PubMed Health⌕ Search

Biomedical subjects

G C Tucker

Publications and source records attributed to G C Tucker.

At least 19 recordsLinked to original sources
PubMed2002

The role of the matrix metalloproteinases during in vitro vessel formation.

Matrix metalloproteinases (MMPs) constitute a large family of extracellular matrix degrading proteases implicated in a number of physiological and pathological processes, including angiogenesis. However, the relative importance of the individual MMPs in vessel formation is poorly understood. Using the three-dimensional rat aortic model, the role of the MMPs in angiogenesis in vitro was investigated both by the use of synthetic MMP inhibitors, and by a study of the expression of nine MMPs and three of their endogenous inhibitors (the TIMPs) during vessel formation. Inhibition of microvessel growth in this model by the MMP inhibitor Marimastat demonstrated the requirement of the MMPs for angiogenesis in both collagen and fibrin matrices (half-maximal inhibition at 5 and 80 nM, respectively). The profile of MMP expression was seen to be modified by both matrix composition and exogenous growth factors. For example, whilst the gelatinase MMP-2 and stromelysin MMP-3 were present at high levels in fibrin culture, the stromelysin MMP-11 and membrane-type-1-MMP were more highly expressed during vessel formation in collagen. The angiogenic basic fibroblast growth factor (bFGF) upregulated the expression of the gelatinases (MMP-2 and MMP-9), the stromelysins (MMP-3, MMP-10 and MMP-11) and the interstitial collagenase MMP-13, whereas vascular endothelial growth factor (VEGF) led to a marked increase in expression of MMP-2 only. Together, the environment-dependent upregulation in expression of a number of MMPs during angiogenesis, and the total inhibition of vessel growth observed at nanomolar concentrations of synthetic MMP inhibitors, suggests a major collective role of these enzymes in angiogenesis, and provides a basis for further development of MMP inhibitors for anti-angiogenic therapy.

Animals↗
PubMed2001

From peptide libraries to optimized nonpeptide ligands in the search for S-farnesyltransferase inhibitors.

A complete 331,776-member library of tetrapeptides made of 24 amino acid building blocks was synthesized robotically on solid phase and subjected to a deconvolution based on the inhibitory potency of the sublibraries in a HPLC assay of the S-farnesyltransferase activity in vitro. One of the non-natural peptide and noncysteine-containing leads Nip-Trp-Phe-His (Nip=p-nitrophenyl-L-alanine) was optimized chemically to give a proteolytically stable pseudopeptide with a 200-fold potency compared with the original lead. The final compound was converted to the C-terminal ethyl ester: p-F-C6H4-CO(CH2)2-CO-Bta-D-Phepsi[CH2NH]His-OEt (Bta = benzothienyl-L-alanine) and shown to behave as a prodrug which was hydrolyzed back to the C-terminal acid following cell penetration. The method confirmed that several structurally original leads can be discovered in large libraries when deconvolution relies upon a highly specific assay and that these leads can be optimized by chemical modification to impart the final compound the desired pharmacological and pharmacokinetic properties.

Alkyl and Aryl Transferases↗
PubMed1999

Relationship between E-cadherin and fibroblast growth factor receptor 2b expression in bladder carcinomas.

E-cadherin is a cell-cell adhesion molecule expressed predominantly by epithelial cells. Reduction or loss of E-cadherin immunoreactivity has been associated with tumour progression in many epithelial cancers, including bladder carcinomas. The fibroblast growth factor receptor 2b (FGFR2b) recognized specifically by FGF7 is expressed only by epithelial cells. Recently, decreased expression of FGFR2b protein and mRNA was found to be associated with tumour progression in bladder carcinomas. The purpose of this investigation was to look for a possible relationship between E-cadherin and FGFR2b expression in bladder carcinomas. As decreased E-cadherin immunoreactivity was found to correlate directly with decreased expression at the mRNA level, the possible relationship between E-cadherin and FGFR2b was investigated at the mRNA level using semi-quantitative RT - PCR in 92 transitional cell carcinomas (TCCs) and four lymph node metastases. All tumours with low E-cadherin expression had low expression of FGFR2b, whereas tumours with low FGFR2b mRNA could express any level of E-cadherin mRNA. The same observation was equally valid for bladder and colon cancer cell lines suggesting that, besides bladder tumours, this relationship could apply to other carcinomas types. These results suggest that a relationship exists between the transcription of the E-cadherin and FGFR2b genes preventing high expression of FGFR2b where expression of E-cadherin is low. We suggest that reduced expression of FGFR2b in conjunction with decreased expression of E-cadherin may contribute to the aggressive behaviour attributable to high grade TCCs.

