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Biomedical subjects

S D Scott

Publications and source records attributed to S D Scott.

At least 19 recordsLinked to original sources
PubMed2006

Hypoxia- and radiation-activated Cre/loxP 'molecular switch' vectors for gene therapy of cancer.

Although a significant negative prognostic factor, tumor hypoxia can be exploited for gene therapy. To maximize targeting within the tumor mass, we have developed synthetic gene promoters containing hypoxia-responsive elements (HREs) from the erythropoietin (Epo) gene as well as radiation-responsive CArG elements from the early growth response (Egr) 1 gene. Furthermore, to achieve high and sustained expression of the suicide gene herpes simplex virus thymidine kinase (HSVtk), our gene therapy vectors contain an expression amplification system, or 'molecular switch', based on Cre/loxP recombination. In human glioma and breast adenocarcinoma cells exposed to hypoxia and/or radiation, the HRE/CArG promoter rapidly activated Cre recombinase expression leading to selective and sustained HSVtk synthesis. Killing of transfected tumor cells was measured after incubation with the prodrug ganciclovir (GCV; converted by HSVtk into a cytotoxin). In vitro, higher and more selective GCV-mediated toxicity was achieved with the switch vectors, when compared with the same inducible promoters driving HSVtk expression directly. In tumor xenografts implanted in nude mice, the HRE/CArG-switch induced significant growth delay and tumor eradication. In conclusion, hypoxia- and radiation-activated 'molecular switch' vectors represent a promising strategy for both targeted and effective gene therapy of solid tumors.

Adenocarcinoma↗
PubMed2005

VP22-mediated intercellular transport for suicide gene therapy under oxic and hypoxic conditions.

During herpes simplex virus type 1 (HSV 1) infection, the tegument protein VP22 is exported from infected cells to the nuclei of surrounding uninfected cells. These intercellular transport characteristics have prompted the exploitation of VP22 fusion proteins for cancer gene therapy, with the goal of maximizing the bystander effect. Since solid tumors contain hypoxic cell populations that are often refractive to therapy, for efficient targeting, it would be optimal if VP22 functioned even at reduced oxygen concentrations. In the present work, VP22 activity under hypoxic conditions was examined for the first time. Plasmid-transfected human glioma U87-MG and U373-MG cells expressing VP22 fused to the green fluorescent protein (GFP) showed protein export to untransfected cells under tumor oxygenation conditions (0-5% O(2)). For suicide gene therapy, VP22 activity was demonstrated under hypoxia by coupling VP22 to the HSV thymidine kinase (HSVtk). In the presence of the prodrug ganciclovir, cell cultures expressing VP22-HSVtk showed a significant increase in toxicity compared with cells transfected with a construct containing HSVtk only, under all tested conditions. To allow effective suicide gene therapy and simultaneous visualization of therapeutic enzyme localization, a triple fusion protein GFP-HSVtk-VP22 was engineered. Functionality of all components was demonstrated under oxia and hypoxia.

Analysis of Variance↗
PubMed2003

Radiogenetic therapy: strategies to overcome tumor resistance.

The aim of cancer gene therapy is to selectively kill malignant cells at the tumor site, by exploiting traits specific to cancer cells and/or solid tumors. Strategies that take advantage of biological features common to different tumor types are particularly promising, since they have wide clinical applicability. Much attention has focused on genetic methods that complement radiotherapy, the principal treatment modality, or that exploit hypoxia, the most ubiquitous characteristic of most solid cancers. The goal of this review is to highlight two promising gene therapy methods developed specifically to target the tumor volume that can be readily used in combination with radiotherapy. The first approach uses radiation-responsive gene promoters to control the selective expression of a suicide gene (e.g., herpes simplex virus thymidine kinase) to irradiated tissue only, leading to targeted cell killing in the presence of a prodrug (e.g., ganciclovir). The second method utilizes oxygen-dependent promoters to produce selective therapeutic gene expression and prodrug activation in hypoxic cells, which are refractive to conventional radiotherapy. Further refining of tumor targeting can be achieved by combining radiation and hypoxia responsive elements in chimeric promoters activated by either and dual stimuli. The in vitro and in vivo studies described in this review suggest that the combination of gene therapy and radiotherapy protocols has potential for use in cancer care, particularly in cases currently refractory to treatment as a result of inherent or hypoxia-mediated radioresistance.

