Low dose radiation response curves, networks and pathways in human lymphoblastoid cells exposed from 1 to 10cGy of acute gamma radiation.

We investigated the low dose dependency of the transcriptional response of human cells to characterize the shape and biological functions associated with the dose-response curve and to identify common and conserved functions of low dose expressed genes across cells and tissues. Human lymphoblastoid (HL) cells from two unrelated individuals were exposed to graded doses of radiation spanning the range of 1-10cGy were analyzed by transcriptome profiling, qPCR and bioinformatics, in comparison to sham irradiated samples. A set of ∼80 genes showed consistent responses in both cell lines; these genes were associated with homeostasis mechanisms (e.g., membrane signaling, molecule transport), subcellular locations (e.g., Golgi, and endoplasmic reticulum), and involved diverse signal transduction pathways. The majority of radiation-modulated genes had plateau-like responses across 1-10cGy, some with suggestive evidence that transcription was modulated at doses below 1cGy. MYC, FOS and TP53 were the major network nodes of the low-dose-response in HL cells. Comparison our low dose expression findings in HL cells with those of prior studies in mouse brain after whole body exposure, in human keratinocyte cultures, and in endothelial cells cultures, indicates that certain components of the low dose radiation response are broadly conserved across cell types and tissues, independent of proliferation status.

Physical mapping of the DDX1 gene to 340 kb 5' of MYCN.

One of the most important prognostic factors in neuroblastoma is amplification of the MYCN gene, which is strongly associated with advanced stages of disease and a poor prognosis. Although the MYCN amplicon sometimes spans more than 1 Mb, no other consistently expressed sequences from the MYCN amplicon have been reported. However, DDX1, a gene encoding a DEAD box protein, was recently mapped to chromosome 2p24 and is frequently co-amplified with MYCN. Therefore, we performed genomic mapping with YACs to determine the physical relationship between DDX1 and MYCN, and whether DDX1 was contained within the core region of amplification. Based on YAC restriction mapping and content analysis, DDX1 maps 340 kb 5' of MYCN, outside the core domain of consistent amplification. Interestingly, we also determined by sequence analysis and detailed restriction mapping that G21, previously isolated as a 'neuroblastoma-specific' cDNA clone from an MYCN amplicon, is a partial cDNA of DDX1. Our data confirm that DDX1 is amplified in some but not all MYCN-amplified tumors, and that it is rearranged in other cases. This suggests that the co-amplification of DDX1 is due to its proximity to MYCN.

Mutational analysis of the adenovirus E2-early promoter in fission yeast Schizosaccharomyces pombe.

The human adenovirus E2-early promoter has a complex architecture consisting of overlapping sequences that constitute the major(+1) and minor(-26) promoters in human cells. In human cells the basal transcription of the major promoter is dependent on 4 cis-acting elements: a TTAAGA motif analogous to the TATA box, two E2F sites that are present as inverted repeats, and an ATF/CREB site. It was also demonstrated that the E2-early promoter was expressed efficiently in the fission yeast Schizosaccharomyces pombe and that the major and minor promoters were differentially utilized with preferential transcription from the -26 promoter. In this report the results of an investigation of the E2-early promoter activity in S. pombe, using an additional group of linkerscan mutants that span the E2 promoter, are presented. The efficient expression of the E2-early promoter in yeast was dependent on all 4 cis-acting elements as monitored by reporter gene expression. However, unlike the situation in human cells, the mutation of the TATA-like element present at -50 bps rendered the -26 promoter inactive and was therefore crucial for the maximal promoter function in S. pombe. As in human cells the wild type promoter activity was seen in S. pombe when the -82 to -92 region was mutated. DNA-protein interaction studies confirmed the presence of ATF and E2F-like transcription factor activities in S. pombe. This report demonstrates the degree of conservation that exists between the transcription apparatus of yeast and man.

The adenovirus EII early promoter has multiple EIA-sensitive elements, two of which function cooperatively in basal and virus-induced transcription.

