Genomic organization and promoter identification of ZNF146, a gene encoding a protein consisting solely of zinc finger domains.

The ZNF146 gene (alias OZF) encodes a protein consisting solely of ten zinc finger motifs. It is amplified and overexpressed in pancreatic carcinomas. To better understand the mechanisms controlling its expression, we have isolated the human ZNF146 gene and performed an initial assessment of its promoter activity. ZNF146 encompasses 25 kb of sequence and consists of four non-coding exons located upstream of a single coding exon. The sequence of proximal 1.4 kb of ZNF146 promoter has a high GC content, is devoid of a TATA box and contains several potential transcriptional elements. This region directs high-level expression of a transfected reporter construct in human cell lines. Analysis of a series of 5'-deletion constructs indicated that the first 80 bp upstream of the potential start site of transcription carry minimal promoter activity whereas the first 550 bp are required for maximal promoter activity.

Cloning, characterization, and chromosome assignment of Zfp146 the mouse ortholog of human ZNF146, a gene amplified and overexpressed in pancreatic cancer, and Zfp260 a closely related gene.

The human OZF gene (ZNF146), located in chromosome band 19q13.1, is amplified and overexpressed in pancreatic carcinomas. It encodes a protein consisting solely of ten Krüppel zinc finger motifs. We report here the isolation and the characterization of the murine OZF cDNA (Zfp146). Comparison of the deduced amino acid sequences between murine, human and bovine cDNAs revealed a strong identity (95%). A closely related gene, Zfp260, was also isolated and characterized. It encodes a putative protein consisting of three vestigial zinc finger motifs followed by ten Krüppel zinc fingers sharing 79% identity and no gap insertion with the Zfp146 zinc fingers. In vitro transcription/translation of both genes led to synthesis of proteins of the predicted size. Co-expression was observed at the mRNA level in eight adult mouse tissues. Two-color FISH revealed co-localization of both genes on mouse chromosome 7 (band B1-B3). The co-expression and co-localization of Zfp146 and Zfp260 together with the close similarity of their zinc finger domains, suggests that both participate in the same regulatory pathway.

The pag gene product, a physiological inhibitor of c-abl tyrosine kinase, is overexpressed in cells entering S phase and by contact with agents inducing oxidative stress.

The human pag gene product is an inhibitor of the c-abl tyrosine kinase and belongs to a new family of proteins. We show here that higher levels of pag gene expression are observed following induction of proliferation and contact with compounds inducing oxidative stress such as diethyl maleate and sodium arsenate. A weaker overexpression is seen in a macrophage cell line using hydrogen peroxide or menadione as inducers. Pag gene expression increases in synchronized cells entering the S phase. This raises the possibility that elevated levels of pag counteract the cytostatic activity of abl. Treatment of growth arrested cells with diethyl maleate and sodium arsenate induces pag gene overexpression, independently of cell proliferation. Thus, enhanced pag gene expression occurs in two cellular events: proliferation and response to oxidative stress.

Identification, nuclear localization, and binding activities of OZF, a human protein solely composed of zinc-finger motifs.

The OZF cDNA was identified in a human mammary cell line and encodes a polypeptide solely composed of ten zinc-finger motifs which belongs to the Kruppel family of zinc-finger proteins. The OZF protein produced in Escherichia coli binds zinc ions, DNA and heparin. These binding activities are characteristic of zinc-finger proteins. Immunochemical analysis using antibodies produced against the recombinant protein detected its expression in human mammary epithelial cells but not in stroma cells, which is consistent with the pattern of expression observed at the RNA level in cell cultures. Western blot analysis demonstrated the expression of a 33-kDa nuclear protein similar in size to the predicted protein and therefore excluded the presence of an additional trans-acting domain. These data establish the unique structure of the OZF protein which is distinct from previously identified zinc-finger proteins. In addition, OZF protein overexpression was found in a tumor cell line, which suggests a possible involvement in carcinogenesis.

