Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements

Despite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumour blocks. Second, hypoxic development is quantified in adjacent sections using immunohistochemical detection of a fluorescently conjugated monoclonal antibody (ELK3-51) to a nitroheterocyclic hypoxia marker (EF5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent (Hoechst 33342 or DiOC7(3)) and immunohistological (PECAM-1/CD31) stains is used to define the anatomical vascular densities and the fraction of blood vessels containing flow. Using a computer-interfaced microscope stage, image analysis software and a 3-CCD colour video camera, multiple images are digitized, combined to form a photo-montage and revisited after each of the three staining protocols. By applying image registration techniques, the spatial distribution of HbO2 saturations is matched to corresponding hypoxic marker intensities in adjacent sections. This permits vascular configuration to be related to oxygen availability and allows the hypoxic marker intensities to be quantitated in situ. © 1999 Cancer Research Campaig

Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis

The evolution of colorectal cancer suggests the involvement of many genes. We performed insertional mutagenesis with the Sleeping Beauty (SB) transposon system in mice carrying germline or somatic Apc mutation. Analysis of common insertion sites (CISs) isolated from 446 tumors revealed many hundreds of candidate cancer drivers. Comparison to human datasets suggested that 234 CIS genes are also deregulated in human colorectal cancers. 183 CIS genes are candidate Wnt targets, and 20 are shown to be novel modifiers of canonical Wnt signaling. We also identified gene mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included members of the FGF signaling cascade. Some 70 genes showed pairwise co-occurrence clustering into 38 sub-networks that may regulate tumor development

A variant of t-PA (T103N, KHRR 296-299 AAAA) that, by bolus, has increased potency and decreased systemic activation of plasminogen

In the accompanying paper, we reported that the properties of decreased plasma clearance rate, increased fibrin specificity, and resistance to inactivation by PAI-1 could be effectively combined in the t-PA variant T103N, KHRR 296-299 AAAA. In the current study we evaluated the in vivo efficacy of this variant as well as variants containing the individual mutations T103N and KHRR 296-299 AAAA. Plasma clearance and in vivo lysis of whole blood and platelet-rich clots were determined in a rabbit arterio-venous shunt model. The T103N containing variants were administered as an intravenous (i.v.) bolus. KHRR 296-299 AAAA and t-PA were infused i.v. over 90 min. The clearance rate of the KHRR 296-299 AAAA variant was similar to t-PA. However, the clearance of the T103N and T103N, KHRR 296-299 AAAA variants were 8 and 6-fold reduced, respectively. Potency of the variants relative to t-PA on whole blood clots ranged from 0.9 (T103N, KHRR 296-299 AAAA) to 1.7 (T103N). Relative potency on platelet-rich clots ranged from 2.4 (T103N) to 4.2 (T103N, KHRR 296-299 AAAA). Fibrinogen concentrations in rabbits 120 min after dosing with a 2.5 mg/kg bolus were: 24, 16, 82, and 77% of initial for t-PA; T103N; KHRR 296-299 AAAA; and T103N, KHRR 296-299 AAAA treatment groups, respectively. These results suggest that the T103N, KHRR 296-299 AAAA variant of t-PA, given as a bolus, could result in greater efficacy, particularly on refractory platelet-rich clots, without inducing the severe systemic lytic state produced by a bolus of a less fibrin specific variant

High resolution analysis of functional determinants on human tissue-type plasminogen activator

Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions

Involvement of residues 296-299 in the enzymatic activity of tissue-type plasminogen activator

The tetra-alanine substitution variant KHRR 296-299 AAAA of tissue-type plasminogen activator (t-PA) was previously shown to have enhanced fibrin specificity and enhanced activity in the presence of fibrin compared with the wild-type form of the molecule. The structural requirements for these alterations in enzymatic activity were investigated by constructing several amino acid substitution variants at each of the positions from 296 to 299 and evaluating their activities under a variety of conditions. Effects on plasminogen activator activity were common among the point mutants at positions 296-299; nearly all had a phenotype similar to the KHRR 296-299 AAAA variant. The greatest effects on enzymatic function were found with multiple substitution variants, but some single charge reversals and proline substitutions had substantial effects. The enhanced fibrin specificity of KHRR 296-299 AAAA t-PA results in less fibrinogenolysis than seen with wild-type t-PA. Approximately four times greater concentration of KHRR 296-299 AAAA compared with wild-type t-PA was required to consume 50% of the fibrinogen in human plasma