Biomedical subjects

Stephanie R McKeown

Publications and source records attributed to Stephanie R McKeown.

8 recordsLinked to original sources

PubMed2006

Gene expression in normal urothelium depends on location within the bladder: a possible link to bladder carcinogenesis.

OBJECTIVES: Clinical studies have shown that more than 70% of primary bladder tumours arise in the area around the ureteric orifice. In this study a genomic approach was taken to explore the molecular mechanisms that may influence this phenomenon. METHODS: RNA was isolated from each individual normal ureteric orifice and the dome biopsy from 33 male patients. Equal amounts of the pooled ureteric orifice and dome mRNAs were labelled with Cy3 and Cy5, respectively before hybridising to the gene chip (UniGEM 2.0, Incyte Genomics Inc., Wilmington, Delaware, USA). RESULTS: Significant changes (more than a twofold difference) in gene expression were observed in 3.1% (312) of the 10,176 gene array: 211 genes upregulated and 101 downregulated. Analysis of Cdc25B, TK1, PKM, and PDGFra with RT-PCR supported the reliability of the microarray result. Seladin-1 was the most upregulated gene in the ureteric orifice: 8.3-fold on the microarray and 11.4-fold by real time PCR. CONCLUSIONS: Overall, this study suggests significant altered gene expression between these two anatomically distinct areas of the normal human bladder. Of particular note is Seladin-1, whose significance in cancer is yet to be clarified. Further studies of the genes discovered by this work will help clarify which of these differences influence primary bladder carcinogenesis.

Adult↗

PubMed2005

Pericytes influence endothelial cell growth characteristics: role of plasminogen activator inhibitor type 1 (PAI-1).

OBJECTIVE: Pericytes, located in close proximity to the underlying endothelium, form an integral component of the microvasculature. These cells are intimately involved in angiogenesis, which is of fundamental importance in many physiological and pathological processes. We evaluated the influence of pericyte-conditioned medium (PCM) on endothelial cell growth characteristics and modulation of endothelial gene expression. METHODS: Migration and tubule formation assays were performed in vitro to determine the effect of PCM on endothelial growth characteristics. cDNA microarray analysis was used to identify alterations in gene expression following exposure of human microvascular endothelial cells (HMEC-1) to PCM. Overexpression of PAI-1 using recombinant protein or transient transfection, and inhibition using an inhibitory antibody against PAI-1, were used to determine whether up- or down-regulation of this gene was responsible for the changes in endothelial cell characteristics observed in response to PCM exposure. RESULTS: We have shown that PCM exerts a dramatic inhibitory influence on endothelial cell migration in vitro. In addition, endothelial cells cultured on Matrigel and exposed to PCM were found to generate significantly fewer angiogenic branches. Microarray analysis of endothelial cells exposed to PCM identified PAI-1 as the gene showing the greatest level of differential expression (3.4-fold induction). Studies using an inhibitory antibody to PAI-1 suggest that induction of this protein by PCM is pivotal to the observed inhibitory influence on the migratory and angiogenic potential of HMEC-1. We further investigated this by overexpressing PAI-1, which was shown to have a potent inhibitory influence on EC migration and angiogenic branching, although the concentration of PAI-1 was clearly important. CONCLUSION: Collectively, these findings suggest that PCM contains a bioactive element(s) that controls both endothelial cell migration and tubule formation in vitro and that these responses may be partially controlled by increased endothelial cell expression of PAI-1.

Blotting, Northern↗

PubMed2005

A cytochrome P450 2B6 meditated gene therapy strategy to enhance the effects of radiation or cyclophosphamide when combined with the bioreductive drug AQ4N.

BACKGROUND: AQ4N is metabolised in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Most solid tumours are known to contain regions of hypoxia whereas levels of CYPs have been found to vary considerably. Enhancement of CYP levels may be obtained using gene-directed enzyme prodrug therapy (GDEPT). We have therefore examined the potential of a CYP2B6-mediated GDEPT strategy to enhance the anti-tumour effect of the combination of AQ4N with radiation or cyclophosphamide (CPA). METHODS: In vitro and in vivo transient transfection of human CYP2B6 +/- CYP reductase (CYPRED) was investigated in RIF-1 mouse tumours. Efficacy in vitro was assessed using the alkaline comet assay (ACA). In vivo, the time to reach 4x the treatment volume (quadrupling time; VQT) was used as the end point. RESULTS: When CYP2B6 was transfected into RIF-1 cells and treated with AQ4N under hypoxic conditions there was a significant increase in DNA damage (measured by the ACA) compared with non-transfected cells. In vivo, a single intra-tumoural injection of a CYP2B6 vector construct significantly enhanced tumour growth delay in combination with AQ4N (100 mg/kg) and 10 Gy X-rays. AQ4N (100 mg/kg) and CPA (100 mg/kg) with CYP2B6 and CYPRED also enhanced tumour growth delay; this effect became significant when the schedule was repeated 14 days later (p = 0.0197). CONCLUSIONS: The results show the efficacy of a CYP2B6-mediated GDEPT strategy for bioreduction of AQ4N; this may offer an additional approach to target radiation- and chemo-resistant hypoxic tumours that should enhance overall tumour control.

Animals↗

PubMed2004

Gene-directed enzyme prodrug therapy: a current assessment.

