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Biomedical subjects

Ian R Hart

Publications and source records attributed to Ian R Hart.

13 recordsLinked to original sources
PubMed2006

Cyclooxygenase-2 inhibition suppresses alphavbeta6 integrin-dependent oral squamous carcinoma invasion.

Worldwide oral squamous cell carcinoma (OSCC) represents about 5.5% of all malignancies, with approximately 30,000 new cases each year in the United States. The integrin alpha(v)beta(6) and the enzyme cyclooxygenase-2 (COX-2) are implicated in OSCC progression and have been suggested as possible therapeutic targets. Each protein also is reported to identify dysplasias at high risk of malignant transformation, and current clinical trials are testing the efficacy of nonsteroidal anti-inflammatory drugs (NSAID) at preventing OSCC development. Given the probable increased expression of alpha(v)beta(6) and COX-2 in OSCC and the inhibition of several integrins by NSAIDs, we investigated whether NSAIDs affected alpha(v)beta(6)-dependent cell functions. We found that expression of both alpha(v)beta(6) and COX-2 was significantly higher in OSCC compared with oral epithelial dysplasias. Neither protein preferentially identified those dysplastic lesions that became malignant. Using OSCC cell lines, modified to express varying levels of alpha(v)beta(6), we assessed the effect of COX-2 inhibition on cell invasion. We found that the COX-2 inhibitor NS398 inhibited specifically alpha(v)beta(6)-dependent, but not alpha(v)beta(6)-independent, OSCC invasion in vitro and in vivo, and this effect was modulated through prostaglandin E(2) (PGE(2))-dependent activation of Rac-1. Transient expression of constitutively active Rac-1, or addition of the COX-2 metabolite PGE(2), prevented the anti-invasive effect of NS398. Conversely, RNA interference down-regulation of Rac-1 inhibited alpha(v)beta(6)-dependent invasion. These findings suggest that COX-2 and alpha(v)beta(6) interact in promoting OSCC invasion. This is a novel mechanism that, given the ubiquity of alpha(v)beta(6) expression by head and neck cancers, raises the possibility that NSAIDs could protect against OSCC invasion.

Animals↗
PubMed2006

A new role for the anti-apoptotic gene A20 in angiogenesis.

A20 is a negative regulator of NF-kappaB activation and thus a potential therapeutic tool for the treatment of diseases where apoptosis and/or inflammatory responses are part of the pathogenic process. Thus, A20 has been shown to improve the long-term outcome of organ transplantation, particularly, the transplantation of islets of Langerhans which may aid the cure of type I diabetes. We now report a new role for A20 in regulating neovascularisation. We used RNA interference to inhibit A20 expression in primary human umbilical vein endothelial cells (HUVECs) and investigated the effect on tubule formation in two in vitro angiogenesis assays, Matrigel and a co-culture assay. Tubule area and tubule length were both reduced following inhibition of A20 expression in HUVECs. These inhibitory effects were particularly evident in the co-culture assay, which incorporates the critical steps of the angiogenic process and ultimately results in the formation of an intricate network of anastomosing tubules that resemble the formed capillary bed: a partial down-regulation of A20 protein (50-60%) resulted in a 28% reduction in tubule area (P < 0.05) and a 26% reduction in tubule length (P < 0.05). A20 may offer a new target in the treatment of human conditions, including cancer, which are characterised by neovascularisation.

Apoptosis↗
PubMed2004

Reduction of TSG101 protein has a negative impact on tumor cell growth.

TSG101 was defined originally as a tumor-suppressor gene, raising the expectation that absence of the encoded protein should lead to increased tumor cell growth and, perhaps, increased tumor cell aggressiveness. We have used the RNA interference (RNAi) technique to downregulate TSG101 in PC3 (prostate cancer) and MDA-MB-231 (breast cancer) cells. An approximately 85% selective downregulation at the protein level was achieved in both cell lines over a period of 12 days as detected by Western blotting. This treatment resulted in inhibition of tumor cell growth, with a decreased level of TSG101 causing partial cell cycle arrest at the G(1)/S boundary and a reduction in the rate at which cells passed from G(2) through mitosis and back into G(1). In both cell lines, the percentage of cells in S-phase was reduced significantly at day 4 after the TSG101 siRNA transfection (27% vs. 41% in MDA-MB-231 cells; 22% vs. 39% in PC3 cells). Additionally, RNAi-mediated downregulation of TSG101 reduced the colony formation capacities of both cancer cell lines. Rather more surprisingly, TSG101 downregulation affected the migratory activity of the MDA-MB-231 cells, independent of any effect on proliferation. Thus, in a Transwell assay, after 4-hr incubation, 36.0% of control MDA-MB-231 cells had migrated to the lower chamber vs. 7.3% of TSG101-downregulated cells (p < 0.001; scrambled control, 36.5%). These results show that the TSG101 gene does not comply with the usual characteristics of a tumor-suppressor gene; rather, its expression may be necessary for activities associated with aspects of tumor progression.

Apoptosis↗
PubMed2004

The integrin cytoplasmic-tail motif EKQKVDLSTDC is sufficient to promote tumor cell invasion mediated by matrix metalloproteinase (MMP)-2 or MMP-9.

