Brain fatty acid-binding protein (B-FABPMalignant gliomas are believed to be derived from the astrocytic cell lineage because they contain bundles of cytoplasmic glial fibrillary acidic protein (GFAP), 1 an intermediate filament protein specifically expressed in differentiated astrocytes.There is an inverse relationship between the number of GFAPpositive cells and aggressive behavior in glioma tumors. Glioblastoma multiforme, the most common and aggressive glioma, often have low GFAP expression, while low grade astrocytomas usually have high levels of GFAP (1-4). In vitro studies directly correlate GFAP expression with a less aggressive behavior (5). Transfection of a GFAP expression vector into GFAP(Ϫ) malignant glioma cells results in decreased cell proliferation and decreased growth in soft agar (6, 7). Conversely, transfection of a GFAP antisense vector into a GFAP(ϩ) line results in undetectable GFAP expression and increased proliferation rate, anchorage-independent growth, and invasiveness (8).We have previously shown that GFAP(ϩ) malignant glioma lines express a second glial cell marker, brain fatty acid-binding protein (B-FABP) (9). Of 15 malignant glioma lines tested, 5 co-expressed B-FABP and GFAP, 8 expressed neither gene, while 2 had low levels of B-FABP and undetectable levels of GFAP. B-FABP is a 15-kDa protein normally found in the radial glial cells of the developing central nervous system as well as in select glial cell populations of the adult brain including glia limitans cells and Bergmann glial cells (10, 11). B-FABP expression has been implicated in the establishment of the radial glial fiber system which serves to guide immature migrating neurons to their correct location in the central nervous system (10, 12). Addition of anti-B-FABP antibody to primary cultures of cerebellar cells prevents both the extension of radial glial processes and the migration of neuronal cells along these processes, suggesting a role for B-FABP in relaying inductive signals required for glial cell differentiation.It is generally believed that radial glial cells are converted into astrocytes once neuronal migration in the developing brain is complete (13). Co-expression of GFAP and B-FABP in the same malignant glioma cells (9) therefore suggests that these tumors are derived from cells that have the potential of expressing proteins that are normally produced at different stages in the glial differentiation pathway. We are studying the regulation of the B-FABP gene in order to identify transcription factors involved in the regulation of glial genes in malignant glioma and understand the basis for the variation in B-FABP expression in different malignant glioma lines. By sequencing and DNase I footprinting, we have identified two NFI-binding sites in the promoter region of the B-FABP gene. We present evidence that a phosphatase specifically expressed in B-FABP(ϩ) cells is responsible for differential expression of the B-FABP gene in malignant glioma lines.

The DEAD box gene, DDX1, is a putative RNA helicase that is co-amplified with MYCN in a subset of retinoblastoma (RB) and neuroblastoma (NB) tumors and cell lines. Although gene amplification usually involves hundreds to thousands of kilobase pairs of DNA, a number of studies suggest that co-amplified genes are only overexpressed if they provide a selective advantage to the cells in which they are amplified. Here, we further characterize DDX1 by identifying its putative transcription and translation initiation sites. We analyze DDX1 protein levels in MYCN/DDX1-amplified NB and RB cell lines using polyclonal antibodies specific to DDX1 and show that there is a good correlation with DDX1 gene copy number, DDX1 transcript levels, and DDX1 protein levels in all cell lines studied. DDX1 protein is found in both the nucleus and cytoplasm of DDX1-amplified lines but is localized primarily to the nucleus of nonamplified cells. Our results indicate that DDX1 may be involved in either the formation or progression of a subset of NB and RB tumors and suggest that DDX1 normally plays a role in the metabolism of RNAs located in the nucleus of the cell.DEAD box proteins are a family of putative RNA helicases that are characterized by eight conserved amino acid motifs, one of which is the ATP hydrolysis motif containing the core amino acid sequence DEAD (Asp-Glu-Ala-Asp) (1-3). Over 40 members of the DEAD box family have been isolated from a variety of organisms including bacteria, yeast, insects, amphibians, mammals, and plants. The prototypic DEAD box protein is the translation initiation factor, eukaryotic initiation factor 4A, which, when combined with eukaryotic initiation factor 4B, unwinds double-stranded RNA (4). Other DEAD box proteins, such as p68, Vasa, and An3, can effectively and independently destabilize/unwind short RNA duplexes in vitro (5-7). Although some DEAD box proteins play general roles in cellular processes such as translation initiation (eukaryotic initiation factor 4A (4)), RNA splicing (PRP5, PRP28, and SPP81 in yeast (8 -10)), and ribosomal assembly (SrmB in Escherichia coli (11)), the function of most DEAD box proteins remains unknown. Many of the DEAD box proteins found in higher eukaryotes are tissue-or stage-specific. For example, PL10 mRNA is expressed only in the male germ line, and its product has been proposed to have a specific role in translational regulation during spermatogenesis (12). Vasa and ME31B are maternal proteins that may be involved in embryogenesis (13,14). p68, found in dividing cells (15), is believed to be required for the formation of nucleoli and may also have a function in the regulation of cell growth and division (16,17). Other DEAD box proteins are implicated in RNA degradation, mRNA stability, and RNA editing (18 -20).The human DEAD box protein gene DDX1 1 was identified by differential screening of a cDNA library enriched in transcripts present in the two RB cell lines Y79 and RB522A (21). The longest DDX1 cDNA insert isolated from this library was 2.4 kb with an ope...

