Potential tumor suppressor NESG1 as an unfavorable prognosis factor in nasopharyngeal carcinoma

Background

Recently we identified nasopharyngeal epithelium specific protein 1 (NESG1) as a potential tumor suppressor in nasopharyngeal carcinoma (NPC). The purpose of this study is to investigate the involvement of NESG1 in tumor progression and prognosis of human NPC.

Methodology/Principal Findings

NESG1 protein expression in NPC was examined. Survival analysis was performed using Kaplan-Meier method. The effect of NESG1 on cell proliferation, migration, and invasion were also investigated.

Results

NESG1 expression was downregulated in atypical hyperplasia and NPC samples compared to normal and squamous nasopharynx tissues. Reduced protein expression was negatively associated with the status of NPC progression. Patients with lower NESG1 expression had a shorter overall survival and disease-free time than did patients with higher NESG1 expression. Multivariate analysis suggested NESG1 expression as an independent prognostic indicator for NPC patient survival. Proliferation, migration, and invasion ability were significantly increased in cell lines following lentiviral-mediated shRNA suppression of NESG1 expression. Microarray analysis indicated that NESG1 participated in multiple pathways, including MAPK signaling and cell cycle regulation. Finally, DNA methylation microarray examination revealed a lack of hypermethylation at the NESG1 promoter, suggesting other mechanisms are involved in suppressing NESG1 expression in NPC.

Conclusion

Our studies are the first to demonstrate that decreased NESG1 expression is an unfavorable prognostic factor for NPC.     

Characterization of sphingolipid long-chain base kinase in Arabidopsis thaliana

Sphingolipid long-chain base (LCB) kinase catalyses the phosphorylation of sphingolipid LCB to form LCB 1-phosphate. Based on sequence identity to a murine sphingosine kinase (murine SPHK1a), we isolated and characterized a LCB kinase-like cDNA in Arabidopsis thaliana. The deduced amino acid sequence of the homologous cDNA shows several regions that are highly conserved in LCB kinases from mouse, yeast, human and Caenorhabditis elegans. These regions are not similar to those of other known kinase families. For a functional identification, the homologous cDNA from A. thaliana was expressed in Escherichia coli, and LCB kinase activity was measured. The recombinant AtLcbk1 protein was found to utilize ATP and sphinganine. These results indicate the first identification of a gene coding for a LCB kinase in plants.     

Proteomic features of potential tumor suppressor NESG1 in nasopharyngeal carcinoma

We previously defined the recently revised NESG1 gene as a potential tumor suppressor in nasopharyngeal carcinoma (NPC). Here, we further used proteomics technology to globally examine NESG1‐controlled proteins in NPC cells. Twenty‐six proteins were found to be deregulated by NESG1 using proteomics analysis while enolase 1 (alpha) (ENO1), heat shock protein 90 kDa beta (Grp94), member 1 (HSP90B1), and cathepsin D (CTSD) proteins were differentially expressed by Western blot. Interestingly, a‐enolase (ENO1), an overexpressed gene in NPC, was confirmed as a NESG1‐regulated protein in NPC cells. Overexpressed ENO1 not only restored cell proliferation and cell‐cycle progression, but also antagonized the regulation of NESG1 to cell‐cycle regulators p21 and CCNA1 expression as well as induced the expression of C‐Myc, pRB, and E2F1 in NESG1‐ovexpressed NPC cells. Real‐time PCR and immunohistochemistry analysis showed that NESG1 expression is negatively correlated with ENO1 expression in NPC tissues. Our observations suggest that ENO1 downregulation plays an important role in NESG1‐induced growth inhibition of NPC cancer cells.     

Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.     

Molecular cloning, identification and characteristics of a novel isoform of carbamyl phosphate synthetase I in human testis

A gene coding a novel isoform of carbamyl phosphate synthetase I (CPS1) was cloned from a human testicular library. As shown by cDNA microarray hybridization, this gene was expressed at a higher level in human adult testes than in fetal testes. The full length of its cDNA was 3831 bp, with a 3149 bp open reading frame, encoding a 1050-amino-acid protein. The cDNA sequence was deposited in the GenBank (AY317138). Sequence analysis showed that it was homologous to the human CPS1 gene. The putative protein contained functional domains composing the intact large subunit of carbamoyl phosphate synthetase, thus indicated it has the capability of arginine biosynthesis. A multiple tissue expression profile showed high expression of this gene in human testis, suggesting the novel alternative splicing form of CPS1 may be correlated with human spermatogenesis.     