Animals↗
PubMed2002

Molecular approaches to chemo-radiotherapy.

Although radiotherapy is used to treat many solid tumours, normal tissue tolerance and inherent tumour radioresistance can hinder successful outcome. Cancer gene therapy is one approach being developed to address this problem. However, the potential of many strategies are not realised owing to poor gene delivery and a lack of tumour specificity. The use of treatment-, condition- or tumour-specific promoters to control gene-directed enzyme prodrug therapy (GDEPT) is one such method for targeting gene expression to the tumour. Here, we describe two systems that make use of GDEPT, regulated by radiation or hypoxic-responsive promoters. To ensure that the radiation-responsive promoter is be activated by clinically relevant doses of radiation, we have designed synthetic promoters based on radiation responsive CArG elements derived from the Early Growth Response 1 (Egr1) gene. Use of these promoters in several tumour cell lines resulted in a 2-3-fold activation after a single dose of 3 Gy. Furthermore, use of these CArG promoters to control the expression of the herpes simplex virus (HSV) thymidine kinase (tk) gene in combination with the prodrug ganciclovir (GCV) resulted in substantially more cytotoxicity than seen with radiation or GCV treatment alone. Effectiveness was further improved by incorporating the GDEPT strategy into a novel molecular switch system using the Cre/loxP recombinase system of bacteriophage P1. The level of GDEPT bystander cell killing was notably increased by the use of a fusion protein of the HSVtk enzyme and the HSV intercellular transport protein vp22. Since hypoxia is also a common feature of many tumours, promoters containing hypoxic-responsive elements (HREs) for use with GDEPT are described. The development of such strategies that achieve tumour targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy.

Combined Modality Therapy↗
PubMed2002

Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy.

We have been developing synthetic gene promoters responsive to clinical doses of ionizing radiation (IR) for use in suicide gene therapy vectors. The crucial DNA sequences utilized are units with the consensus motif CC(A/T)(6)GG, known as CArG elements, derived from the IR-responsive Egr1 gene. In this study we have investigated the parameters needed to enhance promoter activation to radiation. A series of plasmid vectors containing different enhancer/promoters were constructed, transiently transfected into tumor cells (MCF-7 breast adenocarcinoma and U-373MG glioblastoma) and expression of a downstream reporter assayed. Results revealed that increasing the number of CArG elements, up to a certain level, increased promoter radiation-response; from a fold-induction of 1.95 +/- 0.17 for four elements to 2.74 +/- 0.17 for nine CArGs of the same sequence (for MCF-7 cells). Specific alteration of the core A/T sequences caused an even greater positive response, with fold-inductions of 1.71 +/- 0.23 for six elements of prototype sequence compared with 2.96 +/- 0.52 for one of the new sequences following irradiation. Alteration of spacing (from six to 18 nucleotides) between elements had little effect, as did the addition of an adjacent Sp1 binding site. Combining the optimum number and sequence of CArG elements in an additional enhancer was found to produce the best IR induction levels. Furthermore, the improved enhancers also performed better than the previously reported prototype when used in in vitro and in vivo experimental GDEPT. We envisage such enhancers will be used to drive suicide gene expression from vectors delivered to a tumor within an irradiated field. The modest, but tight expression described in the present study could be amplified using a molecular 'switch' system as previously described using Cre/LoxP. In combination with targeted delivery, this strategy has great potential for significantly improving the efficacy of cancer treatment in the large number of cases where radiotherapy is currently employed.

Adenocarcinoma↗
PubMed2002

Novel chimeric gene promoters responsive to hypoxia and ionizing radiation.

