E-Cadherin and EpCAM expression by NSCLC tumour cells associate with normal fibroblast activation through a pathway initiated by integrin αvß6 and maintained through TGFß signalling.

Fibroblasts in the tumour stroma (cancer-associated fibroblasts) influence tumour progression and response to therapeutics; little is known about the mechanisms through which the tumour cell co-opts a normal fibroblast. To study the activation of fibroblasts by tumour cells, a panel of non-small cell lung cancer (NSCLC) cell lines and normal human dermal fibroblasts were co-cultured. A subset of the NSCLC cells induced an activated cancer-associated fibroblast-like fibroblast phenotype defined by induction of fibroblast α-smooth muscle actin expression. Tumour cells that activated fibroblasts were associated with E-Cadherin and EpCAM expression and expression of integrin αvß6. Co-culture of activating tumour cells with fibroblasts resulted in induction of transcripts associated with tumour cell invasion and growth, TGFß1 and TGFBR1, SERPINE-1, BMP6, SPHK1 and MMP9. Fibroblast activation was inhibited by an αvß6/8 integrin blocking antibody (264RAD) and a small molecule inhibitor of the TGF-beta type I receptor activin-like kinase (ALK5) (SB431542), demonstrating that transactivation of the TGFß pathway initiates fibroblast activation. Both integrin and ALK5 antagonists inhibited initiation. Only ALK5 was effective when added after 3 days of co-culture. This suggests that although activation is αvß6-dependent, once fibroblasts are activated alternative TGFß pathway regulators maintain an activation loop. In co-culture activating cells had reduced sensitivity to selumetinib, AZD8931 and afatinib compared with mono-culture. In contrast, non-activating cells were insensitive to selumetinib and AZD8931 in both mono-culture and co-culture. In conclusion NSCLC cell lines, positive for E-Cadherin, EpCAM and αvß6 expression, activate normal fibroblasts through avß6/TGFß signalling in vitro, and influence both gene expression and response to therapeutic agents.

A human monoclonal antibody 264RAD targeting αvß6 integrin reduces tumour growth and metastasis, and modulates key biomarkers in vivo.

αvß6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvß6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvß6, and inhibits binding to all ligands including the latency-associated peptide of TGF-ß. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvß8, but not the integrins α5ß1, αvß3, αvß5 and α4ß1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvß6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-ß-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-ß by NCI-H358 tumour cells in a tumour cell-fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvß6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvß6 integrin, with some activity against αvß8 integrin, that reduces both tumour growth and metastasis.

Infusing liquid anaesthetics into a closed circuit.

Closed circuit anaesthesia offers the user many advantages but practical problems impede its widespread use. When conventional vaporizers are employed adequate amounts of agent cannot be delivered into a totally closed circuit during the early stages of an anaesthetic. Direct injection, or infusion, of liquid anaesthetic into the circuit overcomes this problem. The standard method for injecting agents directly into circuits is that described by Lowe and Ernst. Their system can be approximated to a series of constant rate infusions, as is frequently done for propofol, and forms the basis of our technique. For anaesthesia without nitrous oxide, liquid isoflurane should initially be infused into the circuit at a rate in ml/hr of 14 + 0.4 x weight in kg. After five minutes the infusion rate is reduced to 20% of this value. These rates are altered in the light of measured concentrations and clinical responses.

Structure of the HMG box motif in the B-domain of HMG1.

The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA-binding domains, each of approximately 80 amino acid residues, and an acidic C-terminal tail. Each folded domain represents an 'HMG box', a sequence motif recently recognized in certain sequence-specific DNA-binding proteins and which also occurs in abundant HMG1-like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA-binding structural motif. We have expressed the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H-NMR spectroscopy. There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues. Strikingly, the molecule is L-shaped, the angle of approximately 80 degrees between the two arms being defined by a cluster of conserved, predominantly aromatic, residues. The distinctive shape of the HMG box motif, which is distinct from hitherto characterized DNA-binding motifs, may be significant in relation to its recognition of four-way DNA junctions.