Cadherins↗
PubMed1999

The effect of extracellular pH on angiogenesis in vitro.

The microenvironment of the majority of solid tumours in which new vessels must grow and survive is acidic. Whilst recent reports suggest a role of the low tumour pH in the invasive and metastatic potential of tumour cells, little is known as to its impact on angiogenesis. The three-dimensional in vitro rat aortic ring model was used to study the effects of low extracellular pH (pH(e)) on microvascular growth. The spontaneous angiogenic response in collagen gels was seen to be highly dependent on the pH of the culture medium, with optimal outgrowth at pH 7.4, and a marked delay in microvascular growth at pH 6.9. This inhibition of vascular development was reversible. The absence of similar effects of medium pH on monolayer outgrowths of endothelial cells from rat aortic rings suggested an effect of pH(e) on aspects specific to three-dimensional growth. Vascular endothelial growth factor and basic fibroblast growth factor, whilst having limited effects at pH 7.4, were seen to reduce the time to onset of vessel outgrowth at pH 7.1, and lead to an initial growth rate similar to that observed at pH 7.4 in the absence of growth factors. Thus, the low environmental pH encountered by endothelial cells in solid tumours would not necessarily be detrimental to neovascularisation: a prominent in vitro angiogenic response is still observed at low pH(e) when stimulated by exogenous growth factors, high concentrations of which would be present in vivo.

Journal Article↗
PubMed1998

A zinc chelator inhibiting gelatinases exerts potent in vitro anti-invasive effects.

Matrix metalloproteinases are zinc metalloenzymes involved in remodelling of the extracellular matrix. We compared the anti-invasive properties of a zinc ejector matrix metalloproteinase inhibitor with those of reference compounds (hydroxamic acid-based BB-94 and Ro-31-9790) which form inactive ternary complexes with the enzymes and the catalytic zinc. We show that the compound undecadenedioic acid bis-[[2-(3 H-imidazol-4-yl)-ethyl]-amide] (S 30372) is active against gelatinases, chelates zinc and exhibits enzymatic features compatible with the potential to extract zinc from gelatinases. We then used five invasive cell lines in the Matrigel invasion chamber assay (NIH-3T3 fibroblasts, Lewis lung carcinoma cells, EJ138 and J82 bladder carcinoma and HT1080 fibrosarcoma cells). With the exception of J82 cells which were unaffected by the three inhibitors, all remaining cells were substantially more sensitive to S 30372 in terms of maximal inhibition of invasion attained. This suggests that matrix metalloproteinase inhibitors with zinc chelating/ejecting properties may be more efficient in preventing tumor progression.

3T3 Cells↗
PubMed1997

Activation of MMP-9 by neutrophil elastase in an in vivo model of acute lung injury.

The effect of neutrophil elastase on the functional status of gelatinases was studied in an hamster model developed by intratracheal administration of lipopolysaccharide followed by in situ cell activation with phorbol myristate acetate. This resulted in the production in bronchoalveolar lavage fluids, in addition to the matrix metalloproteinase MMP-9, of a 75 kDa gelatinase associated with collagenolytic activity. Treatment in vivo with an elastase inhibitor abolished the latter activity. Since, in addition, elastase activates in vitro purified MMP-9 gelatinase into a similar 75 kDa entity, these data suggest that elastase may be a physiological activator of MMP-9 in vivo.

