Overexpression of a DEAD Box Protein (DDX1) in Neuroblastoma and Retinoblastoma Cell Lines

Abstract

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...

Identification of Genes Differentially Expressed in Mouse Fetuses from Streptozotocin-induced Diabetic Pregnancy by cDNA Subtraction

“…1). These genes were, SET translocation (SET) [15], DEAD (Asp-Glu-Ala-Asp) box polypeptide 1 (Ddx1) [16], COMM domain containing 3 (Commd3) [17] and BRCA1/BRCA2-containing complex, subunit 3 (Brcc3) [18]. SET has been assumed to play a role in nucleosome assembly [15].…”

Comparative Proteomic Expression Profile in All-trans Retinoic Acid Differentiated Neuroblastoma Cell Line

“…46 The DLD, DDX1, and PSMD11 genes mapped to gain NB regions [50][51][52] The dihydrolipoyl dehydrogenase DLD protein level, which is downregulated in our model system, has been seen to increase in the central nervous system of rats after oxidative stress. 53 The DDX1 gene coding for the DEAD box protein 1, which is downregulated in our model system, has been shown to be overexpressed in a subset of unfavorable NBs and in retinoblastoma cell lines; 54 it has been mapped to chromosome 2p24 and found often co-amplified with the proto-oncogene MYCN in patients with a worse prognosis 55,56 than in patients with only the MYCN gene amplified. 57,58 The role of DDX1 in the tumorigenic process is not known though; it is a putative RNA helicase, predicted to be involved in RNA binding and in the export of mRNA from the nucleus to the cytoplasm.…”

Quantification of MYCN, DDX1, and NAG Gene Copy Number in Neuroblastoma Using a Real-Time Quantitative PCR Assay

“…In addition we determined the copy number of DDX1 and NAG. Both genes are known to be co-amplified in subsets of MYCN amplified tumors (11)(12)(13)(14). The copy numbers of the two latter genes were used to determine a possible correlation of co-amplification with survival and risk to relapse of the patient.…”