Cancer-associated adipocytes promote the invasion and metastasis in breast cancer through LIF/CXCLs positive feedback loop

Cancer-associated adipocytes (CAAs), which are adipocytes transformed by cancer cells, are of great importance in promoting the progression of breast cancer. However, the underlying mechanisms involved in the crosstalk between cancer cells and adipocytes are still unknown. Here we report that CAAs and breast cancer cells communicate with each other by secreting the cytokines leukemia inhibitory factor (LIF) and C-X-C subfamily chemokines (CXCLs), respectively. LIF is a pro-inflammatory cytokine secreted by CAAs, which promotes migration and invasion of breast cancer cells via the Stat3 signaling pathway. The activation of Stat3 induced the secretion of glutamic acid-leucine-arginine (ELR) motif CXCLs (CXCL1, CXCL2, CXCL3 and CXCL8) in tumor cells. Interestingly, CXCLs in turn activated the ERK1/2/NF-κB/Stat3 signaling cascade to promote the expression of LIF in CAAs. In clinical breast cancer pathology samples, the up-regulation of LIF in paracancerous adipose tissue was positively correlated with the activation of Stat3 in breast cancer. Furthermore, we verified that adipocytes enhanced lung metastasis of breast cancer cells, and the combination of EC330 (targeting LIF) and SB225002 (targeting C-X-C motility chemokine receptor 2 (CXCR2)) significantly reduced lung metastasis of breast cancer cells in vivo. Our findings reveal that the interaction of adipocytes with breast cancer cells depends on a positive feedback loop between the cytokines LIF and CXCLs, which promotes breast cancer invasion and metastasis.     

The mechanism of PAK activation. Autophosphorylation events in both regulatory and kinase domains control activity

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both alpha- and betaPAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK N-terminal regulatory domain play no direct role in activation, a phosphorylation of alphaPAK serine 144 or betaPAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck.     

The role of B-cell-specific activator protein in the response of malignant B-1 cells to LPS

Chronic lymphocytic leukemia (CLL) results from the uncontrolled proliferation and accumulation of B-1 cells, many of which demonstrate self-reactivity. The response of B-1 cells to mitogen after undergoing malignant transformation is still unclear. Using our established malignant B-1 cell lines derived from the NZB murine model of human CLL, we investigated the response of malignant B-1 cells to the mitogen LPS. Interestingly, these malignant B-1 cells proliferated initially, but the proliferation rate decreased after a 48-h transition. Prolonged LPS treatment induced apoptosis and pathological differentiation. We studied possible underlying molecular mechanisms and found that the level of the DNA binding protein BSAP (B-cell-specific activator protein) was upregulated by LPS at the initial activation stage, followed by an increase in the apoptotic factor caspase-3 (CPP32) at 48 h and a subsequent decrease of BSAP at 72 h. The pathological differentiation induced by LPS was partially prevented by treatment with antisense BSAP. This study indicates that malignant B-1 cells could be driven to apoptosis and pathological differentiation when activated by the mitogen LPS, and BSAP may be an important factor in regulating these responses.     

Free and conjugated benzoic acid in tobacco plants and cell cultures. Induced accumulation upon elicitation of defense responses and role as salicylic acid precursors

Salicylic acid (SA) is a key endogenous component of local and systemic disease resistance in plants. In this study, we investigated the role of benzoic acid (BA) as precursor of SA biosynthesis in tobacco (Nicotiana tabacum cv Samsun NN) plants undergoing a hypersensitive response following infection with tobacco mosaic virus or in tobacco cell suspensions elicited with beta-megaspermin, an elicitor from Phytophthora megasperma. We found a small pool of conjugated BA in healthy leaves and untreated cell suspensions of tobacco, whereas free BA levels were barely detectable. Infection of plants with tobacco mosaic virus or elicitation of cells led to a rapid de novo synthesis and accumulation of conjugated BA, whereas free BA was weakly induced. In presence of diphenylene iodonium, an inhibitor of superoxide anion formation, SA accumulation was abolished in elicited cells and much higher BA levels were concomitantly induced, mainly as a conjugated form. Furthermore, piperonylic acid, an inhibitor of cinnamate-4-hydroxylase was used as a powerful tool to redirect the metabolic flow from the main phenylpropanoid pathway into the SA biosynthetic branch. Under these conditions, in vivo labeling and radioisotope dilution experiments with [(14)C]trans-cinnamic acid as precursor clearly indicated that the free form of BA produced in elicited tobacco cells is not the major precursor of SA biosynthesis. The main conjugated form of BA accumulating after elicitation of tobacco cells was identified for the first time as benzoyl-glucose. Our data point to the likely role of conjugated forms of BA in SA biosynthesis.     