Animals↗
PubMed1997

Selection of a histidine-containing inhibitor of gelatinases through deconvolution of combinatorial tetrapeptide libraries.

A fully automated peptide synthesizer was used to generate tetrapeptide sublibraries from 24 natural and nonnatural amino acids, from which new inhibitors of gelatinases (matrix metalloproteinases MMP-2 and MMP-9) were selected as potential anticancer drugs. MMP-2 and MMP-9 from mouse Balbc/3T3 fibroblasts conditioned media were assayed in their linear range response by zymography to quantify inhibition at each step of the tetrapeptide library deconvolution. The histidine-epsilon-amino caproic acid-beta-alanine-histidine (His-epsilon Ahx-beta Ala-His) sequence was found to yield optimal inhibition of both MMP-2 and MMP-9. Inhibition by selected tetrapeptides was also evaluated with two other techniques, a native type IV collagen degradation assay and a fluorogenic enzymatic assay, confirming the tetrapeptide potency. The His-epsilon Ahx-beta Ala-His tetrapeptide also inhibited purified human MMP-2 and MMP-9 and the corresponding enzymes present in conditioned media from human tumour cells. Finally, the length of the spacer between the two terminal histidines was found to be crucial to the inhibitory potential. This approach may thus be considered as a-successful strategy to yield specific peptide or pseudopeptide inhibitors, although their potency remains moderate, since it was measured before any chemical optimization was undertaken.

3T3 Cells↗
PubMed1997

Eriochrome Black T, structurally related to suramin, inhibits angiogenesis and tumor growth in vivo.

The polyanionic species suramin is a potential anti-cancer agent of narrow therapeutic index. Among other pharmacological characteristics, suramin is an inhibitor of angiogenesis. We have targeted its angiostatic properties as part of a program to discover less toxic analogs. From screening a series of commercially available compounds, structurally related to suramin and containing a sulfonic acid substituted naphthylamine moiety, we discovered a new lead, Eriochrome Black T (EBT). EBT is a novel inhibitor of angiogenesis, more potent and less toxic than suramin in the chick chorioallantoic membrane assay. EBT was more active than suramin in inhibiting endothelial cell proliferation in primary culture and in inhibiting proliferation of three tumor cell lines, A431, L1210 and M5076 (IC50 10-100 microM). Cell cycle studies on the A431 line showed that both EBT and suramin caused an accumulation of cells in the S phase, EBT being 10-fold more potent. We suggest that this cell cycle perturbation is linked to inhibition of topoisomerase II catalytic activity. EBT was found to be a moderate but significant inhibitor of matrix metalloproteinases (10 microM range), more efficient than suramin. In a s.c. M5076 sarcoma model in mice, EBT had similar efficacy to suramin both by the i.p. or s.c. route and was moreover better tolerated. Combined pharmacological results show that EBT compared favorably with suramin in all assays, and that in ovo and in vivo, EBT is an analog of suramin with diminished toxicity.

Animals↗
PubMed1996

Up-regulated expression of the beta3 integrin and the 92-kDa gelatinase in human HT-144 melanoma cell tumors grown in nude mice.

In situ changes in the repertoire of integrins and proteolytic enzymes have been demonstrated during melanoma metastasis. To investigate whether established human melanoma cell lines, injected into nude mice, could undergo phenotypic changes similar to those observed in in situ lesions, we studied 3 melanoma cell lines of distinct metastatic origin, adherent HT-144 and SK-MEL-2 cells, and non-adherent SK-MEL-1 cells for integrin expression, proteolytic enzyme repertoire and invasive potential after in vitro culture. Heterogeneity in integrin expression, such as elevated levels in alpha(v)beta3 in SK-MEL-1 and SK-MEL-2 cells and low expression in HT-144 cells, correlated with their in vitro invasiveness, since only the adherent HT-144 and SK-MEL-2 cells were able to invade Matrigel, and in addition, secreted a 72-kDa gelatinase. In contrast, no similar correlation could be established in nude mice, as all 3 cell lines, including the non-adherent SK-MEL-1 cells, were tumorigenic when injected s.c., while only HT-144 consistently produced experimental lung metastasis. Immunochemical analysis of the integrin profile in s.c. xenografts revealed over-expression of alpha(v), beta1 and beta3 integrins exclusively in HT-144 cells, as well as increased expression of beta3 in HT-144 cell lung metastases, as confirmed by PCR analysis using species-specific primers, while zymography and Western-blot analysis demonstrated de novo expression of the 92-kDa gelatinase MMP-9 in HT-144 xenografts. Our results highlight a positive correlation between up-regulated beta3 integrin and MMP-9 expression in human HT-144 melanoma cell tumors grown in nude mice.