Despite being an adverse prognostic factor in radiotherapy, hypoxia represents a physiological difference that can be exploited for selective cancer gene therapy. In this study gene therapy vectors responsive to both hypoxia and ionizing radiation (IR) were developed. Gene expression was regulated by novel, synthetic promoters containing hypoxia responsive elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase 1 (PGK1) and the vascular endothelial growth factor (VEGF) genes, and IR-responsive CArG elements from the early growth response (Egr) 1 gene. All chimeric promoters could be activated by hypoxia and/or IR-treatment, and selectively control marker gene expression in human T24 bladder carcinoma and MCF-7 mammary carcinoma cells. Importantly, enhancers containing combinations of HREs and CArG elements were able to respond to both triggering treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo HRE/CArG enhancer could effectively control a suicide gene therapy strategy by selectively sensitizing hypoxic and/or irradiated cells expressing the enzyme horseradish peroxidase (HRP) to the prodrug indole-3-acetic acid (IAA). These data indicate that the use of such chimeric promoters may effectively regulate therapeutic gene expression within the tumor microenvironment in gene therapy strategies aimed at addressing the problem of hypoxia in radiotherapy.

Animals↗
PubMed2001

Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells.

The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.

Base Sequence↗
PubMed2000

Development of synthetic promoters for radiation-mediated gene therapy.

Exposure of cells to ionising radiation results in the activation of specific transcriptional control (CArG) elements within the early growth response 1 (Egr1) gene promoter, leading to increased gene expression. As part of a study investigating the potential use of these elements in radiation-controlled gene therapy vectors, we have incorporated their sequences into a synthetic gene promoter and assayed for the ability to induce expression of a downstream reporter gene following irradiation. In vector-transfected MCF-7 breast adenocarcinoma cells, the synthetic promoter was more effective than the wild-type Egr1 counterpart in up-regulating expression of the reporter gene after exposure to a single 5 Gy dose, and equally effective as the wild-type in U87-MG glioma cells. The level of gene expression achieved using the synthetic promoter was dependent on the inducing radiation dose for both U87-MG and MCF-7 cells, being maximal at 3 Gy and decreasing at 5 and 10 Gy. Furthermore, induction could be repeated by additional radiation treatments. The latter indicates that up-regulation should be additive during fractionated radiotherapy schedules. To demonstrate the potential clinical benefit of such an approach, the synthetic promoters were also shown to drive expression of the herpes simplex virus thymidine kinase gene, leading to enhanced cell killing in the presence of the prodrug ganciclovir (GCV) when compared with cells treated with radiation alone. Our results demonstrate that the synthetic promoter is responsive to low doses of ionising radiation and therefore isolated CArG elements function as radiation-mediated transcriptional enhancers outside their normal sequence context. The continued development and optimisation of such radiation-responsive synthetic promoters is expected to make a valuable contribution to the development of future radiation-responsive vectors for cancer gene therapy.

Adenocarcinoma↗
PubMed2000

A radiation-controlled molecular switch for use in gene therapy of cancer.

Ionising radiation induces the expression of a number of radiation-responsive genes and there is current interest in exploiting this to regulate the expression of exogenous therapeutic genes in gene therapy strategies for cancer. However, the radiation-responsive promoters used in these approaches are often associated with low and transient levels of therapeutic gene expression. We describe here a novel radiation-triggered molecular switching device based on promoter elements from the radiation-responsive Egr-1 gene and the cre-LoxP site-specific recombination system of the P1 bacteriophage. Using this system, a single, minimally toxic dose of radiation induced cre-mediated excision of a lox-P flanked stop cassette in a silenced expression vector and this resulted in amplified levels of CMV-promoter-driven expression of the exogenous tumour-sensitising gene, HSV-tk. This strategy could be used in combination with targeted delivery and tumour-specific promoters to elicit the tumour-targeted and prolonged expression of a variety of tumour-sensitising genes and provide an unprecedented level of control and tumour selectivity.