Integrins promote cellular invasion through a combination of activities, including adhesion to an extracellular matrix ligand, which result in the generation of intracellular signals that lead to changes in cell behavior. Until now, there have been no data that identify a particular region of the cytoplasmic tail of integrin subunits as being responsible specifically for promoting the invasive activity of tumor cells. In this report, we show that amino acids with the sequence EKQKVDLSTDC, which are the C-terminal residues of the integrin beta6 subunit, promote alphavbeta6-dependent invasion in a matrix metalloproteinase (MMP)-9-dependent fashion. This same peptide sequence, when expressed at the cytoplasmic end of the beta3 integrin subunit, was able to enhance alphavbeta3-mediated invasive and enzymatic activity of tumor cells in an MMP-2-dependent fashion. Our results show that these 11 amino acids, when expressed at the C terminus of the beta subunit, are responsible for regulating the activity of invasion-promoting degradative enzymes, whereas the specific MMP involved in this cellular behavior is dependent on the context of the remainder of the beta integrin subunit.

Amino Acid Motifs↗
PubMed2003

Desmosomal proteins, including desmoglein 3, serve as novel negative markers for epidermal stem cell-containing population of keratinocytes.

No single method has been universally adopted for identifying and isolating epidermal stem/progenitor cells, and the emergence of new markers of stem cell populations is worth exploring. Here we report, for the first time, that clusters of basal keratinocytes at the tips of the rete ridges in human palm, previously recognised as a major repository of stem cells, had very low levels of desmoplakin protein and mRNA expression, compared with cells at the sides of the ridges or above the dermal papillae. We found that in populations of palm keratinocytes, selected by their ability to adhere rapidly to type IV collagen, there were significantly reduced levels of desmoplakin and other major desmosome proteins. We then showed that a low desmoglein 3 (Dsg3) expression on the cell surface could be used to enrich for a cell population with high clonogenecity, colony forming efficiency and enhanced proliferative potential, but with a low ability to form the abortive clones, compared with populations with a higher Dsg3 expression. Moreover, stringent sorting of populations showing both beta1 integrin-bright and Dsg3-dull expression enabled even further enrichment of a population containing the putative epidermal stem cells. These findings provide the basis for a new strategy for epidermal stem/progenitor cell enrichment, and encourage further study of the role of desmosomes in stem cell biology.

Cadherins↗
PubMed2003

Lack of plakophilin 1 increases keratinocyte migration and reduces desmosome stability.

Ablation of the desmosomal plaque component plakophilin 1 underlies the autosomal recessive genodermatosis, skin fragility-ectodermal dysplasia syndrome (OMIM 604536). Skin from affected patients is thickened with increased scale, and there is loss of adhesion between adjacent keratinocytes, which exhibit few small, poorly formed desmosomes. To investigate further the influence of plakophilin 1 on keratinocyte adhesion and desmosome morphology, we compared plakophilin 1-deficient keratinocytes (vector controls) with those expressing recombinant plakophilin 1 introduced by retroviral transduction. We found that plakophilin 1 increases desmosomal protein content within the cell rather than enhancing transcriptional levels of desmosomal genes. Re-expression of plakophilin 1 in null cells retards cell migration but does not alter keratinocyte cell growth. Confluent sheets of plakophilin 1-deficient keratinocytes display fewer calcium-independent desmosomes than do plakophilin 1-deficient keratinocytes expressing recombinant plakophilin 1 or keratinocytes expressing endogenous plakophilin 1. In addition electron microscopy studies show that re-expression of plakophilin 1 affects desmosome size and number. Collectively, these results demonstrate that restoration of plakophilin 1 function in our culture system influences the transition of desmosomes from a calcium-dependent to a calcium-independent state and this correlates with altered keratinocyte migration in response to wounding. Thus, plakophilin 1 has a key role in increasing desmosomal protein content, in desmosome assembly, and in regulating cell migration.

3T3 Cells↗
PubMed2003

Combination of microdissection and microarray analysis to identify gene expression changes between differentially located tumour cells in breast cancer.

Comparison of gene expression changes between cancer cells at the periphery and in the centre of breast cancers was performed using a combination of microdissection and microarray analysis. Cancer cells from the two areas were pooled separately from five patients with ductal carcinoma in situ and separately from five patients with frankly invasive cancer. Limited total RNA, 100-200 ng, from this microdissected tissue required use of the Atlas SMART trade mark Probe Amplification Kit to synthesize and amplify cDNA and make (33)P-labelled probes. Probes were then hybridized to Atlas Human Cancer 1.2 Arrays containing 1176 known genes. Triplicate analysis revealed that 22 genes changed their expression levels in the periphery relative to the central region: 15 upregulated and seven downregulated (arbitrary threshold of 1.5-fold or greater). Differences in RNA levels were confirmed by quantitative real-time PCR for two of the genes and by changes in protein levels, detected by immunohistochemistry, for a couple of representative gene products. Thus, changes in gene expression associated with variation in microanatomical location of neoplastic cells can be detected within even small developing tumour masses.