A novel porcine gene, POT1, differentially expressed in the longissimus muscle tissues from Wujin and Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Wujin and Large White pigs. One novel gene differentially expressed was identified through quantitative real time PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 507 amino acids that shares high homology with the protection of telomeres 1 isoform 4 (POT1) of human (86%)-so that this gene can be defined as swine POT1 gene. This gene is structured in 12 exons and 11 introns as revealed by computer-assisted analysis. The tissue expression analysis indicated that the swine POT1 gene is differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, pancreas and spleen. Our experiment is the first to establish the primary foundation for further research on the swine POT1 gene.     

Ubiquitously expressed GPCR membrane-trafficking orthologs

Olfactory receptors are a diverse set of G-protein-coupled receptors (GPCRs) that localize to cellular plasma membranes in the olfactory epithelium. Associated trafficking proteins often assist in targeting these GPCRs to the membrane, facilitating function. One such trafficking protein has been isolated as a mutant defective for both odorant response and proper receptor localization in Caenorhabditis elegans. This gene (ODR-4) allows for functional expression of olfactory receptors in heterologous cells that are otherwise incapable of targeting. We have isolated a full-length human cDNA that is homologous to the C. elegans gene at the protein level across nearly the entire gene by using a novel RecA-based gene enrichment procedure. This sequence is homologous to a family of orthologs that share predicted structural features, indicating a conserved function. The gene was expressed in 41 of 44 human, mouse, and rat tissues, suggesting an important role in trafficking olfactory and other GPCRs.     

The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species.

A recombinant M13 clone (O42) containing a 65 b.p. cDNA fragment from human fetal liver mRNA coding for glyceraldehyde-3-phosphate dehydrogenase has been identified and it has been used to isolate from a full-length human adult liver cDNA library a recombinant clone, pG1, which has been subcloned in M13 phage and completely sequenced with the chain terminator method. Besides the coding region of 1008 b.p., the cDNA sequence includes 60 nucleotides at the 5'-end and 204 nucleotides at the 3'-end up to the polyA tail. Hybridization of pG1 to human liver total RNA shows only one band about the size of pG1 cDNA. A much stronger hybridization signal was observed using RNA derived from human hepatocarcinoma and kidney carcinoma cell lines. Sequence homology between clone 042 and the homologous region of clone pG1 is 86%. On the other hand, homology among the translated sequences and the known human muscle protein sequence ranges between 77 and 90%; these data demonstrate the existence of more than one gene coding for G3PD. Southern blot of human DNA, digested with several restriction enzymes, also indicate that several homologous sequences are present in the human genome.     

The Mouse Homolog of the Bovine Leukemia Virus Receptor Is Closely Related to the δ Subunit of Adaptor-Related Protein Complex AP-3, Not Associated with the Cell Surface

A mouse cDNA (mBLVR1) which was highly homologous to the bovine cDNA of the bovine leukemia virus receptor (BLVR) gene was cloned. The mBLVR1 cDNA, of 4,730 bp, covered nearly the full length of the mRNA (about 5 kb) and included an open reading frame (ORF) encoding a protein of 1,199 amino acids. While the bovine BLVR protein was thought to be a type I transmembrane protein, the deduced protein coded by mBLVR1 did not appear to be a typical transmembrane protein. The ORF of mBLVR1 ended at a site 280 amino acids upstream of the termination codon of the bovine BLVR ORF, so the deduced mouse BLVR protein lacked the corresponding transmembrane and cytoplasmic regions of the predicted bovine BLVR protein. No significant hydrophobic region was found in the mouse protein. Recently, a human cDNA which was highly homologous (69.6% homology) to the mouse BLVR gene was reported. The cDNA encodes the δ subunit of the human adaptor-related protein complex AP-3, which aligned almost collinearly with the mouse BLVR protein. AP-3 and all other related adaptor protein complexes have been shown to be associated with intracellular vesicles but not with the cell surface. Thus, the mouse BLVR homolog appeared to be the mouse AP-3 δ subunit itself or closely related to it, but the bovine BLVR gene seemed slightly different from the adaptor subunit gene family.     

