. Gene expression changes were reproducibly observed in 227 genes representing previously identified chemokines, tumor suppressors, differentiation markers, matrix molecules, membrane receptors, and transcription factors that correlated with neoplasia, including 46 previously uncharacterized genes. Moreover, significant expression of the collagen type XI α1 gene and a novel gene was reproducibly observed in all 9 tumors, whereas these genes were virtually undetectable in their corresponding, adjacent nonmalignant tissues. Conclusions Complementary DNA microarray analysis of human HNSCCAs has produced a preliminary, comprehensive database of tumor-specific gene expression profiles and provided important insights into modeling gene expression changes implicated in carcinogenesis. A large-scale analysis of gene expression carries the future potential of identifying sensitive molecular markers for early tumor detection, prognosis, and novel targets for interceptive therapeutics.

Exposure of cells to arsenicals activates multiple stress pathways resulting in the induction of specific genes whose identity and role in the adaptation to arsenical-induced cellular stress are poorly understood. We report here the identification of a novel gene encoding an arsenite-inducible, cysteine- and histidine-rich RNA-associated protein, AIRAP, that is conserved among mammals, Drosophila and C elegans. Immunochemistry and cell fractionation experiments indicate that, when induced, AIRAP is present in both the nucleus and the cytoplasm, and cross-linking experiments indicate that it associates with RNA in vivo. The expression of a C elegans homologue of AIRAP, aip-1, is also induced by exposure to arsenite, and expression of an aip-1::gfp transgene is most pronounced in hypodermal cells. RNA-mediated interference (RNAi) of aip-1 lowers the resistance of nematodes to arsenite yet does not appear to affect viability under standard growth conditions. These experiments suggest a role for AIRAP/AIP-1 in protecting cells from the toxic effects of arsenite.

TLS (also known as FUS) is an RNA‐binding protein that contributes the N‐terminal half of fusion oncoproteins implicated in the development of human liposarcomas and leukemias. Here we report that male mice homozygous for an induced mutation in TLS are sterile with a marked increase in the number of unpaired and mispaired chromosomal axes in pre‐meiotic spermatocytes. Nuclear extracts from TLS −/− testes lack an activity capable of promoting pairing between homologous DNA sequences in vitro , and TLS −/− mice and embryonic fibroblasts exhibit increased sensitivity to ionizing irradiation. These results are consistent with a role for TLS in homologous DNA pairing and recombination.