The VEGF receptor tyrosine kinase inhibitor, ZD6474, inhibits angiogenesis and affects microvascular architecture within an orthotopically implanted renal cell carcinoma.

The proangiogenic vascular endothelial growth factor-A (VEGF) is essential for the development of new tumor vessels. ZD6474 is a novel inhibitor of VEGF receptor-2 (VEGFR-2) tyrosine kinase activity, which also has additional activity against epidermal growth factor (EGF) receptor tyrosine kinase. The antitumor activity of different schedules of ZD6474 in a clinically relevant, metastasizing, murine renal cell carcinoma (RENCA) model was evaluated in this study. RENCA cells were inoculated into the left kidney of 24 mice (day 0). Daily ZD6474 (50 mg/kg p.o.) treatment was initiated 1 day or 10 days after tumor cell inoculation and continued until day 21. Following treatment, kidney weight and volume were assessed and blood vessel density determined by CD31 staining. Visible metastases in the lungs, spleen, and lymph nodes were quantified using a dissection microscope. In an additional study, animals were treated according to the same regimen and quantitative three-dimensional microvascular corrosion casting was performed to enable detailed assessment of the tumor vascular architecture. Therapy initiated on day 1 or day 10 resulted in a 79% and 59% reduction in primary tumor volume, a 79% and 60% reduction in the number of lung metastases, and a 58% and 59% reduction in vessel density of primary tumors compared with the control group, respectively. Corrosion casting proved a 5.4- and 3.2-fold lower vascular volume compared with untreated tumors, observations that paralleled with significant architectural alterations. In this RENCA model, ZD6474 was a highly active inhibitor of tumor angiogenesis, primary tumor growth and tumor metastasis.

ZD4190: an orally active inhibitor of vascular endothelial growth factor signaling with broad-spectrum antitumor efficacy.

There is evidence that vascular endothelial growth factor (VEGF) contributes to solid tumor growth through the promotion of both angiogenesis and tumor vascular permeability. To abrogate VEGF signaling, we developed a small molecular weight inhibitor of VEGF receptor tyrosine kinase (RTK) activity that was compatible with chronic oral administration. ZD4190, a substituted 4-anilinoquinazoline, is a potent inhibitor of KDR and Flt-1 RTK activity, and VEGF stimulated HUVEC proliferation in vitro. Chronic once-daily oral dosing of ZD4190 to young rats produced a dose-dependent increase in the femoral epiphyseal growth plate area, which may be attributed to the inhibition of VEGF signaling in vivo because vascular invasion of cartilage is a prerequisite to the process of ossification. Once-daily oral dosing of ZD4190 to mice bearing established (approximately 0.5 cm3) human tumor xenografts (breast, lung, prostate, and ovarian) elicited significant antitumor activity and at doses that would not be expected to have any direct antiproliferative effect on tumor cells. Prolonged tumor cytostasis was further demonstrated in a PC-3 xenograft model with 10 weeks of ZD4190 dosing, and upon withdrawal of therapy, tumor growth resumed after a short delay. These observations are entirely consistent with the proposed mode of action. ZD4190 is one of a series of VEGF RTK inhibitors that may have utility in the treatment of a range of histologically diverse solid tumor types.

Design and structure-activity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors.

A series of substituted 4-anilinoquinazolines and related compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptor (Flt and KDR) tyrosine kinase activity. Enzyme screening indicated that a narrow structure-activity relationship (SAR) existed for the bicyclic ring system, with quinazolines, quinolines, and cinnolines having activity and with quinazolines and quinolines generally being preferred. Substitution of the aniline was investigated and clearly indicated that small lipophilic substituents such as halogens or methyl were preferred at the C-4' position. Small substituents such as hydrogen and fluorine are preferred at the C-2' position. Introduction of a hydroxyl group at the meta position of the aniline produced the most potent inhibitors of Flt and KDR tyrosine kinases activity with IC(50) values in the nanomolar range (e.g. 10, 12, 13, 16, and 18). Investigation of the quinazoline C-6 and C-7 positions indicates that a large range of substituents are tolerated at C-7, whereas variation at the C-6 is more restricted. At C-7, neutral, basic, and heteroaromatic side chains led to very potent compounds, as illustrated by the methoxyethoxy derivative 13 (IC(50) < 2 nM). Our inhibitors proved to be very selective inhibitors of Flt and KDR tyrosine kinase activity when compared to that associated with the FGF receptor (50- to 3800-fold). Observed enzyme profiles translated well with respect to potency and selectivity for inhibition of growth factor stimulated proliferation of human umbilical vein endothelial cells (HUVECs). Oral administration of selected compounds to mice produced total plasma levels 6 h after dosing of between 3 and 49 microM. In vivo efficacy was demonstrated in a rat uterine oedema assay where significant activity was achieved at 60 mg/kg with the meta hydroxy anilinoquinazoline 10. Inhibition of growth of human tumors in athymic mice has also been demonstrated: compound 34 inhibited the growth of established Calu-6 lung carcinoma xenograft by 75% (P < 0.001, one tailed t-test) following daily oral administration of 100 mg/kg for 21 days.