The OZF gene encodes a protein consisting essentially of zinc finger motifs.

A novel member of the zinc finger Krüppel family was isolated as a cDNA clone from the human mammary cell line HBL100. It contains an open reading frame consisting of 32 amino acid residues followed by ten zinc finger motifs and was accordingly named OZF (Only Zinc Fingers). Assuming that translation initiation starts at the unique methionine codon, 22 codons downstream of the beginning of the open reading frame as suggested by in vitro translation of OZF mRNA, the OZF protein is 292 residues long and has an estimated molecular mass of 33 kDa. The OZF gene is located in band q13.1 of the long arm of human chromosome 19. It is expressed as a single mRNA in liver, skeletal muscle and mammary cells and as two mRNA species in heart muscle. Absent or very low levels of expression were observed in brain, lung, placenta, kidney and human fibroblasts in culture. Thus, the OZF protein, which consists essentially of a DNA/RNA binding domain, may act as a tissue-specific modulator of gene expression.

Organization and chromosomal assignment of two human PAG gene loci: PAGA encoding a functional gene and PAGB a processed pseudogene.

A cDNA, designated PAG, was recently isolated by differential cloning between an untransformed and a ras-transformed human mammary cell line. Higher levels of expression were found to be associated with cell proliferation. The absence in the pag protein of known consensus sequence, as well as its close relationship with a gene product involved in the differentiation of a mouse erythroleukemia cell line, has suggested that the PAG gene belongs to a family of genes associated with cell proliferation and differentiation. To further characterize this gene, a physical map has been established from a human genomic cosmid library. The PAG gene spans 13 kb of DNA and contains six exons. The promoter region is GC-rich and contains a TFIID motif located 25 nucleotides upstream of the potential site for initiation of transcription and potential recognition sites for a variety of trans-acting factors. Using fluorescence in situ hybridization, the PAG gene was mapped to human chromosome band 1p34.1. A pseudogene was also isolated, sequenced, and mapped to human chromosome band 9p22.

A human cDNA corresponding to a gene overexpressed during cell proliferation encodes a product sharing homology with amoebic and bacterial proteins.

A clone, designated pag, was isolated by differential screening of cDNA libraries made from the untransformed and ras-transformed human mammary epithelial cell line HBL100. This cDNA corresponds to a gene constitutively expressed in most human cells which is induced to higher levels upon serum stimulation in untransformed and ras-transformed HBL100 cells. However, the abundance of the pag transcript is approximately 3-fold higher in transformed as compared to untransformed cells after 7-15 h of serum stimulation. In the promyelocytic leukemia cell line HL60 induced to differentiate the level of pag mRNA starts to decrease between 48 and 72 h following induction. During this period, which represents the commitment phase of differentiation, HL60 cells cease to proliferate. Therefore, in HBL100 and HL60 cells, higher levels of pag gene expression are correlated with cell proliferation. The pag cDNA codes for a 22-kDa protein, devoid of known consensus motifs, and shares 66% homology with a murine gene product (MER5) that is preferentially expressed in erythroleukemia cells during the early period of cell differentiation. In addition, the pag gene product shares approximately 50% identity with a 29-kDa surface antigen of Entamoeba histolytica and a 26-kDa antigen of Helicobacter pylori. Distant relationship was also found with other prokaryotic proteins. The pag cDNA hybridizes to multiple sequences within human and other mammalian genomes and to fewer sequences in chicken and Saccharomyces cerevisiae. Although a true relationship between eukaryotic and prokaryotic genes is difficult to establish, the conservation of pag gene sequences throughout Eukaryotae rather suggests that the pag locus belongs to a new class of genes encoding highly conserved proteins.

Fusion and amplification of two originally non-syntenic chromosomal regions in a mammary carcinoma cell line.

