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R C Wilkins

Publications and source records attributed to R C Wilkins.

15 recordsLinked to original sources
PubMed2007

Evaluating the biological effects of intermittent 1.9 GHz pulse-modulated radiofrequency fields in a series of human-derived cell lines.

Several recent studies have suggested that radiofrequency (RF) fields may cause changes in a variety of cellular functions that may eventually lead to potential long-term health effects. In the present study, we have assessed the ability of non-thermal RF-field exposure to affect a variety of biological processes (including apoptosis, cell cycle progression, viability and cytokine production) in a series of human-derived cell lines (TK6, HL60 and Mono-Mac-6). Exponentially growing cells were exposed to intermittent (5 min on, 10 min off) 1.9 GHz pulse-modulated RF fields for 6 h at mean specific absorption rates (SARs) of 0, 1 and 10 W/kg. Concurrent negative (incubator) and positive (heat shock for 1 h at 43 degrees C) controls were included in each experiment. Immediately after the 6-h exposure period and 18 h after exposure, cell pellets were collected and analyzed for cell viability, the incidence of apoptosis, and alterations in cell cycle kinetics. The cell culture supernatants were assessed for the presence of a series of human inflammatory cytokines (TNFA, IL1B, IL6, IL8, IL10, IL12) using a cytometric bead array assay. No detectable changes in cell viability, cell cycle kinetics, incidence of apoptosis, or cytokine expression were observed in any of RF-field-exposed groups in any of the cell lines tested, relative to the sham controls. However, the positive (heat-shock) control samples displayed a significant decrease in cell viability, increase in apoptosis, and alteration in cell cycle kinetics (G(2)/M block). Overall, we found no evidence that non-thermal RF-field exposure could elicit any detectable biological effect in three human-derived cell lines.

Apoptosis↗
PubMed2006

Increased FITC fluorescence on LPS stimulated neutrophils cultured in whole blood.

Recently, it has been observed that Annexin V labelling of phosphatidylserine (PS) on non-apoptotic cells can vary in different leukocyte populations and with the activation of cells, due to differences in the absolute level of exposed PS. We have also observed changes in the absolute level of Annexin V-FITC intensity, but under conditions where absolute PS expression did not change. In the present study, we have explored the effect of neutrophil cell activation on Annexin V-FITC fluorescence intensity by comparing alternatively labelled matched antibodies against Annexin V. Human venous whole blood was cultured with and without stimulation with lipopolysaccharide (LPS). Apoptosis in the neutrophil and lymphocyte populations was analyzed by flow cytometry and the intensity of FITC labelling was compared to matched fluorochromes conjugated to the same cell surface markers. There was an increase in the intensity of Annexin V-FITC in non-apoptotic neutrophils when stimulated with LPS, which did not correlate with increased apoptosis. Furthermore, CD65-FITC intensity also increased on activated neutrophils. Activated neutrophils exhibited higher amounts of FITC fluorescence that were not associated with changes in extracellular PS expression. This effect appears to be fluorochrome related, likely due to an increase in the pH surrounding activated neutrophils.

Adult↗
PubMed2006

Relative biological effectiveness of 280 keV neutrons for apoptosis in human lymphocytes.

The relative biological effectiveness (RBE) of neutrons varies from unity to greater than ten depending upon neutron energy and the biological endpoint measured. In our study, we examined apoptosis in human lymphocytes to assess the RBE of low energy 280 keV neutrons compared to Cs gamma radiation and found the RBE to be approximately one. Similar results have been observed for high energy neutrons using the same endpoint. As apoptosis is a major process that influences the consequences of radiation exposure, our results indicate that biological effect and the corresponding weighting factors for 280 keV neutrons may be lower in some cell types and tissues.

Apoptosis↗
PubMed2005

Effect of pro-inflammatory cytokines on spontaneous apoptosis in leukocyte sub-sets within a whole blood culture.