Gene-directed enzyme prodrug therapy involves tumor-specific delivery of a gene encoding a drug-metabolizing enzyme allowing metabolism of a non-toxic prodrug to a toxic species directly within tumor cells. This review covers the wide range of enzyme-prodrug combinations currently under preclinical and clinical investigation. Issues of tumor specificity and enhanced cytotoxicity through bystander effects will be discussed.

Animals↗

PubMed2003

Cell cycle checkpoint function in bladder cancer.

BACKGROUND: Cell cycle checkpoints function to maintain genetic stability by providing additional time for repair of DNA damage and completion of events that are necessary for accurate cell division. Some checkpoints, such as the DNA damage G1 checkpoint, are dependent on p53, whereas other checkpoints, such as the decatenation G(2) checkpoint, are not. Because bladder transitional cell carcinomas (TCCs) often contain numerous chromosomal aberrations and appear to have highly unstable genomes, we analyzed cell cycle checkpoint functions in a panel of TCC lines. METHODS: Cell cycle arrest was induced in normal human fibroblasts (NHF1-hTERT) and normal human uroepithelial cells (HUCs), and TCC lines and checkpoint functions were quantified using flow cytometry and fluorescence microscopy. The inducers and checkpoints were ionizing radiation (i.e., DNA damage) (G1 and G2 checkpoints), the mitotic inhibitor colcemid (polyploidy checkpoint), or the topoisomerase II catalytic inhibitor ICRF-193 (decatenation G2 checkpoint). Four of the five TCC lines expressed mutant p53. RESULTS: HUCs had an effective G1 checkpoint response to ionizing radiation, with 68% of cells inhibited from moving from G1 into S phase. By contrast, G1 checkpoint function was severely attenuated (<15% inhibition) in three of the five TCC lines and moderately attenuated (<50% inhibition) in the other two lines. NHF1-hTERT had an effective polyploidy checkpoint response, but three of five TCC lines were defective in this checkpoint. HUCs had effective ionizing radiation and decatenation G2 checkpoint responses. All TCC lines had a relatively effective G2 checkpoint response to DNA damage, although the responses of two of the TCC lines were moderately attenuated relative to HUCs. All TCC lines had a severe defect in the decatenation G2 checkpoint response. CONCLUSION: Bladder TCC lines have defective cell cycle checkpoint functions, suggesting that the p53-independent decatenation G2 checkpoint may cooperate with the p53-dependent G1 checkpoints to preserve chromosomal stability and suppress bladder carcinogenesis.

Carcinoma, Transitional Cell↗

PubMed2003

Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells.

The alkaline Comet assay is a simple, sensitive method for measuring the extent of DNA strand breaks in individual cells. Several modifications to the original assay have been developed to increase its applications. One such modification allows the measurement of DNA cross-links by assessing the relative reduction in DNA migration induced by a strand-breaking agent. Another modification includes the application of fluorescent in situ hybridisation (FISH) to investigate the localisation of specific gene domains within a cell. Although several studies have used these approaches separately, no report to date has combined these two versions of the Comet assay. The current study describes the modification of the Comet assay, to allow both measurement of mitomycin C (MMC)-induced cross-links and the subsequent application of FISH to study repair in the TP53 gene region. RT4 human bladder cancer cells were treated with 0, 5, 50 and 200 microg/ml MMC to study dose response, whilst for cross-link repair studies, they were treated with 50 microg/ml MMC and allowed to repair for up to 24 h. A clear dose response to MMC was displayed, demonstrable by a marked reduction in DNA migration, whilst repair studies showed that MMC-induced cross-links take at least 24 h to repair fully in RT4 cells. For Comet-FISH experiments, the number and location of TP53 hybridisation spots was also recorded for each cell. In dose response experiments, the number of spots per cell, and per Comet tail, decreased as MMC dose increased. In repair experiments, the number of spots, particularly in the Comet tail, increased as repair time increased. Furthermore, our results suggest that repair of the TP53 gene region is most rapid within the first 4 h following MMC treatment. We conclude that the novel experimental protocol presented here has considerable potential in evaluating DNA damage and sequence-related repair responses to cross-linking agents.

Cell Line, Tumor↗

PubMed2003

Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N.

The bioreductive drug, AQ4N, is metabolized under hypoxic conditions and has been shown to enhance the antitumor effects of radiation and chemotherapy drugs. We have investigated the role of cytochrome P450 3A4 (CYP3A4) in increasing the metabolism of AQ4N using a gene-directed enzyme prodrug therapy (GDEPT) strategy. RIF-1 murine tumor cells were transfected with a mammalian expression vector containing CYP3A4 cDNA. In vitro AQ4N metabolism, DNA damage, and clonogenic cell kill were assessed following exposure of transfected and parental control cells to AQ4N. The presence of exogenous CYP3A4 increased the metabolism of AQ4N and significantly enhanced the ability of the drug to cause DNA strand breaks and clonogenic cell death. Cotransfection of CYP reductase with CYP3A4 showed a small enhancement of the effect in the DNA damage assay only. A single injection of CYP3A4 into established RIF-1 murine tumors increased the metabolism of AQ4N, and when used in combination with radiation, three of nine tumors were locally controlled for >60 days. This is the first demonstration that CYPs alone can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only CYP-activated bioreductive agent in clinical trials. Combination with a GDEPT strategy may offer a further opportunity for targeting radiation-resistant and chemo-resistant hypoxic tumor cells.

Animals↗

PubMed2003

Use of the comet-FISH assay to demonstrate repair of the TP53 gene region in two human bladder carcinoma cell lines.

The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy gamma irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after gamma irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.

Comet Assay↗