The FLG/FGFRI gene, encoding a receptor for members of the FGF family, is located at 8p11.2-p12. It is amplified, overexpressed, and not grossly rearranged in the MDA-MB-134 breast carcinoma cell line, whereas other genes from the pericentromeric 8p region are not amplified. The FGF4/HSTFI gene, located at 11q13, is also amplified with a substantial portion of the 11q13 region, but is not overexpressed in MDA-MB-134 cells. In this cell line, amplified sequences constitute a large homogeneously staining region (HSR) which is part of a marker chromosome containing chromosome 8 and chromosome 11 sequences. Using probes for the FGF4/HSTFI and the FLG/FGFRI genes in fluorescence chromosomal in situ hybridization, we show that the HSR contains de novo fused and amplified 11q13 and 8p11-p12 sequences associated in a complex structure containing approximately the same number of FGF4 and FGFRI genes. The significance of this genetic abnormality for MDA-MB-134 cells, and for breast carcinogenesis in general, is unknown, but may underlie a particular type of oncogene activation.

Constitutive overexpression of a 89 kDa heat shock protein gene in the HBL100 human mammary cell line converted to a tumorigenic phenotype by the EJ/T24 Harvey-ras oncogene.

The non tumorigenic human mammary cell line HBL100 has been transformed by the EJ/T24 human bladder carcinoma Harvey(Ha)-ras oncogene. Six cell lines were established from transformed colonies. They all expressed a high level of the ras oncogene and were tumorigenic in athymic nude mice. During an in vivo passage in animals, tumour cells presenting a growth advantage were selected, and some of the tumours revealed an amplification of the transfected ras sequences. Using this model of human cell transformation, we have isolated a cDNA clone corresponding to a heat shock protein gene (hsp89 alpha). This gene, normally transcribed at a higher rate in response to serum stimulation, was found to be constitutively overexpressed in ras-transformed HBL100 cells. In contrast, a closely related hsp gene (hsp89 beta), remained sensitive to serum stimulation, in both untransformed and ras-transformed HBL100 cells. Thus, the regulation of the expression of the hsp89 genes, upon serum stimulation, involves ras-dependent and ras-independent pathways. Constitutive overexpression of the murine homolog of the hsp89 alpha was observed in NIH3T3 cells transformed by the three ras oncogenes, but not with some other oncogenes. Therefore, alteration of the hsp89 alpha gene expression is not a general characteristic of transformed cells, but seems to be linked to ras transformation.

Point mutation at codon 12 of the Ki-ras gene in a primary breast carcinoma and the MDA-MB-134 human mammary carcinoma cell line.

We found an activated Kirsten (Ki)-ras gene in the MDA-MB-134 breast carcinoma cell line by transfection of NIH3T3 cells. Oligonucleotide hybridization demonstrated that this cell line carries a single G to C point mutation at position 12 leading to a glycine-arginine substitution. However, only a fraction of the cell population seems to contain this Ki-ras mutation. Since mutations can occur in cell lines during in vitro culture, we searched in breast carcinoma samples for the presence of single mutations at codon 12, but also for the presence of the double mutation previously found in the H-466B breast carcinoma cell line. Using polymerase chain reaction (PCR), we detected one primary tumour carrying a single mutation at codon 12. No double mutation was found in any of the tumours. These results show that Ki-ras gene mutation could be involved in breast carcinogenesis, albeit at a low frequency.

Two adjacent mutations at position 12 activate the K-ras2 oncogene of a human mammary tumor cell line.

Among 10 human mammary tumor cell lines analyzed for transforming genes by transfection of NIH 3T3 cells, one carcinoma cell line, H-466B, established from an ascitic effusion of a woman with an adenocarcinoma of the breast was scored as positive. The transforming gene was identified as the K-ras2 oncogene. Nucleotide sequencing of exons 1 and 2 of the activated gene revealed two adjacent G----T transversions at the first and second position in codon 12 leading to the replacement of the normally encoded glycine by a phenylalanine. Since the phenylalanine substitution had never been observed in any type of tumor, this raises the question about the frequency as well as the cell type specificity of this K-ras2 activation in mammary tumors.