Pro-inflammatory cytokines are known to affect apoptosis in human peripheral blood cells. Neutrophils, which are an essential component of the immune response and usually undergo apoptosis rapidly, are greatly affected by these cytokines. In this study, the effect of varying concentrations of TNF-alpha, IL-1beta and IL-6 on the apoptotic response of leukocytes and their sub-sets in cultured whole blood were studied over a 48 h culture period. At clinically relevant concentrations, it was found that these pro-inflammatory cytokines reduced the amount of spontaneous apoptosis in neutrophils in culture, but had little effect on the lymphocyte population. Distinct differences in the sensitivity of neutrophils to cytokine-mediated protection against spontaneous apoptosis were apparent when compared to previous studies conducted using purified or enriched neutrophil cultures. IL-1beta, at a dose of 0.01 pg/mL, was observed to significantly inhibit spontaneous neutrophil apoptosis by approximately 90% and 65% at 24 and 48 h of culturing, respectively. This concentration used in whole blood is dramatically lower than that required to elicit similar protection in neutrophil-enriched cell cultures. Higher concentrations of TNF-alpha (1.0 pg/mL) and IL-6 (125 pg/mL) were also found to significantly inhibit neutrophil apoptosis, at levels much lower than previously published using neutrophil-enriched cultures. Furthermore, each cytokine displayed a unique signature with respect to the optimal applied doses required to elicit maximal protection against spontaneous neutrophil apoptosis. These results demonstrate the dramatic differences in cellular responses that exist between neutrophil-enriched cultures and whole blood culture systems, where multiple blood cell types provide a much more complex environment.

Apoptosis↗
PubMed2002

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells.

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.

Apoptosis↗
PubMed2002

The effect of the ratio of CD4+ to CD8+ T-cells on radiation-induced apoptosis in human lymphocyte subpopulations.

PURPOSE: Apoptosis occurs spontaneously in cultured human peripheral blood lymphocytes but is enhanced by exposure to ionizing radiation. Subpopulations of lymphocytes are known to have varying radiosensitivities to radiation-induced apoptosis. The purpose of this study was to examine the radiation-induced apoptotic response of CD4(+) and CD8(+) T-cells incubated as a complete lymphocyte population. MATERIALS AND METHODS: Using a four-colour flow-cytometry method, which measures annexin-V binding to phosphatidyl serine and propidium iodide, spontaneous and radiation-induced apoptosis was measured in the total lymphocyte fraction and in CD4(+) and CD8(+) T-cell subpopulations. RESULTS: It was found that CD8(+) T-cells were more sensitive to radiation-induced apoptosis than CD4(+) T-cells at doses up to 2 Gy. The yield of radiation-induced apoptosis in the total lymphocyte fraction decreased with increasing ratios of CD4(+) to CD8(+) T-cells (CD4/CD8 ratio). By manipulating the CD4/CD8 ratio within lymphocyte cultures, it was found that the CD4/CD8 ratio had a dramatic effect on the yield of spontaneous apoptosis of total lymphocytes fraction and CD4(+) T-cells but not CD8(+) T-cells. CONCLUSION: The CD4/CD8 ratio affects the apoptotic response of human lymphocytes and CD4(+) T-cells.

Adult↗
PubMed1999

DNA distortion and multimerization: novel functions of the glutamine-rich domain of GAGA factor.

GAGA factor (GAF) is an essential protein in Drosophila melanogaster, important for the transcriptional regulation of numerous genes. The effect of GAF on chromatin structure and promoter function has been the subject of much attention, yet little is known of the actual mechanism and the specific contributions of individual GAF domains to its function. The DNA-binding activity of GAF, as specified by the single zinc finger binding domain (Zn), has been examined in some detail; however, the functions of the POZ/BTB and glutamine domain (Q) remain poorly understood. Here, we report three separate activities of the Q domain of GAF; promoter distortion, single-strand binding, and multimerization. In vitro, GAF binding to the hsp70 promoter produces extended DNase I protection and KMnO4 hypersensitivity. These activities require both the Zn domain and Q domain of GAF, and appear independent of the POZ/BTB domain. GAF also has a single-stranded DNA binding affinity, as does the Q-rich region alone. GAF forms multimers both in vitro and in vivo, and the Q domain itself forms multimers. Protein-protein interactions mediated by the Q domain may, therefore, be at least partially responsible for the multimerization capabilities of GAF. We discuss these findings in the context of their possible function in GAF mediated transcriptional regulation.

