Biomedical subjects

W M Gallagher

Publications and source records attributed to W M Gallagher.

14 recordsLinked to original sources

PubMed2006

Novel "plum pudding" gels as potential drug-eluting stent coatings: controlled release of fluvastatin.

This study evaluated novel structural motifs known as "plum pudding" gels as potential drug-eluting stent coatings. Controlled delivery of a HMG-CoA reductase inhibitor (statin) from the intravascular stent surface represents a potential therapeutic modality for prevention of in-stent restenosis (ISR). In this study, gels were comprised of fluvastatin-loaded thermoresponsive microgel particles containing the relatively hydrophilic N-isopropylacrylamide (NiPAAm), mixed with the more hydrophobic N-tert-butylacrylamide (NtBAAm) in different wt/wt ratios: 85/15, 65/35, and 50/50, randomly dispersed in a 65/35 or 85/15 NiPAAm/NtBAAm copolymer matrix. Fluvastatin release from 5 microm copolymer films was greatest from the most hydrophilic systems and least from the more hydrophobic systems. Release from hydrophobic matrices appeared to be via Fickian diffusion, enabling use of the Stokes-Einstein equation to determine diffusion coefficients. Release from hydrophilic matrices was non-Fickian. Eluted drug retained its bioactivity, assessed as selective inhibition of human coronary artery smooth muscle cell proliferation. When stainless steel stent wires were coated (25 microm thickness) with fluvastatin-loaded 65/35 microgels in an 85/15 copolymer matrix, drug elution into static and perfused flow environments followed similar elution profiles. In contrast to elution from copolymer films cast on flat surfaces, diffusion from stent wires coated with hydrophilic and hydrophobic systems both followed Fickian patterns, with slightly larger diffusion coefficients for elution from the flow system. We conclude that manipulation of the relative hydrophobicities of both microgel and matrix components of "plum pudding" gels results in tightly regulated release of fluvastatin over an extended time period relevant to initiation and propagation of ISR.

Anticholesteremic Agents↗

PubMed2005

A potent nonporphyrin class of photodynamic therapeutic agent: cellular localisation, cytotoxic potential and influence of hypoxia.

We have developed a totally new class of nonporphyrin photodynamic therapeutic agents with a specific focus on two lead candidates azadipyrromethene (ADPM)01 and ADPM06. Confocal laser scanning microscopy imaging showed that these compounds are exclusively localised to the cytosolic compartment, with specific accumulation in the endoplasmic reticulum and to a lesser extent in the mitochondria. Light-induced toxicity assays, carried out over a broad range of human tumour cell lines, displayed EC50 values in the micro-molar range for ADPM01 and nano-molar range for ADPM06, with no discernable activity bias for a specific cell type. Strikingly, the more active agent, ADPM06, even retained significant activity under hypoxic conditions. Both photosensitisers showed low to nondeterminable dark toxicity. Flow cytometric analysis revealed that ADPM01 and ADPM06 were highly effective at inducing apoptosis as a mode of cell death. The photophysical and biological characteristics of these PDT agents suggest that they have potential for the development of new anticancer therapeutics.

Apoptosis↗

PubMed2005

Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells.