Antiviral Agents↗
PubMed1999

Prophylactic reinfusion of T cells for T cell-depleted allogeneic bone marrow transplantation.

To increase the graft-vs.-leukemia (GVL) effect while maintaining a low mortality from graft-vs.-host disease (GVHD), we conducted a prospective study of T cell titration for 144 patients (90 related, 54 unrelated) between June 1994 and June 1997. Following infusion of a T cell-depleted marrow graft, predetermined doses of T cells, based on the risk factors for GVHD, were administered up to 3 times if greater than a grade II acute GVHD was not seen. Graft failure occurred in three unrelated recipients (2%). Cumulative grades II-IV acute GVHD were seen in 58 +/- 9% of all recipients; 52 +/- 11% related and 75 +/- 13% unrelated. The incidence of grades II-IV acute GVHD following the third add-back (AB) of T cells 78 median days after marrow infusion was lower than that of the earlier ABs: first AB, 36 +/- 8%; second AB, 32 +/- 11%; third AB, 15 +/- 12% (p < 0.05). Chronic GVHD occurred in 56 +/- 12% of related and 79 +/- 16% of unrelated patients. Six died of acute GVHD and two died of chronic GVHD, with an overall GVHD mortality of 6 +/- 4%. In multivariate analyses, unrelated recipients and patients at low risk for GVHD who received a larger number of T cells were identified as patient groups with significant risk for acute and chronic GVHD (both p < 0.05). Unrelated transplant is also shown to be significant for GVHD-related death (p < 0.01). Relapse-free survival of patients with leukemia was shown to be most dependent on chronic GVHD and grades II-IV acute GVHD (both p < 0.01). Anti-leukemic activity independent of GVHD was not observed.

Acute Disease↗
PubMed1997

Detection of anabolic steroid administration: ratio of urinary testosterone to epitestosterone vs the ratio of urinary testosterone to luteinizing hormone.

Our goal in this study was to determine whether the urinary ratio of testosterone to luteinizing hormone (T/LH) as an indicator of exogenous anabolic steroid (AS) use is superior to the urinary ratio of testosterone to epitestosterone (T/E). After 2 weekly placebo injections, 19 subjects were given testosterone cypionate (TC) injections of 250 or 500 mg/week for 14 weeks followed by 14 weekly placebo injections. Patients were considered to have ceased taking TC if they tested negative 9 weeks after their last injection. For detection of illicit or supraphysiological TC (AS) use, the urinary T/E ratio of > or = 6 yielded a false-negative rate of 46% and a false-positive rate of 4%. However, a urinary T/LH ratio of > or = 30 produced a false-negative rate of only 24% and a false-positive rate of 13%. We conclude that the urinary T/LH ratio of > or = 30 is a more sensitive marker of AS use than the urinary T/E ratio of > or = 6 and remains sensitive for twice as long as urinary T/E.

Adolescent↗
PubMed1997

Testosterone suppression of the HPT axis.

BACKGROUND: Although studies have demonstrated the suppression of normal gonadal function in the experimental setting, the specific mechanisms by which androgenic-anabolic steroids impact male gonadal function remain ill defined. Following 2 consecutive weekly injections of an identically appearing testosterone cypionate (TC) placebo, subjects were randomized to a TC dose of 100 mg/wk, 250 mg/wk, or 500 mg/wk. Following the last weekly injection of active agent the subjects received 12 consecutive weeks of TC placebo injections. RESULTS: Spermatogenesis was impaired by each of the doses of TC employed in this study, but the observed decreases in, sperm count were neither strictly dose dependent nor consistent between individuals treated with the same dose. Basal leuteinizing hormone (LH) and follicle stimulating hormone (FSH) became undetectable 2 weeks after the start of 250 and 500 mg/wk TC injections and were lost within 5 to 6 weeks of starting 100 mg doses. Pituitary gonadotropin responses to leutinizing hormone releasing hormone (LHRH) disappeared more slowly with FSH responses being lost 1 to 3 weeks after the loss of basal FSH activity. Leuteinizing hormone responses to LHRH appeared to be suppressed last, disappearing 4 to 6 weeks after FSH responses to LHRH. CONCLUSIONS: Exogenous testosterone-mediated inhibitory influences on the hypothalamic-pituitary-testicular axis were reversed following the cessation of drug treatment.