Cloning and characterization of a novel member of human β-1,4-galactosyltransferase gene family

By using the EST strategy for identifying novel members belonging to homologous gene families, a novel fulklength cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GalT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1 032 nucleotides (344 amino acids). In view of the homology to memben of the galactosyltransferase gene family and especially the closest relationship toGallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1,4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.     

Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1 beta

A human interleukin 1 beta (IL 1 beta) cDNA probe was utilized to identify a homologous murine cDNA clone. The murine cDNA encodes a 269-residue protein which is 67% homologous to human IL 1 beta. The murine sequence was engineered for expression in mammalian cells and directs the synthesis of biologically active IL 1. This protein, termed murine IL 1 beta, is only 22% homologous with the previously described murine IL 1 sequence. Both IL 1 alpha and IL 1 beta are encoded by single genes, but IL 1 beta mRNA is about fivefold more abundant in a stimulated macrophage cell line.     

Identification of a novel protein interacting with laforin,the EPM2a progressive myoclonus epilepsy gene product

We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy. The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs. Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated. The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed. The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3' untranslated regions, maps to human chromosome 3p22.1. The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs. Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found. The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.     

Structural analysis of the Drosophila rpA1 gene, a member of the eucaryotic ''A'' type ribosomal protein family.

The expression of ribosomal protein (r-protein) genes is uniquely regulated at the translational level during early development of Drosophila. Here we report results of a detailed analysis of the r-protein rpA1 gene. A cloned DNA sequence coding for rpA1 has been identified by hybrid-selected translation and amino acid composition analysis. The rpA1 gene was localized to polytene chromosome band 53CD. The nucleotide sequence of the rpA1 gene and its cDNA have been determined. rpA1 is a single copy gene and sequence comparison between the gene and its cDNA indicates that this r-protein gene is intronless. Allelic restriction site polymorphisms outside of the gene were observed, while the coding sequence is well conserved between two Drosophila strains. The protein has unusual domains rich in Ala and charged residues. The rpA1 is homologous to the "A" family of eucaryotic acidic r-proteins which are known to play a key role in the initiation and elongation steps of protein synthesis.     

Human homeo box-containing genes located at chromosome regions 2q31----2q37 and 12q12----12q13

Four human homeo box-containing cDNAs isolated from mRNA of an SV40-transformed human fibroblast cell line have been regionally localized on the human gene map. One cDNA clone, c10, was found to be nearly identical to the previously mapped Hox-2.1 gene at 17q21. A second cDNA clone, c1, which is 87% homologous to Hox-2.2 at the nucleotide level but is distinct from Hox-2.1 and Hox-2.2, also maps to this region of human chromosome 17 and is probably another member of the Hox-2 cluster of homeo box-containing genes. The third cDNA clone, c8, in which the homeo box is approximately 84% homologous to the mouse Hox-1.1 homeo box region on mouse chromosome 6, maps to chromosome region 12q12----12q13, a region that is involved in chromosome abnormalities in human seminomas and teratomas. The fourth cDNA clone, c13, whose homeo box is approximately 73% homologous to the Hox-2.2 homeo box sequence, is located at chromosome region 2q31----q37. The human homeo box-containing cluster of genes at chromosome region 17q21 is the human cognate of the mouse homeo box-containing gene cluster on mouse chromosome 11. Other mouse homeo box-containing genes of the Antennapedia class (class I) map to mouse chromosomes 6 (Hox-1, proximal to the IgK locus) and 15 (Hox-3). A mouse gene, En-1, with an engrailed-like homeo box (class II) and flanking region maps to mouse chromosome 1 (near the dominant hemimelia gene). Neither of the class I homeo box-containing genes--c8 and c13--maps to a region of obvious homology to chromosomal positions of the presently known mouse homeo box-containing genes.