Tumor-host interactions

A number of malignant tumors interact with the host to cause a syndrome of cachexia, characterized by extensive loss of adipose tissue and skeletal muscle mass, but with preservation of proteins in visceral tissues. Although anorexia is frequently present, the body composition changes in cancer cachexia cannot be explained by nutritional deprivation alone. Loss of skeletal muscle mass is a result of depression in protein synthesis and an increase in protein degradation. The main degradative pathway that has been found to have increased expression and activity in the skeletal muscle of cachectic patients is the ubiquitin-proteasome proteolytic pathway. Cachexia-inducing tumors produce catabolic factors such as proteolysis-inducing factor (PIF), a 24 kDa sulfated glycoprotein, which inhibit protein synthesis and stimulate degradation of intracellular proteins in skeletal muscle by inducing an increased expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. While the oligosaccharide chains in PIF are required to initiate protein degradation the central polypeptide core may act as a growth and survival factor. Only cachexia-inducing tumors are capable of elaborating fully glycosylated PIF, and the selectivity of production possibly rests with the acquisition of the necessary glycosylating enzymes, rather than expressing the gene for the polypeptide core. Loss of adipose tissue is probably the result of an increase in catabolism rather than a defect in anabolism. A lipid mobilizing factor (LMF), identical with the plasma protein Zn-α2-glycoprotein (ZAG) is found in the urine of cachectic cancer patients and is produced by tumors causing a decrease in carcass lipid. LMF causes triglyceride hydrolysis in adipose tissue through a cyclic AMP-mediated process by interaction with a β3-adrenoreceptor. Thus, by producing circulating factors certain malignant tumors are able to interfere with host metabolism even without metastasis to that particular site. © 2004 Wiley-Liss, Inc.

Apoptotic cell‐associated ICAM‐3 in the phagocytic removal of apoptotic cells

Apoptosis is a highly controlled cell death programme that culminates in the exposure of molecular ‘flags’ at the dying cell surface that permit recognition and removal by viable phagocytes. Failure to efficiently remove dying cells can lead to devastating inflammatory and autoimmune disorders. The molecular mechanisms underlying apoptotic cell surface changes are poorly understood. Our previous work has shown an apoptosis-associated functional change in ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) resulting in a molecular ‘flag’ to mediate corpse removal. Here we detail apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. We show ICAM-3 functions to tether apoptotic leukocytes to macrophages via an undefined receptor. Though CD14 has been suggested as a possible receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Furthermore, we demonstrate leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates reduced cell volume throughout apoptosis. This loss of ICAM-3 occurs via shedding of ICAM-3 in microparticles (‘apoptotic bodies’). Such microparticles are potent chemoattractants for macrophages. Notably, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. These data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of apoptosis.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in cell disassembly and the exposure of ‘flags’ at the dying cell surface that permit recognition and removal by viable cells (phagocytes). Efficient phagocytic removal of dying cells is essential to prevent inflammatory and autoimmune disorders. Relatively little is known of the molecular mechanisms underlying changes at the apoptotic cell surface. We have previously shown that ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) undergoes a change of function as cells die so that it acts as a molecular ‘flag’ to mediate corpse removal. Our work seeks to characterise apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. Here we extend earlier studies to show that apoptotic cell-associated ICAM-3 functions, at least minimally, to tether apoptotic leukocytes to macrophages via an undefined receptor. Whilst CD14 has been suggested as a possible innate immune receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Our data additionally indicate, that during apoptosis, leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates with a reduction in cell volume. This reduction in ICAM-3 is explained by cell surface shedding of microparticles (‘apoptotic bodies’) that contain ICAM-3. Such microparticles, released from apoptotic leukocytes, are strongly chemoattractive for macrophages. In addition, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. Taken together these data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of leukocyte apoptosis.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in cell disassembly and the exposure of ‘flags’ at the dying cell surface that permit recognition and removal by viable cells (phagocytes). Efficient phagocytic removal of dying cells is essential to prevent inflammatory and autoimmune disorders. Relatively little is known of the molecular mechanisms underlying changes at the apoptotic cell surface. We have previously shown that ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) undergoes a change of function as cells die so that it acts as a molecular ‘flag’ to mediate corpse removal. Our work seeks to characterise apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. Here we extend earlier studies to show that apoptotic cell-associated ICAM-3 functions, at least minimally, to tether apoptotic leukocytes to macrophages via an undefined receptor. Whilst CD14 has been suggested as a possible innate immune receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Our data additionally indicate, that during apoptosis, leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates with a reduction in cell volume. This reduction in ICAM-3 is explained by cell surface shedding of microparticles (‘apoptotic bodies’) that contain ICAM-3. Such microparticles, released from apoptotic leukocytes, are strongly chemoattractive for macrophages. In addition, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. Taken together these data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of leukocyte apoptosis.

