Expression of eEF1A2 is associated with clear cell histology in ovarian carcinomas: overexpression of the gene is not dependent on modifications at the EEF1A2 locus.

The tissue-specific translation elongation factor eEF1A2 is a potential oncogene that is overexpressed in human ovarian cancer. eEF1A2 is highly similar (98%) to the near-ubiquitously expressed eEF1A1 (formerly known as EF1-alpha) making analysis with commercial antibodies difficult. We wanted to establish the expression pattern of eEF1A2 in ovarian cancer of defined histological subtypes at both the RNA and protein level, and to establish the mechanism for the overexpression of eEF1A2 in tumours. We show that while overexpression of eEF1A2 is seen at both the RNA and protein level in up to 75% of clear cell carcinomas, it occurs at a lower frequency in other histological subtypes. The copy number at the EEF1A2 locus does not correlate with expression level of the gene, no functional mutations were found, and the gene is unmethylated in both normal and tumour DNA, showing that overexpression is not dependent on genetic or epigenetic modifications at the EEF1A2 locus. We suggest that the cause of overexpression of eEF1A2 may be the inappropriate expression of a trans-acting factor. The oncogenicity of eEF1A2 may be related either to its role in protein synthesis or to potential non-canonical functions.

Expression of cellular adhesion molecule 'OPCML' is down-regulated in gliomas and other brain tumours.

The four GPI-anchored cell adhesion molecules that exemplify the IgLON family are most highly expressed in the nervous system and associate to form up to six different heterodimeric 'Diglons' that can modify cell adhesion and inhibit axon migration. Recently, two members, OPCML and LSAMP, were identified as putative tumour suppressor genes in ovarian and renal carcinomas respectively. In this study, we investigated OPCML expression in nonneoplastic brain tissue and 35 brain tumours (18 glioblastoma multiformes, five anaplastic gliomas, five meningiomas, six metastases and one medulloblastoma) and four glioma cell lines using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). OPCML was highly expressed in cerebellum, less so in cerebral cortex, frontal lobe and meninges and was significantly reduced or absent in 83% of brain tumours and all cell lines compared with nonneoplastic whole brain. Two OPCML splice variants have been identified in humans, termed alpha1 and alpha2, but the latter has not been demonstrated in human neural tissues. Using PCR with specific primers, nonneoplastic brain and 3/6 of tested brain tumours expressed both splice variants, whereas the remaining brain tumours only expressed the alpha2 variant. Hypermethylation of the alpha1 OPCML promoter, associated with down-regulation of expression in ovarian tumours, did not correlate with expression levels in the subset of brain tumours tested, implying transcription of OPCML from an alternative promoter or a different mechanism of down-regulation. This study demonstrates that OPCML down-regulation occurs in the majority of brain tumours tested, warranting further investigation of OPCML and other IgLONs in the development and progression of brain tumours.

BARX2 induces cadherin 6 expression and is a functional suppressor of ovarian cancer progression.

The human homeobox BARX2 is located at 11q24-q25, within a minimal region associated with frequent loss of heterozygosity and adverse survival in epithelial ovarian cancer. BARX2 is a transcription factor that regulates transcription of specific cell adhesion molecules in the mouse. We show that BARX2 and cadherin 6 are expressed in normal human ovarian surface epithelium. BARX2 and cadherin 6 both have significantly lower expression in a clinical sample of endometrioid and clear cell ovarian cancers, as compared with serous or mixed mesodermal tumors. In a series of ovarian cancer cell lines, BARX2 expression showed a significant direct correlation with cadherin 6 expression. In OAW42, an ovarian cancer cell line that does not endogenously express BARX2, in vitro transfection of human BARX2 cDNA induced cadherin 6 expression. Transfection of BARX2 into OAW42 inhibited Matrigel invasion, haptotactic cellular migration to a collagen IV signal, and adhesion to collagen IV-coated plates. Our data demonstrate that BARX2 is expressed in the ovarian surface epithelium and has functional suppressor properties in ovarian cancer cells.

