Intracellular patterns of sialophorin expression define a new molecular classification of breast cancer and represent new targets for therapy.

BACKGROUND: Sialophorin is a transmembrane sialoglycoprotein. Normally, the molecule is only produced by white blood cells where it regulates functions such as intercellular adhesion, intracellular signalling, apoptosis, migration and proliferation. METHODS: Normal breast tissue and primary breast tumours were analysed by immunohistochemistry for sialophorin expression. The sialophorin-positive breast cancer cell line MCF7 was engineered to stably express either non-targeted or sialophorin-targeted small interfering RNA (siRNA). Assays were then performed in vitro to assess apoptosis, intracellular adhesion, transendothelial migration and cytotoxicity. An orthotopic mouse model assayed ability to produce tumours in vivo. RESULTS: Normal breast epithelial cells exhibit expression of the N-terminal domain of sialophorin in the cytoplasm but not the nucleus. The majority of these normal cells are also negative for expression of the C-terminal domain. In contrast, malignant breast epithelial cells exhibit N-terminal expression both in the cytoplasm and nucleus and the majority express the C-terminus in the nucleus. Using differential patterns of intracellular expression of the N and C termini of sialophorin, we define six subtypes of breast cancer that are independent of histological and receptor status classification. Targeting sialophorin with siRNA resulted in the MCF7 breast cancer cell line exhibiting increased homotypic adhesion, decreased transendothelial migration, increased susceptibility to apoptosis, increased vulnerability to lysis by natural killer cells and decreased ability to produce tumours in mice. CONCLUSION: Our results indicate that intracellular patterns of sialophorin expression define a new molecular classification of breast cancer and that sialophorin represents a novel therapeutic target.

During U937 monocytic differentiation repression of the CD43 gene promoter is mediated by the single-stranded DNA binding protein Pur alpha.

Human CD43 is an abundant, heavily glycosylated molecule expressed exclusively on the surface of leucocytes. When leucocytes are at rest, CD43 acts to prevent intercellular interaction but during leucocyte differentiation such cell-cell interaction is facilitated by CD43. This change in the function of CD43 is mediated in part by a reduction in its level of expression. Previous studies have implicated proteolytic cleavage events at the cell surface in causing such reduction. Here, we report that, in an in vitro model of leucocyte differentiation, CD43 mRNA levels were also subject to reduction. Specifically, we demonstrated that within 48 h of the cell line U937 being induced to differentiate along the monocytic pathway, CD43 mRNA levels were reduced by 69%. This decline coincided with a decrease in the activity of the CD43 gene promoter mediated by the single-stranded DNA binding protein Pur alpha. Previously, we have demonstrated that Pur alpha mediates induction of the CD11c beta 2 integrin promoter during U937 differentiation. Consequently, Pur alpha represents a potential means by which the induction of pro-adhesive molecules and the repression of anti-adhesive molecules is co-ordinated during leucocyte differentiation.

CD43 gene expression is mediated by a nuclear factor which binds pyrimidine-rich single-stranded DNA.

CD43 is a leukocyte-specific surface molecule which plays an important role both in adhesion and signal transduction. We have identified a site spanning nucleotides +18 to +39 within the human CD43 gene promoter which in vitro is hypersensitive to cleavage by nuclease S1. Repeats of this region are sufficient to activate expression of a heterologous promoter in CD43-positive cell lines. Two nuclear factors, PyRo1 and PyRo2, interact with the hypersensitive site. PyRo1 is a single-stranded DNA-binding protein which binds the pyrimidine-rich sense strand. Mutation analysis demonstrates that the motif TCCCCT is critical for PyRo1 interaction. Replacement of this motif with the sequence CATATA abolishes PyRo1 binding and reduces expression of the CD43 promoter by 35% in Jurkat T lymphocytic cells and by 52% in the pre-erythroid/pre-megakaryocytic cell line K562. However, this same replacement failed to affect expression in U937 monocytic cells or in CEM T lymphocytic cells. PyRo1, therefore, exhibits cell-specific differences in its functional activity. Further analysis demonstrated that PyRo1 not only interacts with the CD43 gene promoter but also motifs present within the promoters of the CD11a, CD11b, CD11c and CD11d genes. These genes encode the alpha subunits of the beta2 integrin family of leukocyte adhesion receptors. Deletion of the PyRo1 binding site within the CD11c gene reduced promoter activity in T lymphocytic cells by 47%. However, consistent with our analysis of the CD43 gene, the effect of this same deletion within U937 monocytic cells was less severe. That PyRo1 binds preferentially to single-stranded DNA and sequences within the CD43 and CD11 gene promoters suggests that expression of these genes is influenced by DNA secondary structure.

