T.Robin Hesketh

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.

Some mitogens cause rapid increases in free calcium in fibroblasts

Quiescent 3T3 fibroblasts grown on microcarrier beads and loaded with the [Ca2+] indicator quin2 had a cytosolic free Ca2+ concentration ([Ca2+]i) of 154 ± 11 nM (SE; n = 32). Stimulation with the mitogens vasopressin, epidermal growth factor (EGF) or prostaglandin F2α (PGF2α) caused a very rapid increase in [Ca2+]i to a maximum of 200–500 nM after 60–90 s. [Ca2+]i declined thereafter to a level above that in quiescent cells which was maintained for at least 15 min. In contrast no immediate effects on [Ca2+]i were detected after the addition of the mitogens insulin or 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). These studies indicate that early changes in [Ca2+]i may be involved in the action on fibroblasts of some, but not all, mitogens.

Intracellular ph and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy.

Photometric fluorescence microscopy has been used to measure intracellular pH (pHi) and free calcium concentrations ([Ca]i) in individual mouse thymocytes and 2H3 rat basophil leukaemic cells containing indicators for pH (quene 1) or calcium (quin 2). The pHi and [Ca]i measurements in individual 2H3 cells and mouse thymocytes and their responses to various stimuli were consistent with the corresponding data obtained from suspensions of these cells measured in a spectrofluorimeter. Photometric fluorescence microscopy of these indicators in individual cells provides a sensitive and fast method of following pHi and [Ca]i responses in individual cells.

Limitations on the use of phenothiazines and local anaesthetics as indicators of calmodulin function in intact cells

The calcium-binding protein calmodulln is thought to regulate a wide range of cellular and enzymic functions [1,2]. The antipsychotic phenothiazine family of drugs inhibits the actions of calcium-calmodulin complexes [3], and this has led to the widespread use of these drugs as indicators of calmodulin involvement in specific biochemical and cellular functions. Drugs with local anaesthetic activity also inhibit calmodulln function in isolated systems [4,5 ]. Among the enzymes regulated by calmodulln is myosin light chain kinase, which controls the activity of cytoskeletal actomyosin ATPase. This complex is thought to be involved in many processes related to cell motility, including cap formation on lymphocytes [6,7], and as part of a study of the latter process we have tested some of the reported calmodulln antagonists. To attribute any effects of the drugs to the inhibition of calmodulin, it is necessary to show that they do not have other independent metabolic effects. In particular we have examined their effects on lymphocyte adenine nucleotide levels, since we had shown that there is a quantitative dependence of cap formation on the ATP level [8]. We have also tested the effects of the drugs on lymphocyte lactate output, since changes in adenine nucleotide levels would be expected to affect the glycolytic flux. The results indicate that each of the drugs tested can reduce lymphocyte ATP levels, greatly reducing the phosphorylation potential, expressed as log [ATP]/ [ADP]. This finding may explain many of the effects

Design of an indicator of intracellular free Na+ concentration using 19F-NMR.

The development is described of an Na+ chelator with appropriate properties for an indicator of intracellular free Na+ concentration ([Na+]i). The new indicator, FCryp-1, is a tribenzo derivative of the parent (2:2:1) cryptand structure, incorporating the same F-substituted dibenzo 19F-NMR reporter group as the free [Ca2+] indicator, 5FBAPTA (Smith, G.A., Hesketh, T.R., Metcalfe, J.C., Feeney, J. and Morris, P.G. (1983) Proc. Natl. Acad. Sci., USA 80, 7178-7182). FCryp-1 has appropriate affinity for Na+ (KNa = 10(1.3) M-1) and selectivity over other intracellular cations (KK; KCa; K Mg less than 10(-1) M(-1)) for a [Na]i indicator. There is an 19F-NMR chemical shift of 2.00 ppm between free FCryp-1 and the Na-FCryp-1 complex which provides a direct read out of free [Na+]. FCryp-1 carries four carboxylate groups to confer aqueous solubility which can be esterified with acetoxymethyl groups to render the indicator membrane permeant. Experiments on pig lymphocytes loaded with FCryp-1 gave an indicated [Na+]i of 13.8 +/- 1.8 mM (n = 4). The FCryp-1 structure can also be readily modified to provide fluorescent [Na+]i indicators.

Loss of a consensus heparin binding site by alternative splicing of latent transforming growth factor-β binding protein-1

Latent transforming growth factor‐β binding protein‐1 (LTBP‐1), plays an important role in controlling localisation and activation of transforming growth factor‐β (TGF‐β). We show that alternative splicing generates a form of mRNA which lacks bases 1277–1435 (termed LTBP‐1Δ53). The 53 amino acids encoded by these bases include the eighth cysteine of the first cysteine repeat and a consensus heparin binding sequence. Sequencing of genomic clones showed that alternative splicing resulted from the use of an intra‐exonic 3′ splice acceptor site. The loss of the heparin binding site implies that LTBP‐1Δ53 will bind to the extracellular matrix less efficiently than LTBP‐1.

Expression of alternatively spliced human latent transforming growth factor β binding protein-1

Latent transforming growth factor β binding protein‐1 (LTBP1) is important in regulating the localisation and activation of transforming growth factor β (TGFβ). Three forms of LTBP1 mRNA have previously been described, LTBP1L, LTBP1S and LTBPΔ53. Here, we have analysed the LTBP1 coding sequence and identified two other spliced forms, LTBP1Δ55 and LTBP1Δ41. LTBP1Δ55 is a short form of LTBP1L which lacks 55 amino acids including two consensus N‐glycosylation sites and LTBP1Δ41 is a form of LTBP1 which lacks the 12th EGF‐like repeat. Furthermore, sequencing of genomic clones showed that splicing to generate LTBP1L occurs using an intra‐exonic 3′ splice acceptor site in the first coding exon of LTBP1S and that LTBP1Δ55 arises from the alternative use of an exonic 3′ splice acceptor site at the end of the following intron. LTBP1Δ41 arises from skipping the exon which encodes the 12th EGF‐like repeat. LTBP1Δ55 and LTBP1Δ41 mRNA are expressed in a wide variety of human tissues but the proportions of each splice form vary in the tissues.

C-fos gene activation in murine thymocytes by a mechanism independent of protein kinase C or a Ca2+ signal

The accumulation of c‐fos mRNA in mouse thymocytes was compared when the cells were stimulated by concanavalin A (Con A), the Ca2+ ionophore A23187 or the phorbol ester, TPA, either separately or by combinations of these mitogens. The c‐fos response to mitogenic concentrations of Con A could not be attributed either to the rise in [Ca2+]i it induces or to activation of protein kinase C. Thus, although Con A causes the breakdown of phosphatidylinositol 4,5‐bisphosphate in these cells, neither of the signals which can be generated by this response was responsible for the activation of the c‐fos gene by Con A. This implies that some other unidentified signal generated by Con A activates the c‐fos gene.

GTPγS activation of proto-oncogene expression in transiently permeabilised Swiss 3T3 fibroblasts

A technique of transient permeabilisation has been used to show that the introduction of guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS), a non‐hydrolysable analogue of GTP, into intact Swiss 3T3 fibroblasts stimulates phosphoinositide hydrolysis, cyclic AMP accumulation and the activation of c‐fos and c‐myc proto‐oncogenes. Of a number of nucleotide triphosphates introduced into the cells, only GTP and its non‐hydrolysable analogues activated inositol phosphate release, suggesting that this response is mediated by guanine nucleotide regulatory (G) protein(s). The data demonstrate that transient permeabilisation provides a method of examining the involvement of G‐proteins in nuclear activation.