Base Sequence↗
PubMed1998

GAGA factor binding to DNA via a single trinucleotide sequence element.

GAGA transcription factor (GAF) is an essential protein in Drosophila , important for the transcriptional regulation of numerous genes. GAF binds to GA repeats in the promoters of these genes via a DNA-binding domain containing a single zinc finger. While GAF binding sites are typically composed of 3.5 GA repeats, the Drosophila hsp70 gene contains much smaller elements, some of which are as little as three bases (GAG) in length. Interestingly, the binding of GAF to more distant trinucleotide elements is relatively strong and not appreciably affected by the removal of larger GA arrays in the promoter. Moreover, a simple synthetic GAG sequence is sufficient to bind GAF in vitro . Here we directly compare the affinity of GAF for different sequence elements by immunoprecipitation and gel mobility shift analysis. Furthermore, our measures of the concentration of GAF in vivo indicate that it is a highly abundant nuclear protein, prevalent enough to occupy a sizable fraction of correspondingly abundant trinucleotide sites.

Animals↗
PubMed1998

Comparison of high dose rate, low dose rate, and high dose rate fractionated radiation for optimizing differences in radiosensitivities in vitro.

Radiotherapy is administered with the assumption that all patients respond similarly to radiation although radiosensitivity does vary from patient to patient, resulting in different degrees of early and late effects. Because the dose given to a patient is limited by the response of normal tissue in the treatment field, it would be beneficial to determine the sensitivity of this normal tissue prior to therapy. Previous studies to predict radiosensitivity have used surviving fractions after a single dose given in vitro, however, differences in cell survival at this low level of kill are not easy to resolve. In this study, we set out to evaluate the use of alternative dose regimens which may better resolve differences in radiosensitivity. We have examined several radiation protocols for predictive value, including survival after high doses (6 Gy) at both high (112 cGy/min) and low (.882 cGy/min) dose rates and after fractionated doses of 2 Gy (6 fractions). A sensitive human fibroblast line (S11358) cultured from a patient showing severe effects after therapy is compared with a cell line (OMB1) cultured from an apparently normal subject. Differences between these cell lines have been compared with those between two human melanoma cell lines (SKMEL3 and HT144) which have shown resistant and sensitive response to radiation in vitro respectively. In both fibroblast and melanoma cell lines, the difference in the survival of normal and sensitive cells increased with increasing dose regardless of whether irradiation was delivered as low dose rate, high dose rate, or as fractionated doses. We propose that radiation doses which more closely mimic clinical treatment are more suitable than surviving fraction after 2 Gy (SF2) for in vitro evaluation of relative radiosensitivities of cell populations.

Adult↗
PubMed1997

Dynamics of potentiation and activation: GAGA factor and its role in heat shock gene regulation.

GAGA factor (GAF) binds to specific DNA sequences and participates in a complex spectrum of chromosomal activities. Products of the Trithorax-like locus (Trl), which encodes multiple GAF isoforms, are required for homeotic gene expression and are essential for Drosophila development. While homozygous null mutations in Trl are lethal, heterozygotes display enhanced position effect variegation (PEV) indicative of the broad role of GAF in chromatin architecture and its positive role in gene expression.The distribution of GAF on chromosomes is complex, as it is associated with hundreds of chromosomal loci in euchromatin of salivary gland polytene chromosomes, however, it also displays a strong association with pericentric heterochromatin in diploid cells, where it appears to have roles in chromosome condensation and segregation. At higher resolution GAF binding sites have been identified in the regulatory regions of many genes. In some cases, the positive role of GAF in gene expression has been examined in detail using a variety of genetic, biochemical, and cytological approaches. Here we review what is currently known of GAF and, in the context of the heat shock genes of Drosophila, we examine the effects of GAF on multiple steps in gene expression.