Animals↗
PubMed1993

Involvement of cell motility in tumor progression.

Since one crucial step in tumor progression consists of the acquisition of invasive and metastatic properties, it is important to analyze the mechanisms used by cancer cells to disperse. Among the possible mechanisms of cell dispersion, cell motility appears as a central phenomenon that still needs to be understood at the molecular level. Our experimental approach to the contribution of cell motility in carcinoma cell dissemination is based on the study of the NBT-II rat bladder carcinoma cell line. The epithelial cell line gives rise to isolated, actively migrating, fibroblast-like cells in response to specific stimuli (collagens and acidic fibroblast growth factor [aFGF]). Analysis of the scattering response indicates that the different stimuli can synergize, leading to increased motility and invasiveness. NBT-II cells have two types of response to aFGF: they can either proliferate or scatter. In addition, the two responses are mutually exclusive, suggesting that the cell status can dictate whether or not tumor cells will disperse after exposure to a scatter factor. Finally, recent studies on the involvement of epithelial-specific cadherins in the process of aFGF-induced cell scattering indicate that a sustained expression of E-cadherin is not sufficient to protect cells from dispersing. In conclusion, our experimental model offers the opportunity to dissect the molecular events leading to tumor cell dissemination.

Animals↗
PubMed1991

Combined effects of extracellular matrix and growth factors on NBT-II rat bladder carcinoma cell dispersion.

Using the rat bladder carcinoma cell line NBT-II we showed that collagens but not laminin and fibronectin were able to induce cell scattering. Acidic fibroblast growth factor and transforming growth factor alpha also promoted NBT-II cell dispersion on glass or tissue culture plastic. We have now further analysed the scatter response to these two growth factors in the presence of extracellular matrix molecules. In the presence of growth factors, no peripheral single-cell dispersion occurred on fibronectin and laminin, although time-lapse video analyses revealed intense cell mingling and motility inside the monolayer forming around NBT-II aggregates. Patterns of strings or files of cells protruding from the monolayer were often observed. The presence of a scattering activity in the complex acellular extracellular matrix deposited by NBT-II cells themselves strongly suggested that substratum conditioning was responsible for this effect. On the other hand, the two growth factors accelerated collagen-mediated NBT-II individual cell dispersion and locomotion in a reversible way. As a marker of cell dissociation, we studied desmosome distribution in aggregate cultures: desmosomes were present in aggregates formed in suspension even in the presence of growth factors, whereas internalization occurred after cell-to-substratum contact. On laminin or fibronectin and in the presence of growth factors, peripheral cells inside the halo of NBT-II aggregates did not exhibit desmosome linkages. These observations suggest that scatter effects per se are dependent on the composition of the extracellular matrix. In particular, on a substratum nonpermissive for direct cell translocation, individual cell dispersion can be replaced by en bloc patterns of migration following substratum conditioning by the cells.

Animals↗
PubMed1991

Amplification of invasiveness in organotypic cultures after NBT-II rat bladder carcinoma stimulation with in vitro scattering factors.