Purification and characterisation of two cancer cachexia factors

Cachexia is a wasting phenomenon that often accompanies malignant disease. Its manifestation is associated with shortened survival and reduced responsiveness to anti-tumour therapy and as yet there is no established, effective amelioratory treatment. The MAC 16 model of cancer cachexia has been shown by many studies to closely mirror the human condition. Thus, cachexia is mediated by the presence of a small, slow-growing solid tumour that is mainly resistant to chemotherapy. In addition, the condition is largely attributable to aberrations in metabolic processes, while weight loss due to anorexia is negligible. Cachexia induced by the MAC 16 tumour, has been shown to be mediated by the production of tumour-derived circulatory catabolic factors, and the further elucidation of the structure of these molecules contributes towards the main content of this report. Thus, a factor with in vitro lipid-mobilising activity has been purified from the MAC 16 tumour, and has been found to have similarities to tumour-derived lipolytic factors published to date. Further work demonstrated that this factor was also purifiable from the urine of a patient with pancreatic cancer, and that it was capable of inducing weight loss in non tumour-bearing mice. Sequence analysis of the homogeneous material revealed an identity to Zn-α-2-glycoprotein, the significance of which is discussed. An additional factor, first detected as a result of its specific reactivity with a monoclonal antibody produced by fusion of splenocytes from MAC 16 tumour-bearing mice with mouse BALB/c myeloma cells, was identified as a co-purificant during studies to isolate the lipolytic factor. Subsequent purification of this material to homogeneity resulted in the determination of 18 of the N-terminal amino acids and revealed the highly glycosylated nature of its structure. Thus, this material (P24) was found to have an apparent molecular mass of 24kD of which 2kD was due to protein, while the remainder (92%) was due to the presence of carbohydrate groups. Sequence analysis of the protein core of P24 revealed an identity with Streptococcal pre-absorbing antigen (PA-Ag) in 11 of the amino acids, and the significance of this is discussed. P24 was shown to induce muscle protein breakdown in vitro and to induce cachexia in vivo, as measured by the depletion of fat (29%) and muscle (14%) tissue in the absence of a reduction of food and water intake. Further studies revealed that the same material was purifiable from the urine of patients with pancreatic cancer and was found to be detectable in the urine of cancer patients with weight loss greater than l.Skg/month. Thus, cachexia induced by the MAC 16 tumour in mice and by malignant disease in humans may be induced by similar mediators. Attempts to isolate the gene for P24 using information provided by the N-terminal protein sequence were unsuccessful. This was probably due to the low abundance o[ the material, as determined by protein purification studies; and the nature of the amino acids of the N-terminal sequence, which conferred a high degree o[ degeneracy to the oligonucleotides designed for the polymerase chain reaction.

Attenuation of muscle atrophy by an N-terminal peptide of the receptor for proteolysis-inducing factor (PIF)

Background: Atrophy of skeletal muscle in cancer cachexia has been attributed to a tumour-produced highly glycosylated peptide called proteolysis-inducing factor (PIF). The action of PIF is mediated through a high-affinity membrane receptor in muscle. This study investigates the ability of peptides derived from the 20 N-terminal amino acids of the receptor to neutralise PIF action both in vitro and in vivo. Methods: Proteolysis-inducing factor was purified from the MAC16 tumour using an initial pronase digestion, followed by binding on DEAE cellulose, and the pronase was inactivated by heating to 80°C, before purification of the PIF using affinity chromatography. In vitro studies were carried out using C2C12 murine myotubes, while in vivo studies employed mice bearing the cachexia-inducing MAC16 tumour. Results: The process resulted in almost a 23?000-fold purification of PIF, but with a recovery of only 0.004%. Both the D- and L-forms of the 20mer peptide attenuated PIF-induced protein degradation in vitro through the ubiquitin-proteosome proteolytic pathway and increased expression of myosin. In vivo studies showed that neither the D- nor the L-peptides significantly attenuated weight loss, although the D-peptide did show a tendency to increase lean body mass. Conclusion: These results suggest that the peptides may be too hydrophilic to be used as therapeutic agents, but confirm the importance of the receptor in the action of the PIF on muscle protein degradation.

Selective in vitro glycosylation of recombinant proteins:semi-synthesis of novel homogeneous glycoforms of human erythropoietin

Background: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure–function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins

Characterisation of ICAM-3 and extracellular vesicles in the clearance of apoptotic cells

Apoptotic cell clearance by phagocytes is a vital part of programmed cell death that prevents dying cells from undergoing necrosis which may lead to inflammatory and autoimmune disorders. Apoptotic cells (AC) are removed by phagocytes, in a process that involves 'find me' and 'eat me' signals that facilitate the synapsing and engulfment of cell corpses. Extracellular vesicles (EV) are shed during apoptosis and promote phagocyte recruitment. Binding of AC is achieved by multiple ligand-receptor interactions. One interesting AC associated ligand is ICAM-3, a highly glycosylated adhesion molecule of the IgSF family, expressed on human leukocytes. On viable cells ICAM-3 participates in initiating immune responses, whereas on AC we show it attracts phagocytes through EV and aids in the binding of AC to the phagocytes. This project aims to characterize the role of ICAM-3 and EV in the clearance of AC and to identify the mechanisms that underlie their function in apoptotic cell clearance. Human B cells induced to apoptosis by UV irradiation were observed during their progression from viable to apoptotic via flow cytometry. The involvement of ICAM-3 in mediating interaction between AC and MØ was assessed. The ability of ICAM3 on EV to mediate chemoattraction was observed using chemotaxis assays. Additionally the anti-inflammatory effect was assessed using LPS-induced TNF-α production that suggested it may have anti-inflammatory effects. Future work in this project will assess the role of ICAM3 on EV from different phases of apoptosis to exert functional effects both in vitro and in vivo.