Walking, cloning, and mapping with YACs in 3q27: localization of five ESTs including three members of the cystatin gene family and identification of CpG islands.

Using yeast artificial chromosomes, we have generated a high-resolution physical map for 2.7 Mb of human chromosomal region 3q27. The YAC clones group into three contigs, one of which has also been linked to the CEPH YAC contig map of human chromosome 3. Fluorescence in sity hybridization has been used to order the contigs on the chromosome and to estimate the distance between them. Expressed sequence tags for five genes, including three members of the cystatin gene family and a gene thought to be involved in B-cell non-Hodgkin lymphoma, have been placed within the YAC contigs, and 12 putative CpG islands have been identified. These YACs provide a useful resource to complete the physical mapping of 3q27 and to begin identification and characterization of further genes that are located there.

Yeast artificial chromosome cloning of the beta-catenin locus on human chromosome 3p21-22.

beta-Catenin has emerged as an important component of the adherens junctions between epithelial cells. As a result of studies of its interaction with the APC gene product, it has been implicated in the development of colorectal cancer. alpha-Catenin, beta-catenin, E-cadherin and APC appear to mediate contact inhibition in epithelia. As part of the study of the organization of the beta-catenin gene, we have isolated yeast artificial chromosomes (YACs) to characterize its intron/exon structure. YAC fluorescence in situ hybridization analysis and polymerase chain reaction analysis of somatic cell hybrid DNAs show that beta-catenin maps in the 3p21-22 region, the location of tumour-suppressor genes deleted in small-cell lung cancer (SCLC) and other disorders. beta-Catenin YACs will provide a source of microsatellite markers useful in loss of heterozygosity studies to assess the importance of beta-catenin deletions in SCLC.

No evidence for involvement of type 1 collagen structural genes in 'genetic predisposition' to alcoholic cirrhosis.

Type 1 collagen is the predominant collagen in cirrhotic livers. Each type 1 collagen molecule contains three subunits, two are identical (the alpha 1 chains) and the sequence of the third (alpha 2) is very similar. They are encoded at the non-synthenic loci, COL1A1 and COL1A2 and restriction site dimorphisms have been described at each locus. Genetic factors have been invoked as a basis for increased susceptibility to alcoholic cirrhosis. One hypothesis is that genetically determined differences in type 1 collagen may be involved in this predisposition. We have examined this by analysing restriction fragment length polymorphisms at each type 1 collagen locus in leucocyte DNA from 56 unrelated patients with alcoholic cirrhosis and 74 local unrelated healthy controls. Based on the presence or absence of these restriction site dimorphisms four possible haplotypes were generated at COL1A1 and COL1A2. We found no significant difference in allele frequencies between alcoholic cirrhotics and controls and, unlike a previous small study, we found no particular haplotype of either gene was associated with alcoholic cirrhosis. Our study provides no evidence for involvement of type 1 collagen structural genes in a genetic predisposition to cirrhosis in alcoholics.

Segregation of structural collagen genes in adolescent idiopathic scoliosis.

The etiology of idiopathic scoliosis remains unknown. The condition results in a characteristic deformity of the spine and surrounding tissues. Both Types I and II collagen are important constituents of the affected tissues, and thus defective collagens are reasonable candidates for the primary abnormality in adolescent idiopathic scoliosis (AIS). Direct analyses of the amount and solubility of collagen have revealed differences between normal individuals and those with AIS. However, these changes may be secondary to the mechanical effects of the spinal deformity. Segregation analysis was done of genetic markers linked to the structural genes encoding Types I and II collagen to test these candidate loci in four pedigrees with dominantly inherited AIS. In one pedigree, markers linked to both of the Type I collagen loci (COL1A1 and COL1A2) were found to be inherited independently of the abnormal phenotype. Two pedigrees were discordant at one of the Type I loci. The condition also segregated independently of the locus for Type II collagen (COL2A1) in three pedigrees. This is evidence against idiopathic scoliosis generally being caused by mutations in the Types I and II collagen genes.

Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16.

Chromosome 21 has often been used as a model system for the development of genome mapping and cloning strategies in humans. In this report methods for systematic chromosome walking, cloning, and mapping are exemplified in the construction of a 1.5-Mb yeast artificial chromosome (YAC) contig encompassing and extending 400 kb beyond each of the genetic loci D21S13 and D21S16. Isolation of insert-terminal sequences from YACs in this contig provides a set of closely spaced physical markers. These have been used to generate a long-range genomic restriction map.