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of vascular endothelial growth factor (VEGF165) to human aortic endothelial cells (HAEC) over an extended time period. Films were prepared using a 50:50 (w/w) mixture of non-crosslinkable and crosslinkable copolymers of the following monomer compositions (w/w): 85:15, N-isopropylacrylamide (NiPAAm):N-tert-butylacrylamide (NtBAAm); and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (ABzPh, crosslinking agent), respectively. After crosslinking by UV irradiation, the ability of films to incorporate a fluorescently labeled carrier protein (FITC-labeled BSA, 1 mg loaded per film), at 4 degrees C, was first established. Incorporation into the matrix was confirmed by the observation that increasing film thickness from 5 to 10 microm increased release from collapsed films at 37 degrees C (1.76 +/- 0.15 and 10.98 +/- 3.38 microg/mL, respectively, at 24 h postloading) and that this difference was maintained at 5 days postloading (1.81 +/- 0.25 and 13.8 +/- 2.3 microg/mL, respectively). Incorporation was also confirmed by visualization using confocal microscopy. When 10-microm films were loaded with a BSA solution (1 mg/mL) containing VEGF165 (3 microg/mL), sustained release of VEGF165 was observed (10.75 +/- 3.11 ng at 24 h; a total of 31.32 +/- 8.50 ng over 7 days). Furthermore, eluted VEGF165 increased HAEC proliferation by 18.2% over control. The absence of cytotoxic species in medium released from the copolymer films was confirmed by the lack of effect of medium (incubated with copolymer films for 3 days) on HAEC viability. In conclusion this study has shown that NiPAAm:NtBAAm copolymers can be loaded with a therapeutic protein and can deliver bioactive concentrations to human vascular endothelial cells over an extended time period.

Acrylic Resins↗

PubMed2004

Oligonucleotide microarray analysis of gene expression in neuroblastoma displaying loss of chromosome 11q.

A number of distinct subtypes of neuroblastoma exist with different genetic abnormalities that are predicative of outcome. Whole chromosome gains are usually associated with low stage disease and favourable outcome, whereas loss of 1p, 3p and 11q, unbalanced gain of 17q and MYCN amplification (MNA) are indicative of high stage disease and unfavourable prognosis. Although MNA and loss of 11q appear to represent two distinct genetic subtypes of advanced stage neuroblastoma, a detailed understanding of how these subtypes differ in terms of global gene expression is still lacking. We have used metaphase comparative genomic hybridization (CGH) analysis in combination with oligonucleotide technology to identify patterns of gene expression that correlate with specific genomic imbalances found in primary neuroblastic tumours and cell lines. The tumours analysed in this manner included a ganglioneuroma, along with various ganglioneuroblastoma and neuroblastoma of different stages and histopathological classifications. Oligonucleotide microarray-based gene expression profile analysis was performed with Affymetrix HU133A arrays representing approximately 14 500 unique genes. The oligonucleotide microarray results were subsequently validated by quantitative real-time PCR, immunohistochemical staining, and by comparison of specific gene expression patterns with published results. Hierarchical clustering of gene expression data distinguished tumours on the basis of stage, differentiation and genetic abnormalities. A number of genes were identified whose patterns of expression were highly correlated with 11q loss; supporting the concept that loss of 11q represents a distinct genetic subtype of neuroblastoma. The implications of these results in the process of neuroblastoma development and progression are discussed.

Biomarkers, Tumor↗

PubMed2003

Extended delivery of the antimitotic agent colchicine from thermoresponsive N-isopropylacrylamide-based copolymer films to human vascular smooth muscle cells.

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of an antimitotic agent to human vascular smooth muscle cells (HASMC) over an extended period of time. Copolymer films were prepared using a 50:50 (w/w) ratio of N-isopropylacrylamide (NiPAAm) monomer to the more hydrophobic N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (0.1 micromol per film) at room temperature. Colchicine release from films was sustained over a 14-day period. At 24 h postloading, the concentration of colchicine in the medium overlying films was 2.12 +/- 0.16 microM; this fell to 0.20 +/- 0.01 microM at 7 days and decreased further to 0.12 +/- 0.01 microM after 14 days. Colchicine released from copolymer films inhibited proliferation when subsequently placed on HASMC: at 0.1 microM, released colchicine reduced proliferation to 18.5 +/- 0.8% of control cells (p < 0.001, n = 9). The antiproliferative effect of released colchicine was comparable to that of native colchicine, as observed in separate experiments. Furthermore, colchicine released from 50:50 polymer films inhibited the proliferation of cells grown in the same environment as the copolymer. Inhibition of cell proliferation was not due to the release of cytotoxic particles from the copolymer because medium incubated with copolymer for 5 days and then applied to HASMC did not alter cell viability. In conclusion, this study demonstrates that 50:50 NiPAAm:NtBAAm copolymers can deliver bioactive concentrations of the antimitotic agent colchicine to human vascular cells over an extended period of time.