Acidic fibroblast growth factor (aFGF) or transforming growth factor-alpha (TGF-alpha), in addition to being mitogenic, induce individual scattering of NBT-II rat bladder carcinoma cell clusters on tissue culture dishes, suggesting that they may contribute to tumor cell dissemination. To assay their scattering potential and their effect on cell invasiveness in a more complex and physiologically relevant model, we analyzed the behavior of NBT-II spheroids confronted with urinary bladder in organotypic cultures. NBT-II spheroids progressively replaced the urothelium at the site of contact with the bladder explant. In the absence of aFGF or TGF-alpha, inserted cells grew in a pattern suggestive of local hyperplasia, with occasional invasive cell protrusions. Exogenous scattering growth factors elicited a more rapid appearance of these protrusions, which were also more numerous. NBT-II cells transfected with cDNA constructs bearing the gene of aFGF, TGF-alpha or the oncogene hst/KFGF were also used. After exogenous or autocrine stimulation of NBT-II cells with the growth factors, a deeper penetration of the bladder wall in the form of nodular outgrowths and clusters of infiltrating cells was always observed. Altogether these observations suggest that the stimulation of NBT-II clusters by scattering/growth factors can promote cell shedding and amplify invasiveness in the complex extracellular environment of bladder tissues.

Animals↗
PubMed1991

Epithelial cell plasticity in neoplasia.

Tumor metastasis is associated with an increase in the plasticity of malignant cells, a phenomenon that is characterized by changes in cell morphology and a decrease in intercellular cohesiveness. The plasticity of cells is correlated with their motility. Therefore, factors that enhance plasticity promote the migration of malignant cells from a primary tumor. Several cytokines that induce the dissociation and dispersal of malignant cells have now been described. By inhibiting the activity of motogenic cytokines, it may be possible to design effective strategies for the treatment of patients with metastatic cancer.

Animals↗
PubMed1990

Collagen-mediated dispersion of NBT-II rat bladder carcinoma cells.

During metastatic spread, locomotion mediated by extracellular matrix components of basement membranes and connective tissues has been invoked as a prerequisite to invasion. We studied the interactions of the rat bladder carcinoma cell line NBT-II with fibronectin, laminin, and collagens (types I, III, IV, and V). They all promoted cell attachment and spreading. To analyze their scatter potential, we studied epithelial outgrowth and/or peripheral cell dispersion from tumor aggregates. All matrix components allowed partial collapse of the aggregate and the appearance of a cellular monolayer forming a halo around the aggregate. No peripheral cell dispersion occurred on fibronectin and laminin. Collagens (especially types I and III) promoted the dispersion of peripheral NBT-II cells with various speeds of locomotion, as revealed by time-lapse videomicroscopy. With the exception of cells at the periphery on collagens, cells inside the halo did not exchange neighbors, migrated transiently as an epithelial sheet during halo formation, and finally remained stationary. These effects were reproduced with NBT-II tumor fragments obtained from nude mice. Tumor cells were linked together with desmosomes (as revealed by immunoreactivity against desmoglein). Migration on collagens correlated with the mechanical disruption of intercellular contacts and consequently with the progressive disappearance of desmoglein immunoreactivity. Immunofluorescence studies also revealed a reduced expression of the epithelium-specific cell adhesion molecule liver cell adhesion molecule after contact with collagens. These results suggest that direct interactions with collagens may favor single cell infiltration by bladder carcinoma.

Animals↗
PubMed1990

Acidic fibroblast growth factor is a modulator of epithelial plasticity in a rat bladder carcinoma cell line.

During normal embryogenesis and neoplastic transformation epithelia change their state of differentiation and degree of cohesiveness. It is thus essential to identify the signals modulating these transitions. We report here that acidic fibroblast growth factor (FGF) induces cells derived from a rat bladder carcinoma to lose their epithelial character and to acquire some properties typical of mesenchymal cells. The structurally related basic FGF did not have such an effect; both factors, however, had a mitogenic activity for these cells. Two distinct populations of receptors for acidic FGF and basic FGF were distinguished by their ligand-binding characteristics. The observations that both acidic and basic FGFs had a mitogenic effect on NBT-II cells and that only acidic FGF caused cell dissociation and dispersion strongly suggest that these two biological activities could be medicated through distinct signaling pathways.

Animals↗