Identification, characterization and leucocyte expression of Siglec-10, a novel human sialic acid-binding receptor.

Here we characterize Siglec-10 as a new member of the Siglec family of sialic acid-binding Ig-like lectins. A full-length cDNA was isolated from a human spleen library and the corresponding gene identified. Siglec-10 is predicted to contain five extracellular Ig-like domains and a cytoplasmic tail containing three putative tyrosine-based signalling motifs. Siglec-10 exhibited a high degree of sequence similarity to CD33-related Siglecs and mapped to the same region, on chromosome 19q13.3. The expressed protein was able to mediate sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates. Using specific antibodies, Siglec-10 was detected on subsets of human leucocytes including eosinophils, monocytes and a minor population of natural killer-like cells. The molecular properties and expression pattern suggest that Siglec-10 may function as an inhibitory receptor within the innate immune system.

Siglec-8. A novel eosinophil-specific member of the immunoglobulin superfamily.

We describe the characterization of siglec-8, a novel sialic acid-binding immunoglobulin-like lectin that is expressed specifically by eosinophils. A full-length cDNA encoding siglec-8 was isolated from a human eosinophil cDNA library. Siglec-8 is predicted to contain three extracellular immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail of 47 amino acids. The siglec-8 gene mapped on chromosome 19q13.33-41, closely linked to genes encoding CD33 (siglec-3), siglec-5, siglec-6, and siglec-7. When siglec-8 was expressed on COS cells or as a recombinant protein fused to the Fc region of human IgG(1), it was able to mediate sialic acid-dependent binding to human erythrocytes and to soluble sialoglycoconjugates. Using specific monoclonal antibodies, siglec-8 could be detected only on eosinophils and hence appears to be the first example of an eosinophil-specific transmembrane receptor.

SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine.

Mice lacking suppressor of cytokine signaling-1 (SOCS1) develop a complex fatal neonatal disease. In this study, SOCS1-/- mice were shown to exhibit excessive responses typical of those induced by interferon gamma (IFNgamma), were hyperresponsive to viral infection, and yielded macrophages with an enhanced IFNgamma-dependent capacity to kill L. major parasites. The complex disease in SOCS1-/- mice was prevented by administration of anti-IFNgamma antibodies and did not occur in SOCS1-/- mice also lacking the IFNgamma gene. Although IFNgamma is essential for resistance to a variety of infections, the potential toxic action of IFNgamma, particularly in neonatal mice, appears to require regulation. Our data indicate that SOCS1 is a key modulator of IFNgamma action, allowing the protective effects of this cytokine to occur without the risk of associated pathological responses.

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival.

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.

Characterization of siglec-5, a novel glycoprotein expressed on myeloid cells related to CD33.

We describe the characterization of siglec-5 (sialic acid-binding Ig-like lectin-5), a novel transmembrane member of the immunoglobulin superfamily, highly related to the myeloid antigen, CD33. A full-length cDNA encoding siglec-5 was isolated from a human activated monocyte cDNA library. Sequencing predicted that siglec-5 contains four extracellular immunoglobulin-like domains, the N-terminal two of which are 57% identical to the corresponding region of CD33. The cytoplasmic tail is also related to that of CD33, containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibitory motif-like motifs. The siglec-5 gene was shown to map to chromosome 19q13.41-43, closely linked to the CD33 gene. When siglec-5 was expressed on COS cells or as a recombinant protein fused to the Fc region of human IgG1, it was able to mediate sialic acid-dependent binding to human erythrocytes and soluble glycoconjugates, suggesting that it may be involved in cell-cell interactions. By using specific antibodies, siglec-5 was found to have an expression pattern distinct from that of CD33, being present at relatively high levels on neutrophils but absent from leukemic cell lines representing early stages of myelomonocytic differentiation. Western blot analysis of neutrophil lysates indicated that siglec-5 exists as a disulfide-linked dimer of approximately 140 kD.

Mechanisms of cyclin-dependent kinase inactivation by progestins.

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of approximately 120 and approximately 200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.

Antiprogestin inhibition of cell cycle progression in T-47D breast cancer cells is accompanied by induction of the cyclin-dependent kinase inhibitor p21.

Progestin antagonists inhibit the proliferation of progesterone receptor-positive cells, including breast cancer cells, by G1 phase-specific actions, but the molecular targets involved are not defined. Reduced phosphorylation of pRB, a substrate for G1 cyclin-dependent kinases (CDKs) in vivo, was apparent after 9 h treatment of T-47D breast cancer cells with the antiprogestins RU 486 or ORG 31710, accompanying changes in S phase fraction. Although the abundance of cyclin D1, Cdk4, and Cdk6 did not decrease cyclin D1-associated kinase activity was reduced by approximately 50% at 9-18 h. Similarly, cyclin E-associated kinase activity decreased by approximately 60% at 12-24 h in the absence of significant changes in the abundance of cyclin E and Cdk2. The CDK inhibitor p21 increased in mRNA and protein abundance and was present at increased levels in cyclin D1 and cyclin E complexes at times when their kinase activity was decreased. Increased p21 protein abundance was observed in another antiprogestin-sensitive cell line, BT 474, but not in two breast cancer cell lines insensitive to antiprogestins. These data suggest increased p21 abundance and concurrent inhibition of CDK activity as a mechanism for antiprogestin induction of growth arrest. Antiprogestin effects on proliferation were markedly reduced after ectopic expression of cyclin D1, indicating that inhibition of cyclin D1 function is a critical element in antiprogestin inhibition of proliferation. However, these data also implicate regulation of cyclin E function in antiprogestin regulation of cell cycle progression.

Cyclin D1 and estrogen receptor messenger RNA levels are positively correlated in primary breast cancer.