Differential display.

Differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) is an extremely powerful method for analyzing differences in gene expression between matched tissue/cell samples. Liang and Pardee originally described the technique in 1992 in their seminal paper (1). DDRT-PCR is now firmly established as a widespread powerful and commonly used method for identifying differentially expressed genes.

A 700-kb physical map of a region of 16q23.2 homozygously deleted in multiple cancers and spanning the common fragile site FRA16D.

We have identified a >600-kb region at 16q23.2 that is homozygously deleted from malignant ovarian ascites using representational difference analysis. Overlapping homozygous deletions were also observed in the colon carcinoma cell line HCT116 and a xenograft established from the small cell lung cancer cell line WX330. This region coincides with that described previously by others as showing loss of heterozygosity in prostate and breast cancers (C. Li et al., Genes Chromosomes Cancer, 24: 175-182, 1999; A. Latil et al., Cancer Res., 57: 1058-1062, 1997; K. Driouch et al., Genes Chromosomes Cancer, 19: 185-191, 1997; A. Iida et al., Br. J. Cancer, 75: 264-267, 1997). In addition, the minimally deleted region spans the common fragile site FRA16D. We have constructed a 700-kb physical map encompassing the deleted region. By fluorescence in situ hybridization of aphidicolin-induced metaphase chromosomes, we have preliminary data to suggest that P1-derived bacterial artificial chromosome clones from the contig lie on both sides of FRA16D. This is confirmed by extensive fluorescence in situ hybridization analysis of the region reported in the accompanying article (M. Mangelsdorf et al., Cancer Res., 60: 1683-1689, 2000) and is consistent with an involvement of this common fragile site in the loss of 16q23.2 material in various cancer types. The minimally deleted region of approximately 210 kb has been characterized using our own markers and public domain markers. Eleven distinct expressed sequences mapped to the region, providing a basis for identifying the predicted tumor suppressor gene in this region.

The Xenopus laevis homologue of the 64-kDa subunit of cleavage stimulation factor.

Cleavage stimulation factor (CstF) is composed of three subunits of 50, 64 and 77 kDa, respectively. We report here the identification of a cDNA clone from Xenopus laevis encoding a homologue of the 64-kDa subunit of human CstF. Comparative sequence analysis reveals that these two proteins are highly conserved with the exception of a unique repeat structure found in the human, but not in the X. laevis, protein. Analysis of expression of this mRNA during X. laevis tadpole development indicates a requirement for this protein throughout all stages of development.

Organization of the region encompassing the human serum amyloid A (SAA) gene family on chromosome 11p15.1.

The four members of the human serum amyloid A protein (SAA) gene family are clustered on human chromosome 11p15.1. Three genes are differentially expressed and encode small apolipoproteins of M(r) 12-19 kDa: SAA1 and SAA2 encode the acute phase SAAs (A-SAAs), and SAA4 encodes the constitutively expressed SAA (C-SAA). A fourth locus, SAA3, is a pseudogene. The human SAA gene family encompasses approximately 150 kb contained on a 900-kb yeast artificial chromosome contig. SAA1 and SAA2 are 15-20 kb apart and are arranged in divergent transcriptional orientations. SAA4 is 9 kb downstream of SAA2 and in the same orientation. SAA3 is 110 kb downstream of SAA4, and its relative orientation could not be determined. All genes known to be in the same human and mouse syntenic linkage group as SAA were mapped within the contig. Interphase FISH was used to orientate the region relative to the centromere: cen-LDHC-LDHA-SAA1-SAA2-SAA4-SAA3-TP H-D11S18- KCNC1-MYOD1-pter.

Characterization, expression and evolution of mouse beta 2-glycoprotein I (apolipoprotein H).

beta 2-glycoprotein I (beta 2I) is a 50kDa serum glycoprotein of ill defined function. Based upon its capacity to bind negatively charged phospholipids a number of possible inhibitory roles for beta 2I have been proposed. We have cloned and sequenced a full length mouse beta 2I cDNA clone and demonstrated that mouse beta 2I does not behave as an acute phase reactant following an experimentally induced inflammation. Phylogenetic analysis of the known mammalian beta 2I homologues has provided evidence that mouse beta 2I is the most divergent and is evolving at a faster rate than beta 2I in other species.