Segregation analysis of the structural genes of the major fibrillar collagens provides further evidence of molecular heterogeneity in type II Ehlers-Danlos syndrome.

Type II Ehlers-Danlos syndrome (EDS) is one of a group of disorders characterized by striking abnormalities of the soft connective tissues. The major fibrillar collagens (types I and III) found in these tissues have important stress-bearing functions and abnormal collagen could therefore account for the clinical features of this condition. We have used a number of restriction site dimorphisms, tightly linked to the structural genes of type I collagen (COL1A1 COL1A2) and type III collagen (COL3A1), to investigate the segregation of corresponding alleles in three pedigrees in which type II EDS was clearly inherited as a dominant trait. Discordant segregation of all three collagen genes was seen in a large pedigree that included 17 affected individuals with the typical phenotype of type II EDS. Thus mutations in neither type I nor type III collagen genes were responsible for the disease in this family. In a second small pedigree discordant segregation of the disease with both type I collagen loci was observed while the concordant segregation seen at COL3A1 could easily have arisen by chance (P = 0.5). The third pedigree was uninformative at all three collagen loci because of inability to discriminate between the parental alleles. These results suggest that there may be molecular heterogeneity of type II EDS since abnormalities of type I collagen have been described in other individuals phenotypically similar to those in our study.

A yeast artificial chromosome contig encompassing the cystic fibrosis locus.

The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.

Segregation of all four major fibrillar collagen genes in the Marfan syndrome.

Linkage markers at or close to the genes encoding the three major fibrillar collagens were used to analyze the segregation of these loci in six pedigrees with dominantly inherited Marfan syndrome. Four pedigrees were discordant at one of the Type I collagen loci (COL1A2), and, of these, two were discordant at the other Type I locus (COL1A1). The Marfan syndrome also segregated independently of the structural loci for Type II and Type III collagen in these two families. This is evidence against the Marfan syndrome being, in general, due to mutations in the major fibrillar collagen genes.

Collagenase secretion by human breast neoplasms: a clinicopathologic investigation.

Evidence is presented that biopsy specimens from fibroadenomas, benign cystic lesions, and carcinomas of the human breast can produce in organ culture a neutral protease capable of digesting type I collagen. This enzyme activity, measured with the use of a radioactive release assay, was characterized as true vertebrate collagenase and occurred in both active and latent (requiring trypsin activation) forms. For the two types of benign breast lesion studied, collagenase secretion was significantly higher from fibroadenomas than from benign cystic tissue. Breast carcinomas, however, exhibited a wide quantitative spectrum of collagenase secretion, encompassing the extremes observed for the benign lesions and showing no correlation with histologic type. These results, while providing a plausible mechanism for the marked collagen degradation seen in disseminating neoplasms, demonstrate that high collagenase secretory activity is not pathognomonic of invasive behavior. The findings, however, indicate disordered regulation of collagenase activity in malignant tumors.

Lethal osteogenesis imperfecta and a collagen gene deletion. Length polymorphism provides an alternative explanation.

A 300 base pair deletion near the 3'-end of the gene encoding Type II (cartilage) collagen has been implicated in the pathogenesis of perinatal lethal osteogenesis imperfecta. We have found similar deletions occurring at a high frequency in normal Asian Indian and West Indian populations generated by a length polymorphism just beyond the 3'-end of the gene. We suggest that this polymorphism provides an alternative explanation of the original results.

Temperature-dependent elaboration of collagenase by the renal adenocarcinoma of the leopard frog, Rana pipiens.

Naturally occurring renal adenocarcinoma in North American leopard frogs, Rana pipiens, metastasize frequently (77%) when these ectothermic animals are kept in a warm environment but not when they are kept cold. We have found that explants of these tumors secrete collagenase, an enzyme capable of dissolving connective tissue fibers and found previously to be closely correlated with metastatic colony-forming capability of murine mammary tumors, and that the amount released sequentially rises and falls as the ambient temperature is shifted between metastasis-permissive and -inhibitory levels. In contrast, normal frog renal tissue has low collagenase output, unaffected by temperature changes.

Degradation of basement membrane collagens by metalloproteases released by human, murine and amphibian tumours.

In this investigation it has been found that naturally-occurring (i.e. indigenous, not transplanted) tumours of diverse organs in a spectrum of vertebrates from frogs to man can secrete enzymes which degrade basement membrane collagens (type IV and V). The enzymes are inhibited by chelating agents (EDTA) but not by other protease antagonists and are, therefore, specific metalloproteases. Individual tumours do not necessarily secrete collagenases active against all collagen types (I, IV and V) and release of these different enzymes does not, therefore, appear to be coordinated. These biochemical findings support those reported for serially transplanted tumour cell lines and provide a plausible mechanism for the destruction of basement membranes and stromal collagen fibres observed morphologically in tumour spread.