Induction of the CD11b gene during activation of the monocytic cell line U937 requires a novel nuclear factor MS-2.

The differentiation of myeloid precursors into mature myelomonocytic cells is characterized by the induction of the gene encoding the beta2 integrin CD11b. The transcription factors Sp1 and PU.1 prime the CD11b promoter, but the nature of the factors responsible for its inducible expression are unknown. In addition to the CD11b gene, the homologous genes encoding CD11a and CD11c also exhibit inducible expression during myeloid differentiation. Therefore, we compared the nucleotide sequences of the CD11a, CD11b, and CD11c gene promoters to identify common elements that might contribute to inducible expression. This analysis identified one such element repeated four times within the CD11b promoter. Mutation of these elements indicated that two, MS-2beta and MS-2gamma, are critical to the induction of the CD11b gene during differentiation of the pro-monocytic cell line U937. Electrophoretic mobility shift assays indicate that MS-2beta and MS-2gamma interact with nuclear factors that are induced during U937 differentiation. These factors are detected at the time the CD11b promoter is activated. The molecular mass of these factors is approximately 28 kDa, and their DNA binding characteristics are indistinguishable from those of the novel nuclear factor MS-2. Taken together, our data indicate that MS-2 mediates induction of the CD11b gene as cells of the monocytic lineage mature. The presence of multiple potential binding sites for MS-2 in the promoter regions of a wide range of genes expressed in mature myeloid cells suggests this factor plays a general role in myeloid differentiation.

The human beta 2 integrin CD18 promoter consists of two inverted Ets cis elements.

To define the minimal promoter responsible for expression of CD18 in myeloid and lymphoid cells, we generated 5' and 3' deletion constructs of a segment extending 785 bp upstream and 19 bp downstream of a major transcription start site and determined their effects on driving expression of the luciferase reporter gene in transfected hematopoietic cell lines. A region extending from nucleotides (nt) -302 to +19 was sufficient for cell-restricted and phorbol ester-inducible expression. DNase I footprinting of this region revealed two adjacent protected segments extending from nt -81 to -68 (box A) and -55 to -41 (box B). When a construct of 47 nt in length containing box A and box B and lacking other 3' or 5' elements was cloned into a promoterless vector, it conferred tissue-specific and phorbol ester-inducible expression. Gel retardation revealed that the protein components of two major protein-DNA complexes that form on both box A and box B and are required for transcriptional activation are members of the Ets oncoprotein family; one is related to the GA-binding protein (GABP), and the other is related to PU.1/Spi-1. The minimal CD18 promoter, lacking TATA, CAAT, and initiator elements and consisting primarily of Ets repeats, may exemplify an emerging class of promoters with which the concerted binding of Ets factors is necessary and sufficient to mediate transcriptional activation through direct recruitment of the basal transcription machinery.

Identification of cell-specific and developmentally regulated nuclear factors that direct myeloid and lymphoid expression of the CD11a gene.

Human CD11a/CD18 is a noncovalently associated heterodimeric receptor expressed exclusively on the surface of lymphocytes and myeloid cells. To begin to understand the mechanisms that direct the expression of the genes encoding this receptor, we have cloned and characterized the promoter region of the CD11a gene and localized cis-acting elements involved in its expression in lymphoid and myeloid cells. One such element is the "LYM" box, which interacts with two sets of DNA-binding activities, one primarily expressed in lymphocytes and preerythroid cells and the other expressed predominantly in myeloid cells. A second element required for expression of the CD11a gene contains the "GAGA" sequence RRRGAGGAAG (R indicates a purine), which interacts with the DNA-binding activities MS-1 and MS-2. MS-1 is expressed exclusively in myeloid cells and probably represents a member of the Ets family of transcription activators. MS-2 is present in epithelial, preerythroid, and lymphoid cells but is only detected in myeloid cells after differentiation. MS-2 also binds to a second element within the CD11a promoter and homologous elements present in the promoter regions of the CD11b and CD43 genes. Since MS-2 interacts with a number of different gene promoters and is developmentally regulated in myeloid cells, it may play a major role in regulating myeloid gene expression.