The CCND1 gene, encoding the cell cycle regulatory protein cyclin D1, maps to chromosome 11q13, a locus that is amplified in about 13% of breast cancers. Because several studies have indicated a relationship between 11q13 amplification and markers of phenotype including estrogen receptor (ER) status, we tested the relationship between CCND1 and ER gene expression in 364 primary breast cancers using Northern blot analysis. Seventy-three % of samples were positive for ER mRNA, and cyclin D1 mRNA levels in the ER-positive group were significantly higher than those in the ER-negative group (P = 0.0001). When the samples were divided into quartiles of cyclin D1 expression, 58% of samples were ER positive in the lowest quartile and 87% in the highest quartile. The tumors expressing the highest levels of cyclin D1 (7%) were all ER positive. Furthermore, ER mRNA levels in the half with lower cyclin D1 mRNA were significantly less than in the half with higher cyclin D1 levels (P = 0.0001). Using simple regression analysis, there was a significant positive correlation between cyclin D1 and ER mRNA levels in the total population (P = 0.0001). This study demonstrates that cyclin D1 mRNA and ER mRNA are positively correlated in primary breast cancer, but the functional relationship between these genes remains to be elucidated.

Expression of the cyclin-dependent kinase inhibitors p16INK4, p15INK4B and p21WAF1/CIP1 in human breast cancer.

Aberrant cyclin expression has been implicated in oncogenesis in a number of human cancers. Since altered function of regulators of cyclin-dependent kinase (CDK) activity other than cyclins, in particular CDK inhibitors, might play a similar role in oncogenesis, we examined the expression and regulation of the CDK inhibitors p16INK4, p15INK4B and p21WAF1/CIP1 in human breast cancer cell lines. Both the INK4 and INK4B genes were homozygously deleted in 3 cell lines, while INK4 alone was deleted in 2 cell lines. A further 2 cell lines displayed loss of an allele at this locus, and in 1 of these the remaining allele contained a mis-sense mutation within the coding region of the p16INK4 protein. The majority of cell lines examined, including 2 normal mammary epithelial cell strains, expressed low levels of INK4 mRNA and low or undetectable levels of INK4B mRNA. However, INK4 mRNA was expressed at high levels in 5 cell lines, and this was associated with deletion or inactivation of the retinoblastoma susceptibility gene product pRB but not with mutation of TP53. No deletions of the WAF1/CIP1 gene were observed, but WAF1/CIP1 mRNA levels were reduced in cell lines with TP53 mutation. Transfection of a p16INK4 expression vector into MDA-MB-231 cells lacking the INK4 gene failed to produce any p16INK4-expressing cell lines, suggesting that such cells were selected against in continuous culture. Despite the frequent deletion of INK4 in breast cancer cell lines, no evidence was obtained for INK4 deletions in DNA from 45 primary breast carcinomas. Thus, homozygous deletion of the INK4 gene appears to be a rare event in primary breast cancer.

Limitations of postextrasystolic potentiation in identifying ischemic myocardium.

To evaluate the usefulness of postextrasystolic potentiation in differentiating ischemic yet viable myocardium from infarcted myocardium, 20 dogs were chronically instrumented with left ventricular pressure gauges, left circumflex coronary artery flow probes, occluders, pacing wires, and ultrasonic segment-length transducers. Ten dogs had acute (1 h) coronary occlusions followed by reperfusion (4 days) and were then killed. Ten more dogs had more prolonged (1 mo) occlusions and were then killed. Timed premature ventricular contractions were induced, and the postextrasystolic beat was evaluated. In ischemic segments that were hypokinetic, postextrasystolic potentiation of shortening occurred in both groups. In ischemic segments that were akinetic or dyskinetic, potentiation of shortening did not occur in either group. Both groups showed recovery of shortening, and histologically normal myocardium was identified in the region between the segments in all animals. Thus, akinetic and dyskinetic segments did not show postextrasystolic potentiation of shortening, even though the tissue was viable and showed functional recovery. Failure to improve shortening after a premature ventricular beta in an ischemic segment does not necessarily indicate nonviable myocardium.

Influence of potassium cardioplegia versus ischemic arrest on regional left ventricular diastolic compliance in humans.

To compare the effects of hypothermic ischemic arrest versus hypothermic potassium cardioplegia, regional left ventricular performance was monitored in 20 adult male patients undergoing saphenous vein bypass operation. Twelve patients received ischemic arrest (Group 1), and 8 received potassium cardioplegia (Group 2). Groups 1 and 2 did not differ in left ventricular ejection fraction (0.62 versus 0.60), number of bypassed vessels (3.7 versus 3.4), mean cross-clamp time (75 versus 63 minutes), or mean cardiopulmonary bypass time (182 versus 170 minutes). Before cardiopulmonary bypass was begun, a pair of ultrasonic crystals was secured in the left ventricular anterior myocardium to measure segment motion and a micromanometer-tipped catheter was placed in the left ventricular chamber. All patients received a saphenous vein bypass graft to a vessel supplying the anterior left ventricular wall in the region of the ultrasonic crystals. Comparison of changes in systolic measurements revealed no significant differences between Groups 1 and 2. After saphenous vein bypass grafting, the left ventricular end-diastolic pressure (11.4 to 17.0 mm HG) and modulus of left ventricular segment stiffness (0.37 to 0.67, p less than 0.02) were elevated in Group 1 but no changes were observed in Group 2 (14.0 to 15.6 mm Hg, and 0.16 to 0.24, respectively). Compared with hypothermic ischemic arrest, hypothermic potassium cardioplegia is not associated with an increased left ventricular diastolic stiffness shortly after saphenous vein bypass grafting in humans.