The putative fifth human serum amyloid A protein (SAA)-related gene "SAA5" is defined by SAA3.

The four well characterised members of the human serum amyloid A protein (SAA) gene family are clustered on chromosome 11p15.1. The acute phase SAA genes, SAA1 and SAA2, are hyperinducible in response to inflammatory stimuli, whereas SAA4 is only minimally induced, and SAA3 is a pseudogene. We recently demonstrated that the GSAA4 sequence, reported by others (Sack, G.H. Jr. and Talbot, C.C. Jr., 1992. Biochem. Biophys. Res. Comm. 183, 362-366), and misidentified as corresponding to the SAA4 locus, maps to the 11p15 region and speculated that it may be in close proximity to a distinct fifth SAA locus: "SAA5". In this report we have used vectorette PCR in combination with direct sequencing and computer based homology searches of the nucleotide sequence databases to establish that the putative fifth SAA-related locus, "SAA5", is defined by SAA3 and therefore does not represent a distinct SAA gene.

The human serum amyloid A protein (SAA) superfamily gene cluster: mapping to chromosome 11p15.1 by physical and genetic linkage analysis.

The human serum amyloid A protein (SAA) family comprises a number of small, hepatically produced, differentially expressed apolipoproteins encoded by genes localized on the short arm of chromosome 11.SAA1 and SAA2 are highly related genes that together encode the acute-phase SAAs; SAA3 is a pseudogene; and SAA4 is a low-level constitutively expressed gene encoding constitutive SAA. We have used a combination of physical and genetic mapping techniques to provide evidence that the SAA gene superfamily comprises a cluster of closely linked genes localized to 11p15.1. Pulsed-field gel electrophoresis placed SAA1 to within 350 kb of the previously linked SAA2 and SAA4 genes. SAA locus-specific polymerase chain reaction amplification from a panel of somatic cell hybrids carrying defined regions of chromosome 11p mapped all four loci to 11p15.1-pter. Fluorescence in situ hybridization analysis using a cosmid probe carrying the SAA2 and SAA4 genes refined the localization of these genes (and SAA1) to 11p15.1. To order SAA3 on the genetic map, a highly polymorphic (CA)n dinucleotide repeat within SAA3 was typed through the CEPH reference families. In accordance with the physical localization of SAAs 1, 2, and 4, SAA3 maps to the 11p15.1 region proximal to the parathyroid hormone (PTH) locus (theta = 0.02; lod = 12.020) and distal to D11S455 (theta = 0.058, lod = 8.274). To provide further evidence of an SAA superfamily gene cluster, an NcoI restriction fragment length polymorphism in the SAA2 gene was also typed through the CEPH reference panel.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of four human serum amyloid A (SAA) protein superfamily genes to chromosome 11p: characterization of a fifth SAA-related gene sequence.

The serum amyloid A proteins (SAAs) are heterogeneous differentially expressed apolipoproteins of M(r) 12-19 kDa. Four SAA loci have been described. Two of the loci (SAA1 and SAA2) encode acute-phase SAAs (A-SAAs), which exhibit a dramatic transient increase in serum concentration in response to inflammatory stimuli; a third locus (SAA3) defines a pseudogene; and a fourth locus (SAA4) encodes a constitutively expressed SAA (C-SAA). Using locus-specific polymerase chain reaction, we have definitively assigned all four well-characterized SAA loci, SAA1, -2, -3, and -4, to chromosome 11p. In addition, we have more fully characterized an ill-defined sequence previously misidentified by others as SAA4, which also maps to chromosome 11p and may represent a fifth SAA locus.

A constitutively expressed serum amyloid A protein gene (SAA4) is closely linked to, and shares structural similarities with, an acute-phase serum amyloid A protein gene (SAA2).