Analysis of the binding sites for the varicella-zoster virus gene 51 product within the viral origin of DNA replication.

The C-terminal 322 amino acids of the varicella-zoster virus (VZV) gene 51 product were expressed in Escherichia coli and shown to bind to specific DNA sequences within the VZV origin of DNA replication. The gene 51 product and its herpes simplex virus type 1 homolog (the UL9 protein) are capable of recognizing identical DNA sequences but the arrangement of binding sites within the origin regions of the two viruses differs. Three binding sites for the VZV gene 51 protein were identified within the VZV origin region and these lie in the same orientation and on the same side of the origin palindromic DNA sequence. DNA replication assays in transfected tissue culture cells demonstrated that the site closest to the palindrome is essential for origin activity whereas the most distal site is dispensable. The middle binding site may play an auxiliary role in DNA synthesis.

Two binding sites for the herpes simplex virus type 1 UL9 protein are required for efficient activity of the oriS replication origin.

Two sites within the short region origin of DNA replication (oriS) in herpes simplex virus type 1 (HSV-1) which bind the product of the UL9 gene have previously been identified. One of these sites (site I) contains an 11 bp sequence which is also present in oriS of varicella-zoster virus, and the other (site II) includes a related element differing in two positions. A third sequence (motif III), which lies close to binding site I, differs from the site I element at only a single position. We have deleted specifically each of these three 11 bp sequences from within functional copies of HSV-1 oriS and have examined the effects on origin activity and binding of the UL9 protein. Gel retardation analyses confirmed the important roles of the regions deleted from sites I and II in interacting with the UL9 protein. In transient replication assays, copies of oriS lacking the site I or II elements exhibited undetectable or residual (4 to 8%) activity respectively. The UL9 protein did not bind to motif III even in the absence of site I sequences, although removal of the motif III sequence caused a small reduction in oriS activity. A single base change which converted the sequence within binding site I to that of motif III was sufficient to abolish both the interaction of the UL9 gene product at this locus and the replicative ability of oriS. Therefore, interaction of the UL9 protein with binding site I is essential for origin activity, but the presence of binding site II is also required for efficient replication.

Binding of the herpes simplex virus type 1 UL9 gene product to an origin of viral DNA replication.

The binding of a herpes simplex virus type 1 (HSV-1) encoded polypeptide to a viral origin of DNA replication has been studied by using a gel retardation assay. Incubation of nuclear extract from HSV-1 infected cells with a labelled origin-containing fragment resulted in the formation of a specific retarded complex, the migration of which was further reduced in the presence of an antibody reactive with the UL9 gene product. Introduction of an additional copy of the UL9 gene, under the control of an immediate early (IE) promoter, conferred the ability to express origin binding activity at the non-permissive temperature upon an HSV-1 ts mutant blocked at the IE stage of infection. Endogenous or exogenous proteolytic activity revealed the presence of a relatively protease-resistant domain which retained sequence-specific DNA binding activity. The C-terminal 317 amino acids of the UL9 gene expressed as a fusion protein in Escherichia coli also bound to the origin. Our results demonstrate that the UL9 gene product binds to a viral origin and that sequence specific recognition and binding are specified by the C-terminal 37% of the polypeptide.

Human kappa-casein as a tumor marker: purification and properties.

To test the efficacy of kappa-casein as a marker of human malignancy, a protein human milk, ostensibly kappa-casein, has been purified and serum levels determined, by RIA, in normal subjects, breast cancer patients, and lactating women. The results do not support claims by other workers for this protein. Concurrent physicochemical characterization of the protein has shown that it is probably lactoferrin and this result casts some doubt on the earlier studies as it is a major, non-beta component of the casein fraction. It also indicates that caution should be exercised when homology between proteins is used as a guide to identity.