Adolescent↗
PubMed1995

Effect of cyclophosphamide administration on the activity and relative content of hepatic P4502D1 in rat.

1. The effects of the administration of the anticancer and immunosuppressive drug, cyclophosphamide, to the rat on hepatic P4502D1 activity and content in the microsomal fraction have been examined. 2. Liver microsomes were obtained from male Hooded Wistar rats administered a single dose (i.p.) of saline or cyclophosphamide (200 mg/kg). Rats receiving cyclophosphamide were killed 1, 4, 7, 10 or 14 days after cyclophosphamide administration. The O-demethylation of dextromethorphan to dextrorphan was used to monitor 2D1 activity. 3. The mean Vmax for dextrorphan formation was reduced significantly (p < 0.0001) 7, 10 and 14 days after cyclophosphamide administration compared with the control group (control, 0.32 +/- 0.07; 7-day, 0.20 +/- 0.08; 10-day, 0.11 +/- 0.02; and 14-day group, 0.15 +/- 0.02 nmol/mg/min). 4. Western blotting revealed that there was a significant reduction (p < 0.0005) in the microsomal relative 2D1 content 10 days after cyclophosphamide administration compared with the control group (control, 1.25 +/- 0.44; and 10-day group, 0.65 +/- 0.14). 5. The activity of reduced nicotinamide adenine dinucleotide phosphate P450 reductase was significantly reduced (p < 0.0001) 7, 10 and 14 days following cyclophosphamide administration (control, 215 +/- 24; 7-day, 102 +/- 20; 10-day, 59 +/- 4 and 14-day group, 76 +/- 8 nmol/mg/min). Cytochrome b5 content was significantly reduced (p < 0.0001) 7 and 10 days following cyclophosphamide administration (control, 0.46 +/- 0.13; 7-day, 0.28 +/- 0.07 and 10-day group, 0.20 +/- 0.03 nmol/mg). 6. The significant reductions in the activity of rat hepatic microsomal 2D1 following cyclophosphamide administration, as seen by the alterations in mean Vmax for dextrorphan formation, do not appear to be due to a single factor, but may result from a combination of several events, including reductions in relative 2D1 content, reduced nicotinamide adenine dinucleotide phosphate P450-reductase activity and cytochrome b5 content.

Animals↗
PubMed1994

Metastatic paraspinal abscess and paraplegia secondary to dental extraction.

An unusual case of metastatic paraspinal abscess and paraplegia is presented. This was a consequence of simple dental extractions and subsequent bacteraemia from a neglected mouth. Neurosurgical intervention and tissue culture yielded organisms almost certainly of dental origin. No alternative primary nidus of infection could be found. The importance of oral health to general health is emphasised.

Abscess↗
PubMed1994

Development of a pregnancy policy for air medical personnel: an administrative approach.

Pregnancy of air medical personnel poses a unique challenge for the administration of air medical services. Pregnant staff vary in their approach to individual pregnancy and their desire to continue flight duties. Administrators are limited to actions that ensure optimal care for the patient regardless of the caregivers' health or condition. Air medical programs must balance what is acceptable for patient care and safety with what are legally acceptable practice restrictions. The Staff For Life helicopter service uses experts in obstetric care to evaluate the pregnant staff members' abilities to perform pre-determined physical duties associated with air medical care. A signed consent form acknowledging risk factors associated with air medical care ensures that the flight staff member has had a frank and honest discussion with her physician. The obstetric evaluation and consent form confirm for program administration a pregnant flight staff member's ability to function within her role and removes all ambiguity from the process.

Air Ambulances↗