The mechanism by which the adenovirus-encoded nuclear oncogene EIA activates transcription of several viral and host promoters is an important issue in the regulation of eucaryotic gene expression and virus-host cell interactions. Identification of cis-acting elements of the promoters and the cognate host transcription factors that are targets for EIA action is crucial for our understanding of the EIA-mediated control of coordinately regulated genes. The adenovirus EII early promoter has a complex architecture and contains two overlapping promoters with start sites at +1 (major promoter) and -26 (minor promoter). The major promoter responds strongly to virus-encoded trans activators EIA and EIV and contains four elements: a TAGA motif analogous to the TATA box, two EIIF sites present in an inverted orientation, and an ATF/CREB site. To determine precisely the roles played by these cis-acting elements in both basal and virus-induced transcription when the promoter is situated in its natural context, we investigated the phenotype of a series of linker scan promoter substitution mutants inserted into the viral chromosome. Promoter constructs harboring linker scan mutations in each element were rebuilt into a novel EIA- adenovirus vector, and transcriptional activity was monitored in virus-infected cells. In the absence of virus-encoded trans activators, basal activity in vivo was dependent on all four cis-acting elements. Surprisingly, a promoter mutant with only one of the two EIIF sites intact could not promote transcription in vivo, suggesting that the two EIIF sites function cooperatively even in basal transcription. Promoters harboring mutations in either of these two EIIF sites also failed to bind to an infection-specific form of EIIF in gel shift assays and competed only very weakly for EIIF binding with the wild-type promoter fragment. The dramatic cooperativity shown by the two inverted EIIF sites of the EII promoter both in vivo and in vitro could reflect simultaneous contact of both sites by the transcription factor EIIF. Furthermore, promoter mutants with mutations in the TAGA motif, the two EIIF sites, and the single ATF site all failed to respond to virus-encoded trans activators. Whereas recent results demonstrate that EIIF activity can be modulated independently by EIV, leading to transactivation of this promoter, our results and those published previously strongly indicate that the three different transcription factors that bind to TAGA, EIIF, and ATF motifs of the EII early promoter are all targets for EIA regulation in vivo. Thus, strong transactivation of the EII early promoter through these multiple EIA-sensitive elements and independently by the recently discovered EIV pathway suggests that the EII early promoter is stringently regulated in virus-infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Co-amplification and concomitant high levels of expression of a DEAD box gene with MYCN in human neuroblastoma.

MYCN gene amplification is strongly correlated with poor prognosis in neuroblastoma (NB), the second most common solid pediatric tumor. However, increased MYCN expression seen in tumors that lack MYCN amplification does not correlate with aggressive clinical behavior. Whereas the MYCN gene spans only 7 kb, the MYCN amplicon has been shown to range in size from 350 kb to more than 1 Mb. Given the large size of the amplicon, it is possible that additional genes are co-amplified in NBs whose expression may contribute to the aggressive phenotype associated with MYCN-amplified tumors. We isolated a cDNA clone from a human NB library that is identical to DDXI, a gene recently reported to be preferentially expressed in two retinoblastoma cell lines that also express high levels of MYCN. DDXI belongs to a family of genes that encode DEAD (Asp-Glu-Ala-Asp) box proteins, putative ATP-dependent RNA helicases implicated in a number of cellular processes involving alterations of RNA secondary structure. We examined the frequency of DDXI amplification in 15 NB cell lines, 1 neuroepithelioma cell line, and 122 NB tumors by Southern blot analyses, and we found that 7 of 10 MYCN-amplified cell lines and 27 of 40 (68%) MYCN-amplified tumors also harbored multiple copies of the DDXI gene. Amplification of DDXI was associated with high levels of DDXI mRNA expression in the NB cell lines and tumors as examined by Northern analysis. Neither DDXI gene amplification nor enhanced expression was observed in tumors or cell lines that lacked MYCN amplification. Because RNA helicases play important roles in both post-transcriptional and translational gene regulation, high levels of DDXI expression consequent to genomic amplification may contribute to the malignant phenotype of a subset of NBs.

Hox gene expression in differentiating human neuroblastoma cells.

Neuroblastoma, a malignancy of early childhood arises in the embryonal neural crest. Neuroblastoma cells are in a state of arrested differentiation; however, they can be induced to differentiate in vitro by retinoic acid. As a first step toward understanding the molecular mechanisms of neuroblastoma differentiation we analyzed the expression pattern of the developmentally important Homeobox genes in cells treated with retinoic acid. The strategy employed involved rapid screening of a cDNA library prepared from retinoic acid treated human LA-N-5 neuroblastoma cells for Homeobox genes by the polymerase chain reaction. Multiple Homeobox genes were amplified from recombinant phage DNA using degenerate primers directed against the conserved homeobox. To date 6 Homeobox genes (HoxC6, HoxC8, HoxD1, HoxD4, HoxD8, and HoxD9) have been identified in the cDNA library prepared from LA-N-5 cells treated with retinoic acid. HoxD1 and HoxC8 are being reported for the first time to be expressed in neuroblastoma cells. Preliminary studies indicate that there is an induction of Homeobox gene expression in differentiating LA-N-5 neuroblastoma cells.

Activation of a dual adenovirus promoter containing nonconsensus TATA motifs in Schizosaccharomyces pombe: role of TATA sequences in the efficiency of transcription.

The role of TATA elements in the expression of a mammalian promoter was investigated in the fission yeast Schizosaccharomyces pombe, by studying the human adenovirus E2-early promoter. This is a unique dual promoter with two nonconsensus TATA elements directing transcription from two cap sites, +1 and -26. A sequence TTAAGA provides the TATA box function for the +1 promoter, whereas a sequence TAAATT, with a closer resemblance to the consensus (TATAA/TA) provides this function for the -26 promoter. Yet, in human cells, the +1 promoter is transcribed about 20 fold more efficiently than the -26 promoter. We found that both promoters are transcribed faithfully in S. pombe with start sites identical or close to those found in human cells. Surprisingly, the relative ratio of expression for the +1 and -26 promoters was exactly reversed in S. pombe cells. This reversal appeared to be due to the relatively weak binding of S. pombe TATA binding protein to the TTAAGA motif, rather than to its rate of dissociation. Furthermore, we show that in S. pombe, promoter expression correlates well with the nucleotide sequence of the TATA element rather than the context in which it is placed. By contrast, it is the context of the TATA element, rather than its nucleotide sequence that appears to be critical for promoter expression in human cells. Our data suggest the existence of one or more additional factors in human cells that permit the utilization of nonconsensus TATA elements. S. pombe appears to lack these factors.