The promoter of the CD11b gene directs myeloid-specific and developmentally regulated expression.

Human CD11b/CD18 (complement receptor type 3) is a member of the beta 2 integrin subfamily which also includes the heterodimers CD11a/CD18 and CD11c/CD18. The CD11 molecules and the common CD18 are the products of different genes that exhibit distinct though overlapping patterns of tissue- and developmental-specific expression. Whereas expression of CD11b and CD11c is almost exclusively restricted to cells of the myeloid lineage, that of CD11a and CD18 is panleukocytic. To begin to understand the mechanisms by which expression of these gene products is restricted to leukocytes and leukocyte subpopulations and to elucidate the mechanisms by which their expression is coordinated, we have cloned and characterized the promoter region of the CD11b gene. A single transcription initiation site has been identified and the region extending 242 base pairs upstream and 71 base pairs downstream of this site has been shown to be sufficient to direct tissue-, cell-, and development-specific expression in vitro, which mimics that of the CD11b gene in vivo. Within this region there are potential binding sites for transcription factors known to be involved in hematopoietic-specific and phorbol ester-inducible gene expression. Further analysis of this region of the CD11b gene and comparison with the promoters of the CD11a, CD11c, and CD18 genes should lead to significant insights into the molecular mechanisms by which these genes are regulated during hematopoietic development and upon activation.

Structure of the human sialophorin (CD43) gene. Identification of features atypical of genes encoding integral membrane proteins.

A human sialophorin (CD43) specific genomic clone was isolated, and a 6.5 kb fragment containing the 4.6 kb sialophorin gene was sequenced. The promoter region contains no TATA or CAAT boxes, but is highly enriched in G and C nucleotides and contains short repeat sequences similar to those found in the promoters of 'housekeeping' genes. S1-nuclease protection and primer-extension experiments established that the sialophorin gene has two major transcription initiation sites. There is a single intron of 378 bp that interrupts the sequence specifying the mRNA 5' untranslated region. The gene is therefore unusual in that the discrete extracellular, transmembrane and intracellular regions of the protein, including repeat sequences in the extracellular region, are not encoded by separate exons. Utilization of alternative polyadenylation signals was previously shown to generate two sialophorin mRNAs of 1.9 and 4.3 kb, which differ in the length of their 3' untranslated regions. Sequence analysis of the gene establishes that a single polyadenylation signal 2301 bp downstream of the first major transcription initiation site and five overlapping polyadenylation signals beginning a further 2290 bp downstream define the 3' termini of the 1.9 and 4.3 kb mRNA species respectively. The gene contains potential Z-DNA structures, Aly sequences, and elements that may be involved in regulating mRNA stability.

Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome.

Sialophorin (CD43) of leukocytes and platelets is a surface sialoglycoprotein that is phenotypically defective on lymphocytes of patients with the X chromosome-linked immunodeficiency Wiskott-Aldrich syndrome. Previous studies with monoclonal antibodies indicate that sialophorin is a component of a T-lymphocyte activation pathway. Here we describe the cDNA cloning and derived amino acid sequence of human sialophorin. The sequence predicts an integral membrane polypeptide with an N-terminal hydrophobic signal region followed by a mucin-like 235-residue extracellular region with a uniform distribution of 46 serine, 47 threonine, and 24 proline residues. This is followed by a 23-residue transmembrane region and a 123-residue C-terminal intracellular region. These latter regions have been highly conserved during evolution; the intracellular region contains a number of potential phosphorylation sites that might mediate transduction of activation signals. The chromosomal location of the sialophorin gene was determined and the implications of this assignment for the pathogenesis of the Wiskott-Aldrich syndrome are discussed.

Deletion analysis of a unique 3' splice site indicates that alternating guanine and thymine residues represent an efficient splicing signal.