Acrylamides↗

PubMed2003

Elevated expression and altered processing of fibulin-1 protein in human breast cancer.

The extracellular matrix protein fibulin-1 suppresses the motility and invasiveness of a variety of tumour cell types in vitro as well as the growth of fibrosarcoma tumours in nude mice. In this study, fibulin-1 protein expression in breast carcinoma specimens and normal breast tissue was evaluated immunohistologically. Fibulin-1 protein expression was also semiquantitatively assessed by immunoblot analysis in a collection of normal breast tissues (n=18), benign tumours (n=5) and breast carcinomas (n=39). In normal breast tissue, fibulin-1 protein expression predominated in the ductal epithelium and underlying myoepithelium, with weaker staining evident in the loose connective surrounding the ducts. Examination of breast carcinomas revealed that the tumour cells also expressed fibulin-1 protein. The level of mature fibulin-1 polypeptide (100 kDa) was higher in the breast carcinoma specimens as compared to normal breast tissue (Mann-Whitney U-test, P=0.0005). In addition to the mature fibulin-1 polypeptide, several smaller sized polypeptides of 55, 50 and 25 kDa were detected using monoclonal antibodies reactive towards an epitope located at the N-terminus of fibulin-1. The immunoreactive 50 kDa polypeptide was detected more frequently in breast carcinoma specimens than in normal breast tissue (chi(2)=17.22, P<0.0001). Furthermore, the ratio of the 50 kDa fragment to the mature fibulin-1 polypeptide correlated with the level of oestrogen receptor alpha (Spearman correlation coefficient, rs=0.49, P<0.003, n=36) and progesterone receptor (rs=0.43, P=0.008, n=36) expression in the tumour specimens. Taken together, these findings indicate that elevated expression and altered processing of fibulin-1 is associated with human breast cancer.

Antibodies, Monoclonal↗

PubMed2001

Human fibulin-4: analysis of its biosynthetic processing and mRNA expression in normal and tumour tissues.

Here, we report the identification of a human orthologue of fibulin-4, along with analysis of its biosynthetic processing and mRNA expression levels in normal and tumour tissues. Comparative sequence analysis of fibulin-4 cDNAs revealed apparent polymorphisms in the signal sequence that could account for previously reported inefficient secretion in fibulin-4 transfectants. In vitro translation of fibulin-4 mRNA revealed the presence of full-length and truncated polypeptides, the latter apparently generated from an alternative translation initiation site. Since this polypeptide failed to incorporate into endoplasmic reticulum membrane preparations, it was concluded that it lacked a signal sequence and thus could represent an intracellular form of fibulin-4. Using fluorescence in situ hybridisation analysis, the human fibulin-4 gene was localised to chromosome 11q13, this region being syntenic to portions of mouse chromosomes 7 and 19. Considering the fact that translocations, amplifications and other rearrangements of the 11q13 region are associated with a variety of human cancers, the expression of human fibulin-4 was evaluated in a series of colon tumours. Reverse transcription-polymerase chain reaction analysis of RNA from paired human colon tumour and adjacent normal tissue biopsies showed that a significant proportion of tumours had approximately 2-7-fold increases in the level of fibulin-4 mRNA expression. Taken together, results reported here suggest that an intracellular form of fibulin-4 protein may exist and that dysregulated expression of the fibulin-4 gene is associated with human colon tumourigenesis.

Amino Acid Sequence↗

PubMed1999

MBP1: a novel mutant p53-specific protein partner with oncogenic properties.