DEAD box proteins are putative RNA helicases that function in all aspects of RNA metabolism, including translation, ribosome biogenesis, and pre-mRNA splicing. Because many processes involving RNA metabolism are spatially organized within the cell, we examined the subcellular distribution of a human DEAD box protein, DDX1, to identify possible biological functions. Immunofluorescence labeling of DDX1 demonstrated that in addition to widespread punctate nucleoplasmic labeling, DDX1 is found in discrete nuclear foci ϳ0.5 m in diameter. Costaining with anti-Sm and anti-promyelocytic leukemia (PML) antibodies indicates that DDX1 foci are frequently located next to Cajal (coiled) bodies and less frequently, to PML bodies. Most importantly, costaining with anti-CstF-64 antibody indicates that DDX1 foci colocalize with cleavage bodies. By microscopic fluorescence resonance energy transfer, we show that labeled DDX1 resides within a Fö rster distance of 10 nm of labeled CstF-64 protein in both the nucleoplasm and within cleavage bodies. Coimmunoprecipitation analysis indicates that a proportion of CstF-64 protein resides in the same complex as DDX1. These studies are the first to identify a DEAD box protein associating with factors involved in 3Ј-end cleavage and polyadenylation of pre-mRNAs. INTRODUCTIONDEAD box proteins are a family of putative RNA helicases found in all cellular organisms and in some viruses. They are characterized by eight conserved amino acid motifs, including the core DEAD (Asp-Glu-Ala-Asp) motif involved in ATP hydrolysis and coupling of ATPase and RNA helicase activity (Pause and Sonenberg, 1992). At least 14 human DEAD box proteins have been identified to date, summarized in the DExH/D protein family database (Jankowsky and Jankowsky, 2000). DEAD box proteins are thought to modulate RNA secondary structure in all cellular processes involving RNA, including transcription, pre-mRNA processing, ribosome biogenesis, RNA export, translation initiation, and RNA degradation (Schmid and Linder, 1992;de la Cruz et al., 1999). Although many of the biological functions of the prokaryotic and lower eukaryotic DEAD box proteins have been identified, DEAD box proteins in higher eukaryotes remain largely uncharacterized.DDX1 is a human DEAD box protein that was identified by differential screening of a cDNA library enriched in transcripts present in two retinoblastoma (RB) cell lines: Y79 and RB522A (Godbout and Squire, 1993). The 2.7-kb DDX1 transcript encodes a protein with a predicted molecular mass of 82.4 kDa (Godbout et al., 1998). In addition to the eight conserved DEAD box family motifs, DDX1 also contains a region with homology to heterogeneous nuclear ribonucleoprotein U (hnRNP U) (Godbout et al., 1994). HnRNP U or scaffold attachment factor A, a protein located in the nuclear matrix, has recently been shown to function as a repressor of RNA polymerase II elongation by inhibiting transcription factor TFIIH-mediated carboxyl-terminal domain phosphorylation (Kim and Nikodem, 1999). Interestingly, the ...

Cytosolic aldehyde dehydrogenase (ALDH) mRNA is present at high levels in the undifferentiated chick retina. Tissue maturation is accompanied by a 20–25× decrease in transcript levels. To determine the spatial and temporal distribution pattern of the ALDH transcript and its encoded protein in the developing retina, in situ hybridization and immunohistochemical analyses were carried out using chick embryos at different stages of development. The ALDH transcript and protein were detected at the earliest stage tested, in the inner layer of the optic cup of stage 14 (day 2) embryos. Both the ALDH transcript and protein were found in the dorsal retina of chick embryos from stage 18 (day 3) to day 16 of incubation. Accumulation of the ALDH protein in the neurites of ganglion cells could readily be detected at early developmental stages. Staining of this ganglion fiber layer was strong in the dorsal retina and could be followed up to and into the optic nerve. By day 11, ALDH mRNA was located primarily in the ciliary margin and in the inner nuclear layer of the dorsal retina. In addition to these areas, the ALDH protein was also found in the inner plexiform and optic nerve fiber layers. These results suggest that environmental or transcriptional factors involved in the regulation of the ALDH gene are restricted to the dorsal retina at early developmental stages and that there is a requirement for the compartmentalization of the ALDH transcript/protein in the undifferentiated chick retina. © 1996 Wiley‐Liss, Inc.

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