The acute-phase reactant serum amyloid A (SAA) is a polymorphic apolipoprotein encoded by a family of highly homologous and closely linked genes: SAA1, SAA2, and SAA3. We have isolated a human genomic cosmid clone containing the gene encoding a fourth, constitutively expressed member of the human SAA superfamily, C-SAA, together with an SAA2*2 (SAA2 beta) gene. The gene encoding C-SAA shares the same 5' to 3' orientation as SAA2*2 and has the characteristic four-exon structure of the other members of the SAA superfamily. The exons of the gene encoding C-SAA share only limited sequence identity with those of SAA1, SAA2, and SAA3; they specify an mRNA, represented by the CS-1 cDNA reported previously by us, which is expressed at low levels (relative to the acute-phase SAAs) in normal and acute-phase liver. The gene encoding C-SAA is located 9 kb downstream of SAA2*2 and therefore occupies the locus that has been identified as containing the SAA4 gene.

Characterization and acute phase modulation of canine apolipoprotein H (beta 2-glycoprotein I).

Apolipoprotein H (ApoH) is a 50 kDa glycoprotein capable of binding to negatively charged phospholipids and is a probable inhibitor of the blood coagulation pathway, platelet aggregation, and platelet prothrombinase activity, as well as being involved in autoimmune disease. We have cloned and sequenced a full length ApoH cDNA clone from a beagle dog liver library. Its derived amino acid sequence shows high cross-species similarity to ApoH from other mammals. Canine ApoH mRNA expression is down regulated during an experimentally induced inflammatory response establishing that it is a negative acute phase reactant.

Characterization and organization of the genes encoding the A-, B- and C-chains of human complement subcomponent C1q. The complete derived amino acid sequence of human C1q.

A partial cDNA clone for the A-chain of human complement subcomponent C1q was isolated from a monocyte library. Use of the A-chain cDNA clone, and a previously characterized B-chain cDNA clone [Reid (1985) Biochem. J. 231, 729-735] allowed the isolation of overlapping cosmid clones that were shown to contain the genes encoding the A-, B- and C-chains of human C1q. The three genes were found to be aligned, 5'----3', in the same orientation, in the order A-C-B on a 24 kb stretch of DNA on chromosome 1p. The A-, B- and C-chain genes are approx. 2.5, 2.6 and 3.2 kb long respectively, and each contains one intron, located within a codon for a glycine residue found half-way along the collagen-like region present in each chain. These glycine residues are located just before the point where the triple-helical portions of the C1q molecule appear to bend when viewed in the electron microscope. Southern-blot analyses indicated that there is only one gene per chain, and preliminary examination of genomic DNA from several C1q-deficient patients showed no evidence for major deletions or insertions within the A-, B- or C-chain genes. The DNA sequence of the coding region of the C-chain gene allows the completion of the entire derived amino acid sequence for the human C1q molecule. The globular, C-terminal, regions of the chains of C1q show a strong similarity in amino acid sequence to the non-collagen-like, C-terminal, regions of the type VIII and type X collagens, indicating structural and evolutionary relationships between these three molecules.

Dog serum amyloid A protein. Identification of multiple isoforms defined by cDNA and protein analyses.

Five distinct serum amyloid A (SAA) cDNA clones have been isolated from a library constructed using hepatic mRNA isolated from an individual beagle dog with canine pain syndrome. This implies the existence of at least three SAA genes in the dog genome. One clone predicts a truncated "amyloid A-like" SAA molecule and offers a possible alternative mechanism for the pathogenesis of secondary amyloidosis. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified by each of the dog cDNA clones. The existence of this peptide in all acute phase dog SAA proteins was confirmed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of acute phase high density lipoprotein and provides supporting evidence for gene conversion as a mechanism for maintaining the homogeneity of the SAA gene family within a species. Analysis of hepatic RNA following induction of an acute phase response shows a dramatic increase in SAA mRNA concentration; the SAA transcripts show a transient increase in size early in inflammation due to an increase in polyadenylation.