The 3' splice site of the second intron (I2) of the human apolipoprotein-AII gene, (GT)16GGGCAG, is unique in that, although fully functional, a stretch of alternating guanine and thymine residues replaces the polypyrimidine tract usually associated with 3' splice junctions. The transient expression of successive 5' deletion mutants has defined the minimum number of nucleotides at the 3' end of apo-AII I2 that are required to direct efficient splicing. Processing in two cell-types, representing apo-AII producing and non-producing tissue was identical; in both, only by removing all the GT repeats did the 3' splice site of apo-AII I2 become completely non-functional. Similar deletion analyses of "classic" 3' splice sites, which conform to the consensus sequence (Y)nNYAG, have indicated that a minimum of 14 nucleotides of the polypyrimidine tract are required for detectable levels of processing to take place. Here we report that the six nucleotides (GT)2GG, which directly replace this tract in a deletion mutant of the 3' splice site of apo-AII I2 are sufficient to direct the splicing process efficiently and correctly.

Dual tissue-specific expression of apo-AII is directed by an upstream enhancer.

Apolipoprotein-AII (apo-AII) is one of a family of evolutionarily related proteins which play a crucial role in lipid transport and metabolism. The serum levels of human apo-AII have been shown to be inversely correlated to the incidence of coronary heart disease and its expression to be limited to the liver and intestine. Here we demonstrate that this dual tissue-specificity involves DNA sequences located in a 259 bp region centred 782 bp upstream from the transcription initiation site. These sequences function in an orientation-independent manner and are absolutely required for transcription from the apo-AII promoter. The regulatory region contains sequences which are homologous to the apo-AI, beta-globin and immunoglobulin gene promoters and to the immunoglobulin heavy-chain enhancer.

A DNA polymorphism of the apoprotein AII gene in hypertriglyceridaemia.

A polymorphism of the apolipoprotein AII gene (on chromosome 1) was investigated using genomic hybridisation analysis. The two common alleles at this locus were defined by MspI restriction fragments of 3.0 kilobase pairs (M3.0) and 3.7 kilobase pairs (M3.7) respectively. The M3.7 allele was significantly more common (P less than 0.02) in Caucasian subjects who were normo-lipaemic (34%, 20/59) than in those who were hypertriglyceridaemic (16%, 16/98). Serum triglyceride levels were measured in 126 Caucasian subjects with different combinations of disease-associated alleles at the ApoAII and ApoCIII gene loci. Mean serum triglyceride levels were found to be significantly higher (P less than 0.05) in subjects with disease-associated alleles of both the ApoCIII and ApoAII genes, compared with subjects with a disease-associated allele of one or neither locus.

Comparison of the human apolipoprotein genes. Apo AII presents a unique functional intron-exon junction.

The structure and function of the apolipoproteins are of interest because of their central role in lipid metabolism. We report the complete primary structure of the human apo AII and CIII genes. These, like apo AI, contain three introns located at conserved positions. This may reflect their evolutionary and functional relationship. Indeed, computer-aided analysis shows that these apolipoproteins and apo AIV (rat), CI, CII and E contain homologous amino acid sequences. In the case of apo AI, AII and CIII, such sequences are encoded by equivalent exons. Finally, the apo AII gene presents a unique intron-exon junction sequence 5' (G-T)16G-G-G-C-A-G 3' that, although departing considerably from the accepted consensus (Py)15X-Py-A-G, appears to function efficiently both in vivo and in a transient expression system.

Human apolipoproteins AI, AII, CII and CIII. cDNA sequences and mRNA abundance.

The structure and function of the genes encoding the polypeptide components of plasma lipoproteins are of interest because of the central role they play in the regulation of lipid metabolism. We have now completed our previous studies on the human apoAI gene and furthermore isolated and sequenced cDNA clones for apoAII , CII and CIII. The nucleotide sequences show the signal peptides of apoAII , CII and CIII to be 18, 22 and 20 amino acids in length, respectively, and in addition that prepro apoAII bears a classical propeptide structure of 5 amino acids. The amino acid homology detected between apoCII and pro- apoAI is discussed, as is the gene arrangement of the 5' non-coding region of apoAI mRNA. The relative liver mRNA levels of the 4 apolipoproteins analysed in this study have been estimated and compared with their corresponding plasma products. The data reported here provide an essential basis for further studies of structural and functional alleles of apo AI, AII, CII and CIII genes.