Adenocarcinoma↗
PubMed2003

The role of E-cadherin in low-grade ductal breast tumourigenesis.

Grade I invasive ductal breast carcinomas have a specific pattern of genetic aberrations, namely gain of 1q and loss of 16q. This pattern is very similar to the changes seen in invasive lobular breast carcinomas (ILCs). The gene on 16q involved in ILC is known to be E-cadherin (CDH1). This study has investigated whether the same gene is responsible for grade I invasive ductal carcinoma (IDC), using allele imbalance analysis, mutation screening, and immunohistochemistry (IHC). The data suggest that despite the shared pattern of genetic aberrations seen in grade I IDC and ILC, CDH1 is not the target gene in low-grade ductal tumourigenesis.

Alleles↗
PubMed2003

Application of differential display, with in situ hybridization verification, to microscopic samples of breast cancer tissue.

The technique of differential display (DD) has been used widely to identify potentially interesting overexpressed or repressed genes in a variety of compared samples. When used in studying tissue samples, it inevitably confronts problems of limited amount of input material and cell-type heterogeneity. We report here the application of in situ hybridization as a method of confirmatory test for DD as well as definition of cell type expressing differential cDNA. This procedure employed material derived from a single case of human mammary, grade III, infiltrating ductal carcinoma, using free-hand microdissection, where we have compared gene expression profiles in invasive tumour with those in adjacent normal tissue. A total of 21 cDNAs were found to be differentially expressed between the two tissue types; 11 upregulated in the tumour sample and 10 upregulated in the normal sample. Six cDNAs were utilized as probes for in situ hybridization analysis of a further five cases of comparably staged breast cancer. One of these clones, 11AT1, which was found to be homologous to Hsc70, was shown to be overexpressed in tumour cells relative to adjacent normal stroma and to benign glandular epithelium in all five cases; an increase in expression was further confirmed at protein level by immunohistochemistry. The study demonstrated the applicability of in situ hybridization as a screening test in DD strategy for studying tissue material and a reasonable technique combination of identifying changes in gene expression associated with tumour development.

Base Sequence↗
PubMed2002

Rapid solid phase synthesis and biodistribution of 18F-labelled linear peptides.

A rapid method for radiolabelling short peptides with 18F ( t(1/2)=109.7 min) for use in positron emission tomography (PET) was developed. Linear peptides (13mers) were synthesised using solid phase peptide synthesis and 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. The peptides were assembled on a solid-phase polyethylene glycol-polystyrene support using the "hyper acid labile" linker xanthen-2-oxyvaleric acid and were labelled in situ with 4-[19F]- or 4-[18F]fluorobenzoic acid. Optimum coupling of 4-[19F]fluorobenzoic acid to the peptidyl resin was achieved within 2 min using N-[(dimethylamino)-1 H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]- Nmethylmethanaminium hexafluorophosphate N-oxide (HATU/DIPEA), and optimum cleavage was achieved within 7 min using trifluoroacetic acid/phenol/water/Triisopropylsilane at 37 degrees C. The linear peptides were rapidly labelled with 4-[18F]fluorobenzoic acid with an overall radiochemical yield of 80%-90% (decay corrected), a radiochemical purity of >95% without HPLC purification and an overall synthesis time of 20 min. This novel method was used to label peptides containing the arginine-glycine-aspartic acid (RGD) motif, the binding site of many integrins. In vitro studies showed that the fluorobenzoyl prosthetic group had no deleterious effect on the ability of these peptides to inhibit the binding of human cells via integrins. Biodistribution studies in tumour-bearing mice showed that although the linear peptides were rapidly removed from the circulation by the liver and kidneys, there was a transient and non-RGD-dependent accumulation in the tumour of both the test and the control peptides. The use of more selective peptides with a longer half-life in the circulation combined with this rapid labelling technique will significantly enhance the application of peptides in PET.

Amino Acid Sequence↗
PubMed2002

Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis.

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.

Amino Acid Motifs↗
PubMed2002

Effectiveness of a cardiology review course for internal medicine residents using simulation technology and deliberate practice.

BACKGROUND: Objective evaluations of residents' clinical skills reveal serious deficits. PURPOSE: To develop, implement, and evaluate outcomes from a review course in cardiology bedside skills for internal medicine residents. METHODS: We used a 1-group pretest-posttest design with historical comparisons. The study was conducted at the University of Miami School of Medicine as part of the internal medicine residency program from July 1999 to June 2000. A total of 67 2nd- and 3rd-year medicine residents received an educational intervention involving deliberate practice using simulation technology. A total of 155 4th-year medical students in one intervention and one comparison group (n = 53) served as historical comparisons. Outcome measures were a reliable computer-delivered pretest and posttest that evaluate cardiology bedside skills. RESULTS: Residents who received the review course and medical students who received a comparable educational intervention showed large and statistically significant pretest-to-posttest improvement in bedside skills. These 2 groups are also significantly and substantially different at posttest from a comparison group of 4th-year medical students that did not receive a specific educational intervention. CONCLUSION: Educational interventions using simulation technology that engage learners in deliberate practice of clinical skills produce large improvements in a relatively short time, with little faculty involvement.

Cardiology↗