Animals↗
PubMed1995

Distribution of GAGA protein on Drosophila genes in vivo.

GAGA protein binds specific CT.GA-rich DNA sequences in vitro, and many of these sequences are required for transcription in vivo. GAGA protein has been implicated in the transcription of numerous Drosophila genes, including hsp70, hsp26, actin 5C, and Ubx. Here, we examine the in vivo distribution of GAGA protein on a number of Drosophila genes that do and do not have CT-rich sequences by use of a UV cross-linking technique. Prior to heat shock, GAGA protein is associated with the promoter regions of the uninduced hsp70 and hsp26 genes. Upon heat shock induction, GAGA protein is recruited to their transcription units with its distribution coincident with that of RNA polymerase II. The recruitment of GAGA protein to the hsp70 gene after an instantaneous heat shock occurs in a 5' to 3' manner with kinetics similar to RNA polymerase. GAGA protein has been shown to disrupt nucleosome both in vivo and in vitro. We propose that GAGA protein may function in vivo both by binding constitutively to its high-affinity binding sites and by spreading through the induced gene opening the chromatin structure allowing polymerase to elongate efficiently.

Actins↗
PubMed1995

No effect of 60 Hz electromagnetic fields on MYC or beta-actin expression in human leukemic cells.

Epidemiological studies have shown weak correlations between exposure to extremely low-frequency electromagnetic fields (ELF EMFs) and the incidence of several cancers, particularly childhood leukemias, although negative studies have also been reported. These observations have prompted a broad range of in vitro cellular studies in which effects of ELF EMFs have been observed. However, no reported response has been replicated widely in independent laboratories. One potentially important response is the rapid activation of proto-oncogenes and other genes in human leukemic (HL60) cells and a wide variety of other eukaryotic cells, because of the role of these genes in cell proliferation. We describe quantitative Northern analysis of MYC and beta-actin mRNAs from HL60 cells exposed to fields under conditions very similar to those reported previously to activate these genes, namely 60 Hz sinusoidal magnetic fields of 0.57, 5.7 or 57 microT for 20 min. In addition we have used a new design of field-exposure system and introduced a number of other modifications to the protocol to optimize any response. We have also developed a novel method providing enhanced accuracy for the quantitative measurement of mRNA. No significant effect of ELF EMFs on gene expression was observed using any of these systems and analytical methods.

Actins↗
PubMedDate not supplied

Prediction of radiosensitivity by measurement of X-ray induced apoptosis in human blood using the comet assay.

BACKGROUND: The aim of this work was to determine whether levels of radiation-induced apoptosis in human peripheral leukocytes could be used as a predictor of radiosensitivity. MATERIALS AND METHODS: Peripheral blood was obtained from venous blood and exposed to 0-3 Gy of X-rays. Apoptosis levels were measured at 4, 24, 48 and 72 hours after exposure using the neutral comet assay. Intra-individual apoptotic response was measured using repeated blood samples from four healthy individuals. Inter-individual variation was investigated in whole blood, granulocytes and mononuclear cells from 8 radiotherapy patients (4 demonstrating a radiosensitive response and 4 demonstrating a normal response to radiation exposures). RESULTS: Amongst the four healthy individuals there was both inter- and intra-individual variation of about the same magnitude. However, when comparing the apoptotic response of the radiosensitive and normal patients, consistent trends were observed at all X-ray doses for all of the patients. CONCLUSION: This indicates that apoptosis has some potential as a predictive assay, however, large intra-individual variation exists. More studies are required to investigate the causes of intra-individual variation and how it might be minimized.

Aged↗