Using a yeast two-hybrid screening strategy with a common tumour-derived p53 mutant as bait, we identified several mutant p53-interacting partners including the known proteins wild-type (wt) p53, hUBC9 and GBP/PIAS1. In addition, a novel protein partner was identified which we have termed MBP1, for Mutant p53-Binding Protein 1. MBP1 is a new member of the emerging fibulin gene family, which currently comprises fibulin-1, fibulin-2 and S1-5. Expression of MBP1 mRNA is differentially regulated both temporally during development of the mouse embryo and in a tissue-specific manner within the adult. Specific interaction between MBP1 and mutant p53 was illustrated by both two-hybrid analysis in yeast and co-immunoprecipitation in mammalian cells. MBP1 displayed the following order of binding specificity towards different p53 forms: H175 > G281 > H273 > or = W248>wt p53. Thus, MBP1 appears to bind preferentially to p53 mutants of the 'structural' rather than 'contact' class, reflecting a potential bias towards those mutants having a significant alteration in conformation from that assumed by wt p53. We propose that MBP1 is the product of a candidate oncogene as rates of both neoplastic transformation and tumour cell growth were shown to be significantly enhanced when the protein is ectopically overexpressed. Furthermore, MBP1 may play a role in determining if a 'gain of function' effect is seen with certain p53 mutants.

Amino Acid Sequence↗

PubMed1999

p53-oriented cancer therapies: current progress.

Nearly twenty years after the initial discovery of p53, we are now in an ideal position to exploit our vast knowledge of p53 biology in the creation of novel cancer therapies. Disruption of p53 function through mutation, or other means, occurs very frequently in human cancer. Loss of p53 function has been linked with unfavourable prognosis in a large number of tumour types, as indicated by more aggressive tumours, early metastasis and decreased survival rates. Many different avenues of research have converged upon p53 to highlight this protein as being one of the foremost cellular responders to stress, in particular to DNA damage. Huge advances have been made in understanding the complex role p53 plays in the regulation of apoptosis and cell cycle arrest. This review is not meant to be a comprehensive description of p53 biology, but rather serves to highlight current progress in the development of p53-oriented cancer therapies. These may be categorised into three basic strategies: gene replacement therapy using wild-type p53, restoration of p53 function by other means and, finally, targeting of the p53 dysfunction itself. Rapid progress is expected to be made regarding the identification of conventional pharmaceutical agents which either work in a p53-independent manner or act preferentially in p53 defective cells. Gene replacement therapy with wild-type p53 also holds considerable potential for obtaining clinically relevant results quickly. The other forms of cancer therapies based around p53 are much further behind in the developmental process, but may prove to more efficacious in the long run, especially in terms of specificity. As with many other fields, the innovation of successful p53-oriented cancer therapies is only limited by our understanding of p53 biology and the creative use of such knowledge.

Animals↗

PubMed1997

hMLH1 expression and cellular responses of ovarian tumour cells to treatment with cytotoxic anticancer agents.

Loss of expression of the hMLH1 and hPMS2 subunits of the MutL alpha-mismatch repair complex is a frequent event (9/10) in independent cisplatin resistant derivatives of a human ovarian carcinoma cell line. However, only hMLH1 mRNA is decreased in these MutL alpha-deficient lines. No alterations in the levels of the hMSH2 and hMSH6 (GTBP) subunits of the MutS alpha-complex are observed. An increase in the proportion of ovarian tumours negative for the hMLH1 subunit is observed in samples taken at second look laparotomy after chemotherapy (36%: 4/11), compared to untreated tumours (10%: 4/39). No significant difference is observed for hMSH2, hMSH6 or hPMS2. Furthermore, cisplatin and doxorubicin-resistant ovarian lines deficient in hMLH1 expression are cross-resistant to 6-thioguanine and the methylating agent N-methyl-N-nitrosourea (MNU). Depletion of O6-alkylguanine-DNA-alkyltransferase (ATase) activity confers only limited increased sensitivity to MNU. Thus the mismatch repair deficient lines retain DNA damage tolerance even after ATase depletion. The hMLH1 deficient lines also lose ability to engage G1 and G2 cell cycle arrest after cisplatin damage. Together these data suggest that loss of hMLH1 expression may be a high frequency event following exposure of ovarian tumour cells to cisplatin and may be critically involved in the development of drug resistance. Thus, the hMLH1 status of these cells appears to be highly correlated with the ability to engage cell death and cell cycle arrest after DNA damage induced by cisplatin.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Identification of p53 genetic suppressor elements which confer resistance to cisplatin.