Ribosomal DNA Intergenic Spacer of the Swimming Crab, Charybdis japonica

We have determined the full sequence of the ribosomal DNA intergenic spacer (IGS) of the swimming crab, Charybdis japonica, by long PCR for the first time in crustacean decapods. The IGS is 5376 bp long and contains two nonrepetitive regions separated by one long repetitive region, which is composed mainly of four subrepeats (subrepeats I, II, III, and IV). Subrepeat I contains nine copies of a 60-bp repeat unit, in which two similar repeat types (60 bp-a and 60 bp-b) occur alternatively. Subrepeat II consists of nine successive repeat units with a consensus sequence length of 142 bp. Subrepeat III consists of seven copies of another 60-bp repeat unit (60 bp-c) whose sequence is complementary to that of subrepeat I. Immediately downstream of subrepeat III is subrepeat IV, consisting of three copies of a 391-bp repeat unit. Based on comparative analysis among the subrepeats and repeat units, a possible evolutionary process responsible for the formation of the repetitive region is inferred, which involves the duplication of a 60-bp subrepeat unit (60 bp-c) as a prototype. Received: 13 April 1999 / Accepted: 2 August 1999     

Evidence for a regulatory role of cholesterol superlattices in the hydrolytic activity of secretory phospholipase A2 in lipid membranes

We have conducted a detailed study of the effect of membrane cholesterol content on the initial hydrolytic activity of Crotalus durissus terrificus venom phospholipase A2 (sPLA2) in large unilamellar vesicles of cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and cholesterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC) at 37 degrees C. The activity was monitored by using the acrylodan-labeled intestinal fatty acid binding protein and HPLC. In contrast to conventional approaches, we have used small cholesterol concentration increments ( approximately 0.3-1.0 mol %) over a wide concentration range (e.g., 13-54 mol % cholesterol). In both membrane systems examined, the initial hydrolytic activity of sPLA2 is found to change with cholesterol content in an alternating manner. The activity reaches a local minimum when the membrane cholesterol content is at or near the critical cholesterol mole fractions (e.g., 14.3, 15.4, 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol) predicted for cholesterol regularly distributed in either hexagonal or centered rectangular superlattices. According to the sterol regular distribution model [Chong, P. L.-G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10069-10073; Liu et al. (1997) Biophys. J. 72, 2243-2254], the extent of lipid superlattices is maximal at the critical cholesterol mole fractions, at which the membrane free volume is minimal. Thus, our present data can be taken to indicate that the initial hydrolytic activity of sPLA2 is governed by the extent of cholesterol superlattice. These data provide the first functional evidence for the formation of cholesterol superlattices in both saturated (e.g., DMPC) and unsaturated (e.g., POPC) liquid-crystalline phospholipid bilayers. The data also illustrate the functional importance of cholesterol superlattice and demonstrate a new type of regulation of sPLA2. Furthermore, upon binding to cholesterol/POPC large unilamellar vesicles, the intrinsic fluorescence intensity of sPLA2 shows an alternating variation with cholesterol content, exhibiting a minimum at the critical cholesterol mole fractions. This result suggests that either the number of sPLA2 bound to lipid vesicles or the conformation of membrane-bound sPLA2 or both vary with the extent of the cholesterol superlattice in the plane of the membrane.     

Site-specific characterization of the N-linked glycans of murine prion protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions

The murine prion protein PrP gene encodes a protein of 254 amino acids with two consensus sites for Asn-linked glycosylation at codons 180 and 196. A partial site-specific study of the N-linked glycans from hamster PrP has previously been carried out by mass spectrometry [Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prusiner, S. B. (1993) Biochemistry 32, 1991-2002] and revealed that the glycosylation at Asn-181 (equivalent to mouse 180) is heterogeneous, comprising over 30 glycoforms. The identification of the glycosylated peptide spanning Asn-197 was not reported. Recent technical advances in electrospray mass spectrometry now provide the sensitivity to detect low femtomole quantities of glycopeptides with >5000 mass resolution and 30 ppm mass measurement [Medzihradszky, K. F., Besman, M. J., and Burlingame, A. L. (1998) Rapid Commun. Mass Spectrom. 12, 472-478]. This performance coupled with stepwise exoglycosidase digestion has been employed to establish the differential nature of the structural complexity (glycoforms) of the glycans at Asn-180 and Asn-196 from a single strain infected with the ME7 strain. Some sixty structures have been found characterized by neutral and sialylated bi-, tri-, and tetraantennary complex-type bearing outer-arm alpha(1-3)-fucosylation (the Lewisx and sialyl-Lewisx epitopes), core alpha(1,6) fucosylation, and the presence of terminal HexNAc residues. The Lewisx trisaccharide is the major nonreducing structure at Asn-180, and significant amounts of both Lewisx and sialyl Lewisx epitopes are observed at Asn-196. The abundance of the Lewisx and sialyl Lewisx epitopes on murine PrPSc may indicate a role for these structures in the normal function of PrPC or the pathophysiology of PrPSc.