Up-regulation of HOXC6, HOXD1, and HOXD8 homeobox gene expression in human neuroblastoma cells following chemical induction of differentiation.

An early event in the pathogenesis of neuroblastoma (NB), a tumor derived from embryonal neural crest tissue, appears to be the arrested differentiation of neuroblasts. However, NB cells can be induced to differentiate in vitro with numerous chemicals including retinoic acid (RA) and dibutyryl cyclic AMP (db-cAMP). One family of transcription factors, encoded by the homeobox (HOX) genes, plays a crucial role in Drosophila, Xenopus, and mammalian embryonic differentiation and development. We have previously identified six HOX genes (HOXC6, HOXC8, HOXD1, HOXD4, HOXD8, and HOXD9), by a sensitive PCR-based approach, in a cDNA library prepared from the human LA-N-5 NB cell line induced to differentiate with RA. In this report, we studied the regulation of these six HOX genes in a series of NB cell lines chemically induced to differentiate. Untreated NB cells express low or undetectable levels of HOX mRNA, and HOXC8 remains undetectable in the induced cells. However, a significant induction of HOXC6, HOXD1, and HOXD8 expression is seen in the RA-treated NB cell lines, albeit with different patterns and degree of up-regulation. db-cAMP treatment also induced HOXC6 and HOXD8 expression in two of the three NB cell lines analyzed. Low levels of HOXD4 and HOXD9 induction were observed in two and one RA-treated NB cell line, respectively. Up-regulation of HOXC6, HOXD1, and HOXD8 expression in human NB cells, chemically induced to differentiate, appears to be associated with maturation toward a differentiated neuronal phenotype.

The biological effects of antisense N-myc expression in human neuroblastoma.

Although N-myc amplification and overexpression are believed to play an important role in determining the clinical behavior of neuroblastoma (NB), the exact function of N-myc in NB cell growth and differentiation remains unknown. To better understand the function of N-myc, an established human NB cell line was transfected with N-myc antisense (AS) complementary DNA in an effort to down-regulate N-myc gene expression. Five clones expressing AS N-myc RNA have been maintained in culture for over 2 years. Compared to control cells, a 30-69% decrease in the quantity of N-myc protein was demonstrated by Western blot analysis in 4 of the 5 AS clones. All 5 of the AS clones exhibited a 50-75% decrease in colony formation in soft agar assays compared to control cells. In addition, all 5 AS clones expressed a 3.2-kilobase protein kinase C-alpha transcript, whereas this message was not detected by Northern blot analysis in any of the control clones. These results suggest that N-myc may play an important role in NB cell growth and that antisense N-myc expression is associated with an induction of protein kinase C-alpha RNA expression. Further characterization of the AS clones may provide insight into the function of N-myc and may thus lead to a better understanding of the role that N-myc plays in determining the clinical behavior of this childhood neoplasm.

E2F site activates transcription in fission yeast Schizosaccharomyces pombe and binds to a 30-kDa transcription factor.

The mammalian transcription factor E2F binds to several cellular proteins including Rb, p107, cyclin A, cyclin E, and p33cdk2 protein kinase in a stage-specific manner during cell cycle. Its recognition sequence, TTTCGCGC, is present in two of the human adenovirus early promoters and in several promoters of cellular genes whose products are implicated in the control of cell proliferation. These observations suggest that E2F may play an important role in cell-cycle regulation and prompted us to ask whether E2F-like activities are present in yeast. We found that the E2F motif can function as an activating sequence in Schizosaccharomyces pombe when cloned upstream of a reporter gene. Consistent with this, the expression of adenovirus E2 promoter in S. pombe was dependent on both E2F motifs of this promoter. A protein, spE2F, that binds to the E2F site was partially purified from S. pombe using DNA-affinity chromatography. The binding specificity of this protein was compared to that of human E2F using a number of mutant E2F sites as competitors. These studies showed that spE2F recognizes a sequence closely related to the E2F site. Ultraviolet cross-linking and Southwestern blot studies indicated that the molecular size of spE2F is 30 kDa. Previous studies have shown that a cis-acting element, ACGCGTNA, also called MluI cell cycle box, or MCB, is critical for the regulated expression of cell cycle related genes both in fission and budding yeast. In S. pombe, the cdc10 gene product binds to this element and controls the cell cycle related genes. Electrophoretic mobility shift assays and molecular size determination studies indicated that spE2F is different from that encoded by cdc10. Thus, our studies suggest that spE2F is a novel transcription factor. We discuss these results in light of recent observations about the periodically expressed genes involved in the cell cycle progression in yeast.