Loss of p53 function is associated with the acquisition of cisplatin resistance in the human ovarian adenocarcinoma A2780 cell line. Selection for cisplatin resistance of A2780 cells was used to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human or murine TP53 cDNA. Six GSEs were identified, encoding either dominant negative mutant peptides or antisense RNA molecules which corresponded to various regions within the TP53 gene. Both types of GSE induced cisplatin resistance when introduced individually into A2780 cells. Expression of antisense GSEs led to decreased intracellular levels of p53 protein. One sense GSE induced loss of p53-mediated activities such as DNA damage induced cell cycle arrest and apoptosis. A synthetic peptide, representing the predicted amino acid sequence of this GSE, conferred resistance to cisplatin when introduced into A2780 cells and inhibited the sequence specific DNA binding activity of p53 protein in vitro. Overall, these results directly indicate that inactivation of p53 function confers cisplatin resistance in these human ovarian tumour cells. We have identified short structural domains of p53 which are capable of independent functional interactions and highlighted the efficacy of this approach to discriminate biologically active GSEs from a random fragment library.

Animals↗

PubMed1996

Microsatellite instability, apoptosis, and loss of p53 function in drug-resistant tumor cells.

We have examined microsatellite instability and loss of p53 function in human tumor cell line models of acquired anticancer drug resistance. We observe acquisition of an RER(+) phenotype in cell lines selected for resistance to cisplatin or doxorubicin. The majority of independent cisplatin-resistant sublines are RER(+), whereas the parental line shows no evidence of microsatellite instability. Microsatellite mutations in random, nonselected subclones of a cislatin-resistant line are observed in the absence of further drug exposure, suggesting that the RER(+) phenotype is a stable phenotype rather than being transiently induced by DNA damage. Furthermore, a cisplatin-resistant derivative shows reduction in a G:T mismatch recognition activity compared to the parental line. Independent lines selected by multiple exposure to cisplatin show resistance factors of up to a 5-fold by clonogenic assay and have reduced cisplatin-induced apoptosis. The resistant lines that are RER(+) show evidence of loss of p53-dependent functions, as measured by a loss of radiation-induced G(1) arrest and reduced CIP1 mRNA. Induced loss of p53 function by transfection of mutant TP53 does not cause a detectable RER(+) phenotype. We speculate that tolerance of DNA damage and expansion of cells with an RER(+) phenotype may select for reduced ability to engage apoptosis and loss of p53 function.

Antibiotics, Antineoplastic↗

PubMed1996

Structural basis of p21H-ras molecular switch inhibition by a neutralizing antibody.

The ras oncogene product p21 functions as a molecular switch in the early section of the signal transduction pathway that is involved in cell growth and differentiation. When the protein is in its GTP-complexed form it is active in signal transduction, whereas it is inactive in its GDP-complexed form. The transforming activity of p21ras is neutralized by the mouse monoclonal antibody Y13-259, possibly by preventing GDP-GTP exchange. A molecular model of the variable fragment of Y13-259 has been derived using a knowledge-based prediction approach and computer-assisted modeling techniques. An analysis of this model while complexed with p21ras/(GDP) indicated that the two molecular switch regions are constrained by complex formation. Antibody binding inhibits GDP-GTP exchange through a mechanism of steric hindrance. Having identified necessary bound sites for inhibition, and explored their electrostatic properties, it should be possible to proceed with the design of antibody mimics as therapeutic agents in cancer control.

Amino Acid Sequence↗

PubMed1980

The inability to judge: the malaise of our time.

A certain Alice in Wonderland mentality pervades the current times as society cautions us not to be judgmental. But when virtue and vice are indistinguishable, Christian living and growth are precluded.

Christianity↗